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					                                                                Contents


Sponsors		......................................................................................................................................	4
Chairman message	......................................................................................................................	5
                .
Local Organizing Committee	 ......................................................................................................	6
                          .
Scientific Advisory Committee	 ...................................................................................................	7
                             .
Student Poster Evaluation Committee	.......................................................................................	7
General Information	.....................................................................................................................	8

Program Day 1, Wednesday, August 15th	.................................................................................	10	
           Abstracts	for	VIC	Workshops	and	Opening	Conference	..................................................	14	

Program for Day 2, Thursday, August 16th	...............................................................................	18	
           Abstracts	for	Presentations	Day	2	....................................................................................	21

Program for Day 3, Friday, August 17th	....................................................................................	26
                                      .
           Abstracts	for	Presentations	Day	3	....................................................................................	31

Program for Day 4, Saturday, August 18th	................................................................................	35	
           Abstracts	for	Speakers	Day	4	...........................................................................................	40

Program for Day 5, Sunday, August 19th	..................................................................................	44
           Abstracts	for	Speakers	Day	5	...........................................................................................	49


Abstracts for Posters Presentations	........................................................................................	52	

Author index 	............................................................................................................................	146

Cross references for key words	 .............................................................................................	153
                              .

Cross reference for species	.....................................................................................................158	

List of participants		..................................................................................................................	159
4




    Sponsors
	      Dear	Veterinary	Immunologists,
	
	      The	Brazilian	Society	for	Immunology	welcomes	you	to	the	8th	International	Veterinary	Immunology	
Symposium.	Participants	from	more	than	35	countries	congregate	in	Ouro	Preto	to	share	knowledge	and	
experience.	


	      The	scientific	program,	the	heart	of	the	8th	IVIS,	is	the	result	of	the	efforts	of	the	Scientific	Advisory	
Committee,	of	the	Chairs	for	the	scientific	sessions	and	of	the	Local	Organizing	Committee.	We	are	truly	
thankful	for	our	colleagues’	insights	and	time,	which	produced	a	comprehensive	and	exciting	program.

	      We	wish	all	participants	a	very	fruitful	meeting.




                                                                      Isabel	K.	F.	de	Miranda	Santos
                                                                                          Chair

                                                                             Beatriz	Rossetti	Ferreira
                                                                                       Vice-Chair
6




                                Local Organizing Committee


                               Isabel K.F. de Miranda Santos
      Faculdade	de	Medicina	de	Ribeirão	Preto,	Universidade	de	São	Paulo,	Ribeirão	Preto
                                Beatriz Rossetti Ferreira
      Escola	de	Enfermagem	de	Ribeirão	Preto,	Universidade	de	São	Paulo,	Ribeirão	Preto
                                    Maristela Martins Camargo
             Instituto	de	Ciências	Biomédicas,	Universidade	de	São	Paulo,	São	Paulo
                                Rosangela Zacarias Machado	
    Faculdade	de	Ciências	Agrárias	e	Veterinárias,	Universidade	Estadual	Paulista,	Jaboticabal	
                                 Alexandre Barbosa Reis	
            Faculdade	de	Farmácia,	Universidade	Federal	de	Ouro	Preto,	Ouro	Preto
                               Alexandre Rodrigues Caetano	
                      Embrapa	Recursos	Genéticos	e	Biotecnologia,	Brasília
                              Aline Aparecida Rezende Rodrigues	
                                      Vallée	SA,	São	Paulo
                                Ana Paula Junqueira-Kipnis	
           Faculdade	de	Medicina	Veterinária,	Universidade	Federal	de	Goiás,	Goiânia
                                   Itabajara da Silva Vaz Jr.
        Centro	de	Biotecnologia,	Universidade	Federal	do	Rio	Grande	do	Sul,	Porto	Alegre
                                   José Roberto Kfoury Jr.	
            Faculdade	de	Medicina	Veterinária,	Universidade	de	São	Paulo,	São	Paulo
                                 Lygia Maria Friche Passos	
            Faculdade	de	Medicina	Veterinária,	Universidade	Federal	de	Minas	Gerais
                                    Maria Julia B. Flaminio
                                    Cornell	University,	Ithaca
                                    Ricardo T. Fujiwara
                            Fundação	Oswaldo	Cruz,	Belo	Horizonte
                                 Ricardo Tostes Gazzinelli
                            Fundação	Oswaldo	Cruz,	Belo	Horizonte
                                       Valeria M.F. Lima	
                            Universidade	Estadual	Paulista,	Araçatuba
                                                                                          7




                                Scientific Advisory Committee


    Bruce N. Wilkie	                   University	of	Guelph,	Guelph,	Canada
    Caroline Fossum	                   SUAS,	Uppsala,	Sweden
    Chieko Kai	                        University	of	Tokyo,	Tokyo	Japan
    Jayne C. Hope	                     Institute	for	Animal	Health,	Compton,	UK
    Falko Steinbach	                   VLA,	EVIG,	Addlestone,	UK
    Isabelle Schwartz-Cornil	          INRA,	Jouy-en-Josas,	France
    Jan Naessens	                      VIC,	ILRI,	Nairobi,	Kenya
    Javier Dominguez	                  INIA	,	Madrid,	Spain
    Joan K. Lunney	                    ARS-USDA,	Beltsville,	USA
    Mark Jutila	                       MSU,	Bozeman,	USA
    Paul Wood                          Pfizer,	West	Ryde,	Australia
    Ricardo T. Gazzinelli	             FioCruz,	Belo	Horizonte,	Brazil
    Wendy C. Brown	                    WSU,	Pullman,	USA
    Wilmar Dias da Silva	              UENF,	Campos	de	Goitacazes,	Brazil




                         Student Poster Evaluation Committee


    Beatriz Rossetti Ferreira	         Chair,	University	of	São	Paulo,	Brazil
    Ana Paula Junqueira-Kipnis	        Federal	University	of	Goiás,	Brazil
    Joan K. Lunney	                    USDA-ARS,	USA
    David Artis	                       University	of	Pennsylvania,	USA
    Ildiko van Rhijn	                  Utrecht	University,	The	Netherlands
    Isabelle Oswald	                   National	Agricultural	Research	Institute,	France
    Juan Anguita	                      University	of	Massachusetts,	USA
    Maristela Martins Camargo	         University	of	São	Paulo,	Brazil
    Preben Boysen	                     National	Veterinary	Institute,	Norway	
    Ricardo Fujiwara	                  Oswaldo	Cruz	Foundation,	Brazil	
    Waithaka Mwangi	                   Texas	A&M,	USA
	   Rosangela Zacarias Machado         Universidade	Estadual	Paulista
8




                                              General Information


    n	Identification   Badges	are	required	for	access	to	all	symposium	activities.
    n	The Registration Desk	is	located	at	the	entrance	to	the	Poster	Exhibit	Hall	(Salão	Diamante).
      o	Staff	are	ready	to	help	you	and	are	identified	by	their	badges	and	uniforms	(white	shirt	with	the	
        symposium	logo).
    n	Participant Materials include	identification	badge,	a	book	and	CD	with	the	complete	program	and	
      abstracts	and	a	certificate	of	participation.	
    n	Transportation	from	Ouro	Preto	to	the	Convention	Center	located	at	the	Estalagem	das	Minas	Gerais	
      Hotel.
      o	The	shuttles	from	Ouro	Preto	to	the	Convention	Center	are	offered	by	the	event	organizers	and	
        operated	by	Blumar	/	Master	Turismo.
      o	All	Shuttles	are	identified	by	signs	with	the	8th	IVIS	LOGO.

      o	The	distance	between	Ouro	Preto	and	the	Convention	center	is	about	7	Kilometers	(5	Miles).

      o	The	shuttle	routes	will	depart	from	the	following	locations	and	by	the	following	schedule.

        Route	1	–	Grande	Hotel	de	Ouro	Preto.
        Route	2	–	Colonial	Hotel.
        Route	3	–	Pousada	do	Mondego.
        Route	4	–	Casa	dos	Contos	Hotel	and	Pousada	dos	Sinos.
        Route	5	–	Tiradentes	Square	(Praça	Tiradentes,	in	the	Historical	Center)	and	Pousada	Sinhá	
        Olímpia;
      o	Schedules	for	the	shuttles:
        August	15th	(Wednesday)
         o	Departure:	08:00h	/	08:20h	/	08:40h	/	09:10h	/	09:20h	/	10:00h

         o	Return:	19:30h	/	19:50h	/	20:10h	/	20:25h	/	20:40h	/	20:50h	/	21:15h	/	21:30h


        August	16th	(Thursday)
         o	Departure:	07:30h	/	07:50h	/	08:10h	/	08:40h	/	08:50h	/	09:30h

         o	Return:	19:00h	/	19:20h	/	19:40h	/	19:55h	/	20:10h	/	20:20h	/	20:40h	/	21h00h


        August	17th	(Friday)
         o	Departure:	07:30h	/	07:50h	/	08:10h	/	08:40h	/	08:50h	/	09:30h

         o	Return:	18:30h	/	18:50h	/	19:10h	/	19:25h	/	19:40h	/	19:50h	/	20:00h	/	20:20h	/	20:40h	/	21:00h	/	


           21:20h	/	21:40h	/	22:00h
        August	18th	(Saturday)
         o	Departure:	07:30h	/	07:50h	/	08:10h	/	08:40h	/	08:50h	/	09:30h

         o	Return:	18h00h	/	18:20h	/	18:40h	/	18:55h	/	19:10h	/	19:20h	/	19h40h	/	20:00h


        August	19th	(Sunday)
         o	Departure:	07:40h	/	08:00h	/	08:20h	/	08:50h	/	09:00h	/	09:30h

         o	Return:	19:00h	/	19:20h	/	19:40h	/	19:55h	/	20:10h	/	20:20h	/	20:40h	/	21:00h


      o	A taxi ride	(one	way)	between	the	Convention	Center	and	the	main	hotels	in	the	Ouro	Preto	
        Historical	Center	costs	approximately	R$	25,00	(USD	13.00).
                                                                                                                9




n	The  Media desk	is	located	in	the	Salão	Ágata	(lower	floor)	and	is	open	daily	from	07:50	to	18:00h.	
  Speakers	are	kindly	requested	to	deliver	their	files	at	least	2	hours	in	advance	of	their	presentation	or,	
  in	the	case	of	sessions	beginning	at	8:30	am,	the	day	before.
n	Lunch
  o	Box	lunches	with	fruit	juice	will	be	sold	at	the	convention	center	for	R$	11,20	(approximately	USD	
    5.60).	Soft	drinks,	beer,	coffee,	tea	and	mineral	water	will	be	sold	separately.
  o	A	buffet	with	salads	and	hot	dishes	is	available	at	the	restaurant	located	at	the	Hotel	Estalagem	das	
    Minas	Gerais	(600	m	from	the	Convention	Center).	Each	meal	costs	R$	22,00	(approximately	USD	
    11.00)	with	juice,	soft	drinks	or	mineral	water	or	R$	28,00	(approximately	USD	14.00)	with	beer.	The	
    restaurant	has	limited	seating	capacity.
n	Lost   and Found	items	will	be	handled	by	the	Registration	Desk.
n	Bulletin   Boards	for	Messages	and	Announcements	are	located	in	the	Poster	Exhibit	Hall.
n	Internet	is	complimentary	and	wireless	access	is	available	at	the	Convention	Center.	Computers	
  linked	to	internet	are	located	in	the	Salão	Ágata	(lower	floor).
n	Blumar Agency	desk	is	located	in	the	Salão	Ágata	(lower	floor).	Transfers	from	Ouro	Preto	to	Belo	
  Horizonte	may	be	scheduled	with	Blumar	Agency.
n	Certificates
  o	Participation	certificates	will	be	included	in	the	Symposium	materials	handed	out	at	registration.

  o	Certificates	for	poster	presentations	will	be	delivered	by	the	Symposium	staff	during	the	Poster	
    Sessions.
  o	Certificates	for	speakers	at	plenary	and	concurrent	sessions	will	be	delivered	by	the	Session	Chairs.
Day 1 - Wednesday, August 15th




                                                                   Day 1 - August 15th - Wednesday

                                 	 8:30	-	20:30	h	    	 	 	 	 	 	 	 Registration	and	Poster	setup	(all	posters)

                                 	 11:00	-	18:30	h	   Veterinary	Immunology	Committee	(VIC)	Workshops	             SALÃO OURO
                                 	                	   All	registered	participants	are	welcome.

                                 	 19:30	-	20:30	h	   Opening of the 8th IVIS and	                                 SALÃO OURO
                                                      Opening Conference

                                 	        20:30	h	    Welcome	reception	                                          SALÃO DIAMANTE
                                                                                                      11




                                                                                                      Day 1 - Wednesday, August 15th
                                                                                         SALÃO OURO
	 1:00	-	13:00	h	 VIC Workshop: NK cells
1
	               	 Chairs:	Anne	K.	Storset	-	Norway	and	Armin	Saalmüeller	-	Austria	

	 11:00	-	11:50	h	 Basic Functions and Definitions
	              	 Anne	K.	Storset
	              	 Norwegian	School	of	Veterinary	Science,	Norway
                 NK cells - a brief overview. The role of NK cells in the immune system
                 Definitions of NK cells.
	              	 Preben	Boysen
	              	 National	Veterinary	Institute,	Norway
                 Characteristics and functions of natural killer cells in the cow.
		             	 Abstract	and	Poster	PR170,	VA211
	              	 Reginaldo	Bastos
	              	 Washington	State	University,	USA
                 Interaction of natural killer cells, monocytes and dendritic cell populations
                 in cattle
                 Abstract	and	Poster	VA219
		             	 Tim	Connelley
	              	 University	of	Edinburgh,	UK
                 In vitro maintenance of bovine NK cells by culture with Theileria-infected
                 lymphocytes
                 Abstract	and	Poster	AP127
	              	 Maša	Pintarič
	              	 University	of	Veterinary	Medicine	Vienna,	Austria
                 Synergistic effects of IL-2, IL-12 and IL-18 on cytolitic activity and IFN-γ
                 production of porcine natural killer cells
               	 Abstract	and	Poster	AP132
	 11:50	-	12:10	h	 NK- cells: Role in infections
	                	 Bryce	Buddle
	                	 AgResearch,	New	Zealand
                   Interactions between NK cells and Mycobacterium bovis.
		             	 Daniela	Dantas	Moré
	              	 University	of	São	Paulo,	Brazil
                 Tick infestations affect subpopulations of peripheral blood lymphocytes
                 Abstract	and	Poster	PR157
	 12:15	-	12:50	h	 NK cell receptors	
	              	 Shirley	Ellis
                 Institute	for	Animal	Health	at	Compton,	UK	
	              	 KIR receptors in cattle
		             	 Liliana	Jaso-Friedmann
	              	 Georgia,	USA
                 A novel PRR on teleost NK cells and its function in antibacterial immunity
		             	 Harris	Lewin
	              	 University	of	Illinois	at	Urbana-Champaign,	USA
                 Organization and Evolution of Cattle ULBP Genes
	 12:50	-	13:00	h	 Conclusions
	              	 Armin	Saalmüeller
	              	 University	of	Veterinary	Medicine	Vienna,	Austria
	              	 Summing up and future perspectives
	 13:00	-	14:00	h Lunch
                 12
Day 1 - Wednesday, August 15th




                                                                                                                        SALÃO OURO
                                 14:00	-	16:00h		 VIC Workshop: Comparative MHC: levels of diversity and mechanisms
                                                  involved in its generation
                                 	              	 Chairs:	Shirley	Ellis	-	UK	and	Declan	McKeever	-	UK

                                 	              	 Shirley	Ellis
                                 	              	 Institute	for	Animal	Health,	UK;	chair	of	ISAG/VIC	Comparative	MHC	
                                 	              	 nomenclature	committee
                                                  Introduction and background
                                 	              	 Wendy	Brown	
                                 	              	 Washington	State	University,	USA
                                                  Direct detection of antigen-specific CD4+ T cells in peripheral blood with
                                                  bovine DRB3 tetramers
                                                  Abstract	HC001
                                 	              	 Rebecca	Baxter
                                 	              	 Roslin	Institute,	UK
                                                  Quantifying the contribution of BoLA-DRB3 to immune responses in a cattle
                                                  cross-population
                                                  Abstract	and	Poster	AP126
                                 	              	 Chris	Davies
                                 	              	 Washington	State	University,	USA
                                                  A million years of evolution in the bovine MHC class I region
                                 	              	 Keith	Ballingall
                                 	              	 Moredun	Research	Institute,	UK
                                                  Characterisation of diversity within the class I and II regions of four ovine MHC
                                                  haplotypes
                                                  Hirohide	Uenishi	
                                 	              	 NIAS,	Japan
                                                  Differences of genomic structure among haplotypes of the SLA region
                                                  Abstract	and	Poster	IG018
                                 	 16:00	-	16:30	h	 Coffee Break

                                 16:30	-	18:30	h	 	 VIC Toolkit Workshop: The generation, maintenance and availability of
                                                  reagents and technologies to study immune function in veterinary species	
                                 	              	 Chairs:	Gary	Entrican	-	UK	and	Victor	Rutten	-	The	Netherlands
                                 	 16:30	-	16:40	h	 Gary	Entrican	
                                 	              	 Moredun	Institute,	UK	
                                 	              	 Victor	Rutten	
                                 	              	 Utrecht	University,	Netherlands	
                                                  Introduction and Workshop Objectives
                                 	 16:40	-	17:00	h	 Jim	Kaufman
                                 	              	 University	of	Cambridge,	UK
                                                  The BBSRC/SEERAD Immunological Toolbox
                                 	              	 Abstract	TK001
                                                                                                  13




                                                                                                  Day 1 - Wednesday, August 15th
	 17:00	-	17:20	h	 Cynthia	Baldwin
	              	 University	of	Massachusetts,	USA
                 The US Veterinary Immune Reagent Network
                 Abstract	and	Poster	TK004
		17:20	-	17:40	h	 Edwin	Tijhaar
	                	 Utrecht	University,	Netherlands
                   The Cytokine Centre: Developing Tools for Research in Veterinary Immunology
                   Abstract	and	Poster	TK007
		17:40	-	18:00	h	 Harry	Dawson
	                	 USDA-ARS,	Maryland,	USA
                   The Porcine Immunology and Nutrition Resource Database
	                	 Abstract	and	Poster	TK005
		18:00	-	18:30	h Open Discussion
	
	              	 Tool Kit Workshop poster viewing: Friday 17 August, 8:30 – 10:30h




                                                                                     SALÃO OURO
	 19:30	-	19:45	h	 Opening of the 8th IVIS
                 Isabel	de	Miranda	Santos,	Chair	8th	IVIS

	 19:45	-	20:30	h		 Opening Conference
                 Speaker:	Julio	Scharfstein	
	              	 Federal	University	of	Rio	de	Janeiro,	Brazil
                 Bradykinin B2 receptors of dendritic cells: an innate pathway that promotes
                 development of CD8 effector T cells.




                                                                             SALÃO DIAMANTE
	       20:30	h	 Welcome Reception
                 14
Day 1 - Wednesday, August 15th



                                                                   AbstrACts for PresentAtions And tool Kit Posters


                                                                                      ViC WorKsHoP ComPArAtiVe mHC

                                  HC001. IMPROVED SENSITIVITy ENABLES DETECTION                                   T	 cells	 in	 PBMC.	To	 improve	 the	 sensitivity	 of	 detecting	 antigen	
                                   OF ANTIGEN-SPECIFIC CD4+ T CELLS STAINED WITH                                  specific	T	cells	in	peripheral	blood,	we	adopted	a	procedure	that	
                                    BOVINE DRB3 CLASS II TETRAMERS DIRECTLy IN                                    enriches	 tetramer	 positive	 cells	 prior	 to	 FACS	 analysis.	The	 tet-
                                      PERIPHERAL BLOOD MONONUCLEAR CELLS                                          ramers	were	formed	by	binding	biotinylated	DRB3/DRA	heterodi-
                                                                                                                  mers	 to	 phycoerythin	 (PE)-labeled	 streptavidin.	 PBMC	 obtained	
                                   WEnDy	C	BRoWn1,	WAITHAKA	MWAnGI2,	yAn	ZHUAnG1,	
                                                                                                                  from	calves	immunized	with	the	30-mer	F2-5	epitope	of	the	major	
                                           GUy	H	PALMER1,JUnZo	noRIMInE1
                                                                                                                  surface	protein	(MSP)1a	of	A.	marginale	were	labeled	with	DRB3	
                                      1Veterinary	Microbiology	and	Pathology,	Washington	State	                   *1101	tetramer	linked	to	the	minimal	peptide	epitope	F2-5B.	Cells	
                                      University,	Pullman,	WA	99164;	2Veterinary	Pathobiology,	                   were	washed	and	incubated	with	FITC-Mab	specific	for	CD4,	with	
                                         Texas	A	&	M	University,	College	Station,	TX	77843                        Alexa-fluor-647-conjugated	Mabs	specific	for	CD14,	CD8,	CD21,	
                                       We	 have	 previously	 reported	 the	 use	 of	 BoLA	 DRB3	 *1101	           and	 TcR	 delta	 chain,	 and	 with	 7-AAD	 (which	 labels	 dead	 cells)	
                                 and	 *1201	 tetramers	 to	 enumerate	 T	 cells	 specific	 for	 peptide	          and	 tetramer-positive	 cells	 were	 selected	 by	 binding	 to	 anti-PE	
                                 epitopes	 of	 the	 cattle	 pathogen	 Anaplasma	 marginale	 in	 popu-             magnetic	 beads.	 Cells	 were	 then	 analyzed	 by	 four-color	 flow	
                                 lations	 of	 immune	 lymphocytes	 stimulated	 with	 antigen	 ex	 vivo	           cytometry.	 This	 method	 permitted	 detection	 of	 0.004	 to	 0.1%	 of	
                                 for	at	least	one	week	(Norimine	et	al.,	Immunogenetics,	58:726).	                CD4+	T	cells	that	were	tetramer-positive	and	obtained	at	different	
                                 However,	because	the	frequency	of	MHC	class	II	restricted	T	cells	               times	after	immunization	and	boosting.
                                 is	 very	 low,	 the	 tetramers	 were	 unable	 to	 directly	 detect	 CD4+	


                                                                ViC tool Kit:AbstrACts for PresentAtions And Posters

                                                                  Poster PresentAtions: fridAy, August 17tH, 8:30-10:30H

                                       tK001. THE BBSRC/SEERAD IMMUNOLOGICAL                                      molecules,	we	screened	commercially	available	monoclonal	antibod-
                                                      TOOLBOx                                                     ies	 (mAbs)	 for	 reactivity	 to	 conserved	 epitopes,	 by	 flow	 cytometry.	
                                 JIM	KAUFMAn1,	PETE	KAISER1,	JAynE	HoPE1,	PAUL	SoPP1,	                            Equine	 whole	 blood	 leukocytes	 and	 peripheral	 blood	 mononuclear	
                                  SHIRLEy	ELLIS1,	DUnCAn	HAnnAnT2,	JULIA	KyDD2,	CoLIn	                            cells	 (PBMC)	 were	 stained	 with	 mAbs	 to	 defined	 equine	 leukocyte	
                                     MCInnES3,	DECLAn	MCKEEVER3,GARy	EnTRICAn3                                    surface	antigens,	with	the	mAbs	of	interest	or	with	isotype	controls.	
                                                                                                                  The	whole	blood	was	labeled	in	PBS	at	room	temperature,	while	the	
                                    1Institute	for	Animal	Health,	Compton,	Berkshire,	RG20	7NN;	                  PBMC	were	stained	in	PBS-BSA-NaN3	at	4°C.	The	cytometry	gates	
                                    2Animal	Health	Trust,	Lanwades	Park,	Kentford,	Newmarket,	                    were	 defined	 on	 the	 expression	 of	 equine	 CD172	 (monocytes	 and	
                                 Suffolk	CB8	7UU;	3Moredun	Research	Institute,	Pentlands	Science	                 granulocytes),	CD14	(monocytes)	and	CD5	(lymphocytes).	The	fol-
                                                Park,	Bush	Loan,	Midlothian,	EH26	0PZ	                            lowing	mAbs	labeled	less	than	1%	cells	from	either	gate:	anti-mouse	
                                       Progress	in	understanding	and	utilising	the	immune	response	of	            CD8	 (clone	 KT14),	 human	 CD11a	 (HI111)	 and	 ruminant	 γδ-TCR	
                                 livestock	animals	to	pathogens	and	vaccines	has	long	been	limited	               (CACTB6A)	and	activation	molecule	2	(CACT63A).	MAbs	specific	to	
                                 by	 lack	 of	 appropriate	 reagents.	 There	 is	 a	 particular	 gap	 in	 suit-   mouse	CD4	(YTS19-1.1),	F4/80	(Cl:A3-1,	macrophages)	and	Ly-6G	
                                 able	reagents	to	identify,	isolate	and	manipulate	immune	molecules	              (1A8,	neutrophils)	labeled	horse	leukocytes,	but	very	differently	from	
                                 (such	as	cytokines)	and	immune	cell	populations	(including	antigen-              mouse	 cells.	 These	 reagents	 are	 unlikely	 to	 recognize	 homologue	
                                 presenting	 cells	 and	 antigen-specific	 T	 cells).	 The	 Immunological	        molecules.	However,	the	anti-human	CD49d	mAb	9F10	usually	rec-
                                 Toolbox	 consortium,	 funded	 by	 BBSRC	 and	 SEERAD,	 addresses	                ognized	more	than	50	%	lymphocytes	and	monocytes.	Interestingly,	
                                 the	problem	by	a	co-ordinated	effort	to	develop	reagents	(other	than	            the	 anti-human	 CD38	 mAb	 HIT2	 labeled	 15	 to	 45%	 whole	 blood	
                                 those	 for	 functional	 genomics)	 necessary	 to	 study	 protective	 and	        lymphocytes,	but	up	to	twice	as	many	PBMC.	Similarly,	lymphocytes	
                                 pathological	responses	in	horses,	cattle,	sheep,	pigs	and	chickens.	             were	stained	by	mAbs	3G8	(5-19%)	and	CD28.2	(0.5-7%),	to	human	
                                 The	consortium	is	organised	and	led	by	three	institutes	with	a	history	          CD16	and	CD28,	only	in	the	presence	of	sodium	azide.	The	weak-
                                 of	work	in	reagent	development:	the	Institute	for	Animal	Health,	the	            ness	or	the	absence	of	staining	of	whole	blood	leukocytes	was	prob-
                                 Animal	Health	Trust	and	the	Moredun	Research	Institute.	Currently,	              ably	due	to	antigen	internalization.	The	anti-human	CD16,	CD38	and	
                                 a	 large	 number	 of	 people	 contribute	 to	 the	 consortium	 at	 various	      CD49d	 mAbs	 could	 recognize	 homologue	 molecules	 in	 the	 horse.	
                                 levels,	working	on	several	species	and	in	areas	of	technology.	These	            Biochemical	characterization	(by	immunoprecipitation)	and	staining	
                                 include	sheep,	horses,	cattle,	chickens,	development	of	tetramer	and	            of	leukocyte	sub-populations	are	currently	underway	to	confirm	their	
                                 co-stimulatory	 reagents	 (including	 for	 pigs),	 and	 website/database	        identity.
                                 development.	A	 website	 backed	 by	 a	 growing	 database	 has	 been	            Key words:	 leukocyte	 antigens,	 monoclonal	 antibodies,	 cross-
                                 constructed	(http://www.immunologicaltoolbox.co.uk/).	                           species	reactivity
                                 Key words:	 leukocyte	 antigens,	 monoclonal	 antibodies,	 cross-                Species:	domestic	horse	(Equus	caballus)
                                 species	reactivity                                                               Session:	VIC	Tool	Box	workshop
                                 Species:	all
                                                                                                                       tK003. THE IMMUNOLOGICAL TOOLBOx: OVINE
                                        tK002. SCREENING OF COMMERCIALIZED                                                            REAGENTS
                                      MONOCLONAL ANTIBODIES FOR CROSS-SPECIES                                              SEAn	WATTEGEDERA,	DonnA	WATSon,	
                                     REACTIVITy ON EQUINE WHOLE BLOOD AND PBMC                                       CATHERInE	JEPSon,	DAVID	DEAnE,	KEITH	BALLInGALL,	
                                             CATHERInE	MéRAnT,DAVID	W	HoRoHoV                                        DECLAn	MCKEEVER	CoLIn	MCInnES,GARy	EnTRICAn
                                    Department	of	Veterinary	Science,	University	of	Kentucky,	108	                 Moredun	Research	Institute,	Pentlands	Science	Park,	Bush	Loan,	
                                     Gluck	Equine	Research	Center,	Lexington,	KY	40546,	USA.	                                         Midlothian,	EH26	0PZ.		
                                                    catherine.merant@uky.edu                                                   sean.wattegedera@moredun.ac.uk
                                       In	 order	 to	 find	 new	 reagents	 against	 equine	 leukocyte	 mark-           Investigation	of	immune	responses	in	sheep	has	been	restricted	
                                 ers,	 lymphocyte	 sub-populations	 and	 costimulatory	 or	 adhesion	             by	 a	 lack	 of	 appropriate	 reagents	 compared	 to	 those	 available	 to	
                                                                                                                                                                       15




                                                                                                                                                                       Day 1 - Wednesday, August 15th
study	immune	responses	in	laboratory	rodents	and	in	humans.	The	                    tK005. THE PORCINE IMMUNOLOGy AND NUTRITION
objective	 of	 this	 project	 is	 to	 develop	 tools	 and	 reagents	 to	 allow	                  RESOURCE DATABASE
more	detailed	studies	of	immune	responses	in	sheep,	with	particular	                   HARRy	D	DAWSon,	CATHERInE	A	GUIDRy,	VAnDAnA	
reference	 to	 responses	 that	 control	 viral	 infections.	 The	 SEERAD	                     VAnGIMALLA,	JoSEPH	F	URBAn	JR
component	 of	 the	 Immunological	 Toolbox	 funds	 a	 post	 at	 MRI	 to	
assist	in	the	development	of	tools	and	reagents	to	study	immunol-                    United	States	Department	of	Agriculture,	Agricultural	Research	
ogy	in	sheep.	The	MRI	project	has	focussed	on	the	development	of	                    Service,	Beltsville	Human	Nutrition	Research	Center,	Nutrient	
reagents	and	techniques	that	facilitate	studies	on	immune	activation	                Requirements	and	Functions	Laboratory,	Beltsville,	Maryland,	
and	immune	regulation.	This	includes:                                                                          USA	20705.		
                                                                                                       harry.dawson@ars.usda.gov
   1.	Expression	of	ovine	recombinant	cytokines	and	other	immu-
nomodulatory	molecules	such	as	Indolamine	2,3-dioxygenase.                               Diverse	 genomics-based	 databases	 have	 developed	 to	 facili-
                                                                                  tate	research	with	human	and	rodent	models.		Current	porcine	gene	
    2.	 Development	 of	 quantitative	 assays	 to	 measure	 cytokine	             databases,	 however,	 lack	 the	 nutritional	 and	 immunological	 orien-
production	at	the	molecular	and	protein	level.                                    tation	 and	 robust	 annotation	 to	 design	 effective	 molecular	 tools	 to	
    3.	 Generation	 and	 characterisation	 of	 a	 flock	 of	 MHC	 class	 I-       study	relevant	pig	models.		To	address	this	need,	a	comprehensive	
typed	sheep.                                                                      literature-based	 survey	 was	 conducted	 that	 first	 identified	 genes	
     Progress	to	date	 has	been	the	 expression	 of	 ovine	 IL-1β	 and	           related	 to	 nutrition	 and	 which	 were	 associated	 with	 macro-	 and	
ovine	IL-8	as	markers	of	innate	immunity.	Mice	have	been	immun-                   micronutrient	 metabolism,	 atherosclerosis,	 diabetes,	 and	 obesity.	     	
ised	 with	 these	 cytokines.	 Five	 monoclonal	 antibodies	 (mabs)	 to	          It	 also	 selected	 genes	 related	 to	 immunology	 and	 associated	 with	
IL-8	and	two	mabs	to	IL-1β	are	currently	being	characterised.	The	                allergy	 and	 asthma;	 chemokines,	 cytokines,	 and	 their	 receptors;	
established	 cytokine	 expression	 technology	 at	 MRI	 has	 also	 been	          dendritic,	 mast	 and	 NK	 cells;	 type	 1	 IFN-induced	 proteins;	 inflam-
used	to	express	bovine	IL-4	and	IL-10,	generating	added	value	from	               mation;	toll	receptor	signaling	pathways;	and	B	and	T	cell	activation	
the	Toolbox	consortium	with	our	partners	at	IAH.	Reciprocally,	mabs	              and	 development.	 The	 process	 identified	 3,035	 candidate	 genes	
produced	 to	 bovine	 cytokines	 at	 IAH	 have	 been	 incorporated	 into	         used	to	select	potential	porcine	homologues	by	searching	multiple	
assays	to	measure	ovine	cytokines.	Various	expression	systems	for	                online	sources	of	porcine	gene	information.	We	then	cloned	full	or	
recombinant	cytokines	have	also	been	investigated	with	our	collabo-               partial	 length	 sequences	 for	 85	 missing	 high	 priority	 target	 genes,	
rators	 at	AHT.	 Quantitative	 real-time	 RT-PCR	 has	 been	 developed	           and	developed	real	time	PCR	assays	for	1,350high	priority	genes	of	
to	determine	the	kinetics	of	mRNA	expressing	ovine	IFN-γ	(inflam-                 particular	interest.	As	a	result,	the	database	also	contains	compre-
matory)	and	IL-10	(regulatory)	in	antigen-specific	ovine	T	cells.	This	           hensive	 information	 on	 antibody	 availability	 and	 published	 porcine	
has	been	compared	to	protein	expression	as	measured	by	ELISA.	                    gene	and	protein	expression	data.	This	unique	database	links	gene	
Sheep	 homozygous	 for	 four	 MHC	 class	 I	 haplotypes	 have	 been	              expression	to	gene	function,	identifies	related	gene	pathways,	and	
established.	Mice	have	been	immunised	with	leukocytes	from	these	                 connects	to	other	porcine	gene	databases.	
MHC-defined	 sheep	 and	 fusions	 are	 being	 conducted	 to	 produce	             Key words:	cytokines,	reagents
haplotype-specific	 mabs.	The	Toolbox	initiative	has	both	added	 to,	             Species:	swine
and	benefited	from,	the	existing	infrastructure	in	ovine	immunology	
at	MRI.	                                                                            tK006. SWINE TOOLKIT PLANS AND PROGRESS FOR
                                                                                     THE US VETERINARy IMMUNE REAGENT NETWORK
tK004. US VETERINARy IMMUNE REAGENT NETWORK:                                        JoAn	K	LUnnEy1,	PATRICIA	BoyD1,	DAnTE	ZARLEnGA2,	
           PRIORITIZATION & PROGRESS                                              FEDERICo	ZUCKERMAnn3,	WILLIAM	SCHnITZLEIn3,	JoAnnA	
    C	BALDWIn*1,	SJ	BLACK1,	J	LUnnEy2,	H	LILLEHoJ2,	J	                                LABRESH4,	BETTInA	WAGnER5,	CynTHIA	BALDWIn6
                       LABRESH3,	                                                    1APDL,	BARC,	USDA,	Beltsville,	MD;2	BFGL,	BARC,	USDA,	
    D	HoRoHoV4,	J	HAnSEn5,	n	MILLER6,	E	BEnGTEn6,	G	                                 Beltsville,	MD;	3Univ.	Illinois-Urbana,	IL;	4Kingfisher	Biotech,	
          CHInCHAR6,M	WILSon6,	B	WAGnER7                                              St.	Paul,	MN;	5Cornell	University,	Ithaca	NY;	6University	of	
    1University	of	Massachusetts,	Amherst,	MA,	2USDA-ARS,	                                            Massachusetts,	Amherst	MA.		
 Beltsville,	MD,	3Kingfisher	Biotech,	Minneapolis,	MN,	4University	                                   jlunney@anri.barc.usda.gov
 of	Kentucky,	Lexington,	KY,	5Western	Fisheriers	Res	Ctr	USGS,	                         The	 US	 Veterinary	 Immune	 Reagent	 Network	 (http://www.
 Seattle,	WA,	6University	of	Mississippi	Medical	Center,	Jackson,	                umass.edu/vetimm/)	was	established	to	address	the	lack	of	immu-
                 MS,	7Cornell	University,	Ithaca,	NY                              nological	reagents	specific	for	ruminant,	porcine,	poultry,	equine	and	
      Immunological	 reagents	 including	 recombinant	 cytokines	 and	            aquaculture	species	and	accordingly	has	set	a	minimum	goal	of	20	
chemokines	 and	 monoclonal	 and	 polyclonal	 antibodies	 (Ab)	 that	             reagents	 per	 species	 group.	 Current	 plans	 are	 to	 produce	 sets	 of	
identify	 the	 major	 leukocyte	 subsets	 (T	 and	 B	 lymphocytes,	 NK	           immune-related	reagents:	recombinant	cytokines	and	chemokines;	
cells,	 macrophages,	 dendritic	 cells,	 neutrophils),	 that	 react	 with	        monoclonal	antibodies	(mAb)	to	them	and	their	receptors;	and	mAb	
cytokines/chemokines	 and	 their	 receptors,	 and	 react	 with	 other	            that	identify	the	major	leukocyte	surface	antigens,	the	CD	antigens,	
important	receptors	that	modulate	immune	function	such	as	toll-like	              the	T	cell	receptors	(TCR)	and	the	Toll-like	receptors	(TLR).	These	
receptors	are	used	to	evaluate	changes	during	disease	including	the	              entities	 are	 needed	 to	 evaluate	 changes	 in	 the	 immune	 system	 of	
causes	of	immune-pathology.	They	also	allow	scientists	to	evaluate	               a	 diseased	 or	 vaccinated	 animal	 and	 to	 test	 as	 potential	 biothera-
host	 responses	 to	 vaccination.	 Finally,	 they	 provide	 the	 means	 to	       peutics.	 For	 the	 US	 Swine	 Toolkit	 portion	 of	 this	 initiative,	 we	 first	
manipulate	or	modulate	immune	responses	either	to	enhance	pro-                    collated	a	list	of	existing	swine	reagents	so	that	our	priority	list	for	
tective	immune	responses	to	vaccines	or	to	reduce	immune-system-                  new	 reagents	 could	 be	 developed.	 This	 priority	 list	 and	 plan	 was	
mediated	pathology.	A	broad	community	effort	began	in	the	US	18	                  based	on:	1)	importance	for	swine	immune	studies;	2)	significance	
months	 ago	 with	 the	 target	 species	 ruminants	 including	 cattle	 and	       to	other	toolkit	efforts;	3)	availability	of	swine	sequence	information;	
sheep,	 swine,	 poultry	 including	 chickens	 and	 turkeys,	 horses	 and	         and	 4)	 likelihood	 of	 developing	 the	 respective	 protein/mAb.	 Since	
catfish	and	trout.	The	project	directors	are	coordinating	their	efforts	          many	 swine	 cytokine	 and	 CD	 reagents	 are	 available,	 our	 priority	
with	other	international	groups	and	are	continually	revising	the	pri-             focused	 on	 anti-TCRαβ,	 and	 on	 chemokines	 and	 their	 receptors.	
oritization	list	and	seeking	input	from	scientists	working	with	these	            Efforts	are	also	underway	to	produce	bioactive	IFN-α,	IL-7,	IL-13	and	
species.	A	list	of	currently	targeted	reagents	and	progress	regarding	            IL-15	and	relevant	mAb.	In	addition,	since	an	anti-CD45RO	mAb	has	
these	will	be	presented.	                                                         not	 been	 produced	 from	 traditional	 efforts,	 a	 peptide	 immunization	
                                                                                  protocol	is	now	being	tested.	Before	making	anti-TLR	mAb,	the	cross	
Key words:	 reagents,	 monoclonal	 antibodies,	 cytokines,	 CD	
                                                                                  reactivity	of	known	anti-human	counterparts	will	be	tested	to	reduce	
molecule
                                                                                  duplication	of	effort.	Our	overall	goal	is	to	produce	reagents	that	will	
Species:	other
                                                                                  function	in	ELISA,	ELISpot,	flow	cytometric	and	immunohistochemi-
                                                                                  cal	applications.	Products	developed	in	this	proposal	will	be	openly	
                 16
Day 1 - Wednesday, August 15th



                                 available	to	collaborators	and	will	be	made	commercially	available	             Institute,	Institute	for	Animal	Health,	Compton	and	the	Animal	Health	
                                 using	 non-exclusive	 licenses.	 These	 reagents	 are	 expected	 to	            Trust	(AHT;	see	accompanying	abstracts	and	posters).
                                 benefit	a	large	group	of	researchers	including	veterinary	immunolo-                  The	aims	of	the	equine	component	of	this	project	are	to:	
                                 gists,	 pathologists,	 microbiologists	 and	 scientists	 using	 swine	 as	 a	
                                                                                                                     1.	generate	antibodies	against	equine	molecules	of	key	immu-
                                 biomedical	 model	 for	 humans.	 This	 project	 was	 funded	 by	 USDA	
                                                                                                                 nological	interest;	
                                 NRICGP	and	ARS.
                                                                                                                     2.	develop	a	rapid	technique	to	identify	MHC	class	I	B2	positive	
                                 Key words:	tool	kit
                                                                                                                 horses;	
                                 Species:	swine
                                                                                                                     3.	construct	tetramers	of	the	equine	MHC	class	I	B2	gene	and	
                                      tK007. THE CyTOKINE CENTER: DEVELOPING                                     CTL	target	peptide	in	Equine	herpesvirus-1	gene	64.
                                     TOOLS FOR CyTOKINE RESEARCH IN VETERINARy                                        Progress	 has	 been	 made	 in	 all	 three	 areas.	 Firstly,	 DNA	
                                                   IMMUNOLOGy                                                    sequences	 encoding	 a	 selection	 of	 equine	 cytokines	 have	 been	
                                                                                                                 cloned	 into	 prokaryotic	 (pET21A	 Novagen	 and	 pGEX-3X	 GE	
                                   EDWIn	J	TIJHAAR1,2,	DAPHnE	VAn	HAARLEM1,	JUDITH	
                                                                                                                 Healthcare)	 and	 eukaryotic	 (pcDNA3IgHG1;	 Wagner	 et	 al	 2003)	
                                 HEnDRIKS1,	DARSHAnA	MoRAR1,	AUREL	nEGREA1,	VICToR	
                                                                                                                 expression	 vectors.	 Polyclonal	 antiserum	 to	 TNFα	 and	 IL-15	 have	
                                                     PMG	RUTTEn1
                                                                                                                 been	produced	and	additional	immunisations	with	CD14	are	under-
                                     Division	of	Immunology,	Department	of	Infectious	Diseases	                  way.	 Stable	 CHO	 cell	 lines	 which	 express	 IL6	 and	 RANTES	 have	
                                    and	Immunology,	Faculty	of	Veterinary	Medicine	Utrecht1	and	                 been	produced	in	collaboration	with	Dr	Wagner	(Cornell	University)	
                                    Cell	Biology	and	Immunology	Group,	Wageningen	University,	                   and	recombinant	protein	will	be	used	for	monoclonal	antibody	pro-
                                                   Wageningen2,	The	Netherlands.		                               duction.	Antibodies	against	bovine	cytokines	from	our	IAH	colleagues	
                                                       edwin.tijhaar@wur.nl                                      have	been	screened	against	equine	leucocytes	and	different	eukary-
                                       Research	in	veterinary	immunology	is	frequently	hampered	by	              otic	expression	systems	investigated	with	our	MRI	colleagues.	For	
                                 the	lack	of	cytokine	reagents.	To	improve	this	situation,	the	Cytokine	         our	second	aim,	a	PCR	which	amplifies	the	B2	gene	from	genomic	
                                 Center	 was	 established	 with	 the	 primary	 aim	 to	 produce	 tools	 for	     and	complementary	DNA	of	A3	homozygous	ponies	has	been	devel-
                                 cytokine	 research	 in	 the	 following	 species:	 cat,	 chicken,	 cow,	 dog,	   oped	 and	 its	 specificity	 is	 currently	 being	 tested	 in	 heterozygous	
                                 horse,	 man,	 mouse,	 pig,	 rat,	 and	 sheep.	 Due	 to	 the	 large	 number	     animals.	Thirdly,	the	B2	gene	has	been	cloned	and	fragment	cloning	
                                 of	 cytokines	 that	 are	 of	 interest,	 a	 technology	 platform	 was	 devel-   of	gene	64	is	underway	to	identify	immunogenic	regions.	
                                 oped	 to	 optimize	 the	 process	 of	 cloning,	 subcloning,	 and	 expres-            The	 reagents	 which	 are	 emerging	 from	 the	 BBSRC-funded	
                                 sion	of	cytokine-genes	and	the	generation	of	mono-	and	polyclonal	              Immunological	 Toolbox	 (http://www.immunologicaltoolbox.co.uk/),	
                                 antibodies	 against	 the	 different	 cytokines.	 The	 IL4-,	 IL5-,	 IL6-,	      together	 with	 the	 USDA-funded	 project	 (http://www.umass.
                                 IL10-,	 IFNgamma-,	 GMCSF-,	 TNFalpha-	 and	 TGFbeta1-genes	 of	                edu/vetimm/)	 and	 the	 initial	 assembly	 from	 the	 Equine	 Genome	
                                 most	of	the	species	mentioned	above	have	been	cloned.	The	IL4-,	                Sequencing	Project	(Broad	Institute,	Boston,	USA	(www.broad.mit.
                                 IFNgamma-,	 GMCSF-	 and	TNFalpha-genes	 have	 been	 expressed	                  edu/ftp/pub/assemblies/mammals/horse/)	will	provide	equine	immu-
                                 in	 bacterial	 and	 mammalian	 expression	 systems	 and	 the	 corre-            nologists	with	a	substantially	wider	panel	of	reagents	to	enhance	the	
                                 sponding	proteins	have	been	purified	and	refolded	when	necessary.	              characterisation	of	innate	and	adaptive	immune	responses	induced	
                                 These	 recombinant	 cytokines	 were	 subsequently	 used	 to	 produce	           by	infection	or	vaccination	against	endemic	and	emerging	viral	dis-
                                 polyclonal	antibodies	in	chickens	and	rabbits	and	to	generate	mono-             eases	at	the	molecular	and	cellular	levels.
                                 clonal	(mouse)	antibodies.	The	mono-	and	polyclonal	antibodies	are	                  Wagner,	B.	Robeson	J.	McCracken	M.,	Wattrang,	E.,	Anctzak,	
                                 used	to	develop	highly	sensitive	cytokine	detection	assays.	The	cur-            D.F.	2005.	Horse	cytokine	/	IgG	fusion	proteins	–	mammalian	expres-
                                 rent	state-of-the-art	will	be	presented.	                                       sion	of	biologically	active	cytokines	and	a	system	to	verify	antibody	
                                 Key words:	cytokine,	antibodies,	TNF,	IFN                                       specificity	 to	 equine	 cytokines.	 Vet.	 Immunol.	 Immunopathol.	
                                 Species:	all	species                                                            105:1-14.
                                                                                                                 Key words:	tetramers;	equine	MHC	class	I	B2;	PCR;	TNFα;	IL-15;	
                                     tK008. GENERATION OF NOVEL REAGENTS: THE                                    CD14;	IL6;	RANTES
                                         CHICKEN IMMUNOLOGICAL TOOLBOx                                           Species:	equine
                                  MUHAMMAD	IqBAL,	LISA	RoTHWELL,	ZHIGUAnG	WU,	UDAy	
                                   PATHAnIA,	SUCHARITHA	BALU,	LAI	SHAn	KWonG,	PAUL	                              tK010. U.S. VETERINARy IMMUNE REAGENT NETWORK:
                                   SoPP,	JAynE	HoPE,	JoHn	yoUnG,	JIM	KAUFMAn,PETE	                                           POULTRy REAGENTS UPDATE
                                                       KAISER                                                                   LILLEHoJ,	HyUn	AnD	HonG,	yEonG	
                                     Institute	for	Animal	Health,	Compton,	Berkshire	RG20	7NN,	                   Animal	Parasitic	Diseases	Laboratory,	USDA-ARS,	Beltsville,	MD	
                                                             England,	UK                                                                    20705,USA	
                                                                                                                        A	major	obstacle	to	advance	poultry	immunology	and	disease	
                                     tK009. THE IMMUNOLOGICAL TOOLBOx: EQUINE                                    research	 is	 the	 lack	 of	 adequate	 immunological	 reagents	 specific	
                                                     REAGENTS                                                    for	 poultry.	 Although	 many	 immunological	 reagents	 which	 detect	
                                     JULIA	H	KyDD*,	CARL	RoBInSon,	RUTH	CASE,	KELLy	                             cell-surface	 antigens	 of	 immune	 system	 are	 commercially	 avail-
                                   SAUnDERS,	nICoLA	WRIGHT,	SALLy	DEBEnHAM,	SHIRLEy	                             able,	there	is	very	few	monoclonal	antibodies	(mAb)	and	polyclonal	
                                                ELLIS#,DUnCAn	HAnnAnT*                                           antibodies	which	can	identify	the	major	chicken	cytokines	and	che-
                                                                                                                 mokines,	 as	 well	 as	 their	 receptors.	 	The	 new	 initiative	 to	 address	
                                      Animal	Health	Trust,	Lanwades	Park,	Kentford,	Newmarket,	                  general	lack	of	immunological	reagents	for	veterinary	animal	species	
                                       Suffolk	CB8	7UU.	*Current	address:	School	of	Veterinary	                  including	fish	was	funded	by	the	USDA-CSREES	National	Research	
                                  Medicine	&	Science,	University	of	Nottingham,	Sutton	Bonington,	               Initiative	grant	in	2006.	This	project	represents	a	broad	community	
                                      Loughborough,	Leicestershire	LE12	5RD,	United	Kingdom.	                    plan	to	begin	to	systematically	address	the	immunological	reagent	
                                  #
                                    Institute	for	Animal	Health,	Compton,	Newbury,	Berkshire	RG20	               gap	for	the	U.S.	veterinary	immunology	research	community	for	the	
                                                          7NN,	United	Kingdom.	                                  following	 groups:	 ruminants,	 swine,	 poultry,	 horses,	 and	 aquacul-
                                                      julia.kydd@nottingham.ac.uk                                ture	species.	The	goal	of	this	project	is	to	develop	20	reagents	per	
                                       Progress	in	the	characterisation	of	immune	responses	to	patho-            each	species	group	including	antibodies	that	function	in	ELISA	and	
                                 gens	of	livestock	species	and	the	horse	has	been	limited	by	the	lack	           ELISpot	assays,	for	intracellular	staining,	for	blocking	function	and	
                                 of	appropriate	reagents	and	this	in	turn	has	hindered	the	identifica-           signaling,	for	flow	cytometric	analysis,	as	well	as	for	immunochem-
                                 tion	 of	 protective	 immune	 responses	 and	 their	 stimulation	 by	 vac-      istry	using	tissue	sections.	Chicken	full-length	genes	encoding	IL-2,	
                                 cination.	 The	 Immunological	 Toolbox	 aims	 to	 redress	 this	 problem	       IL-15,	 IL-16,	 IL-17,	 IFN-γ,	 TNFSF15,	 NK-lysin,	 LPS-induced	 TNF-
                                 through	 a	 collaborative	 initiative	 involving	 the	 Moredun	 Research	       like	 factor	 (LITAF),	 IL-4,	 IL-10,	 IL-18,	 lymphotactin,	 CCL4,	 CCL20,	
                                                                                                                                                                   17




                                                                                                                                                                   Day 1 - Wednesday, August 15th
and	 IL-12	 have	 been	 cloned	 and	 proteins	 are	 being	 expressed	 for	       Veterinary	Immune	Reagent	Network).
antibody	production.	                                                            Key words:	 Recombinant	 proteins;	 cell	 surface	 molecules;	 T-cell	
Key words:	cell-surface	antigens;	commercial	antibodies;	cytokines;	             receptor	chains;	CD	molecules;	cytokine	receptors
chemokines;	receptors.                                                           Species:	Ruminants;	Equine;	Swine;	Avian;	Fish
Species:	Avian
                                                                                   tK012. BOVINE: PROGRESS AND PLANS WITHIN THE
       tK011. ExPRESSION OF RECOMBINANT                                              U.S. VETERINARy IMMUNE REAGENT NETWORK
 IMMUNOGLOBULINS AND CELL SURFACE MOLECULES                                          CynTHIA	BALDWIn1,	EDWARD	HUDGEnS1,	DAnnIELLE	
   FOR SIx SPECIES: CATTLE, PIG, HORSE, CHICKEN,                                     ToMPKInS1,	JoAnnA	LABRESH2,	BETTInA	WAGnER3.
                CATFISH AND TROUT.
                                                                                  1University	of	Massachusetts,	Amherst	,	MA;	2Kingfisher	Biotech,	
 BETTInA	WAGnER1,	JULIA	M.	HILLEGAS1,	RICHARD	ICoM1,	                                     Minneapolis,	MN;	3Cornell	University,	Ithaca,	NY
 JoAn	K.	LUnnEy2,	EVA	BEnGTEn3,	noRMAn	W.	MILLER3,	
                                                                                        In	 an	 effort	 to	 overcome	 the	 lack	 of	 immunological	 reagents	
  HyUn	LILLEHoJ2,	JoHn	D	HAnSEn4,	CynTHIA	BALDWIn5
                                                                                 targeted	for	ruminants,	thereby	improving	research	into	bovine	and	
 1Cornell	University,	Ithaca	NY;	2APDL,	BARC,	USDA,	Beltsville,	                 ovine	immunology	and	disease,	the	bovine	component	of	the	“U.S.	
 MD;	3University	of	Mississippi,	Jackson,	MS;	4Western	Fisheries	                Veterinary	Immune	Reagent	Network”	has	isolated	and	sequenced	
  Research	Center,	Seattle,	WA;	5University	of	Massachusetts,	                   the	complete	coding	sequence	for	a	large	number	of	genes.	These	
                            Amherst	MA	                                          include	 the	 chemokines	 and	 cytokines	 IL-1β,	 IL-2,	 IL-4,	 IL-5,	 IL-6,	
      The	US	Veterinary	Immune	Reagent	Network	seeks	to	develop	                 IL-7,	 IL-8,	 IL-12p35,	 IL-12p40,	 IL-13,	 IL-17,	 IL-18,	 IL-23,	 IFN-γ,	
new	 tools	 for	 molecules	 of	 the	 immune	 system	 in	 veterinary	 spe-        IFN-a	 A,	 IFN-β,	 TNF-a,	 CXCL9	 (MIG),	 CXCL10	 (IP-20),	 CXCL11	
cies	 (cattle,	 pig,	 horse,	 chicken	 and	 fish).	 Recombinant	 proteins	       (I-TAC),	 CCL2	 (MCP2),	 CCL5	 (RANTES),	 and	 CCL11	 (eotaxin).	
for	 immunoglobulins	 and	 various	 cell	 surface	 molecules,	 including	        Polymorphisms	for	some	of	these	genes	among	cattle	breeds	have	
T-cell	 receptor	 chains,	 CD	 molecules	 and	 cytokine	 receptors	 have	        been	investigated.	Genes	cloned	for	cell	surface	molecules	include:	
been	 expressed	 at	 Cornell	 University.	 Two	 expression	 mammalian	           the	chemokine	rcceptors	CCR7,	CCR5,	CXCR3	and	CXCR5	and	the	
systems	are	used;	(1)	an	IgG	fusion	protein	and	(2)	an	IL-4	fusion	              cytokine	receptors	IL-23R	and	IL-10Rβ,	the	TCR	γ (6	constant	region	
protein	 system.	 All	 proteins	 are	 expressed	 in	 Chinese	 Hamster	           genes	 and	 8	 variable	 genes),	 TCRd (1	 constant	 and	 4	 variable),	
Ovary	 (CHO)	 cells.	 Purified	 recombinant	 proteins	 are	 then	 submit-        TCRa and	β	(1	constant	gene	each),	and	finally	CD40	and	CD40L.	
ted	 to	 the	 University	 of	 Massachusetts	 at	Amherst	 for	 monoclonal	        The	genes	have	been	cloned	 into	expression	 vectors	and	proteins	
antibody	production.	The	first	recombinant	proteins	that	have	been	              will	 be	 expression	 in	 mammalian	 cells	 (cell-surface	 molecules)	 or	
expressed	 with	 this	 system	 were	 T-cell	 receptor	 constant	 region	         yeast	 (chemokines	 and	 cytokines)	 and,	 subsequently,	 the	 proteins	
domains.	 These	 include	 the	 bovine	 TCRγ	 and	 TCRd	 proteins	 and	           used	 for	 monoclonal	 antibody	 (mAb)	 production	 and/or	 evaluated	
the	 catfish	 TCRa	 and	 TCRγ	 constant	 regions.	 TCR	 genes	 of	 the	          for	 bioactivity.	 The	 C-domain	 of	 both	 TCRγ	 and	 TCRd	 have	 been	
horse,	pig	and	trout	are	being	processed.	Other	molecules	that	have	             expressed	 (Cornell)	 and	 mAb	 production	 to	 TRDC	 UMass	 begun.	
been	expressed	with	the	fusion	protein	system	include	equine	CD40	               This	 is	 a	 pilot	 study	 to	 determine	 whether	 expression	 of	 individual	
and	 the	 FceRI	 a-chain.	 Expression	 cloning	 and	 protein	 production	        C-domains	 will	 yield	 a	 conformationally-preserved	 molecule	 and	 if	
is	 ongoing	 for	 cattle	 IL-23R	 and	 IL-10R,	 pig	 IL-4Ra	 and	 IL-13Ra1,	     successful	will	be	followed	by	TRBC	and	TRAC.
equine	 CD23,	 CD25,	 CD28	 and	 the	 IgD	 heavy	 chain,	 and	 chicken	          Key words:	 antibodies;	 chemokines;	 cytokines;	 chemokine	
IL-2Ra	and	CXCR4.                                                                rcceptors;	cytokine	receptors;	TCR
     This	work	is	supported	by	USDA	Grant	#2005-01812	(The	US	                   Species:	Ruminants




                                                             oPening ConferenCe

    brAdyKinin b2 reCePtors of dendritiC                                         G-protein	 coupled	 bradykinin	 B2	 receptors	 (B2R).	The	 premise	 that	
   Cells: An innAte PAtHWAy tHAt Promotes                                        this	 endogenous	 signaling	 pathway	 may	 stimulate	 type-1	 adaptive	
    deVeloPment of Cd8 effeCtor t Cells                                          responses	 was	 recently	 confirmed	 in	 the	 subcutaneous	 model	 of	
                                                                                 Trypanosoma	 cruzi	 infection.	 Analysis	 of	 the	 dynamics	 of	 parasite-
                         JULIo	SCHARFSTEIn
                                                                                 evoked	 edema	 formation	 revealed	 that	 activation	 of	 TLR2/neutro-
              Federal	University	of	Rio	de	Janeiro,	Brazil                       phils	 drives	 the	 influx	 of	 plasma	 proteins,	 such	 as	 kininogens,	 into	
      Strategically	 positioned	 in	 peripheral	 tissues,	 immune	 sentinel	     peripheral	tissues.	After	docking	to	sulfated	proteoglycans,	the	sur-
cells	such	as	macrophages	and	mast	cells,	sense	microbes	and/or	                 face-associated	 bound	 kininogens	 are	 turned	 into	 facile	 substrates	
their	 products	 through	 different	 types	 of	 pattern-recognition	 recep-      for	T.	cruzi	cysteine	proteases.	Once	liberated,	the	short-lived	kinin	
tors.	Upon	secretion	of	cytokines	and	chemokines,	inflammation	is	               peptides	potently	activate	DCs	via	B2R,	converting	these	APCs	into	
rapidly	 amplified	 through	 cooperative	 involvement	 of	 the	 microvas-        Th1	 inducers.	 Intensity	 of	 the	 innate	 signals	 conveyed	 by	 kinins	 is	
culature,	of	circulating	leukocytes,	peripheral	neurons	and	dendritic	           tightly	controlled	by	the	activity	of	kinin-degrading	metallopeptidases,	
cells	(DCs).	Owing	to	disturbances	of	endothelium	barrier	function,	             e.g.	 Angiotensin	 Converting	 Enzyme	 (ACE/CD143).	 Exploring	 the	
the	 plasma	 proteins	 diffuse	 into	 extravascular	 tissues,	 allowing	 for	    knowledge	about	mechanisms	underlying	kinin	generation	and	deg-
proteolytic	generation	of	short-lived	proinflammatory	peptides,	such	            radation,	we	recently	developed	a	vaccination	scheme	that	protects	
as	 complement	 activation	 peptides	 and	 the	 kininogens	 (i.e.,	 kinin-       mice	 from	 lethal	 T.	 cruzi	 infection.	An	 important	 lead	 coming	 from	
precursor	 proteins).	 While	 much	 emphasis	 has	 been	 placed	 on	             these	 vaccination	 studies	 was	 the	 evidence	 that	 bradykinin	 steers	
analysis	 of	 the	 pro-inflammatory	 activity	 of	 complement	 peptides,	        development	of	type-1	effector/memory	CD8+	T	cells	through	modu-
the	 innate	 role	 of	 kinins	 (eg.	 bradykinin,	 BK)	 was	 long	 overlooked.	   lation	of	the	DC/T	cell	interface.	In	summary,	lessons	taken	fr			om	
A	 few	 years	 ago,	 however,	 we	 reported	 that	 the	 nonapeptide	 bra-        studies	of	the	mechanisms	underlying	bradykinin-adjuvanticity	may	
dykinin	 (BK)	 is	 a	 potent	 inducer	 of	 dendritic	 cell	 (DC)	 maturation,	   stimulate	 development	 of	 new	 vaccine	 formulations	 against	 veteri-
driving	 IL-12-dependent	 Th1	 responses	 through	 the	 activation	 of	          nary	diseases	caused	by	virus	and/or	intracellular	parasites.	
Day 2 - Thursday, August 16th




                                                                   Day 2 - August 16th - Thursday

                                	 7:45	-	18:00	h	    	 	 	 	 	 	 	 	 	 Registration	and	Poster	Setup

                                	 8:30	-	10:30	h	    Plenary	Session:	Immunogenetics	                    SALÃO OURO

                                	 10:45	-	13:15	h	   Plenary	Session:	Bacterial	Diseases	                SALÃO OURO

                                	 14:00	-	16:00	h	   Plenary	Session:	Reproduction,	Stress,	Nutrition	   SALÃO OURO

                                	 16:30	-	17:30	h	   Pfizer	Award	for	Distinguished	                     SALÃO OURO
                                	                	   Veterinary	Immunologist

                                	 17:30	-	18:00	h	   Introduction	to	the	9th	IVIS;	                      SALÃO OURO
                                	                	   Introduction	to	Incoming	President	of	VIC

                                	 18:00	-	19:30	h	   Plenary	Session:	Skin,	Mucosae,	Mammary	Gland	      SALÃO OURO
                                	
                                                                                                         19




                                                                                           SALÃO OURO
	 8:30	-	10:30	h      Plenary Session: Immunogenetics and genomics of hosts; genomics of
                      pathogens
	                   	 Chairs:	Fuad	Iraqi	-	Israel	and	Elizabeth	J.	Glass	-	UK

                      Speakers
	   8:30	-	9:00	h	 Alberto	Davila
                      Oswaldo	Cruz	Foundation,	Brazil




                                                                                                         Day 2 - Thursday, August 16th
                      Exploring the genome of Trypanosoma vivax: towards markers for diagnosis
                      and typing and relevance to immunity and disease resistance in livestock
	   9:00	-	9:30	h	 James	Womack
	                   	 Texas	A&M,	USA
                      Bovine Genome Sequencing Update: Discovering Variation in the TLR Gene Family
	 9:30	-	10:00	h	 Adrian	Smith
	                   	 Institute	for	Animal	Health,	UK
                      Parasite genetics and the search for protective antigens
	 10:00	-	10:30	h	 Allan	Crawford
	                   	 AgResearch,	New	Zealand
                      Transcriptomics: Its use in understanding resistance to nematode parasite
                      infection in sheep
10:30-10-45           Coffee Break

1
	 0:45	-	13:15	h	 Plenary Session: Immune Responses in Bacterial and Viral Diseases;
                  Prions and BSE
	                   	 Chairs:	Wendy	Brown	-	USA	and	Paul	Wigley	-	UK	

                      Speakers
	 10:45	-	11:15	h	 Don	M.	Estes
	                	 University	of	Texas	Medical	Branch,	USA
                   Tuberculosis vaccine development and immunity in the neonatal calf model
	 11:15	-	11:45	h     Arthur	Summerfield
	                   	 Institute	of	Virology	and	Immunoprophylaxis	Switzerland
                      Immune Responses during infections with Foot and Mouth Disease Virus
	 12:00	-	12:30	h	 Paul	Wigley
	                   	 University	of	Liverpool,	UK
                      Salmonella and the macrophage: The Immunobiology of systemic avian salmonellosis
	 12:15	-	12:45	h Wendy	Brown
	                   	 Washington	State	University,	USA
                      Immunoproteomic analysis of the protective outer membrane fraction of
                      Anaplasma marginale
	 12:45	-	13:15	h	 yasmin	Belkaid
	                   	 NIH,	USA
                      Role and origins of regulatory T cells during parasitic infections
	 13:15	-	14:00	h Lunch
                20




                                                                                                                       SALÃO OURO
                                1
                                	 4:00	-	16:00	h Plenary Session: Immunoendocrinology, Stress and Immunology of Re
                                                 production and Neonates; Microbial Flora, Nutrients and the Immune
                                                 Response
                                	              	 Chairs:	Gary	Entrican	-	UK	and	José	Roberto	Kfoury	Jr.	-	Brazil
                                                  Speakers
                                	 14:00	-	14:30	h	 Antonio	La	Cava
Day 2 - Thursday, August 16th




                                	              	 UCLA,	USA
                                                 Cross-talk between neuroendocrine and immune system: the case of leptin
                                		14:30	-	15:00	h	 Harry	Dawson
                                	                	 ARS-USDA,	USA
                                                   Regulation of porcine hepatic and pulmonary-associated immune responses
                                                   by vitamin A
                                		15:00	-	15:30	h	 Harris	Lewin
                                	                	 University	of	Illinois,	USA
                                                   Good Embryos Gone Bad: A Transcription Profile of What Goes Wrong
                                		15:30	-	16:00	h	 Joseph	Urban
                                	                	 ARS-USDA,	USA
                                                   Use of parasitic infection to explore dietary components that regulate
                                                   appropriate mucosal immune responses
                                	 16:00	-	16:30	h Coffee break

                                16:30	-	17:30	h	 Pfizer Award for Distinguished Veterinary Immunologist
                                	              	 Introduction	by	Wayne	Hein,	Chair	of	Award	Selection	Committee,	New	Zealand	
                                	              	 Award	presented	by	Paul	Wood,	Pfizer,	Australia
                                                  Speaker:
                                	              	 John	E.	Butler
                                	              	 University	of	Iowa,	USA
                                                 Diversity, Frontiers and Careers in Immunology

                                17:30	-	18:00	h Introduction to the 9th IVIS
                                	              	 Dr.	Takashi	onodera,	Japan	
                                	              	 Introduction	to	Incoming	President	of	VIC	
                                	              	 Introduction	by	Jan	naessens,	Kenya

                                	 8:00	-	19:30	h Plenary Session: Immunology of the Mucosae, Skin and of the Mammary
                                1
                                                 Gland; Mastitis
                                	              	 Chair:	Michael	P.	Murtaugh	-	USA	
                                                  Speakers
                                	 18:00	-	18:30	h	 Lorraine	Sordillo
                                	              	 Michigan	State	University,	USA
                                                 Mammary Immunity and Susceptibility to Mastitis: role of oxidant stress
                                	 18:30	-	19:00	h	 Els	Meeusen
                                	              	 Monash	University,	Australia
                                                 Structure and protection of the ruminant lung
                                	 19:00	-	19:30	h	 Adrian	Smith
                                	              	 Institute	for	Animal	Health,	UK
                                                 Structural and cellular aspects of immunity to pathogens in the gut
                                                                                                                                                                21


      PlenAry session: immunogenetiCs And genomiCs of Hosts; genomiCs of PAtHogens.

  ALBERTo	DAVILA	(oSWALDo	CRUZ	FoUnDATIon,	BRAZIL)	                            against	 each	 other.	 The	 process	 first	 involved	 identification	 of	 two	
  exPloring tHe genome of tryPAnosomA                                          antigenically	distinct	strains	of	E.	maxima,	one	of	which	was	selected	
                                                                               for	resistance	to	the	drug	robenidine.	Next,	these	parent	strains	were	
ViVAx: toWArds mArKers for diAgnosis And
                                                                               used	to	establish	a	co-infection	which	results	in	the	generation	of	a	
  tyPing And releVAnCe to immunity And
                                                                               genetic	mixture	of	offspring	as	a	result	of	sexual	recombination.	The	
     diseAse resistAnCe in liVestoCK
                                                                               progeny	 of	 the	 cross	 were	 exposed	 to	 the	 double-selective	 barrier	
                                                                               of	drug	exposure	and	immunity	(to	the	drug-resistant	strain)	which	
          JAMES	WoMACK	(TExAS	A&M,	USA)	BoVInE	                                kills	both	parental	genotypes	and	any	irrelevant	recombinants.	The	
  genome sequenCing uPdAte: disCoVering                                        surviving	parasites	have	recombined	all	the	loci	required	to	escape	
                                                                               the	double	barrier	and	we	term	these	“relevant	recombinants”	(RR).	
     VAriAtion in tHe tlr gene fAmily
                                                                               Analysis	of	the	inheritance	patterns	of	AFLP	fragments	from	multiple	
     	Approximately	one	year	ago,	the	Bovine	Genome	Sequencing	                independent	populations	of	RR,	unselected	progeny	of	the	cross	and	
Project	(http://genome.gov/12512284)	released	the	third	version	of	            single-barrier	 selected	 populations	 revealed	 36	 polymorphic	 DNA	




                                                                                                                                                                Day 2 - Thursday, August 16th
the	 bovine	 genome	 assembly,	 Btau_3.1,	 which	 is	 a	 7.15X	 mixed	         markers	 that	 were	 consistently	 selected	 according	 to	 the	 immune	
assembly	that	combines	whole	genome	shotgun	(WGS)	sequence	                    barrier.	 These	 markers	 group	 into	 five	 regions	 of	 the	 parasite	
with	BAC	sequence.	The	sequence,	predominantly	from	a	Hereford	                genome	which	are	under	scrutiny	to	identify	genes	responsible	for	
female,	 is	 available	 in	 GenBank,	 EMBL,	 and	 DDBJ.	 Sequencing	           highly	effective	strain-specific	immunity.	
skims	 for	 single	 nucleotide	 polymorphism	 (SNP)	 discovery	 were	
                                                                                    These	 results	 have	 several	 implications:	 Firstly,	 that	 just	 five	
generated	from	random	shotgun	libraries	from	individual	animals	of	
                                                                               genomic	 regions	 (<1	 Mbp)	 are	 under	 strong	 immune	 selection	 out	
Holstein,	Angus,	Brahman,	Limousin,	and	Jersey	breeds.
                                                                               of	a	60	Mbp	genome	implies	that	only	a	small	number	of	genes	are	
      Toll-like	receptors	(TLRs)	play	a	crucial	role	in	innate	immunity	       responsible	 for	 protection.	 The	 simultaneous	 selection	 of	 all	 five	
in	vertebrates	as	well	as	in	insects	where	they	were	discovered.		The	         regions	in	all	parasite	populations	suggests	that	high	level	protection	
recognition	of	microbial	elements	by	different	members	of	the	gene	            requires	stimulation	with	at	least	one	antigen	in	each	region	(i.e.	5+	
family	initiates	signal	transduction	pathways	leading	to	the	expres-           antigens).	Nonetheless,	the	ability	to	identify	each	region	has	sub-
sion	of	specific	genes	important	to	the	innate	immune	response	and	            stantially	reduced	the	complexity	of	the	antigen	discovery	process.	
to	direct	paths	to	antigen-specific	acquired	immunity.		A	large	body	          Finally,	where	the	ability	to	select	appropriate	phenotypes	exists,	this	
of	data	is	emerging	suggesting	roles	of	single	nucleotide	polymor-             approach	may	be	applicable	to	the	search	for	protective	antigens	in	
phisms	 (SNPs)	 in	 human	 TLR	 genes	 in	 susceptibility	 to	 infectious	     other	parasitic	pathogens.
and	inflammatory	diseases.	
     We,	along	with	others,	have	mapped	the	bovine	TLR	family	of	10	
                                                                                     ALLAn	CRAWFoRD	(AGRESEARCH,	nEW	ZEALAnD)
genes	to	their	respective	chromosome	positions	which	correspond	
to	expected	locations	relative	to	conserved	synteny	in	humans.		We	             trAnsCriPtomiCs: its use in understAnding
have	 begun	 a	 search	 for	 bovine	 SNPs	 in	 coding	 regions	 of	 TLR	       resistAnCe to nemAtode PArAsite infeCtion
genes,	utililzing	data	available	from	the	bovine	genome	sequencing	                            in sHeeP
project	and	also	by	resequencing	the	coding	regions	of	all	genes	in	               ALLAn	M	CRAWFoRD,	oRLA	M	KEAnE,	CRISTInA	DIEZ-
10	animals	representing	10	different	breeds	of	cattle.		                                      TASCon,		JoHn	C	MCEWAn
      We	 sequenced	 approximately	 35	 kb	 of	 coding	 sequence	 in	          AgResearch	Invermay	Research	Centre,	Puddle	Alley,	Private	Bag	
eight	of	the	ten	bovine	TLR	genes	from	10	animals.		More	than	250	                             50034,	Mosgiel,	New	Zealand.
SNPs	were	revealed,	49	of	which	account	for	non-synonomous	cod-
                                                                                      Nematode	parasite	infections	of	the	alimentary	tract	are	the	larg-
ing.		SNP	distribution	is	not	uniform	with	TLR-4	and	10	revealing	13	
                                                                               est,	most	pervasive	health	problem	for	sheep	and	goats	produced	in	
SNPs	per	kb	of	coding	sequence.	 	As	expected,	most	SNPs	were	
                                                                               a	 grazing	 environment.	 Over	 the	 last	 40	 years	 chemical	 drenches	
discovered	in	Bos	Taurus/Bos	Indicus	comparisons.		
                                                                               have	been	the	major	therapy	for	control	of	nematode	parasite	infec-
      We	 compared	 SNP	 discovery	 from	 genome	 sequence	 data-              tions,	however,	drenches	are	now	failing.	One	of	the	most	promis-
bases	and	resequencing	animals	from	diverse	breeds.		As	expected	              ing	alternative	means	of	control	has	been	to	breed	sheep	resistant	
at	 this	 early	 stage	 of	 the	 bovine	 SNP	 project,	 most	 SNPs	 for	TLR	   to	 parasite	 infection.	 Parasite	 resistance	 is	 a	 moderately	 heritable	
genes	are	not	yet	available	from	the	databases.                                trait	 and	 the	 benefits	 of	 measuring	 parasite	 burden	 in	 lambs	 and	
                                                                               including	this	information	in	a	production	index	for	breeding	is	now	
                                                                               well	 documented.	 Measuring	 parasite	 burden	 involves	 measuring	
     ADRIAn	SMITH	(InSTITUTE	FoR	AnIMAL	HEALTH,	UK)	                           parasite	 eggs	 in	 faeces	 from	 each	 animal.	 This	 is	 understandably	
        PArAsite genetiCs And tHe seArCH                                       an	 unpopular	 chore	 with	 sheep	 breeders,	 made	 doubly	 so	 by	 the	
            for ProteCtiVe Antigens                                            low	 repeatability	 of	 each	 measurement	 necessitating	 duplicate	 or	
                                                                               triplicate	samples	having	to	be	taken	over	3	days.
     Enteric	Immunology,	Institute	for	Animal	Health,	Compton,	
                                                                                    The	 appeal	 of	 a	 DNA	 test	 to	 identify	 those	 sheep	 resistant	 to	
                    Berkshire	RG20	7NN,	UK	
                                                                               parasites	is	obvious,	especially	as	in	addition	to	saving	faecal	sam-
                                                                               pling	it	would	mean	animals	could	be	tested	early	in	life	and	saved	
                                                                               from	a	potentially	debilitating	parasite	challenge.	QTL	searches	have	
      Development	 of	 effective	 sub-unit	 vaccines	 is	 dependent	           not	been	encouraging	in	that	only	QTL	with	quite	small	effects	have	
on	 selection	 of	 antigens	 capable	 of	 inducing	 protective	 immunity.	     been	identified.	
Unfortunately,	 responses	 against	 most	 antigens	 are	 not	 protective	           We	 have	 therefore	 tried	 another	 gene	 discovery	 option	 which	
and	 new	 strategies	 are	 required	 for	 antigen	 selection,	 especially	     complements	 the	 QTL	 approach,	 and	 examines	 gene	 expression	
with	 antigenically	 complex	 pathogens.	 For	 example,	 the	 genomes	         using	arrays	of	cDNA	sequences	on	glass	slides.	The	same	resistant	
of	parasitic	pathogens	encode	many	thousands	of	potential	antigens	            and	susceptible	selection	lines	or	breeds	of	sheep	have	been	used	
and	large	subsets	of	these	induce	responses	in	the	infected	host.	To	          to	compare	gene	expression.	The	majority	of	this	presentation	con-
avoid	the	problem	of	interpreting	response-based	assays	we	have	               cerns	what	we	have	learnt	from	gene	expression	studies	in	parasite	
developed	an	approach	based	upon	parasite	genetics,	selective	bar-             resistant	and	susceptible	Perendale	sheep	and	how	this	compares	
riers	 and	AFLP-based	 fingerprinting.	The	 essential	 features	 of	 this	     with	studies	in	other	animal	systems.		
approach	will	be	illustrated	with	the	intestinal	apicomplexan	parasite	
Eimeria	 maxima.	 This	 parasite	 induces	 extremely	 strong	 immunity	
against	 rechallenge	 infection	 but	 immunity	 is	 strain-specific,	 i.e.	
field	or	laboratory	strains	can	be	identified	that	do	not	cross-protect	
                22


                                      PlenAry session: immune resPonses in bACteriAl And VirAl diseAses; Prions And bse

                                  Don	M	ESTES	(UnIVERSITy	oF	TExAS	MEDICAL	BRAnCH,	                                           immunobiology of systemiC AViAn
                                                        USA)	                                                                         sAlmonellosis
                                   tuberCulosis VACCine deVeloPment And                                                 PAUL	WIGLEy1,	CLAIRE	JoHnSTon1,	LUCy	CHAPPELL2,	
                                    immunity in tHe neonAtAl CAlf model                                                   PETE	KAISER2,	RICHARD	BEAL2,	ADRIAn2	SMITH,	
                                                                                                                                         PAUL	BARRoW3
                                    ARTHUR	SUMMERFIELD	(InSTITUTE	oF	VIRoLoGy	AnD	                                     1	Department	of	Veterinary	Pathology,	University	of	Liverpool,	
                                          IMMUnoPRoPHyLAxIS	SWITZERLAnD)	                                                             Leahurst,	Neston,	CH64	7TE,	UK.	
                                                                                                                     2	Division	of	Immunology,	Institute	for	Animal	Health,	Compton,	UK	
                                 immune resPonses during infeCtions WitH                                                 3	School	of	Veterinary	Medicine	and	Science,	University	of	
                                      foot And moutH diseAse Virus                                                                   Nottingham,	Sutton	Bonnington,	UK.
                                                                                                                           Systemic	salmonellosis	in	the	chicken	is	caused	primarily	by	the	
                                      Foot-and-mouth	 disease	 (FMD)	 represents	 one	 of	 the	 most	                two	avian	specific	serovars	Salmonella	enterica	serovar	Gallinarum	
Day 2 - Thursday, August 16th




                                economically	 important	 diseases	 of	 farm	 animals.	 Although	 suc-                and	 serovar	 Pullorum.	 Infection	 with	 S.	 Gallinarum	 results	 in	 Fowl	
                                cessful	 eradication	 programs	 based	 on	 vaccination	 and	 stamping	               Typhoid,	a	severe	and	acute	form	of	disease	with	high	mortality	rates	
                                out	policies	has	been	applied	in	developed	countries,	the	virus	is	still	            in	 birds	 of	 all	 ages.	 S.	 Pullorum	 causes	 Pullorum	 Disease,	 which	
                                endemic	in	large	parts	of	Africa,	South	Asia	and	South	America.	The	                 causes	 high	 mortality	 in	 young	 chicks,	 accompanied	 by	 distinctive	
                                basis	for	the	constant	threat	caused	by	this	virus	is	the	extreme	rate	              white	diarrhoea,	but	frequently	leads	to	persistent	infection	without	
                                of	replication,	short	incubation	time,	contagiosity,	virus	tenacity	and	             clinical	disease	in	older	birds	resulting	in	reproductive	tract	infection	
                                a	high	mutation	rate	resulting	in	constant	antigenic	changes.	Thus,	                 and	transmission	of	infection	to	eggs	and	chicks.
                                although	protective	immune	responses	against	FMD	virus	(FMDV)	
                                are	rapid	and	efficacious,	the	virus	has	the	capacity	to	overrun	the	                     Interaction	with	the	immune	system	during	infection	with	these	
                                immune	system.	The	basis	for	this	is	not	only	a	particular	rapid	rate	               serovars	has	three	distinct	phases.	Phase	one	is	invasion	through	
                                of	 replication	 but	 also	 the	 ability	 of	 FMDV	 to	 shut	 down	 the	 cellu-      the	gastrointestinal	tract	and	initial	exposure	to	the	innate	immune	
                                lar	 protein	 synthesis,	 including	 IFN	 type	 I,	 through	 the	 viral	 Lpro	 in	   system.	The	second	phase	is	the	establishment	of	systemic	infection	
                                susceptible	epithelial	cell	cultures.	This	appears	important	for	virus	              leading	 to	 persistence,	 immune	 clearance	 or	 death	 of	 the	 infected	
                                evolution,	as	FMDV	is	quite	sensitive	to	the	action	of	IFN.	Despite	                 host.	The	final	phase	is	infection	of	the	reproductive	tract	and	egg	
                                this,	 innate	 immune	 responses	 can	 be	 detected	 in	 vivo	 indicating	           infection.
                                that	the	effect	of	Lpro	is	not	absolute.	In	vitro	for	example,	the	porcine	                Data	 from	 in	 vitro	 models	 has	 previously	 suggested	 that	 the	
                                epithelial	 cell	 line	 PK-15	 has	 some	 degree	 of	 resistance	 against	           invasion	 process	 of	 both	 S.	 Gallianrum	 and	 S.	 Pullorum	 does	 not	
                                FMDV	involving	the	triggering	of	the	RNA	helicase	mda-5	and	IFN	                     induce	the	expression	of	proinflammatory	cytokines	or	chemokines,	
                                type	 I	 receptor	 signalling.	 Furthermore,	 the	 virus	 can	 induce	 IL-6	         unlike	gastrointestinal	associated	serovars	such	as	S.	Typhimurium	
                                responses	in	these	cells.	Of	interest	is	the	interaction	of	FMDV	with	               or	S.	Enteritidis.	Following	oral	infection,	neither	S.	Gallinarum	nor	S.	
                                DC,	in	particularly	plasmacytoid	DC,	which	results	in	the	release	of	                Pullorum	induce	gut-associated	inflammation	or	pathology	whereas	
                                relatively	 large	 quantities	 of	 IFN.	 The	 cells	 do	 not	 allow	 complete	       S.	 Typhimurium	 and	 S.	 Enteritidis	 induce	 an	 influx	 of	 heterophils	
                                replication	of	FMDV,	but	current	data	indicate	that	RNA	replication	in	              (PMNs)	and	severe	damage	to	the	ileum.	Comparison	of	the	cyto-
                                the	cytoplasm	and	autophagosomal	delivery	to	endosomes	contain-                      kine	 and	 chemokines	 expression	 profile	 following	 infection	 with	 S.	
                                ing	TLR7	would	represent	the	mechanism	by	which	these	cells	are	                     Enteritidis	and	S.	Pullorum	in	chicks	reveals	that	although	there	is	no	
                                activated.	Such	responses	are	amplified	when	the	virus	is	presented	                 difference	between	serovars	in	the	expression	of	IL-6	and	IL-1β	in	
                                in	form	of	immune	complexes,	which	neutralize	the	virus	for	epithelial	              the	ileum,	and	indeed	little	change	over	controls,	there	is	significant	
                                cell	infection,	indicating	a	participation	of	innate	immune	responses	               increases	in	expression	of	CXC	chemokines	following	S.	Enteritidis	
                                also	during	secondary	immune	responses.	Recent	research	has	also	                    infection	 whilst	 there	 is	 downregulation	 of	 expression	 following	 S.	
                                focussed	on	novel	FMD	vaccines,	which	provide	protection	through	                    Pullorum	 infection.	 These	 differences	 are	 most	 pronounced	 with	
                                innate	 immune	 defences,	 particularly	 beneficial	 for	 the	 situation	 of	        CXCLi1,	 the	 major	 chemokines	 associated	 with	 heterophil	 recruit-
                                emergency	vaccination.	                                                              ment,	 and	 indicate	 that	 S.	 Pullorum	 does	 not	 induce	 inflammation	
                                      With	 respect	 to	 adaptive	 immune	 responses	 against	 FMDV	 a	              during	invasion	for	the	gut.	The	absence	of	flagella	in	S.	Pullorum	is	
                                large	 amount	 of	 data	 has	 accumulated.	As	 a	 cytolytic	 virus	 infec-           likely	to	be	as	major	factor	in	this.
                                tion	 mainly	 neutralizing	 antibodies	 principally	 control	 FMDV.	 Their	                The	 interaction	 with	 macrophages	 is	 crucial	 to	 the	 establish-
                                development	is	T	helper	cell-dependent	and	also	dependent	on	DC	                     ment	of	systemic	infection	in	the	spleen	or	liver.	Salmonella	deficient	
                                activity.	 Even	 secondary	 in	 vitro	 antibody	 responses	 require	 both	           in	 their	 ability	 to	 survive	 in	 macrophages,	 (e.g.	 following	 mutation	
                                IL-2	and	BAFF.	The	role	of	cytotoxic	T	cells	are	less	clear	and	their	               in	 the	 SPI2	 type	 III	 secretion	 system)	 are	 completely	 attenuated	
                                triggering	could	be	of	use	for	the	clearance	of	“carrier”	cattle	in	which	           in	 their	 ability	 to	 cause	 disease.	 Host	 genetic	 background	 has	 a	
                                the	virus	persists	in	epithelial	cells	of	the	dorsal	palate.	                        substantial	influence	on	the	course	of	infection.	Macrophages	from	
                                       A	major	challenge	for	the	development	of	novel	vaccines,	which	               Salmonella-resistant	inbred	chicken	lines	are	more	efficient	at	clear-
                                are	not	based	on	the	requirement	of	growing	live	virus,	is	the	efficient	            ing	 intracellular	 Salmonella	 and	 express	 key	 cytokines,	 including	
                                and	rapid	induction	of	neutralizing	antibodies.	As	a	general	rule,	this	             Th1-associated,	 more	 rapidly	 and	 at	 a	 higher	 magnitude	 following	
                                is	achieved	with	vaccines	containing	intact	capsids,	enabling	efficient	             challenge.	 Resistant	 line	 birds	 survive	 S.	 Gallinarum	 challenge,	
                                cross-linking	B	cell	receptors	and	presentation	of	conformational	epi-               whereas	susceptible	line	birds	show	high	mortality.	Clearance	of	S.	
                                topes.	Considering	that	FMDV	enters	through	the	mucosal	surfaces	                    Gallinarum,	 determined	 using	 the	 attenuated	 9R	 vaccine	 strain,	 is	
                                of	 the	 upper	 respiratory	 tract,	 and	 that	 conventional	 vaccines	 can-         primarily	mediated	through	a	Th1	response	with	peak	antigen-spe-
                                not	prevent	local	virus	replication	with	establishment	of	persistence,	              cific	splenic	T	cell	proliferation	and	interferon-γ	expression	coincid-
                                another	new	focus	is	the	development	of	vaccines	with	the	capac-                     ing	with	bacterial	clearance.	Adoptive	transfer	of	T	cells	also	gives	
                                ity	to	induce	mucosal	immune	responses.	Despite	these	important	                     partial	protection	to	challenge.	In	contrast	to	the	‘kill	or	be	cleared’	
                                impulses	coming	from	immunological	research,	successful	vaccines	                    nature	of	S.	Gallinarum,	S.	Pullorum	is	able	to	persist	within	splenic	
                                will	 not	 only	 need	 to	 confer	 rapid	 protection	 against	 several	 of	 the	     macrophages	with	limited	clinical	disease.	Infection	induces	a	strong	
                                seven	known	serotypes	but	will	also	need	to	be	constantly	adapted	                   antibody	 response,	 but	 cellular	 responses	 are	 slow	 to	 develop.	 S.	
                                to	circulating	antigenic	variants.	                                                  Enteritidis	or	S.	Typhimurium	infection	results	in	peak	expression	of	
                                                                                                                     both	 interferon-γ	 and	 IL-18	 around	 14	 days	 post	 challenge	 which	
                                                                                                                     coincides	 with	 subsequent	 clearance.	 In	 contrast	 expression	 of	
                                         PAUL	WIGLEy	(UnIVERSITy	oF	LIVERPooL,	UK)	                                  these	 cytokines	 is	 limited	 following	 S.	 Pullorum	 infection,	 though	
                                     sAlmonellA And tHe mACroPHAge: tHe                                              some	 expression	 of	 IL-4	 can	 be	 detected,	 something	 not	 found	 in	
                                                                                                                                                                   23


other	 serovars.	 This	 suggests	 that	 S.	 Pullorum	 may	 modulate	 the	         cells,	as	numbers	of	CD4+	cells	in	both	the	spleen	and	reproductive	
immune	 response	 favouring	 a	 Th2	 response	 rather	 than	 the	 Th1	            tract	fall	to	around	a	third	of	the	prior	level	prior	to	the	start	of	the	lay-
response	associated	with	clearance.                                               ing	period.	As	laying	becomes	established	CD4+	numbers	and	T	cell	
      S.	Pullorum	persists	in	low	numbers	in	the	spleen	and	liver	of	             function	begin	to	recover	leading	to	a	fall	in	bacterial	numbers.
hens	until	the	start	of	the	egg	laying	period	where	a	recrudescence	
of	 systemic	 infection	 and	 spread	 to	 the	 reproductive	 tract	 occurs.	         WEnDy	BRoWn	(WASHInGTon	STATE	UnIVERSITy,	USA)	
This	coincides	with	a	drop	in	T	cell	activity	both	to	specific	antigenic	
or	 mitogenic	 stimulation.	 	 We	 hypothesise	 that	 during	 the	 carrier	             immunoProteomiC AnAlysis of tHe
state	 the	 intracellular	 bacteria	 and	 the	 immune	 system	 reach	 a	             ProteCtiVe outer membrAne frACtion of
‘stalemate’.	The	loss	of	T	cell	function	and	presumably	interferon-γ	                         AnAPlAsmA mArginAl
stimulation	of	macrophges	allows	the	Salmonella	to	‘breakout’	of	the	                 Rickettsial	 pathogens	 in	 the	 genera	 Anaplasma	 and	 Ehrlichia	
stalemate	multiply	and	spread.	Our	recent	studies	suggest	that	the	               cause	acute	infection	in	immunologically	naïve	hosts	and	are	major	
lack	 of	 function	 may	 be	 a	 consequence	 of	 a	 large	 fall	 in	 T	 helper	   causes	of	tick-borne	disease	in	animals	and	humans.		Immunization	




                                                                                                                                                                   Day 2 - Thursday, August 16th
   PlenAry session: immunoendoCrinology, stress And immunology of reProduCtion And
             neonAtes; miCrobiAl florA, nutrients And tHe immune resPonse



with	Anaplasma	marginale	purified	outer	membranes	induces	com-                     United	States	Department	of	Agriculture,	Agricultural	Research	
plete	 protection	 against	 anaplasmosis	 in	 75%	 of	 animals,	 whereas	            Service,	Beltsville	Human	Nutrition	Research	Center,	Diet,	
immunization	 with	 the	 well-studied	 and	 immunodominant	 major	                Genomics	and	Immunology	Laboratory,	Beltsville,	Maryland,	20705	
surface	 proteins	 MSP1,	 MSP2,	 MSP3,	 MSP4,	 and	 MSP5	 has	 pro-                                            USA
vided	 little	 or	 no	 protection.	 	 The	 completed	 genome	 sequence	 of	             Pigs	infected	with	Ascaris	suum	or	controls	were	given	100	µg	
A.	 marginale	 facilitated	 the	 identification	 of	 subdominant	 and	 less	      (LD)	or	1,000	µg	(HD)	all-trans	retinoic	acid	(ATRA)/kg	body	weight	
abundant	 immunogenic	 proteins	 in	 the	 outer	 membrane	 fraction,	             in	corn	oil	or	corn	oil	alone	per	os	on	–1,	1,	and	3	days	after	inocula-
using	 two	 approaches.	 First,	 two-dimensional	 electrophoresis	 and	           tion	 (DAI)	 with	 infective	 eggs.		 Plasma	 aspartate	 animotransferase	
immunoblotting	of	the	outer	membrane	fraction	with	immune	serum	                  increased	in	pigs	given	LD-ATRA,	while	IL-4	and	IL-12p70	increased	
identified	 numerous	 antigenic	 protein	 spots.	 Analysis	 of	 individual	       in	infected	pigs	given	ATRA	at	7	DAI.		Treatment	with	ATRA	augmented	
proteins	excised	from	the	gels	by	liquid	chromatography	and	tandem	               the	increase	in	bronchial-alveolar	lavage	eosinophils	observed	at	7	
mass	spectrometry	identified	21	novel	antigens.	Of	particular	interest	           and	14	DAI	.	A	quantitative	real	time	RT-PCR	array	was	designed	to	
is	the	finding	that	three	proteins	from	the	type	IV	secretion	system	             test	the	hypothesis	that	ATRA	would	enhance	robust	gene	expres-
(TFSS),	conjugal	transfer	protein,	VirB9,	and	VirB10	were	antigenic.	             sion	following	parasite	infection.	Infected	pigs	had	increased	levels	
TFSS	 proteins	 form	 channels	 and	 are	 responsible	 for	 secretion	 or	        of	 hepatic	 mRNA	 for	 T	 helper	 2	 (Th2)-associated	 cytokines,	 mast	
cell-to-cell	transfer	of	molecules	and	DNA-protein	complexes	in	other	            cell	markers,	and	T	regulatory	(Treg)	cells,	while	infected	pigs	given	
gram-negative	bacterial	pathogens	These	proteins	were	expressed	                  ATRA	 had	 higher	 levels	 of	 hepatic	 IL4,	 IL13,	 CCR3	 and	 CCR4	
and	shown	to	stimulate	IgG2,	and	CD4+	T	cell	proliferation	and	IFN-g	             ligands,	 and	 TPSB1	 compared	 to	 controls.		 Gene	 expression	 for	
production,	responses	associated	with	protective	immunity	in	outer	               Th1-associated	 markers	 (IFNG,	 IL12B,	 and	 TBX21),	 the	 CXCR3	
membrane	 vaccinates.	A	 second	 approach	 to	 more	 directly	 screen	            ligand	(CXCL9),	IL1B,	and	the	putative	Treg	marker	TNFRSF18	was	
for	antigens	recognized	by	T	lymphocytes	involved	in	vitro	transcrip-             increased	by	LD-ATRA	in	infected	animals.		Expression	of	IL4,	IL13,	
tion	and	translation	(IVTT)	of	ORFs	encoding	proteins	predicted	to	               IL1B,	IL6,	CCL11,	and	CCL26	was	increased	in	the	lungs	of	infected	
be	 localized	 on	 the	 outer	 or	 inner	 membrane	 or	 to	 have	 a	 signal	      pigs	treated	with	ATRA.		Thus,	ATRA	augments	a	diverse	Th1-,	Th2-,	
peptide.	 PCR	 products	 of	 selected	 ORFs	 engineered	 to	 express	             and	inflammation-associated	response	in	the	liver	and	lungs	of	swine	
antibody-binding	 sequence	 tags	 were	 amplified	 and	 expressed	                infected	with	Ascaris.
using	IVTT.	As	proof	of	principal,	VirB9	and	outer	membrane	protein	
(OMP)7,	OMP8,	and	OMP9,	known	to	stimulate	T	cell	responses	in	
outer	 membrane	 vaccinates,	 were	 expressed	 by	 IVTT	 and	 affinity	                    HARRIS	LEWIn	(UnIVERSITy	oF	ILLInoIS,	USA)
purified	by	binding	to	anti-tag	antibody	coupled	to	protein-G	bound	                good embryos gone bAd: A trAnsCriPtion
beads,	 and	 the	 beads	 were	 added	 to	 APC	 and	 used	 to	 stimulate	                  Profile of WHAt goes Wrong
immune	CD4+	T	cell	proliferation.	This	novel	technology	can	be	used	
                                                                                   	RoBIn	E	EVERTS1,	SADIE	L	SMITH2,		AnTHony	RAZZAK1,	
to	rapidly	screen	a	large	number	of	proteins	from	a	given	pathogen	
                                                                                   PASCALE	CHAVATTE-PALMER3,	ISABELLE	HUE3,	CHERyL	A	
for	 recognition	 by	 both	 antibody	 and	T	 lymphocytes	 if	 the	 genome	
                                                                                  GREEn1,	RoSAnE	oLIVEIRA1,	SAnDRA	L	RoDRIGUEZ-ZAS1,	
sequence	is	available.
                                                                                  x	CInDy	TIAn2,	xIAnGZHonG	yAnG2,	JEAn-PAUL	REnARD3,	
                                                                                                     HARRIS	A	LEWIn1,4			
                     yASMIn	BELKAID	(nIH,	USA)                                    1Department	of	Animal	Sciences,	University	of	Illinois,	Urbana,	IL,	
   role And origins of regulAtory t Cells                                         USA,		2Center	for	Regenerative	Biology,	University	of	Connecticut,	
         during PArAsitiC infeCtions                                               Storrs,	CT,	USA,	3Biologie	du	Développement	et	Reproduction,	
                                                                                    INRA,	Jouy	en	Josas,	France	4,	Institute	for	Genomic	Biology,	
                                                                                      University	of	Illinois	at	Urbana-Champaign,	Urbana,	IL,	US
                   AnTonIo	LA	CAVA		(UCLA,	USA)	                                                              h-lewin@uiuc.edu
  Cross-tAlK betWeen neuroendoCrine And                                                 Somatic	 cell	 nuclear	 transfer	 (SCNT)	 is	 a	 unique	 model	 sys-
     immune system: tHe CAse of lePtin                                            tem	 to	 explore	 nuclear	 reprogramming	 in	 early	 embryos,	 and	 to	
                                                                                  identify	 the	 critical	 genes	 and	 pathways	 that	 produce	 anomalies	 in	
                	HARRy	DAWSon	(ARS-USDA,	USA)	                                    placental	 and	 fetal	 development.	 	 Cloning	 cattle	 using	 SCNT	 has	
                                                                                  several	advantages	as	a	model	system,	including	high	success	rate	
     regulAtion of PorCine HePAtiC And
                                                                                  of	 blastocyst	 development	 (30%-50%),	 relatively	 high	 frequency	 of	
  PulmonAry-AssoCiAted immune resPonses                                           live	offspring	(up	to	20%)	and	well-established	embryology.	At	least	
               by VitAmin A                                                       66%	of	cattle	embryos	are	lost	by	day-60	of	development.		Placentas	
                24


                                from	 cloned	 cattle	 typically	 show	 extensive	 abnormalities,	 such	 as	        evolutionary	biologists.	For	veterinarians	understanding	such	diver-
                                increased	 size	 and	 fewer	 placentomes.	 	 Of	 those	 SCNT-derived	              sity	is	of	more	than	academic	interest	and	is	important	to	animal	health	
                                clones	that	develop	to	term,	~40%	have	Large	Offspring	Syndrome,	                  and	essential	to	the	profession.	The	genetic	loci	encoding	the	T	cell	
                                which	 is	 characterized	 by	 enlarged	 organs,	 hydrops	 of	 fetus,	 leth-        receptors	and	kappa	and	lambda	light	chains	are	highly	conserved	
                                argy,	and	respiratory	problems.		Large	Offspring	Syndrome	is	similar	              in	 higher	 vertebrates	 but	 major	 differences	 occur	 in	 the	 immuno-
                                to	human	Beckwith-Wiedemann	Syndrome,	which	is	associated	with	                    globulin	heavy	chain	locus.	Most	mammals	have	a	genome	capable	
                                imprinting	defects.		These	data	indicate	problems	in	reprogramming	                of	expressing	all	of	the	major	antibody	isotypes	although	there	are	
                                of	the	somatic	nucleus	during	the	process	of	SCNT.		We	have	con-                   major	 differences	 in	 subclass	 diversification	 of	 IgA	 and	 IgG	 and	 in	
                                ducted	a	series	of	experiments	to	identify	the	metabolic	and	cellular	             the	heavy	chain	variable	locus.	There	are	also	important	differences	
                                pathways	that	cause	the	death	of	cloned	cattle	embryos	and	fetuses.	        	      in	 how	 these	 are	 used	 to	 form	 the	 antibody	 repertoire.	 Differences	
                                Transcriptomic	analysis	of	placentae	derived	from	SCNT	clones,	IVF	                include	 the	 site	 of	 repertoire	 diversification,	 the	 mechanisms	 used,	
                                and	artificial	insemination	(AI)	was	performed	at	different	stages	of	             the	time	of	class-switch	recombination	and	perhaps	even	the	number	
                                development	in	order	to	understand	how	the	cloning	process	leads	                  of	B	cell	subsets.	Studies	on	immunological	diversity	teach	that	cau-
                                to	placental	defects	and	LOS	in	cattle	produced	by	SCNT	and	IVF.	           	      tion	is	needed	in	extrapolating	paradigms	from	mouse-based	studies	
                                For	these	studies,	microarrays	consisting	of	either	7,872	cDNAs	or	                to	other	species.
Day 2 - Thursday, August 16th




                                13,257	70-mer	oligonucleotides	were	used	to	analyze	gene	expres-                         	     Diversity	 among	 mammalian	 immune	 systems	 also	
                                sion	in	individual	d-7	blastocysts,	d-25	embryonic	disk	and	extraem-               extends	to	the	transmission	of	immunity	from	mother	to	young	and	
                                bryonic	tissues,	d-75	fetal	tissues,	and	term	placentomes.	We	have	                in	the	maturity	of	the	offspring’s	immune	system	at	the	time	of	birth.	
                                demonstrated	 massive	 reprogramming	 of	 nuclear	 genes	 in	 SCNT	                Diversity	 of	 this	 type	 allows	 certain	 species	 to	 be	 used	 as	 models	
                                embryos	and	identified	approximately	50	genes	that	are	differentially	             and	tools	to	address	hypotheses	and	solve	problems	that	cannot	be	
                                expressed	in	cloned	blastocysts	as	compared	to	blastocysts	derived	                addressed	in	rodent	models.	Examples	involve	the	study	of	naturally	
                                by	 AI.	 These	 data	 reveal	 candidate	 genes	 and	 cellular	 pathways	           occurring	 premature	 marsupials	 and	 isolator	 piglets.	 Both	 can	 be	
                                for	embryonic	mortality	in	clones,	and	for	placental	defects	in	those	             useful	to	address	major	issues	within	the	“critical	window”	of	neona-
                                clones	that	survive	to	term.	We	have	shown	that	the	defects	in	clones	             tal	immune	development.	These	include	the	role	of	maternal	regula-
                                that	lead	to	abnormalities	in	placental	development	are	likely	to	be	in	           tory	 factors,	 the	 impact	 of	 gut	 colonization	 and	 the	 early	 exposure	
                                the	formation	and	functioning	of	extraembryonic	tissues,	which	are	                to	 pathogen	 and	 the	 development	 of	 tolerance	 to	 dietary	 antigens	
                                necessary	 for	 normal	 placentation.	 	 We	 propose	 that	 the	 placental	        and	commensal	gut	flora.		Many	of	these	factors	are	believed	to	be	
                                defects	are	pathway-dependent,	may	be	lethal	or	compensated	for	                   important	to	the	hygiene	hypothesis	in	which	early	exposure	to	bac-
                                depending	on	the	reprogrammability	and	extent	of	successful	repro-                 teria	and	parasites	can	later	effect	the	development	of	allergies	and	
                                gramming	 of	 the	 donor	 somatic	 nucleus.	 	 Our	 studies	 may	 lead	 to	        inflammatory	bowel	disorders.	Addressing	these	issues	opens	new	
                                new	techniques	for	improving	cloning	efficiency	and	a	greater	under-               frontiers	for	immunology.	Regardless	of	how	pervasive	model	build-
                                standing	 of	 nuclear	 reprogramming	 and	 stem	 cell	 differentiation.	    	      ing	has	become	in	training	immunology	students,	one	must	remain	
                                Additionally,	 our	 work	 provides	 insight	 into	 the	 processes	 of	 early	      cognizant	 that	 while	 models	 provide	 useful	 information	 many	 are	
                                development,	implantation,	and	placental	pathologies.                              wrong	so	the	best	model	is	the	species	itself.	Therefore	the	need	to	
                                                                                                                   characterize	the	immune	system	of	each	species	will	persist.
                                                 JoSEPH	URBAn	(ARS-USDA,	USA)                                            	   Veterinary	 species	 also	 provide	 new	 frontiers	 in	 medi-
                                    use of PArAsitiC infeCtion to exPlore                                          cal	science.	These	involve	engineering	of	farm	animals	to	produce	
                                     dietAry ComPonents tHAt regulAte                                              immunoglobulins	 to	 fill	 the	 dwindling	 supply	 of	 IVIG	 as	 well	 as	 to	
                                   APProPriAte muCosAl immune resPonses                                            produce	 targeted	 antibodies	 for	 immunotherapy	 in	 cases	 where	 in	
                                                                                                                   vitro	production	is	too	low.	Veterinary	species	can	also	be	used	as	
                                                                                                                   disease	models	in	which	rodent	models	fail.
                                Pfizer AWArd for distinguisHed VeterinAry                                                	     Old	 biologists	 often	 become	 old	 philosophers,	 usually	
                                              immunologist                                                         to	 the	 benefit	 of	 younger	 scientists.	 They	 are	 able	 to	 share	 years	
                                                                                                                   of	 experience,	 ponder	 mistakes	 and	 even	 successes	 and	 pass	
                                          JoHn	E	BUTLER	(UnIVERSITy	oF	IoWA,	USA)                                  that	 information	 on	 the	 young	 investigators.	 “Six	 commandments”	
                                                                                                                   fundamental	 to	 bulding	 a	 successful	 career	 in	 immunology	 will	 be	
                                       diVersity, frontiers And CAreers in
                                                                                                                   discussed.	The	three	topics	comprising	this	lecture	are	all	intertwined	
                                                   immunology                                                      and	are	relevant	to	career	building.
                                     Immunological	diversity	has	long	been	of	academic	interest	to	



                                  PlenAry session: immunology of tHe muCosAe, sKin And of tHe mAmmAry glAnd; mAstitis

                                 LoRRAInE	SoRDILLo	(MICHIGAn	STATE	UnIVERSITy,	USA)                                sive	 accumulation	 of	 ROS	 can	 lead	 to	 a	 condition	 referred	 to	 as	
                                  mAmmAry immunity And susCePtibility to                                           oxidative	 stress	 that	 plays	 a	 central	 role	 in	 mediating	 uncontrolled	
                                                                                                                   inflammatory	 responses	 and	 causes	 tissue	 injury.	 Data	 generated	
                                     mAstitis: role of oxidAnt stress
                                                                                                                   thus	 far	 establishes	 a	 strong	 correlation	 between	 oxidative	 stress,	
                                      Mastitis	causes	significant	economic	losses	to	the	world’s	dairy	            such	as	that	associated	with	the	periparturient	period,	and	exagger-
                                producers.	 	 Dairy	 cattle	 are	 more	 susceptible	 to	 mastitis	 during	         ated	inflammatory	responses	of	bovine	mammary	endothelial	cells.	             	
                                the	 periparturient	 period.	 	 	 	 The	 incidence	 of	 mastitis	 with	 respect	   Host	tissues	do	have	several	enzymes	and	small	molecules	that	can	
                                to	lactation	stage	is	directly	related	to	changes	in	the	composition,	             reduce	 ROS	 to	 less	 reactive	 metabolites	 and	 it	 is	 this	 antioxidant	
                                magnitude,	 and	 efficiency	 of	 the	 mammary	 gland	 defense	 system.	            capability	 that	 will	 help	 to	 protect	 cells	 from	 the	 damaging	 effects	
                                There	 exist	 numerous	 genetic,	 physiological,	 and	 environmental	              of	oxidative	stress.			For	example,	the	degree	of	vascular	oxidative	
                                factors	that	can	compromise	host	defense	mechanisms	during	the	                    stress	 can	 be	 controlled	 by	 several	 important	 antioxidant	 seleno-
                                functional	 transitions	 of	 the	 mammary	 gland.	 	 For	 example,	 physi-         proteins,	 including	 glutathione	 peroxidase	 (GPX)	 and	 thioredoxin	
                                ological	 stresses	 associated	 with	 rapid	 differentiation	 of	 secretory	       reductase	 (TrxR).	 These	 antioxidant	 enzymes	 can	 either	 directly	
                                parenchyma,	 intense	 mammary	 gland	 growth,	 and	 the	 onset	 of	                reduce	harmful	ROS	or	play	a	role	in	redox	regulation	of	intracellular	
                                milk	 synthesis	 and	 secretion	 are	 accompanied	 by	 a	 high	 energy	            signaling	processes	that	control	pro-inflammatory	gene	responses.	            	
                                demand	 and	 an	 increased	 oxygen	 requirement.	 Increased	 oxygen	               This	paper	will	outline	some	of	the	molecular	pathways	targeted	by	
                                metabolism	 augments	 the	 production	 of	 oxygen-derived	 reactants,	             GPX	and	TrxR	that	can	influence	excessive	inflammatory	responses	
                                collectively	 termed	 reactive	 oxygen	 species	 (ROS).	 	 The	 exces-             of	bovine	mammary	endothelial	cells.	The	prospects	for	controlling	
                                                                                                                                                                    25


the	 duration	 and	 severity	 of	 mastitis	 by	 manipulating	 these	 critical	         The	 intestine	 is	 the	 largest	 immune	 compartment	 of	 the	 body	
host	antioxidant	defense	mechanisms	are	discussed.                               and	 is	 the	 site	 of	 residence	 or	 portal	 of	 entry	 for	 many	 pathogenic	
                                                                                 microorganisms.	 However,	 constant	 exposure	 of	 the	 gut	 to	 foreign	
                                                                                 material	derived	from	food	and	resident	non-harmful	microorganisms	
     18:30–19:00H	-	ELS	MEEUSEn	(MonASH	UnIVERSITy,	
                                                                                 complicates	the	rules	of	engagement	for	the	immune	system,	neces-
                         AUSTRALIA)
                                                                                 sitating	development	of	tight	regulatory	networks.	Focusing	on	stud-
struCture And ProteCtion of tHe ruminAnt                                         ies	 with	 the	 highly	 immunogenic	 apicomplexan	 protozoan	 Eimeria	
                 lung                                                            vermiformis	 I	 will	 explore	 aspects	 of	 the	 enteric	 immune	 response	
                                                                                 to	 infectious	 challenge,	 including	 the	 requirements	 for	 different	
    Animal	Biotechnology	Research	Laboratories,	Department	of	
                                                                                 lymphoid	 structures,	 the	 cellular	 interactions	 that	 mediate	 effective	
              Physiology,	Monash	University,	Australia	
                                                                                 immunity	and	the	regulation	of	pathology.		
                 e.meeusen@med.monash.edu.au
                                                                                       Effective	immunity	against	primary	infection	with	E.	vermiformis	
      Protection	of	the	lung	from	infection	and	injury	is	primarily	regu-
                                                                                 is	mediated	by	induction	of	a	rapid	Th1-type	response	that	is	depen-
lated	 by	 mechanical	 and	 innate	 defence	 mechanisms	 that	 prevent	
                                                                                 dent	on	co-ordinated	induction	in	both	the	Peyers	patches	(PP)	and	
pathogens	and	obnoxious	substances	from	reaching	the	more	vul-
                                                                                 the	mesenteric	lymph	nodes	(MLN).	The	timing	of	Th1-induction	in	




                                                                                                                                                                    Day 2 - Thursday, August 16th
nerable	lower	respiratory	tract.	Cells	that	line	and	inhabit	the	upper	
                                                                                 both	MLN	and	gut	was	dependent	on	the	presence	of	PP	suggesting	
and	lower	respiratory	tract	have	a	crucial	role	in	sensing	pathogenic	
                                                                                 a	level	of	cooperation	between	immune	responses	induced	in	these	
organisms	for	the	generation	of	a	protective	immune	response,	but	
                                                                                 distinct	 lymphoid	 structures.	The	 delay	 in	Th1-induction	 was	 attrib-
also	 in	 suppressing	 unnecessary	 inflammation	 that	 may	 interfere	
                                                                                 uted	to	the	late	arrival	of	a	broad	range	of	dendritic	cell	(DC)	subsets	
with	 the	 lung’s	 primary	 function	 of	 gas	 exchange.	 The	 structure	 of	
                                                                                 in	the	MLN	and	a	substantial	reduction	of	CD8a-CD11bhi	B220-	Th1-
the	lungs	of	large	animals	is	distinct	from	smaller	laboratory	models	
                                                                                 response	promoting	DC	in	PP-deficient	mice.	The	effective	TCRaβ+	
in	a	number	of	aspects	that	influence	the	type	of	innate	and	adaptive	
                                                                                 CD4+	 T	 cell	 expressed	 IFNγ	 required	 expression	 of	 the	 IFNγR	 on	
response	that	will	be	generated	following	infection.	This	presentation	
                                                                                 stromal	cells	rather	than	any	bone-marrow	derived	immune	cell	sub-
will	give	an	overview	of	the	different	immune	mechanisms	active	at	
                                                                                 set	but	this	interaction	also	induced	the	enteric	pathology	associated	
distinct	levels	of	the	ruminant	lung	and	how	this	may	have	implica-
                                                                                 with	 infection.	 Hence,	 although	 IFNγ	 is	 an	 essential	 component	 of	
tions	for	the	design	of	respiratory	vaccines.
                                                                                 control	of	intracellular	pathogens	in	the	gut	this	cytokine	also	drives	
                                                                                 life-threatening	 pathologies.	 Hence,	 proinflammatory	 infection	 con-
    19:00–19:30H	-	ADRIAn	SMITH	(InSTITUTE	FoR	AnIMAL	                           trol	is	tempered	by	the	rapid	induction	of	regulatory	responses	and	
                        HEALTH,	UK)                                              with	E.	vermiformis	this	is,	at	least	in	part,	mediated	by	the	activities	
      struCturAl And CellulAr AsPeCts of                                         of	TCRγd+	T	cells.	In	the	absence	of	these	cells	the	TCRaβ+	T	cell	
       immunity to PAtHogens in tHe gut                                          mediated	pathologies	are	more	severe	and	result	in	overt	bleeding	
                                                                                 into	the	intestine	of	infected	TCRγ-/-	mice.
Enteric	Immunology,	Institute	for	Animal	Health,	Compton,	Berkshire	
                          RG20	7NN,	UK	
                                                                   Day 3 - August 17th - Friday

                              	 7:45	-	18:00	h	     Registration

                              	   8:30	-	11:00	h	   Poster	Viewing:		Themes	1	–	5	                  SALÃO DIAMANTE

                              	 11:00	-	13:00	h	    Plenary	Session:	Antigen	Presentation,	           SALÃO OURO
Day 3 - Friday, August 17th




                              	                	    Effector	Cells,	Immunoregulation

                              	 14:00	-	16:00	h	    Concurrent	Session	2:	Bacterial	and	              SALÃO OURO
                              	                	    Viral	Diseases	Prions

                              	 14:00	-	16:00	h	    Concurrent	Session	7:	Comparative	Immunology	   SALÃO AMETISTA

                              	 14:30	-	16:30	h	    Concurrent	Session	9:	Innate	immunity,	         SALÃO TURMALINA
                              	                	    Inflammation,	Adjuvants;	Memory,
                              	                	    Acquired	Immunity,	Vaccines

                              	 14:30	-	16:30	h	    Concurrent	Session	10:	Clinical	Immunology	     SALÃO ESMERALDA
                              	                	    Immunopathology

                              	 17:00	-	19:00	h	    Plenary	Session:	Comparative	Immunology	          SALÃO OURO
                                                                                                      27



                                                                                   SALÃO DIAMANTE

	 08:30	-	11:00	h	 Poster Viewing
                 Coffee Break at 10:00h, during poster session
                 1. Immunogenetics and Genomics: posters	IG001-	IG026
                 2. Immune Responses in Bacterial and Viral Diseases; Prions and BSE:
                 posters	BV027	–	BV077
                 3. Immunoendocrinology; and Stress; Immunology of Reproduction and Neonates;
                 Microbial Flora, Nutrients and the Immune Response: posters	ER078-ER094
                 4. Immunology of the Mucosae and Skin and of the Mammary Gland; Mastitis:
                 posters	SM095-SM118
                 5. Antigen Presentation and Dendritic Cells; Effector Cells, B and T cells, NK and
                 NK T cells; Immunoregulatory cells: posters	AP119-AP140




                                                                                      SALÃO OURO
	 1:00	-	13:00	h Plenary Session: Antigen Presentation, Dendritic Cells; Effector Cells, B
1
                 and T cells, NK and NK T cells; Immunoregulation
	              	 Chairs:	Marc	Jutila -	USA	and	Isabelle	Schwartz-Cornil	-	France




                                                                                                      Day 3 - Friday, August 17th
                 Speakers
	 11:00	-	11:30	h	 Ken	McCullough
	              	 Institute	of	Virology	and	Immunoprophylaxis,	Switzerland
	              	 Porcine Dendritic Cells: at the Front Line of Pathogen Attack
	 11:30	-	12:00	h	 Gregory	Borhach
	              	 University	of	Idaho,	USA
                 Bovine T Regulatory Cells
	 12:00	-	12:30	h	 Anne	K.	Storset
	              	 Norwegian	School	of	Veterinary	Science,	Norway
                 NK cells; general characteristics and immunity to infection
	 12:30	-	13:00	h	 JoAnn	Flynn
	              	 University	of	Pittsburgh	School	of	Medicine,	USA
                 IL-17-producing gamma delta T cells and Tuberculosis
	 13:00	-	14:00	h Lunch
1
	 4:00	-	16:00	h Concurrent Session #2: Immune Responses in Bacterial and Viral
                 Diseases; Prions and BSE
                 Chairs:	Tracey	J.	Coffey	-	UK	and	Joan	Lunney	-	USA
	              	 Speakers
	 14:00	-	14:30	h	 Juergen	Richt
	              	 USDA,	USA
                 Prion Knockout cattle as a model to study prion disease
	       14:30	h	 Sandra	Sommer	
	              	 Michigan	State	University,	USA
                 Mycobacterium Paratuberculosis Suppresses CD40 Signaling Induced
                 IL-12p40 and iNos Gene Expression In Bovine Monocyte-Derived Macrophages
                 Abstract	and	Poster	BV030
             28



                              	       14:45	h	 Charles	J.	Czuprynski
                              	              	 University	Of	Wisconsin-Madison,	USA
                                               Prothrombotic Effects of Haemophilus Somnus on Bovine Endothelial
                                               Cells and Platelets
                                               Abstract	and	Poster	BV037
                              	 15:00	-	15:30	h	 Kristien	Van	Reeth
                              	              	 Ghent	University,	Belgium
                                               Can Immunity To H1N1 Influenza Protect Against an H5N1 Avian Influenza Virus? -
                                               Experiments in Pigs as a Model for Humans
                              	       15:30	h	 David	M.	Haig
                              	              	 Moredun	Research	Institute,	UK
                                               IL-15, TNF-a and the Autoimmune Pathogenesis of Malignant Catarrhal Fever
                              	              	 Abstract	and	Poster	BV075
                              	       15:45	h	 Elizabeth	J.	Glass
                              	              	 Roslin	Institute,	UK
                                               Transcriptomic Analysis of the Chicken Anaemia Virus (CAV)-Induced
                                               Immunosuppression
                                               Abstract	and	Poster	BV067




                                                                                                           SALÃO AMETISTA
Day 3 - Friday, August 17th




                              1
                              	 4:00	-	16:00	h Concurrent Session #7: Comparative Immunology
                                               Chairs:	Christopher	J.	Secombes	-	UK	and	Robert	Kammerer	-Germany
                                               Speakers
                              	 14:00	-	14:30	h	 Gregory	Warr
                              	              	 Hollings	Marine	Laboratory,	USA
                                               Innate and Adaptive Immunity in a Marine Shrimp
                              	 14:30	-	15:00	h	 Robert	Miller
                              	                	 University	of	New	Mexico,	USA
                                                 The marsupial immune system: novel adaptations and convergent evolution
                              	       15:00	h	 Takayuki	Kubota
                              	              	 National	Institute	of	Animal	Health,	Japan
                                               Gene expression of chicken interleukine-4 by baculovirus
                                               Abstract	and	Poster	CI206
                              	       15:15	h	 olivia	J	Holland
                              	              	 University	of	Auckland,	New	Zealand
                                               MHC population structure in the New Zealand brushtail possum
                                               Abstract	and	Poster	CI203
                              	       15:30	h	 Robert	Kammerer
                              	              	 LIFE	Center,	LMU,	Germany
                                               The carcinoembryonic antigen (CEA) family in placental mammals of the
                                               superordinal clade Laurasiatheria
                                               Abstract	and	Poster	CI202
                              	       15:45	h	 Harry	D.	Dawson
                              	              	 ARS-USDA,	USA
                                               A Comparative Analysis of the Porcine, Murine, and Human Immune Systems
                                               Abstract	and	Poster	CI205
                                                                                                            29




                                                                                SALÃO TURMALINA
1
	 4:30	-	16:30	h Concurrent Session #9: Innate immunity, Inflammation and Adjuvants;
                 Memory, Acquired Immunity and Vaccines
                 Chairs:	Thomas	Jungi	-	Switzerland	and	Falko	Steinbach	-	UK
                 Speakers
	 14:30	-15:00	h	 Jan	Rombout	
	              	 Wageningen	University,	The	Netherlands
                 Phylogeny and Ontogeny of Innate Immunity.
	 15:00	-	15:30	h	 Reuben	Harris	
	              	 University	of	Minnesota,	USA
                 APOBEC3 proteins in artiodactyls constitute an innate retrovirus defense mechanism.
	 15:30-16:00	h	 Falko	Steinbach	
	              	 Veterinary	Laboratories	Agency,	UK
                 Dendritic cells and their long road to clinical application.
	       16:00	h	 Reginaldo	G.	Bastos	
	              	 Washington	State	University,	USA
                 Interaction of natural killer cells, monocytes and dendritic cell populations in cattle.
                 Poster	and	Abstract	VA219




                                                                                                            Day 3 - Friday, August 17th
	       16:10	h	 oliver	Bruhn	
	              	 University	of	Kiel,	Denmark
                 An equine a-defensin: gene transcription, recombinant expression and
                 characterization of the structure and function
                 Poster	and	Abstract	232
	       16:20	h	 Javier	Dominguez	
	              	 National	Inst.	For	Agricultural	and	Food	Technology,	Spain
                 Targeting to sialoadhesin receptor improves antigen presentation to T cells
                 Poster	and	Abstract	VA243



                                                                                SALÃO ESMERALDA
14:30	-	16:30	h Concurrent Session #10: Clinical Immunology and Immunopathology
                 Chair:	Maria	Julia	Flaminio	-	USA	
                 Speakers
	 14:30	-	15:00	h	 Jean-Pierre	Lavoie
	              	 University	of	Montreal,	Canada
                 Th2-type cytokines and neutrophils: are they playing a role in heaves?
	 15:00	-	15:30	h	 Dennis	Hickstein
	              	 National	Institutes	of	Health,	USA
                 Correction of the phenotype in canine leukocyte adhesion deficiency using stem
                 cell transplant and gene therapy: Canine Leukocyte Adhesion Deficiency Model
                 for New Approaches to LAD
	 15:30	-	16:00	h	 Cornelia	Deeg
	              	 Lugwig	Maximilians	University,	Germany
                 A proteomic approach to the pathogenesis of spontaneous equine recurrent uveitis
             30



                              	 16:00	-	16:30	h	 Ricardo	Tostes	Gazzinelli
                              	              	 Oswaldo	Cruz	Foundation,	Brazil
                                               Towards an anti-amastigote vaccine for canine leishmaniasis: experimental
                                               and pre-clinical trials

                              	 16:00	-	17:00	h Coffee Break

                                                                                                                         SALÃO OURO
                              1
                              	 7:00	-	19:00	h Plenary Session: Comparative Immunology
                                                Chairs:	Maristela	Martins	Camargo	-	Brazil	and	Martin	Bilej	-	Czech	Republic	
                                                Speakers
                              	 17:00	-	17:30	h	 Jean-Marc	Reichhart
                              	              	 Institute	of	Molecular	and	Cellular	Biology,	France
                                               Evolution of the innate immune system, lessons from the Drosophila model
                              	 17:30	-	18:00	h	 Christopher	J.	Secombes
                              	                	 University	of	Aberdeen,	UK
                                                 How much have we learnt about the cytokine network of fish?
                              	 18:00	-	18:30	h	 Jim	Kaufman
                              	              	 Biotechnology	and	Biological	Sciences	Research	Council,	UK
                                               Co-evolution between MHC genes determines alternative immune strategies
                                               in vertebrates
Day 3 - Friday, August 17th




                              	 18:30	-	19:00	h	 John	Butler
                              	              	 University	of	Iowa,	USA
                                               Piglet models in studies on antibody repertoire development.
                                                                                                                                                                 31


               PlenAry session. ANTIGEN PRESENTATION, DENDRITIC CELLS; EFFECTOR CELLS, B AND
                              T CELLS, NK AND NK T CELLS; IMMUNOREGULATION.

                    KEn	MCCULLoUGH		                                            genesis	of	this	infection.		Evidence	from	murine	and	human	animal	
    (InSTITUTE	oF	VIRoLoGy	AnD	IMMUnoPRoPHyLAxIS,	                              models	suggests	that	regulatory	T	cells	(Tregs)	are	induced	by	expo-
                      SWITZERLAnD)                                              sure	to	SAgs.		Recently,	we	studied	the	effects	of	exposing	bovine	
 PorCine dendritiC Cells: At tHe front line                                     peripheral	 blood	 mononuclear	 cells	 (PBMCs)	 to	 a	 physiologically	
                                                                                relevant	dose	(5	ng/ml)	of	SE	type	C1	(SEC1)	for	up	to	10	days.	We	
           of PAtHogen AttACK
                                                                                observed	 that	 SEC1	 caused	 initial	 proliferation	 of	 CD4+	 and	 CD8+	
   KEnnETH	C	MCCULLoUGH,	CARoLE	BALMELLI,	oLIVER	                               T	cells	at	similar	rates.	However,	in	prolonged	cultures,	nearly	all	T	
    BAUHoFER,	LAUREnCE	GUZyLACK-PIRIoU,	nICoLAS	                                cell	proliferation	occurred	independently	of	Vβ	expression,	although	
    RUGGLI,	ARTUR	SUMMERFIELD,	ISABELLE	E	VInCEnT                               CD8+	T	cells	proliferated	more	vigorously.	Expression	of	the	CD25,	
Research	Department,	Institute	of	Virology	and	Immunoprophylaxis,	              CD152	 genes	 increased	 concurrently	 with	 a	 decreased	 expres-
         Sensemattstrasse	293,	CH-3147	Mittelhäusern                            sion	 of	 IL-2.	 	 IL-10	 and	TGF-β	 gene	 expression	 induced	 by	 SEC1	
     Efficient	 immune	 defence	 function	 is	 dependent	 on	 the	 role	        occurred	 within	 the	 CD4+CD25+	 T	 cell	 subpopulation.	 	 Expression	
played	 by	 dendritic	 cells	 (DC),	 particularly	 the	 interaction	 between	   of	Foxp3	also	increased	as	determined	by	measuring	mRNA	levels	
conventional	 (“myeloid”)	 DC	 (cDC)	 and	 plasmacytoid	 DC	 (pDC),	            and	 by	 use	 of	 a	 bovine	 Foxp3-specific	 monoclonal	 antibody.	 	This	
together	 with	 other	 monocytic	 cells.	 This	 determines	 the	 outcome	       effect	coincided	with	an	up-regulation	of	CD152	and	down-regulation	
of	immune	response	development,	but	the	host	defence	capacity	is	               of	IL-2	transcription,	characteristic	of	Tregs.	SEC1-stimulated	CD4+	T	
also	open	to	manipulation	by	viral	pathogens	infecting	DC.	The	man-             cells	were	immunosuppressive	in	vitro	and	suppressed	the	prolifera-
ner	by	which	different	viruses	interfere	with	DC	function	depends	on	           tion	of	naïve	PBMCs	in	response	to	heat-killed-fixed	S.	aureus	in	an	
both	the	virus	and	the	subset	of	DC	involved.                                   IL-10	and	TGF-β	dependant	manner.		Activated	CD8+	T	cells	were	
                                                                                also	immunosuppressive	in	this	assay,	although	the	effect	was	not	
      Classical	 swine	 fever	 virus	 (CSFV)	 is	 a	 monocytotropic	 RNA	       mediated	by	IL-10	or	TGF-β.		These	results	suggest	1)	the	activation	
virus	 infecting	 cDC	 and	 pDC.	 The	 viral	 non-structural	 Npro	 protein	    of	 a	 bovine	 cell	 population	 characteristic	 of	 Tregs,	 2)	 SAgs	 induce	
antagonizes	the	Type	I	interferon	(IFN)	induction	pathway,	promoting	           Tregs	in	the	bovine	model,	and	3)	induction	of	these	cells	in	vivo	could	
proteasomal	degradation	of	interferon	regulatory	factor	(IRF)3.	With	
                                                                                contribute	to	mastitis	and	persistent	infections	in	dairy	animals.	
CSFV	variants	lacking	Npro	will	induce	IFNa	production	by	means	of	
their	dsRNA	replicative	intermediates.	Infection	of	pDC	by	wild	type	
virus	also	results	in	IFNa	induction,	probably	because	the	Npro	does	                           ANNE K. SToRSET
not	interfere	with	the	IRF7	whichis	more	active	pDC.	This	ability	of	the	       (NoRWEGIAN SCHooL oF VETERINARy SCIENCE, NoRWAy)




                                                                                                                                                                 Day 3 - Friday, August 17th
virus	to	inhibit	cDC	production	of	IFNa,	while	augmenting	the	IFNa	                 nK Cells; generAl CHArACteristiCs And
production	 by	 pDC	 would	 lead	 to	 an	 exaggerated	 pDC	 response,	
                                                                                            immunity to infeCtion
relating	to	the	immunopathological	characteristics	of	the	disease.
                                                                                      Natural	killer	cells	were	first	known	in	the	early	1970s	as	large	
      The	ssDNA	virus	–	porcine	circovirus	type	2	(PCV2)	–	is	also	
                                                                                granular	 lymphocytes	 that	 could	 spontaneously	 kill	 tumour	 cells	
immunomodulatory.	 In	 contrast	 to	 CSFV,	 PCV2	 does	 not	 replicate	
                                                                                -	 often	 ignored	 as	 disturbing	 sources	 of	 background	 in	 assays	 of	
in	DC,	but	accumulates	to	high	levels	both	in	vitro	and	in	vivo.	This	
                                                                                cancer	immunology.	As	the	nature	of	these	cells	became	clear	they	
accumulation	is	dependent	on	virus	capsid	protein,	but	independent	
                                                                                attracted	increasing	attention,	and	their	ability	to	kill	cells	that	lack	
of	 virus	 replication.	 Nevertheless,	 the	 presence	 of	 PCV2	 in	 cDC	
                                                                                MHC	class	I	was	regarded	as	their	most	important	feature.	Studies	
does	 not	 interfere	 with	 processing	 of	 other	 antigens.	 In	 contrast,	
                                                                                of	NK	cells	and	their	receptors	exploded	in	the	1990s,	and	today	they	
elevated	IL-10	production	is	observed.	This	alone	would	not	explain	
                                                                                are	recognised	as	central	players	of	the	rodent	and	human	immune	
the	 immunoregulatory	 characteristics	 of	 PCV2-induced	 diseases.	
                                                                                systems.	 As	 NK	 cells	 have	 now	 been	 assigned	 roles	 exceeding	
PCV2	 will	 impair	 and	 even	 abrogate	 “danger”	 recognition	 by	 cells	
                                                                                that	of	cancer	and	transplantation	immunology,	they	have	became	
of	the	innate	defences,	a	property	dependent	on	the	viral	genome,	
                                                                                increasingly	interesting	for	veterinary	immunologists.	
particularly	the	dsDNA	replicative	form.	The	DC	family	represents	a	
critical	central	element	in	the	efficient	functioning	of	immune	defense	              NK	cells	are	involved	in	defence	mechanisms	against	several	
generation	and	maintenance.	While	certain	viruses	can	interfere	with	           microbial	 infections,	 and	 they	 have	 a	 role	 in	 shaping	 the	 adaptive	
the	efficient	functioning	of	these	cells,	immune	defences	can	also	be	          immune	 response,	 being	 of	 potential	 interest	 in	 vaccine	 develop-
enhanced	–	by	vaccination.	This	also	requires	interaction	between	              ment.	In	primates,	they	also	have	a	role	in	placental	development.	
cDC	 or	 pDC,	 towards	 building	 the	 immune	 defensive	 barrier	 for	         NK	 cells	 belong	 to	 the	 innate	 immune	 system	 and	 work	 through	
protecting	 the	 host.	 Targeting	 DC	 has	 high	 potential	 for	 improved	     germline	encoded	receptors.	Families	of	receptors	with	both	inhibi-
vaccination	strategies,	protecting	the	host	against	manipulation	of	its	        tory	 and	 activating	 functions	 have	 been	 the	 hallmark	 of	 NK	 cells,	
immune	defenses	by	the	viral	pathogen	in	question.	The	central	ele-             although	most	of	these	receptors	may	also	be	expressed	on	other	
ment	for	success	is	the	efficient	targeting	of	DC,	offering	as	it	does	         leukocytes.	 The	 cytokine	 and	 chemokine	 producing	 capabilities	 of	
high	potential	for	improved	vaccines	both	now	and	in	the	future.                NK	cells	is	important	in	their	immunoregulatory	functions.
                                                                                      At	present,	there	is	no	consensus	phenotypic	definition	of	NK	
                                                                                cells	across	species.	But	very	recently	the	surface	expression	of	the	
                       GREGoRy BoRHACH                                          natural	cytottoxicity	receptor,	NKp46,	has	been	suggested	as	a	defi-
                   (UNIVERSITy oF IDAHo, USA)                                   nition	criterion.	The	presence	of	the	gene	for	this	receptor	in	several	
                  BOVINE T REGULATORy CELLS                                     veterinary	species	makes	this	proposal	an	important	stimulant	to	the	
   boVine regulAtory t Cells induCed by A                                       development	of	the	NK	cell	field	in	veterinary	immunology.	
   stAPHyloCoCCAl suPerAntigen in Vitro                                               NK	 cells	 in	 cattle	 and	 their	 functions	 have	 been	 studied	 for	
   KEUn	SEoK	SEo1,	yonG	Ho	PARK2,	WILLIAM	C	DAVIS3,	                            some	years,	and	although	this	field	is	still	in	its	infancy,	possible	roles	
                GREGoRy	A	BoHACH1                                               in	 several	 infections	 have	 been	 described.	 Bovine	 NK	 cells	 have	
    1Dept	Microbiol,	Molecular	Biol	&	Biochem,	Univ,	of	Idaho,	                 showed	 reactions	 against	 cells	 infected	 with	 intracellular	 bacteria	
  Moscow,	ID,	USA;	2Dept	Microbiol,	Seoul	National	Univ,	Seoul,	                and	may	also	be	of	importance	in	parasitic	infections.
 Korea;	3Dept	Vet	Microbiol,	&	Pathology,	Washington	State	Univ,	
                        Pullman,	WA,	USA	                                                            JoANN FLyNN
                      	gbohach@uidahoedu                                         (UNIVERSITy oF PITTSBURGH SCHooL oF MEDICINE, USA)
     Staphylococcal	superantigens	(SAgs),	including	staphylococcal	                il-17-ProduCing gAmmA deltA t Cells And
enterotoxins	 (SEs)	 are	 expressed	 by	 a	 high	 percentage	 of	 bovine	                        tuberCulosis
mastitis	 isolates,	 suggesting	 that	 these	 toxins	 contribute	 to	 patho-
             32


                                    ConCurrent session #2. IMMUNE RESPONSES IN BACTERIAL AND VIRAL DISEASES; PRIONS AND BSE


                                                         JUERGEN RICHT                                              Since	no	humans	have	immunity	to	the	H5	influenza	virus	hae-
                                                           (USDA, USA)                                        magglutinin	(HA),	the	appearance	of	a	highly	pathogenic	(HP)	H5N1	
                              Prion KnoCKout CAttle As A model to study                                       avian	 influenza	 virus	 (AIV)	 in	 humans	 is	 of	 major	 concern.	 On	 the	
                                                                                                              other	hand,	the	H5N1	virus	shares	its	neuraminidase	(NA)	subtype	
                                           Prion diseAse
                                                                                                              with	the	endemic	human	H1N1	viruses	and	cell-mediated	immunity	
                                 JA	RICHT1,	An	HAMIR1,	C	SoTo2,	J	RoBL3,	y	KURoIWA3                           to	the	relatively	conserved	internal	viral	proteins	may	also	contribute	
                                  1National	Animal	Disease	Center,	Ames,	IA.,	USA;	2UTMB,	                    to	protection.	We	use	the	pig	model	of	influenza	to	study	the	extent	
                                 Galveston,	TX.,	USA;	3Hematech	Inc.,	Sioux	Falls,	S.D.,	USA                  of	 cross-protection	 between	 influenza	 viruses	 with	 unrelated	 HA	
                                    Prion	 diseases,	 such	 as	 bovine	 spongiform	 encephalopathy	           subtypes	and	the	underlying	immune	mechanisms.	In	this	lecture	I	
                              (BSE)	in	cattle	and	Creutzfeldt–Jakob	disease	(CJD)	in	human,	are	              will	discuss	recent	data	about	the	effect	of	prior	infection	with	H1N1	
                              caused	by	propagation	of	a	misfolded	form	of	normal	cellular	prion	             swine	influenza	virus	on	challenge	with	a	low	pathogenic	(LP)	H5N1	
                              protein,	PrPC.	Disruption	of	PrPC	expression	in	the	mouse,	a	species	           AIV.	Influenza	virus	naïve	pigs	were	inoculated	with	H1N1	and	H5N1	
                              that	does	not	naturally	contract	prion	diseases,	results	in	no	appar-           at	a	4-week	interval	(H1N1-H5N1	group)	or	with	H5N1	only	(H5N1	
                              ent	developmental	abnormalities,	and	in	resistance	to	mouse	prion	              challenge	 control	 group).	 We	 examined	 serum	 antibody	 titres	 to	
                              disease.		However,	the	impact	of	the	ablation	of	PrPC	function	in	a	            both	viruses	in	haemagglutination	inhibition	(HI),	virus	neutralisation	
                              natural	host	species	of	prion	diseases	is	unknown.		Here,	we	report	            (VN)	and	neuraminidase	inhibition	(NI)	tests,	influenza	virus	specific	
                              the	generation	and	characterization	of	PrPC-deficient	cattle.		At	over	         lymphoproliferative	 responses	 of	 peripheral	 blood	 mononuclear	
                              24	months	of	age,	the	cattle	are	clinically,	physiologically,	histopatho-       cells,	and	clinical	and	virological	protection	against	H5N1	challenge.	
                              logically	 and	 immunologically	 normal,	 indicating	 that	 “loss	 of	 func-    Before	challenge,	the	H5N1	challenge	control	pigs	were	negative	in	
                              tion”	of	endogenous	bovine	PrPC	does	not	directly	cause	BSE	and	                all	serological	assays.	Pigs	of	the	H1N1-H5N1	group	had	HI	and	VN	
                              that	PrPC	function	itself	is	generally	dispensable	for	normal	animal	           antibodies	to	H1N1	only,	and	NI	antibodies	to	both	H1N1	(mean	titre	
                              development.		Furthermore,	the	knockout	(KO)	cattle	are	resistant	to	           80)	and	H5N1	(mean	titre	61).	This	group	also	showed	20-fold	higher	
                              prion	propagation	in	vitro	by	protein	misfolding	cyclic	amplification.	     	   lymphoproliferative	 indexes	 to	 H5N1	 than	 the	 challenge	 control	
                              The	KO	cattle	could	be	a	useful	model	for	prion	research	as	a	natural	          group.	The	H5N1	challenge	produced	mild	to	moderate	clinical	signs	
                              host	species	of	prion	diseases	and	provide	prion	protein-free	bovine	           in	all	challenge	control	pigs	and	the	virus	was	isolated	from	the	nasal	
                                                                                                              mucosa	(4/9	pigs),	trachea	(7/9	pigs)	and	lungs	(9/9	pigs)	of	most	
                              products	for	bio-industries.
                                                                                                              pigs.	Pigs	of	the	H1N1-H5N1	group,	in	contrast,	showed	complete	
Day 3 - Friday, August 17th




                                                                                                              clinical	 protection	 and	 strongly	 reduced	 virus	 isolation	 rates.	 Our	
                                                    KRISTIEN VAN REETH                                        data	indicate	that	immunity	to	an	H1N1	influenza	virus	may	partially	
                                                (GHENT UNIVERSITy, BELGIUM)                                   protect	pigs	from	avian	H5N1	influenza.	Cross-protection	was	clearly	
                                   studies on tHe immune resPonse to                                          independent	 of	 antibody	 to	 the	 viral	 HA,	 and	 may	 be	 mediated	 by	
                                 influenzA in Pigs: lessons for PAndemiC                                      antibody	to	the	N1	NA	and/or	cross-reactive	cell-mediated	immunity.	
                                           influenzA VACCines                                                 Cross-reactive	N1	antibodies	were	recently	shown	to	afford	partial	
                                                                                                              protection	against	challenge	with	HP	H5N1	in	mice.	The	pig	is	a	valu-
                                      VAn	REETH	K1,	BRAECKMAnS	D	1,	Cox	E	2,	DE	                              able	model	for	further	studies	on	the	mechanisms	and	modalities	of	
                                                 VLEESCHAUWER	A	1                                             cross-protection	between	H1N1	and	H5N1.	Such	studies	will	reveal	
                                   Laboratory	of	Virology	(1)	and	Immunology	(2),	Faculty	of	                 whether	increasing	levels	of	immunity	to	H1N1	in	humans	could	be	a	
                                       Veterinary	Medicine,	Ghent	University,	Belgium                         possible	pandemic	strategy	against	H5N1.




                                                                    ConCurrent session #7. COMPARATIVE IMMUNOLOGy

                                                    GREGoRy WARR                                                                      RoBERT MILLER
                                          (HoLLINGS MARINE LABoRAToRy, USA)                                                   (UNIVERSITy oF NEW MExICo, USA)
                                innAte And AdAPtiVe immunity in A mArine                                          tHe mArsuPiAl immune system: noVel
                                                sHrimP                                                           AdAPtAtions And ConVergent eVolution



                                                    ConCurrent session #9. INNATE IMMUNITy, INFLAMMATION AND ADJUVANTS;
                                                                MEMORy, ACQUIRED IMMUNITy AND VACCINES

                                                  JAN RoMBoUT                                                 At	 least	 two	 of	 the	 human	 proteins,	APOBEC3F	 and	APOBEC3G,	
                                     (WAGENINGEN UNIVERSITy, THE NETHERLANDS                                  can	effectively	inhibit	the	replication	of	HIV-1.	However,	HIV-1	usually	
                                     PHylogeny And ontogeny of innAte                                         neutralizes	this	host	defense	through	Vif,	which	triggers	APOBEC3	
                                                immunity                                                      ubiquitination	and	degradation.	We	have	discovered	an	APOBEC3F-
                                                                                                              like,	double	deaminase	domain	protein	from	three	artiodactyls	(cattle,	
                                                                                                              pigs	 and	 sheep).	 Like	 their	 human	 counterparts,	 APOBEC3F	 and	
                                                      REUBEN HARRIS                                           APOBEC3G,	the	artiodactyl	APOBEC3F	proteins	are	DNA	cytosine	
                                              (UNIVERSITy oF MINNESoTA, USA)                                  deaminases	that	locate	predominantly	to	the	cytosol	and	can	inhibit	
                                  APobeC3 Proteins in ArtiodACtyls                                            the	replication	of	HIV-1	and	MLV.	Retrovirus	restriction	is	attributable	
                               Constitute An innAte retroVirus defense                                        to	deaminase-dependent	and	-independent	mechanisms,	as	deami-
                                              meCHAnism                                                       nase-defective	 mutants	 still	 retain	 significant	 anti-retroviral	 activity.	
                                                                                                              However,	unlike	human	APOBEC3F	and	APOBEC3G,	the	artiodac-
                                Department	of	Biochemistry,	Molecular	Biology	and	Biophysics,	                tyl	APOBEC3F	proteins	have	an	active	amino	terminal	DNA	cytosine	
                                      University	of	Minnesota,	Minneapolis,	MN	55455	                         deaminase	domain,	which	elicits	a	broader	dinucleotide	deamination	
                                   The	APOBEC3	proteins	are	unique	to	mammals.	Many	inhibit	                  preference,	and	they	are	resistant	to	HIV-1	Vif.	These	data	indicate	
                              retrovirus	infection	through	a	cDNA	cytosine	deamination	mechanism.	            that	DNA	cytosine	deamination,	subcellular	localization	and	retrovi-
                                                                                                                                                             33


rus	restriction	activities	are	conserved	in	mammals,	whereas	active	          Key words:	 innate	 immunity,	 virus	 restriction,	 DNA	 cytosine	
site	location,	local	mutational	preferences	and	Vif	susceptibility	are	       deamination
not.	 Together,	 these	 studies	 indicate	 that	 some	 properties	 of	 the	   Species:	ruminants
mammal-specific,	 APOBEC3-dependent	 retroelement	 restriction	
system	are	necessary	and	conserved,	but	others	are	simultaneously	                                FALKo STEINBACH
modular	 and	 highly	 adaptable.	 Interestingly,	 artiodactyls	 appear	 to	             (VETERINARy LABoRAToRIES AGENCy, UK)
have	an	APOBEC3	genomic	organization	more	like	rodents	than	that	
                                                                                 dendritiC Cells And tHeir long roAd to
of	primates.	This	has	contributed	to	a	model	for	an	overall	dynamic	
expansion	of	the	APOBEC3	locus	in	mammals.                                                CliniCAl APPliCAtion




                      ConCurrent session #10. CLINICAL IMMUNOLOGy AND IMMUNOPATHOLOGy

                    JEAN-PIERRE LAVoIE                                        Since	 gammaretroviral	 vectors	 have	 led	 to	 insertional	 activation	 of	
             (UNIVERSITy oF MoNTREAL, CANADA)                                 nearby	oncogenes	and	leukemia	in	previous	gene	therapy	trials,	we	
  tH2-tyPe CytoKines And neutroPHils: Are                                     carried	out	gene	therapy	in	the	CLAD	model	using	a	vector	based	on	
                                                                              foamy	virus	(FV).		Four	of	five	CLAD	dogs	receiving	non-myeloabla-
       tHey PlAying A role in HeAVes?
                                                                              tive	conditioning	with	200	cGy	TBI	and	infusion	of	autologous	CD34+	
                                                                              hematopoietic	 stem	 cells	 transduced	 by	 the	 FV	 vector	 expressing	
                         DENNIS HICKSTEIN                                     canine	CD18	had	complete	reversal	of	the	CLAD	phenotype,	which	
                            (NIH, USA)                                        was	sustained	two	years	following	treatment.		In	vitro	assays	dem-
  CorreCtion of tHe PHenotyPe in CAnine                                       onstrated	 correction	 of	 the	 lymphocyte	 proliferation	 and	 neutrophil	
leuKoCyte AdHesion defiCienCy using stem                                      adhesion	defects	that	characterize	CLAD.		There	were	no	genotoxic	
Cell trAnsPlAnt And gene tHerAPy: CAnine                                      complications	and	integration	site	analysis	demonstrated	polyclonal	
                                                                              marking	by	transduced	cells.		These	results	suggest	that	FV	vectors	
leuKoCyte AdHesion defiCienCy model for
                                                                              will	be	effective	in	treating	human	hematopoietic	diseases	such	as	
         neW APProACHes to lAd
                                                                              LAD.		These	studies	indicate	that	new	therapeutic	approaches	are	
      Children	 with	 the	 genetic	 immunodeficiency	 leukocyte	 adhe-        becoming	available	to	treat	LAD	now	20	years	after	the	initial	cloning	
sion	 deficiencyor	 LAD	 have	 heterogeneous	 molecular	 defects	 in	         of	the	CD18	cDNA.
the	 leukocyte	 integrin	 CD18	 molecule	 and	 suffer	 life-threatening	




                                                                                                                                                             Day 3 - Friday, August 17th
bacterial	infections	due	to	the	inability	of	their	leukocytes	to	adhere	      Dennis D. Hickstein, M.D.
and	migrate	to	sites	of	infection.		Hematopoietic	stem	cell	transplant	       Senior	Investigator	
remains	 the	 only	 curativetherapy	 for	 LAD,	 however	 the	 toxicity	 of	   Experimental	Transplantation	and	Immunology	
this	treatment	has	limited	its	use	in	genetic	diseases	such	as	LAD.	          Center	for	Cancer	Research,	National	Cancer	Institute	
We	tested	new	stem	cell	transplant	and	gene	therapy	approaches	               National	Institutes	of	Health	
to	 LAD	 in	 a	 canine	 model	 of	 LAD.	 Matched	 littermate	 transplant	     Bldg	10	CRC,	Room	3-3142	(3	East	Labs)	
following	 a	 non-myeloablative	 conditioning	 regimen	 with	 200	 cGy	       10	Center	Drive,	MSC	1203
total	body	irradiation	resulted	in	reversal	of	the	phenotype	of	CLAD	
with	 minimal	 toxicity.	 However,	 the	 optimal	 therapy	 for	 LAD	 would	
                                                                                                  CoRNELIA DEEG
involve	 gene	 therapy	 since	 no	 donor	 is	 required	 and	 graft	 versus	
                                                                                      (LUGWIG MAxIMILIANS UNIVERSITy, GERMANy)
host	disease	is	not	an	issue.	We	first	evaluated	ex	vivo		gammaretro-
viral-mediated	gene	therapy	using	two	non-myeloablative	condition-             A ProteomiC APProACH to tHe PAtHogensis
ing	regimens	-200	cGy	TBI	or	10	mg/kg	busulfan	-	with	or	without	              of sPontAneous equine reCurrent uVeitis
post-transplant	immunosuppression.		Six	of	11	treated	CLAD	dogs	
achieved	therapeutic	levels	of	CD18+	leukocytes.		Conditioning	with	                          RICARDo ToSTES GAzzINELLI
either	TBI	 or	 busulfan	 allowed	 long-term	 engraftment	 and	 immune	                   (oSWALDo CRUz FoUNDATIoN, BRAzIL)
suppression	was	not	required	for	efficacy.		The	percentage	of	CD18+	
leukocytes	increased	over	6-8	months	to	levels	ranging	from	0.72%	              toWArds An Anti-AmAstigote VACCine for
to	8.37%	at	one-year	follow-up	in	the	6	dogs.		These	levels	resulted	           CAnine leisHmAniAsis: exPerimentAl And
in	 reversal	 of	 the	 severe	 CLAD	 phenotype.	 	 Linear	 amplification-                 Pre-CliniCAl triAls
mediated-PCR	 assays	 indicated	 polyclonality	 of	 insertion	 sites.	



                                          PlenAry session. COMPARATIVE IMMUNOLOGy



                 JEAN-MARC REICHHART                                                                      CoUNCIL, UK)
   (INSTITUTE oF MoLECULAR AND CELLULAR BIoLoGy,                                  Co-eVolution betWeen mHC genes
                       FRANCE)
                                                                              determines AlternAtiVe immune strAtegies
   eVolution of tHe innAte immune system,                                                  in VertebrAtes
     lessons from tHe drosoPHilA model
                                                                                                        JoHN BUTLER
                   CHRISToPHER J SECoMBES                                                         (UNIVERSITy oF IoWA, USA)
                 (UNIVERSITy oF ABERDEEN, UK)
                                                                                   Piglet models in studies on Antibody
      HoW muCH HAVe We leArnt About tHe                                                  rePertoire deVeloPment
          CytoKine netWorK of fisH?                                                 The	 piglet	 was	 selected	 for	 studies	 on	 antibody	 repertoire	
                                                                              development	 because	 no	 maternal	 antibodies	 cross	 the	 placenta	
                 JIM KAUFMAN,                                                 to	 influence	 fetal	 development	 and	 because	 their	 precosial	 nature	
(BIoTECHNoLoGy AND BIoLoGICAL SCIENCES RESEARCH                               allows	 them	 to	 be	 raised	 in	 isolator	 units	 in	 which	 environmental	
             34


                              influences	are	controlled	by	the	experimenter.	Their	use	as	models	             conserved	and	similar	to	those	in	other	mammals.
                              required	characterization	 of	their	B	and	T	cell	repertoires	and	their	               	     The	isolator	piglet	model	has	allowed	us	to	study	events	
                              development.	Discoveries	made	during	this	characterization	process	             in	 antibody	 repertoire	 development	 that	 occur	 during	 the	 “critical	
                              contributed	to	the	accumulating	information	on	the	diversity	of	anti-           window”.	Studies	have	shown	that	the	development	of	the	adaptive	
                              body	systems	in	other	vertebrates	and	on	the	mechanisms	involved	               immune	system	in	this	species	depends	on	gut	colonization	acting	
                              in	the	development	of	antibodies	repertoires.                                   through	ligands	they	produce	that	are	recognized	by	receptors	of	the	
                                    	     Swine	 possess	 the	 same	 five	 isotypes	 of	 immunoglobu-         innate	immune	system.	During	the	“critical	window”	piglets	are	espe-
                              lins	as	most	all	mammals	but	have	greatly	diversified	their	IgG	(Cγ)	           cially	susceptible	to	immune	dysregulation	of	the	type	produced	by	
                              genes	of	which	at	least	eleven	are	expressed.	In	addition,	there	are	           pathogens	like	porcine	reproductive	and	respiratory	syndrome	virus	
                              alternatively	spliced	forms	so	pigs	can	potentially	express	more	than	          (PRRSV).		PRRSV	interferes	with	normal	development	of	adaptive	
                              20	Cγ	variants.	Especially	interesting	is	IgG3	that	is	expressed	in	an	         immunity	by	selective	expansion	of	a	minor	subset	of	pre-immune	B	
                              antigen-independent	 manner	 in	 the	 ileal	 Peyers	 patches	 and	 mes-         cells	bearing	hydrophobic,	germline-encoded	HCDR3s.	These	are	not	
                              enteric	lymph	node.	Class	switch	recombination	occurs	early	in	fetal	           believed	to	contribute	anti-viral	antibodies.	This	diversion	is	believed	
                              life	and	piglets	are	born	with	B	cells	expressing	all	major	isotypes.	          to	be	due	to	a	putative	B	cell	superantigen	such	that	one-third	of	all	
                              Unique	to	swine	is	the	use	of	four	VH	genes,	2	DH	segments	and	                 B	cells	in	isolator	piglets	can	be	of	this	type.	This	is	not	dissimilar	to	
                              one	 JH	 segment	 to	 form	 >80%	 of	 the	 pre-immune	 repertoire.	This	        patients	with	myeloma	and	we	believe	this	diversion	accounts	for	the	
                              user-friendly	system	allows	diversification	of	the	antibody	repertoire	         delay	in	the	development	of	effective	adaptive	immunity.
                              to	 be	 readily	 monitored	 by	 quantitative	 clonal	 hybridization.	 Unlike	
                              the	heavy	chain	locus,	the	kappa,	lambda,	Vβ	and	Vd	loci	are	greatly	
Day 3 - Friday, August 17th
                                    Day 4 - August 18th - Saturday

	 7:45	-	18:00	h	    Registration

	 8:30	-	10:30	h	    Plenary	Session:	Immunoparasitology	                    SALÃO OURO

	 11:00	-	13:00	h	   Plenary	Session:	Mediators;	Immunoglobulins	            SALÃO OURO	
	                	   and	Fc	Receptors;	Signal	Transduction

	 14:00	-	18:00	h	   Concurrent	Session	4:	Mucosae,	Skin	and	              SALÃO AMETISTA
	                	   Mammary	Gland

	 14:00	-	18:00	h	   Concurrent	Session	8:	Mediators	of	Recruitment	and	     SALÃO OURO
	                	   Function	of	Cells	of	the	Immune	System;
	                	   Immunoglobulins;	Signal	Transduction

	 14:30	-	18:00	h	   Concurrent	Session	6:	Immunoparasitology	             SALÃO TURMALINA




                                                                                             Day 4 - Saturday, August 18th
	 14:30	-	18:00	h	   Concurrent	Session	5:	Antigen	Presentation;	          SALÃO ESMERALDA
	                	   Effector	Cells;	Immunoregulatory	Cells
                      36



                                                                                                                            SALÃO OURO
                                    8:30	-	10:30	h Plenary Session: Immunoparasitology: Immune Responses to Protozoa,
                                                   Helminths and Ectoparasites
                                                   Chairs:	Jan	naessens	-	Kenya	and	Elisabeth	A.	Innes	-	UK
                                                   Speakers
                                	    8:30	-	9:10	h	 David	Artis
                                	                	 University	of	Pennsylvania,	USA
                                                   A critical role for intestinal epithelial cells in regulating CD4 T cell responses
                                                   and immunity to parasitic infection in the gastrointestinal tract
                                	    9:10	-	9:50	h	 Alex	Loukas
                                	                	 Queensland	Institute	of	Medical	Research
                                                   Using the dog as a model for developing a human hookworm vaccine
                                	 9:50	-	10:30	h	 Declan	McKeever
                                	                	 Royal	Veterinary	College
                                                   CTL immunity as a driver for diversity in Theileria parva parasites
                                    10:30	-	11:00	h Coffee Break

                                    11:00	-	13:00	h Plenary Session: Mediators of Recruitment and Function of Cells of the
                                                    Immune System; Signal Transduction and Gene Expression
                                                   Chairs:	Jayne	C.	Hope	-	UK	and	Cynthia	Baldwin	-	USA
                                                   Speakers
                                	 11:00	-	11:30	h	 Stephanie	Widdison
                                	                	 Institute	for	Animal	Health,	UK
                                                   Macrophages and Mycobacteria: what’s the attraction?
                                		11:30	-	12:00	h	 Jim	Harris
                                	                	 University	of	Dublin,	Ireland
                                                   TH1-Th2 polarisation and the control of intracellular mycobacteria by Autophagy
                                		12:00	-	12:30	h	 Cynthia	Baldwin
                                	                	 University	of	Massachusetts-Amherst,	USA
                                                   Control of gamma delta T cell IFN-γ responses: the role of the TCR and
Day 4 - Saturday, August 18th




                                                   co-receptor WC1
                                		12:30	-	13:00	h	 Jayne	Hope
                                	                	 Institute	for	Animal	Health-Compton,	UK
                                                   Dendritic cells, cytokines and mycobacteria
                                    13:00	-	14:00	h Lunch



                                                                                                                     SALÃO AMETISTA
                                    14:00	-	17:30	h Concurrent Session #4: Immunology of the Mucosae and Skin and of the
                                                    Mammary Gland; Mastitis
                                                   Chairs:	Douglas	Bannermann	-	USA	and	Theo	A.	niewold	-	The	Netherlands
                                                   Speakers
                                	 14:00	-	14:30	h	 Michael	Murtaugh
                                	                	 University	of	Minnesota,	USA
                                                   Genomic Analysis of Mucosal Immunobiology in the Porcine Small Intestine
                                	 14:30	-	15:00	h	 Eric	Cox
                                	                	 Ghent	University,	Belgium
                                                                                                            37



                   Mucosal responses against fimbrial antigens of enterotoxigenic E. coli in pigs
	         15:00	h	 Theo	niewold
	               	 Catholic	University	of	Leuven,	Belgium
                  The in vivo early transcriptional intestinal response to rotavirus infection
                  in germ-free piglets
                  Abstract	and	Poster	SM107
	         15:15	h	 Anne	Goubier
	               	 Merial,	France
                  Colostrum from sows vaccinated with an inactivated PCV2 vaccine contains
                  antigen specific leukocytes
                  Abstract	and	Poster	SM111
	 15:30	-	16:00	h	 Coffee Break
	 16:00	-	16:30	h	 Isabel	K.	Ferreira	de	Miranda	Santos
	               	 University	of	São	Paulo,	Brazil
                  Sequential morphology and gene expression profiles of cutaneous reactions to
                  tick antigens in bovines
                  Abstract	and	Poster	SM096,	SM097,	SM098
	         16:30	h	 Arthur	Summerfield
	               	 Institute	of	Virology	and	Immunoprophylaxis	Switzerland
                  Modulation of peripheral dendritic cells towards mucosa-type dendritic cells
                  by all-trans retinoic acid
                  Abstract	and	Poster	SM109
	         16:45	h	 Masahiro	yasuda
	               	 University	of	Miyazaki,	Japan
                  Dynamics of B-cell repertoire in sheep jejunal and ileal Peyer’s patch single follicles
                  Abstract	and	Poster	SM101
    17:00	-	17:30	h Finish


                                                                                           SALÃO OURO
	 4:00	-	18:00	h Concurrent Session #8: Mediators of Recruitment and Function of Cells
1
                 of the Immune System; Fc Receptors and Immunoglobulins; Signal




                                                                                                            Day 4 - Saturday, August 18th
                 Transduction and Gene Expression in cells of the immune system
                   Chairs:	Victor	Rutten	-	The	Netherlands	and	Ildiko	van	Rhijn	-The	Netherlands
                   Speakers
	 14:00	-	14:30	h	 Ildiko	Van	Rhijn
	                	 Utrecht	University,	The	Netherlands
                   Trafficking and diversity of bovine gamma delta T cells
	 14:30	-	15:00	h	 Mark	Jutila
	               	 Montana	State	University,	USA
                  Gene expression in bovine gamma/delta T cell subsets with distinct migratory patterns
		15:00	-	15:30	h	 Jean	Pierre	Scheerlinck
	               	 The	University	of	Melbourne,	Australia
                  Regulation of cell trafficking in a single lymph node
    15:30	-	16:00	h Coffee Break
	 16:00	-	16:30	h	 Efrain	Guzman
	               	 Institute	for	Animal	Health,	Compton,	UK
                  Identification and characterization of cattle MHC class I chain related (MIC)
	 16:30	-	17:00	h	 Eric	Denkers
                      38



                                	               	 Cornell	University,	USA
                                                  Host responses to Toxoplasma gondii infections
                                	 17:00	-	17:30	h	 yvette	van	Koocyk
                                	               	 Vrije	Universiteit	Medical	Center,	The	Netherlands
                                                  Interaction between neutrophils and dendritic cells
                                	 17:30	-	18:00	h	 Doug	Bannermann
                                	                	 USDA-ARS,	USA
                                                   The role of cytokines in dictating the outcome of mastitis




                                                                                                                SALÃO TURMALINA
                                1
                                	 4:30	-	18:00	h Concurrent Session #6: Immunoparasitology: Immune Responses to
                                                 Protozoa, Helminths and Ectoparasites
                                                  Chairs:	David	Artis	-	USA	and	Misao	onuma	-	Japan	
                                                  Speakers
                                	 14:30	-	14:55	h	 Albert	Mulenga
                                	                	 Texas	A&M,	USA
                                                   Understanding how the tick attachment phase is initiated; current status and future
                                	 14:55	-	15:20	h	 Juan	Anguita
                                	               	 University	of	Massachusetts,	USA
                                                  Host-vector-pathogen interactions mediated by the multifaceted tick saliva
                                                  protein, Salp15
                                	 15:20	-	15:33	h	 Satoru	Konnai
                                	               	 Hokkaido	University,	Japan
                                                  Suppression of proliferation and cytokine expression by HeLIS, a tick salivary
                                                  gland-derived protein of Haemaphysalis longicornis. Abstract and Poster PR195
                                	 15:33	-	15:46	h	 Sandra	Maruyama
                                	                	 University	of	São	Paulo,	Brazil
                                                   Transcriptomes of Ticks Fed on Resistant and Susceptible Cattle: Genes
                                                   Affected By Host Immune Responses. Abstract and Poster PR149
Day 4 - Saturday, August 18th




                                	 15:46	-	16:00	h	 Elizabeth	J.	Glass
                                	                	 Roslin	Institute,	UK
                                                   TLR Expression in Bovine Monocytes derived from cattle breeds with
                                                   differing susceptibility to tropical Theileriosis
                                                   Abstract	and	Poster	PR158
                                    16:00	-	16:30	h Coffee Break
                                	 16:30	-	16:55	h	 Will	Goff
                                	               	 Washington	State	University,	USA
                                                  The Bovine Spleen: Interactions Between Dendritic Cells and both NK Cells
                                                  and Gamma-Delta T-Cells in the Immunologic Control of Hemoparasitic Infections
                                	 16:55	-	17:20	h	 Mark	Wilson
                                	               	 National	Institutes	of	Health,	USA
                                                  Regulation of Th2 responses- Cooperative mechanisms controlling acute
                                                  inflammation and chronic immunopathology
                                	 17:20	-	17:33	h	 Preben	Boysen	
                                	                	 Norwegian	School	of	Veterinary	Science	
                                                   Bovine natural killer cells act as primary responders in the early stages of Neospora
                                                   caninum-infected cattle.
                                                   Poster	and	Abstract	PR170
                                                                                                         39



	 17:33	-	17:46	h	 Valéria	M.F.	Lima
	                	 São	Paulo	State	University,	Brazil
                   Leishmania vaccine-induced immune response in dogs from an endemic area
                   of canine visceral leishmaniasis
                   Abstract	and	Poster	PR189
	 17:46	-	18:00	h	 Harry	Dawson
	             	 USDA-ARS,	Maryland,	USA
                Feeding probiotic bacteria to swine enhances immunity to Ascaris suum
	             	 Abstract	and	Poster	PR172




                                                                                SALÃO ESMERALDA
1
	 4:30	-	18:00	h Concurrent Session #5: Antigen Presentation and Dendritic Cells;
                 Effector Cells, B and T cells, NK and NK T cells; Immunoregulatory cells
                 Chairs:	D.	Mark	Estes	-	USA	and	Serge	Muyldermans	-	Belgium	

                 Speakers
	 14:30	-	15:00	h	 Serge	Muyldermans
	             	 Flanders	Institute	for	Biotechnology	Belgium
                Nanobody technology and camelid Immunoglobulins
		15:00	-	15:30	h	 Waithaka	Mwangi
	             	 Texas	A&M	University,	USA
                Enhancing Vaccine Efficacy by Directed Priming of CD4+ and CD8+ T Lymphocytes
	 15:30	-	16:00	h	 Imre	Kacskovics
	             	 Eötvös	Loránd	University	Hungary
                The role of the neonatal Fc receptor in IgG catabolism and homeostasis:
                IgG metabolism in bovine FcRn transgenic mice
	 16:00	-	16:30	h Coffee Break
	 16:30	-	17:00	h	 Shirley	Ellis
	                	 Biotechnology	and	Biological	Sciences	Research	Council,	UK
                   Complexity in the cattle CD94/NKG2 gene families




                                                                                                         Day 4 - Saturday, August 18th
	       17:00h	 Amanda	Stalker
	             	 Royal	Veterinary	College,	UK
                Identification of circulating lineage-negative type-I IFN producing plasmacytoid
                dendritic cell-like cells in the bovine blood
                Poster	and	Abstract	AP122	
	       17:15h	 Maša	Pintarič
	             	 University	of	Veterinary	Medicine	of	Vienna,	Austria
                Synergistic effects of IL-2, IL-12 and IL-18 on cytolitic activity and IFNγ production
                of porcine natural killer cells
                Poster	and	Abstract	AP132
	       17:30h	 Gervásio	Bechara
	             	 São	Paulo	State	University,	Brazil
                Draining lymph node APCs in the resistance of goats to Amblyomma cajennense
                (Fabricius, 1787) nymphs
                Poster	and	Abstract	AP125	
	       17:45h	 Aad	Hoek	
	             	 Utrecht	University,	The	Netherlands
                Characterization of bovine regulatory cells.
                Poster	and	Abstract	AP124
                      40


                                          PlenAry session. IMMUNOPARASITOLOGy: IMMUNE RESPONSES TO PROTOZOA, HELMINTHS AND
                                                 ECTOPARASITES CHAIRS: JAN NAESSENS - KENyA AND ELISABETH A. INNES - UK


                                      DAVID	ARTIS	(UnIVERSITy	oF	PEnnSyLVAnIA,	USA)                              infections.	Susceptibility	to	infection	and	expression	of	proinflamma-
                                  A CritiCAl role for intestinAl ePitHeliAl                                      tory	cytokines	also	resulted	in	the	development	of	severe	intestinal	
                                                                                                                 inflammation	 in	 infected	 IEC-ikkβ-/-	 mice.	 	 Gene	 profiling	 of	 IEC	
                                 Cells in regulAting Cd4 t Cell resPonses
                                                                                                                 responses	during	Trichuris	infection	revealed	a	critical	role	for	epi-
                                 And immunity to PArAsitiC infeCtion in tHe
                                                                                                                 thelial-derived	TSLP	in	regulating	intestinal	dendritic	cell	responses.	    	
                                           gAstrointestinAl trACt
                                                                                                                 Taken	together,	these	studies	implicate	the	IEC-intrinsic	NF-κB	path-
                                      Intestinal	epithelial	cells	(IEC)	that	line	the	mucosal	surface	pro-       way	in	governing	dendritic	cell	and	CD4	T	cell	responses	following	
                                vide	an	essential	barrier	between	the	host	and	resident	or	invading	             exposure	to	intestinal	nematode	parasites.		Harnessing	the	functions	
                                microorganisms.	In	vitro	studies	have	shown	that	following	exposure	             of	 IECs	 will	 be	 an	 important	 consideration	 in	 the	 design	 of	 novel	
                                to	 enteric	 pathogens	 including	 intestinal	 nematodes,	 IEC	 exhibit	         therapeutics	and	mucosal	vaccines.
                                NF-κB	activation	and	expression	of	innate	immune	response	genes.	
                                However,	the	role	of	IEC	in	influencing	innate	and	adaptive	immune	
                                responses	to	intestinal	nematodes	in	vivo	is	unknown.	To	test	this,	                  ALEx	LoUKAS	(qUEEnSLAnD	InSTITUTE	oF	MEDICAL	
                                mice	with	IEC-specific	deletions	in	IKKβ	(IEC-ikkβ-/-)	were	generated	                                 RESEARCH)
                                using	Cre-lox	technology.		IKKβ	is	a	critical	kinase	required	for	clas-           using tHe dog As A model for deVeloPing A
                                sical	 NF-κB	 activation.	 	 Mice	 were	 infected	 with	 Trichuris	 muris,	 a	            HumAn HooKWorm VACCine
                                natural	 pathogen	 of	 mice	 that	 lives	 partially	 embedded	 within	 host	
                                IEC	of	the	large	intestine.		Persistent	chronic	infections	are	promoted	
                                                                                                                         	DECLAn	MCKEEVER	(RoyAL	[DICK]	SCHooL	oF	
                                by	 T	 helper	 type	 1	 (Th1)	 responses,	 while	 host	 resistance	 is	 criti-
                                                                                                                                   VETERInARy	STUDIES)
                                cally	dependent	on	CD4	T	cells	that	produce	the	Th2	cytokines	IL-4	
                                and	IL-13.		Therefore,	infection	provides	a	model	system	to	inves-                 Ctl immunity As A driVer for diVersity in
                                tigate	 the	 regulation	 of	 CD4	 T	 cell-dependent	 immune	 responses	                   tHeileriA PArVA PArAsites
                                in	 the	 gut.	 	 While	 wild	 type	 mice	 mounted	 protective	Th2	 cytokine	
                                responses	and	eradictated	Trichuris,	IEC-ikkβ-/-	animals	exhibited	a	
                                                                                                                 STEPHAnIE	WIDDISon	(InSTITUTE	FoR	AnIMAL	HEALTH,	UK)	
                                polarized	IFN-γ	and	IL-17	cytokine	response	and	harbored	chronic	



                                  PlenAry session. MEDIATORS OF RECRUITMENT AND FUNCTION OF CELLS OF THE IMMUNE SySTEM; SIGNAL
                                                              TRANSDUCTION AND GENE ExPRESSION.

                                           CHemoKines, mACroPHAges And                                           of	mycobacteria.		Conversely,	the	Th2	cytokines	IL-4	and	IL-13	inhibit	
                                                  myCobACteriA                                                   autophagy	in	murine	and	human	macrophages.		Inhibition	of	starva-
                                                                                                                 tion-induced	autophagy	by	IL-4	and	IL-13	is	dependent	on	signaling	
                                         JIM	HARRIS	(UnIVERSITy	oF	DUBLIn,	IRELAnD)
                                                                                                                 via	 the	 Akt-pathway,	 while	 inhibition	 of	 IFN-γ-induced	 autophagy	
                                 tH1-tH2 PolArisAtion And tHe Control of                                         is	Akt-independent.	 Induction	 of	 autophagy	 leads	 to	 increased	 kill-
                                intrACellulAr myCobACteriA by AutoPHAgy                                          ing	of	intracellular	mycobacteria	by	macrophages	and	this	effect	is	
                                JAMES	HARRIS1,	SERGIo	S	DE	HARo2,	SHARon	S	MASTER2,	                             abrogated	 by	 the	 addition	 of	 IL-4	 or	 IL-13.	 	 Thus,	 the	 induction	 of	
                                 MonICA	DELGADo,	ESTEBAn	RoBERTS,	JoSEPH	KEAnE1,	                                autophagy	in	macrophages	can	influence	the	fate	of	phagocytosed	
                                                  VoJo	DERETIC2		                                                mycobacteria,	but	these	effects	are	inhibited	by	IL-4	and	IL-13.		This	
                                                                                                                 represents	a	novel	pathway	by	which	Th-1/Th2	polarization	can	influ-
                                    1Department	of	Clinical	Medicine,	Trinity	Centre	for	Health	
                                                                                                                 ence	to	outcome	of	mycobacterial	infection.
                                Sciences,	St.	James’s	Hospital,	Dublin	8;	2Department	of	Molecular	
Day 4 - Saturday, August 18th




                                  Genetics	and	Microbiology,	University	of	New	Mexico	School	of	
                                             Medicine,	Albuquerque,	NM	87131,	USA.	                                  CynTHIA	BALDWIn	(UnIVERSITy	oF	MASSACHUSETTS-
                                      Autophagy	 is	 a	 major	 intracellular	 pathway	 for	 the	 lysosomal	                          AMHERST,	USA)
                                degradation	of	long-lived	cytoplasmic	macromolecules	and	damaged	                     Control of gamma delta t Cell Ifn-γ
                                or	 surplus	 organelles.	 	 Under	 conditions	 of	 amino-acid	 or	 specific	         resPonses: tHe role of tHe tCr And Co-
                                growth	factor	deprivation,	autophagy	degrades	stable	long-lived	pro-                             reCePtor WC1
                                teins	and	enables	cell	survival	by	supplying	anabolic	needs.		Recently,	
                                                                                                                   	JAynE	HoPE	(InSTITUTE	FoR	AnIMAL	HEALTH-CoMPTon,	
                                autophagy	has	also	been	linked	with	innate	and	adaptive	immunity	
                                                                                                                                           UK)
                                against	intracellular	pathogens,	including	Mycobacterium	tuberculo-
                                sis,	which	is	able	to	survive	within	macrophages	by	blocking	fusion	
                                of	 the	 phagosome	 with	 lysosomes.	 	 Induction	 of	 autophagy	 with	                     dendritiC Cells, CytoKines And
                                amino	 acid	 starvation	 or	 IFN-γ	 enables	 macrophages	 to	 overcome	                             myCobACteriA
                                this	phagosome	maturation	block	and	inhibit	the	intracellular	survival	




                                ConCurrent session #4. IMMUNOLOGy OF THE MUCOSAE AND SKIN AND OF THE MAMMARy GLAND; MASTITIS



                                            ERIC	Cox	(GHEnT	UnIVERSITy,	BELGIUM)                                   	MICHAEL	MURTAUGH	(UnIVERSITy	oF	MInnESoTA,	USA)
                                     “muCosAl resPonses AgAinst fimbriAl                                              “genomiCs of immune resPonses in tHe
                                      Antigens of enterotoxigeniC e. COLI”                                                    intestinAl muCosA”
                                                                                                                                                                 41


 ConCurrent session #8. MEDIATORS OF RECRUITMENT AND FUNCTION OF CELLS OF THE IMMUNE SySTEM;
  FC RECEPTORS AND IMMUNOGLOBULINS; SIGNAL TRANSDUCTION AND GENE ExPRESSION IN CELLS OF THE
                                       IMMUNE SySTEM.

        ILDIKo	VAn	RHIJn	(UTRECHT	UnIVERSITy,	THE	                             regulAtion of Cell trAffiCKing in A single
                      nETHERLAnDS)                                                            lymPH node
trAffiCKing And diVersity of boVine gAmmA
              deltA t Cells                                                          EFRAIn	GUZMAn	(InSTITUTE	FoR	AnIMAL	HEALTH,	
  ILDIKo	VAn	RHIJn,	VICToR	RUTTEn,	BRyAn	CHARLESTon,	                                               CoMPTon,	UK)
                    WILLEM	VAn	EDEn,	AD	KoETS                                     identifiCAtion And CHArACterizAtion of
      Tissue-specific	distribution	of	γδ	cells	with	limited	TCR	diversity	         CAttle mHC ClAss i CHAin relAted (miC)
is	a	common	phenomenon	in	species	with	a	low	percentage	of	γδ	T	
                                                                                 EFRAIn	GUZMAn,	CRISTInA	DE	JUAn	SAnJUAn,	SHIRLEy	
cells	like	humans	and	mice.		Using	pseudoafferent	lymph	duct	can-
                                                                                                       ELLIS
nulation	in	cattle	(Bos	taurus),	we	showed	that	large	numbers	of	γδ	T	
cells,	but	not	αβ	T	cells,	are	constitutively	present	in	pseudoafferent	          Institute	for	Animal	Health.	Division		of	Immunology.	Compton,	
lymph	draining	bovine	skin.		The	cells	did	not	have	an	activated	phe-                          Newbury.	RG20	7NN.	United	Kingdom.	
notype	as	exemplified	by	the	absence	of	surface	expression	of	MHC	                                  efrain.guzman@bbsrc.ac.uk
class	II	and	costimulatory	molecules.		The	level	of	γδ	T	cell	egress	                In	 humans,	 major	 histocompatibility	 complex	 (MHC)	 class	 I	
was	enough	to	deplete	all	γδ	T	cells	from	the	skin	within	46	hours.	     	     chain-related	(MIC)	molecules	show	homology	with	classical	human	
As	this	massive	γδ	T	cell	migration	was	observed	during	14	days,	              leukocyte	 antigen	 (HLA)	 molecules,	 but	 they	 do	 not	 combine	 with	
constant	replenishment	of	these	cells	must	have	taken	place.		The	             β2	 microglobulin,	 do	 not	 bind	 peptide	 and	 are	 not	 expressed	 on	
reason	and	mechanism	of	this	extensive	trafficking	is	unknown,	but	            circulating	lymphocytes.	Mapping	studies	in	humans	have	identified	
the	data	suggest	that	γδ	T	cells	in	tissues	fulfill	more	than	exclusively	     seven	MIC	loci	(MICA-MICG),	of	which	only	MICA	and	MICB	encode	
local	functions.		                                                             expressed	transcripts.	The	predicted	domain	structure	of	MIC	prod-
      Subsequent	 analysis	 of	 δ	 	 chain	 sequences	 of	 these	 skin-        ucts	is	similar	to	that	of	classical	MHC	class	I	molecules,	including	
derived,	trafficking	γδ	T	cells,	as	well	as	δ	chain	sequences	of	γδ	T	         three	external	domains	(α1-3)	a	transmembrane	and	a	cytoplasmic	
cells	isolated	from	lymph	node,	spleen,	small	intestine,	large	intes-          domain.	MIC	proteins	are	expressed	in	response	to	stress,	on	the	
tine,	and	blood,	showed	no	preferential	usage	of	certain	Vδ	segments	          cell	 surface	 of	 freshly	 isolated	 gastric	 epithelium,	 endothelial	 cells	
in	any	of	these	tissues,	which	is	quite	different	from	the	situation	in	       and	fibroblasts	and	engage	with	the	activating	natural	killer	receptor	
mice	 and	 humans.	 	 A	 wide	 variety	 of	 δ	 chain	 CDR3	 lengths	 was	      NKG2D,	which	is	found	on	many	cells	within	the	immune	system.	We	
observed		in	each	of	the	bovine	tissues	tested	and	was	confirmed	by	           have	identified	two	transcribed	cattle	MIC	genes	which	differ	by	a	21	
spectratyping.		The	highly	variable	CDR3	length	appeared	to	be	due	            nucleotide	indel	in	the	α2	domain,	and	there	is	evidence	for	at	least	
to	the	use	of	up	to	four	diversity	(D)	segments	per	bovine	δ	chain.		A	        four	 more	 bovine	 MIC	 genes	 or	 pseudogenes.	 Transcribed	 cattle	
high	number	of	Vδ	segments,	in	combination	with	the	use	of	up	to	              MIC	genes	were	found	to	have	65%	identity	at	the	nucleotide	level	to	
four	out	of	five	D	segments,	and	the	possibility	of	using	non-template	        their	human	homologues,	and	45%	identity	at	the	amino	acid	level.	
encoded	(N)	nucleotides	on	either	side	of	these,	makes	the	potential	          Transcription	of	cattle	MIC	was	found	in	stomach,	lung	and	intestine	
bovine	δ	chain	repertoire	much	bigger	than	any	known	TCR	chain.	               tissues,	but	not	in	the	liver	or	heart.	We	have	constructed	MIC	tetra-
                                                                               mers	and	used	them	to	stain	circulating	lymphocytes.	Staining	of	γδ	
     The	 reason	 and	 mechanism	 of	 γδ	 T	 cell	 trafficking	 and	 the	
                                                                               T	cells	and	αβ	CD8+	T	cells	was	detected,	but	αβ	CD4+	T	cells	did	
implications	of	the	high	δ	chain	diversity	remains	subject	to	further	
                                                                               not	stain,	as	seen	in	humans.	We	also	show	activation	of	γδ	T	cells	
studies.		
                                                                               by	MIC	using	an	interferon-γ	ELIspot	assay.	These	results	indicate	
Key words:	γδ	T	cells;	δ	chain	repertoire;	skin;	lymph	node;	spleen;	          that	there	are	at	least	two	functional	MIC	genes	in	cattle	which	differ	
small	 intestine;	 large	 intestine;	 blood;	 pseudoafferent	 lymph	 duct	     by	a	21	nucleotide	indel,	and	show	the	presence	of	several	additional	
cannulation                                                                    MIC	genes	or	pseudogenes.	
Species:	ruminants




                                                                                                                                                                 Day 4 - Saturday, August 18th
                                                                               Key words:	 MHC	 class	 I	 chain-related	 molecules;	 γδ	 T	 cells;	 αβ	
                                                                               CD8+	T	cells;	αβ	CD4+	T	cells;	interferon-γSpecies:	ruminants
      MARK	JUTILA	(MonTAnA	STATE	UnIVERSITy,	USA)
   gene exPression in boVine gAmmA/deltA                                                 ERIC	DEnKERS	(CoRnELL	UnIVERSITy,	USA)
   t Cell subsets WitH distinCt migrAtory
                                                                                    Host resPonses to toxoPlAsmA gondii
                  PAtterns
                                                                                                infeCtions
      Abstract.	Analysis	of	global	gene	expression	in	immune	cells	
has	provided	unique	insights	into	immune	function	and	response	to	
infection.	 Recently,	 microarray	 and	 serial	 analysis	 of	 gene	 expres-         yVETTE	VAn	KooCyK	(VRIJE	UnIVERSITEIT	MEDICAL	
sion	(SAGE)	techniques	have	been	applied	to	the	study	of	γd	T	cell	                           CEnTER,	THE	nETHERLAnDS
function	in	humans,	rodents	and	cattle.	We	applied	these	approaches	               interACtion betWeen neutroPHils And
to	the	study	of	bovine	tissue-restricted	CD8+	and	CD8-	(WC1+)	γd	                            dendritiC Cells
T	cell	subsets	isolated	from	the	blood,	and	total	γd	T	cells	and	aβ	T	
cells	 isolated	 from	 mucosal	 lymphatics	 prior	 to	 and	 following	 oral	      Molecular	Cell	Biology,	Vumc,	v.d.	Boechorststraat	7,	1081	BT	
Salmonella	serovar	Typhimurium	infection	in	calves.	These	studies	                                Amsterdam,	The	Netherlands.		
provided	new	insights	into	the	function	of	bovine	γd	T	cells	in	general	                              y.vankooyk@vumc.nl
and	differences	in	mucosal	(CD8+)	and	peripheral	(CD8-)	γd	T	cell	                   Dendritic	cells	(DC)	are	specialized	in	the	recognition	of	patho-
subsets.	Common	to	both	subsets	was	expression	of	many	myeloid	                gens	and	play	a	pivotal	role	in	the	control	of	immunity.		Yet	DC	are	
cell	 associated	 genes,	 such	 as	 receptors	 for	 pathogen	 associated	      also	important	for	homeostatic	control	recognizing	self	antigens	and	
molecular	patterns	(PAMPs).	We	found	that	PAMPs	prime	γd	T	cells	              tolerizing	its	environment,	indicating	that	the	nature	of	the	antigen	it	
to	 more	 robustly	 respond	 to	 downstream	 cytokine	 and/or	 antigen	        recognizes	may	steer	a	DC	towards	immunity	or	tolerance.	C-type	
signals.	The	nature	of	the	priming	response	and	examples	of	potent	            lectin	 receptors	 expressed	 by	 DC	 are	 involved	 in	 the	 recognition	
priming	agents	will	be	discussed.                                              and	capture	glycosylated	self	antigens	or	pathogens.	To	date	seven	
                                                                               different	C-type	lectins	have	been	identified	on	DC.	It	is	now	becom-
                                                                               ing	 clear	 that	 these	 C-type	 lectin	 receptors	 may	 not	 only	 serve	 as	
	JEAn	 PIERRE	 SCHEERLInCK	 (THE	 UnIVERSITy	 oF	 MEL-                         antigen	receptor	recognizing	pathogens	to	allow	internalisation	and	
BoURnE,	AUSTRALIA)                                                             antigen	presentation,	but	may	also	function	in	the	recognition	of	self	
                      42


                                antigen,	or	as	adhesion	molecules	and	signaling	molecules.	We	have	                 between	PMN	and	DC	allowing	proper	antigen	delivery.	Both	Mac-1	
                                studied	in	great	detail	the	function	and	the	glycan	specificity	of	the	             and		CEACM1	have	been	identified	as	glycoproteins	on	neutrophils	
                                DC-specific	C-type	lectin	DC-SIGN.	DC-SIGN	recognizes	high	man-                     that	interact	with	DC-SIGN	and	regulate	DC	neutrophil	interactions.
                                nose	structure	and	non-sialylated	Lewis	antigens	(Lex,	Ley,	Leb	and	                      Understanding	the	diversity	of	C-type	lectins	being	expressed	
                                Lea)	which	are	expressed	on	many	pathogens,	such	as	the	envelope	                   on	DC	as	well	as	their	carbohydrate	specific	recognition	profile	will	
                                protein	 gp120	 of	 HIV-1,	 and	 many	 other	 viral	 envelope	 glycopro-            be	 instrumental	 to	 understand	 DC	 pathogen	 recognition	 in	 many	
                                teins,	but	also	on	the	cell	wall	component	ManLam	of	Mycobacteria.	                 pathogenic	 disorders,	 as	 well	 as	 the	 regulation	 of	 cellular	 interac-
                                Targeting	of	these	pathogens	to	DC-SIGN	however	leads	to	immune	                    tions	of	DC	that	are	essential	in	the	control	of	immunity.
                                escape.	These	findings	hint	to	a	function	of	DC-SIGN	in	recognizing	
                                glycosylated	self	antigen	to	tolerize	it	environment.
                                                                                                                                  DoUG	BAnnERMAnn	(USDA-ARS,	USA)
                                     To	date	little	is	know	on	the	specificity	by	which	C-type	lectins	
                                interact	 with	 self-glycoproteins.	 Lewis	 antigens	 are	 recognized	 on	              tHe role of CytoKines in diCtAting tHe
                                glycoproteins	 present	 on	 PMNs	 and	 mediate	 a	 cellular	 interaction	                        outCome of mAstitis




                                    ConCurrent session #6. IMMUNOPARASITOLOGy: IMMUNE RESPONSES TO PROTOZOA, HELMINTHS AND
                                                  ECTOPARASITES CHAIRS: DAVID ARTIS - USA; MISAO ONUMA - JAPAN



                                               ALBERT	MULEnGA	(TExAS	A&M,	USA)                                        tHe boVine sPleen: interACtions betWeen
                                  understAnding HoW tHe tiCK AttACHment                                                 dendritiC Cells And botH nK Cells And
                                   PHAse is initiAted; Current stAtus And                                              gAmmA-deltA t-Cells in tHe immunologiC
                                                    future                                                              Control of HemoPArAsitiC infeCtions
                                              A	MULEnGA,	AB	MARIA,	R	KHUMTHonG
                                   Texas	A	&	M	University,	Department	of	Entomology,	College	of	                       MARK	WILSon	(nATIonAL	InSTITUTES	oF	HEALTH,	USA)
                                  agriculture	&	Life	Sciences,	2475	TAMU,	College	Station,	Texas	                    immunoPAtHology during sCHistosomiAsis:
                                                               77843                                                  treg-deriVed il-10 Controls inflAmmAtion
                                       The	tick	feeding	style	of	lacerating	host	tissue	to	create	its	feed-              whIle Il-13rα2 CrItICally Controls
                                ing	 site	 and	 then	 sucking	 up	 blood	 from	 the	 hematoma	 that	 forms	                immunoPAtHology And fibrosis.
                                in	the	wounded	area	should	under	normal	circumstances	stimulate	                    MARK	SWILSon,	ELDAD	ELnEKAVE,	MARGARET	M	MEnTInK-
                                tissue	repair	response,	which	will	ultimately	stop	bleeding.	However,	              KAnE,	JoHn	T	PESCE,	THIRUMALAI	R	RAMALInGAM,	RoBERT	
                                ticks	ensure	a	full	blood	meal	by	secreting	a	cocktail	of	potent	phar-                   W	THoMPSon,	ALLEn	CHEEVER,	THoMAS	Wynn
                                macologically	 active	 enzymes	 that	 collectively	 disarm	 the	 host’s	
                                                                                                                     	Laboratory	of	Parasitic	Diseases,	National	Institute	of	Allergy	and	
                                tissue	repair	mechanism.	Since	the	mammalian	host’s	tissue	repair	
                                                                                                                             Infectious	Diseases.	National	Institutes	of	Health.	
                                response	 to	 prevent	 further	 bleeding	 is	 expected	 to	 be	 swift,	 it	 is	
                                logical	to	imagine	that	the	tick	will	be	ready	to	evade	host	defense	                      The	 development	 of	 Immunopathology;	 such	 as	 airway	 hyper	
                                responses	upon	penetration	of	host	skin	for	it	to	successfully	feed.	To	            responsiveness	 (AHR)	 in	 asthmatics	 and	 fibrosis	 during	 schisto-
                                understand	how	the	tick	is	able	to	initiate	its	feeding	process,	we	are	            somiasis	 often	 result	 from	 superfluous	 inflammation,	 yet	 the	 tran-
                                using	the	Lone	Star	tick,	Amblyomma	americanum	and	bovine	model.	                   sition	 from	 inflammation	 to	 resultant	 immunopathology	 is	 poorly	
                                Towards	discovery	of	molecular	signaling	cascades	that	trigger	and/                 understood.	 This	 study	 aimed	 to	 dissect	 critical	 mediators	 during	
Day 4 - Saturday, August 18th




                                or	 facilitate	 the	 tick	 attachment	 and	 formation	 of	 its	 feeding	 lesion,	   Th2-driven	inflammation,	following	infection	or	airway	allergen	provo-
                                suppressive	 subtractive	 hybridization,	 high	 throughput	 sequencing	             cation,	which	mediate	down-stream	pathology	and	to	subsequently	
                                and	validation	of	differential	expression	by	cDNA	dot	blot	hybridiza-               identify	 the	 regulatory	 mechanisms	 that	 control	 them.	 To	 separate	
                                tion	were	performed	on	A.	americanum	ticks	that	had	attained	appe-                  inflammation	from	immunopathology,	we	used	two	in-vivo	models-	a	
                                tence	and	were	exposed	to	feeding	stimuli.	This	approach	allowed	                   model	of	asthma	and	Schistosomiasis,	both	of	which	present	defini-
                                for	identification	of	40	genes	that	are	up	regulated	before	ticks	begin	            tive	 cellular	 inflammation	 and	 downstream	 pathological	 changes.	
                                to	penetrate	the	host	skin.	Among	the	40	genes,	we	have	identified	                 We	 observed	 an	 accumulation	 of	 IL-10+	 regulatory	 T	 cells	 (Treg),	
                                antimicrobial	 peptides,	 insulin-like	 growth-factor	 binding	 proteins,	          using	IL-10gfp	reporter	mice	and	Fox	p3	expression	in	the	lungs	fol-
                                lipocalin/histamine	 binding	 protein	 and	 an	 extracellular	 matrix	 met-         lowing	 allergen	 challenge	 or	 in	 the	 liver	 following	 infection.	 In	 the	
                                taloprotease	inducer.	Putative	biological	functions	of	these	proteins	              absence	of	IL-10	overwhelming	inflammation	with	exacerbated	Th2	
                                suggest	that	they	are	involved	in	mediation	of	molecular	mechanism	                 responses,	as	well	as	the	emergence	of	Th1	responses,	develops.	
                                that	underpin	formation	and	maintenance	of	the	tick-feeding	lesion.	                Paradoxically,	despite	increased	inflammation	in	IL-10-/-	mice	pathol-
                                Quantitative	real	time	RT-PCR	analysis	has	shown	these	genes	are	                   ogy	was	reduced.	In	both	the	lung	and	the	liver,	IL-13R	alpha	2,	an	
                                up	regulated	in	the	tick	salivary	glands	within	the	first	24	hrs	of	the	            endogenous	 neutralizer	 of	 IL-13,	 was	 elevated.	 To	 test	 the	 role	 of	
                                tick	 initiating	 its	 feeding	 lesion.	 Research	 to	 determine	 molecular	        IL-13R	 alpha	 2	 in	 regulating	 Th2	 associated	 pathology	 we	 gener-
                                interactions	of	these	genes	at	the	tick-host	interface	is	ongoing.                  ated	mice	deficient	in	both	IL-13R	alpha	2	and	IL-10	(IL-13R	alpha	
                                                                                                                    2-/-IL-10-/-).
                                                                                                                         We	discovered	that	the	absence	of	IL-13R	alpha	2,	on	an	IL-10-/-	
                                    JUAn	AnGUITA	(UnIVERSITy	oF	MASSACHUSETS,	USA)
                                                                                                                    background,	increased	lung	and	liver	pathology,	compared	to	WT	or	
                                    Host-VeCtor-PAtHogen interACtions                                               IL-10-/-	mice,	indicating	that	IL-13Rα2	is	a	key	regulator	of	pathology.	
                                  mediAted by tHe multifACeted tiCK sAliVA                                          This	 study	 presents	 a	 novel	 two-pronged	 model	 controlling	 inflam-
                                               Protein, sAlP15                                                      mation	and	immunopathology	by	IL-10-secreting	Treg	cells	and	IL-
                                                                                                                    13Rα2,	respectively,	which	could	be	exploited	to	control	a	variety	of	
                                      WILL	GoFF	(WASHInGTon	STATE	UnIVERSITy,	USA)                                  important	Th2-dominant	diseases.
                                                                                                                                                                   43


       ConCurrent session #5. ANTIGEN PRESENTATION AND DENDRITIC CELLS; EFFECTOR CELLS, B AND
        T CELLS, NK AND NK T CELLS; IMMUNOREGULATORy CELLS. CHAIRS: D. MARK ESTES-USA AND SERGE
                                         MUyLDERMANS- BELGIUM
      SERGE	MUyLDERMAnS	(FLAnDERS	InSTITUTE	FoR	                                  scytosis	 in	 several	 mucosal	 layers	 and	 is	 involved	 in	 the	 maternal	
               BIoTECHnoLoGyBELGIUM)                                              immune	transport.	Most	recently,	it	has	been	shown	that	FcRn	binds	
       nAnobody teCHnology And CAmelid                                            albumin	 and	 protects	 it	 from	 degradation	 just	 as	 it	 does	 IgG.	 Both	
                                                                                  IgG	and	albumin	bind	FcRn	at	low	pH	at	distinct	sites.	We	cloned	and	
              immunoglobulins
                                                                                  characterized	the	bovine	FcRn	(bFcRn)	alpha	chain	and	detected	its	
                                                                                  expression	in	various	epithelial	cells	that	is	involved	in	IgG	secretion.	
      WAITHAKA	MWAnGI	(TExAS	A&M	UnIVERSITy,	USA)                                 We	 have	 also	 shown	 that	 this	 receptor	 is	 expressed	 in	 the	 bovine	
   enHAnCing VACCine effiCACy by direCted                                         capillary	endothelial	cells	and	involved	in	IgG	homeostasis	in	cattle.
   Priming of Cd4+ And Cd8+ t lymPHoCytes                                               In	order	to	study	the	regulation	of	the	bovine	FcRn	heavy	chain	
                                                                                  gene	and	analyze	its	role	in	IgG	and	albumin	metabolism,	we	gener-
                                                                                  ated	 and	 characterized	 transgenic	 mice	 carrying	 a	 102	 kb	 bovine	
      IMRE	KACSKoVICS	(EöTVöS	LoRánD	UnIVERSITy	                                  genomic	 fragment,	 encoding	 the	 bFcRn.	A	 bacterial	 artificial	 chro-
                       HUnGARy)	                                                  mosome	containing	the	bFcRn	alpha-chain	gene	(bFCGRT)	with	its	
   tHe role of tHe neonAtAl fC reCePtor                                           44	 kb	 5’	 and	 50	 kb	 long	 3’	 flanking	 sequences	 was	 microinjected	
  in igg CAtAbolism And HomeostAsis: igg                                          into	 fertilized	 mouse	 oocytes.	 Two	 of	 the	 transgenic	 lines	 gener-
metAbolism in boVine fCrn trAnsgeniC miCe                                         ated,	showed	copy	number	related	and	integration	site	independent	
                                                                                  bFcRn	 expression	 based	 on	 Northern	 and	 Western	 blot	 studies.	
BALáZS	BEnDER1,	LILLA	BoDRoGI1,	JUDIT	CERVEnAK5,	ZITA	                            Pharmacokinetic	 studies	 showed	 that	 the	 half-lives	 of	 the	 injected	
SCHnEIDER2,	BALáZS	MAyER2,	yAoFEnG	ZHAo3,	LEnnART	                                mouse	and	human	IgG	were	significantly	longer	in	transgenic	mice	
 HaMMarströM3,	andré	EggEn4,	ZsuZsanna	BősZE1,	                                   compared	 to	 wild-type	 animals.	 These	 data	 indicate	 that	 bovine	
                 IMRE	KACSKoVICS5                                                 FcRn	 heavy	 chain	 is	 indeed	 expressed	 in	 the	 mouse	 endothelial	
  1Agricultural	Biotechnology	Center,	Gödöllő,	Hungary;	2Faculty	                 cells,	formed	a	functional	receptor	and	protected	IgG	from	degrada-
     of	Veterinary	Science,	Szent	István	University,	Budapest,	                   tion.	Our	results	underline	the	feasibility	of	creating	BAC	transgenic	
    Hungary;	3	Division	of	Clinical	Immunology,	Department	of	                    mouse	models	of	economically	important	bovine	genes.
  Laboratory	Medicine,	Karolinska	Institute,	Stockholm,	Sweden;	
     4	INRA,	UR339,	Laboratoire	de	Génétique	biochimique	et	
                                                                                        SUPPoRTED	By	THE	GRAnTS	oTKA	T049015,	oMFB	
    de	Cytogénétique,	Jouy-en-Josas,	France;	5Department	of	
                                                                                               1605/1606/2002,	GAK-CALVES05
    Immunology,	Faculty	of	Science,	Eötvös	Loránd	University,	
                        Budapest,	Hungary
                                                                                        SHIRLEy	ELLIS	(BIoTECHnoLoGy	AnD	BIoLoGICAL	
     IgG	 has	 the	 longest	 survival	 time	 in	 the	 circulation	 of	 the	 Ig	
                                                                                              SCIEnCES	RESEARCH	CoUnCIL,	UK)
classes	 and	 the	 lowest	 fractional	 catabolic	 rate.	 The	 MHC	 class	 I	
related	Fc	receptor	for	IgG	(FcRn),	which	is	composed	of	the	FcRn	                  ComPlexity in tHe CAttle Cd94/nKg2 gene
heavy	 chain	 and	 the	 beta2-microglobulin	 (b2m),	 protects	 IgG	 from	                           fAmilies
intracellular	catabolic	degradation,	plays	important	roles	in	IgG	tran-




                                                                                                                                                                   Day 4 - Saturday, August 18th
                                                                  Day 5 - August 19th - Sunday

                             	   8:30	-	11:00	h	   Poster	Viewing	Themes	6-10	                        SALÃO DIAMANTE

                             	 11:00	-	13:30	h	    Mini-symposium:	Canine	Visceral	Leishmaniasis		      SALÃO OURO

                             	 14:00	-	16:00	h	    Concurrent	Session	1:	Immunogenetics	                SALÃO OURO

                             	 14:00	-	16:00	h	    Concurrent	Session	3:	Stress;	Reproduction;	       SALÃO TURMALINA
                             	                	    Immunoendocrinology;	Microbial	Flora,	Nutrients

                             	 16:30	-	17:00	h	    Student	Awards	                                      SALÃO OURO

                             	 17:00	-	19:30	h	    Plenary	Session:	Innate	Immunity,	Inflammation,	     SALÃO OURO
                             	                	    Adjuvants;	Memory,	Acquired	Immunity,	Vaccines
Day 5- Sunday, August 19th
                                                                                                       45




                                                                                   SALÃO DIAMANTE
	 8:30	-	11:00	h	 Poster Viewing
                Coffee	Break	at	10:00	h,	during	poster	session
                6. Immunoparasitology: Immune Responses to Protozoa, Helminths and Ectoparasites;
                Canine Visceral Leishmaniasis: Posters PR141-PR196
                7. Comparative Immunology; Immunoecology: Posters CI197-CI206
                8. Mediators of Recruitment and Function of Cells of the Immune System; Fc Receptors
                and Immunoglobulins; Signal Transduction and Gene Expression in cells of the
                immune system: Posters MI207-MI210
                9. Innate immunity, Inflammation and Adjuvants; Memory, Acquired Immunity and
                Vaccines: Posters VA211-VA250
                10. Clinical Immunology and Immunopathology: Posters IP251-IP281




                                                                                      SALÃO OURO
1
	 1:00	-	13:30	h Mini-symposium Canine Visceral Leishmaniasis: Immunology and
                 Vaccines
                Chairs:	olindo	Martins	Filho	-	Brazil	and	Javier	Moreno	-	Spain	
                Speakers
	             	 Alexandre	Barbosa	Reis
	             	 Federal	University	of	Ouro	Preto,	Brazil
                Systemic and compartmentalized Immune Responses in Canine Visceral
                Leishmaniasis
	             	 Javier	Moreno
	             	 Center	for	Biological	Investigations,	Spain
                Cytokine profiles in Canine Visceral Leishmaniasis
	             	 Washington	Luis	dos	Santos
	             	 Oswaldo	Cruz	Foundation,	Brazil
                Cell Migration in Tissues of Dogs Infected with Leishmania chagasi
		            	 olindo	Assis	Martins	Filho	
	             	 Oswaldo	Cruz	Foundation,	Brazil
                Advances in flow cytometric serology to distinguish Leishmania	(Leishmania)	chagasi	
	             	 infected from Leishmune ®-vaccinated dogs
		            	 Dr.	Gérard	Marie	Papierok
	             	 Bio	Veto	Test-VIRBAC,	France
                Vaccinal interests of purified excreted-secreted antigens of Leishmania infantum
                against canine visceral leishmaniasis: explanation of success of experimental and
                efficacy field trials
                                                                                                       Day 5 - Sunday, August 19th




	 13:00	-	14:00	h	 Lunch
             46



                                                                                                                     SALÃO OURO

                              1
                              	 4:00	-	16:00	h Concurrent Session #1: Immunogenetics and Genomics
                                                Chairs:	William	ollier	-	UK	and	Alexandre	Caetano	-	Brazil

                                                Speakers
                              	 14:00	-	14:30	h	 Joan	K.	Lunney	
                              	               	 Agricultural	Research	System,	USDA,	USA
                                                Comparative immune responses of pigs to infection with Salmonella enterica serovars
                                                of food safety (Typhimurium) and animal health (Choleraesuis) importance.	
                              	       14:30	h	 John	Williams	
                              	               	 Parco	Tecnologico	Padano,	Italy
                                                Identification of polymorphisms in bovine genes with immune function.	
                              	               	 Abstract	and	Poster	IG007
                              	      14:40		h	 Dirk	Werling	
                              	               	 Royal	Veterinary	College,	UK	
                                                Novel approaches to enhance disease resistance in ruminants? -Breeding for geo
                                                graphically important TLR SNPs. The	Ruminant	TLR	Consortium.	
                              	               	 Abstract	and	Poster	IG003
                              	       14:50	h	 Elizabeth	Glass	
                              	               	 Roslin	Institute,	UK
                                                Monocytes from disease resistant and susceptible cattle display distinct transcriptome
                                                profiles during Theileria annulata infection.	
                              	               	 Abstract	and	Poster	IG011
                              	 15:00	-	15:30	h	 William	ollier	
                              	               	 University	of	Manchester,	UK
                                                Immunogenetic risk factors contributing to Canine Diabetes.	
                              	       15:30	h	 Hirohide	Uenishi
                              	               	 Natl.	Institute.	of	Agrobiological	Sciences,	Japan
                                                Difference of genomic structure among haplotypes of swine leukocyte antigen region.	
                                                Abstract	and	Poster	IG019
                              	        15:40h	 Carlos	Prudêncio	
                              	               	 Federal	University	of	Uberlândia,	Brazil
                                                Identification of Boophilus microplus phagotopes from phage displayed peptide libraries.	
                                                Abstract	and	Poster	IG022
                              	       15:50	h	 Tomoko	Eguchi-Togawa	
                              	               	 Natl.	Institute.	of	Agrobiological	Sciences,	Japan
                                                Genomic analysis revealed the duplication model of porcine CD1 genes during evolution.	
                              	               	 Abstract	and	Poster	IG020
                              	 16:00	-	16:30	h Coffee Break



                                                                                                                  SALÃO AMETISTA
Day 5 - Sunday, August 19th




                              	 4:00	-	16:00	h Concurrent Session #3: Immunoendocrinology; and Stress; Immunology
                              1
                                               of Reproduction and Neonates; Microbial Flora, Nutrients and the
                                               Immune Response
                                              	 Chairs:	nicola	Lacetera	-	Italy	and	Isabelle	P.	oswald	-	France

                                                Speakers
                              	       14:00	h	 José	Roberto	Kfoury
                                                                                                       47



	              	 University	of	São	Paulo,	Brazil
                 Influence of hormones in the expression of indoleamine-2,3 dioxigenase in
                 cultured cells from bovine placenta
                 Abstract	and	poster	ER081
	 14:15	-	14:45	h	 Gary	Entrican
	              	 Moredun	Research	Institute,	UK
                 The effect of pregnancy on maternal immunity in sheep
	 14:45	-	15:15	h	 Isabelle	oswald
	              	 INRA,	France
                 Effect of some food contaminants, the mycotoxins, on the immune response of the pig
	       15:15	h	 Katsuro	Hagiwara
	              	 Rakuno	Gakuen	University,	Japan
                 Colostral CD8 positive cell is a potent producing cell for IFN-gamma
                 Abstract	and	Poster	ER079
	       15:30	h	 Jesús	Hernández
	              	 CIAD,	Mexico
                 Vitamin E modulates the expression of Th2 cytokines in porcine PBMC
	              	 Abstract	and	Poster	ER086
	      	15:45	h	 Amanda	A.	Adams
	              	 Gluck	Equine	Research	Center,	USA
                 Contribution of body condition score and percent body fat to the inflammatory
                 response in aged horses
                 Abstract	and	Poster	ER090
		16:00	-	16:30	h Coffee Break



                                                                                        SALÃO OURO
	 16:30	-	17:00	h Student Awards
	              	 Chairs:	Beatriz	R.	Ferreira	–	Brazil	and	Joan	K.	Lunney	-	USA

1
	 7:00	-	19:30	h Plenary Session Innate immunity, Inflammation and Adjuvants; Memory,
                 Acquired Immunity and Vaccines
                 Chairs:	Phillip	Griebel	-	Canada	and	Dirk	Werling	-	UK	

                 Speakers
	 17:00	-	17:30	h	 Gordon	MacPherson
	              	 Oxford	University,	UK
                 The Role of Dendritic Cells at mucosal surfaces and regulation of inflammation
	 17:30	-	18:00	h	 Volker	Gerdts
	              	 Vaccine	and	Infectious	Disease	Organization,	Canada
                 Strategies to link innate and adaptive immunity when designing vaccine adjuvants
		18:00	-	18:30	h	 Pat	Shewen
	              	 University	of	Guelph,	Canada
                 Challenges in Development of Mucosal Vaccines
                                                                                                       Day 5 - Sunday, August 19th




		18:30	-	19:00	h	 Sarah	Doyle
	                	 Trinity	College,	Dublin
                   TLR Signaling in Infection and Inflammation
	 19:00	-	19:30	h	 Steve	Reiner
	              	 University	of	Pennsylvania,	USA
                 Specifying the T cell fates required for immunity: Asymmetric Division of a
                 T Lymphocyte in the Initiation of Adaptive Immunity
             48



                                                                                                          9th IVIS: JAPAN, 2010

                              ThE 9 Th INTERNATIONAL VETERINARy IMMUNOLOgy SyMpOSIUM

                              Dear	Fellow	Veterinary	Immunologists;

                              A	very	warm	welcome	to	the	9th	International	Veterinary	Immunology	
                              Symposium	(or	the	9th	IVIS).

                              Japanese	Association	of	Veterinary	Immunologists	would	like	to	cordially	
                              invite	you	to	the	9th	IVIS	scheduled	on	August	16-20,	2010	at	Tower	
                              Hall	Funabori	(Edogawa-ku)	in	Tokyo.

                              We	wish	to	call	for	papers	on	various	scientific	themes	of	interest	
                              related	with	all	aspects	of	veterinary	immunology.	We	sincerely	hope	
                              that	the	9th	IVIS	will	become	an	exciting,	useful	and	memorable	meeting	
                              for	all	participants	to	discuss	recent	development	of	veterinary	
                              immunology	and	future	prospect.

                              The	venue	for	the	9th	IVIS	is	located	in	the	center	of	Tokyo	with	easy	
                              access	from	the	Tokyo	Central	Station,	Narita	International	Airport	and	
                              Haneda	Domestic	Airport	by	efficient	transportation	system.	The	region	
                              offers	various	traditional	culture	events,	excellent	cuisine,	and	
                              leisure	facilities,	as	well.	We	are	sure	that	you	will	be	satisfied	from	
                              both	scientific	and	social	aspects.

                              Similarly	as	the	previous	IVIS	meetings,	the	9th	IVIS	is	organized	two	
                              days	ahead	of	the	International	Congress	of	Immunology,	which	will	be	
                              held	in	Kobe	from	August	22-27,	for	the	convenience	of	participants	
                              coming	for	both	meetings.

                              We	look	forward	to	seeing	you	in	Tokyo.	



                                                                                    Takashi	Onodera,	Chairperson	of	the	9th	IVIS
Day 5 - Sunday, August 19th




                                                                                                aonoder@mail.ecc.u-tokyo.ac.jp
                                                                                                 http://WWW.frc.a.u-tokyo.ac.jp/
                                                                                                                                                          49


                  mini-symPosium CAnine VisCerAl leisHmAniAsis. IMMUNOLOGy AND VACCINES

      ALExAnDRE	BARBoSA	REIS	(FEDERAL	UnIVERSITy	oF	                      not	 only	 against	 experimental	 L’infantum	 infection	 (Study	 on	 18	
                  oURo	PRETo,	BRAZIL)	                                    DOGS,(1))	but	also	against	visceral	leishmanisis	in	the	field	(study	
 systemiC And ComPArtimentAlized immune                                   on	414	dogs)	(2).
resPonses in CAnine VisCerAl leisHmAniAsis                                    The	 good	 results	 obtained	 with	 this	 vaccinal	 candidate	 are	
                                                                          due	to	the	double	immune	response	specifically	directed	against	Li	
	                                                                         ESAp:	an	anti-Li	ESA	Ig	G2	production	and	a	cell	mediated
                                                                                immune	response	(Th1	response).	Both	responses	have	a	spe-
                                                                          cific	role	against	the	parasite.
            JAVIER	MoREno	(CEnTER	FoR	BIoLoGICAL	
                    InVESTIGATIonS,	SPAIn)	                                     About	 the	 humoral	 response,	 the	 specific	 Ig	 G2	 antibodies	
                                                                          induce	a	loss	of	promastigote’s	and	amastigote’s	viability	and	prolif-
        CytoKine Profiles in CAnine VisCerAl                              eration	in	vitro	(an	inhibition	of	these	2	parasitical	forms	proliferation	
                   leisHmAniAsis                                          “in	vitro”).
                                                                              Leishmania	 viability	 was	 reduced	 to	 50%	 (after	 contact	 with	
        	WASHInGTon	LUIS	DoS	SAnToS	(oSWALDo	CRUZ	                        serum	of	immunized	dogs)	and	no	proliferation	was	evidenced	after	
                   FoUnDATIon,	BRAZIL)                                    30	min	of	contact	of	Leishmania	with	pure	immunized	dog(s)	serum.	
Cell migrAtion in tissues of dogs infeCted                                Immune	and	control	dogs	sera	were	tested	by	a	standard	ELISA	pro-
         WitH leisHmAniA CHAgAsi                                          cedure	for	Ig	G2	antibodies	levels	to	Li	ESAp.	For	the	experimental	
                                                                          study	and	the	field	trial,	all	vaccinated	dogs	showed	increased	anti-Li	
                                                                          ESAp	IgG2	reactivity	after	immunization.
         oLInDo	ASSIS	MARTInS	FILHo	(oSWALDo	CRUZ	
                                                                                The	 cellular	 immune	 response	 was	 characterized	 by	 the	 acti-
                    FoUnDATIon,	BRAZIL)
                                                                          vation	 of	 macrophages	 in	 response	 to	 higher	 IFN-g	 production	 by	
      AdVAnCes in floW CytometriC serology                                Th1	 lymphocytes.	 This	 response	 was	 studied	 by	 several	 methods	
      to distinguisH leisHmAniA (leisHmAniA)                              and	 especially	 by	 the	 significant	 enhanced	 antileishmanial	 activity	
       CHAgAsi infeCted from leisHmune ®-                                 of	 canine	 macrophages	 co-cultivated	 with	 autologus	 lymphocytes	
                 VACCinAted dogs                                          (in	response	to	higher	IFN-g	production	by	T	cells).	Anti-leishmanial	
                                                                          activity	 increased	 significantly	 after	 vaccine	 administration	 (51,1	 ±	
                                                                          20,6%,	P<	0,01)	and	was	higher	after	the	booster	(60,7	±	10,4%,	P<	
    GéRARD	MARIE	PAPIERoK	(InSTITUTE	PASTEUR,	FRAnCE)
                                                                          0,01)	 in	 field	 trial.	 Furthermore	 we	 demonstrated	 that	 the	 lympho-
    VACCinAl interests of Purified exCreted-                              cytes	of	dogs	immunised	with	Li	ESAp	co-incubated	with	Leishmania	
    seCreted Antigens of LEISHMANIA INFANTUM                              infected	 macrophages	 produce	 IFN-g	 resulting	 in	 NO-mediated	
     AgAinst CAnine VisCerAl leisHmAniAsis:                               amastigote	apoptosis	(3).
    exPlAnAtion of suCCess of exPerimentAl                                     All	results	during	the	experimental	trial	(1)	agree	with	the	results	
           And effiCACy field triAls                                      obtained	in	feld	trial	(2).
    GéRARD-MARIE	PAPIERoK	,	CHRISToPHE	HUGnET;	GILLES	                        Today	we	understand	the	roles	of	Li	ESAp	for	a	good	protection	
           BoURDoISSEAU,	JEAn-LoUP	LESMERE                                against	visceral	leishmaniasis	infection	in	dogs	and	have	a	specific	
   	Bio	Veto	Test	Laboratory	83500	La	Seyne	sur	Mer;	Clinique	            method	 of	 distinguishing	 between	 naturally	 infected	 animals	 from	
  vétérinaire,	26160,	La	Begude	de	Mazenc,	France;	Service	de	            vaccinated	 dogs	 (detection	 of	 specific	 Ig	 G2	 antibodies).	 Another	
   Parasitologie,	ENV	de	Lyon,	69280	Lyon,	France;	Institut	de	           way	 of	 research	 appears	 :	 the	 study	 of	 Li	 ESAp	 and	 especially,	
Recherche	pour	le	Développement,	UR	008,	911	Avenue	Agropolis,	           among	them,	the	identification	of	the	main	specific	antigen	able	to	
          BP	64501,	34394	Montpellier	cedex	5,	France	                    induce	the	LIESAp	immunopropective	response.
                         gpapiero@bvt.fr                                       (1)	 “Protection	 against	 experimental	 visceral	 leishmaniasis	
     We	have	recently	demonstrated	that	the	combination	of	naturally	     infection	in	dogs	immunized	with	purified	excreted	secreted	antigens	
excreted-secreted	antigens	from	culture	supernatant	of	Leishmania         of	Leishmania	infantum	promastigotes.
infantum	 promastigotes	 (Li	 ESAp)	 as	 vaccine	 antigen	 in	 formula-        JL.	LEMESRE	et	al,	Vaccine	23	(2005)	2825-2840
tion	with	muramyl	dipeptide	(MDP)	has	the	capacity	to	protect	dogs	



                                ConCurrent session #1. IMMUNOGENETICS AND GENOMICS


      JoAn	K.	LUnnEy	(AGRICULTURAL	RESEARCH	SySTEM,	                            Salmonella	infections	cause	food	safety	concerns	for	humans	as	
                        USDA,	USA)                                        well	as	production	problems	for	swine.	Our	team	has	used	suppres-
  ComPArAtiVe immune resPonses of Pigs                                    sion	hybridization	(SSH),	long	oligonucleotide	Qiagen	and	Affymetrix	
  to infeCtion WitH sAlmonellA enteriCA                                   porcine	GeneChip®	arrays,	and	real	time	gene	expression	(Q-PCR)	
seroVArs of food sAfety (tyPHimurium) And                                 to	 understand	 the	 host	 response	 to,	 and	 control	 of,	 S.	 enterica	
                                                                          serotype	 Typhimurium	 (ST)	 as	 compared	 to	 S.	 enterica	 serotype	
 AnimAl HeAltH (CHolerAesuis) imPortAnCe.
                                                                                                                                                          Day 5 - Sunday, August 19th




                                                                          Choleraesuis	(SC).	We	identified	differentially	expressed	(DE)	genes	
 J	K	LUnnEy1,	S	M	D	BEARSon2,	y	F	WAnG3,	J	J	UTHE2,3,	L	                  in	mesenteric	lymph	nodes	(MLN)	and	lungs	of	pigs	with	acute	[8,	24,	
qU3,	o	P	CoUTURE3,	S	H	ZHAo4,	D	KUHAR1,	D	nETTLETon5,	                    48	hours	post-inoculation	(hpi)],	and	chronic	stages	[7,	21	days	(dpi)]	
              J	C	DEKKERS3,	C	K	TUGGLE3                                   of	infection.	The	SSH	analyses	identified	several	genes	involved	in	
       1	Animal	Parasitic	Diseases	Laboratory,	BARC,	USDA-ARS,	           heat	shock	response	and	cytoskeletal	rearrangements.	Hierarchical	
      Beltsville,	MD	20705,	USA;	2	National	Animal	Disease	Center,	       gene	cluster	and	pathway	analyses	of	the	microarray	data	revealed	
      USDA-ARS,	Ames,	IA	USA;	3	Department	of	Animal	Science,	            that	host	protein	translation	was	repressed	by	both	pathogens,	with	
    2255	Kildee	Hall,	Iowa	State	University,	Ames,	IA	USA;	4	Key	Lab	     an	especially	strong	transcriptional	response	at	48	hpi	with	SC.	A	high	
     of	Ag	Animal	Genetics,	Breeding,	and	Reproduction,	Huazhong	         proportion	of	significantly	up-regulated	DE	genes	in	both	infections	
     Agricultural	Univ.,	Wuhan,	PR	China;	5	Department	of	Statistics,	    are	involved	in	pathways	for	immune	T	helper	1	(Th1)	differentiation,	
           124	Snedecor	Hall,	Iowa	State	University,	Ames,	IA             innate	 immune/inflammatory	 responses	 and	 antigen	 processing.	
             50


                              Gene	expression	induction	was	weaker	and	occurred	earlier	in	ST	                diabetes.	 	 There	 is	 no	 strong	 evidence	 for	 a	 canine	 equivalent	 of	
                              (24	 hpi)	 as	 compared	 to	 SC.	 In	 SC	 the	 response	 was	 maximal	 at	      human	type	2	diabetes	despite	canine	obesity.		Adult	onset	IDD	is	
                              48	hpi	but	continued	to	be	elevated	at	7	dpi.	This	differential	tran-           the	most	common	type	of	canine	diabetes	and	there	is	evidence	for	
                              scriptional	response	was	mirrored	by	interferon	gamma	and	tumor	                insulitis	and	an	immune	mediated	beta-cell	destructive	process.
                              necrosis	factor	serum	protein	levels	as	well	as	bacterial	load	in	the	                Whilst	 IDD	 can	 occur	 in	 many	 breeds,	 some	 (e.g	 Samoyed	
                              lymph	nodes.	Apoptosis	and	antigen	presentation/dendritic	cell	func-            and	Tibetan	Terriers)	are	particularly	predisposed,	whilst	others	(e.g	
                              tion	 pathways	 were	 down-regulated	 at	 8	 hpi	 for	 ST.	 Cluster	 analy-     Boxer	 and	 German	 Shepherds)	 appear	 protected.	 	 These	 breed	
                              ses,	confirmed	by	Q-PCR	analyses,	revealed	that	many	DE	genes	                  differences	 in	 susceptibility	 suggest	 a	 genetic	 component	 to	 IDD	
                              grouped	into	a	specific	induced	sub-cluster	are	known	NFkB	targets.	            aetiology	in	this	species.		Canine	IDD	is	therefore	likely	to	represent	
                              Suppression	of	NFkB	signaling	from	24	to	48	hpi	may	allow	ST	to	                a	 complex	 condition	 where	 both	 multiple	 susceptibility	 genes	 and	
                              elude	an	anti-bacterial	inflammatory	reaction.	Studies	are	underway	            environmental	factors	interact	to	trigger	disease.
                              to	study	Salmonella-cell	culture	invasion	further	using	IPEC	J2	epi-
                              thelial	 cells	 derived	 from	 porcine	 small	 intestine.	 We	 propose	 that	        We	 have	 examined	 this	 hypothesis	 by	 determining	 whether	
                              NFkB	suppression	in	antigen	presenting	cells	may	be	a	mechanism	                genes	 previously	 shown	 to	 be	 associated	 with	 immune	 mediated	
                              by	 which	 ST	 eludes	 a	 strong	 inflammatory	 response,	 thus	 setting	       human	 diabetes	 also	 represent	 good	 candidate	 genes	 in	 canine	
                              the	stage	for	establishing	a	carrier	status	in	pigs.	For	SC	infections,	        diabetes	 and	 confer	 risk/resistance.	 	 To	 pursue	 this	 investigation	
                              there	was	a	strong	NFkB-dependent	transcriptional	response	and	a	               we	 have	 examined	 candidate	 gene	 polymorphisms	 and	 haplotype	
                              more	intense	and	extended	up-regulation	of	porcine	immune	gene	                 associations	for	a	wide	range	of	candidate	genes	of	known	immuno-
                              expression,	potentially	resulting	in	clearance	of	the	SC	infection.             logical	function	in	well	characterised	canine	IDD	cases	(>500)	and	
                                                                                                              controls	(>1,000).
                                                                                                                   Studies	 examining	 MHC	 (DLA)	 Class	 II	 haplotypes	 revealed	
                                   WILLIAM	oLLIER	(UnIVERSITy	oF	MAnCHESTER,	UK)                              clear	 risk	 associations	 with	 DLA*-DRB1*009-DQA1*001	 and	 DLA-
                               immunogenetiC risK fACtors Contributing                                        DRB1*015-DQA1*006	 haplotypes	 and	 resistance	 with	 the	 DLA-
                                         to CAnine diAbetes                                                   DQA1*004-DQB1*013	haplotype.		These	associations	appeared	to	
                                                                                                              relate	closely	with	breed	predisposition.
                               WER	oLLIER1,	LJ	KEnnEDy1,	AD	SHoRT1,	LJ	DAVIDSon2,	A	
                                 BARnES3,	n	FRETWELL4,	C	JonES4,	B	CATCHPoLE5                                      Other	 immunologically	 related	 candidate	 genes	 examined,	
                                                                                                              included	 CTLA-4,	 IL-4	 and	 IL-10.	 	 SNPs	 were	 identified	 in	 these	
                                1.CIGMR,	The	University	of	Manchester,	UK;	2.Dept.	Clinical	                  genes	 using	 dHPLC	 screening	 and	 DNA	 sequencing.	 	 Confirmed	
                                 Veterinary	Science,	Cambridge,	UK;	3.Faculty	of	Veterinary	                  SNP’s	 were	 genotyped	 in	 cases	 and	 controls	 using	 Sequenom	
                               Sciences,	University	of	Liverpool,	UK;	4.Waltham	Centre	for	Pet	               mass-array	technology.		SNP	haplotype	analysis	revealed	both	IDD	
                               Nutrition,	Leicester,	UK;	5.Royal	Veterinary	College,	Hatfield,	UK             susceptibility	 and	 resistance	 haplotypes.	 	 These	 results	 and	 their	
                                   Diabetes	Mellitus	occurs	spontaneously	in	domestic	dogs	and	               implications	for	canine	ID	will	be	presented	and	discussed.
                                                                                                      	
                              virtually	 all	 affected	 dogs	 become	 dependent	 on	 insulin	 therapy.	       Key words:	 diabetes;	 DLA;	 Class	 II	 haplotypes;	 CTLA-4;	 IL-4;	 IL-
                              Canine	diabetes	can	be	classified	into	a	number	of	types	including	             10
                              congenital,	hormonal	(dioestrous)	and	adult	onset	insulin	dependent	            Species:	canine



                                ConCurrent session #3. IMMUNOENDOCRINOLOGy; AND STRESS; IMMUNOLOGy OF REPRODUCTION AND
                                             NEONATES; MICROBIAL FLORA, NUTRIENTS AND THE IMMUNE RESPONSE

                                 GARy	EnTRICAn	(MoREDUn	RESEARCH	InSTITUTE,	UK)                                              	ISABELLE	oSWALD	(InRA,	FRAnCE)
                                   tHe effeCt of PregnAnCy on mAternAl                                          effeCt of some food ContAminAnts, tHe
                                             immunity in sHeeP                                                myCotoxins, on tHe immune resPonse of tHe
                                                                                                                                  Pig



                                     PlenAry session innAte immunity, inflAmmAtion And AdjuVAnts; memory, ACquired
                                                                immunity And VACCines

                                    GoRDon	MACPHERSon	(oxFoRD	UnIVERSITy,	UK)                                    strAtegies to linK innAte And AdAPtiVe
                                tHe role of dendritiC Cells At muCosAl                                             immunity WHen designing VACCine
                               surfACes And regulAtion of inflAmmAtion                                                         AdjuVAnts
                                    GoRDon	MACPHERSon,	SIMon	MILLInG,	ULF	yRLID
                                   Dendritic	cells	(DC)	migrate	constitutively	from	the	intestine	to	               	PAT	SHEWEn	(UnIVERSITy	oF	GUELPH,	CAnADA)	
                              draining	mesenteric	lymph	nodes.	These	DC	comprise	at	least	three	                                    muCosAl VACCines
                              phenotypically	and	functionally	distinct	subsets.	In	contrast	to	some	
                              current	dogma,	these	DC	are	not	constitutively	suppressed	but	are	the	
                              strongest	activators	of	naive	T	cells	we	have	found.	Such	activated	T	                     SARAH	DoyLE	(TRInITy	CoLLEGE,	DUBLIn)
Day 5 - Sunday, August 19th




                              cells	secrete	a	mixture	of	Th1	and	Th2	cytokines.	Immuno-modula-                           tlr signAling in infeCtion And
                              tory	agents	that	are	potential	intestinal	adjuvants	–	R848	(a	TLR7/8	                              inflAmmAtion
                              ligand,	LPS,	E.	coli	heat	labile	toxin	and	schistosome	egg	antigen)	
                              have	been	used	to	investigate	changes	in	intestinal	DC	associated	
                              with	the	switch	from	tolerance	to	active	immunity.	Our	results	suggest	             STEVE	REInER	(UnIVERSITy	oF	PEnnSyLVAnIA,	USA)	
                              that	this	switch	is	not	associated	with	any	conspicuous	changes	in	
                              the	numbers	or	properties	of	migrating	DCs.		                                       sPeCifying tHe t Cell fAtes required
                                                                                                                for immunity: AsymmetriC diVision of A t
                                                                                                                lymPHoCyte in tHe initiAtion of AdAPtiVe
                                  VoLKER	GERDTS	(VACCInE	AnD	InFECTIoUS	DISEASE	
                                             oRGAnIZATIon,	CAnADA)	                                                            immunity
                                                                                                                                                           51



 Abramson	Family	Cancer	Research	Institute,	and	Department	of	              segregation	of	determinants	appears	to	be	coordinated	by	prolonged	
  Medicine,	University	of	Pennsylvania,	Philadelphia,	PA	19104              interaction	between	the	T	cell	and	its	antigen-presenting	cell	prior	to	
      A	hallmark	of	mammalian	immunity	is	the	heterogeneity	of	cell	        division.		Additionally,	the	first	two	daughter	T	cells	display	phenotypic	
fate	 that	 exists	 among	 pathogen-experienced	 lymphocytes.	 	 I	 will	   and	functional	indicators	of	being	differentially	fated	toward	effector	
present	evidence	that	a	dividing	T	lymphocyte	initially	responding	to	a	    and	memory	lineages.		These	results	suggest	a	mechanism	in	which	
microbe	exhibits	unequal	partitioning	of	proteins	that	mediate	signal-      a	 single	 lymphocyte	 can	 apportion	 diverse	 cell	 fates	 necessary	 for	
ing,	cell	fate	specification,	and	asymmetric	cell	division.	Asymmetric	     adaptive	immunity.




                                                                                                                                                           Day 5 - Sunday, August 19th
                                                       Abstracts for Posters


1. Immunogenetics and Genomics
                       .
   Posters	IG001-IG026		 .........................................................................................................................	53

2. Immune Responses in Bacterial and Viral Diseases; Prions and BSE
   Posters	BV027–BV077		.......................................................................................................................	62	

3. Immunoendocrinology; and Stress; Immunology of Reproduction
   and Neonates; Microbial Flora, Nutrients and the Immune Response
   Posters	IE078-ER094		.........................................................................................................................	79	

4. Immunology of the Mucosae and Skin and of the Mammary Gland; Mastitis
   Posters	SM095-SM118		.......................................................................................................................	85

5. Antigen Presentation and Dendritic Cells; Effector Cells, B and T cells,
   NK and NK T cells; Immunoregulatory cells
   Posters	AP119-AP140	.........................................................................................................................	93

6. Immunoparasitology: Immune Responses to Protozoa, Helminths and
   Ectoparasites; Canine Visceral Leishmaniasis
   Posters	PR141-PR196		.....................................................................................................................	100

7. Comparative Immunology; Immunoecology
   Posters	CI197-CI206		........................................................................................................................ 	118

8. Mediators of Recruitment and Function of Cells of the Immune System;
   Fc Receptors and Immunoglobulins; Signal Transduction and
   Gene Expression in cells of the immune system
   Posters	MI207-MI210		.......................................................................................................................	121

9. Innate immunity, Inflammation and Adjuvants; Memory,
   Acquired Immunity and Vaccines
                        .
   Posters	VA211-VA250	.......................................................................................................................	123	

10. Clinical Immunology and Immunopathology
                        .
  		Posters	IP251-IP281		......................................................................................................................	136	
                                                    1. IMMUNOGENETICS: POSTERS IG001-IG026


   ig001. IDENTIFICATION OF NOVEL BOVINE DH GENES                                    gens	and	will	be	valuable	tools	for	subunit	vaccine	development	and	
     MADHURI	KoTI1,	GALInA	KATAEVA2,	AZAD	K	KAUSHIK1                                 comparative	studies	of	MHC	diversity	and	evolution.	
1Department	of	Molecular	and	Cellular	Biology,	University	of	Guelph,	                Key words:	Sheep,	MHC,	haplotypes
        Canada;	2	McMaster	University,	Hamilton,	Canada	                             Species:	ruminants
                      mkoti@uoguelph.ca
      Our	laboratory	earlier	demonstrated	that	limited	germline	sequence	                ig003. NOVEL APPROACHES TO ENHANCE DISEASE
divergence	 both	 at	 the	 heavy	 and	 light	 chain	 restricts	 combinatorial	             RESISTANCE IN RUMINANTS? - BREEDING FOR
diversity	 in	 cattle,	 similar	 to	 other	 species	 such	 as	 chicken,	 pig	 and	           GEOGRAPHICALLy IMPORTANT TLR SNPS
sheep.	 One	 of	 the	 most	 important	 characteristics	 of	 bovine	 antibody	        THE	RUMInAnT	TLR	ConSoRTIUM:	DW	BURT1,	TJ	CoFFEy3,	S	
repertoire	 is	 that	 an	 extensive	 CDR3H	 length	 heterogeneity	 exists	 in	       CHAnG2,	EJ	GLASS1,	D	HAIG2,	JC	HoPE3,	o	JAnn1,	J	SALT1,	C	
bovine	antibodies	that	is	evident	from	fetal	B-cell	stage.	The	generation	                           WARKUP4,	D	WERLInG6
of	an	exceptionally	long	CDR3H	(up	to	61	amino	acids)	with	multiple	                       1Roslin	Institute	(Edinburgh);	2Moredun	Research	Institute	
cysteine	residues	and	somatic	hypermutations	contribute	to	antibody	                     (Edinburgh);	3Institute	for	Animal	Health	(Compton);	4Genesis	
diversification	in	cattle.	Partial	characterization	of	bovine	DH-gene	locus	            Faraday	(Edinburgh);	5Pfizer	Ltd	(Sandwich);	6Royal	Veterinary	
in	 our	 laboratory	 demonstrated	 the	 presence	 of	 both	 short	 and	 long	                                    College	(London)
germline	 DH	 genes	 with	 the	 potential	 to	 directly	 encode	 49	 codons.	
We	have	now	characterized	the	bovine	DH-gene	locus	from	cattle	that	                       Opportunistic	infections	resulting	from	intensive	husbandry	of	live-
demonstrates	polymorphism	apart	from	the	fact	that	bovine	DH	genes	                  stock	have	become	one	of	the	major	problems	in	modern	animal	pro-
are	organized	in	distinct	sub	clusters.                                              duction.	As	bacterial	resistance	to	antibiotics	is	expected	to	escalate,	
                                                                                     novel	 approaches	 to	 disease	 prevention	 will	 need	 to	 be	 established.	
     (Supported	by	NSERC	research	grant).                                            One	 approach	 includes	 breeding	 for	 disease	 resistance	 by	 selecting	
Key words:	Antibody	Diversity,	CDR3H,	DH	genes                                       the	‘fittest’	innate	immune	system.	The	innate	immune	system	recog-
Species:	Ruminants                                                                   nises	 pathogens	 by	 means	 of	 pattern	 recognition	 receptors,	 such	
                                                                                     as	 Toll-like	 receptors	 (TLRs).	 These	 interact	 with	 various	 microbial	
  ig002. GENERATION, HAPLOTyPE CHARACTERISATION                                      components	and	induce	a	specific	innate	immune	response.	Several	
   AND PRACTICAL APPLICATIONS OF AN MHC DEFINED                                      polymorphisms	in	TLR	genes	have	been	described	for	the	human	and	
              SHEEP RESOURCE FLOCK.                                                  murine	 system	 that	 influence	 the	 abilities	 of	 affected	 TLRs	 to	 recog-
                                                                                     nise	pathogen-derived	molecules	-	rendering	individuals	more	or	less	
 KEITH	BALLInGALL1,	DESPoInA	MILTIADoU1,	MARA	RoCCHI1,	                              susceptible	to	infection.	The	first	nucleotide	polymorphisms	(SNPs)	in	
                 DECLAn	MCKEEVER	1,2                                                 ruminant	 TLRs	 were	 characterised	 in	 bovine	 TLR4,	 the	 receptor	 for	
 1	Moredun	Research	Institute,	Pentlands	Science	Park,	Midlothian,	                  gram-negative	bacteria.	Analysis	of	codon-based	models	of	selection	
    UK;	2	Royal	(Dick)	School	of	Veterinary	Studies,	Easter	Bush	                    identifies	many	sites	in	TLR4	under	positive	selection	in	the	region	261	
                 Veterinary	Centre,	Midlothian,	UK	                                  to	 375	 residues.	This	 region	 is	 located	 in	 the	 middle	 of	 the	 extracel-
                  keith.ballingall@moredun.ac.uk	                                    lular	 domain	 between	 clusters	 of	 leucine-rich-repeats	 (LRRs)	 and	 is	
      In	jawed	vertebrates	the	T	cell	receptor	binds	to	foreign	pathogen	            predicted	to	be	the	ligand	binding	domain.	We	would	expect	a	higher	
fragments	 in	 association	 with	 classical	 self	 major	 histocompatibility	        frequency	of	nonsynonymous	SNPs	to	map	to	his	domain.	Indeed,	the	
complex	 (MHC)	 molecules.	 This	 recognition	 event	 is	 fundamental	               first	nonsynonymous	SNPs	map	to	this	region.
to	 the	 induction	 and	 immune-mediated	 control	 of	 infectious	 disease.	     	         We	have	cloned	and	mapped	ten	bovine	TLRs,	and	are	currently	
However,	 extensive	 allelic	 diversity	 within	 the	 genes	 encoding	 the	          in	the	progress	of	functionally	characterising	these	TLRs.	In	addition,	
MHC	molecules	is	a	significant	constraint	to	long	term	studies	of	T	cell	            TLR	genes	from	sheep	are	also	being	cloned	and	characterised.	TLRs	
immunology	in	out-bred	species	since	they	rely	on	the	undefined	MHC	                 from	different	cattle	and	sheep	breeds	are	currently	been	analysed	for	
haplotype	 carried	 by	 each	 experimental	 animal.	 	 This	 “MHC	 restric-          the	 presence	 of	 synonymous	 and	 non-synonymous	 SNPs.	 Our	 data	
tion”	led	to	the	development	of	inbred	animal	models	with	identical	and	             suggest	a	heterogeneity	in	extracellular	regions	of	TLR	genes,	which	
well	characterised	MHC	regions	that	are	now	used	in	the	majority	of	                 may	be	advantageous	to	promote	a	specific	disease	resistance,	and	
infectious	disease	and	basic	immunological	research.		However,	data	                 represent	a	new	approach	to	select	disease	resistance	-	based	on	a	
derived	from	studies	in	model	organisms	such	as	the	laboratory	mouse	                geographical	 distribution	 of	 TLR	 SNPs.	 Breeding	 of	 ruminants	 with	
are	 often	 not	 directly	 transferable	 to	 the	 original	 target	 species	 such	   TLRs	that	confer	a	‘fitter’	innate	 immune	 system	resulting	 in	disease	
as	human.		Infectious	disease	research	in	livestock	on	the	other	hand	               resistance	could	play	a	major	role	in	the	future	of	the	farming	industry	
has	the	advantage	of	being	able	to	study	the	disease	and	the	mecha-                  both	in	the	UK	and	worldwide.
nisms	of	immunity	within	the	target	species.		However	the	problem	of	                     Funding	 and	 support	 from	 the	 BBSRC,	 Genesis	 Faraday	 and	
MHC	restriction	remains.	To	address	this	we	have	generated	an	MHC	                   Pfizer	Research.
defined	resource	flock	which	is	based	around	four	common	but	diverse	
sheep	haplotypes.		As	well	as	a	large	cohort	of	MHC	heterozygous	ani-                Key words:	 Pattern	 recognition	 receptors,	 TLR,	 Innate	 immune	
mals	we	have	also	purpose	bred	MHC	homozygous	animals	covering	                      response,	Genetic	resistance
each	of	the	four	haplotypes.		In	addition	to	providing	a	valuable	cellular	          Species:	ruminants
resource	for	analysis	of	T	cell	responses,	these	homozygous	animals	
also	allow	the	unambiguous	molecular	genetic	characterisation	of	the	                        ig004. THE DISCOVERy OF QTL FOR MASTITIS
four	target	MHC	haplotypes.		Here	we	describe	the	characterization	of	                          RESISTANCE IN NORDIC DAIRy CATTLE.
the	transcribed	MHC	class	I	and	class	II	genes	carried	by	these	distinct	              nICoLA	HASTInGS1,	AnA	FERnAnDEZ2,	MoGEnS	S.	LUnD3,	
haplotypes.		This	provides	substantial	new	 information	on	allelic	 and	                 GoUTAM	SAHAnA3,	Bo	THoMSEn3,	nInA	SCHULMAn4,	
haplotype	diversity	in	sheep.		The	MHC	defined	flock	is	available	for	                  JoHn	L	WILLIAMS5,	LEnA	AnDERSSon-EKLUnD6,	HALDJA	
collaborative	studies	of	T	cell	responses	to	infectious	livestock	patho-                            VIInALASS7,JoHAnnA	VILKKI4
54


        1Roslin	Institute,	Roslin,	Midlothian,	EH25	9PS,	U.K.;	2Mejora	                     TCRβ	chain	sequences	generated	from	cDNA	studies	completed	in	our	
       Genética	Animal,	SGIT-INIA,	Crta.	Coruña	Km.7.5,	28040	Madrid,	                      laboratory	have	identified	more	than	30	Vβ	genes	that	are	not	present	
     Spain;	3Danish	Institute	of	Animal	Sciences,	Blichers	Alle,	8830	Tjele,	               in	the	current	bovine	genome	assembly,	indicating	that	the	full	genomic	
       Denmark;	4MTT,	Biotechnology	and	Food	Research,	Genomics,	                           Vβ	 repertoire	 remains	 undefined.	 In	 contrast,	 comparison	 to	 cDNA	
      31600	Jokioinen,	Finland;	5Parco	Tecnologico	Padano,Via	Einstein,	                    and	EST	data	suggests	that	the	full	Jβ	repertoire	is	represented	in	the	
     Polo	Universitario,	Lodi	26900,	Italy;	6Department	of	Animal	Breeding	                 genome	 assembly.	 Combination	 of	 the	 cDNA	 and	 genomic	 analyses	
     and	Genetics,	Swedish	University	of	Agricultural	Sciences,	Box	7023,	                  suggest	that	the	functional	bovine	TCRβ	gene	repertoire	is	composed	
      S-750	07	Uppsala,	Sweden;	7Estonian	University	of	Life	Sciences,	                     of	 over	 100	 Vβ	 genes,	 3	 Dβ	 genes	 and	 17	 Jβ	 genes.	The	 results	 of	
                     Kreutzwaldi	64,	51014	Tartu,	Estonia.                                  this	work	indicate	that	the	bovine	TCRβ	gene	repertoire	is	the	largest	
           Mastitis	remains	the	most	costly	disease	affecting	the	European	                 yet	 characterised.	The	 dramatic	 expansion	 of	 membership	 of	 certain	
     dairy	cattle	industry.	The	frequency	of	clinical	mastitis	is	increasing	due	           Vβ	subfamilies	and	comparison	to	the	human	and	murine	TCRβ	gene	
     to	an	unfavourable	genetic	correlation	between	increased	productivity	                 repertoires	raises	interesting	questions	concerning	the	forces	influenc-
     and	mastitis	resistance.	This	and	because	the	trait	has	a	low	heritabil-               ing	the	evolution	of	this	immunologically	important	locus.
     ity	 make	 selective	 breeding	 for	 mastitis	 resistance	 difficult.	 Mapping	        Key words: TCR,	Genome
     of	 quantitative	 trait	 loci	 (QTL)	 in	 livestock	 using	 existing	 population	      Species:	ruminant
     structures	 is	 restricted	 to	 the	 traits	 that	 are	 recorded	 in	 the	 breeding	
     schemes.	The	Nordic	countries	have	traditionally	paid	particular	atten-                    ig006. SNPS IN BOVINE CANDIDATE GENES FOR
     tion	 to	 recording	 health	 traits,	 such	 as	 mastitis,	 in	 dairy	 cattle.	 The	       MEDIATING RESISTANCE TO INFESTATIONS WITH THE
     extensive	 recording	 in	 the	 Nordic	 herds	 provides	 a	 unique	 resource	                               CATTLE TICK
     for	mapping	of	loci	affecting	functional	traits	especially	clinical	mastitis.	
     Using	the	Nordic	herd	records	and	DNA	samples	from	the	herds,	which	                             AnTonIo	R	R	ABATEPAULo1,	ISABEL	K	F	DE	
     included	the	three	breeds	Finnish	Ayrshire,	Swedish	Red	and	White,	                           MIRAnDA	SAnToS1,	DAnIELA	D	MoRé1,	WAnESSA	A	
     and	Danish	Red,	we	analysed	five	chromosomes	for	QTL	affecting	clin-                               CARVALHo1,ALExAnDRE	R	CAETAno2
     ical	mastitis	(CM)	and	somatic	cell	score	(SCS).	Joint	single-trait	and	                 1Dept.	of	Biochemistry	and	Immunology,	Ribeirão	Preto	Medical	
     two-trait	analyses	were	performed	using	variance	components	models.	                   School,	University	of	São	Paulo,	Ribeirão	Preto,	SP,	Brazil;	2Embrapa	
     QTL	affecting	CM	and/or	SCS	segregate	on	BTA9,	11,	14	and	18	while	                         Recursos	Genéticos	e	Biotecnologia,	Brasília,	DF,	Brazil.		
     a	QTL	on	BTA29	could	not	be	confirmed.	However,	our	analyses	con-                                               abatepaulo@usp.br
     firmed	at	least	two	linked	QTL	on	BTA9,	one	that	mainly	affects	CM	and	                     Introduction
     a	second	that	primarily	affects	SCS.	In	order	to	obtain	accurate	QTL	
                                                                                                  Ticks	 are	 hematophagous	 arthropods	 that	 cause	 serious	 losses	
     positioning	on	BTA9	we	improved	the	level	of	information	in	the	analy-
                                                                                            in	animal	productivity	and	health,	especially	in	tropical	environments.	
     sis	by	employing	a	number	of	methods	to	increase	the	marker	density	
                                                                                            Alternatives	 to	 acaricides,	 the	 current	 method	 of	 control,	 are	 needed	
     and	quality	of	the	mapping	information.	In	developing	new	markers	we	
                                                                                            because	 of	 the	 resulting	 contamination	 of	 the	 environment	 and	 food	
     used	 in	 silico	 and	 molecular	 biological	 techniques.	 The	 methods	 we	
                                                                                            products,	and	loss	of	effectiveness	ensuing	from	development	of	resis-
     employed	 included	 mining	 the	 bovine	 genome	 for	 new	 microsatellite	
                                                                                            tance	by	the	ticks.	Bovine	hosts	express	breed-specific,	heritable,	con-
     markers	and	re-sequencing	of	genes	and	ESTs	for	new	SNPs.	Using	
                                                                                            trasting	phenotypes	(susceptible:	S	or	resistant:	R)	when	exposed	to	
     this	 new	 marker	 information	 along	 with	 previously	 reported	 markers	
                                                                                            larvae	of	Rhipicephalus	microplus.	Bos	indicus	breeds	are	significantly	
     from	 USDA	 MARC	 (http://www.marc.usda.gov/genome/cattle/cattle.
                                                                                            more	 resistant	 than	 Bos	 taurus	 breeds,	 while	 animals	 in	 segregating	
     html)	we	created	dense	radiation	hybrid	(RH)	and	linkage	maps.	The	
                                                                                            populations	derived	from	crosses	between	these	groups	show	varying	
     new	maps	were	used	to	fine-map	the	QTL	using	a	linkage/linkage	dis-
                                                                                            levels	of	resistance.	This	suggests	a	polygenic	basis	for	the	trait	and	
     equilibrium	mapping	approach.
                                                                                            offers	 an	 opportunity	 to	 identify	 specific	 genes/mutations	 associated	
     Key words:	Mastitis,	Resistance,	QTL                                                   with	tick	resistance.	
     Species:	ruminant
                                                                                                  Objectives:	Examine	genomic	sequences	of	candidate	genes,	pre-
                                                                                            viously	determined	to	be	differentially	expressed	in	tick-infested	skin,	
       ig005. GENOMIC AND CDNA ANALySIS OF THE BOVINE                                       for	 the	 presence	 of	 breed-specific	 Single	 Nucleotide	 Polymorphisms	
                   TCRβ GENE REPERTOIRE                                                     (SNPs)	 by	 comparing	 tick-resistant	 and	 tick–susceptible	 bovine	
                T	ConnELLEy,	J	AERTS,	A	LAW,	WI	MoRRISon                                    breeds
      1Division	of	Clinical	Veterinary	Sciences,	University	of	Edinburgh,	                       Methods
     Edinburgh,	United-Kingdom.	Dept.	of	Bioinformatics,	Roslin	Institute,	                      Genomic	DNA	from	16	resistent	hots	(Nelore)	and	16	susceptble	
                Roslin	Biocentre,	Midlothian,	UK,	EH25	9RS.                                 hots	 (HPB)	 was	 PCR-amplified	 with	 specific	 primers	 designed	 to	
           The	diversity	of	the	TCR	repertoire	that	is	integral	to	effective	αβ	            generate	an	amplicon	anchored	on	two	exons	and	spanning	at	least	
     T-cell	function	is	generated	by	somatic	recombination	of	variable	(V),	                one	 intron.	 The	 PCR	 fragments	 were	 purified	 with	 ExoSapitTM	 and	
     diversity	(D	-	β	chains	only)	and	joining	(J)	gene	segments	of	the	com-                sequenced	in	an	ABI	3100	DNA	analyzer.
     ponent	α	and	β	chains.	To	facilitate	the	development	of	techniques	to	                      Results	and	Discussion
     examine	bovine	T-cell	responses	we	undertook	an	extensive	analysis	
                                                                                                 We	 looked	 for	 breed-specific	 SNPs	 by	 comparing	 genomic	
     of	genomic	and	cDNA	data	to	characterise	the	bovine	TCRβ	gene	rep-
                                                                                            sequences	from	Nelore	(N	=	16)	and	Holstein	(N	=	16)	for	candidate	
     ertoire.	Analysis	 of	 the	 third	 bovine	 genome	 assembly	 demonstrates	
                                                                                            genes	(TGF-α,	IFN-γ,	IP-10,	TNF-α,	MIP-1α,	IGF-1	and	MCP-1)	we	had	
     that	 although	 the	 TCRB	 locus	 is	 still	 incomplete,	 the	 TCRβ	 gene	
                                                                                            previously	determined	to	be	differentially	expressed	in	the	tick-infested	
     repertoire	 in	 cattle	 is	 significantly	 larger	 than	 that	 of	 either	 humans	
                                                                                            skin	 of	 resistant	 hosts.	A	 total	 of	 40	 SNPs	 were	 found,	 at	 least	 five	
     or	 mice.	 Most	 notably,	 several	 of	 the	 Vβ	 subfamilies,	 such	 as	 Vβ1,	
                                                                                            SNPs	per	gene,	and	several	alleles	were	found	to	be	breed-specific.	
     10	 and	 13	 have	 undergone	 extensive	 duplication,	 containing	 35,	 16	
                                                                                            The	results	provide	information	that	will	allow	for	association	studies	in	
     and	40	members	respectively.	Much	of	this	expansion	appears	to	be	
                                                                                            composite/segregating	populations	resulting	from	crosses	of	B.	taurus	
     due	 to	 duplication	 of	 ‘cassettes’	 containing	 multiple	 genes	 leading	 to	
                                                                                            and	B.	indicus	breeds	to	ascertain	if	these	markers	are	associated	with	
     tandemly	arranged	duplicates	incorporating	members	of	two	or	more	
                                                                                            causal	mutations	which	confer	tick	resistance	to	B.	indicus	cattle.	
     subfamilies	(e.g.	Vβ18-Vβ17-Vβ2-Vβ10).	In	all,	130	Vβ	genes	distrib-
     uted	 over	 24	 subfamilies	 have	 been	 identified	 in	 the	 genome	 so	 far.	             Supported	by	FAPESP,	CNPq,	The	World	Bank	and	Vallée	SA.
     Compared	 to	 other	 species	 examined,	 the	 bovine	 genome	 also	 has	               Key words:	SNP,	Bovine	gene,	Resistance,	Tick
     expanded	 Dβ	 and	 Jβ	 gene	 repertoires	 due	 to	 triplicate	 rather	 than	           Species:	ruminants
     duplicate	 copies	 of	 the	 DJC	 region	 of	 the	 TCRB	 locus.	 The	 majority	
     (126/130)	of	the	Vβ	genes	and	all	of	the	Dβ,	Jβ	and	Cβ	genes	present	                        ig007. IDENTIFICATION AND CONFIRMATION OF
     in	 the	 genome	 assembly	 are	 located	 of	 4	 large	 scaffolds,	 3	 of	 which	
                                                                                                 POLyMORPHISMS IN BOVINE GENES WITH IMMUNE
     have	been	mapped	to	chromosome	4.	Extensive	analysis	of	over	900	
                                                                                                                   FUNCTION
                                                                                                                                                                      55


A	CAPRERA2,	C	CAMBULI1,	R	CAPoFERRI1,	CGoRnI2,	SVIoLInI	                        of	inflammatory	cytokines	and	in	particular	a	remarkable	induction	of	
         F	PAnZITTA2,	B	LAZZERI2,JL	WILLIAMS2                                   multiple	members	of	the	chemokine	gene	family.
 IDRA	Laboratory	ISLS1-PTP2,	Polo	Universitario,	via	Einstein,	Lodi	            Key words: innate	immunity,	mastitis,	chemokine,	infection.
                         26900	Italy.                                           Species: ruminants
      The	identification	of	the	genetic	variations	controlling	phenotypes,	
including	immune	function,	can	be	achieved	by	a	combination	of	link-             ig009. STRAIN SPECIFIC AND COMMON PATTERNS OF
age	mapping,	association	studies	or	candidate	gene	approaches.		The	            GENE ExPRESSION IN MACROPHAGES INFECTED WITH M.
availability	 of	 the	 draft	 bovine	 genome	 sequence,	 progress	 towards	               PARATUBERCULoSIS ISOLATES.
its’	annotation	and	information	on	putative	single	nucleotide	polymor-              E	KABARA*1,	C	KLoSS2,	M	WILSon2,	S	SREEVATSAn3	,	H	
phisms	 provides	 considerable	 new	 information	 to	 identify	 positional	                      JAnAGAMA3,	P	CoUSSEnS2	
candidate	genes.		However,	from	the	2.2	million	bovine	SNPs	identified	
                                                                                   1Department	of	Biochemistry;	2Center	for	Animal	Functional	
from	the	genome	sequencing	project,	up	to	now,	few	have	been	con-
                                                                                Genomics,	Department	of	Animal	Science,	Michigan	State	University,	
firmed.		Therefore,	the	identification	and	validation	of	polymorphisms	
                                                                                  East	Lansing,	MI.;	3Center	for	Animal	Health	and	Food	Safety,	
in	the	functional	or	regulatory	regions	of	the	genes	that	are	putatively	
                                                                                           University	of	Minnesota,	Minneapolis,	MN.
involved	in	regulating	immune	function	remains	an	important	task.
                                                                                      Mycobacterium	 avium	 subspecies	 paratuberculosis	 (MAP)	 is	 an	
      We	are	interested	in	the	role	of	various	immune	genes	in	the	con-         intracellular	pathogen	that	causes	an	economic	burden	to	the	US	dairy	
trol	of	mycobacterial	infections	in	cattle.		Therefore	we	have	selected	a	      industry	estimated	at	over	one	billion	dollars	annually.		A	hallmark	of	
panel	of	genes	involved	in	macrophage	function	and	are	examining	the	           MAP	 infection	 is	 survival	 in	 host	 macrophages,	 cells	 that	 normally	
level	of	polymorphism	in	these	genes		The	starting	point	is	to	identify	        destroy	 ingested	 microbes.	 	 As	 with	 other	 mycobacteria,	 survival	
putative	polymorphisms	in	target	genes	using	bovine	sequence	infor-             in	 macrophages	 appears	 to	 be	 a	 key	 determinant	 of	 pathogenesis	
mation.		A	software	application	has	been	developed	to	locate	putative	          associated	 with	 MAP	 infections.	 	 Previously,	 we,	 and	 others,	 have	
SNPs	available	from	DB	SNP	within	target	genes	or	sequences	using	              demonstrated	that	infection	with	both	MAP	and	the	closely	related,	but	
positional,	ePCR	and	BLAST	approaches.		The	presence	of	SNPs	is	                non-pathogenic	Mycobacterium	avium	subspecies	avium	(MAA)	have	
then	confirmed	by	direct	sequencing	prior	to	determining	allele	frequen-        profound	 effects	 on	 macrophage	 gene	 expression.	 	 Based	 on	 these	
cies	in	a	panel	of	animals	from	a	range	of	genetically	diverse	breeds.          studies,	we	hypothesized	that	different	strains	of	MAP	would	have	both	
Key words:	Polymorphisms,	SNP                                                   common	and	strain-specific	effects	on	macrophage	cell	gene	expres-
Species:	ruminants                                                              sion.	 	 To	 test	 this	 hypothesis,	 we	 have	 now	 studied	 the	 effect	 of	 10	
                                                                                different	 MAP	 strains	 on	 macrophage	 gene	 expression	 profiles,	 with	
   ig008. GENOMIC RESPONSES OF THE BOVINE                                       the	ultimate	goal	of	relating	gene	expression	differences	to	virulence	
                                                                                and	genetics	of	the	MAP	strains.		Our	initial	data	analysis	suggests	that	
MAMMARy GLAND AND EPITHELIAL CELLS TO ACUTE LPS
                                                                                there	are	over	120	macrophage	genes	whose	expression	is	generally	
                 CHALLENGE
                                                                                altered	following	infection	with	any	strain	of	MAP.	Our	data	has	been	
  DE	KERR1,	M	LATSHAW1,	R	PAREEK1,	J	ZHEnG1,	JP	BonD2                           further	 scrutinized	 via	 mixed-model	 analysis	 to	 investigate	 potential	
  1Department	of	Animal	Science;2Department	of	Microbiology	and	                strain-specific	 and/or	 host	 origin-specific	 differences	 in	 MAP-macro-
     Molecular	Genetics	University	of	Vermont,	Burlington	VT                    phage	interactions	and	remove	the	specific	effects	of	each	animal	used	
                                                                                in	the	study.		Examples	include	genes	typically	associated	with	immune	
       The	 early	 innate	 immune	 response	 to	 bacterial	 entry	 into	 the	
                                                                                response,	such	as	IL-1a,	IL-8,	and	CCL-3.		Expression	patterns	of	other	
mammary	gland	is	thought	critical	in	determining	the	outcome	of	mas-
                                                                                genes,	 not	 typically	 associated	 with	 immune	 responses,	 such	 as	 an	
titis.	The	goal	of	identifying	animals	with	enhanced	genetic	resistance	
                                                                                ADP-ribosyltransferase,	were	also	affected	by	infection	with	all	strains	
to	mastitis	will	depend	on	greater	knowledge	of	the	genes	induced	in	
                                                                                of	MAP.		This	study	also	revealed	transcription	level	changes	in	several	
response	to	pathogens.	We	have	used	Affymetrix	GeneChips	to	profile	
                                                                                previously	unknown	genes	that	may	be	important	for	the	protection	of	
the	 acute	 genomic	 response	 to	 an	 LPS	 challenge	 in	 lactating	 cows	
                                                                                the	pathogen	and	thus	merit	further	investigation.			Upon	hierarchical	
and	 in	 cultured	 mammary	 epithelial	 cells.	 Sources	 of	 RNA	 included	
                                                                                clustering	using	fold-change	data,	MAP	strains	isolated	from	human,	
biopsy	 samples	 from	 LPS-challenged	 (1	 ug/gland)	 and	 contralateral	       cattle	 and	 sheep	 showed	 little	 initial	 host	 species	 similarity,	 but	 two	
control	mammary	glands	of	3	lactating	cows	4	h	post-challenge;	and	             supershedder	 strains	 clustered	 tightly	 together,	 perhaps	 suggesting	
from	 LPS-stimulated	 (50	 ng/ml)	 or	 control	 cultures	 of	 primary	 mam-     these	two	strains	have	very	similar	effects	on	bovine	macrophage	cells	
mary	epithelial	cells	3	h	post-LPS.	The	cells	were	previously	obtained	         and	potentially	suggesting	a	relation	between	bacterial	phenotype	and	
and	 cryopreserved	 from	 3	 different	 cows.	 We	 identified	 75	 immune-      host	reaction	to	the	pathogen.
associated	genes	that	were	differentially	regulated	(74	up,	1	down)	in	
response	 to	 LPS	 in	 the	 tissue	 biopsy	 samples	 (P	 ≤0.01,	 fold-change	   Key words:	 Pathogen-host	 interaction,	 microarray	 analysis,	 Johne’s	
(FC)	≥	2.0).	Induced	genes	included	a	remarkable	17	members	of	the	             disease,	Crohn’s	disease
                                                                                Species:	ruminants
CCL,	CXCL,	and	CX3CL	family	of	chemokines.	Also	induced	were,	3	
inflammatory	 cytokines	 (IL1b,	 IL6,	 TNF),	 3	 acute	 phase	 (HP,	 SAA3,	
LTF),	 2	 antimicrobial	 (BNBD-4,	 LAP),	 and	 6	 members	 of	 the	 S100A	       ig010. KNOCK-DOWN OF BOVINE LEUKEMIA VIRUS TAx
calcium	binding	proteins.	The	central	role	of	the	NF-kB	complex	was	             By RNAI AND ITS EFFECTS ON HOST GENE ExPRESSION
illustrated	by	induction	of	NFKB1	and	RELB,	and	interestingly,	a	con-            RoSAnE	oLIVEIRA1,	ALLISon	SoMMERS1,	RoBIn	E	EVERTS1,	
cordant	upregulation	of	3	members	of	the	I-kB	family	that	appears	to	                              HARRIS	A	LEWIn1,2
indicate	a	negative	modulation.	The	cell	culture	response	revealed	44	
immune-associated	genes	differentially	regulated	(P≤0.01,	fold-change	          1Department	of	Animal	Sciences	and	2Institute	for	Genomic	Biology,	
(FC)	≥	2.0;	all	up)	in	response	to	LPS	stimulation.	The	gene	list	from	the	        University	of	Illinois	at	Urbana-Champaign,	Urbana,	IL,	USA
cultured	cells	indicated	a	close	reflection	of	the	in	vivo	response	in	that	          Bovine	Leukemia	Virus	(BLV)	tax	protein	is	a	transcription	trans-
34	upregulated	genes	were	common	between	the	tissue	and	cell	culture	           activator	of	host	cell	genes	that	modulates	cell	growth	and	proliferation.	       	
experiments.	Commonly	upregulated	genes	include	10	members	of	the	              By	 interfering	 with	 the	 transcription	 of	 host	 cell	 genes,	 tax	 expres-
CCL,	CXCL,	and	CX3CL	family	of	chemokines,	three	S100	genes	(A8,	               sion	 is	 believed	 to	 be	 an	 essential	 first	 step	 in	 the	 dysregulation	 of	
A9,	A12),	 and	 the	 inflammatory	 cytokines,	 and	 NF-kB	 related	 genes	      homeostasis	leading	to	the	transformation	of	B-lymphocytes.		To	better	
previously	mentioned.	Immune-associated	genes	upregulated	only	in	              understand	the	role	of	BLV	tax	in	driving		lymphoproliferation	and	cell	
the	LPS	treated	cells	included	three	additional	inflammatory	cytokines	         transformation	in	the	host,	BLV	tax	gene	expression	in	a	BLV-infected	
(IL1A,	 CSF1,	 CSF2)	 and	 the	 enzymes	 urinary	 plasminogen	 activator	       bovine	 lymphoblastoid	 cell	 line	 (BL3*)	 was	 knocked	 down	 by	 RNA	
(PLAU)	 and	 nitric	 oxide	 synthase	 2A	 (NOS2A).	 These	 studies	 have	       interference	 (RNAi).	 	 Gene	 expression	 profiling	 was	 then	 performed	
revealed	 a	 rapid,	 robust	 genomic	 response	 of	 the	 bovine	 mammary	       using	a	13,257-element	cattle	oligonucleotide	microarray.		A	tax-spe-
gland	to	a	small	quantity	of	E.	coli	LPS.	The	genomic	response	appears	         cific	small-interfering	RNA	(siRNA)	and	control	scrambled	siRNA	were	
dominated	by	activation	of	the	NF-kB	complex	leading	to	upregulation	           used	 for	 transfections.	 	Transfection	 efficiency	 was	 analyzed	 by	 flow	
56


     cytometry	 of	 cells	 transfected	 with	 a	 GFP-construct,	 and	 tax	 mRNA	                ig012. COMPARATIVE GENOME ANALySIS OF
     levels	were	assayed	by	quantitative	PCR.		Six	biological	replicates	of	                            TRyPANOTOLERANCE QTL
     the	knock-down	were	performed	with	both	BL3*	and	BL3º	(uninfected	                      JoSEPH	nGAnGA1,2,	MABEL	IMBUGA2,FUAD	A	IRAqI3
     parental	cell	line).		Each	treatment	comprised	5	or	6	technical	replicates	
     to	provide	enough	RNA	for	the	microarrays	(total	of	132	transfections).	  	       	1International	Livestock	Research	Institute,	P.	O.	Box	30709,	Nairobi,	
     The	average	transfection	and	knock-down	efficiencies	were	92.9%	and	              Kenya;	2Jomo	Kenyatta	University	of	Agriculture	and	Technology,	P.O.	
     72.25%,	respectively.	RNA	isolated	from	each	treatment	was	pooled,	                  Box	62000,	Nairobi,	Kenya;	3Department	of	Human	Microbiology,	
     and	 four	 independent	 transcriptome	 comparisons	 were	 carried	 out	            Sackler	Faculty	of	Medicine,	Tel	Aviv	University,	Ramat	Aviv,	Tel	Aviv	
     using	microarray	analysis	(total	of	46	slides).		The	different	compari-                                       69978,	Israel.
     sons	allowed	the	identification	of	genes	specifically	knocked	down	by	                  	Certain	breeds	of	domestic	ruminants	show	remarkable	resistance	
     tax-siRNA,	as	well	as	cell	line-specific	and	off-target	effects.	                 to	the	effects	of	African	trypano-somosis.	Unlike	susceptible	animals,	
           	     In	BL3*	cells	treated	with	tax-siRNA,	186	genes	were	found	           trypanotolerant	 animals	 control	 parasitemia	 and	 do	 not	 show	 severe	
     to	be	differentially	expressed	after	removal	of	off-target	effects	(t-test,	      anaemia	 or	 production	 loss.	 Identification	 of	 trypanotolerance	 genes	
     false	discovery	rate,	P-value<0.2).		The	genes	affected	by	tax	knock-             in	 cattle	 is	 hampered	 by	 cost	 and	 breeding	 time.	 Marked	 differences	
     down	were	mined	for	affected	pathways	and	functions	using	Ingenuity	              between	 inbred	 strains	 of	 mice	 in	 their	 response	 to	 T.	 congolense	
     Pathway	 Analysis.	 	 Among	 the	 canonical	 pathways	 significantly	             infection	 can	 be	 exploited	 in	 the	 analysis	 of	 the	 genetic	 basis	 of	 the	
     affected	by	the	BLV	tax	knock-down	were	ERK/MAPK	signaling,	oxi-                  infection.	Murine	trypanotolerance	QTLs	have	been	identified	on	chro-
     dative	 phosphorylation	 and	 glutathione	 metabolism.	 	Analysis	 of	 the	       mosome	 17,	 5	 and	 1,	 and	 designated	 as	 Tir1,	 2	 and	 3,	 respectively.	
     distribution	of	genes	according	to	Gene	Ontology	processes	revealed	              Tir1	 and	 2	 have	 been	 fine	 mapped	 to	 a	 confidence	 interval	 of	 1	 cM.	
     that	critical	pathways	in	cell	growth	(e.g.	cell	signaling,	cell	cycle	and	       In	order	to	find	the	mouse	homologous	region	on	the	bovine	genome,	
     cell	death/apoptosis)	are	affected	directly	or	indirectly	by	BLV	tax	and/         nucleotide	 sequence	 across	 95%	 CI	 of	 Tir2	 and	 3	 were	 used	 in	 the	
     or	other	virally-encoded	genes.		On	the	basis	of	these	results,	a	model	          selection	 of	 candidate	 genes.	 Homologous	 sequences	 were	 used	 in	
     for	 BLV-induced	 lymphoprolferation	 and	 transformation	 is	 proposed.	    	    the	definition	of	synteny	relationships	and	subsequent	identification	of	
     We	 postulate	 that	 persistent	 lymphocytosis	 in	 BLV-infected	 cattle	 is	     the	shared	disease	response	genes.	The	homologous	genes	within	the	
     caused	by	tax-induced	dysregulation	of	a	self-renewing	population	of	             human	genome	were	then	identified	and	aligned	to	the	bovine	radiation	
     pre-B	 lymphocytes.	 	 Transformation	 leading	 to	 lymphosarcoma	 is	 a	         hybrid	map	in	order	to	identify	the	mouse/bovine	homologous	regions.	
     rare	event	that	is	caused	by	secondary	mutations	in	the	infected	B	cell	          This	revealed	homology	between	murine	and	bovine	QTL	on	Tir3	while	
     precursor.	 	 Our	 results	 demonstrate	 the	 power	 of	 RNAi	 coupled	 with	     the	region	on	Tir2	is	linked	to	innate	immune	response.	
     microarray	 analysis	 for	 dissecting	 the	 genetic	 and	 cellular	 processes	    Key words:	Trypanosomosis,	Quantitative	trait	loci,	Homology,	single	
     leading	to	cell	transformation	by	retroviruses.                                   nucleotide	polymorphism
     Key words:	Bovine	Leukemia	Virus,	Tax	gene,	RNA	interference                      Species:	ruminants
     Species:	ruminants
                                                                                        ig013. ExPRESSION VARIATION OF TLR AS CANDIDATE
                                                                                        GENES UNDERLyING TRyPANOTOLERANCE QTL IN MICE
           ig011. MONOCyTES FROM DISEASE RESISTANT
            AND SUSCEPTIBLE CATTLE DISPLAy DISTINCT                                                     JoSEPH	nGAnGA1,2,FUAD	A.	IRAqI3
           TRANSCRIPTOME PROFILES DURING THEILERIA                                     	1International	Livestock	Research	Institute,	P.	O.	Box	30709,	Nairobi,	
                     ANNULATA INFECTION.                                               Kenya;	2Jomo	Kenyatta	University	of	Agriculture	and	Technology,	P.O.	
                                                                                          Box	62000,	Nairobi,	Kenya;	3Department	of	Human	Microbiology,	
           K	JEnSEn1,	R	TALBoT2,	D	WADDInGTon1,EJ	GLASS1
                                                                                        Sackler	Faculty	of	Medicine,	Tel	Aviv	University,	Ramat	Aviv,	Tel	Aviv	
     1Roslin	Institute,	Roslin,	Midlothian,	EH25	9PS,	UK;	2ARK-Genomics,	                                          69978,	Israel.
               Roslin	Institute,	Roslin,	Midlothian,	EH25	9PS,	UK
                                                                                             After	QTL	mapping	and	physical	representation	of	the	particular	
            The	 tick-borne	 protozoan	 parasite	 Theileria	 annulata	 causes	 a	      chromosomal	 fragment	 spanning	 trypanosomosis	 resistance	 loci	
                                                                                 	
     debilitating	and	often	fatal	disease	of	cattle	called	tropical	theileriosis.	     Tir2	 and	 3	 in	 mice,	 possible	 candidate	 genes	 were	 selected.	 These	
     The	disease	has	a	global	economic	impact	on	livestock	production	as	              appeared	 to	 be	 linked	 to	 the	 innate	 immune	 response.	 Plausible	 try-
     it	is	endemic	in	many	areas	of	the	world	from	the	Mediterranean	basin	            panotolerance	candidate	genes	within	the	loci	include	TLR1,	5	and	6.	
     to	China.		Control	strategies	have	so	far	failed	to	eradicate	T.	annulata	        TLRs	are	critical	in	the	regulation	of	pro-inflammatory	cytokine	secre-
     or	its	vector.		An	attractive	alternative	control	strategy	is	to	breed	for	       tions.	In	an	effort	to	find	an	association	between	the	genes	and	the	dis-
     combined	 resistance	 and	 productivity	 in	 cattle	 by	 using	 pre-existing	     ease,	expression	patterns	of	TLR	genes	mapping	to	trypanotolerance	
     genetic	resistance.		We	have	identified	a	Bos	indicus	breed	of	cattle	            QTL	were	investigated	using	quantitative	real	time	PCR.	Susceptible	
     that	originates	from	Pakistan,	the	Sahiwal,	which	is	resistant	to	tropical	       and	resistant	mice	infected	with	T.	congolense	portrayed	diverse	TLR	
     theileriosis.		Our	current	studies	aim	to	identify	the	genes	underlying	          expression	patterns.	Up	regulation	of	different	TLR	seemed	to	coincide	
     the	resistance	of	Sahiwal	cattle	to	T.	annulata.		                                with	up	regulation	of	IL-10	and	TNF	in	susceptible	and	resistant	strains	
           	     T.	annulata	principally	infects	bovine	macrophages	and	the	           respectively.	 T.	congolense	 infection	 therefore	 induces	 a	 response	
     pathology	of	the	disease	is	associated	with	the	intramacrophage	stage	            characterized	 by	 changes	 in	 TLR	 1,	 5	 and	 6	 expression	 in	 liver	 and	
     of	 the	 parasite.	 	Therefore	 we	 have	 focussed	 our	 studies	 on	 macro-      spleen	 tissues	 which	 appear	 to	 regulate	 cytokine	 profiles	 in	 mice.	
     phages	and	monocytes	derived	from	resistant	(Sahiwal)	and	susceptible	            These	phenomena	may	be	responsible	for	the	diverse	disease	pathol-
     (Holstein-Friesian,	B.	taurus)	breeds	of	cattle.		We	have	undertaken	a	           ogy	evident	in	different	mouse	strains	and	may	account	similar	trends	
     global	analysis	of	the	transcriptional	response	of	Sahiwal	and	Holstein-          observed	in	livestock	breeds.
     Friesian	 derived	 monocytes	 to	 T.	 annulata	 infection	 using	 a	 bovine	      Key words:	 Qtl,	 Expression	 Variation,	 Toll	 Like	 Receptors,	
     macrophage	 specific	 microarray	 developed	 in	 our	 laboratory.	 	 This	        Interleukins
     approach	has	identified	over	60	expressed	genes	that	exhibit	breed-               Specie:	other	(mice)
     specific	 differential	 expression	 either	 in	 resting	 monocytes	 or	 during	
     T.	annulata	infection,	which	may	be	related	to	T.	annulata	resistance.	       	
                                                                                            ig014. IMMUNOGLOBULIN ALLOTyPE-DEFINED
     Many	of	the	differentially	expressed	genes	are	cell-surface	expressed	
     proteins,	e.g.	prion	protein	and	ICAM1,	which	are	involved	in	the	inter-
                                                                                         PEDIGREED RABBITS AND GENES OF IMMUNOLOGICAL
     action	of	macrophages	with	other	immune	cells.		Further	analysis	of	the	               INTEREST IN 2x RABBIT GENOME ASSEMBLIES
     pathways	leading	to	variation	in	expression	of	these	molecules,	may	                                  (WORKSHOP)
     reveal	the	underlying	causal	genes	for	resistance	and	susceptibility	to	                                         RoSE	G	MAGE
     T.	annulata,	and	provide	new	approaches	for	disease	control.                        Molecular	Immunogenetics	Section,	Laboratory	of	Immunology,	
     Key words:	 macrophages,	             disease	    resistance,	    protozoan,	     NIAID,	NIH,	Bethesda,	MD	20892	USA		This	research	was	supported	
     transcriptome                                                                           by	the	Intramural	Research	Program	of	the	NIH,	NIAID.	
     Species:	ruminants                                                                                       rmage@niaid.nih.gov		
                                                                                                                                                                        57


      Although	 genomic	 sequence	 of	 the	 rabbit	 is	 currently	 “unfin-        like	all	marsupials	give	birth	to	relatively	immature	young	so	we	inves-
ished”	at	2x,	deeper	7x	coverage	is	expected	soon.	The	January	2006	              tigated	the	timing	of	TCR	expression	in	ontogeny.	We	found	that	some	
document	 “Increasing	 sequence	 coverage	 from	 2x	 to	 high	 coverage	          TCRs	 are	 expressed	 as	 early	 as	 postnatal	 day	 2,	 which	 appears	 to	
(6-7x)	for	selected	mammalian	species,”	that	recommended	rabbit	be	               precede	 thymus	 development.	 Within	 each	 TCR	 isotype	 there	 is	 dif-
sequenced	more	deeply,	describes	the	NIAID	allotype-defined	rabbits	              ferential	expression	of	V	subgroups	at	different	times	of	development,	
at	p.14.	These	animals	represent	a	valuable	resource	for	future	SNP	              consistent	 with	 changes	 in	 repertoire	 diversity	 during	 development.	
discovery.	 They	 have	 polymorphisms	 of	 a	 variety	 of	 immune	 system	        Overall,	the	content	and	genomic	organization	of	conventional	TCRs	
genes	including	variants	(allelic	allotypes)	of	the	VH,	CH,	and	CL	regions	       in	the	opossum	is	similar	to	what	is	known	for	placental	mammals	with	
of	antibody	molecules.		The	colony	also	contains	descendants	of	rab-              a	great	potential	to	express	diversified	antigen	receptors.	In	addition,	a	
bits	formerly	at	the	Basel	Institute	for	Immunology,	including	the	mutant	        fifth	TCR	could	represent	a	novel	specialized	subset	of	T	cells	present	
VH1a2-deleted	Alicia,	CK1	splicing	defective	Basilea,	and	several	VH-             in	marsupials.
CH	recombinant	heavy	chain	types.	These	rabbits	are	now	available	to	
interested	individuals,	particularly	to	sites	where	breeding	colonies	can	        Key words:	T	Cell,	TCR,	Opossum,	marsupial
be	established.	A	relational	database	(computer	program	4D)	contains	             Species:	others
more	than	45	years	of	breeding	records	and	other	information	about	
animals	in	the	colony.	The	whole	genome	shotgun	(WGS)	“unfinished	                  ig016. GENERATION OF PAIRED IMMUNE RECEPTORS
oryctolagus	 cuniculus	 database”	 of	 2,076,044,328	 letters	 in	 719,158	         By GENE CONVERSION WITHIN THE CANINE CEA GENE
sequences	 (ACCESSION	 	 	AAGW00000000).	 has	 serious	 gaps,	 yet	
                                                                                                       FAMILy.
the	 information	 has	 already	 proven	 useful	 for	 immunological	 as	 well	
as	in	silico	studies.	At	the	Broad	Institute,	the	DNA	of	the	“Thorbecke	             RoBERT	KAMMERER1,2,	TAnJA	PoPP1,	STEFAn	HäRTLE3,	
Inbred	 Rabbit”	 chosen	 for	 sequencing	 was	 found	 by	 preliminary	                 BERnHARD	B	SInGER4,WoLFGAnG	ZIMMERMAnn1
sequencing	 to	 have	 less	 heterozygosity	 than	 outbred	 NZW	 from	 the	        1Tumor	Immunology	Laboratory,	LIFE	Center,	Klinikum	Grosshadern,	
same	company	(Covance).	Ancestors	of	this	strain	accepted	skin	grafts	             LMU,	Munich,	Germany;	2Institute	for	Molecular	Immunology,	GSF	
after	20	generations	of	inbreeding.	When	I	typed	serum	samples	from	               National	Research	Center	for	the	Environment	and	Health,	Munich,	
such	animals	in	1995,	6/12	were	heterozygous	for	the	VH1a	types	a1	                Germany;	3Institute	for	Animal	Physiology,	LMU,	Munich,	Germany;	
and	a2	and	for	the	linked	C	gamma	hinge	region	d11/12	types.	Two	of	               4Institute	for	Anatomy,	University	Hospital	Essen,	Essen,	Germany
12	rabbits	were	also	heterozygous	for	CK1	allotypes	(b4/b5).	A	search	
for	 VH	 genes	 in	 the	 Trace	 Archive	 of	 oryctolagus	 cuniculus	 (WGS)	             Immune	cell	surface	receptors	sharing	highly	similar	extracellular	
finds	 perfect	 matches	 at	 	 http://www.ncbi.nlm.nih.gov/blast/mmtrace.         domains	and	having	counteracting	(activating	and	inhibitory)	signaling	
shtml,	to	previously	published	sequences	of	both	VH1a1	and	VH1a2.	                properties	are	called	“paired	immune	receptors”.	There	is	evidence	for	
Selective	maintenance	of	heterozygosity	reported	in	wild	rabbit	popula-           paired	immune	receptors	within	the	KIR	(Killer	cell	Ig-like	Rezeptors)	
tions,	 also	 occurred	 at	 the	 heavy	 chain	 locus	 during	 inbreeding.	The	    and	the	Ly49	receptor	families,	that	the	inhibitory	receptors	are	ances-
genomic	sequences	of	the	hinge	region	and	rabbit	CK	were	not	found	               tral	and	the	activating	receptors	evolved	from	the	inhibitory	receptors	
although	there	are	some	VK	sequences	in	the	trace	archive.	Links	at	              by	mutation	to	counteract	pathogen	attack.	This	view	is	supported	by	
the	 Rabbit	 Genome	 Resources	 site	 http://www.ncbi.nlm.nih.gov/proj-           the	 finding,	 that	 the	 murine	 cytomegalovirus	 (MCMV)-encoded	 m157	
ects/genome/guide/rabbit/	to	searches	for	genes	in	the	assemblies	of	             glycoprotein	 binds	 to	 the	 NK	 cell	 inhibitory	 receptor	 Ly49	 and	 sup-
the	2x	WGS	sequence	at	Ensembl	and	UCSC	(BLAT)	are	valuable	for	                  press	anti-viral	immune	response,	while	mice	expressing	the	activating	
discovering	 predicted	 mRNA	 sequences	 and	 exon	 boundaries	 useful	           receptor	Ly49H	are	resistant	to	MCMV	infection.	More	recently,	paired	
in	designing	primers	for	quantitative	reverse	transcriptase	PCR.	A	full	          immune	receptors	were	also	found	within	the	Siglec	family.	This	pair	
rabbit	genome	sequence	will	aid	discovery	of	genetic	contributions	to	            of	receptors	has	undergone	concerted	evolution	via	gene	conversion	
animal	and	human	disease	susceptibilities.                                        in	 multiple	 primate	 species,	 however	 the	 different	 directions	 of	 gene	
Key words:	Pedigreed	NIAID	Rabbits,	immunoglobulin	allotypes,	2x	to	              conversion	between	inhibitory	and	activation	receptors	argues	against	
7x	coverage,	genome	assemblies                                                    a	 pathogen-driven	 evolution.	 We	 have	 analyzed	 the	 evolution	 of	
Species: rabbit                                                                   paired	 immune	 receptors	 within	 the	 carcinoembryonic	 antigen	 (CEA)	
                                                                                  gene	family.	One	of	the	primordial	members	of	the	CEA	family	is	the	
                                                                                  ITIM-bearing	CEACAM1,	which	both	regulates	immune	responses	and	
  ig015. GENOMIC ORGANIZATION AND ExPRESSION OF                                   serves	as	a	cellular	receptor	for	pathogens,	in	human,	mice	and	cattle.	
   T CELL RECEPTORS (TCR) IN THE SOUTH AMERICAN                                   In	 the	 dog,	 the	 CEACAM1	 gene	 gave	 rise	 to	 a	 recent	 expansion	 of	
                    OPOSSUM                                                       the	CEA	family,	similar	to	that	previously	found	in	humans	and	mice.	
  ZULy	E	PARRA,	MICHELLE	L	BAKER,	JonATHAn	TRUJILLo,	                             However,	while	the	murine	and	human	CEACAM1-related	CEACAMs	
APRIL	LoPEZ,	ALAnA	SHARP,	JEnnIFER	HATHAWAy,RoBERT	D	                             are	 predominantly	 secreted	 and	 GPI-anchored,	 respectively,	 in	 the	
                        MILLER                                                    dog,	 most	 CEACAMs	 represent	 ITAM-bearing	 transmembrane	 pro-
 Department	of	Biology,	University	of	New	Mexico,	Albuquerque	NM	                 teins.	 The	 N-domain	 of	 one	 of	 these	 proteins,	 CEACAM28,	 exhibits	
                           87131	USA.                                             nearly	 complete	 sequence	 identity	 with	 the	 ligand-binding	 N-domain	
                                                                                  of	 CEACAM1	 but	 with	 antagonizing	 signaling	 motifs.	 Phylogenetic	
      T	cells	play	an	important	role	in	regulation	and	effector	functions	
in	the	adaptive	immune	response	of	all	jawed	vertebrates.	There	are	              analyses	of	the	canine	CEACAMs	suggested	that	the	high	sequence	
two	subsets	of	T	cells	according	to	their	antigen	receptors:	aβT	cells	           similarity	of	the	N-domains	is	due	to	a	partial	gene	conversion,	in	which	
and	γdT	cells.	Here	we	present	an	analysis	of	the	complete	genomic	               the	 inhibitory	 receptor	 CEACAM1	 converted	 the	 activating	 receptor	
content	and	organization	of	the	TCR	loci	in	a	model	marsupial	species.	           CEACAM28.	Comparison	of	nonsynonymous	and	synonymous	substi-
The	 South	 American	 opossum	 (Monodelphis	 domestica)	 is	 used	 as	            tutions	indicates	that	the	CEACAM28	N-domain	is	under	the	strongest	
model	for	both	UV	induced	melanoma	and	is	reservoir	for	the	parasite	             purifying	selection	of	all	canine	CEACAM1-related	CEACAMs.	In	addi-
that	causes	Chagas	disease	in	humans.	Better	understanding	of	opos-               tion,	CEACAM28	shows	a	similar	expression	pattern	in	resting	immune	
sum	T	cell	diversity	would	improve	their	utility	for	such	research.	We	           cells	 as	 CEACAM1.	 However,	 upon	 activation	 CEACAM28	 is	 down-
found	that	the	organization	and	complexity	of	the	opossum	TCRa	and	               regulated	in	T	cells	while	CEACAM1	is	upregulated.	Thus	CEACAM1	
d	 loci	 are	 similar	 to	 that	 of	 human,	 with	 TCRd	 nested	 within	 TCRa.	   and	CEACAM28	are	the	first	paired	immune	receptors	identified	within	
Similarly,	the	opossum	TCRβ	locus	resembles	that	of	humans,	but	with	             the	 CEA	 gene	 family,	 most	 likely	 involved	 in	 the	 fine	 tuning	 of	T	 cell	
two	 additional	 clusters	 of	 gene	 segments.	The	 opossum	TCRγ	 locus	          responses.	The	direction	of	gene	conversion	accompanied	by	a	purify-
has	a	translocon	organization,	different	from	that	of	human	where	it	is	          ing	selection	suggests	the	possibility	that	the	generation	of	CEACAM28	
clustered.	As	we	reported	recently,	opossums	have	a	fifth	TCR	(TCRµ)	             was	pathogen-driven.
which	does	not	have	a	homologue	in	placental	mammals.	TCRµ	occu-                  Key words:	 cell	 surface	 molecules,	 costimulation,	 comparative	
pies	a	distinct	locus	from	those	encoding	the	conventional	TCRs	and	it	
                                                                                  immunology,	carcinoembryonic	antigen
has	a	cluster	rather	than	the	usual	translocon	organization.	Opossums	
                                                                                  Species:	canine
58


         ig017. GENETIC ANALySIS OF PORCINE TLR GENES                                   class	I	genes	from	two	different	haplotypes.	The	two	SLA	haplotypes	
      InGRID-MARIA	BERGMAn1,	AMELIE	JoHAnSSon1,	CARoLInE	                               belonged	to	a	single	Landrace	individual	in	which	microsatellite-based	
        FoSSUM2,	LEIF	AnDERSSon3,	4,	InGER	EDFoRS-LILJA1                                genotyping	 suggested	 alteration	 of	 the	 SLA	 region	 in	 regard	 to	 the	
                                                                                        reference	H01	haplotype.	For	each	of	the	two	relevant	segments	we	
         1School	of	Pure	and	Applied	Natural	Sciences,	University	of	                   constructed	a	contig	of	bacterial	artificial	chromosome	(BAC)	using	a	
        Kalmar,	Kalmar,	Sweden,	2	Department	of	Biomedical	Sciences	                    library	made	with	genomic	DNA	from	the	selected	individual.	Southern	
      and	Veterinary	Public	Health,	and	3Department	of	Animal	Breeding	                 blot	 analysis	 of	 the	 genomic	 DNA	 and	 the	 BAC	 containing	 contigs	
      and	Genetics,	Swedish	University	of	Agricultural	Sciences,	Uppsala,	              indicated	the	relevance	of	contig	assembly.	BAC	sequences	revealed	
       Sweden,	4Department	of	Medical	Biochemistry	and	Microbiology,	                   the	increment	of	classical	class	I	genes	of	six	and	two	in	the	respec-
                    Uppsala	University,	Uppsala,	Sweden	                                tive	SLA	haplotypes.	RT-PCR	with	primers	to	amplify	specifically	each	
                        Ingrid-Maria.Bergman@hik.se                                     classical	class	I	locus	indicated	that	the	number	of	expressed	SLA-1	
           Quantitative	 trait	 loci	 (QTLs)	 influencing	 leukocyte	 numbers	 and	     or	 SLA-3	 genes	 were	 seven,	 at	 least,	 in	 the	 individual,	 whereas	 two	
     functions	and	other	immune	related	parameters	have	been	identified	                SLA-1	 or	 SLA-3	 genes	 were	 expressed	 in	 each	 chromosome	 of	 the	
     on	pig	chromosomes	8	(SSC8)	and	1	(SSC1).	TLRs	1,	2,	4,	6,	and	10	                 H01	haplotype.	The	process	of	the	duplication	of	SLA	classical	class	
     are	among	the	candidate	genes	mapped	to	these	regions.	TLR	poly-                   I	 genes	 were	 estimated	 using	 DNA	 transposon-like	 sequences,	 and	
     morphisms	 associated	 with	 susceptibility	 to	 infectious	 diseases	 have	       characteristic	microsatellite	repeats	adjacent	to	the	duplicated	class	I	
     been	found	in	man	and	pig1.	Several	of	these	polymorphisms	are	single	             genes.	 Furthermore,	 sequencing	 of	 the	 genomic	 region	 from	 DRA	 to	
     nucleotide	substitutions,	but	a	polymorphic	microsatellite	with	influence	         DOB	 in	 SLA	 class	 II	 region	 revealed	 the	 copy	 number	 variance	 also	
     on	in	vitro	response	has	also	been	found	upstream	the	human	TLR2	                  in	DRB	genes	between	haplotypes.	This	study	revealed	the	difference	
     gene2.	 The	 aim	 of	 this	 project	 is	 to	 explore	 genetic	 polymorphism	 in	   of	structure	among	SLA	haplotypes	both	in	the	class	I	and	II	regions,	
     porcine	 TLR	 genes	 and	 to	 relate	 these	 to	 functional	 studies	 in	 vitro	   and	implied	difficulty	of	estimation	of	SLA	haplotypes	in	commercial	pig	
     and	in	vivo.	Primers	were	designed	for	the	upstream	region	of	porcine	             breeds	by	genotyping	of	SLA	genes	per	se.	We	propose	an	alternative	
     TLR2	and	a	microsatellite	(HIK10)	was	identified	approximately	100	bp	             method	of	estimation	of	haplotypes	using	microsatellite	markers	distrib-
     upstream	translation	start.	Two	sets	of	pigs	were	used	to	establish	a	             uted	throughout	the	entire	SLA	region,	and	demonstrate	an	example	
     genetic	linkage	map	including	TLRs	1,	2,	6	and	10:	47	backcross	(BC2)	             of	 the	 significant	 association	 between	 antibody	 responses	 after	 vac-
     offspring	 of	 a	 BC1	 boar,	 originating	 from	 a	 Wild	 Boar	 (W)	 x	 Swedish	   cination	against	opportunistic	infections	and	particular	SLA	haplotypes	
     Yorkshire	 (Y)	 pedigree,	 and	 191	 Swedish	 Landrace	 (L)	 x	 Y	 crosses	        reconstructed	by	microsatellite	markers	in	pigs.
     after	 six	 L	 boars.	Also,	 to	 compare	 TLR	 polymorphism	 amongst	 dif-         Key words:	MHC,	SLA,	genomics,	copy	number	variation
     ferent	 breeds,	 additional	 tissue	 samples	 have	 been	 collected.	 The	         Species:	swine
     families	 were	 genotyped	 for	 the	 KIT	 gene	 and	 14	 microsatellites	 on	
     SSC8.	A	 linkage	 map	 was	 constructed	 using	 the	 CRI-MAP	 software.	             ig019. COMPARATIVE TRANSCRIPTOMIC ANALySIS OF
     Based	on	recombination	between	markers,	the	relative	order	Sw1101	                          PRV-HOST CELL INTERACTIONS IN PIG
     –	Sw1037	–	SJA7,	SJA10,	SJA11	(TLR1,	6,	10)	–	Sw444	–	HIK2,	HIK3,	
     HIK4	–	KIT	–	S0086	–	Sw1679	–	SJ108	(IL8)	–	S0069	–	S0225	–	HIK10	                       FLoRI	LAUREnCE1,	MARIAnI	VALEnTInA2,	CHARDon	
     was	determined.	Several	non-synonymous	SNPs	in	TLR1,	2,	4,	5	and	                            PATRICK1,	LEMonnIER	GAéTAn1,	LEFEVRE	
     6	have	been	found	in	a	study	comprising	11	pig	breeds3.	These	as	well	                         FRAnçoIS3,RoGEL-GAILLARD	CLAIRE1
     as	 the	 polymorphic	 microsatellites	 in	 pig	 IL8,	 within	 the	 TLR1-TLR6-         1INRA	CEA,	Laboratoire	de	Radiobiologie	et	Etude	du	Génome,	
     TLR10	 cluster,	 and	 upstream	TLR2	 will	 enable	 further	 studies	 of	 the	         Jouy-en-Josas,	France;	2Parco	Tecnologico	Padano,	Lodi,	Italy;	
     influence	of	these	candidate	genes	on	immune	related	traits.                        3INRA,	Laboratoire	de	Virologie	et	Immunologie	Moléculaires,	Jouy-
          References	                                                                                            en-Josas,	France
         1.	 Muneta	 et	 al.	 2003.	 J	 Interferon	 Cytokine	 Research	 23:	                   The	pseudorabies	(PrV)	is	the	etiologic	agent	of	the	Aujezsky	dis-
     583-590                                                                            ease.	The	disease	was	eradicated	thanks	to	vaccination	but	the	PrV	is	
                                                                                        still	an	excellent	model	to	set	up	in	vitro	systems	to	study	interactions	
         2.	Yim	et	al.	2004.	FEMS	Immunology	and	Medical	Microbiology	                  between	 Herpesviruses	 and	 host	 cells	 due	 to	 efficient	 infection	 and	
     40:163-169                                                                         propagation	 in	 cell	 cultures.	 In	 vivo	 the	 first	 target	 cells	 are	 mucous	
          3.	Shinkai	et	al.	2006.	Immunogenetics	58:324-330                             epithelial	cells	and	immature	dendritic	cells	(iDCs)	that	further	activate	
     Key words:	TLR,	Gene,	Polymorphism,	Innate	immunity                                adaptive	 immune	 response.	 The	 dialog	 between	 PrV	 and	 these	 two	
     Species:	swine                                                                     cell	types	are	expected	to	differ.	The	aim	of	our	work	was	to	analyze	
                                                                                        these	 differences	 during	 time	 course	 of	 infection	 and	 we	 performed	
       ig018. DIFFERENCE OF GENOMIC STRUCTURE AMONG                                     a	 transcriptome	 approach	 that	 allowed	 us	 to	 simultaneously	 analyze	
       HAPLOTyPES OF SWINE LEUKOCyTE ANTIGEN REGION                                     cellular	 and	 viral	 gene	 expression.	 Epithelial	 PK-15	 cells	 and	 swine	
                                                                                        iDCs	that	were	in	vitro	differentiated	from	blood	monocytes	using	IL-4	
      HIRoHIDE	UEnISHI1,3,	MAIKo	TAnAKA2,3,	TAKASHI	AWATA1,3,	
                                                                                        and	GM-CSF	were	infected	by	the	PrV.	Infected	or	mock-infected	cells	
                 ASAKo	AnDo4,PATRICK	CHARDon5
                                                                                        were	collected	0,	1,	2,	4,	8	and	12	post	infection	(pi)	for	PK-15	or	0,	
        1National	Institute	of	Agrobiological	Sciences	(NIAS),	Tsukuba,	                12,	 18	 and	 24	 hours	 pi	 for	 iDCs.	Total	 RNA	 transcripts	 were	 labeled	
          Ibaraki,	Japan;	2Institute	of	Society	for	Techno-Innovation	of	               and	hybridized	onto	DNA	chips	comprising	80	PrV	amplicons	covering	
        Agriculture,	Forestry	and	Fisheries	(STAFF-Institute),	Tsukuba,	                the	 whole	 viral	 genome	 and	 a	 set	 of	 1663	 cellular	 genes,	 including	
      Ibaraki,	Japan;	3Animal	Genome	Research	Program,	NIAS/STAFF,	                     420	 genes	 mapping	 to	 the	 extended	 major	 histocompatibility	 (MHC)	
         Tsukuba,	Ibaraki	Japan;	4Tokai	University	School	of	Medicine,	                 locus,	 73	 immune	 genes	 outside	 the	 MHC	 and	 1170	 randomly	 cho-
          Isehara,	Kanagawa,	Japan;	5Institut	National	de	Recherche	                    sen	 genes.	 In	 PK-15	 cells,	 a	 high	 increase	 in	 viral	 gene	 expression	
     Agronomique	(INRA),	Commisariat	a	l’Energie	Atomique	(CEA),	Jouy	                  was	found	4	hours	pi	and	most	viral	genes	were	detected	differentially	
                                 en	Josas,	France		                                     expressed	12	h	pi	in	both	cell	types.	No	early	global	cellular	gene	shut	
                               huenishi@affrc.go.jp                                     off	occurred	and	the	highest	number	of	differentially	expressed	cellular	
          Swine	 major	 histocompatibility	 complex	 (MHC),	 also	 designated	          gene	was	observed	8h	pi	in	PK-15	cells.	The	results	showed	that	MHC	
     as	 swine	 leukocyte	 antigen	 (SLA)	 region,	 is	 thought	 to	 have	 strong	      class	I	genes	were	down-regulated	in	both	infected	cell	types	and	that	
     association	 with	 traits	 related	 to	 disease	 resistance.	 Elucidation	 of	     MHC	class	II	genes	were	also	down-regulated	in	iDCs.	Genes	that	are	
     association	between	alleles	of	SLA	genes	and	the	traits	requires	pre-              involved	in	other	pathways	such	as	apoptosis,	protein	metabolism	and	
     cise	 knowledge	 of	 the	 genomic	 structure	 of	 this	 highly	 polymorphic	       modification	were	also	identified	as	differentially	expressed.	Real	time	
     region,	 and	 an	 efficient	 genotyping	 method	 for	 the	 SLA	 genes.	 The	       quantitative	PCR	experiments	confirmed	the	down-regulation	of	MHC	
     entire	 genomic	 region	 of	 SLA	 was	 recently	 elucidated	 in	 a	 particular	    class	Ia	genes,	as	well	as	TAP1,	TAP2,	LMP2	and	LMP7	all	involved	in	
     haplotype	named	H01.	However,	it	has	been	suggested	that	the	num-                  class	I	antigen	presentation	pathway,	the	down-regulation	of	cyclophilin	
     ber	of	SLA	loci	varies	according	to	the	SLA	haplotypes.	To	clarify	such	           A	and	the	up-regulation	of	TNFA	in	infected	PK-15	cells.	Validation	of	
     variance,	we	sequenced	the	genomic	segment	encoding	SLA	classical	                 the	down-regulation	of	class	II	antigen	presentation	pathway	in	infected	
                                                                                                                                                                        59


iDCs	 is	 in	 progress.	The	 present	 comparative	 study	 will	 provide	 new	       swine	industry	today,	costing	U.S.	pork	producers	at	least	$560	million	
data	on	time-dependant	differences	according	to	the	host	cell	during	               annually.	 Despite	 substantial	 research	 efforts	 the	 exact	 components	
PrV	infection.	                                                                     of	a	protective	anti-PRRSV	immune	response	are	still	not	known,	thus	
Key words:	PrV,	transcriptome,	MHC,	dendritic	cells                                 we	are	testing	alternate	approaches	to	evaluate	immunity	and	genetic	
Species:	swine                                                                      resistance	 to	 PRRSV.	 We	 used	 host	 genomics	 to	 compare	 different	
                                                                                    lines	 of	 pigs	 and	 look	 for	 factors	 that	 correlated	 with	 PRRSV	 resis-
ig020. GENOMIC ANALySIS REVEALED THE DUPLICATION                                    tance/susceptibility.	 Viremia,	 weight	 change,	 and	 rectal	 temperature	
  MODEL OF PORCINE CD1 GENES DURING EVOLUTION                                       at	 0,	 4,	 7,	 and	 14	 days	 post-PRRSV	 infection	 (dpi)	 were	 recorded	
                                                                                    and	 genetic	 differences	 detected	 (Petry	 et	 al.,	 2005).	 We	 evaluated	
 ToMoKo	EGUCHI-oGAWA1,	2,	TAKEyA	MoRoZUMI1,	3,	MAIKo	
                                                                                    immune	gene	expression	in	RNA	from	frozen	lung	and	bronchial	lymph	
  TAnAKA1,	3,	HIRoKI	SHInKAI,1,	3	nAoHIKo	oKUMURA1,	3,	
                                                                                    node	 (BLN)	 tissue	 of	 the	 7	 highest	 and	 lowest	 responders	 per	 line,	
 KoHEI	SUZUKI1,	3,	TAKASHI	AWATA1,	2,HIRoHIDE	UEnISHI1,	2
                                                                                    and	 from	 each	 of	 their	 control	 littermates,	 as	 well	 as	 serum	 cytokine	
 1Animal	Genome	Research	Program;	2Division	of	Animal	Sciences,	                    protein	 levels.	 Genetic	 analyses	 of	 this	 data	 indicated	 that	 levels	 of	
    National	Institute	of	Agrobiological	Sciences,	2-1-2	Kannondai,	                interleukin-8	 (IL8)	 may	 be	 predictive	 of	 resistance.	 Additionally,	 low	
   Tsukuba,	Ibaraki	305-8602,	Japan,	3Second	Research	Division,	                    (not	the	expected	high)	levels	of	serum	interferon-gamma	(IFNG)	after	
STAFF-Institute,	446-1	Ippaizuka,	Kamiyokoba,	Tsukuba,	Ibaraki	305-                 infection	may	be	associated	with	a	PRRSV	resistant	phenotype.	These	
                              0854,	Japan	                                          data	are	critical	for	genetic	association	studies	to	fine	map	candidate	
               egutomo@affrc.go.jp	(T.	Eguchi-Ogawa)                                genes	and	determine	causative	alleles	of	PRRSV	resistance/suscepti-
      CD1	 is	 an	 MHC	 class	 I-like	 protein	 that	 presents	 lipid	 antigens	    bility.	Further	genetic	studies	are	required	to	affirm	these	associations.	
to	 T	 cell	 receptors.	 To	 clarify	 the	 variety	 and	 genomic	 structure	 of	    For	direct	immunity	studies	we’ve	assessed	immune	gene	expression	
porcine	 CD1	 gene,	 we	 constructed	 a	 bacterial	 artificial	 chromosome	         in	lung,	BLN,	and	tonsil	samples,	and	protein	expression	in	serum,	col-
(BAC)	contig,	and	determined	470,187	bp	of	the	region	encoding	the	                 lected	from	pigs	infected	for	over	200	days	after	PRRSV	infection.	We	
CD1	genes.	We	identified	16	genes	in	this	region	and	newly	identified	              compared	pigs	that	apparently	cleared	the	viral	infection	in	the	first	28	
CD1A2,	CD1B,	CD1C,	CD1D,	and	CD1E	in	addition	to	formerly	identi-                   dpi	to	pigs	that	even	at	150	dpi	have	evidence	of	long	term	persistent	
fied	 porcine	 CD1	 gene,	 CD1.1	 (CD1A1,	 homolog	 of	 human	 CD1A).	              PRRSV	infection.	Results	show	that	there	is	up	regulation	of	expression	
RT-PCR	 analysis	 showed	 CD1A1,	 CD1B,	 CD1D	 and	 CD1E	 were	                     of	IFNG	associated	T	helper	1	(Th1)	markers	from	14	to	84	dpi;	regula-
expressed,	and	suggested	both	group	1	(CD1A1,	CD1B,	CD1E)	and	                      tory	IL10	and	apoptosis	associated	markers	are	also	increased	early.	
group	2	(CD1D)	CD1	genes	are	functioned	in	pig.	Tyrosine-containing	                To	 date,	 however,	 no	 significant	 differences	 between	 persistent	 and	
motif	involved	in	CD1	intracellular	trafficking	was	conserved	in	the	C	             non-persistent	PRRSV	infected	pigs	have	been	discovered	in	immune	
termini	of	porcine	CD1B	and	CD1D.	The	C	terminus	of	porcine	CD1b	                   gene	expression;	serum	protein	expression	studies	are	underway.	We	
shared	a	similar	motif	with	human	CD1d,	and	porcine	CD1d	had	similar	               hope	to	reveal	differential	protein	expression	associated	with	PRRSV	
motif	with	that	of	human	CD1b;	therefore,	porcine	CD1d	may	comple-                  clearance.	Overall,	by	combining	these	diverse	approaches,	we	expect	
ment	the	function	of	human	CD1b	in	pigs.	Southern	blot	hybridization	               to	 develop	 new	 hypotheses	 about	 protective	 anti-PRRSV	 responses	
with	 several	 breeds	 of	 pig	 genome	 was	 performed	 using	 the	 exon	 4	        and	to	identify	novel	regulatory	pathways	that	would	stimulate	PRRSV	
sequence	 encoding	 the	 C-like-domain	 [D3]	 of	 CD1.1	 as	 a	 probe.	             immunity.	Supported	by	USDA	ARS	and	NRI	PRRS	CAP1	funds.
Although	slight	difference	in	restriction	sites	was	observed	among	the	             Key words:	 Porcine	 Reproductive	 and	 Respiratory	 Syn,	 resistance/
breeds,	 we	 confirmed	 that	 no	 more	 than	 six	 CD1	 genes	 existed	 on	
                                                                                    susceptibility,	immune	gene	expression,	cytokine	regulation.
the	porcine	genome.	Genomic	sequence	analysis	showed	that	porcine	
                                                                                    Species:	swine
CD1	 genes	 were	 located	 in	 clusters	 between	 KIRREL	 and	 olfactory	
receptor	 (OR)	 genes,	 as	 observed	 in	 humans,	 although	 they	 were	
divided	into	two	regions	by	a	region	encoding	OR	genes.	Comparison	                     ig022. IDENTIFICATION OF BOOPHILUS MICROPLUS
of	the	genomic	structure	encoding	CD1	genes	in	pigs	with	other	mam-                      PHAGOTOPES FROM PHAGE DISPLAyED PEPTIDE
mals	 showed	 that	 separation	 of	 the	 CD1	 gene	 cluster	 by	 ORs	 was	                                 LIBRARIES.
observed	 only	 in	 pigs.	 To	 investigate	 the	 process	 of	 evolution	 of	 the	
                                                                                    CARLoS	RoBERTo	PRUDEnCIo1,	ALInE	APARECIDA	REZEnDE	
region	where	the	porcine	CD1	genes	are	located,	we	identified	charac-
                                                                                      RoDRIGUES,	GUILHERME	RoCHA	LIno	SoUZA1,	JULIAnA	
teristic	repetitive	sequences	commonly	found	close	to	the	human	and	
                                                                                       FRAnCo	ALMEIDA1,	AnA	PAULA	PERES	FRESCHI,	RonE	
porcine	 CD1A	 genes.	 By	 estimation	 of	 the	 time	 of	 CD1A	 duplication	
                                                                                     CARDoSo1,	FAUSTo	EMÍLLIo	CAPPARELLI1,	LUIZ	RICARDo	
by	 using	 the	 repetitive	 sequences,	 we	 conclude	 CD1A	 duplication	 in	
                                                                                                         GoULART1	
the	 porcine	 genome	 might	 occur	 after	 the	 divergence	 of	 the	 human	
and	porcine.	We	constructed	the	schematic	model	of	porcine	CD1	and	                     1Instituto	de	Genética	e	Bioquímica	-	Universidade	Federal	de	
OR	gene	duplication,	which	indicated	that	the	unique	split	structure	of	                               Uberlândia	-	MG;	2	-	Vallée	S/A.	
the	CD1	cluster	in	the	pig	had	been	established	before	the	shuffling	of	                                   crprudencio@gmail.com
the	OR	genes	in	the	artiodactyl	lineage.	This	analysis	of	the	genomic	                    The	 ticks	 cause	 serious	 economic	 losses	 to	 animal	 production	
sequence	of	the	porcine	CD1	family	will	contribute	to	our	understand-               worldwide,	in	the	order	of	billions	of	dollars.	Phage	display	techniques	
ing	of	the	evolution	of	mammalian	CD1	genes.                                        have	been	widely	employed	to	map	the	epitope	structures	which	have	
Key words:	CD1                                                                      served	 as	 the	 basis	 for	 developing	 molecular	 vaccines.	 In	 the	 pres-
Species:	swine                                                                      ent	study,	we	applied	this	technique	to	map	the	epitopes	of	Boophilus	
                                                                                    microplus	 and	 directly	 evaluated	 the	 immune	 responses	 in	 mice	
 ig021. SWINE IMMUNITy AND GENETIC RESISTANCE TO                                    to	 verify	 immunogenicity	 of	 the	 selected	 phage-displayed	 epitopes	
PORCINE REPRODUCTIVE AND RESPIRATORy SyNDROME                                       (phagotopes).	 Seven	 phage-displayed	 random	 peptide	 libraries	 were	
              VIRUS (PRRSV) INFECTION                                               biopanned	in	different	situations	of	stringency	with	the	purified	IgY	of	
                                                                                    chiken	anti-B.	microplus	hyperimmune	serum	and	the	selected	phage	
   JoAn	K	LUnnEy1,	DEREK	PETRy2,3,	RoDGER	JoHnSon2,	
                                                                                    clones	 were	 sequenced	 and	 analyzed.	 Some	 of	 the	 inserts	 of	 the	
    DAnIEL	KUHAR1,	RAMon	MoLInA4,	JAnE	CHRISToPHER-
                                                                                    selected	 phagotopes	 showed	 a	 good	 match	 with	 the	 known	 proteins	
     HEnnInGS5,	JEFFREy	ZIMMERMAn4,,RRR	RoWLAnD6
                                                                                    of	B.	microplus.	Others,	which	did	not	match	with	any	known	proteins,	
 1APDL,	BARC,	USDA,	Beltsville,	MD;	2Univ.	of	Nebraska,	Lincoln,	                   but	 shared	 extensive	 homology	 with	 each	 other,	 were	 clustered	 and	
   NE;	3Triumph	Foods;	4Iowa	State	University,	Ames	IA;	5South	                     classified	as	the	conformational	epitopes	of	B.	microplus.	To	evaluate	
 Dakota	State	University;	6Kansas	State	University,	Manhattan,	KS.		                the	potential	of	using	these	phagotopes	as	effective	vaccines,	several	
                   jlunney@anri.barc.usda.gov                                       phage	clones	were	chosen	to	immunize	mice.	The	serum	raised	by	the	
      Current	 vaccines	 are	 only	 partially	 effective	 against	 Porcine	         phage	clones	clearly	recognized	tick	proteins	indicating	that	the	phago-
Reproductive	 and	 Respiratory	 Syndrome	 (PRRS)	 virus	 infection	                 tope-induced	 immune	 responses	 were	 antigen-specific.	 The	 present	
because	they	elicit	a	weak	immune	response	that	is	not	fully	protec-                work	 demonstrates	 that	 the	 whole	 epitope	 profile	 can	 be	 obtained	
tive.	 PRRS	 is	 the	 most	 economically	 significant	 disease	 facing	 the	        through	screening	the	phage	displayed	peptide	libraries	with	the	hyper-
60


     immune	 serum	 and	 reveals	 the	 potential	 of	 using	 epitope-displaying	          play	low	reproductive	efficiency.	In	order	for	ticks	to	efficiently	infest	
     phages	as	peptide	vaccines.                                                          their	 hosts,	 they	 must	 produce	 cement-like	 proteins	 that	 ensure	
          Finacial	support:	Finep,	Vallée	S/A,	Cappes.                                    attachment	and	blood-feeding.	Our	hypothesis	is	that	different	lev-
                                                                                          els	of	host	anti-tick	immunity	affect	gene	expression	in	cattle	ticks.	
     Key words:	epitope	profile,	B.	microplus,	Phage	Display,	Vaccine.
                                                                                          The	objective	is	to	evaluate	if	and	how	the	expression	of	cement-like	
     Species:	other
                                                                                          proteins	 is	 affected	 by	 immune	 responses	 of	 tick-susceptible	 	 and	
                                                                                          resistant	bovine	hosts.	
        ig023. CONSTRUCTION OF AN ANTIBODIES LIBRARy
                                                                                                Methods	 and	 Results:	 cDNA	 libraries	 were	 constructed	 with	
        (SCFV) FOR SELECTION AND CHARACTERIZATION OF
                                                                                          SMART	 (Clontech-BD)	 technology	 and	 mRNA	 from	 salivary	 gland	
                BOOPHILUS MICROPLUS ANTIGENS                                              of	 nymphs,	 male	 and	 female	 ticks	 fed	 on	 susceptible	 or	 resistant.	
       GUILHERME	RoCHA	LIno	DE	SoUZA1,	CARLoS	RoBERTo	                                    Clones	were	randomly	selected	for	PCR	amplification	and	the	DNA	
     PRUDEnCIo1,	RonE	CARDoSo1,	JULIAnA	FRAnCo	ALMEIDA1,	                                 inserts	were	sequenced	and	analyzed	with	bioinformatic	tools	that	
     FAUSTo	EMÍLLIo	CAPPARELLI1,	AnA	PAULA	PEREZ	FRESCHI1,	                               trim	 ESTs	 of	 primer	 and	 vector	 sequences,	 clusterize	 them	 into	
        AnDRéA	qUEIRoZ	MARAnHÃo2,	MARCELo	DE	MACEDo	                                      contigs	 and	 confront	 them	 against	 the	 NCBI	 non-redundant	 (NR)	
     BRÍGIDo2,	LIZIAnE	MARIA	DE	LIMA3,	MARCELo	BEMqUERER4,	                               protein	database	and	a	private	one	containing	sequences	for	Acari.	
                      LUIZ	RICARDo	GoULART1                                               Developmental	stages	obtained	from	susceptible	hosts	(RmS)	gen-
          1Instituto	de	Genética	e	Bioquímica	-	Universidade	Federal	                     erated	4418	ESTs,	while	those	from	resistant	hosts	(RmR)	generated	
         de	Uberlândia-	MG;	2Departamento	de	Imunologia	molecular-                        2875	 ESTs.	The	 ESTs	 combined	 from	 all	 developmental	 stages	 of	
     Universidade	de	Brasília	(UnB)-	DF;	3Empresa	Brasileira	de	Pesquisa	                 RmS	presented	618	ESTs	(expected	574)	similar	to	cement	proteins,	
      Agropecuária,	Centro	Nacional	de	Pesquisa	de	Algodão,	Campina	                      while	ESTs	from	RmR	exhibited	numbered	only	322	(expected	365;	
       Grande-PB;	4Instituto	de	Ciências	Biológicas,	Departamento	de	                     P=0.003,	χ2	test).	Males	were	the	developmental	stage	presenting	
       Bioquímica	e	Imunologia	Universidade	Federal	de	Minas	Gerais	                      the	highest	expression	of	ESTs	similar	to	cement-like	proteins	(596),	
                                  (UFMG)-	MG	                                             but	 there	 were	 no	 significant	 differences	 between	 RmS	 and	 RmR	
                               grlino@gmail.com                                           males.	However,	RmS	females	and	nymphs	contain	more	ESTs	sim-
                                                                                          ilar	to	cement	proteins	than	RmN,	there	being	54	ESTs	from	female	
          The	Boophilus	microplus	tick	is	one	of	the	most	important	arthro-
                                                                                          RmS	 (expected	 36)	 and	 14	 ESTs	 from	 female	 RmR	 (expected	 30;	
     pods	that	can	parasitize	bovines,	causing	great	damages	to	the	world	
                                                                                          P<0.001),	279	ESTs	from	nymphs	RmS	(expected	162)	and	29	ESTs	
     livestock	through	direct	and	indirect	effects.	The	application	of	chemi-
                                                                                          from	nymphs	RmR	(expected	114;	P<0.001).
     cal	products	is	the	principal	method	of	controlling	this	parasite,	but	in	
     function	of	the	disadvantages	of	this	practice,	the	use	of	vaccines	is	a	                  Conclusions:	Our	data	indicate	that	the	host	immune	response	of	
     good	alternative,	because	they	are	residue	free,	specific	and	present	               tick-resistant	 cattle	 negatively	 affects	 expression	 of	 genes	 coding	 for	
     lower	possibility	of	developing	resistance.	With	the	objective	of	select-            cement-like	proteins	in	R.	microplus	and	that	this	can	be	preventing	the	
     ing	and	characterizing	new	proteic	targets	as	vaccine	against	ticks,	we	             attachment	and	feeding	of	ticks	on	these	hosts.
     developed	 a	 combinatorial	 antibody	 library	 (scFv),	 expressed	 in	 the	              Financial	Support:	CNPq,	FAPESP	and	Vallée	SA.	
     capsid	 of	 bacteriophages,	 produced	 from	 chickens	 immunoglobulins	
                                                                                          Key words:	Rhipicephalus	microplus,	transcriptome,	salivary	glands,	
     diversity	 previously	 sensitized	 with	 total	 larval	 and	 adults	 proteins	 of	
                                                                                          cement-like	proteins,	vaccine.
     the	Boophilus	microplus.	These	antibodies	(scFv)	recognize	a	protein	
                                                                                          Species:	others
     of	the	total	larval	and	adult’s	extract	of	the	proximally	80	kDa,	by	west-
     ern	 blotting	 tests,	 and	 the	 sequence	 of	 these	 reactive	 proteins	 was	
     checked	by	N-terminal	sequencing.	The	band	corresponded	to	a	GP80	                       ig025. CONSERVATION OF PEPTIDES 4822 AND 4823
     protein	with	high	similarity	(91%)	and	shown	huge	quantities	in	parasite	                 CONSTITUENTS OF SyNTHETIC VACCINE SBM7462
     eggs	of	adults	and	larval	stages.	With	the	objective	of	selecting	mimo-                  AGAINST RHIPICEPHALUS (BOOPHILUS) MICRoPLUS
     topes	 of	 B.	 microplus	 by	 phage	 displayed	 epitope	 characterization,	                AnA	PAULA	PEConICK,	FLáVIA	ARAúJo	GIRÃo,	
     the	recombinant	antibodies	with	major	frequency	were	submitted	to	a	                       SIDIMAR	SoSSAI,	MARInA	qUADRIo	RAPoUSo	
     selection	against	a	constricted	peptide	library	displayed	on	phages.	The	              BRAnCo	RoDRIGUES,	BREno	SoUZA	SALGADo,	CARLoS	
     cross	reactivity	was	confirmed	by	the	recognition	of	a	recombinant	anti-                 HEnRyqUE	SoUZA	E	SILVA,	CARLA	LEITE	MEDEIRoS,	
     body	(scFv)	to	a	clone	expressing	the	motif	similar	to	GP80	sequence	                HUGo	GUIEIRo	RIBEIRo	RoCHA,	KARLoS	HEnRIqUE	M	KALKS,	
     obtained	from	peptide	library	selection.		This	work	confirms	the	GP80	                            JoAqUÍn	HERnAn	PATARRoyo
     as	a	vaccine	candidate	and	represents	the	selection	efficiency	of	new	
                                                                                              Laboratory	of	Biology	and	Control	of	Haematozoa	and	Vectors,	
     vaccine	targets	by	phage	displayed	libraries	methodologies	to	control	
                                                                                           Institute	of	Biotechnology	Applied	to	Agriculture	and	Animal	Science	
     B.	microplus.
                                                                                            (BIOAGRO/Veterinary	Departament),	Federal	University	of	Viçosa,	
          Financial	support:	CNPq                                                                              36571-000,	Viçosa,	MG,	Brazil.	
     Key words:	 Phage	 display,	 Antibody	 libraries,	 Peptide	 libraries,	                                          jpatarro@ufv.br
     Boophilus	microplus.	                                                                      Rhipicephalus	(Boophilus)	microplus	is	one	of	the	most	important	
     Species:	other                                                                       parasite	of	cattle	in	Central	and	South	America	and	Australia	from	an	
                                                                                          economical	 point	 of	 view.	 Vaccines	 derived	 from	 Bm86	 glycoprotein	
        ig024. ExPRESSION OF CEMENT-LIKE PROTEINS IN                                      have	a	great	potential	of	non-chemical	control	of	ticks.	The	SBm7462	
      TRANSCRIPTOMES FROM TICKS FED ON RESISTANT AND                                      is	a	synthetic	vaccine	derived	from	Bm86	and	has	three	immunogenic	
                    SUSCEPTIBLE CATTLE.                                                   epitopes:	 4822	 (a.a.	 398-411),	 4824	 (a.a.	 123-145)	 and	 4823	 (a.a.	
                                                                                          21-35).	 The	 knowledge	 about	 the	 conservation	 of	 the	 bm86	 gene	 is	
        MARUyAMA	SRC1,	GARCIA	GR1,	BRAnDÃo	LG1,	RIBEIRo	
                                                                                          very	important	to	evaluate	efficiency	of	SBm7462.	Twenty	six	R.	(B.)	
        JMC2,	AnDERSon	JM2,	VALEnZUELA	JG2,	FERREIRA	BR1,	
                                                                                          microplus	 strains	 from	 Argentina,	 Colombia,	 Uruguay	 and	 various	
                    DE	MIRAnDA	SAnToS	IKF1	
                                                                                          regions	 from	 Brazil	 were	 analyzed	 for	 the	 bm86.	 Two	 fragments	 of	
       1Departamento	de	Bioquímica	e	Imunologia,	FMRP-USP,	Ribeirão	                      cDNA	 were	 amplified,	 fragment	A	 (among	 the	 nucleotides	 39	 –	 438)	
               Preto,	Brasil;	2NIAID-NIH,	Rockville-MD,	USA.                              and	fragment	C	(among	the	nucleotides	839	-	1600).	They	were	cloned	
           Introduction	and	Objectives:	Rhipicephalus	(Boophilus)	micro-                  into	 the	 pGEM-T®	 vector	 and	 four	 clones	 were	 sequenced	 for	 each	
     plus,	the	cattle	tick,	causes	enormous	losses	for	animal	production	                 population.	The	nucleotides	and	deduced	amino	acid	sequences	were	
     and	health.	Ticks	induce	immune	responses	in	their	hosts,	indicat-                   compared	 with	 the	 bm86	 and	 bm95	 genes.	 The	 analysis	 was	 made	
     ing	 that	 their	 immunobiological	 control	 is	 possible.	 Bovines	 pres-           through	alignment	of	multiple	sequences	by	the	program	BioEdit	ver-
     ent	different	phenotypes	related	to	intensity	of	tick	infestations	and	              sion	 7.0.5.3	 and	 the	 polymorphisms	 verification	 for	 visual	 inspection.	
     those	 phenotypes	 are	 mediated	 by	 qualitatively	 distinct	 immune	               The	 results	 demonstrated	 the	 genetic	 conservations	 of	 the	 peptides	
     responses.	Ticks	fed	on	resistant	bovines	do	not	feed	well	and	dis-                  4823	 and	 4822	 for	 analysed	 samples.	 Inside	 of	 the	 gene	 bm86,	 the	
                                                                                                                                                                    61


amino	acids	variability	was	of	5,49%	and	3,89%	compared	with	Bm86	                  library	 in	 lambda	 phages	 with	 the	 SMART	 cDNA	 kit	 (Clontech).	 The	
and	Bm95,	respectively.	                                                            resulting	 plaques	 were	 randomly	 selected	 for	 PCR	 amplification	 and	
Key words:	peptides,	vaccine,	Rhipicephalus	(Boophilus)	microplus.                  the	DNA	inserts	were	mass	sequenced	and	analyzed	by	bioinformat-
Species:	other                                                                      ics.	A	cDNA	library	was	constructed	from	salivary	glands	of	both	ticks.	
                                                                                    Randomly	selected	clones	were	sequenced	and	a	total	of	1803	were	
                                                                                    analyzed	 by	 bioinformatics	 programs.	 The	 sequences	 were	 grouped	
  ig026. COMPARATIVE ANALySIS OF TRANSCRIPTOMES
                                                                                    into	 867	 clusters,	 which	 were	 confronted	 against	 the	 following	 data-
    OF SALIVARy GLANDS FROM TICKS, AMBLyoMMA                                        bases:	NCBI	non-redundant	protein,	GO	and	KOG.	About	90%	of	the	
     CAJENNESE AND RHIPICEPHALUS SANGUINEUS.                                        mRNA	 sequences	 showed	 significant	 similarity	 to	 known	 proteins	 in	
 AnATRIELLo	E1,	FERREIRA	BR1,	BRAnDÃo	LG1,	VALEnZUELA	                              the	non-redundant	protein	database	by	the	NCBI	blastx	program	and	
   JG2,	RIBEIRo	JM2,	SILVA	JS1,DE	MIRAnDA	SAnToS	IKF1	                              appeared	 to	 be	 coding	 for	 functional	 predited	 proteins,	 whereas	 the	
  1Dept.	Biochemistry	and	Immunology,	USP	–	Ribeirão	Preto	–	SP,	                   remaining	 10%	 had	 no	 similar	 sequences	 and	 may	 represent	 novel	
   Brazil;	2Vector	Molecular	Biology	Unit,	Laboratory	of	Malaria	and	               genes.
         Vector	Research,	NIAID,	NIH	–	Bethesda	–	MD,	USA                                Discussion:	 Among	 the	 predicted	 protein	 sequences,	 we	 found	
      Introduction	and	objectives:	Ticks	are	hematophagous	arthropod	               similarities	to	cements,	protease	inhibitors,	anticoagulants,	metallopro-
vectors	 of	 disease.	 Vaccines	 are	 an	 alternative	 for	 their	 control,	 the	   teases,	anti-inflammatory	molecules,	and	potent	immunosuppressants.	
premise	 being	 that	 infestations	 with	 these	 parasites	 stimulate	 host	        A	comparative	analysis	of	the	two	libraries	led	to	identification	of	sev-
immune	responses,	which	are	implicated	in	their	rejection.	The	tick’s	              eral	transcripts	that	are	common	to	both	species	of	ticks,	suggesting	
salivary	glands	are	important	for	acquiring	blood	meals	and	for	counter-            that	cross-reactive	protective	antigens	may	obtained.	
ing	the	host’s	defenses.	In	order	to	elucidate	the	biology	of	the	tick-host	             Financial	support:	FAPESP,	CNPq	and	Vallée	SA.
interface	and	discover	protective	antigens	for	a	multicomponent	vac-                Key words:	 Dog	 Tick,	 Horse	 TickTranscriptomes,	 Comparative	
cine,	transcriptomes	of	salivary	glands	of	the	horse	tick,	A.	cajennense,	          Analysis,	Salivary	Glands,	Vaccine
and	dog	tick,	R.	sanguineus,	are	being	analyzed.                                    Species:		other
     Methods	 and	 Results:	 Salivary	 glands	 from	 partially	 fed	 female	
ticks	 were	 used	 to	 obtain	 mRNA	 in	 order	 to	 construct	 a	 PCR-based	
           2. IMMUNE RESPONSES IN BACTERIAL AND VIRAL DISEASES; PRIONS AND BSE: POSTERS BV027-BV077



  bV027. LONG-TERM STAPHyLOCOCCAL ENTEROTOxIN                                     MHCII	 and	 CD69	 but	 were	 negative	 for	 CD1a,	 CD11a,	 CD11c,	 and	
   C1 ExPOSURE INDUCES SOLUBLE FACTOR MEDIATED                                    CD172a.	Importantly,	25%	of	cells	lacked	CD14.	Furthermore,	SSMs	
                IMMUNOSUPPRESSION.                                                developed	 long	 dendrites	 on	 their	 surface,	 suggesting	 they	 are	 den-
 KEUn	SEoK	SEo1,	SAnG	Un	LEE1,	yonG	Ho	PARK2,	WILLIAM	                            dritic	cells.	Pinocytosis	by	SSMs	was	significantly	higher	than	that	by	
    C	DAVIS3,	LAWREnCE	K	Fox4,	GREGoRy	A	BoHACH1                                  naïve	 monocyte.	 Upon	 stimulation,	 transcription	 of	 proinflammatory	
                                                                                  cytokines	(IL-1α,	β,	IL-6,	TNF-α)	and	chemokines	(CXCL1,	2,	3,	and	
 1Department	of	Microbiology,	Molecular	Biology	and	Biochemistry,	
                                                                                  6,	CCL2	and	5)	rapidly	increased	(within	24	h)	but	gradually	decreased	
University	of	Idaho,	Moscow,	ID	83844;	2Department	of	Microbiology,	
                                                                                  later.	However,	transcription	of	β	chemokines	(CCL3,	CCL8,	SDF-1),	
      College	of	Veterinary	Medicine	and	School	of	Agricultural	
                                                                                  responsible	for	mononuclear	cell	migration,	was	sustained.	Consistent	
  Biotechnology,	Seoul	National	University,	Seoul,	151-742,	Korea;	
                                                                                  with	these	data,	in	vitro	cell	migration	assays,	showing	vigorous	early	
3Department	of	Veterinary	Microbiology	and	Pathology,	Washington	
                                                                                  migration	 of	 granulocytes,	 followed	 by	 subsequent	 mononuclear	 cell	
     State	University,	Pullman,	WA	99164,	USA;	4Department	of	
   Veterinary	Medicine,	Washington	State	University,	Pullman,	WA	                 migration,	demonstrated	that	temporally	differential	cell	migration	was	
                             99164,	USA                                           mediated	by	SSM-derived	chemokines.	These	results	suggest	that	the	
                                                                                  stimulation	 by	 SAgs	 differentiate	 peripheral	 monocyte	 into	 dendritic	
      Regulatory	T	cells	(Tregs)	help	control	development	and	maintenance	        cells.	These	findings	provide	further	insight	into	the	potential	roles	of	
of	 protective	 immunity	 and	 can	 lead	 to	 aberrant	 immune	 responses	
                                                                                  APCs	during	SAg-mediated	immunomodulation.
to	 some	 pathogens.	 Several	 lines	 of	 evidence	 suggest	 that	 Tregs	 are	
induced	by	exposure	to	superantigens	(SAgs)	in	vitro	or	in	vivo.	In	this	         Key words:	Dendritic	cells;	chemokines;	macrophages	
study,	 bovine	 PBMCs	 were	 exposed	 in	 vitro,	 to	 a	 relatively	 low	 dose	   Species:	ruminants
(5	ng/ml)	of	staphylococcal	enterotoxin	C1	(SEC1)	for	up	to	10	days.	
Upon	stimulation,	CD4+	and	CD8+	T	cells	initially	proliferated	at	similar	         bV029. EVALUATION OF THE AG85 SPECIFICITy IN THE
rates.	Subsequently,	from	days	6	through	10,	most	CD4+	and	CD8+	T	                HUMORAL AND CELLULAR IMMUNE RESPONSE IN BOVINE
cells	proliferated	regardless	of	Vβ	specificity,	but	proliferation	of	CD8+	                        TUBERCULOSIS.
T	 cells	 occurred	 more	 vigorously.	 Transcription	 of	 CD25	 and	 CD152	
genes	 increased,	 while	 that	 of	 IL-2	 decreased.	 γd	 T	 cells	 appeared	     	EDIAnE	B	SILVA,	MARIA	I	MoURA,	MARCo	A	M	SILVA,	ARIoLDo	
unresponsive.	 An	 increase	 in	 the	 transcription	 of	 IL-10	 and	 TGF-β	          CARVALHo	JR,	AnDRé	KIPnIS,	AnA	P	JUnqUEIRA-KIPnIS	
genes	in	SEC1	stimulated	cultures	was	attributed	to	the	CD4+CD25+	                  Departamento	de	Imunologia,	Laboratório	de	Imunopatologia	das	
T	cell	subpopulation.		Expression	of	Foxp3	mRNA	also	increased,	and	                   Doenças	Infecciosas,	Federal	University	of	Goiás	–	UFG.	
was	accompanied	by	up-regulation	of	CD152	and	down-regulation	of	                                  edianeveterinária@hotmail.com
IL-2	transcription,	suggesting	that	cells	in	this	subpopulation	are	Tregs.	             Bovine	tuberculosis	caused	by	Mycobacterium	bovis	affect	bovine,	
Functionally,	SEC1-stimulated	CD4+	T	cells	suppressed	the	prolifera-              equine	and	several	other	domestic	animals	as	well	as	human	beings.	
tion	 of	 naïve	 PBMCs	 in	 response	 to	 heat-killed-fixed	 S.	 aureus.	 The	    The	world	presents	around	50	millions	of	infected	animals	that	directly	
suppression	 was	 partially	 mediated	 by	 IL-10	 and	 TGF-β,	 another	
                                                                                  affects	 milk	 and	 meat	 productivity.	 	 Although	 the	 cellular	 immune	
characteristic	of	certain	types	of	Tregs.	The	CD8+	T	cell	population	also	
                                                                                  response	is	the	main	protective	response	elicited,	some	proteins	from	
suppressed	naïve	PBMCs	through	another	mechanism	not	mediated	
                                                                                  M.	bovis	induce	antibodies	formation	and	therefore	could	be	used	as	a	
by	IL-10	or	TGF-β.	These	results	provide	further	insight	into	potential	
                                                                                  disease	markers.	The	detection	of	cellular	immune	response	by	nitric	
mechanisms	by	which	SAgs	could	contribute	to	evasion	of	the	immune	
                                                                                  oxide	production	(NO)	helps	to	understand	the	macrophage	activation	
response,	affecting	the	outcome	of	infection	or	colonization.
                                                                                  and	the	presence	of	specific	antibodies	could	determine	the	evolution	
Key words:	Superantigen,	Regulatiory	T	cell,	Foxp3                                of	the	infection.	In	order	to	identify	the	specificity	of	NO	production	by	
Species:	bovines                                                                  mononuclear	cells	against	Ag85,	tuberculin	and	BCG	among	a	Brazilian	
                                                                                  herd,	it	was	used	13	cattle,	adults	Holstein	which	were	diagnosed	as	
  bV028. PHENOTyPIC AND FUNCTIONAL ANALySIS OF                                    tuberculosis	 positive	 using	 intra	 dermal	 	 tuberculin	 test	 (ITT)	 and	 six	
STAPHyLOCOCCAL SUPERANTIGEN-STIMULATED BOVINE                                     negative	control	animals.	Twelve	ml	of	blood	were	collected	in	heparin	
                  MONOCyTES                                                       tubes.	The	peripheral	blood	mononuclear	cells	(PBMC)	were	obtained	
   Joo	yoUn	PARK1,2,	KEUn	SEoK	SEo2,	WILLIAM	C	DAVIS3,	                           and	incubated	with	recombinant	Ag85a	(20	µg/ml),	tuberculin	(5	µg/ml)	
         LAWREnCE	K	Fox1,,GREGoRy	A	BoHACH2                                       or	BCG	(10	µg/ml).	Twenty	eight	serum	samples	were	analyzed	in	an	
  1Department	of	Veterinary	Medicine,	Washington	State	University,	               ELISA	 (20	 from	 TB	 positive	 cattle	 and	 8	 samples	 from	 TB	 negative	
Pullman,	Washington	99164;	2Department	of	Microbiology,	Molecular	                ones).	 In	 the	 evaluation	 of	 NO	 production	 there	 were	 no	 differences	
Biology,	and	Biochemistry,	University	of	Idaho,	Moscow,	Idaho	83844;	             between	 the	 PBMC	 cultures	 from	 infected	 animals	 stimulated	 with	
 3Department	of	Veterinary	Microbiology	and	Pathology,	Washington	                Ag85,	(5,72	nM	±	3,19),	tuberculin	(5,77	nM	± 3,11),	and	BCG,	(7,73	
            State	University,	Pullman,	Washington	99164                           nM	 ±	 5,99)	 when	 compared	 to	 the	 control	 group	 [Ag85	 (10,13	 nM	 ±	
                                                                                  10,49),	tuberculin	(7,32	nM	±	3,02),	BCG	(8,65	nM	±	4,90)].	Evaluating	
     Staphylococcal	 enterotoxins	 are	 prototype	 microbial	 superanti-
                                                                                  the	 humoral	 immune	 response,	 it	 was	 observed	 higher	 levels	 of	 IgG	
gens	(SAgs)	which	elicit	extensive	T	cell	proliferation	in	an	MHC	class	II	
                                                                                  among	 ITT-positive	 cattle	 (1,18	 ±	 0,13)	 when	 compared	 to	 the	 ITT	
(MHCII)	dependent	manner.	Although	antigen	presenting	cells	(APCs)	
provide	crucial	signals	to	T	cells	through	MHCII	and	other	co-stimulatory	        negative	 cattle	 (0,89	 ±	 0,10)	 (p<0,05).	 	 Therefore	 evaluation	 of	 	 NO	
surface	molecules,	little	is	known	about	their	phenotypes	and	functions	          production	 can	 not	 be	 used	 for	 the	 	 discrimination	 between	 naturally	
during	SAgs-induced	activation.	In	this	study,	bovine	peripheral	blood	           infected	 bovines	 from	 healthy	 ones.	 In	 contrast,	 humoral	 immune	
mononuclear	cells	were	exposed	to	a	low	dose	(5	ng/ml)	of	staphylo-               response	with		recombinant	Ag85	is	a	good	candidate	to	be	used	in	the	
coccal	enterotoxin	C1	(SEC1)	for	up	to	10	days.	The	characteristics	of	           diagnosis	of	M.	bovis	infection.		
SEC1-stimulated	monocytes	(SSM)	were	analyzed	by	flow	cytometry	                  Key words:	Micobacterium	bovis,	nitric	oxide,	recombinant	antigen
and	quantitative	real-time	PCR.	SSM	highly	expressed	CD40,	CD11b,	                Species:	ruminants
    bV030. MyCoBACTERIUM PARATUBERCULoSIS                                        not	yet	known.		In	the	previous	studies,	we	found	that	the	expression	
  SUPPRESSES CD40 SIGNALING INDUCED IL-12P40 AND                                 of	tumor	necrosis	factor	(TNF)-α	and	its	receptors	closely	associated	
INOS GENE ExPRESSION IN BOVINE MONOCyTE-DERIVED                                  with	 disease	 progression	 in	 sheep	 experimentally	 infected	 with	 BLV.	    	
                 MACROPHAGES.                                                    Interestingly,	we	found	a	conflicting	role	of	TNF–a	in	sheep	experimen-
                                                                                 tally	 infected	 with	 BLV.	 In	 the	 early	 phase	 of	 infection,	TNF-a	 mRNA	
SAnDRA	SoMMER,	CHARLES	B	PUDRITH,	CHRIS	CoLVIn,PAUL	
                                                                                 expression	was	significantly	up-regulated	in	BLV-resistant	sheep;	how-
                  M	CoUSSEnS	
                                                                                 ever,	 down-regulation	 of	 TNF-a	 was	 found	 in	 susceptible	 sheep.	 	 In	
  Dept.	of	Animal	Science,	Molecular	Pathogenesis,	Michigan	State	               contrast,	TNF-a	strongly	induced	the	proliferative	response	of	periph-
                 University,	East	Lansing,	MI,	USA.                              eral	 blood	 mononuclear	 cells	 (PBMCs)	 in	 sheep	 with	 higher	 level	 of	
      Mycobacterium	avium	ssp.	paratuberculosis	(MAP),	the	causative	            BLV	in	the	late	phase	of	infection.		In	order	to	investigate	the	different	
agent	of	Johne`s	disease,	is	a	facultative	intracellular	pathogen,	resid-        TNF-a-induced	responses,	we	examined	the	TNF-a-induced	prolifera-
ing	in	subepithelial	macrophages.	Clearance	of	MAP	critically	depends	           tive	responses	and	the	expression	levels	of	two	distinct	TNF	receptors	
upon	 an	 appropriate	 pro-inflammatory	 and	 cytotoxic	 Th-1	 immune	           of	PBMCs	derived	from	cattle	with	different	stage	of	BLV-infection	(AL	
response	leading	to	activation	and/or	lysis	of	persistently	infected	mac-        or	PL).		The	proliferative	response	of	PBMC	isolated	from	those	cattle	
rophages	to	promote	bacterial	killing.	Work	in	vivo	has	shown,	that	the	         with	 PL	 in	 the	 presence	 of	 recombinant	 bovine	TNF-a	 (rTNF-a)	 was	
appropriate	Th-1	immune	response	occurring	early	in	MAP	infection	is	            significantly	higher	than	those	from	AL	and	uninfected	cattle.	The	cells	
lost,	followed	by	an	ineffective,	antibody-mediated	Th-2	response.	Our	          from	PL	cattle	expressed	significant	higher	mRNA	levels	of	TNF	recep-
overall	hypothesis	is	that	once	MAP	persists	within	naïve	macrophages,	          tor	type	II	(TNF-RII)	than	those	from	AL	and	BLV-infected	cattle.		No	
it	reduces	the	ability	of	infected	macrophages	to	react	to	normal	T	cell	        difference	was	found	in	TNF-RI	mRNA	levels	among	the	animals.		Most	
signaling,	failing	to	be	activated	and	destroy	MAP,	and	failing	to	properly	     cells	 expressing	TNF-RII	 in	 PL	 cattle	 were	 CD5+	 or	 sIgM+	 cells	 and	
signal	T	cells	to	respond.	To	test	this	hypothesis,	we	investigated	the	         these	cells	showed	resistance	to	TNF-a-induced	apoptosis.		Moreover,	
effect	of	MAP	infection	on	CD40	signaling,	a	main	pathway	used	by	T	             there	were	significant	positive	correlations	between	the	TNF-RII	mRNA	
cells	to	activate	macrophages.		Our	recent	studies	demonstrate	that	a	           levels	with	either	the	changes	in	provirus	load	and	the	TNF-a-induced	
short-lived	response	of	bovine	monocyte-derived	macrophages	(MDM)	               proliferation.		These	data	suggest	that	imbalance	in	the	expression	of	
to	MAP	infection	in	vitro	is	apparently	followed	by	a	block	in	the	ability	      TNF	 receptors	 could	 at	 least	 in	 part	 contribute	 to	 the	 progression	 of	
of	infected	cells	to	respond	normally	to	subsequent	external	activation.	        lymphocytosis	in	BLV	infection.
We	have	demonstrated	by	using	Q-RT-PCR	that	normal	MDM	respond	
to	 CD40	 ligand	 (CD40L)	 stimulation	 by	 up-regulation	 of	 immune	           Key words:	BLV,	TNF-a,	TNF-receptors,	cell	proliferation
response	genes,	including	those	encoding	IL-6,	TNF-α,	iNOS,	and	IL-              Species:	ruminants
12p40.	Consistent	with	these	results,	western	blot	analyses	indicated	
that	 CD40L	 stimulation	 causes	 a	 rapid,	 but	 short-lived	 activation	 of	          bV032. EARLy LOCAL IMMUNE RESPONSES TO
JNK,	ERK1/2	and	p38	MAPK.	Studies	with	specific	inhibitors	revealed	                    MyCOBACTERIAL 70 KD HEAT-SHOCK PROTEIN
that	 the	 CD40L-mediated	 increase	 in	 IL-6	 and	 IL-8	 gene	 expression	                           VACCINATION
is	dependent	upon	activation	of	ERK1/2	and	JNK,	while	increases	in	               FEMKE	BRoERE2,	WILLEM	VAn	EDEn2,VICToR	RUTTEn2	,	AD	
IL-12p40	and	iNOS	gene	expression	are	dependent	upon	activation	of	
                                                                                                      KoETS1,	2
p38.	Once	infected	with	MAP,	however,	MDM	cells	fail	to	up-regulate	
the	expression	of	iNOS	and	IL-12p40	encoding	genes	in	response	to	                   1Department	of	Farm	Animal	Health,	P.O.	Box	80.165,	Faculty	
CD40L,	 whereas	 the	 expression	 of	 the	 other	 tested	 genes,	 such	 as	         of	Veterinary	Medicine,	Utrecht	University,	3508	TD	Utrecht,	The	
IL-8	 and	 TNF-α	 is	 not	 repressed.	 Using	 flow	 cytometric	 analysis	 we	        Netherlands;	2Immunology	Division,	Department	of	Infectious	
determined,	that	failure	of	infected	macrophages	to	respond	to	CD40L	              Diseases	and	Immunology,	P.O.	Box	80.165,	Faculty	of	Veterinary	
was	 not	 due	 to	 down-regulation	 of	 CD40	 on	 the	 cell	 surface	 of	 MAP	      Medicine,	Utrecht	University,	3508	TD	Utrecht,	The	Netherlands
infected	 MDM.	 Western	 blot	 analysis	 also	 revealed	 that	 interference	          Paratuberculosis	is	a	chronic	granulomatous	inflammation	of	the	
with	CD40L-mediated	increases	in	gene	expression	does	not	appear	                small	intestine	of	cattle	and	other	ruminants,	caused	by	infection	with	
to	 be	 due	 to	 prevention	 of	 p38,	 ERK1/2,	 or	 JNK	 activation,	 suggest-   Mycobacterium	avium	ssp.	paratuberculosis	(MAP).	The	disease	can	
ing	the	block	is	downstream	of	these	kinases.	Continuing	studies	are	            be	found	in	ruminant	herds	worldwide,	causing	substantial	economic	
underway	to	uncover	the	mechanism	responsible	for	MAP	interference	              losses	at	farm	level	due	to	premature	culling	and	production	losses.
with	CD40	signaling	in	infected	macrophages.
                                                                                       We	 have	 documented	 previously	 that	 mycobacterial	 heat-shock	
Key words:	 Johne´s	 disease,	 macrophage,	 CD40	 signaling,	                    proteins	 (Hsp)	 are	 dominant	 antigens	 in	 various	 stages	 of	 bovine	
mycobacteria                                                                     paratuberculosis.	Especially	the	70	kD	Hsp	(Hsp70)	induces	cell	medi-
Species:	ruminants                                                               ated	 responses	 in	 natural	 infection.	 Furthermore,	 recombinant	 MAP	
                                                                                 Hsp70has	 been	 shown	 to	 be	 a	 successful	 subunit	 vaccine	 against	
  bV031. IMBALANCE OF TUMOR NECROSIS FACTOR                                      bovine	 paratuberculosis.	 Surprisingly	 the	 main	 hallmark	 of	 vaccina-
RECEPTORS DURING PROGRESSION IN BOVINE LEUKEMIA                                  tion	 induced	 immunity	 was	 antibody	 production	 rather	 then	 T	 cell	
                VIRUS INFECTION                                                  immunity.
SAToRU	KonnAI1,	TATSUFUMI	USUI1,	MAnABU	IKEDA2,	JUnKo	                                 To	 explore	 the	 immunological	 mechanisms	 of	 induction	 of	 early	
  KoHARA3,	KoSUKE	oKADA2,	KAZUHIKo	oHASHI1,MISAo	                                cellular	responses	at	the	local	draining	lymphnodes	within	days	after	
                      onUMA1                                                     vaccination	 we	 adopted	 a	 murine	 model.	 Mice	 were	 vaccinated	 with	
  1	Department	of	Disease	Control,	Graduate	School	of	Veterinary	                Hsp70	 or	 OVA	 in	 DDA,	 comparable	 to	 the	 cattle	 vaccine	 and	 BrdU	
Medicine,	Hokkaido	University,	Sapporo,	Hokkaido	060-0818,	Japan;		              incorporation	was	measured	by	flowcytometry	4	and	7	days	after	vacci-
 2	Faculty	of	Agriculture,	Iwate	University,	Morioka,	Iwate	020-8550,	           nation.	In	addition	lymph	node	cells	and	splenocytes	were	restimulated	
Japan;	3	Hokkaido	Animal	Research	Center,	Shintoku,	Hokkaido	081-                in	vitro	to	address	the	functional	differentiation	of	the	immune	response	
                             0038,	Japan.	                                       as	measured	by	cytokine	profile	and	antibody	production.
                    konnai@vetmed.hokudai.ac.jp                                        Enhanced	 BrdU	 incorporation	 was	 observed	 in	 draining	 lym-
      Bovine	 leukemia	 virus	 (BLV),	 the	 genus	 Deltaretrovirus,	 family	     phnodes	 of	 mice	 that	 were	 immunized	 with	 Hsp70	 compared	 to	
Retroviridae,	is	the	causative	agent	of	enzootic	bovine	leukosis	(EBL),	         OVA	treated	mice	7	days	after	immunization.	No	differences	in	BrdU	
which	is	the	most	common	neoplastic	disease	of	cattle.		The	progres-             incorporation	 were	 observed	 in	 non-draining	 lymphnodes	 or	 at	 day	
sion	 of	 BLV	 infection	 is	 divided	 into	 three	 stages:	 aleukemic	 (AL),	   4	after	immunization.	In	addition,	in	vitro	B	cell	restimulation	showed	
persistent	lymphocytosis	(PL)	and	lymphosarcoma	(LS).		Although	the	             an	enhanced	antigen	specific	B	cells	response	in	the	draining	lymph	
pathogenesis	of	infection	clearly	involves	immunoregulatory	host	fac-            nodes	only	after	Hsp70	vaccination	at	day	7,	whereas	B	cells	isolated	
tors,	including	expression	of	the	cytokines,	the	exact	mechanism	of	the	         from	OVA	treated	mice	did	not	produce	antibodies	in	an	antigen	spe-
disease	progression	from	AL	to	PL	or	PL	to	LS	in	BLV-infected	cattle	is	         cific	fashion.	
64


           Similar	to	the	immunization	outcome	in	cattle	in	the	murine	model	                     KAy-S	SAUTER,	MARIJA	BRCIC,,THoMAS	W	JUnGI	
     there	is	a	preferential	activation	of	B	cell	activity	following	subcutane-           Institute	of	Veterinary	Virology,	University	of	Bern,	Bern,	Switzerland	
     ous	 Hsp70/DDA	 vaccination.	Therefore,	 the	 murine	 model	 presented	                                    thomas.jungi@ivv.unibe.ch
     in	this	study	offers	a	convenient	means	to	study	the	mechanism	lead-
                                                                                               The	 concept	 that	 cells	 of	 the	 innate	 immune	 system	 recognize	
     ing	to	this	immuneresponse	bias	which	is	opposite	to	Hsp70	immune	
                                                                                         pathogen-associated	 microbial	 patterns	 (PAMPs)	 by	 pattern	 recogni-
     responses	in	natural	infection	and	yet	confers	protective	immunity	to	
                                                                                         tion	receptors	(PRRs)	has	received	wide	acceptance	in	recent	years.	
     paratuberculosis.
                                                                                         An	important	family	of	PRR	is	that	of	Toll-like	receptors	(TLR).	Some	
     Key words:	 mycobacterium	 avium	 ssp.	 Paratuberculosi,	 heat	 shock	              of	 these,	 e.g.	 TLR2,	 TLR4	 or	 TLR5	 mediate	 a	 response	 to	 bacterial	
     protein,	vaccination                                                                PAMPs	and	 may	 be	 implicated	 in	 mastitis.	 Rapid	 progress	 has	 been	
     Species:	ruminants                                                                  made	 with	 regard	 to	 tissue	 distribution,	 agonist	 specificity,	 signalling	
                                                                                         pathways	and	effector	functions	mediated	by	TLRs.	However,	knowl-
     bV033. IMMUNOGENICITy OF A CHIMERIC SUBCELLULAR                                     edge	is	restricted	to	species	such	as	human	or	mouse,	and	whether	
     VACCINE IN OVINE BRUCELLOSIS                                                        knowledge	acquired	in	these	species	can	be	extrapolated	to	those	of	
                                                                                         veterinary	interest	is	largely	unknown.	This	is	due	to	the	facts	that	in	
         SILVIA	M	ESTEIn1*,	MARÍA	A	FIoREnTIno2*,	FERnAnDo	
                                                                                         these	 species	 the	 knockout	 technology	 and	 TLR-specific	 antibodies	
           A	PAoLICCHI3,	JULIAnA	CASSATARo3*	GUILLERMo	
                                                                                         are	generally	unavailable,	and	the	siRNA	knockdown	methodology	cre-
         GIAMBARToLoMEI3*,	VAnESA	ZyLBERMAn4*,	CARLoS	A	
                                                                                         ates	artefacts	such	as	interferon	type	I	induction.	To	gain	insights	into	
                 FoSSATI3*,	FERnAnDo	A	GoLDBAUM4*                                        the	interaction	of	bacterial	pathogens	with	PRRs	we	started	a	program	
        1Laboratorio	de	Inmunología,	Facultad	de	Ciencias	Veterinarias,	                 generating	 HEK293	 cells	 being	 deficient	 for	 endogenous	 (human)	
      UNCPBA,	(7000)	Tandil,	Buenos	Aires,	Argentina.	Tel./fax:	+54	2293	                TLRs	 but	 stably	 transduced	 with	 bovine	 TLR	 members.	 Here,	 IL-8	
      43	9850;	2Laboratorio	de	Bacteriología,	Área	de	Producción	Animal,	                production	as	a	result	of	an	engagement	of	transduced	whole	bovine	
     INTA-Balcarce;	3Laboratorio	de	Inmunogenética,	Hospital	de	Clínicas	                TLR2	 or	 whole	 bovine	TLR4	 with	 or	 without	 MD-2	 is	 reported.	TLR4	
     “José	de	San	Martín”,	Facultad	de	Medicina.	UBA,	Bs.	As.,	Argentina,	               is	 part	 of	 the	 LPS	 receptor	 complex,	 together	 with	 CD14	 and	 MD-2.	
       Instituto	de	Estudios	de	la	Inmunidad	Humoral	(IDEHU-CONICET),	                   Cells	 stably	 transduced	 with	 TLR4	 failed	 to	 respond	 to	 LPS	 unless	
      Facultad	de	Farmacia	y	Bioquímica,	UBA,	Buenos	Aires,	Argentina,	                  cotransduced	with	bovine	or	porcine	MD-2.	Doubly	transduced,	cloned	
      4Fundación	Instituto	Leloir,	Buenos	Aires,	Argentina.	*CONICET,	Bs.	               cells	 reacted	 to	 LPS	 by	 IL-8	 production,	 in	 decreasing	 order,	 in	 the	
                                   As.	Argentina.                                        presence	of	foetal	bovine	serum,	human	AB	serum	and	human	serum	
                                                                                         albumin	(HSA).	The	partial	response	in	the	presence	of	HSA	was	com-
                             silmares@vet.unicen.edu.ar                                  plemented	by	the	addition	of	highly	purified	soluble	human	CD14,	but	
            	Brucella	ovis	causes	an	infectious	disease	in	sheep	characterized	          not	by	the	addition	of	highly	purified	human	lipopolysaccharide	binding	
     by	epididymitis	and	infertility	in	rams	and	abortion	in	ewes.	Vaccination	          protein.	 Stimulation	 by	 partial	 LPS	 structures	 being	 recognized	 in	 a	
     programs	are	the	only	viable	means	for	the	control	of	B.	ovis	in	coun-              species-specific	 manner	 suggested	 that	 cells	 resembled	 the	 reactiv-
     tries	with	a	high	incidence.	B.	melitensis	Rev.1	is	considered	the	best	            ity	of	bovine	monocyte-derived	macrophages	but	reacted	in	a	manner	
     vaccine	for	the	prophylaxis	of	ovine	brucellosis	but	has	important	dis-             distinct	from	humans,	mice	or	hamsters.	TLR2	is	engaged	in	recogni-
     advantages.	Accordingly,	 research	 is	 under	 way	 to	 develop	 effective	         tion	 of	 various	 PAMPs	 expressed,	 e.g.,	 by	 gram-positive	 bacteria.	A	
     subcellular	vaccines.	Detergent-extracted	recombinant	Omp31	from	B.	                clone	 was	 generated	 that	 responded,	 in	 decreasing	 order,	 to	 Staph.	
     melitensis	was	previously	identified	as	a	protective	immunogen	against	             aureus-derived	 peptidoglycan,	 to	 Palm3-Cys,	 lipoteichoic	 acid,	 heat-
     B.	 ovis	 in	 rams.	 Moreover,	 our	 previous	 results	 demonstrate	 that	 a	       killed	 Listeria	 monocytogenes	 and	 LPS	 from	 E.	 coli	 (TLR4	 agonist).	
     chimera	of	Brucella	lumazine	synthase	(BLS)	that	contains	an	immu-                  The	 response	 of	 this	 clone	 to	 causative	 agents	 of	 mastitis	 is	 under	
     nodominant	epitope	of	Omp31	delivered	as	a	protein	(BLS-Omp31)	or	                  study.	These	stably	transduced	clones	might	be	useful	when	searching	
     plasmidic	 DNA	 vaccine	 (pCIbls-omp31)	 conferred	 protection	 against	            for	antibodies	specific	for	or	crossreactive	with	bovine	TLR	members,	
     B.	ovis	infection	in	mice.	In	this	work,	we	evaluated	the	immunogenic-              and	when	analysing	the	interaction	of	bovine	bacterial	pathogens	with	
     ity	 of	 these	 vaccines	 in	 rams	 using	 different	 strategies	 of	 immunisa-     TLR-expressing	cells.
     tion.	 Seventy	 rams	 four-five-	 months-old	 were	 randomly	 distributed	          Key words:	TLR4,	TLR2,	MD-2,	stable	transduction
     in	 following	 groups	 of	 immunisation:	 G1)	 BLS-Omp31	 in	 Freund´s	             Species:	ruminants
     incomplete	adjuvant	(FIA),	G2)	BLS-Omp31	in	QUIL	A	adjuvant,	G3)	
     pCIbls-omp31	 with	 electroporation	 (INOVIO,	 Norway),	 G4)	 pCIbls-                    bV035. PROTECTIVE ROLE FOR γδ T CELLS IN THE
     omp31	without	electroporation,	G5)	Prime-boost,	G6)	HS	(hot	saline)	                      INNATE IMMUNE RESPONSE AGAINST BRUCELLA
     extract	 in	 FIA,	 G7)	 PBS.	Animals	 were	 vaccinated	 three	 times,	 four	                              ABoRTUS
     weeks	 apart.	 G5	 received	 four	 injections	 (DNA	 prime-protein	 boost).	
                                                                                              JERoD	A	SKyBERG,	MARK	A	JUTILA,DAVID	W	PASCUAL
     Rams	were	bled	after	each	immunization.	Antibody	responses	in	serum	
     were	 evaluated	 in	 indirect	 ELISA	 against	 BLS-Omp31.	 Immunisation	                  Department	of	Veterinary	Molecular	Biology;	Montana	State	
     with	the	protein	chimera	formulated	with	adjuvant	induced	IgG	specific	                          University;	Bozeman,	Montana,	59718	USA
     antibodies	significantly	higher	than	chimerical	DNA	vaccine.	However,	                    γd	T	 cells	 have	 been	 postulated	 to	 act	 as	 a	 first	 line	 of	 defense	
     humoral	 immune	 response	 was	 enhanced	 in	 electroporated	 sheep.	               against	 infectious	 disease,	 particularly	 intracellular	 pathogens,	 and	
     Combination	of	a	plasmid	DNA	priming	step	followed	by	a	boost	with	                 appear	to	be	an	important	link	between	the	innate	and	adaptive	immune	
     the	homologous	protein	resulted	in	improved	humoral	antigen-specific	               responses..		While	γd	T	cells	represent	a	small	percentage	of	circulating	
     response.	 These	 results	 indicate	 that	 the	 chimerical	 subunit	 vaccine	       T	cells	in	adult	humans	and	mice,	γd	T	cells	constitute	a	major	subset	of	
     was	immunogenic	in	rams	and	would	be	considered	as	potential	vac-                   lymphocytes	in	adult	ruminants	and	make	up	a	majority	(up	to	70%	of	
     cine	in	ovine	brucellosis.	                                                         circulating	lymphocytes)	in	neonatal	calves,	suggesting	an	enhanced	
          References                                                                     importance	of	this	subset	in	the	immune	response	of	ruminants.	Indeed	
                                                                                         past	studies	have	intimated	that	bovine	γd	T	cells	play	a	pivotal	early	
           Blasco	J.M.	1990.	Brucella	ovis,	In:	Nielsen	K.	Duncan	J.R.,	edi-             role	in	the	response	to	the	intracellular	pathogen	Mycobacterium	bovis.	           	
     tors.	Animal	 Brucellosis.		 	Estein	 S.M.	 et	 al.,	 	 2003,	 Microbes	 Infect.	   Previous	work	conducted	with	humans	has	shown	that	γd	T	cells	expand	
     Estein	 S.M.	 et	 al.,	 2004,	 Vet.	 Microbiol.	 Cassataro,	 J.	 et	 al.,	 2007,	   in	the	peripheral	blood	of	patients	infected	with	Brucella	and	that	γd	T	
     Vaccine.	Cassataro	J.	et	al.,		2007,	Clinical	and	Vaccine	Immunology.	              cells	are	active	against	Brucella	in	vitro.		However,	the	role	of	γd	T	cells	
     Key words:	ovine	brucellosis,	subcellular	vaccines,	rams,	chimeric                  in	experimental	brucellosis	models	has	yet	to	be	studied.		Due	to	the	
     Species:	ruminants                                                                  abundance	of	γd	T	cells	in	ruminants,	along	with	the	established	role	of	
                                                                                         γd	T	cells	in	controlling	intracellular	infections,	we	hypothesize	that	γd	
     bV034. INTERACTION OF BOVINE TOLL-LIKE RECEPTORS                                    T	cells	may	be	an	important	resource	to	resolve	Brucella	infections	of	
      WITH MOLECULAR PATTERNS AS ASSESSED By STABLy                                      cattle.		Here	we	report	that	γd	T	cell-deficient	(TCR	γd	-/-)	mice	are	more	
                                                                                         susceptible	 to	 B.	 abortus	 infection	 than	 age	 matched	 C57BL/6	 mice	
                    TRANSDUCED CELLS
                                                                                                                                                                        65


at	one	week	post-infection.	B.	abortus-infected	TCRγd-/-	mice	had	sig-             		1Dept.	Pathobiological	Sciences,	University	of	Wisconsin-Madison,	
nificantly	greater	splenomegaly	attributed	to	increased	brucellae	than	             Madison	WI	53706;		2Center	for	Molecular	Medicine	and	Infectious	
did	C57BL/6	wild-type	mice.		An	increase	in	γd	T	cells	was	observed	               Diseases,	Virginia-Maryland	Regional	College	of	Veterinary	Medicine,	
in	the	spleens	of	B.	abortus-infected	C57BL/6	mice	which	peaked	at	                  Virginia	Polytechnic	Institute	and	State	University,	Blacksburg,	VA.
two	weeks	post-infection,	and	occurred	concomitantly	with	diminished	                    Haemophilus	 somnus	 (also	 known	 as	 Histophilus	 somnii)	 is	 an	
brucellae.		Intracellular	cytokine	analysis	of	splenic	lymphocytes	from	           important	pathogen	of	cattle	and	sheep	that	can	cause	respiratory	and	
infected	C57BL/6	mice	revealed	that	the	CD8-	γd	T	cells	are	the	major	             reproductive	 infections,	 and	 an	 acute	 thrombomeningoencephalitis	
source	 of	 IL-17	 and	 TNF-α	 amongst	 lymphocytes	 during	 B.	 abortus	          (TME).	A	commonality	of		H.	somnus			infections	is	vasculitis	in	both	
infection.		These	results	indicate	a	protective	role	for	γd	T	cells,	possibly	     large	and	small	blood	vessels.	We	have	been	involved	in	a	series	of	
via	IL-17	and/or	TNF-a,	and	that	role	of	bovine	γd	T	cells	in	the	immune	          investigations	 of	 mechanisms	 related	 to	 the	 development	 of	 vasculi-
response	to	Brucella	warrants	further	investigation.                               tis.	Previous	work	in	our	laboratory	showed	that	H.	somnus	binds	to,	
Key words:	Brucella,	γδ	T	cells,	Immunity                                          but	 is	 not	 internalized,	 by	 bovine	 endothelial	 cells.	 This	 process	 can	
Species: ruminants                                                                 result		in	endothelial	cell	death	by	apoptosis.	We	also	found		that	bovine	
                                                                                   platelets	are	activated	by	H.	somnus,	and	can	in	turn	amplify	the	apop-
 bV036. CONSTRUCTION OF A BOVINE HERPESVIRUS 5                                     totic	 response	 of	 endothelial	 cells.	 More	 recent	 studies	 have	 looked	
                                                                                   at	 earlier	 events	 in	 the	 response	 of	 endothelial	 cells	 to	 H.	 somnus,	
 WITH A DELETION ON THE GENE VIRION HOST SHUTOFF
                                                                                   and	 how	 platelets	 might	 influence	 these	 responses.	 Endothelial	 cells	   	
     MARCoS	S	PRoFES1,	FRAnS	AM	RIJSEWIJK1,	PAULo	M	                               incubated	with	H.	somnus	produce	the	inflammatory	mediators	IL-1β	
                RoEHE1,	AnA	C	FRAnCo1                                              and	TNF-a,	 and	 express	 increased	 amounts	 of	 tissue	 factor	 on	 their	    	
 1Laboratório	de	Virologia,	Departamento	de	Microbiologia,	Instituto	              surface.	 They	 also	 produce	 less	 thrombomodulin,	 and	 therefore	 are	
 de	Ciências	Básicas	da	Saúde,	UFRGS;	Rua	Sarmento	Leite,	500,	                    less	able	to	activate	protein	C.	The	sum	of	these	changes	would		be	
                 CEP	90050-170,	Porto	Alegre,	RS.	                                 expected	 to	 augment	 thrombus	 formation.	 Incubation	 	 of	 endothelial	
                    marcos_s_p@yahoo.com.br                                        cell		monolayers	with	H.	somnus	caused	cytoskeletal	alterations	that	
       Bovine	herpesvirus	type	5	(BHV-5)	is	a	member	of	the	sub-fam-               decreased	 electrical	 resistance	 and	 increased	 monolayer	 perme-
ily	Alphaherpesvirinae,	of	the	family	Herpesviridae,	and	is	one	of	the	            ability.	 We	 have	 evidence	 that	 phosphorylation	 of	 myosin	 light	 chain	
major	causes	of	bovine	viral	encephalitis	in	Brazil.	All	alphaherpesvi-            kinase	 is	 an	 important	 step	 in	 	 this	 process.	 When	 bovine	 platelets	
ruses	carry	a	homolog	of	the	virion	host	shutoff	(vhs)	protein	encoded	            that	had	been	activated	by	incubation	with	H.	somnus	were	incubated	
by	the	UL41	gene	of	prototype	alphaherpesvirus:	herpes	simplex	virus	              with	endothelial	cells,	the	latter	increased	their		surface	expression	of	
                                                                                   ICAM-1,	E-selectin	and	tissue	factor,	and		their	production	of	several	
1.	The	vhs	protein	is	present	in	the	viral	tegument	and	upon	infection	of	
                                                                                   inflammatory	mediators	(IL-1β,	MCP-1	and	MIP-1a).			To	our	surprise	
the	host	cell	initiates	degradation	of	mRNAs.	Consequently,	vhs	down-
                                                                                   we	observed	that	some	activated	platelets	were	internalized	by	bovine	
regulates	 protein	 synthesis	 and	 impairs	 the	 expression	 of	 molecules	
                                                                                   endothelial	 cells.	Activation	 of	 bovine	 platelets	 by	 H.	 somnus	 can	 be	
such	 as	 MHC	 class	 I.	 vhs	 also	 interferes	 with	 the	 interferon	 type	 I	
                                                                                   prevented	 by	 inhibitors	 of	 the	 platelet	 activating	 factor	 receptor.	 We	
responses	of	infected	cells	and	blocks	the	activation	of	dendritic	cells.	
                                                                                   believe	this	does	not	reflect	a	role	of	platelet	activating	factor	per	se	in	
These	effects	of	vhs	interfere	with	the	immune	responses	of	the	host	
                                                                                   the	process.	Rather	it	appears	to	identify	a	role	for	phosphorylcholine	
against	the	infection	virus.	Considering	this,	vhs	negative	alphaherpes-
                                                                                   on	 the	 lipooligosaccharide	 of	 H.	 somnus.	 H.	 somnus	 cells	 that	 lack	
viruses	 may	 be	 less	 pathogenic	 and	 may	 induce	 a	 stronger	 immune	
                                                                                   phosphorylcholine	do	not	cause	platelet	aggregation.	
response,	characteristics	which	could	make	them	strong	candidates	for	
the	development	of	attenuated	vaccines.	Aiming	the	construction	of	an	                  These	studies	illustrate	dynamic	interactions	between	H.	somnus	
attenuated	BHV-5	recombinant	that	could	also	be	used	as	differential	              and		several	of	the	cellular	elements	of	the	vasculature	(i.e.	platelets	
vaccine,	we	describe	here	the	deletion	of	the	UL41	gene	from	the	BHV-              and	 endothelial	 cells).	 It	 is	 hoped	 that	 further	 investigation	 of	 these	
5	 genome	 of	 triple	 deletion	 mutant	 of	 BHV-5	 (EVI88/95	 gI/gE/US9-).	       interactions	will	help	us	better	understand	the	pathogenic	mechanisms	
For	this,	the	upstream	(5´)	and	downstream	(3´)	regions	of	the	UL41	               of		vasculitis	in	H.	somnus	infected	animals.		
gene	 of	 EVI88/95	 were	 amplified	 and	 each	 one	 was	 cloned	 into	 the	       Key words:	endothelial,	platelet,	thrombus,	vasculitis
pCR2.1-TOPO	 vector.	 After	 that,	 the	 3´	 region	 was	 sub-cloned	 into	        Species:	ruminants
the	vector	with	the	5´	region	and	the	orientation	of	the	fragments	was	
verified	by	restriction	enzyme	analyses.	One	clone	with	both	regions	in	               bV038. EVALUATION OF NOVEL COMBINATIONS OF
the	same	orientation	as	in	the	viral	genome	was	chosen	to	insert	the	                 ADJUVANTS AND IMMUNOMODULATORS FOR BOVINE
enhanced	 green	 fluorescent	 protein	 gene	 (EGFP)	 under	 the	 control	                        TUBERCULOSIS VACCINES
of	the	immediate	early	ie1/2	promoter	of	human	cytomegalovirus.	The	
EGFP	gene	will	be	cloned	between	the	5´and	3´	fragments.	After	this,	                 BRyCE	M	BUDDLE1,	MICHEL	DEnIS1,	D	nEIL	WEDLoCK1,	H	
the	construct	will	be	co-transfected	with	EVI88/95	gI/gE/US9-	genomic	                    MARTIn	VoRDERMEIER2,	R	GLyn	HEWInSon2	
DNA	 into	 bovine	 cells	 to	 allow	 the	 recombination	 and,	 consequently,	        1AgResearch,	Hopkirk	Research	Institute,	Palmerston	North,	New	
the	deletion	of	the	UL41	gene.	The	co-transfection	will	be	performed	                   Zealand;	2Veterinary	Laboratory	Agency,	Weybridge,	UK		
using	embryonic	bovine	trachea	cells	applying	the	calcium	phosphate	                               bryce.buddle@agresearch.co.nz
method.	Mutants	expressing	EGFP	in	the	UL41	locus	will	be	selected	                     Our	previous	studies	have	shown	that	vaccination	of	cattle	with	a	
under	the	UV	microscope	and	plaque	purified	and	multiplied.	The	DNA	               combination	of	BCG	and	a	tuberculosis	protein	vaccine	(Mycobacterium	
of	one	of	the	recombinants	will	be	analyzed	by	restriction	endonuclease	           bovis	culture	filtrate	protein,	CFP)	induced	better	protection	than	BCG	
analysis	and	partial	sequencing,	and	further	characterized	by	studying	            vaccine	alone.	Further	studies	are	now	required	to	optimise	the	adju-
it’s	in	vitro	growth	properties.	This	recombinant	virus	will	eventually	be	        vant	and	immunostimulant	used	in	the	vaccine	to	maximise	protection	
used	in	future	animal	experiments	in	order	to	establish	its	immunogenic	           against	 bovine	 tuberculosis	 and	 minimise	 vaccination	 site	 reactions.	
properties.                                                                        The	 adjuvant	 used	 in	 the	 current	 study	 was	 dimethyldioctyldecyl	
Key words:	 Bovine	 herpesvirus	 5,	 virion	 host	 shutoff,	 immune	               ammonium	chloride	(DDA),	which	has	been	used	in	a	Mycobacterium	
evasion                                                                            paratuberculosis	protein	vaccine	to	protect	cattle	against	Johne’s	dis-
Species:	ruminants                                                                 ease.	The	three	immunostimulants	to	be	tested,	all	potent	stimulants	
                                                                                   of	 Th-1	 type	 immune	 responses,	 included	 a	 synthetic	 mycobacterial	
                                                                                   Phosphatidylinositol	Mannoside	(PIM),	Monophosphoryl	Lipid	A	(MPL)	
  bV037. PROTHROMBOTIC EFFECTS OF HAEMOPHILUS
                                                                                   and	 a	 synthetic	 Lipopeptide	 (LP).	 Sixty	 calves,	 5-6	 months	 old	 were	
    SOMNUS ON BOVINE ENDOTHELIAL CELLS AND
                                                                                   randomly	divided	into	six	groups	comprising:	
                  PLATELETS
                                                                                        1.	   Non-vaccinated	
   ERICA	BEHLInG-KELLy1,	CHRISToPHER	J	KUCKLEBURG1,	
     SHAADI	F	ELSWAIFI2,	THoMAS	J	InZAnA2,CHARLES	J	                                    2.	   BCG	alone	
                      CZUPRynSKI1                                                       3.	   BCG	+	M.	bovis	CFP/DDA/PIM
66


          4.	   BCG	+	M.	bovis	CFP/DDA/MPL                                               lowest	potency.	Therefore	tuberculins	of	different	sources	may	give	dif-
          5.	   BCG	+	M.	bovis	CFP/DDA/LP                                                ferent	results	and	the	overall	assay	performance	may	be	improved	by	
                                                                                         optimizing	tuberculin	concentrations.	
          6.	   BCG	+	DDA/MPL
                                                                                         Key words:	 tuberculosis,	 daignosis,	 Bovigam®,	 tuberculin	 early	
           BCG	vaccine	(106	CFU/dose)	was	administered	subcutaneously	
                                                                                         secretory	antigenic	target	6,	culture	filtrate	protein	10
     on	one	occasion,	while	the	M.	bovis	CFP	or	the	adjuvant/immunostimu-
                                                                                         Species:	ruminants
     lant	vaccines	were	administered	subcutaneously	at	Weeks	0,	3	and	6.	
     All	calves	were	challenged	intratracheally	with	a	low	dose	of	M.	bovis	at	
     14	weeks	after	the	initial	vaccination	and	were	euthanized	and	exam-                      bV040. DETECTION OF ELEVATED LEVELS OF
     ined	for	tuberculous	lesions	at	15	weeks	after	challenge.	The	highest	                INTERLEUKIN-10 IN CATTLE FOLLOWING A TUBERCULIN
     level	 of	 protection	 against	 challenge	 with	 M.	 bovis	 was	 observed	 in	                            SKIN-TEST
     groups	vaccinated	with	the	combination	of	BCG	plus	a	M.	bovis	CFP	                        M	CoAD,	Ao	WHELAn,	SG	RHoDES,	DJ	CLIFFoRD,	RG	
     vaccine,	 with	 significant	 reductions	 in	 four	 to	 five	 lesion	 parameters	                   HEWInSon,	HM	VoRDERMEIER
     in	the	lungs	and	lymph	nodes.	The	most	promising	immunostimulant	
                                                                                            TB	Research	Group,		Veterinary	Laboratories	Agency;	Woodham	
     was	 the	 synthetic	 lipopeptide,	 with	 only	 one	 of	 10	 animals	 from	 this	
                                                                                                      Lane,	Addlestone,	Surrey	KT15	3RW,	UK
     group	showing	lung	lesions	and	two	animals	with	lymph	node	lesions.	
     DDA	proved	to	be	a	valuable	adjuvant	for	a	M.	bovis	protein	vaccine	                     Bovine	tuberculosis	(BTB)	is	a	disease	of	economic	and	zoonotic	
     by	 promoting	 protective	 immune	 responses,	 while	 inducing	 minimal	            importance	 caused	 by	 the	 bacterial	 pathogen	 Mycobacterium	 bovis.	
     vaccination	 site	 reaction.	 Combining	 the	 adjuvant/	 immunostimulant	           Control	 of	 BTB	 in	 the	 UK	 centres	 on	 the	 application	 of	 the	 Single	
     vaccine	 plus	 BCG	 vaccine	 appeared	 to	 negate	 the	 protective	 effects	        Intradermal	Comparative	Tuberculin	Test	(SICTT)	and	the	subsequent	
     of	BCG.	The	different	levels	of	protection	observed	amongst	the	five	               slaughter	of	animals	testing	positive	(reactors).	
     vaccinated	groups	did	not	relate	to	post-vaccination	interferon-gamma	                   In	 this	 study	 we	 selected	 naturally	 infected	 field	 cattle	 of	 differ-
     (IFN-γ)	levels	released	from	bovine	PPD-stimulated	blood	cultures.		                ent	ages	and	breeds	and	housed	them	in	secure	accommodation	and	
     Key words:	Bovine,	tuberculosis,	vaccine                                            investigated	the	effects	of	repeat	skin	testing.	During	the	study	period,	
     Specie:	ruminants                                                                   animals	were	subjected	to	repeat	skin	testing	at	a	minimum	interval	of	
                                                                                         60	days	in	line	with	EU		field	practice	(mean	number	of	tests	per	animal	
        bV039. EFFECTS OF CULTURE CONDITIONS AND                                         =	2.9).	Regular	blood	samples	were	taken	for	cytokine	testing	and	ani-
                                                                                         mals	were	thoroughly	examined	at	post-mortem	with	tissues	examined	
     TUBERCULIN SOURCE ON INTERFERON-γ PRODUCTION IN
                                                                                         by	culture	and	histopathology	for	the	presence	of	BTB.	
     WHOLE BLOOD CULTURES FROM MyCOBACTERIUM BOVIS
                    INFECTED CATTLE                                                            Of	the	30	cattle	tested,	21	(70%)	showed	a	drop	of	at	least	3mm	
                                                                                         between	 the	 disclosing	 and	 final	 SICTT.	 Eight	 (27%)	 of	 these	 reac-
      IREnE	SCHILLER1,	RAy	WATERS2,	MARTIn	VoRDERMEIER3,	
                                                                                         tors	 became	 inconclusive	 under	 the	 standard	 test	 interpretation.	 Of	
     MITCHELL	PALMER2,	TEKLU	EGnUnI3,	RoLAnD	HARDEGGER1,	
                                                                                         fourteen	 animals	 that	 have	 so	 far	 been	 culled,	 all	 have	 been	 shown	
          AnnIKA	KyBURZ1,	ALEx	RAEBER1,	BRUno	oESCH1
                                                                                         to	be	positive	for	BTB.	These	data	suggest	that	animals	may	become	
        1Prionics	AG,	Schlieren,	Switzerland;	2National	Animal	Disease	                  desensitised	to	bovine	PPD	with	repeat	testing	(although	it	is	accepted	
         Center,	Ames,	United	States;	3Veterinary	Laboratory	Agency,	                    that	the	study	animals	would	have	been	removed	from	the	herd	follow-
                           Addlestone,	Great	Britain		                                   ing	their	disclosing	test	result).
                         irene.schiller@prionics.com
                                                                                               Investigations	 into	 a	 possible	 mechanism	 for	 this	 effect	 have	
           The	 BOVIGAM®	 interferon	 (IFN)	 -	 γ	 assay	 constitutes	 an	 ante-         shown	 that	 production	 of	 the	 anti-inflammatory	 cytokine	 Interleukin-
     mortem,	in	vitro	laboratory-based	tuberculosis	test	and	is	widely	used	             10	 increases	 following	 the	 SICTT.	 We	 did	 not	 observe	 a	 consistent	
     complementary	to	the	tuberculin	skin	test.	The	assay	is	performed	in	               decrease	 in	 Interferon-γ	 production	 following	 peaks	 in	 IL-10.	 Work	is	
     two	 stages:	 firstly,	 whole	 blood	 is	 cultured	 with	 antigens	 stimulating	    continuing	 to	 look	 at	 the	 possibility	 that	 numbers	 of	 IL-10	 producing	
     blood	 leucocytes	 to	 produce	 IFN-γ	 which	 is	 quantified	 by	 ELISA	 in	 a	     cells	 do	 not	 fall	 sufficiently	 before	 the	 next	 SICTT	 thus	 suppressing	
     second	 step.	 Environmental	 conditions	 before	 and	 during	 the	 cultur-         the	 delayed-type	 hypersensitivity	 response	 which	 would	 normally	 be	
     ing	of	the	leucocytes	influence	the	efficacy	of	in	vitro	IFN-γ	production.	         observed	in	BTB	infected	cattle.
     Optimal	conditions	are	therefore	essential.	In	this	study	we	analyzed	
                                                                                         Key words:	Tuberculosis;	Cattle;	Interleukin-10;	Skin-test
     the	effect	of	stimulation	vessel	geometry,	temperatures	during	stimula-
                                                                                         Species:	ruminants
     tion	and	the	stability	of	antigens	stored	at	different	temperatures.	Blood	
     from	 experimentally	 infected	 cattle	 and	 from	 tuberculosis-negative	
     cattle	was	stimulated	in	24-well	tissue	culture	trays	(standard),	48-well	             bV041. TRUNCATED E2 GLICOPROTEIN ExPRESSION
     and	96-well	culture	plates	with	the	following	antigens:	Purified	protein	              IN CHO-K1 CELLS TO PRODUCE A SUBUNIT VACCINE
     derivate	from	Mycobacterium	bovis	(PPD-B)	and	from	Mycobacterium	                                      AGAINST BVDV
     avium	(PPD-A),	a	fusion	protein	from	early	secretory	antigenic	target	6	              SEBASTIán	M	CHIAVEnnA1,	AGUSTÍn	oSTACHUK1,	AnDREA	
     (ESAT-6)	and	culture	filtrate	protein	10	(CFP-10),	and	pokeweed	mito-                   PECoRA1,	M	SUSAnA	LEVy2,	MARInA	J	DUS	SAnToS1,	
     gen.	 Stimulation	 was	 equally	 efficient	 in	 all	 three	 plate	 formats.	 The	                   AnDRéS	WIGDoRoVITZ1
     results	with	specific	antigens	correlate	with	mitogen	induced	stimula-
                                                                                                1Instituto	de	Virología,	CICVyA,	INTA-Castelar,	Argentina;	
     tion.	CO2	is	not	required	during	incubation,	as	cultures	from	an	incuba-
     tor	 with	 5%	 CO2	 produced	 similar	 amounts	 of	 IFN-γ	 as	 without	 CO2.	                             2Biogénesis-Bago	,	Argentina	
     However,	the	temperature	used	for	stimulation	was	critical:	Stimulation	                                  schiavenna@cnia.inta.gov.ar
     at	 37	 °C	 and	 33	 °C	 were	 equally	 efficient,	 but	 a	 culture	 temperature	         Bovine	Viral	Diarrhea	Virus	(BVDV)	is	responsible	for	worldwide	
     of	 29	 °C	 reduced	 IFN-γ	 production	 significantly.	At	 25	 °C	 and	 22	 °C	     economic	 losses	 in	 cattle	 population	 mainly	 due	 to	 reproductive	 fail-
     no	 stimulation	 was	 detectable.	Antigens	 are	 usually	 stored	 at	 2-8	 °C	      ure.	 Control	 of	 fetal	 BVDV	 infection	 through	 vaccination	 is	 critical	 to	
     (tuberculins)	or	at	-80	°C	(recombinant	proteins)	until	usage.	We	tested	           avoid	the	appearance	of	persistently	infected	calves	at	birth.	Current	
     in	 parallel	 antigen	 storage	 of	 recombinant	 proteins	 (ESAT-6:CFP-10	          vaccines	use	inactivated	virus	as	immunogen.	However,	viral	antigen	
     fusion	 protein,	TB10.4,	TB27.4,	 MPB83)	 at	 4	 °C	 for	 24h	 or	 at	 20	 °C	      production	in	cell	cultures	is	difficult	and	may	affect	the	effectiveness	
     for	8h	prior	to	use	in	cell	culture.	Our	results	show	that	antigens	may	            of	 the	 resulting	 vaccines.	 To	 overcome	 this	 problem,	 we	 decided	 to	
     be	stored	at	either	of	these	conditions	without	affecting	the	efficacy	of	          develop	a	truncated	version	of	BVDV	E2	glicoprotein	expressed	in	a	
     stimulation.	Finally,	we	compared	the	activities	of	tuberculins	from	five	          mammalian	cell	system	to	be	tested	as	immunogen	for	production	of	
     different	 sources	 in	 naturally	 infected	 cattle	 (n	 =	 10).	 Matched	 PPD-     an	experimental	subunit	vaccine.	Here	we	report	the	development	of	a	
     B	and	PPD-A	tuberculins	were	used	at	eight	dilutions	each.	Relative	                Chinese	Hamster	Ovary	(CHO-K1-E2T)	cell	line	which	is	stably	trans-
     potency	 30	 (RP30)	 was	 defined	 as	 the	 tuberculin	 concentration	              fected.	 It	 expresses	 a	 truncated	 version	 of	 E2	 (E2T).	 Recombinant	
     required	 to	 induce	 30%	 of	 the	 peak	 response	 values.	 RP30	 differed	        E2T	 lacks	 the	 transmembrane	 domain	 of	 the	 native	 protein	 and	 it	 is	
     by	a	factor	of	more	than	10	between	the	PPD-B	with	the	highest	and	                 fused	to	a	secretion	peptide	to	allow	recovery	of	the	recombinant	pro-
                                                                                                                                                                      67


tein	in	cell	culture	supernatants.	Transcriptional	fusion	to	a	C-terminal	          dairy	 cattle	 infected	 with	 MAP	 develop	 regulatory	 T	 cells	 capable	 of	
histine	 tag	 also	 permits	 its	 purification	 by	 immobilized	 metal	 affinity	   producing	 IL-10	 (Tr1)	 or	TGFβ	 (Th3),	 thereby	 limiting	 peripheral	 and	
chromatography	 (IMAC).	 CHO-K1-E2T	 supernatants	 were	 analyzed	                  tissue-specific	TH1	 immune	 responses.	 In	 support	 of	 this	 hypothesis,	
and	a	recombinant	protein	of	the	expected	electrophoretic	mobility	was	             our	 recent	 studies	 demonstrated	 that	 stimulation	 of	 peripheral	 blood	
detected	using	a	monoclonal	antibody	against	E2	(CA3)	as	probe.The	                 mononuclear	 cells	 (PBMCs)	 from	 subclinical	 MAP-infected	 cattle	
E2T	was	purified	from	culture	supernatant	by	IMAC	and	it	was	quanti-                results	 in	 enhanced	 expression	 of	 IL-10	 mRNA.	 Furthermore,	 IL-10	
fied	using	a	colorimetric	assay.	This	protein	was	used	as	standard	for	             suppresses	IFN-γ	expression	in	MAP	antigen-reactive	effector	T	cells,	
cuantification	of	the	E2T	obtained	in	each	cell	culture	using	an	ELISA	             suggesting	 that	 IL-10	 up	 regulation	 favors	 MAP	 proliferation	 in	 mac-
assay.	 Preliminary	 results	 obtained	 using	 a	 guinea-pig	 experimental	         rophages.	 Depletion	 studies	 in	 MAP-infected	 cattle	 also	 revealed	
model	indicate	the	ability	of	the	recombinant	E2T	to	induce	BVDV-spe-               that	 the	 MAP-responsive	 and	 IL-10	 producing	 T	 cell	 population	 is	
cific	antibodies.	Further	testing	is	currently	being	conducted	to	obtain	           likely	 CD4+	 and	 CD25+.	 In	 order	 to	 test	 our	 hypothesis,	 we	 have	
a	more	detailed	characterization	of	the	immunogenic	properties	of	this	             first	begun	to	profile	bovine	regulatory	T	cells	in	healthy	cows	(n	=	4).	
BVDV	subunit	antigen.                                                               Using	 primary	 and	 secondary	 monoclonal	 antibodies	 including	 anti	
Key words:	BVDV,	Vaccine,	Mammalian	system                                          (aCD4,	 aCD25,	 aCD45RO,	 aCD8,	 and	 aIL-10,	 dual	 flow	 cytometric	
Species:	ruminants                                                                  analyses	of	PBMCs	were	performed.	Preliminary	results	indicate	that	
                                                                                    resting	bovine	PBMCs	are	composed	of	2.7%	of	CD4+CD25+	T	cells,	
                                                                                    28.9%	of	CD4+CD45RO+	T	cells,	3.3%	of	CD4+IL-10+	T	cells,	3.0%	
     bV042. BLS PROTEIN AS BRV ANTIGEN DELIVERy
                                                                                    of	 CD8+CD25+	 T	 cells,	 26.8%	 of	 CD8+CD45RO+	 T	 cells,	 13.3%	 of	
         SySTEM IN A SUCKLING MICE MODEL.
                                                                                    CD25+CD45RO+	T	 cells,	 and	 3.3%	 of	 CD25+IL-10+	T	 cells.	 Further	
    DEMIAn	BELLIDo1	,	PATRICIo	o	CRAIG2,	MARInA	V	                                  analysis	of	regulatory	T	cells	in	MAP	infected	and	healthy	control	cattle	
MoZGoVoJ1,	DIEGo	D	GonZALEZ1,	AnDRES	WIGDoRoWITZ1,	                                 is	in	progress	using	additional	antibodies,	such	as	aFoxp3	(a	transcrip-
     FERnAnDo	GoLDBAUM2,	MARIA	J	DUS	SAnToS1                                        tion	factor	specific	for	regulatory	T	cells	capable	of	differentiating	them	
    1Laboratorio	de	Biología	Molecular,	Instituto	de	Virologia,	INTA	               from	 Th2	 cells),	 and	 three-color	 flow	 cytometry.	 Characterization	 of	
    Castelar,	Argentina;	2Laboratorio	de	Inmunología	Estructural	y	                 MAP-responsive	regulatory	T	cells	is	essential	to	determine	their	role	
           Molecular.	Fundación	Instituto	Leloir,	Argentina	                        in	limiting	appropriate	TH1	immune	responses	in	MAP-infected	cattle	
         1Corresponding	Author:	demianbellido@gmail.com                             and	determining	their	role	in	progression	to	clinical	disease.
      Brucella	 spp	 lumazine	 synthase	 (BLS)	 presents	 a	 very	 resistent	       Key words:	 Paratuberculosis,	 Regulatory	 T	 cells,	 Mycobacterium	
and	high	order	decameryc	structure.	It	allows	the	fusion	of	peptides	in	            avium	paratuberculosis,	Cattle
its	10	N-terminus,	giving	chimeric	proteins	with	high	order	arrangement	            Species: ruminants
that	are	able	to	induce	efficient	immune	responses.	The	objective	of	this	
work	is	to	produce	stable	fusion	proteins	when	bigger	protein	domains	               bV044. CLONING AND CHARACTERIZATION OF BIGHORN
are	inserted	in	the	BLS	structure	and	to	evaluate	the	immune	response	                SHEEP INFLAMMATORy CyTOKINES INTERLEUKIN-1β,
induced	 by	 the	 chimeric	 protein.	 We	 believe	 that	 	 Bovine	 Rotavirus	          TUMOR NECROSIS FACTOR-α, AND INTERLEUKIN-8.
(BRV)	 VP8	 protein	 is	 an	 accurate	 protein	 to	 be	 fused	 to	 BLS	 based	
on	its	physical,	chemical	and	immunological	properties.	The	inner	part	              CARoLInE	n	HERnDon,	RoHAnA	P	DASSAnAyAKE,	WEIGUo	
of	 VP8,	 VP8d,	 is	 a	 high	 structured	 soluble	 domain	 that	 preserve	 the	         LIU,	WILLIAM	J	FoREyT,SUBRAMAnIUM	SRIKUMARAn	
sialic	acid	binding	site	and	the	relevant	epitopes	wich	induce	neutral-               Department	of	Veterinary	Microbiology	and	Pathology,	Washington	
izing	antibodies.	The	present	work	shows	that	the	BLS-VP8d	chimera	                          State	University,	Pullman,	WA	99164-7040,	USA.			
is	able	to	efficiently	stimulate	the	immune	system.	The	immunological	                                  cherndon@vetmed.wsu.edu
properties	of	the	chimeric	protein	and	its	capability	for	inducing	passive	               Pneumonia	caused	by	the	Gram	negative	bacterium	Mannheimia	
protection	 were	 tested	 in	 a	 suckling	 mice	 model.	These	 results	 were	       haemolytica	is	a	devastating	disease	to	the	bighorn	sheep	population	
compared	 with	 those	 obtained	 with	 the	 single	 VP8d	 protein.	 Dams	           in	North	America.		Although	M.	haemolytica	can	cause	pneumonia	in	
immunized	with	the	chimeric	protein	gave	birth	to	pups	that	after	chal-             both	 domestic	 and	 bighorn	 sheep,	 the	 latter	 are	 more	 susceptible	 to	
lenge	 with	 BRV	 C486	 [P1]G6	 showed	 100%	 protection	 level;	 on	 the	          this	 disease	 than	 are	 domestic	 sheep.	 	 Pro-inflammatory	 cytokines	
other	hand,	pups	born	to	dams	immunized	with	VP8d	presented	a	level	                have	been	implicated	in	the	pathogenesis	of	a	number	of	lung	diseases	
of	protection	of	30%.	The	antibody	pattern	was	also	significatly	different	         of	both	humans	and	animals.	Studies	in	cattle	have	shown	that	alveolar	
in	both	experimental	 groups,	 as	it	 was	 evaluated	 by	 ELISA	 in	 serum	         macrophages,	 upon	 stimulation	 with	 the	 lipopolysaccharide	 (LPS)	 of	
and	milk.	                                                                          M.	 haemolytica,	 secrete	 pro-inflammatory	 cytokines	 IL-8,	 IL-1β,	 and	
Key words:	 BLS	 (Brucella	 Lumazine	 Synthase),	Bovine	 Rotavirus,	                TNF-a.	 	 IL-8	 attracts	 polymorphonuclear	 leukocytes	 (PMNs)	 into	 the	
VP8,	sukcling	mice	model                                                            lungs,	 and	 IL-1β	 and	 TNF-a	 enhance	 the	 expression	 of	 β2-integrins	
Species:	ruminants                                                                  by	 PMNs.	 	 Enhanced	 expression	 of	 β2-integrins	 on	 PMNs	 results	 in	
                                                                                    enhanced	cytolysis	by	leukotoxin.	We	hypothesize	that	bighorn	sheep	
    bV043. CHARACTERIZATION OF MyCoBACTERIUM                                        are	more	susceptible	to	M.	haemolytica	pneumonia	than	are	domestic	
  AVIUM SUBSP. PARATUBERCULoSIS ANTIGEN-SPECIFIC                                    sheep	because	of	an	enhanced	pro-inflammatory	cytokine	response.	             	
                REGULATORy T CELLS.                                                 The	 long	 term	 goal	 of	 this	 study	 is	 to	 compare	 the	 pro-inflammatory	
                                                                                    cytokine	secretion	by	alveolar	macrophages	of	bighorn	and	domestic	
    DEnISE	E	DE	ALMEIDA,	CHRISToPHER	J	CoLVIn,PAUL	M	
                                                                                    sheep	 in	 response	 to	 M.	 haemolytica	 infection.	 Characterization	 of	
                       CoUSSEnS
                                                                                    the	 cytokine	 response	 of	 bighorn	 and	 domestic	 sheep	 necessitates	
    	Molecular	Pathogenesis	Laboratory,	Dept.	of	Animal	Science,	                   the	cloning	and	sequencing	of	cDNA	encoding	IL-8,	IL-1β,	and	TNF-
         Michigan	State	University,	East	Lansing,	MI,	USA.                          α,	 which	 forms	 the	 objective	 of	 this	 study.	 	 Bighorn	 sheep	 alveolar	
      Johne’s	disease	is	caused	by	Mycobacterium	avium	subspecies	                  macrophages	and	peripheral	blood	mononuclear	cells	were	stimulated	
paratuberculosis	 (MAP)	 and	 is	 one	 of	 the	 most	 costly	 infectious	 dis-      with	LPS	from	Escherichia	coli.		Total	RNA	was	extracted	and	cDNA	
eases	in	dairy	cattle.	MAP	is	an	intracellular	pathogen	that	survives	in	           was	 made	 by	 RT-PCR.	 The	 PCR-amplified	 cDNA	 was	 cloned	 into	 a	
host	intestinal	macrophages	leading	to	chronic	enteritis	in	ruminants.	             mammalian	expression	vector	and	sequenced.	The	cDNA	of	bighorn	
Following	 initial	 exposure	 to	 MAP,	 a	T	 helper	 (TH)	 1	 response	 devel-      sheep	IL-1β	encodes	266	amino	acids	and	exhibits	99%,	92%,	60%,	
ops.	However,	during	the	late	subclinical	phase	of	MAP	infection,	this	             and	59%	identity	to	ovine,	bovine,	human,	and	murine	IL-1β,	respec-
pro-inflammatory	TH1	response	is	lost,	and	an	antibody-mediated	TH2	                tively.		The	cDNA	of	bighorn	sheep	TNF-α	encodes	235	amino	acids	
response	becomes	predominate,	but	is	ineffective.	The	immunological	                and	exhibits	96%,	88%,	and	76%	identity	to	ovine,	bovine,	and	human	
mechanisms	responsible	for	suppression	of	MAP-specific	TH1	immune	                  TNF-α,	 respectively.	 	 The	 cDNA	 of	 bighorn	 sheep	 IL-8	 encodes	 102	
responses	are	unknown.	However,	understanding	these	mechanisms	                     amino	acids	and	exhibits	98%,	94%,	and	80%	identity	to	ovine,	bovine,	
is	critical	to	accurately	diagnose	Johne’s	disease	and	for	development	             and	human	IL-8,	respectively.		The	cloned	cytokines	are	currently	being	
of	effective	therapies	and	vaccines.	The	hypothesis	of	this	study	is	that	          tested	in	bioassays.		Availability	of	IL-1β,	TNF-a,	and	IL-8	will	help	in	
68


     the	characterization	of	the	pro-inflammatory	cytokine	response	of	big-               ceptible	murine	cell-line	susceptible	to	Lkt-induced	cytolysis,	we	have	
     horn	and	domestic	sheep	in	M.	haemolytica-induced	pneumonia.                         previously	 demonstrated	 that	 CD18	 mediates	 Lkt-induced	 cytolysis.	          	
     Key words:	bighorn,	inflammatory,	cytokine,	cloning                                  However,	 in	 that	 study,	 bovine	 CD18	 was	 expressed	 as	 a	 heterodi-
     Species:	ruminants                                                                   mer	with	murine	CD11a	which	precluded	the	elucidation	of	the	role	of	
                                                                                          bovine	CD11a.		Therefore,	the	objective	of	this	study	was	to	precisely	
                                                                                          identify	the	role	of	bovine	CD11a	and	CD18	in	Lkt-binding	and	cytolysis	
      bV045. ExPRESSION OF IL-8 AND MIP-1α CHEMOKINES
                                                                                          of	target	cells.		cDNA	for	bovine	CD11a	and	CD18,	either	individually	
     IN HELA CELLS INFECTED WITH CAMPyLoBACTER FETUS
                                                                                          or	together,	was	transfected	into	the	human	embryonic	kidney	cell-line	
                      SUBSP. VENEREALIS                                                   (HEK-293)	 which	 does	 not	 express	 any	 β2-integrins.	 	 Transfectants	
     APC	CoTToRELLo1,	TM	ALVES2,	MM	MACHADo3,	BS	ARAUJo4,	                                stably	 expressing	 monomeric	 CD11a,	 CD18,	 or	 heterodimeric	 LFA-1	
             EFB	STAnCIoLI5,	RL	SAnToS6,	AP	LAGE6                                         (CD11a/CD18)	on	their	cell	surface	were	selected	by	flow	cytometric	
      1Post	Doc	–	Escola	de	Veterinária	–	UFMG;	2PhD	student	–	Escola	                    analysis	 with	 monoclonal	 antibodies	 specific	 for	 bovine	 CD11a	 or	
       de	Veterinária	–	UFMG;	3Master	student	–	Escola	de	Veterinária	–	                  CD18.	 In	 Lkt-binding	 assays,	 all	 three	 transfectants,	 but	 not	 the	 par-
     UFMG;	4Grad	student	–	Escola	de	Enfermagem	–	UFMG;	5Professor	                       ent	 cells,	 effectively	 bound	 Lkt.	 	 However,	 Lkt-induced	 cytolysis	 was	
          –	ICB	–	UFMG;	6Professor	–	Escola	de	Veterinária	–	UFMG                         observed	only	with	transfectants	expressing	monomeric	CD18	or	LFA-
            Bovine	 genital	 campylobacteriosis,	 caused	 by	 Campylobacter	              1.	Furthermore,	intracellular	[Ca2+]i	elevation	following	exposure	to	Lkt,	
     fetus	 subsp.	 venerealis	 (Cfv),	 is	 a	 venereal	 disease	 responsible	 for	       which	is	acknowledged	as	an	indication	of	Lkt-receptor	interaction,	was	
     economical	losses	in	countries	like	Brazil,	where	artificial	insemination	           seen	 only	 with	 transfectants	 expressing	 monomeric	 CD18	 or	 LFA-1.	         	
     is	still	restricted	to	a	small	percentage	of	herds.	Although	there	are	sev-          Taken	together,	these	results	clearly	indicate	that	it	is	the	CD18	subunit	
     eral	previously	published	studies	on	the	host	humoral	response	against	              that	 is	 involved	 in	 the	 Lkt-induced	 cytolysis	 of	 target	 cells.	 	Although	
     this	organism,	no	information	is	available	on	the	interaction	of	Cfv	with	           CD11a	binds	to	Lkt,	it	is	not	involved	in	the	events	leading	to	intracel-
     host	 epithelial	 cells	 in	 vitro	 or	 in	 vivo	 as	 well	 as	 about	 chemokines	   lular	[Ca2+]i	elevation	and	cytolysis.	The	LFA-1	expression	in	the	trans-
     involved	 in	 the	 innate	 immune	 response.	 The	 aim	 of	 this	 study	 was	        fectants	was	stable	for	longer	periods	than	either	one	of	the	subunits	
     to	 evaluate	 the	 kinetics	 expression	 of	 two	 inflammatory	 chemokines,	         suggesting	that	association	of	the	two	subunits	was	necessary	for	their	
     IL-8	and	MIP-1α,	in	HeLa	cells	infected	with	Cfv.	The	strains	used	in	               stable	expression.	
     this	 study	 were	 Cfv	 NCTC	 10354	 and	 strain	 PN,	 which	 was	 isolated	         Key words:	Mannheimia	haemolytica,	Leukotoxin,	Receptor,	C18
     seven	days	after	vaginal	inoculation	of	a	heifer	with	Cfv	NCTC	10354.	               Species: ruminants
     These	strains	were	incubated	with	cells	for	0.5,	1,	2,	4,	6,	8,	and	12	
     hours.	After	RNA	extraction	and	cDNA	synthesis,	PCR	for	amplification	                    bV047. DETECTION OF ANTIBODIES TO oVINE
     of	IL-8	(IL-8F-	5’CTT	GGC	AGC	CTT	CCT	GAT	TT	–	3’,	IL-8R-	5’	TCA	                     HERPESVIRUS-2 INTERLEUKIN-10HOMOLOGUE IN SHEEP-
     AAA	ACT	TCT	CCA	CAA	CC	3’)	and	MIP-1a	(MIP-1aF	-	5’ACC	ATG	                               ASSOCIATED MALIGNANT CATARRHAL FEVER
     GCT	CTC	TGC	AAC	CA-3’,	MIP-1aR-5’TGT	GGA	GGT	CAC	ACG	CAT	
     GTT-3’)	was	performed.	IL-8	mRNA	transcription	was	evident	in	HeLa	                      RoBIn	L	CISSELL1,	SHAHIRA	ABDEL	WAHAB2,	RoBERT	L	
     cells	 within	 half	 an	 hour	 of	 exposure	 to	 Cfv.	 Induction	 was	 maximal	                     DonnELL3,STEPHEn	A	KAnIA2
     after	 2	 and	 8	 hours	 of	 incubation	 for	 PN	 and	 NCTC	 10354	 strains,	          University	of	Tennessee	Veterinary	Teaching	Hospital,	Knoxville;	
     respectively.	The	PN	strain	induced	higher	levels	of	IL-8	mRNA	(P	<	                 2Department	of	Comparative	Medicine;	3Department	of	Pathobiology;	
     0.05)	when	compared	to	NCTC	10354	from	half	an	hour	up	to	4	hours	                           1Comparative	and	Experimental	Medicine	Program
     after	infection.	MIP-1a	expression	occurred	later	in	cells	infected	by	PN	                 Interleukin-10	 (IL-10)	 interferes	 with	 monocyte	 and	 macrophage	
     strain,	with	maximal	induction	12	hours	after	infection.	Infection	of	HeLa	          activation	of	Th1	helper	lymphocyte	production	of	nitric	oxide	and	syn-
     cells	with	the	NCTC	10354	strain	did	not	resulted	in	induction	of	MIP-               thesis	of	various	inflammatory	mediators.		An	IL-10homologue	(vIL-10)	
     1α	in	the	period	studied.	This	later	expression	of	MIP-1α	compared	to	               is	produced	by	several	herpes	viruses	and	is	hypothesized	to	help	the	
     IL-8	was	expected	since	it	acts	as	chemoattractant	for	monocytes	and	                virus	 down	 regulate,	 and	 thus	 evade,	 host	 immune	 responses.	 	 The	
     macrophages,	which	is	in	good	agreement	with	the	histological	findings	              gammaherpes	rhadinovirus	ovine	herpesvirus-2	(OvHV-2)	encodes	an	
     in	infected	heifers.	IL-8	expression	was	higher	(P	<	0.05)	than	MIP-1α	              IL-10	like	molecule	highly	homologous	to	mammalian	IL-10.		This	virus	
     expression	in	the	majority	of	incubation	times	studied	for	both	strains.	            causes	 sheep-associated	 malignant	 catarrhal	 fever	 (MCF),	 the	 most	
     In	conclusion,	these	data	suggest	that	these	chemokines	are	important	
                                                                                          common	form	of	MCF	in	the	United	States.		MCF	is	a	lymphoprolifera-
     in	the	initial	immune	response	against	C.	fetus	subsp.	venerealis.
                                                                                          tive	and	inflammatory	syndrome	which	has	delayed	clinical	presenta-
     Key words:	C.	fetus	subsp.	Venerealis,	chemokines	expression,	HeLa	                  tion	 hypothesized	 to	 be	 influenced	 by	 the	 production	 of	 vIL-10.	 	 For	
     cells                                                                                this	 study,	 the	 gene	 encoding	 vIL-10,	 Ov2.5	 ORF,	 was	 amplified	 by	
     Species:	ruminants                                                                   PCR,	cloned,	sequenced,	and	a	predicted	30	amino	acid	segment	from	
                                                                                          the	amino	terminus		synthesized.		This	synthetic	peptide	was	used	to	
          bV046. MONOMERIC ExPRESSION OF BOVINE β2-                                       develop	a	novel	direct	enzyme-linked	immunosorbant	assay	(ELISA)	to	
           INTEGRIN SUBUNITS CLARIFIES THEIR ROLE IN                                      detect	isotype-specific	antibodies	to	OvHV-2	vIL-10.		Work	to	date	indi-
         MANNHEIMIA HAEMOLyTICA LEUKOTOxIN-INDUCED                                        cates	that	lambs	do	not	have	detectable	levels	of	maternally	derived	
                         CyTOLySIS.                                                       antibody	to	vIL-10	during	the	first	few	weeks	of	age.		Ewes,	which	are	
                                                                                          refractory	 to	 clinical	 infection,	 generally	 have	 high	 levels	 of	 antibody	
         RoHAnA	P	DASSAnAyAKE,SUBRAMAnIAM	SRIKUMARAn
                                                                                          to	vIL-10.		A	weak	correlation	exists	between	the	vIL-10	ELISA	and	a	
       Department	of	Veterinary	Microbiology	and	Pathology,	Washington	                   commercially	 available	 competitive-inhibition	 ELISA	 (CI-ELISA).	 	 We	
              State	University,	Pullman,	WA	99164-7040,	USA.		                            believe	 the	 vIL-10	 ELISA	 will	 refine	 the	 ability	 to	 identify	 ruminants	
                       ssrikumaran@vetmed.wsu.edu                                         exposed	to	sheep-associated	MCF,	provide	an	important	tool	for	deter-
           β2-integrins	are	leukocyte-specific	integrins	that	facilitate	homing	          mining	the	role	of	vIL-10	in	disease	pathogenesis,	and	may	contribute	
     into	 areas	 of	 inflammation,	 phagocytosis,	 antigen	 presentation,	 and	          toward	 the	 development	 of	 a	 new	 vaccine	 strategy	 for	 the	 control	 of	
     cytotoxicity.		They	are	expressed	on	the	cell-surface	as	a	heterodimer	              malignant	catarrhal	fever.	
     composed	of	α	and	β	subunits.	The	common	β	subunit,	CD18,	associ-                    Key words:	viral	IL-10,	Malignant	Catarrhal	Fever,	Ovine	herpesvirus-
     ates	with	3	distinct	α	chains,	CD11a,	CD11b,	and	CD11c	to	give	rise	                 2,	IL-10homologue
     to	 3	 different	 β2-integrins:	 CD11a/CD18	 (lymphocyte	 function-associ-           Species:	ruminants
     ated	 antigen	 1,	 LFA-1),	 CD11b/CD18	 (complement	 receptor	 3,	 CR3	
     or	 Mac-1);	 CD11c/CD18	 (complement	 receptor	 4,	 CR4).	 	 Previous	
                                                                                                 bV048. IMMUNOPROTEOMIC ANALySIS OF THE
     studies	by	us	and	others	identified	β2-integrins	as	the	receptors	for	leu-
     kotoxin	(Lkt)	which	is	the	most	important	virulence	factor	produced	by	                     PROTECTIVE OUTER MEMBRANE FRACTION OF
     Mannheimia	(Pasteurella)	haemolytica,	the	primary	bacterial	pathogen	                                ANAPLASMA MARGINALE
     of	bovine	respiratory	disease	complex.		By	rendering	an	Lkt-non-sus-                                              WEnDy	C	BRoWn	
                                                                                                                                                                    69


  Department	of	Veterinary	Microbiology	and	Pathology,	Washington	                   subsp	venerealis	and	2	specific	for	C.	fetus	subsp	fetus.	None	of	the	
                State	University,	Pullman,	WA	99164	                                 clones	were	reactive	against	C.	sputorum	biovar	sputorum	LMG	6647.	
                     wbrown@vetmed.wsu.edu                                           All	clones	recognized	a	protein	with	molecular	mass	of	approximately	
      Rickettsial	 pathogens	 in	 the	 genera	 Anaplasma	 and	 Ehrlichia	            148	kDa	from	lised	C.	fetus	subsp.	venerealis	NCTC	10354.
cause	 acute	 infection	 in	 immunologically	 naïve	 hosts	 and	 are	 major	         Key words:	Monoclonal	antibodies,	C.	fetus	subsp.	venerealis,	cattle,	
causes	 of	 tick-borne	 disease	 in	 animals	 and	 humans.	 	 Immunization	          bovine	genital
with	Anaplasma	marginale	purified	outer	membranes	induces	complete	                  campylobacteriosis
protection	against	anaplasmosis	in	75%	of	cattle,	whereas	immunization	              Species:	ruminants
with	the	well-studied	and	immunodominant	major	surface	proteins	such	
as	MSP2	has	provided	little	or	no	protection.		The	completed	genome	                 bV050. CHARACTERIZATION OF THE IMMUNE RESPONSE
sequence	of	A.	marginale	facilitated	the	identification	of	subdominant	                 INDUCED By LIPOARABINOMANNAN (LAM) FROM
and	 less	 abundant	 immunogenic	 proteins	 in	 the	 outer	 membrane	                    MyCoBACTERIUM SP. IN VACCINATED CALVES
fraction,	 using	 two	 approaches.	 First,	 two-dimensional	 electrophore-
sis	 and	 immunoblotting	 of	 the	 outer	 membrane	 fraction	 with	 immune	              JoLLy	A,	STEMPLER	A,	CoLAVECCHIA	S,	FERnAnDEZ	E,	
bovine	 sera	 identified	 numerous	 antigenic	 protein	 spots.	Analysis	 of	                                  MUnDo	S			
individual	proteins	excised	from	the	gels	by	liquid	chromatography	and	                 Facultad	de	Ciencias	Veterinarias,	Universidad	de	Buenos	Aires	
tandem	mass	spectrometry	identified	21	novel	antigens.	Of	particular	                      Paratuberculosis,	caused	by	Mycobacterium	avium	subsp.	para-
interest	 is	 the	 finding	 that	 three	 proteins	 from	 the	 type	 IV	 secretion	   tuberculosis	(Map),	is	a	chronic	granulomatous	enteritis	in	cattle.	LAM	
system,	 conjugal	 transfer	 protein,	 VirB9,	 and	 VirB10	 were	 antigenic.	        is	the	most	abundant	polysaccharide	in	mycobacteria	cell	wall	and	also	
TFSS	 proteins	 form	 channels	 and	 are	 responsible	 for	 secretion	 or	           the	major	immunodominant	surface	antigen.		Currently	available	vac-
cell-to-cell	transfer	of	molecules	and	DNA-protein	complexes	in	other	               cines	do	not	provide	fully	protection	(1).	LAM	vaccines	were	tested	in	
gram-negative	bacterial	pathogens,	but	have	not	been	studied	as	vac-                 laboratory	animals	with	promising	results	(2).	In	order	to	evaluate	the	
cine	antigens.	These	proteins	were	expressed	and	shown	to	stimulate	                 use	of	LAM	as	a	vaccine	in	cattle,	this	work	characterizes	the	immune	
IgG2,	and	CD4+	T	cell	proliferation	and	IFN-γ	production,	responses	                 response	of	calves	immunized	with	LAM.	
associated	with	protective	immunity	in	outer	membrane	vaccinates.	A	
                                                                                          Mycobacterium	 avium	 subsp.	 avium	 were	 sonicated	 (S)	 and	 a	
second	 approach	 to	 more	 directly	 screen	 for	 antigens	 recognized	 by	
                                                                                     glycolipid	 fraction	 containing	 LAM	 (L)	 was	 obtained	 through	 phenol-
T	lymphocytes	involved	an	in	vitro	transcription	and	translation	(IVTT)	
                                                                                     chloroform	 extraction.	 Both	 extracts	 were	 emulsified	 with	 Freund’s	
of	 ORFs	 encoding	 proteins	 predicted	 to	 be	 localized	 on	 the	 outer	 or	
                                                                                     Incomplete	Adjuvant	(FIA)	and	used	to	immunize	3	months-calves.	Two	
inner	membrane	or	to	have	a	signal	peptide.	PCR	products	of	selected	
                                                                                     doses	of	immunogen	were	inoculated	at	day	0	and	35	(2	mg	of	total	
ORFs	 engineered	 to	 express	 antibody-binding	 sequence	 tags	 were	
                                                                                     carbohydrates	each)	in	S	group	(n	=	4)	and	L	group	(n	=	6).	Another	
amplified	and	expressed	using	IVTT.	As	proof	of	principal,	VirB9	and	
                                                                                     group	(n	=	3)	was	kept	as	negative	control	and	received	PBS-FIA	(AC	
outer	membrane	protein	(OMP)7,	OMP8,	and	OMP9,	known	to	stimu-
                                                                                     group).	Serum	samples	were	evaluated	at	day	0	and	60.	ELISA	was	
late	T	cell	responses	in	outer	membrane	vaccinates,	were	expressed	
                                                                                     performed	to	detect	total	and	isotypes	anti-LAM	antibodies	induced	by	
by	IVTT	and	affinity	purified	by	binding	to	anti-tag	antibody	coupled	to	
                                                                                     vaccination.	Cellular	immune	response	was	evaluated	in	vivo	at	day	60	
protein-G	bound	beads,	and	the	beads	were	added	to	APC	and	used	
                                                                                     by	Intradermal	reaction	test	(IDR),	using	bovine	PPD.	
to	stimulate	immune	CD4+	T	cell	proliferation.	This	novel	technology	
can	be	used	to	rapidly	screen	a	large	number	of	proteins	from	a	given	                    Anti-LAM	 antibodies	 were	 detected	 in	 all	 vaccinated	 calves.	 In	
pathogen	 for	 recognition	 by	 both	 antibody	 and	 T	 lymphocytes	 if	 the	        L	group,	83.3%	of	calves	showed	titers ≥2000,	whereas	all	calves	in	
genome	sequence	is	available.                                                        group	S	had	titers	≥2000.	Isotype	profile	analysis	is	shown	in	table	1.	
Key words:	 mass	 spectrometry,	 Anaplasma	 marginale,	 outer	                            Table	1.	Anti-LAM	antibodies	Profiles
membranes,	T	lymphocytes                                                                               IgM            IgG1       IgG2        IgA
Species:	ruminants
                                                                                      L	group        ≤0,001       0,177±0,030    ≤0,04       ≤0,04
                                                                                      S	group     0,247	±0,310 0,448±0,110 0,477±0,260       ≤0,07
  bV049. PRODUCTION AND CHARACTERIZATION OF
 MONOCLONAL ANTIBODIES AGAINST CAMPyLoBACTER                                              Results	are	expressed	as	DO	mean	value	after	AC	group	mean	
           FETUS SUBSP. VENEREALIS                                                   value	substraction	±	standard	error

   TELMA	MARIA	ALVES1,	LUIZ	GUILHERME	DIAS	HEnEInE2,	                                     Serum	samples	dilution	was	analyzed	at	1/100
     BáRBARA	SILVEIRA	ARAúJo1,	LUCIAnA	MARIA	SILVA3,	                                     No	positive	reactions	were	detected	by	IDR	in	L	vaccinated	group,	
   PATRÍCIA	CoTA	CAMPoS2,	MáRCIA	SILVA	HERMoGEnES3,	                                 while	all	S	vaccinated	calves	reacted.	
                 AnDREy	PEREIRA	LAGE1*                                                    Our	 results	 demonstrate	 that	 immunization	 with	 LAM	 is	 able	 to	
  1Laboratório	de	Bacteriologia	Aplicada	-	Núcleo	de	Pesquisa	em	                    induce	 a	 specific	 humoral	 immune	 response	 without	 interfering	 with	
 Saúde	Animal	-	Departamento	de	Medicina	Veterinária	Preventiva	                     IDR	test	usually	used	for	bovine	Tuberculosis	diagnosis.	It	has	been	
  -	Escola	de	Veterinária	-	Universidade	Federal	de	Minas	Gerais	-	                  described	that	IgG1	is	the	predominant	isotype	in	the	immune	response	
Belo	Horizonte	-	Minas	Gerais	–	Brazil;	2Laboratório	de	Imunologia	e	                to	LAM	of	infected	animals	(3).	Our	results	indicate	that	the	same	iso-
Bioprodutos	-	Fundação	Ezequiel	Dias,	Belo	Horizonte	Minas	Gerais	                   type	profile	is	obtained	when	LAM	is	inoculated	as	a	purified	antigen.		
 -	Brazil;		3Laboratório	de	Biologia	Celular	e	Molecular	-	Fundação	                      These	results	could	support	further	investigations	evaluating	LAM	
        Ezequiel	Dias,	Belo	Horizonte	Minas	Gerais	-	Brazil.                         as	a	candidate	for	a	Paratuberculosis	subunit	vaccine.	
      Myeloma	cells	Sp2/0-Ag14	and	spleen	cells	from	BALB/c	mouse	                        References
immunized	 with	 sonicated	 Campylobacter	 fetus	 subsp.	 venerealis	
                                                                                          1.	Uzonna	JE,	Vaccine	2003.		
NCTC	10354	were	fused	with	polyethylene	glycol	(PEG)	for	the	pro-
duction	 of	 monoclonal	 antibodies	 (MAb’s).	 Clones	 were	 obtained	 by	                2.	Hamasur	B,	Clin	Exp	Immunol	2004.	
limiting	 dilution	 and	 screened	 for	 specific	 MAb’s	 to	 C.	 fetus	 subsp.	           3.	Koets	AD,	Infect	and	Immun	2001.
venerealis	 NCTC	 10354	 by	 indirect	 ELISA	 and	 western	 blot	 against	           Key words:		(cattle),	(Paratuberculosis),	(LAM),	(subunit	vaccine)
a	panel	of	bacteria:	C.	fetus	subsp.	venerealis	NCTC	10354,	C.	fetus	                Species:	ruminants
subsp	fetus	ADRI	1812,	C.	sputorum	biovar	sputorum	LMG	6647,	C.	lari	
NCTC	11352,	and	Arcobacter	skirrowii	LMG	6621	for	the	ELISA	and	C.	
fetus	subsp.	venerealis	NCTC	10354	and	C.	sputorum	biovar	sputorum	                  bV051. BOVINE ROTAVIRUS VACCINES: IMMUNOGENICITy
LMG	6647	for	the	western	blotting.	Fifteen	clones	producing	MAb’s	anti	              OF TWO STRAINS OF BOVINE ROTAVIRUS IN CATTLE AND
–	C.	fetus	subsp.	venerealis	of	the	IgM	(1)	and	IgG	(14)	classes	were	                                 GUINEA PIGS
further	screened	for	species-specificity.	Four	clones	of	the	15	obtained	                VIVIAnA	PARREño1,	DAnIELA	RoDRIGUEZ1,	MERCEDES	
were	producers	of	species	specific	MAb’s:	2	were	specific	for	C.	fetus	                IZUEL2,	JoRGE	FILIPPI2,	LAURA	MARAnGUnICH3,	VIRGInIA	
70


       LóPEZ4,	FERnAnDo	FERnánDEZ1,	RoDoLFo	BELLInZonI2,	                                  a	decrease	in	number	of	positive	bulls	for	the	brucellosis	diagnosis	(2	
                         MARÍA	M.	VEnA2                                                    positive	animals).	On	the	other	hand,	when	used	the	seminal	plasma,	
         1Instituto	de	Virologia,	INTA,	Castelar;	2Biogenesis	Bagó	S.A.;	                  the	number	of	animals	with	positive	diagnosis	has	increased	(7	positive	
                           3UNTREF;	4UBA,		Argentina.		                                    animals).	This	study	results	highlight	the	immediate	necessity	to	also	
                        maria.vena@biogenesisbago.com                                      investigate	antibodies	to	Brucella	in	seminal	plasma.
           Bovine	 Rotavirus	 (BRV)	 is	 a	 main	 cause	 of	 severe	 diarrhea	 in	              Key	words:	Seminal,	Bovine,	Brucellosis,	Bulls
     neonatal	calves,	worldwide.	 In	Argentina	BRV	was	diagnosed	as	the	                        Species:	ruminants
     cause	of	neonatal	diarrhea	in	71	and	58%	of	the	outbreaks	registered	
     in	beef	and	dairy	farms,	respectively.	The	BRV	strain	typed	as	P[5]G6	
     was	 prevalent	 in	 beef	 herds,	 while	 P[11]	 was	 the	 prevalent	 P-type	
                                                                                             bV053. EFFICIENCy OF AN INDIRECT ELISA USING TWO
     (71%),	associated	in	similar	proportions	with	variants	of	G6	and	G10,	                   ANTIGEN PREPARATIONS OF BRUCELLA CANIS FOR
     in	 dairy	 herds	 (Garaioechea	 et	 al,	 2006).	 Prevention	 strategies	 are	                          IMMUNODIAGNOSIS
     based	 on	 the	 vaccination	 of	 pregnant	 cows	 in	 order	 to	 transfer	 high	          MARIA	Z	D	oLIVEIRA1,3*,	SonGELI	M	FREIRE3,	RoBERTo	
     titers	of	maternal	antibodies	(Ab)	to	the	calf	through	colostrum	intake.	               MEyER3,	LARA	KEID4,	STELLA	M	BARRoUIn-MELo1,2,	PALIS,	
     Three	experimental	vaccines	containing	10^7	FFU/dose	of	UK	P[5]G6,	                                              PAULo	
     or	B223	P[11]G10	or	both	strains	formulated	in	oil	adjuvant	were	tested	                   1Laboratório	de	Infectologia	Veterinária,	Escola	de	Medicina	
     together	with	a	commercial	vaccine	(Rotatec	J5®)	in	beef	cattle	(with	                 Veterinária,	Universidade	Federal	da	Bahia	(UFBA),	Av.	Ademar	de	
     low		Ab	titer	due	to	natural	RV	infection)	and	seronegative	guinea	pigs.	             Barros,	500,	Salvador,	Bahia,	Brasil,	CEP:40170-000;	2Departamento	
     Immune	response	was	evaluated	by	ELISA	and	virus	neutralization	test	                    de	Patologia	e	Clínicas,	Escola	de	Medicina	Veterinária,	UFBA;	
     (VN).	Bovine	trial	consisted	of	two	vaccinations	and	serum	sampling	at	               3Laboratório	de	Imunologia	e	Biologia	Molecular,	Instituto	de	Ciências	
     0,	30,	60	and	90	dpv.	whereas	guinea	pigs	received	one	or	two	doses	                  da	Saúde,	UFBA,	AvReitor	Miguel	Calmon	S/N	Vale	do	Canela	Salva
     of	 vaccine	 and	 were	 sampled	 weekly	 until	 60	 dpv.	The	 kinetic	 of	 RV	        dor,Bahia,Brasil,CEP40.110-100;	4Faculdade	de	Medicina	Veterinária	
     Ab	 responses	 by	 ELISA,	 mostly	 directed	 to	 VP6,	 was	 similar	 among	                        e	Zootecnia	da	Universidade	de	São	Paulo.	
     all	the	vaccines	tested	in	both	species,	with	seroconvertion	registered	                                     zoraida.vet@gmail.com
     at	60	dpv	in	cattle	and	at	21	dpv	in	guinea	pigs.	All	vaccinated	bovines	
     developed	VN	Abs	against	UK	independently	of	the	vaccine	formula-                            Introduction:	 Canine	 brucellosis	 is	 a	 zoonotic	 bacterial	 infection	
     tion,	 indicating	 the	 previous	 contact	 with	 that	 strain.	 In	 contrast	 only	   whose	clinical	diagnosis	is	difficult	to	perform	due	to	frequent	asymp-
     the	 groups	 vaccinated	 with	 vaccines	 containing	 B223	 RV	 developed	             tomatic	 cases.	 The	 disease	 is	 important	 for	 Public	 Health.	 Human	
     a	VN	Ab	response	to	that	strain,	suggesting	minimum	field	exposure	                   infections	 have	 been	 reported,	 being	 laboratory	 technicians	 and	 dog	
     to	 P[11]G10.	 In	 guinea	 pigs	 optimal	 Ab	 response	 was	 obtained	 by	            owners	the	most	exposed	to	B.	canis.	Having	a	worldwide	distribution,	
     experimental	 and	 commercial	 vaccines.	 The	 VN	 responses	 in	 naïve	              the	 infection	 represents	 a	 great	 cause	 of	 economic	 losses	 in	 breed-
     guinea	pig	allowed	to	demonstrated	that	there	is	no	cross	protection	                 ing	 kennels,	 since	 it	 causes	 abortion	 and	 infertility	 in	 dogs.	 Despite	
     between	UK	and	B223	strains	confirming	that	in	order	to	obtain	protec-                molecular	 techniques	 have	 been	 recently	 described,	 blood	 culture	
     tion	 against	 the	 prevalent	 RV	 types	 circulating	 in	 Argentina	 all	 P[5],	     is	 still	 considered	 the	 only	 routine	 definitive	 test	 for	 the	 diagnosis	 of	
     P[11],	G6	and	G10	types	should	be	included	in	the	vaccine.		                          canine	infection.	Different	serological	tests	are	routinely	used,	but	there	
                                                                                           is	a	need	for	a	fast,	reliable	and	low	cost	immunological	test	to	improve	
     Key words:	 bovine	 rotavirus,	 Cattle	 immune	 response,	 Guinea	 pig	
                                                                                           the	 immunodiagnosis	 and	 surveillance	 studies	 of	 canine	 brucellosis.	
     immune	response,	vaccines
                                                                                           The	aim	of	this	study	was	to	compare	two	antigen	preparations	made	
     Species:	ruminants;	other
                                                                                           from	 a	 culture	 of	 B.	 canis	 in	 ELISA	 tests	 using	 sera	 from	 dogs	 with	
                                                                                           distinct	profiles	of	infection,	defined	as	positive	and	negative.	Material	
         bV052. INVESTIGATION THE SEROPREVALENCE OF                                        and	 Methods:	 A	 B.	 canis	 strain,	 isolated	 from	 aborted	 placenta	 and	
          BOVINE BRUCELLOSIS USING BLOOD SERUM AND                                         fetuses	 of	 a	 bitch,	 was	 used	 for	 producing	 two	 antigen	 preparations:	
                       SEMINAL PLASMA                                                      a	heat	soluble	bacterial	extract	and	a	sonically	obtained	extract.	After	
       DAnILo	G	JUnqUEIRA	JúnIoR,	GILSon	P	MoRAES,	AnnA	                                   centrifugation	 steps,	 the	 soluble	 fractions	 of	 both	 preparations	 were	
     MCLIMA,	GERMAno	K	FARIA,	FERnAnDA	K	MARqUES,	PAULo	R	                                 applied	in	two	indirect	ELISA	tests,	performed	with	sera	from	85	dogs,	
                   oLIVEIRA,	CéSAR	A	GARCIA                                                for	detection	of	B.	canis-specific	IgG.	Fifty	one	from	these	85	animals	
         Faculdade	de	Medicina	Veterinária	da	Universidade	Federal	de	                     were	diagnosed	positive	for	canine	brucellosis,	by	blood	cultures	and	
                          Uberlândia-FAMEV-UFU		                                           serology	in	agar-gel	immunodiffusion	test	(AGID).	Thirty	four	of	these	
                        dan_hp2002@yahoo.com.br	                                           dogs	were	healthy	negative	animals,	with	no	history	of	breeding.	The	
                                                                                           optical	 density	 readings	 were	 used	 to	 calculate	 specificity,	 sensitivity	
           Brucellosis	 is	 an	 important	 public	 health	 problem	 is	 a	 zoonotic	       and	accuracy	of	the	ELISAs	by	ROC	analysis.	Results:	The	sensitivity,	
     disease	that	causes	economic	loss,	and	seen	all	over	the	world.	The	                  specificity	and	accuracy	were	of	100,	84.30	and	90.6%	for	the	ELISA	
     infection	 caused	 by	 members	 of	 genus	 Brucella,	 as	 male	 as	 female	           test	with	sonically	obtained	extract,	and	of	91.2,	100%	and	94.4%	for	
     bovine,	induces	humoral	and	cellular	immune	response.	The	humoral	                    the	 ELISA	 test	 with	 heat	 soluble	 extract,	 respectively.	 Conclusions:	
     response	against	natural	infection	characterized	by	almost	simultane-                 Both	ELISA	tests	using	the	different	antigen	preparations	obtained	from	
     ous	increasing	of	antibodies	(Ig)	concentration	of	kinds	IgM	and	IgG.	In	
                                                                                           a	wild	B.	canis	were	considered	applicable	for	serodiagnosis	of	canine	
     these	animals,	the	Ig	of	IgM	kind	reduces,	tending	towards	disappear-
                                                                                           brucellosis,	showing	good	specificity	and	sensitivity.
     ing	in	a	few	weeks	after	the	infection.	On	the	other	hand,	the	Ig	of	IgG	
     kind	established	continues	in	chronically	infected	animals.	The	official	             Key words:	Brucella	canis,	Immunodiagnostics,	ELISA,	Dog
     tests	 in	 Brazil	 are	 based	 in	 these	 antibody	 detections	 only	 in	 bovine	     Species:	canine	
     blood	serum.	Tamponated	acidified	antigen	test	(TAA	or	Rose-Bengal,	
     RB)	detects	the	IgG1	while	the	tube	agglutination	with	2-mercaptoeta-                   bV054. INHIBITION OF EHRLICHIA CANIS INFECTION By
     nol	(2-ME)	detects	IgG.	Although,	the	favored	place	of	Brucella	in	tes-                            INTERFERON GAMMA IN VITRo
     ticles	and	accessory	glans	in	male	bovine	can	form	low	serum	Ig	titles;	                     ToMoKo	TAJIMA1,	MAKoTo	WADA1,MISAo	onUMA2	
     which	makes	extremely	hard	the	diagnosis	by	serological	techniques	
     of	triage.	The	law	doesn´t	establish	any	test	for	Ig	detection	in	semen	               1Laboratory	of	Veterinary	Microbiology,	Graduate	School	of	Life	and	
     of	reproductive	bulls.	The	purpose	of	this	study	was	to	investigate	the	               Environmental	Science,	Osaka	Prefecture	University,	Osaka,	Japan;	
     seroprevalence	of	brucellosis	using	blood	serum	and	seminal	plasma	                     2Laboratory	of	Infectious	Disease,	Graduate	School	of	Veterinary	
     from	71	bulls	provided	of	8	properties	in	Uberlandia	County	–	MG	State,	                        Medicine,	Hokkaido	University,	Sapporo,	Japan		
     Brazil.	This	work	has	compared	the	results	of	two	tests	for	diagnosis	of	                         e-mail	address:	tajima@vet.osakafu-u.ac.jp
     bovine	brucellosis,	applying	the	tests	of	RB	(of	triage)	and	2-ME	(con-                    Ehrlichia	 canis	 infects	 to	 macrophages-monocytes	 of	 dog	 and	
     firmatory).	The	results	found	in	these	two	tests	present	a	negative	cor-              cause	 canine	 monocytic	 ehrlichiosis,	 a	 persistent	 infection	 that	 con-
     relation,	considering	that:	when	used	the	blood	serum,	it	has	presented	              tinues	 for	 a	 long	 period	 even	 after	 treatment	 with	 antibiotics.	 	 We	
                                                                                                                                                                     71


previously	 reported	 that	 expression	 of	 mRNA	 for	 interferon-γ	 (IFN-γ)	           Altogether	our	data	showed	that	mucosal	CD4+	T	cells	and	B	cells	
was	 detected	 in	 peripheral	 blood	 mononuclear	 cells	 (PBMC)	 from	 E.	        constitute	a	site	of	active	replication	of	the	virus	in	chronically	infected	
canis-infected	dogs	within	three	days	after	infection	and	continued	for	           cats.	 Our	 results	 emphasize	 the	 role	 of	 GALT	 in	 FIV	 pathogenesis,	
more	than	50	days.	To	determine	the	role	of	IFN-γ	in	E.	canis	infection,	          further	 reinforcing	 the	 homologies	 observed	 between	 FIV	 and	 HIV	
E.	canis	was	cultured	with	white	blood	cells	(WBC)	or	PBMC	from	E.	                infection.
canis-infected	 dogs.	 	 Two	 SPF	 female	 beagle	 dogs	 were	 inoculated	         Key words: FIV,	chronic	infection,	CD134,	GALT,	p24,	reservoir
intravenously	 with	 E.	 canis	 Oklahoma	 strain	 and	 the	 infection	 was	        Species: felines
confirmed	by	PCR	using	peripheral	blood	DNA	of	these	dogs	14	days	
post-inoculation	 (DPI).	 	 Blood	 was	 drawn	 from	 each	 dog	 on	 16	 DPI,	
                                                                                   bV056. MICROARRAy ANALySIS OF SySTEMIC PIG INNATE
and	isolated	WBC	or	PBMC	were	added	to	E.	canis-inoculated	DH82	
cells.		WBC	and	PBMC	from	an	uninfected	beagle	dog	were	used	as	                    IMMUNE RESPONSES AFTER ExPERIMENTAL INFECTION
negative	controls.		The	cultured	cells	were	collected	daily,	stained,	and	              WITH ACTINOBACILLUS PLEUROPNEUMONIAE
incidence	 of	 inclusion-positive	 cells	 was	 calculated	 by	 counting	 cells	       KERSTIn	SKoVGAARD,	SHILA	MoRTEnSEn,	KARIn	TARP	
under	 microscope.	 Incidence	 of	 inclusion-positive	 cells	 was	 signifi-          PoULSEn,	GREGERS	JUnGERSEn,	PETER	M	H	HEEGAARD
cantly	reduced	in	the	culture	with	WBC	or	PBMC	from	E.	canis-infected	                 National	veterinary	Institute,	Technical	University	of	Denmark,	
dogs	but	not	with	those	from	uninfected	dog.		Such	reduction	of	inclu-                       Bülowsvej	27,	DK-1790	Copenhagen	V,	Denmark		
sion-positive	cells	was	inhibited	by	addition	of	anti-dog	IFN-γ	antibody.	    	                              pmhh@vet.dtu.uk
On	the	other	hand,	cultured	supernatant	was	collected	and	assayed	for	
IFN-γ	by	ELISA.		Though	IFN-γ	was	detected	in	all	supernatant	from	                      Actinobacillus	 pleuropneumoniae	 (Ap)	 is	 the	 causative	 agent	
cultures	with	WBC	or	PBMC	from	infected	dogs,	the	concentration	was	               of	 porcine	 pleuropneumonia,	 a	 highly	 infectious	 respiratory	 disease.	
reduced	when	the	cells	were	cultured	with	anti-dog	IFN-γ	antibody.		To	            The	role	of	the	porcine	innate	immune	response	in	the	pathogenesis	
examine	the	direct	effect	of	IFN-γ,	recombinant	canine	IFN-γ	(r-IFN-γ)	            and	 clinical	 outcome	 of	 this	 infection	 remains	 poorly	 understood.	 In	
was	added	to	E.	canis-inoculated	DH82	cells	and	cultured.		Significant	            this	study	we	examined	the	innate	transcriptional	response	in	the	liver,	
reduction	 in	 incidence	 of	 inclusions	 is	 observed	 in	 E.	 canis-infected	    14	hours	after	inoculation	with	Ap	using	oligonucleotide	microarrays.	
DH-82	cells	cultured	with	r-IFN-γ.		Present	results	showed	an	impor-                    Specific	pathogen	free	pigs	(8-10-week-old)	were	challenged	with	
tant	role	of	IFN-γ	in	the	immunity	of	E.	canis	infection.	Counterbalance	          Ap	serotype	5B	(isolate	L20).	Microarrays	were	used	to	reveal	genes	
between	IFN-γ	activity	and	E.	canis	growth	may	lead	to	establishment	              being	 differentially	 expressed	 in	 liver	 tissue	 from	 infected	 animals	
of	persistent	infection.	Enhancement	of	IFN-γ	production	may	be	effec-             (N	=	10)	versus	liver	samples	from	non-infected	animals	(N	=	5).	
tive	to	prevent	persistent	infection	in	dogs.                                            The	microarray	used	in	this	study	(POM-4)	is	a	focused	low	density	
Key words:	Ehrlichia	canis,	interferon	gamma                                       in-house	 spotted	 microarray	 comprising	 immunologically	 relevant	 oli-
Species:	canine                                                                    gonucleotide	probes	corresponding	to	genes	coding	for	interferons	and	
                                                                                   interleukins	(and	receptors),	chemokines	(and	receptors),	acute	phase	
   bV055. INTESTINAL CD134+ T AND B LyMPHOCyTES:                                   proteins,	 apoptosis-related	 factors	 and	 sequences	 with	 relevance	 to	
   SITES FOR ACTIVE FIV REPLICATION IN CHRONICALLy                                 Toll-like	 receptors	 and	 their	 intracellular	 signalling	 pathways.	 Probes	
                    INFECTED CATS                                                  for	different	house	keeping	genes	are	also	included	for	normalisation	
                                                                                   purposes.	The	 microarray	 holds	 384	 different	 oligonucleotides	 repre-
    H	EL	GARCH1,	A	GoUBIER1,	H	PoULET1,	y	MUELLE,	S	
                                                                                   senting	more	than	200	different	immune-related	genes.
RICHARD1,	L	FoREST1,	L	CHAPAT1,	C	AnDREonI1,V	JUILLARD1
                                                                                        A	total	of	61	genes	were	found	to	be	significantly	(P<	0.05;	fold	
1Merial	S.A.S.,	Discovery	Research,	254	rue	Marcel	Mérieux,	69342	
                                                                                   change	>2)	differentially	expressed	in	liver	tissue	from	infected	animals	
                      Cedex	07	Lyon,	France
                                                                                   compared	 to	 control.	 Of	 these	 genes,	 36	 were	 expressed	 at	 a	 lower	
      Feline	Immunodeficiency	Virus	(FIV)	is	a	major	pathogen	of	cats,	            level	and	24	genes	were	expressed	at	a	higher	level.	
responsible	 for	 an	 acquired	 immuno-deficiency	 syndrome	 (AIDS),	
                                                                                        Among	the	differentially	expressed	genes	were	several	to	belong-
comparable	 to	 Human	 Immunodeficiency	 Virus	 (HIV)-associated	
                                                                                   ing	to	the	acute	phase	proteins	(APP).	C-reactive	protein,	fibrinogen,	
AIDS	in	humans.	Therefore,	not	only	is	FIV	a	major	issue	in	veterinary	
                                                                                   haptoglobin	and	serum	amyloid	A	were	up-regulated	and	alpha-1	acid	
medicine,	but	it	also	provides	a	natural	model	to	study	lentivirus	patho-
                                                                                   glycoprotein,	albumin,	apolipoprotein	A-1,	transferrin,	and	transthyretin	
genesis.	Entry	of	HIV	and	FIV	in	host	cells	follows	a	two-step	model	
                                                                                   were	 down-regulated	 in	 liver	 samples	 of	 infected	 animals	 compared	
in	 which	 binding	 to	 a	 primary	 receptor	 induces	 conformational	 rear-
                                                                                   to	 control	 animals.	 In	 addition,	 the	 expression	 of	 genes	 encoding	
rangements	 exposing	 the	 co-receptor	 binding	 site.	 Both	 viruses	 use	
                                                                                   pro-inflammatory	 mediators	 of	 the	 innate	 immune	 response	 includ-
CXCR4	as	a	co-receptor	but	differ	in	their	primary	receptor	(CD4	for	
                                                                                   ing	TNF-alpha,	 IFN-gamma,	 IL-1,	 IL-10	 and	 IL-18	 was	 also	 changed	
HIV	and	CD134	for	FIV)	and	in	their	tropism,	restricted	to	CD4+	T	cells	
                                                                                   significantly.	
for	HIV	and	broader	for	FIV.	Gut-associated	lymphoid	tissue	(GALT)	is	
a	reservoir	for	the	virus	and	a	site	of	CD4+	T	cells	depletion	in	chroni-               Gene	 expression	 differences	 of	 the	 four	 up-regulated	APPs	 (C-
cally	HIV-infected	humans,	but	its	role	in	FIV	pathogenesis	needs	to	be	           reactive	protein,	fibrinogen,	haptoglobin	and	serum	amyloid	A)	as	well	
investigated	further.	                                                             as	three	of	the	down-regulated	APPs	(apolipoprotein	A-1,	transferrin,	
     Our	 objectives	 were	 to	 characterize	 FIV-induced	 immune	 disor-          and	transthyretin)	were	verified	by	quantitative	real-time	PCR.
der	and	FIV	target	population	in	GALT	of	chronically	infected	cats.	We	                 Thus,	the	immune	focused	porcine	microarray	POM-4	allowed	the	
therefore	developed	a	method	to	isolate	GALT	immune	cells	and	ana-                 study	 of	 hundreds	 of	 immune	 related	 genes	 in	 a	 single	 analysis	 and	
lyzed	 immunological	 changes	 and	 virus	 multiplication	 through	 detec-         a	 number	 of	 genes	 were	 significantly	 up-	 or	 down-regulated	 in	 liver	
tion	of	p24.	We	first	observed	a	depletion	of	mucosal	CD4+	T	cells	and	            samples	according	to	infection	status	and	reflecting	a	systemic	innate	
B	cells.	As	in	HIV	infection,	this	depletion	is	preferentially	observed	in	        immune	response	to	the	infection.	
GALT	effector	sites	(intestinal	epithelium	and	lamina	propria),	as	com-            Key words:	pig	host	response,	microarrays,	innate	immunology
pared	 to	 the	 inductive	 compartments	 (mesenteric	 lymph	 nodes	 and	           Species:	swine
Peyer’s	Patches).	By	focusing	on	CXCR4	and	CD134	expressing	cells,	
we	observed	a	preferential	depletion	of	mucosal	CD134+	B	cells	and	
                                                                                          bV057. PROTECTION AGAINST LAWSONIA
CD4+CD134+	 T	 cells,	 especially	 in	 those	 co-expressing	 CD134	 and	
CXCR4.	Whether	these	depletions	were	associated	with	the	induction	
                                                                                    INTRACELLULARIS RE-INFECTION DID NOT CORRELATE
of	apoptosis	was	not	investigated.	                                                 WITH HUMORAL OR CELL-MEDIATED IMMUNE RESPONSE
     To	 further	 characterize	 FIV	 replication	 in	 the	 gut	 of	 chronically	      1ULLA	RIBER,	1ToRSTEn	S	BoUTRUP,	1LIEn	THI	nGUyEn,	
infected	cats,	the	expression	of	p24	was	analyzed	by	flow	cytometry	in	                 1JEAnnE	T	JAKoBSEn,	1TIM	K	JEnSEn,	1PETER	MH	
mucosal	immune	cells.	We	observed	that	p24	positive	cells	were	within	                         HEEGAARD,	1GREGERS	JUnGERSEn
the	CD134+	cell	population	and	were	preferentially	observed	in	GALT	                	1National	Veterinary	Institute,	Technical	University	of	Denmark,	27,	
effector	compartments,	a	T-cell	rich	site.	                                                    Bülowsvej,	DK-1790	Copenhagen	V,	Denmark
72


           Background:	 Lawsonia	 intracellularis	 is	 an	 obligate	 intracellular	         isotypes	 ratio	 in	 sows	 from	 a	 herd	 naturally	 infected	 with	 B.	 suis	 in	
     bacterium	causing	proliferative	enteropathy	(PE)	in	pigs.	Clinical	signs	              pregnant	(group	I),	non-pregnant:	irregular	oestrus	return	(group	II)	and	
     of	 affected	 pigs	 after	 weaning	 include	 diarrhoea	 and	 uneven	 weight	           females	that	had	aborted	(group	III).	All	the	animals	shared	the	same	
     gain.	                                                                                 environment.	Sera	samples	were	taken	and	reproductive	variables	like	
          Experimental	design:	Three	groups	of	pigs	were	established:	“Re-                  pregnancy,	 abortions	 and	 infertility	 were	 controlled,	 simultaneously,	
     infected	pigs”	(Re-i;	N=8)	received	a	primary	L.	intracellularis	infection	            during	the	experience.	IgG	isotype	was	determined	by	Indirect	ELISA	
     at	5	weeks	of	age	by	oral	inoculation	with	at	least	1010	bacteria	in	an	               with	mouse	anti	porcine	IgG1	and	IgG2	in	320	female	pigs.	The	differ-
     infected	mucosa	homogenate.	After	3	weeks	the	infection	was	cleared	                   ences	between	groups	were	analyzed	with	the	unpaired	t	test	using	the	
     by	Tiamulin	treatment	for	one	week,	and	at	12	weeks	of	age,	the	pigs	                  Graph	Pad	InStat	statistical	software.	The	IgG1/IgG2	ratio	from	group	
     were	 re-infected	 with	 L.	 intracellularis.	 “Challenge	 control	 pigs”	 (Cc;	       I	[Median	(Range):	0.28	(0.05-0.72)]	was	significantly	lower	from	that	
     N=10)	were	not	infected	at	5	weeks	of	age,	but	received	the	Tiamulin	                  of	group	II	[1.21	(0.41-4.10)]	and	group	III	[0.90	(0.53-1.35)]	(p<0.0005	
     treatment	 and	 a	 (primary)	 infection	 at	 12	 weeks	 of	 age.	 “Treatment	          and	 p<0.0001,	 respectively).	 These	 results	 agree	 with	 the	 functional	
     control	 pigs”	 (Tc;	 N=4)	 did	 not	 receive	 the	 re-infection	 after	 antibiotic	   and	phenotypic	differences	previously	found	in	these	animals.	We	can	
     treatment.                                                                             conclude	that	a	low	IgG1/IgG2	ratio	or,	in	other	words,	a	isotype	balance	
          Protection	against	re-infection:	While	faecal	excretion	of	L.	intra-              favoring	IgG2,	would	be	associated	to	a	protective	immune	response	
     cellularis	was	detected	by	PCR	from	6	to	21	days	after	primary	inocula-                leading	 to	 the	 absence	 of	 reproductive	 alterations	 and/or	 to	 a	 good	
     tions	at	5	weeks	(Re-i	and	Tc)	or	12	weeks	(Cc)	of	age,	excretion	of	L.	               reproductive	performance.	These	results	contribute	to	a	better	under-
     intracellularis	 was	 not	 detected	 following	 re-infection.	 In	 addition,	 PE	      standing	of	disease	control	mechanisms	and	vaccine	responses.
     was	only	observed	in	the	primary	infected,	Cc,	pigs	at	necropsy	and	                   Key words:	 IgG	 Isotypes,	 Brucella	 suis,	 Sows,	 Reproductive	
     histopathological	evaluation.	                                                         performance
          Immune	 response:	 Acute	 phase	 protein	 measurements	 were	                     Species:	swine
     employed	to	characterise	the	magnitude	and	extent	of	immediate	tissue	
     destruction	by	the	infection	as	a	measure	of	the	capacity	of	the	animal	                     bV059. PRRSV-INDUCED IMMUNE DySREGULATION
     to	handle	the	infection.	Lawsonia	specific	IgG	in	serum	(ELISA)	was	                        SELECTS B CELLS WITH HyDROPHOBIC HCDR3S FOR
     measured	in	all	pigs,	but	a	boost	was	not	obvious	in	the	Re-i	group.	                                        ExPANSION
           IFN-γ	was	measured	in	whole	blood	and	ileocaecal	lymph	node	                          CAITLIn	D	LEMKE1,	JoHn	E	BUTLER2,	PATRICK	WEBER2,	
     (ILN)	 cell	 cultures	 after	 in	 vitro	 stimulation	 with	 sonicated	 L.	 intracel-          MAREK	SInKoRA3,	AMy	VInCEnT4,KELLy	D	LAGER4
     lularis,	 followed	 by	 quantification	 of	 produced	 IFN-γ	 by	 ELISA.	 Only	
                                                                                            	                1Robarts	Institute,	London,	Ontario,	CAN;	2Dept	of	
     low	levels	of	antigen	specific	IFN-γ	responses	were	detected	in	whole	
                                                                                                Microbiol.,	Univ.	of	Iowa,	USA;	3Czech	Academy	of	Science,	Novy	
     blood	cultures,	while	the	levels	were	higher	in	ILN	cell	cultures.	Flow	
                                                                                                Hradek,	CZ;	4National	Animal	Disease	Center,	Ames,	Iowa,	USA
     cytometric	measurement	of	T-	cell	proliferation	after	in	vitro	stimulation	
     with	sonicated	L.	intracellularis	revealed	proliferative	CD4+	responses	                     Germfree	 isolator	 piglets	 infected	 with	 PRRS	 virus	 (PRRSV)	
     in	all	3	groups	of	pigs.	Differences	in	cell	subset	phenotypes	in	blood	               develop	 lymphoid	 hyperplasia,	 extreme	 hypergammaglobulinemia,	
     samples	and	in	cell	preparations	from	ileum	identified	some	transloca-                 deposition	of	immunoglobulin	complexes	of	all	isotypes	in	the	kidney	
     tion	of	CD4+	and	CD8+	cells	into	the	affected	tissues.                                 and	autoimmunity	(Lemke	et	al	2004).		Hypergammaglobulinemia	of	all	
                                                                                            major	isotypes	was	seen	in	the	bronchial-alveolar	lavage	(BAL)	as	well	
           Conclusion:	Protection	against	re-infection	was	clearly	observed	
                                                                                            as	serum.	Analysis	of	VH	usage	revealed	the	lack	of	repertoire	diversi-
     by	 lack	 of	 faecal	 excretion	 of	 L.	 intracellularis	 and	 absence	 of	 PE.	
                                                                                            fication	while	spectratypic	analyses	showed	that	clones	having	certain	
     Differences	in	immune	response	due	to	this	protection	against	infec-
                                                                                            HCDR3	lengths	were	preferentially	expanded.	Interestingly,	the	same	
     tion	 could	 not	 clearly	 be	 identified	 from	 the	 parameters	 measured	 in	
     this	experiment                                                                        clones	 were	 expanded	 in	 most	 tissues	 examined	 in	 contrast	 to	 the	
                                                                                            pattern	 observed	 in	 SIV-infected	 littermates	 or	 sham	 controls.	These	
     Key words:	Lawsonia,	protection,	immunity,	pigs                                        same	 expanded	 clones	 expressed	 IgG	 and	 IgA	 and	 sometimes	 IgM.	
     Species:	swine                                                                         Sequence	analyses	of	HCDR3s	in	PRRSV-infected	animals	showed	a	
                                                                                            strong	bias	favoring	sequences	in	the	hydrophobic	region	of	the	hydro-
         bV058. IGG ISOTyPES RATIO AND REPRODUCTIVE                                         pathicity	profile.	A	high	proportion	share	the	AMVLV	and	related	motifs	
        PERFORMANCE IN SOWS NATURALLy INFECTED WITH                                         encoded	by	the	germline	sequence	of	reading	frame	3	of	DHA	(Butler	
                        BRUCELLA SUIS                                                       et	al	2007).	Since	the	spectratypic	pattern	of	cells	from	the	BAL	and	the	
      ARESTEGUI	MB1,	GUALTIERI	CAS1,	ToRIonI	DE	ECHAIDE	S2,	                                tracheo-bronchial	lymph	nodes	were	identical,	we	considered	B	cells	
      AGUIRRE	n2,	DELGADo	G3,	PERALTA	L1,	BESSo	R1,	GEnTILE	                                from	the	BAL	to	be	representative	of	lymph	nodes	associated	with	the	
                      n1,	SCHARoVSKy	oG4                                                    lung.	B	cells	in	BAL	were	significantly	elevated	and	most	all	retained	
                                                                                            CD2,	a	marker	of	naïve	B	cells.	Additional	phenotypic	analyses	of	BAL	
        1Cátedra	de	Sueros	y	Vacunas,	3Cátedra	de	Obstetricia,	Fac.	
                                                                                            lymphocytes	indicated	that	aβ	T	cells	were	greatly	expanded	and	that	
        Cs.	Veterinarias,	Casilda;	2INTA,	EEA	Rafaela;	4Inst.	Genética	
                                                                                            both	aβ	and	γd	T	cells	displayed	activation	markers.	We	suggest	that	in	
     Experimental,	Fac.	Cs.	Médicas,	Consejo	de	Investigaciones,	U.N.R.,	
                                                                                            isolator	piglets,	PRRSV	infection	results	in	a	proper	T	cell	response	but	
                              Rosario,	Argentina.		
                                                                                            acts	as	a	B	cell	superantigen	that	causes	dysregulation	of	the	normal	
                          maresteg@fveter.unr.edu.ar
                                                                                            anti-viral	immune	response.
           Differential	immunoglobulin	(Ig)	isotype	expression	allows	diverse	
     isotype-related	functions,	which	can	be	related	to	infection	or	to	protec-                  Lemke,	C.D.,	J.S.	Haynes,	R.	Spaete,	D.	Adolphson,	A.	Vorwald,	
     tion,	depending	upon	the	prevalent	Ig	isotype.	It	has	been	confirmed	                  K.D.	Lager	and	J.E.		        Butler.	 	 2004.	 J.	 Immunol.	 172:	
     the	differences	in	biological	functions	for	different	IgG	isotypes	of	the	             1916-1925
     pig.	 In	 previous	 studies	 we	 have	 shown,	 in	 a	 farm	 naturally	 infected	            Butler,	J.E.	,	C.D.	Lemke,	P.	Weber,	A.	Vincent,	M.	Sinkora	and	
     with	B.	suis,	that	B.	suis	antibody	concentration	was	associated	with	                 K.D.	Lager.	2007	J.	Immunol.	178	(in	press)
     an	 increased	 individual	 animal	 risk	 of	 non-pregnancy	 and	 abortion.	            Key words:	Immune	dysregulation;	virus;	HCDR3;	superantigen
     Porcines	from	herds	with	diverse	levels	of	reproductive	success,	that	                 Species:	swine
     were	seropositive	(S+)	for	brucellosis	by	BPA,	2ME,	FPA,	C-ELISA	and	
     CF	(complement	fixation)	analysis,	had	a	higher	risk	of	abortion	than	
                                                                                                 bV060. TRANSFECTION OF PORCINE SIALOADHESIN
     seronegative	 (S-)	 animals.	 In	 this	 study,	 we	 aimed	 to	 investigate	 the	
     host–parasite	relationship	within	the	pregnant	host,	in	order	to	increase	
                                                                                                INTO A MURINE MACROPHAGE CELL-LINE RENDERS IT
     the	understanding	of	the	factors	that	influence	the	infection	dynamics;	                          PERMISSIVE FOR PRRSV REPLICATION
     also,	we	studied	if	the	IgG1/IgG2	serological	relationship	is	associated	              SUDARVILI	SHAnTHALInGAM1,	WEIGUo	LIU1,	KEVIn	SnEKVIK1,	
     to	the	type	of	response	to	Brucella	infection	and	its	role	in	infections	at	             ASIT	PATTnAIK2,	FERnAnDo	A	oSoRIo2,SUBRAMAnIAM	
     pregnancy	 and	 reproductive	 performance.	 We	 studied	 the	 IgG1/Gg2	                                     SRIKUMARAn1
                                                                                                                                                                           73


1Department	of	Veterinary	Microbiology	and	Pathology,	Washington	                    PCV2	 isolate	 Imp1010-Stoon	 (AF055392),	 from	 a	 Canadian	 PMWS	
State	University,	Pullman,	WA	99164-7040,	USA	and	2Department	of	                    case,	 and	 is	 identical	 in	 an	 isolate	 from	 a	 healthy	 Swedish	 SPF	 pig	
Veterinary	and	Biomedical	Sciences,	University	of	Nebraska,	Lincoln,	                herd	(EF184220).	However,	the	corresponding	PCV1	motif	differ	at	8	
                        NE	68583-0905,	USA.	                                         nucleotide	positions	and	the	motif	in	the	PCV2	isolate	N1	(EF184226),	
                   Contact	sudar@vetmed.wsu.edu                                      from	the	first	Swedish	PMWS	case,	differ	at	two	positions.	In	the	pres-
      Sialoadhesin	 is	 a	 macrophage-specific	 adhesion	 molecule.	 	 It	 is	       ent	study,	we	designed	ODNs	representing	this	motif	from	Stoon,	N1	or	
the	 prototypic	 member	 of	 the	 sialic	 acid	 binding	 immunoglobulin-like	        PCV1.	The	inhibitory	effect	was	tested	in	cultures	of	porcine	peripheral	
lectin	 (siglec)	 family,	 and	 hence	 referred	 to	 as	 siglec-1,	 and	 also	 as	   blood	mononuclear	cells	stimulated	with	ODN	2216,	pcDNA3	plasmid	
CD169.		It	binds	sialylated	ligands	on	other	hematopoietic	cells,	pre-               or	 poly	 I:C.	 Both	 Stoon	 and	 N1	 motifs	 decreased	 IFN-a	 produced	 in	
dominantly	neutrophils,	but	also	monocytes,	natural	killer	cells,	B-cells	           response	 to	 ODN	 2216	 and	 pcDNA3	 but	 not	 to	 poly	 I:C.	 The	 PCV1	
and	cytotoxic	T	lymphocytes.		It	is	a	non-phagocytic	receptor,	but	may	              motif	also	decreased	induction	of	IFN-a	production	to	pcDNA3	but	was	
aid	 in	 phagocytosis.	 	 Other	 functions	 of	 sialoadhesin	 are	 not	 clear.	  	   less	effective	in	decreasing	IFN-a	production	induced	by	ODN	2216.	
Earlier	studies	have	identified	sialoadhesin	as	the	cell-surface	protein	            Using	 a	 secondary	 structure-predicting	 program,	 an	 ability	 to	 form	
on	 porcine	 alveolar	 macrophages	 (PAMs)	 that	 mediates	 internaliza-             hairpin-like	structures	was	found	in	the	motifs	from	all	three	isolates.	
tion	of	porcine	reproductive	and	respiratory	syndrome	virus	(PRRSV).	            	   However,	while	the	N1	motif	formed	the	most	stable	hairpin,	the	hair-
In	 these	 studies,	 transfection	 of	 porcine	 sialoadhesin	 into	 a	 porcine	      pin	formed	by	the	PCV1	motif	was	the	least	stable.	The	PCV1	motifs	
kidney	 cell-line	 (PK-15)	 rendered	 it	 susceptible	 to	 PRRSV	 infection.	    	   inability	to	form	a	stable	hairpin	structure	may	contribute	to	its	reduced	
However,	the	internalized	virus	did	not	replicate.		We	hypothesized	that	            inhibition	 of	 ODN	 2216	 induced	 IFN-α	 production,	 but	 still	 it	 remains	
replication	 of	 PRRSV	 requires	 porcine	 sialoadhesin	 as	 well	 as	 addi-         to	 establish	 if	 this	 discrepancy	 is	 of	 importance	 for	 the	 difference	 in	
tional	macrophage-specific	factors.		The	objective	of	this	study	was	to	             virulence	between	PCV1	and	PCV2.	
clone	the	cDNA	encoding	porcine	sialoadhesin,	transfect	into	a	murine	               Key words:	IFN-a,	circoviruses	
macrophage	cell-line,	and	determine	the	permissiveness	of	the	trans-                 Species:	swine
fectant	cell-line	for	PRRSV	replication.
      	    Total	RNA	from	PAMs	was	extracted	and	cDNA	was	made	
                                                                                     bV062. MICE VACCINATION WITH VAPA: CHALLENGE WITH
by	RT-PCR.	The	PCR-amplified	cDNA	for	sialoadhesin	was	cloned	into	
                                                                                      RHoDoCoCCUS EqUI IS FOLLOWED By PRODUCTION OF
a	 mammalian	 expression	 vector	 and	 sequenced.	 Four	 independent	
clones	were	sequenced.	Comparison	of	our	sequence	data	(Genbank	                                       TH1 CyTOKINES.
accession	 no.	 DQ176853)	 with	 that	 published	 previously	 (Genbank	                 ALInE	F	oLIVEIRA1,	SAnDRo	G	SoARES1,MARIA-CRISTInA	
accession	no.	NM_214346)	revealed	15	amino	acid	difference,	which	                                        RoqUE-BARREIRA1
likely	represents	polymorphism.		The	cDNA	for	sialoadhesin	was	trans-                1Department	of	Cellular	and	Molecular	Biology.	School	of	Medicine	of	
fected	 into	 a	 mouse	 macrophage	 cell-line.	 	 The	 transfectants	 were	                                Ribeirão	Preto,	USP-SP.
labeled	with	a	monoclonal	antibody	to	sialoadhesin,	and	subjected	to	                      Introduction	and	Objectives:	Rhodococcus	equi	is	the	most	impor-
fluorescence-activated	cell	sorting.		We	obtained	51	single	cell	clones	             tant	cause	of	bacterial	infection	in	young	foals	all	over	the	world,	and	
which	expressed	porcine	sialoadhesin	on	the	cell	surface.	Of	these,	17	              has	 emerged	 as	 an	 opportunistic	 pathogen	 in	 immunocompromised	
clones	continued	to	express	porcine	sialoadhesin	on	their	surface,	to	               humans.	 The	 severe	 pneumonia	 caused	 by	 R.	 equi,	 constitutes	 an	
varying	 degrees.	Three	 of	 these	 clones	 stably	 expressing	 sialoadhe-           important	economic	and	public	health	problem,	generating	the	interest	
sin	were	found	to	be	susceptible	to	PRRSV	infection.		The	clone	AA9	                 in	the	development	of	efficient	vaccines	against	the	bacteria.	The	VapA	
was	 tested	 further	 to	 determine	 its	 ability	 to	 support	 PRRSV	 replica-      protein	corresponds	to	an	important	virulence	factor	of	R.	equi	(Jain	et	
tion.		AA9	cells	support	the	replication	of	PRRSV.		The	titer	of	PRRSV	              al.,	 Mol.	 Microbiol.	 50:	 115-28,	 2003),	 and	 is	 considered	 a	 protective	
obtainable	 in	AA9	 cells	 is	 between	 2x105	 and	 2x106	TCID50/ml.	 	This	         immunogen.	 We	 have	 developed	 an	 attenuated	 Salmonella	 enterica	
cell-line	should	be	valuable	for	large	scale	propagation	of	PRRSV	for	               strain	expressing	the	VapA	protein,	whose	administration	to	mice	has	
vaccine	production.		                                                                induced	protection	against	R.	equi	infection,	as	demonstrated	by	higher	
Key words:	PRRSV,	Sialoadhesin,	Receptor,	Trasfectant	                               survival	rates	and	bacterial	clearance	in	comparison	with	the	non-vac-
Species:	swine                                                                       cinated	animals.	This	protection	was	associated	with	detection	of	high	
                                                                                     levels	 of	 serum	 specific	 antibodies,	 predominantly	 of	 IgG2a	 isotype	
  bV061. INHIBITION OF IFN-α PRODUCTION By PORCINE                                   (Oliveira	et	al.,	Micobes	Infect.	9(3):	382-90,	2007).	Moreover,	in	vitro	
           CIRCOVIRUS-DERIVED DNA MOTIFS                                             antigen	stimulation	of	cells	obtained	from	vaccinated	mice	resulted	in	
                                                                                     high	production	of	IL-12p40,	TNF-a,	IL-1β,	and	IFN-γ,	whereas	no	IL-4	
  TAnJA	LöVGREn,	FRIDA	HASSLUnG	WIKSTRöM,	LISBETH	
                                                                                     was	detected.	Since	Th1	immune	response	is	extremely	important	to	
FUxLER,	SIRJE	TIMMUSK,	EVA	WATTRAnG,	CARoLInE	FoSSUM
                                                                                     achieve	protection	against	rhodococcosis,	the	present	study	aimed	to	
    Dept.	of	Biomedicine	and	Veterinary	Public	Health,	Sect	of	                      evaluate	the	profile	of	cytokines	produced	in	vivo	by	immunized	mice	
 Immunology,	Swedish	University	of	Agricultural	Sciences,	Uppsala,	                  following	challenge	with	virulent	R.	equi	strain.
                             Sweden.		
                                                                                           Methods	and	Results:	Groups	of	BALB/c	mice	were	orally	immu-
                    tanja.lovgren@bvf.slu.se
                                                                                     nized	with	two	doses	of	1x109CFU	of	S.	enterica	Typhimurium	vapA+	
       Porcine	circoviruses	are	small,	non-enveloped	viruses	with	single-            or	vapA-.	A	third	group	of	mice	received	only	PBS.	Two	weeks	after	the	
stranded,	 circular	 DNA	 genomes	 of	 around	 1760	 nucleotides.	 There	            last	immunization,	all	mice	were	intravenously	challenged	with	a	sub-
are	 two	 types	 of	 porcine	 circoviruses;	 type	 1	 (PCV1)	 and	 2	 (PCV2).	       lethal	dose	of	virulent	R.	equi.	On	days	2,	4,	8,	and	10	post-challenge,	
PCV1	 has	 not	 been	 associated	 to	 disease	 whereas	 PCV2	 has	 been	             groups	of	4	mice	were	sacrificed	and	their	spleens,	livers,	and	lungs	
proposed	 to	 be	 the	 causative	 agent	 of	 postweaning	 multisystemic	             were	removed.	Tissues	homogenates	were	used	for	cytokines	quan-
wasting	 syndrome	 (PMWS)	 and	 porcine	 dermatitis	 and	 nephropathy	               tization	 by	 ELISA.	 The	 vaccinated	 mice	 (inoculated	 with	 S.	 enterica	
syndrome	(PDNS).	There	are	several	isolates	of	PCV2	and	while	the	                   Typhimurium	 vapA+),	 in	 contrast	 with	 the	 control	 groups	 (inoculated	
sequence	identity	between	PCV2	and	PCV1	is	around	80%,	the	iden-                     with	 S.	 enterica	 Typhimurium	 vapA-	 strain	 or	 PBS),	 produced	 higher	
tity	 between	 PCV2	 isolates	 is	 more	 than	 96%.	The	 immune	 defence	            levels	 of	 IL-12p40	 and	 IFN-γ	 in	 all	 organs	 analyzed,	 whereas	 lower	
against	viruses	depend	strongly	on	type	1	interferon	(IFN-a/β)	produc-               levels	of	TNF-a	and	IL-4	were	detected.	
tion	by	leukocytes	responding	to,	for	example,	viral	nucleic	acids.	In	the	
PCV2	 genome,	 several	 motifs	 inducing	 production	 of	 IFN-α	 but	 also	               Conclusion:	Mice	protection	against	R.	equi	infection	provided	by	
a	20-nucleotide	motif	(nucleotides	1481-1500)	that	inhibits	production	              vaccination	with	S.	enterica	Typhimurium	vapA+	is	associated	with	a	Th1	
of	 IFN-α	 has	 been	 identified.	A	 phosphodiester	 oligodeoxynucleotide	           biased	specific	immune	response.	The	immunization	using	attenuated	
(ODN)	 representing	 this	 motif	 inhibits	 IFN-a	 production	 induced	 by	          recombinant	Salmonella	strains	as	vector	for	the	delivery	of	heterolo-
the	stimulatory	ODN	2216,	plasmid	DNA	or	DNA	virus	(Aujeszky’s	dis-                  gous	antigens	had	already	been	shown	to	induce	a	Th1-type	response	
ease	virus),	but	not	that	induced	by	double-stranded	RNA	(polyI:C)	or	               preferentially	(Lange	et	al.,	Infect.	Immun.	72:	4924-28,	2004).
RNA-virus	(Sendai	virus).	The	inhibitory	motif	was	first	identified	in	the	               Financial	Support:	FAPESP	and	CNPq.
74


     Key words:	 vaccines,	 Rhodococcus	 equi,	 Salmonella	 enterica,	                 VP2	 and	 the	 N-terminal	 region	 of	 VP2.	Antibody	 to	 epitopes	 on	 VP2	
     VapA	protein                                                                      (both	 native	 and	 recombinant	 forms)	 persisted	 longer	 post-infection	
     Species:	equine                                                                   (>105	days)	than	antibodies	specific	for	epitopes	on	other	fragments.	
                                                                                       Our	data	also	suggest	that	B	cell	epitopes	within	C-terminus	of	VP1	and	
     bV063. RECOMBINANT PROTEIN M DETECTS ANTIBODIES                                   N-terminus	of	VP2	contribute	to	a	large	proportion	of	the	total	reactivity	
     INDUCED By STREPToCoCCUS EqUI STRAINS ISOLATED                                    of	 recombinant	 VP1	 and	 VP2,	 respectively,	The	 reactivity	 in	 enzyme	
                FROM CASES OF STRANGLES                                                linked	 immunosorbent	 assay	 (ELISA)	 of	 individual	 linear	 epitopes	 or	
         CARInA	M	MoRAES1*,	AnDRéA	S	R	RoCHA1,	LUAnA	A	                                a	recombinant	antigen	engineered	to	express	a	mosaic	of	these	linear	
       DUMMER1,	ALCEU	G	S	JUnIoR1,	CARLoS	E	W	noGUEIRA2,	                              epitopes,	showed	variable	correlation	with	other	assays.		Importantly	
         AGUEDA	P	C	VARGAS3,	FáBIo	P	L	LEITE4,	FABRÍCIo	R	                             however,	 the	 reactivity	 in	 ELISA	 of	 combined	 VP1	 and	 VP2	 recom-
              ConCEIçÃo1	,	CARLoS	GIL-TURnES1-2	                                       binant	 proteins	 correlated	 well	 with	 a	 range	 of	 native	 antigen-based	
                                                                                       serological	assays	using	sera	from	12	field	horses.	This	study	provides	
          1CENBIOT/	UFPel;	2FV/UFPel;	3DVP/UFSM;	4DMP/UFPel	
                                                                                       promising	candidates	for	development	of	an	ERAV	ELISA	capable	of	
                      carinamoraes@terra.com.br
                                                                                       detecting	antibodies	elicited	during	infection	of	the	natural	host.
           Strangles	is	a	contagious	disease	of	the	anterior	respiratory	tract	
     of	Equidæ	caused	by	Streptococcus	equi.	Asymptomatic	carriers	are	                Key words:	equine,	respiratory	virus,	antibody,	ELISA
     responsible	for	the	maintenance	of	the	infection	in	the	herds,	and	are	           Species:	equine
     recognized	 only	 by	 serological	 or	 microbiological	 methods.	 Vaccines	
     used	in	its	control	induce	low	protection	levels,	generally	below	50	%.	           bV065. CELLULAR AND FUNCTIONAL CHANGES IN THE
     S.	equi	protein	M	(SeM)	is	considered	the	mdost	promising	protecting	             REPRODUCTIVE TRACT ASSOCIATED WITH ONSET-OF-LAy
     antigen	against	the	infection.	In	this	research	we	studied	the	antigenic-                           IN CHICKENS.
     ity	of	two	commercial	vaccines	in	use	in	southern	Brazil	and	of	other	ten	                         CLAIRE	E	JoHnSTon,PAUL	WIGLEy
     monovalent	vaccines	prepared	with	strains	of	S.	equi	recovered	from	
     clinical	cases	of	Strangles.	All	the	vaccines	had	the	same	concentra-              Department	of	Veterinary	Pathology,	Faculty	of	Veterinary	Science,	
     tion	of	inactivated	antigen	and	used	Aluminum	Hydroxide	as	adjuvant.	                          University	of	Liverpool,	United	Kingdom.	
     Isogenic	 Balb-c	 mice	 were	 randomly	 grouped	 and	 vaccinated	 with	                                 c.e.johnston@liv.ac.uk
     1/20th	of	an	equine	dose	of	the	respective	vaccine	on	days	0	and	14.	                   Infection	 of	 the	 chicken	 reproductive	 tract	 and	 transmission	 to	
     Serum	antibodies	collected	on	days	0,	14,	28,	56	and	70	were	titrated	by	         developing	eggs	can	result	in	disease	in	the	progeny	and	infection	of	
     ELISA	using	recombinant	SeM	as	antigen	(0,12	µg/well).	Recombinant	               eggs	for	human	consumption.	Salmonella	enterica	serovar	Enteritidis,	
     SeM	was	produced	in	our	laboratory	by	a	transformed	E.	coli	harboring	            for	 example,	 is	 an	 important	 food-borne	 zoonosis,	 with	 poultry	 meat	
     the	SeM	gene	obtained	by	PCR	(GenBank	accession	Nº	U73162).	The	                  and	 eggs	 being	 a	 major	 source	 of	 infection	 in	 humans.	 In	 the	 U.K.,	
     amplicon	 was	 cloned	 in	 the	 pAE	 vector,	 expressed	 in	 competent	 E.	       vaccination	 of	 laying	 hens	 and	 improved	 biosecurity	 has	 resulted	 in	
     coli	pLyss,	and	the	protein,	purified	by	affinity	chromatography	(his-tag)	       a	decline	in	S.	Enteritidis	infection,	but	throughout	the	rest	of	Europe	
     using	AKTAPrimeTM	 Plus	 (GE	 Healthcare),	 was	 detected	 by	 Western	           infection	remains	prevalent.	Systemic	infection	with	S.	Enteritidis	can	
     Blot.	All	 the	 vaccines	 induced	 antibodies	 against	 SeM,	 although	 the	      result	in	colonisation	of	the	ovary	and	oviduct	leading	to	contamination	
     titers	 differed	 among	 them.	 Mean	 seroconversions	 induced	 by	 the	          of	both	the	yolk	and	albumen.	As	yet	however,	the	precise	mechanism	
     experimental	vaccines	varied	between	3.1	and	9.2,	while	those	of	the	             of	 this	 transmission	 is	 unclear.	 It	 is	 now	 well-known	 that	 systemic	
     commercial	vaccines	were	1.7	and	3,3.		Seronconversions	induced	by	               immunosuppression	 occurs	 at	 the	 point-of-lay,	 and	 in	 S.	 Pullorum-
     the	experimental	vaccines	were	higher	than	those	of	the	commercial	               infected	hens	this	immunosuppression	is	associated	with	infection	of	
     vaccines.	Our	results	showed	that	there	are	quantitative	differences	in	          the	reproductive	tract.	Chickens	may	carry	S.	Pullorum	asymptomati-
     the	immunogenicity	of	different	strains	of	S.	equi,	and	that	this	property	       cally	in	splenic	macrophages	for	many	months.	However,	upon	sexual	
     may	be	useful	in	the	selection	of	vaccinal	strains.	                              maturity,	 T-cell	 responsiveness	 significantly	 decreases	 in	 the	 spleen	
                                                                    	
          Financial	support:	CNPq	(474509/2004-4);	FAPERGS	(0523299)	                  and	numbers	of	S.	Pullorum	increase.	Furthermore,	Salmonellae	can	
     Key words:	 M	 protein,	 Strangles,	 Streptococcus	 equi,	 recombinant	           be	recovered	from	the	reproductive	tract	and	developing	eggs.	As	yet,	
     protein                                                                           very	little	is	known	about	the	local	cellular	changes	which	may	occur	in	
     Species:	equine                                                                   the	reproductive	tract	during	this	immunosuppressed	state	and	which	
                                                                                       may	facilitate	Salmonella	transmission	to	the	egg.	Studies	have	shown	
        bV064. MAPPING THE ANTIBODy RESPONSE OF THE                                    macrophages,	CD4+	and	CD8+	T	lymphocytes	to	be	present	through-
        NATURAL HOST TO EQUINE RHINITIS A VIRUS CAPSID                                 out	the	reproductive	tract	of	hens,	with	B	cells	mainly	restricted	to	the	
                         PROTEINS.                                                     oviduct.
      FAn	LI1,	BREnDAn	S	CRABB2,	MICHAEL	J	STUDDERT1,	JAMES	                                 In	order	to	characterise	the	cellular	and	functional	changes	associ-
                  R	GILKERSon1,	CARoL	A	HARTLEy1*                                      ated	with	immunosuppression	at	the	point-of-lay,	we	have	carried	out	a	
                                                                                       detailed	immunohistochemical	and	gene	expression	study	of	the	repro-
     1School	of	Veterinary	Science,	The	University	of	Melbourne,	Victoria	
       3010,	Australia;	2The	Walter	and	Eliza	Hall	Institute	of	Medical	               ductive	tract-associated	immune	system	of	hens.	Between	weeks	15	
                Research,	Parkville,	Victoria	3052,	Australia.	                        and	24	post-hatch,	we	measured	the	change	in	immune	cell	numbers	
                          carolah@unimelb.edu.au                                       in	the	ovary,	infundibulum	and	magnum	by	immunohistochemistry,	and	
                                                                                       linked	this	with	systemic	changes	in	the	spleen.	Furthermore,	we	have	
          Equine	rhinitis	A	virus	(ERAV)	is	a	significant	pathogen	of	horses	          begun	 to	 functionally	 characterise	 these	 cells,	 measuring	 expression	
     and	is	also	closely	related	to	Foot-and	mouth	disease	virus	(FMDV).	              of	an	array	of	cytokines	and	chemokines.	Thus,	these	studies	provide	
     We	have	evidence	showing	50%	of	horses	become	infected	with	ERAV	                 important	information	on	the	local	immunology	of	the	reproductive	tract	
     by	 5	 years	 of	 age,	 and	 it	 appears	 many	 of	 these	 seroconversions	 to	
                                                                                       in	laying	hens	and	the	nature	of	the	point-of-lay	immunosuppression.	
     ERAV	may	occur	coincident	with	1-2	year	old	horses	entering	training	
                                                                                       Also,	characterising	these	cells	will	provide	an	insight	into	the	role	of	
     stables.	Despite	this,	knowledge	of	the	prevalence	and	importance	of	
                                                                                       different	cell-types	in	future	Salmonella	infection	studies	and	how	we	
     ERAV	infections	remains	limited	largely	due	to	the	absence	of	a	simple,	
                                                                                       can	enhance	immunity	in	order	to	prevent	transmission	to	the	develop-
     robust	diagnostic	assay.		Efforts	to	develop	suitable	antigens	for	such	
                                                                                       ing	egg.
     an	assay	have	concentrated	on	mapping	the	antigenic	structure	of	the	
     ERAV	capsid	and	the	kinetics	of	the	humoral	immune	response	to	those	             Key words:	 Reproductive	 tract,	 Immunosuppression,	 Cytokines,	
     proteins.	 The	 antigenicity	 of	 recombinant	 full-length	 and	 fragmented	      Salmonella
     ERAV	capsid	proteins	expressed	in	E.	coli,	have	been	used	to	map	the	             Species:	avian
     antibody	response	of	sera	from	experimentally	infected	and	naturally-
     exposed	horses.	We	found	that	each	of	the	five	experimentally	infected	           bV066. CLONING, ExPRESSION AND CHARACTERIZATION
     horses	 examined	 produced	 antibodies	 that	 reacted	 against	 recombi-             OF NUCLEOCAPSID PROTEIN FROM INFECTIOUS
     nant	proteins	encompassing	the	C-terminal	region	of	VP1,	full-length	                    BRONCHITIS VIRUS IN ESCHERICHIA CoLI
                                                                                                                                                                         75


  ALIAnDRA	M	GIBERTonI,	CAMILA	C	FERnAnDES,	MARIA	DE	                                signalling	 responses	 to	 its	 own	 benefit.	Two	 highly-passaged	 attenu-
        FATIMA	S	MonTASSIER,HéLIo	J	MonTASSIER                                       ated	 cloned	 isolates	 and	 three	 VP3	 CAV	 mutant	 viruses	 were	 also	
  Laboratório	de	Virologia	e	Imunologia,	Departamento	de	Patologia	                  used	in	this	study	with	the	aim	of	elucidating	the	interactions	of	each	of	
     Veterinária;	Faculdade	de	Ciências	Agrárias	e	Veterinárias,	                    these	proteins	with	the	host	cell.	VP3	mutations	resulted	in	lower	lev-
 Universidade	Estadual	Paulista,	14884-900,	Jaboticabal,	SP,	Brazil.	                els	of	transcripts	encoding	signalling,	transcription	and	mitosis	related	
 Projeto	Financiado	pela	Fundação	de	Auxílio	à	Pesquisa	do	Estado	                   proteins.	Overall,	these	data	extend	our	understanding	of	how	broadly	
 de	São	Paulo	(FAPESP),	processos	nº	05/54275-4	e	nº	01/14950-3.	                    CAV	alters	the	regulation	of	host	gene	products	and	highlight	the	virus’	
           aligiber@fcav.unesp.br;	heliojm@fcav.unesp.br                             ‘need’	to	utilise	the	host	cell	machinery	to	replicate	itself.
      The	 avian	 infectious	 bronchitis	 virus	 (IBV)	 is	 the	 etiologic	 agent	   Key words: transcriptome,	chicken	anaemia	immunosuppression
of	 avian	 infectious	 bronchitis,	 which	 causes	 an	 acute,	 highly	 conta-        Species: avian
gious	 disease	 in	 chickens	 that	 affects	 the	 respiratory,	 reproductive	
and	 renal	 systems.	 IBV	 is	 the	 prototype	 of	 the	 Coronaviridae	 family	             bV068. CLONING AND ExPRESSION OF S1
and	 is	 distributed	 worldwide,	 influencing	 negatively	 the	 production	 of	       GLyCOPROTEIN OF INFECTIOUS BRONCHITIS VíRUS (IBV)
the	 poultry	 industry,	 so	 that	 rapid	 and	 accurate	 diagnosis	 is	 required	             IN SACCHAROMyCES CEREVISIAE
for	the	adoption	of	the	most	efficient	control	measures.	The	IBV	RNA	                AP	oLIVEIRA,	AM	GIBERTonI,	MFS	MonTASSIER,	AG	CAETAno,	
genome	encodes	three	major	structural	proteins:	the	spike	protein	(S),	                                  H	J	MonTASSIER
the	membrane	protein	(M)	and	the	nucleocapsid	protein	(N).	The	latter	
containing	409	amino	acids	and	a	molecular	mass	of	50	kDa,	is	highly	                Laboratório	de	Virologia	e	Imunologia.	Departamento	de	Microbiologia	
conserved	 among	 different	 IBV	 strains	 and	 is	 abundantly	 produced	                -	Faculdade	de	Ciências	Agrárias	e	Veterinárias,	Universidade	
during	infection,	which	makes	it	an	ideal	antigenic	target	for	the	immu-              Estadual	Paulista,	Rod.	Prof.	Paulo	D.	Castellane,	s/n.	Jaboticabal,	
nodiagnosis	 of	 IBV	 infection.	 This	 study	 aims	 to	 clone	 and	 express	         SP,	Brasil,	Projeto	Financiado	pela	Fundação	de	Auxílio	à	Pesquisa	
the	 IBV	 N	 protein,	 using	 the	 vector	 pET28a(+)	 and	 Escherichia	 coli	          do	Estado	de	São	Paulo	(FAPESP),	processos	nº	02/08649-1	e	nº	
as	host-cells	and	to	characterize	immunochemically	this	recombinant	                        01/14950-3	e	bolsa	de	estudos	concedida	pela	CAPES.		
protein.	Firstly,	the	N	protein	gene	from	M41	strain	of	IBV	was	amplified	                                   heliojm@fcav.unesp.br
using	primers	containing	specific	restriction	sites	and	flanking	the	open	                 The	infectious	bronchitis	virus	(IBV)	is	one	of	the	most	important	
reading	frame	of	this	gene	by	reverse	transcription	-	polymerase	chain	              respiratory	 pathogens,	 causing	 an	 infectious	 disease	 in	 chickens,	
reaction	(RT-PCR).	The		amplicon			was	cloned	into	the	pGEM-T	Easy	                  which	 is	 characterized	 by	 severe	 losses	 on	 the	 productive	 perfor-
(Promega)	plasmid	was	digested	with	NdeI	and	xho	I,	gel-purified	for	                mance	of	the	poultry	flocks.	High	genetic	and	antigenic	variations	are	
subcloning	 into	 the	 Escherichia	 coli	 expression	 plasmid	 pET28a(+)	            common	features	of	IBV,	bringing	serious	difficulties	for	the	control	of	
(Novagen),	using	T4	DNA	ligase.	The	expression	of	the	His-Tag-N	gene	                this	disease.	In	addition	to	this,	such	genetic	and	antigenic	modifica-
fused	protein	was	induced	by	the	addition	of	isopropyl-1-thio-	D-galac-              tion	affects	mainly	the	S1	glycoprotein	and	particularly	its	neutralizing	
toside	(IPTG),	and	the	recombinant	protein	was	detected	and	charac-                  antigenic	sites.	The	diagnosis	and	immunoprophylaxis	of	IBV	requires	
terized	by	SDS-PAGE	and	Western-Blotting,	which	demonstrated	the	                    the	 search	 for	 more	 effective	 serological	 reagents	 and	 immunoges,	
presence	of	the	N	protein	with	approximately	54	kDa	and	carrying	the	                respectively.	Thus,	we	carried	out	this	study	aiming	to	clone	the	entire	
poly-histidine	tag	and	mostly	of	the	viral	N	protein	epitopes,	since	both	           open	reading	frame	of	S1	gene	from	M	41	strain	of	IBV	into	pYES2.1/
the	anti-His6	MAb	and	the	chicken	polyclonal	antibodies	from	chickens	               V5-His-TOPO	vector	and,	following	cloning	in	Escherichia	coi	and	sub-
hyperimmunized	against	IBV,	reacted	with	this	recombinant	protein.	In	               cloning	in	Saccharomyces	cerevisiae,	to	induce	the	expression	of	this	
conclusion,	 our	 results	 indicated	 that	 the	 E.	 coli	 -	 expressed	 N	 IBV	     protein	in	host	yeast	cells.	The	S1	recombinat	protein	was	successfully	
protein	has	high	homology	to	the	viral	N	protein	and	could	be	a	good	                expressed	and	characterized	as	a	protein	with	approximate		molecular	
source	of	antigen	to	develop	immunodiagnostic	assays	for	the	specific	               weight	of	87	kDa	in	PAGE-SDS	and	carrying	specific	reactivity	against	
detection	of	antibodies	in	chickens	infected	with	IBV.                               anti-His6	 MAb	 or	 polyclonal	 antibodies	 of	 laying	 hens	 that	 had	 been	
Key words: avian	infectious	bronchitis	virus,	recombinant	nucleoprotein,	            immunized	with	M41	strain	of	IBV	was	detected	in	the	Western-blotting	
Escherichia	coli,	expression                                                         analysis.	 Therefore,	 such	 recombinant	 polypeptide,	 due	 to	 the	 pres-
Species: avian                                                                       ence	 of	 common	 epitopes	 with	 the	 M41	 strain	 of	 IBV,	 may	 have	 the	
                                                                                     potential	to	be	used	successfully	in	the	imuno-diagnosis	of	IBV	and	in	
                                                                                     immunization	against	this	virus.	
 bV067. TRANSCRIPTOMIC ANALySIS OF THE CHICKEN
 ANAEMIA VIRUS (CAV)-INDUCED IMMUNOSUPPRESSION.                                      Key words:	S1	Glycoprotein,	Infectious	Bronchitis	Vírus	(IBV),	Cloning	
                                                                                     and	Expression,	Saccharomyces	cerevisiae
    EFSTATHIoS	S	GIoTIS1,2,5,	DAVE	W	BURT2,	ALISTAIR	nJ	                             Species:	avian
   SCoTT1,5,	ALISon	DoWnInG3,	RICHARD	T	TALBoT3,	LISA	
  RoTHWELL4,	PETE	KAISER4,	ELIZABETH	J	GLASS2,	DAnIEL	
                         ToDD1,5
                                                                                         bV069. CONSTRUCTION AND ExPRESSION OF SCFV
                                                                                          ANTIBODy FRAGMENT SPECIFIC FOR INFECTIOUS
 1Department	of	Veterinary	Sciences,	Queen’s	University	of	Belfast;	
                                                                                             BRONCHITIS VIRUS IN ESCHERICHIA CoLI
  2Department	of	Genetics	and	Genomics,	Roslin	Institute,	Roslin,	
  Midlothian;	3Ark	Genomics.	Roslin;	4Institute	for	Animal	Health,	                     ALInE	G	CAETAno,	ALIAnDRA	M	GIBERTonI,	MARIAnA	C	M	
 Compton,	Berkshire;	5Agrifood	and	Biosciences	Institute,	Stormont,	                              GonçALVES,HéLIo	J	MonTASSIER
                             Belfast.	                                               Laboratório	de	Virologia	e	Imunologia.	Departamento	de	Microbiologia	
       Chicken	 anaemia	 virus	 (CAV)	 is	 an	 unusually	 small	 virus	 that	            -	Faculdade	de	Ciências	Agrárias	e	Veterinárias,	Universidade	
causes	 severe	 anaemia	 and	 immunosuppression	 in	 young	 chick-                    Estadual	Paulista,	Rod.	Prof.	Paulo	D.	Castellane,	s/n.	Jaboticabal,	
ens.	 Such	 effects	 reduce	 the	 efficiency	 of	 routine	 vaccinations	 while	       SP,	Brasil,	Projeto	Financiado	pela	Fundação	de	Auxílio	à	Pesquisa	
aggravating	the	effects	of	other	pathogens	in	chicken	populations,	con-                do	Estado	de	São	Paulo	(FAPESP),	processos	nº	02/08649-1	e	nº	
stituting	a	serious	economic	threat	to	poultry	industry.	The	replication/                   01/14950-3	e	bolsa	de	estudos	concedida	pela	CAPES.		
pathogenicity	of	CAV	relies	on	the	expression	of	only	a	single	structural	                   alinegcaetano@yahoo.com.br	/	heliojm@fcav.unesp.br
protein	 VP1	 and	 two	 non-structural	 proteins	 VP2	 and	 VP3	 (apoptin).	               A	Bronchitis	Virus	(IBV)	was	produced	at	high	level	in	Escherichia	
The	function	of	each	individual	protein	is	as	yet	unclear.	This	study	has	           coli.	The	codifying	genes	functional	single-chain	Fv	antibody	fragment	
used	transcriptional	profiling	to	identify	pathways	that	are	dramatically	           (scFv)	 specific	 for	 the	 H120	 vaccine	 strain	 of	 Infectious	 for	 variable	
modulated	after	48	hr	of	infection	in	an	in	vitro	model	(MDCC-MSB1	                  regions	of	immunoglobulin	(Ig)	heavy	chain	(VH)	and	light	chain	(VL)	
cells)	and	after	4	days	of	experimental	infection	(1	day	old	chickens-	in	           were	amplified	from	spleen	RNA	extracts	collected	from	SPF	chickens	            	
vivo	model),	using	Affymetrix	chicken	oligonucleotide	arrays.	Changes	               immunized	against	IBV,	using	two	specific	pair	of	primers		by	RT-PCR	
in	 transcript	 levels	 between	 infected	 and	 uninfected	 cells	 indicate	 a	      and	these	amplicons	were	connected	to	a	flexible	oligo-peptide	linker,	
dysfunction	 in	 the	 Mitogen	Activated	 Protein	 kinases	 (MAPK)	 and	 T	           using	another	specific	set	of	primers	and	PCR.	The	final	construct	of	
cell	receptor	signalling	cascades	implying	that	CAV	might	subvert	host	              this	recombinant	gene	(VH-linker-VL),	which	is	able	to	codify	the	ScFv	
76


     antibody	 fragments,	 was	 inserted	 into	 a	 phagemid	 pCANTAB	 5E	 fol-                        CM	APPoLInáRIo,	AM	MAZInI,	J	MEGID
     lowed	 by	 panning	 -	 selection	 with	 the	 Recombinant	 Phage	Antibody	         Universidade	Estadual	Paulista	“Júlio	de	Mesquita	Filho”-	Campus	de	
     System	(RPAS)	against	purified	IBV	particles.	The	scFv	gene	inserted	                   Botucatu	Faculdade	de	Medicina	Veterinária	e	Zootecnia	
     into	 the	 recombinant	 phagemid	 pCANTAB	 5E	 –	 H11	 and	 harvested	                   Departamento	de	Higiene	Veterinária	e	Saúde	Pública		
     from	a	positive	clone	carrying	anti-IBV	specificity	was	subcloned	into	                                  jane@fmvz.unesp.br
     pET28a	fused	to	N-terminal	His-tag	sequence	in	frame	and,	following	
     IPTG	induction,	it	was	overexpressed	in	E.	coli	BL21.	The	presence	of	                  The	 use	 of	 immunomodulators,	 antiviral	 drugs	 and	 interferon	 in	
     scFv	anti-IBV	antibodies	was	determined	by	sodium	dodecyl	sulfate-                rabies	 was	 reported	 by	 some	 authors.	 	 Propionibacterium	 acnes	 (P.	
     polyacrylamide	 gel	 eletrophoresis	 (SDS-PAGE)	 followed	 by	 Western	           acnes),	 as	 immunomodulator,	 has	 been	 evaluated	 in	 rabies	 virus	
     blotting	analyses.	A	protein	fraction	with	the	expected	molecular	weight	         infected	 mice	 and	 researchers	 observed	 greater	 survival	 rates	 in	
     of	approximately	32KDa	and	carrying	the	poly-histidine	tag	was	recog-             animals	 treated	 with	 the	 immunomodulator	 and	 Fuenzalida	 Palacios	
     nized	by	anti-His6	MAb.	This	monoclonal	scFv	antibody	produced	here	              antirabies	vaccine.	The	percentage	of	survival	was	correlated	to	higher	
     combined	specifically	with	the	subunit	1	of	spike	glycoprotein	(S1)	of	           positivity	to	fluorescent	antibody	test	(FAT)		in	lymphoid	tissues	in	the	
     IBV	and	did	not	react	with	other	virus	structural	proteins.	Thus,	such	           infected	vaccinated	mice	submitted	to	P.acnes,	followed		by	the	group	
     recombinant	 antibody	 fragment	 could	 be	 useful	 to	 characterize	 more	       submitted	 to	 P.acnes	 suggesting	 	 macrophage	 activation	 induced	 by	
     precisely	the	conformational	structure	of	S1	glycoprotein	and	its	immu-           P.acnes	 	 and	 viral	 sequester	 inside	 the	 someones.	 Considering	 the	
     nogenicity,	as	well	as	its	variability,	since	this	protein	carries	the	major	     possibility	of	virus	persistence	in	spleen	of	infected	mice	and	the	sub-
     virus-neutralizing	 epitopes	 and	 suffers	 preferentially	 more	 variability	    stitution	of	Fuenzalida	Palacios	by	VERO	antirabies	vaccine	in	Brazil	
     during	virus	evolution.                                                           the	objective	of	this	work	was	to	compare	the	percentage	of	positivity	
                                                                                       to	FAT	in	spleen	of	rabies	virus	infected	mice	submitted	or	not	to	antira-
     Key words:	 Infectious	 Bronchitis	 Virus,	 Monoclonal	 scFv	 antibody,	          bies	VERO	or	Fuenzalida	Palacios	vaccine		in	different	moments	post	
     Recombinant	antibody,	Escharichia	coli                                            inoculation.	This	trial	was	conducted	using	16	experimental	groups	with	
     Species:	avian                                                                    40	 mice	 in	 each,	 divided	 in	 2	 subgroups,	 being	 8	 using	 Fuenzalida	
                                                                                       Palacios	 vaccine	 and	 8	 using	 VERO	 vaccine.	Animals	 were	 infected	
       bV070. TLR7 AND INOS RNA ExPRESSION IN SPLEEN                                   by	intramusculary	route	and	after	24	hours	received	or	not	anti-rabies	
     CELLS OF COCONUT MEAL-FED BROILERS AFTER A VIRAL                                  vaccine.	P.	acnes	was	used	isolated	or	in	association	with	vaccine	in	
                    CHALLENGE WITH IBVD                                                one,	 two	 or	 three	 doses.After	 immunomodulator	 treatment,	 6	 mices	
         SUSAn	D	EICHER1,	TSAnG	L	LIn2,	CHInG	C	WU2	ToDD	J	                            from	each	group	were	killed	after	5d,	10d,	14d	and	21d	post	infection	
                  APPLEGATE3,JoHn	A	PATTERSon3                                         and	 spleen	 and	 brain	 collected.	 The	 material	 was	 submitted	 to	 FAT.	
       1USDA-ARS,	2Department	of	Comparative	Pathobiology,3School	                     The	percentage	of	lethality	was	evaluated	in	a	number	of	8	mice	from	
       of	Veterinary	Medicine	and	Department	of	Animal	Science,	Purdue	                each	 group	 maintained	 in	 a	 separate	 cage,	 observed	 for	 symptoms	
                      University,	West	Lafayette,	IN.	USA	                             and	death	during	30	days.Clinical	signs	and	death	were	observed	7-12	
                             spruiett@purdue.edu                                       d	after	virus	inoculation.	Greater	percentage	of	positivity	was	observed	
                                                                                       in	spleen,	comparatively	to	brain,	in	almost	all	groups	and	moments.	
            Coconut	 meal	 is	 immunomodulating	 in	 part	 because	 of	 its	 lau-      The	positivity	was	not	related	to	treatment	with	P.	acnes	or	antirabies	
     ric	 acid	 content.	 Whether	 this	 dietary	 immunomoulator	 can	 enhance	        vaccine.	 Rabies	 virus	 was	 detected	 in	 spleen	 and	 brain	 before	 the	
     innate	immunity	of	chickens	to	circumvent	or	combat	a	viral	challenge	            presence	of	clinical	signs	and	positivity	was	also	detected	21	d	after	
     has	not	been	studied.	Chickens	are	frequently	raised	in	confinement	              virus	inoculation	suggesting	rabies	virus	persistence	in	agreement	to	
     buildings,	but	a	growing	trend	for	pasture	raised	birds	is	evident.	The	          authors	that	consider	vírus	replication	and	persistence	in	macrophages	
     objective	of	this	work	was	to	determine	the	effect	of	coconut	meal	on	            correlated	with	a	longer	incubation	period	with	possibility	of	subsequent	
     innate	 immune	 function	 of	 pasture	 and	 confinement	 raised	 chickens	        virus	replication	.
     without	 and	 with	 Infectious	 Bursal	 Disease	 (IBDV).	 	 Sixty	 day-old	
     broiler	chickens	were	assigned	to	either	a	coconut	meal	diet	or	a	stan-           Key words:	 rabies,	 macrophages,	 Propionibacterium	 acnes,	
     dard	corn/soy	diet	containing	antibiotics	in	each	of	two	studies.		The	           Fluorescent	Body	Test
     first	study	was	conducted	with	pasture	raised	broilers	and	the	second	            Species:	other
     with	broilers	in	typical	confinement	housing.		After	receiving	the	diets	
     for	4	weeks,	birds	were	challenged	orally	with	4.1	x	103	EID50	IBDV	                  bV072. PRODUCTION OF TNF-A AND IL-6, ANTIBODy
     virus.		Six	chickens	from	each	dietary	group	were	euthanized	at	0,	24	                 RESPONSE, AND BACTERIAL RECOVERy, DURING
     and	 96	 h	 after	 challenge	 and	 spleens	 harvested	 for	 determination	 of	                  LEPTOSPIROSIS INFECTION
     toll-like	 receptor	 (TLR)	 7	 and	 inducible	 nitric	 oxide	 synthase	 (iNOS)	      *M	MARInHo,C	SILVA,	VMF	LIMA,	G	F	MACHADo,J	R	PEIRo,	
     RNA	expression.	Toll-like	receptor	7	increased	(P	<	0.05)	following	the	                                  SHV	PERRI
     viral	challenge	in	both	pasture	raised	and	confinement	raised	chickens	            Departamento	de	Apoio	Produção	e	Saude	Animal,	Faculdade	de	
     and	was	greater	(P	<	0.05)	for	the	coconut	diet	at	96	h.		The	expres-              Odontologia	de	Araçatuba,	Universidade	Estadual	Paulista	Júlio	de	
     sion	 of	 iNOS	 in	 the	 spleen	 cells	 did	 not	 change	 in	 the	 confinement	                            Mesquita	Filho	
     raised	birds	(P	>	0.10),	but	was	initially	greater	in	the	pasture	raised	                            *mmarinho@fmva.unesp.br
     birds,	suggesting	exposure	to	bacterial	pathogens	in	the	environment.	        	
     iNOS	decreased	by	1.67	fold	by	96	h	compared	to	0h	for	birds	fed	the	                   The	 aims	 of	 the	 present	 study	 were	 to	 investigate	 the	 humoral	
     corn/soy	diet	and	decreased	by	more	than	10,000	fold	for	coconut	meal	            and	cellular	response	kinetics	in	leptospirosis.	It	was	observed	that	the	
     fed	birds.		iNOS	was	less	(P	<	0.05)	in	the	birds	fed	the	coconut	diet	           presence	of	the	tumor	necrosis	factor-alpha	(TNF-a)	and	interleukin-6	
     than	for	the	corn/soy	fed	birds	at	96	h	post	challenge.	These	data	show	          (IL-6)	were	associated	to	the	production	of	antibodies	and	the	bacterial	
     the	viral	challenge	enhanced	TLR7	expression	by	96	h	after	challenge	             recovery,	and	the	compromising	of	both	in	the	immunopathogenesis	of	
     and	was	affected	by	dietary	treatment	by	that	time.		Expression	of	iNOS	          leptospirosis	in	an	experimental	infection	of	Balb/c	mice	inoculated	with	
     was	variably	affected	by	the	viral	challenge	in	birds	raised	under	differ-        Leptospira	 interrogans	 serovar	 canicola.	 The	 analysis	 of	 the	 results	
     ent	conditions	and	fed	different	diets.		These	results	will	be	useful	for	        showed	higher	levels	of	TNF	it	and	IL-6	in	the	initial	phase	of	the	infec-
     producers	in	determining	the	risks	and	benefits	when	seeking	possible	            tion,	 period	 where	 it	 was	 observed	 the	 greatest	 bacterial	 clearence.	
     immune	 modulators	 to	 replace	 antibiotics.	 This	 study	 was	 funded	 in	      However,	 when	 comparing	 the	 bacterial	 recovery	 with	 the	 kinetics	
     part	by	Tropical	Traditions.                                                      of	 the	 production	 of	 antibodies,	 the	 results	 revealed	 a	 proportionally	
                                                                                       inverted	kinectics	to	the	production	of	antibodies.	we	concluded	that	in	
     Key words:	TLR7,	iNOS,	Coconut	meal,	IBVD                                         leptospirosis	there	is	a	greater	mobilization	of	the	activity	of	the	cellular	
     Species:	avian                                                                    immune	response,	mainly	in	the	initial	phase	of	the	infectious	process,	
                                                                                       for	posterior	involvement	of	the	humoral	response.	This	fact	could	be	
         bV071. FLUORESCENT ANTIBODy TEST IN SPLEEN                                    associated	 to	 some	 inhibitory	 factor	 which	 could	 be	 responsible	 for	
          OF RABIES VIRUS INFECTED MICE SUBMITTED TO                                   the	selective	suppression	of	the	cellular	immune	response	and	that	as	
                  PRoPIoNIBACTERIUM ACNES                                              much	the	TNF-α	as	the	IL-6	could	be	associated	to	the	immunopatho-
                                                                                                                                                                          77


genesis	of	the	disease.	We	concluded	that	in	leptospirosis	there	is	a	                    bV075. IL-15, TNF-α AND THE AUTOIMMUNE
greater	 mobilization	 of	 the	 activity	 of	 the	 cellular	 immune	 response,	        PATHOGENESIS OF MALIGNANT CATARRHAL FEVER
mainly	in	the	initial	phase	of	the	infectious	process,	and	that	as	much	             DAVID	M	HAIG,	IAn	AnDERSon,	DAVID	DEAnE,	SAnDI	SWA1,	
the	TNF-α	as	the	IL-6	could	be	associated	to	the	immunepathogenesis	                               x-q	WEI2,	GEoRGE	RUSSELL
of	the	disease.
                                                                                      Moredun	Research	Institute,	Edinburgh,	1Cancer	Research	UK,	
Key words:	 cytokines,	 cellular	 immune	 response,	 Balb/c	 mice,	                           London,	U.K.,	2University	of	Cardiff,	Wales.
leptospirosis	
                                                                                         Malignant	 catarrhal	 fever	 (MCF)	 is	 a	 fatal	 lymphoproliferative	
Species:	other
                                                                                   disease	 of	 cattle,	 deer,	 pigs	 and	 bison.	 It	 is	 caused	 by	 either	 of	 the	
                                                                                   γ-herpesviruses	AlHV-1	or	OvHV-2	that	do	not	cause	disease	in	their	
        bV073. EFFECT OF BACILLUS CEREUS AND                                       reservoir	hosts	(wildebeest	and	sheep	respectively)	but	cause	disease	
      SACCHARoMyCES BoULARDII ON THE HUMORAL                                       in	 susceptible	 species	 characterised	 by	 lymphocyte	 accumulation	
            RESPONSE OF VACCINATED MICE                                            and	 areas	 of	 necrosis	 in	 multiple	 tissues.	 We	 believe	 that	 the	 tissue	
     TALITA	BAnDEIRA	RooS1,	AG	SAnToS2,	G	FISCHER2,	T	                             destruction	 in	 MCF	 is	 due	 to	 the	 indiscriminate	 cytotoxic	 activity	 of	
              VIDoR3,	FPL	LEITE4,	C	GIL-TURnES                                     lymphocytes	activated	as	a	consequence	of	infection.	The	pathology	
                                                                                   of	MCF	is	similar	to	that	seen	in	some	autoimmune	diseases	involving	
1FV/UFPel,		2CENBIOT/UFPel,	3Laboratório	de	Virologia,	FV/UFPel,	
                                                                                   dysregulated	expression	of	IL-15	(and	consequently	TNF-a)	that	main-
              4Laboratório	de	Imunologia,	IB/UFPel
                                                                                   tains	 active	 cytotoxic	 cells.	 In	 a	 rabbit	 model	 of	AlHV-1	 and	 OvHV-2	
      Probiotics	have	some	kind	o	effect	on	both	the	innate	and	acquired	          infection,	we	discovered	that	there	is	abundant	expression	of	IL-15	and	
immune	responses,	probably	due	to	the	acceleration	of	the	formation	               TNF-α	in	tissues	affected	by	MCF.	The	IL-15-producing	cells	appeared	
of	memory	clones,	increasing	the	specific	immune	response.	However,	               to	 be	 predominantly	 non-T	 non-B	 lymphocytes.	 Large	 granular	 lym-
studies	on	the	effect	of	probiotics	on	immunity,	are	scarce.	The	objec-            phocytes	 (LGL),	 which	 are	 virus-infected	 cells	 obtained	 from	 MCF-
tive	of	this	research	was	to	evaluate	the	effect	of	Bacillus	cereus	var.	          affected	 tissues	 in	 culture,	 did	 not	 produce	 IL-15	 but	 did	 respond	 to	
toyoi	 and	 of	 Saccharomyces	 boulardii	 on	 the	 humoral	 response	 of	          the	 cytokine	 in	 bioassays.	 Experiments	 to	 deplete	 IL-15	 in	 vivo	 with	
mice	to	an	inactivated,	oil	adjuvanted	Bovine	Herpes	Virus-5	vaccine.	             an	IL-15	receptor	protein	gave	equivocal	results.	This	could	be	due	to	
Isogenic	Balb-c	 mice	 were	randomly	 grouped.	The	animals	 were	 fed	             the	 recent	 discovery	 that	 such	 treatment	 can	 enhance	 IL-15	 activity	
with	 a	 commercial	 feed	 free	 of	 antibacterials	 supplemented	 with	 S.	       rather	than	block	it.	Other	experiments	are	under	way	to	block	TNF-α	
boulardii	(1x107	UFC	gr-1)	for	group	A,	B.	cereus	var.	toyoi	(1x106	viable	        with	Etanercept®,	a	TNF-α	receptor	protein.	We	conclude	that	IL-15	is	
spores	gr-1)	for	group	B	and	without	supplementation	for	group	C.	The	             produced	in	abundance	in	MCF	and	that	cytotoxic	cells	maintain	their	
animals	were	vaccinated	on	days	0,	28	and	114	with	1/20th	of	a	bovine	             active	 phenotype	 in	 the	 presence	 of	 IL-15,	 contributing	 to	 the	 tissue	
dose.	Blood	was	collected	from	the	orbital	sinus	on	days		0,	42,	114	              damage	seen	in	MCF.	
and	 144.	 The	 humoral	 response	 was	 quantified	 by	 ELISA	 using	 the	         Key words:	 Herpesvirus,	 autoimmunity,	 interleukin-15,	 malignant	
homologous	virus	as	antigen.	Results	were	transformed	in	seroconver-               catarrhal	fever
sions	 dividing	 the	 actual	 absorbencies	 by	 that	 of	 day	 0	 of	 the	 same	   Species:	rabbit
animal.	Mean	seroconversions	on	days	42,	114	e	144	of	group	A	were	
8.9,	8.5	and	9.9,	those	of	groups	B	were	10.1,	7.2	and	7.7,	and	those	             bV076. IMMUNE RESPONSES AGAINST MEASLES VIRUS IN
of	 group	 C	 were	 8.2,	 6.7	 e	 7.0,	 respectively.	 Seroconversions	 of	 the	                CyNOMOLGUS MONKEyS
animals	 that	 received	 probiotics	 were	 significantly	 (P	 <	 0.01)	 higher.	         HIRoKI	SATo,	FUMIo	KoBUnE,	yASUSHI	AMI,	MISAKo	
The	fact	that	the	response	in	supplemented	animals	remained	higher	                                    yonEDA,CHIEKo	KAI
even	 after	 suspending	 probiotic	 administration	 suggests	 that	 	 the	
immuomodulation	was	triggered	at	the	beginning	of	its	administration.	             Laboratory	Animal	Research	Center,	Institute	of	Medical	Science,	The	
We	 concluded	 that	 both	 probiotics	 enhanced	 the	 humoral	 response	                            University	of	Tokyo,	Tokyo,	Japan
against	the	vaccine.                                                                      Measles	virus	(MV)	induces	profound	suppression	of	the	immune	
                                                                                   response	during	and	for	weeks	after	acute	infection.	On	the	other	hand,	
     Supported	by	CNPq	(474509/2004-4)	and	FAPERGS	(0523299)	
                                                                                   virus-specific	immune	responses	that	mediate	viral	clearance	and	con-
Key words:	Probiotics,	Immunomodulation,	BHV-5                                     fer	long-lasting	immunity	are	efficiently	generated.	To	investigate	this	
Species:	other	(mice)                                                              paradox	 we	 studied	 the	 immune	 responses	 in	 cynomolgus	 monkeys	
                                                                                   against	 MV	 infection.	 Cynomolgus	 monkeys	 experimentally	 infected	
  bV074. A NEW ADJUVANT ENHANCES THE PR OF THE                                     with	wild-type	MV	(MV-HL)	showed	marked	leukopenia	associated	with	
COMMERCIAL INFLUENZA VACCINE IN THE FERRET MODEL                                   a	steady	reduction	in	CD4+	T	cell	numbers	for	18	days	post	inoculation.	
   CyRIL	J	MARTEL1,	TRInE	H	JEnSEn1,	LARS	P	nIELSEn2,	                             Transient	expression	of	interferon	and	interleukin	(IL)-6	were	observed	
     BIRGITTE	VIUFF1,	ELSE-MARIE	AGGER3,	MERETE	                                   in	the	serum	between	four	and	six	days	post	inoculation,	indicating	that	
BLIxEnKRonE-MØLLER1,	PETER	AnDERSEn3,	BEnT	AASTED1	                                MV	replication	induced	early	antiviral	response.	IL-10	levels	increased	
                                                                                   after	11	days	post	inoculation,	suggesting	that	the	prolonged	immuno-
    1Institute	of	Veterinary	Pathobiology,	Faculty	of	Life	Sciences,	
                                                                                   suppression	observed	during	measles	infection	is	due	to	the	inhibitory	
    University	of	Copenhagen,	Denmark,	2Department	of	Virology,	
                                                                                   effects	of	IL-10	on	type	1	CD4+	T	cells.	Interestingly,	serum	levels	of	IL-
    Statens	Serum	Institute,	Copenhagen,	Denmark,	3Department	
                                                                                   8	showed	considerable	variation	that	was	characteristic	of	three	peaks	
    of	Immunology	of	Infectious	Diseases,	Statens	Serum	Institute,	
                                                                                   at	day	3,	5-6,	and	11	post	inoculation.	IL-8	mRNA	in	peripheral	blood	
                         Copenhagen,	Denmark
                                                                                   mononuclear	cells	peaked	at	day	2,	4,	and	9	post	inoculation,	which	
      DDA-TDB	 is	 a	 cationic	 liposome-based	 adjuvant	 known	 to	 pro-          correlated	with	IL-8	serum	protein	levels.	Although	the	first	IL-8	serum	
duce	 a	 very	 substantial	 CMI	 and	 at	 the	 same	 time	 a	 strong	 humoral	     protein	peak	was	the	highest	of	the	three	protein	peaks	observed,	the	
response,	desirable	for	a	high	number	of	disease	targets.	We	tested	               IL-8	 mRNA	 peak	 was	 lower	 than	 estimated	 from	 the	 serum	 protein.	
the	effect	of	this	adjuvant	when	combined	to	a	commercially	available	             In	 vitro	 study	 using	 a	 respiratory	 epithelial	 cell	 line	 revealed	 that	 MV	
inactivated	 influenza	 vaccine.	 When	 challenged	 with	 H1N1	 A/New	             particle	 induces	 IL-8	 production	 via	 binding	 to	 and/or	 incorporation	
Caledonia/20/99,	 ferrets	 immunized	 with	 the	 adjuvanted	 vaccine	 dis-         into	 cells	 without	 virus	 replication.	 These	 suggested	 that	 rapid	 IL-8	
played	a	much	stronger	humoral	response	and	lower	viral	titers	than	               secretion	at	day	three	post	inoculation	is	primarily	induced	by	a	small	
the	 ones	 that	 received	 only	 the	 regular	 vaccine.	 Gamma-interferon	         amount	of	circulating	MV	particles	that	arose	from	respiratory	epithelial	
production,	assessed	by	both	RT-PCR	and	flow	cytometry,	and	pathol-                cells.	 The	 cynomolgus	 monkey	 is	 valuable	 for	 the	 study	 of	 measles	
ogy	studies	on	the	upper	and	lower	respiratory	tract	confirmed	those	              pathogenesis,	the	mechanism	of	long-term	immune	memory,	and	for	
findings.	This	study	indicates	that	DDA/TDB	has	a	strong	potential	to	             the	development	of	a	new	measles	vaccine	that	would	still	be	effective	
be	used	as	an	adjuvant	for	inactivated	influenza	vaccines.	                        in	the	presence	of	maternal	neutralizing	antibodies.
Key words:	Vaccine,	adjuvant,	influenza,	ferret,	model	                            Key words:	measles	virus,	cytokine,	immunosuppression,	cynomolgus	
Species:	other	(human	and	ferret)                                                  monkey
78


     Species:	other	(monkeys)                                                         immune	 response	 with	 various	 antigens.	 Specific	 range	 of	 adjuvant	
                                                                                      based	on	metabolisable	oils	can	be	used	to	reduce	local	and	general	
               bV077. VETERINARy VACCINE ADJUVANTS                                    reactions	linked	to	reactive	antigens	association	to	mineral	oil.	A	sec-
                                                                                      ond	 generation	 of	 adjuvant	 suitable	 for	 veterinary	 vaccines	 is	 based	
      L	DUPUIS1,	S	DEVILLE1,	J	AUCoUTURIER1,	S	ASCARATEIL1,	A	
                                                                                      on	 nanoparticles	 with	 a	 new	 immunostimulant	 and	 combined	 their	
                          LAVAL2,V	GAnnE3
                                                                                      immunostimulating	 properties	 to	 induce	 a	 positive	 synergistic	 effect.	    	
      1Tour	KupkaC	7	boulevard	Franck	Kupka	92039	Paris	La	Defense	                   Different	 mechanisms	 of	 action	 can	 explain	 the	 efficacy	 of	 adjuvant:	   	
      cedex,	France,	2National	veterinary	school	of	Nantes,	Atlanpole-La	             -The	 depot	 effect	 where	 the	 emulsion	 entraps	 the	 antigens	 and	
       Chantrerie-BP	40706	Nantes	cedex	03	France,	3SEPPIC	China	                     induce	 a	 slow	 release	 of	 it	 at	 the	 injection	 site.	 Inflammatory	 reac-
        Room	510	Jin	Tai	Building	58	South	Moa	Ming	Road.	Shanghai	                   tion	 correlated	 with	 the	 induction	 of	 an	 immune	 response	 can	 be	
                                200020	China.                                         also	 observed.	 -The	 recruitment	 of	 immuno-competent	 cells	 by	
          The	development	of	efficacious	and	safe	vaccine	is	more	and	more	           micro	 diffusion	 of	 the	 droplets	 via	 the	 lymphatic	 system,	 or	 by	
     linked	to	the	selection	of	an	appropriate	adjuvant.	Specific	adjuvants	for	      the	 facilitation	 of	 the	 antigen	 uptake	 by	 antigen	 presenting	 cells.	
     veterinary	 accines	 ave	 o	 e	 elected	 ccording	 o	 arious	 riteria	ike	 he	
                v         h     t b s         a         t v       c       l t         -Adjuvants	can	also	enhance	the	humoral	and	cell	mediated	immune	
     target	species,	kind	of	antigen,	the	type	and	duration	of	immune	response	       response.	 Therefore,	 the	 choice	 of	 the	 adjuvant	 should	 be	 done	
     needed	for	protection.	There	is	no	known	universal	adjuvant	formula.	        	   according	 to	 several	 criteria	 with	 the	 goal	 to	 obtain	 a	 good	 balance	
     Most	commonly	used	adjuvants	in	veterinary	vaccines	are	oil	adjuvants	           between	 safety	 and	 immunogenicity.	 For	 example,	 a	 well	 tolerated	
     and	aluminum	hydroxide.	Oil	adjuvants	are	generally	water	in	oil	for-            adjuvant	should	be	recommended	for	use	with	a	crude	bacterial	extract	
     mulations	 inducing	 strong	 and	 long	 term	 immunity.	Adjuvants	 based	        and	also	for	use	with	LPS,	both	of	which	are	reactive.	Conversely,	a	
     on	mineral	oils	are	known	to	be	efficacious	but	can	sometimes	induce	            recombinant	viral	protein	can	be	a	weak	immunogen	thus	requiring	a	
     local	reactions	with	reactive	antigens.	Multiphasic	emulsions	have	also	         strong	adjuvant.
     proved	their	efficacy	in	vaccine	as	they	can	induce	short	and	long	term	         Key words:	vaccine,	adjuvant,	Montanide	
                                                                                      Species:	all	species
                 3. IMMUNOENDOCRINOLOGy, AND STRESS, IMMUNOLOGy OF REPRODUCTION AND NEONATES,
                      MICROBIAL FLORA, NUTRIENTS AND THE IMMUNE RESPONSE: POSTERS ER078 - ER094


 er078. EFFECTS OF BOVINE RECOMBINANT LEPTIN ON                                    after	parturition,	but	the	CD4	positive	cells	showed	no	change	during	
  PROLIFERATION OF HEAT SHOCKED LyMPHOCyTES IN                                     the	 observation	 period.	To	 clarify	 the	 IFN-γ	 expressing	 colostral	 lym-
                   DAIRy COWS                                                      phocytes,	magnetic	separation	technology	(Dynabeads,	DYNAL)	was	
                                                                                   employed	to	sort	the	lymphocytes	(CD4,	CD8	and	γd-T)	from	the	colos-
     nICoLA	LACETERA,	GIoRGInA	KUZMInSKy,	UMBERTo	
                                                                                   tral	cells.	These	positively	selected	lymphocytes	have	been	examined	
    BERnABUCCI,	PATRIZIA	MoRERA,	LoREDAnA	BASIRICò,	
                                                                                   for	 IFN-γ	 mRNA	 expression	 by	 RT-real	 time	 PCR	 (RtPCR).	 RtPCR	
                 ALESSAnDRo	nARDonE
                                                                                   analysis	 showed	 a	 potent	 expression	 of	 IFN-γ	 gene	 in	 CD8	 positive	
 Dipartimento	di	Produzioni	Animali,	Università	della	Tuscia,	Viterbo,	            cells	 and	 the	 gene	 expression	 was	 higher	 than	 CD4	 or	 γd-T	 positive	
                                Italy		                                            cells.	These	results	suggest	that	CD8	positive	T	cells	in	colostrum	play	
                          nicgio@unitus.it                                         a	role	in	IFN-γ	producing	cells.
      We	 have	 previously	 reported	 that	 heat	 shock	 alters	 mitogen	          References:
driven	 proliferation	 and	 gene	 expression	 of	 leptin	 and	 leptin	 recep-           1.	Hagiwara,	K.,	S.	Kataoka.,	H,	Yamanaka.,	K,	R,	Kirisawa	and	H,	
tors	in	peripheral	blood	mononuclear	cells	(PBMC)	of	dairy	cows,	and	              Iwai.	2000.	Detection	of	Cytokines	in	Bovine	colostrum.	Vet.	Immunol.	
that	under	conditions	of	elevated	temperatures	PBMC	proliferation	in	              Immunopathol.	76:	183-190.
response	to	mitogens	is	positively	correlated	with	leptin	mRNA.	Present	
                                                                                   Key words:	IFN-γ,	,	γδ-T	cell,	CD8+	T	cell,	colostrum
study	was	carried	out	to	establish	whether	addition	of	bovine	recombi-
                                                                                   Species:	ruminants
nant	leptin	to	bovine	PBMC	cultured	under	elevated	temperatures	may	
counteract	the	impairment	of	the	proliferative	response	of	these	cells	
to	 concanavalin	 A	 (ConA).	 Six	 Holstein	 early	 pregnant	 heifers	 were	         er080. THE EFFECTS OF LOW LEVELS OF DIETARy
utilised	as	blood	donors.	PBMC	were	cultured	under	39	or	42	°C	for	                COBALT ON SELECT PARAMETERS OF THE SPECIFIC AND
65	 hours,	 in	 the	 presence	 of	 the	 following	 concentrations	 of	 bovine	        NON-SPECIFIC IMMUNE RESPONSES OF GOATS
recombinant	 leptin:	 0,	 9,	 18,	 27,	 or	 150	 ng/ml.	 ConA	 was	 added	 to	         EUGEnE	H	JoHnSon*,	KHALID	AL-HABSI,RASHILD	M	AL-
PBMC	cultures	at	concentrations	of	2.5	or	0.25	µg/ml.	Additional	wells	                                   BUSAIDy
were	also	arranged	to	contain	the	five	concentrations	of	leptin	without	
                                                                                   Department	of	Animal	and	Veterinary	Sciences,	College	of	Agricultural	
ConA.	Cultivation	of	PBMC	at	39	°C	was	intended	to	mimic	conditions	
                                                                                   and	Marine	Sciences,	Sultan	Qaboos	University,	P.O.	Box	34,	Al-Khod	
of	normothermia,	whereas	cultivation	at	42	°C	was	realised	to	simulate	
                                                                                                               123,	Oman	
conditions	of	hyperthermia.	Addition	of	leptin	in	absence	of	ConA	did	
                                                                                                        *ejohnson@squ.edu.om
not	 affect	 proliferation	 of	 PBMC.	 Proliferation	 of	 PBMC	 under	 39	 °C	
was	positively	affected	by	the	highest	concentration	of	leptin	(150	ng/                   The	trace	element	cobalt	(Co)	is	synthesized	by	rumen	bacteria	to	
ml)	only	when	a	suboptimal	concentration	of	ConA	was	utilized	(0.25	               produce	 vitamin	 B12	 which	 assists	 the	 enzymes	 methylmalonyl-coen-
µg/ml).	These	results	are	in	line	with	those	already	reported	for	other	           zyme	A	mutase	in	the	formation	of	glucose	and	methionine	synthase	
species.	Exposure	of	PBMC	to	42	°C	was	responsible	for	decreased	                  needed	for	methane,	acetate	and	methionine	synthesis.	Goats,	in	con-
proliferation	 only	 when	 the	 suboptimal	 concentration	 of	 ConA	 was	          trast	to	sheep	have	been	reported	to	be	rather	resistant	to	low	levels	
added	to	culture	media,	and	under	these	conditions,	addition	of	leptin	            of	dietary	Co.	However,	in	Oman,	we	have	described	in	goats,	fed	low	
did	 not	 interfere	 with	 the	 negative	 effects	 of	 elevated	 temperature	      levels	of	dietary	cobal,	a	commonly	occurring	condition	referred	to	as	
on	 PBMC	 proliferation.	 Verification	 of	 the	 hypothesis	 that	 addition	 of	   hepatic	 lipidosis,	 as	 well	 as	 	 anemia,	 poor	 weight	 gains,	 reductions	
leptin	may	counteract	the	negative	effects	of	elevated	temperatures	on	            in	 their	 serum	 protein	 levels,	 a	 	 decrease	 in	 their	 apparent	 nutrient	
PBMC	proliferation	provided	negative	results.	Reduced	expression	of	               digestibility	 coefficients	 and	 poorer	 meat	 quality.	 The	 present	 study	
leptin	receptors	due	to	heat	shock	is	likely	to	explain	present	findings.          expands	on	these	observations	in	order	to	ascertain	whether	goats	fed	
                                                                                   low	levels	of	Co	exhibit	alterations	in	select	parameters	of	their	non-
Key words:	leptin,	bovine,	heat	shock,	lymphocyte	proliferation
                                                                                   specific	and	specific	immune	systems.		In	the	first	phase	of	this	study	
Species:	ruminants
                                                                                   we	 utilized	 twenty,	 ten-week	 old,	 newly	 weaned	 male	 Batinah	 goats.	
                                                                                   They	were	randomly	divided	into	two	groups,	namely	a	control	(n=10)	
   er079. COLOSTRAL CD8 POSITIVE CELL IS A POTENT                                  and		a	treated	group	(n	=	10).	Each	group	was	housed	in	separate	pens	
             PRODUCING CELL FOR IFN-γ                                              and	fed	a	diet	of	150	g/day	of	a	specially	formulated	concentrate	and	
       KATSURo	HAGIWARA,	MAyUMI	DoMI,	JUnICHI	AnDo                                 Rhodegrass	hay	ad	libitum	containing	0.12	mg/kg	and	0.1	mg/kg	DM	of	
                        Rakuno	Gakuen	University		                                 Co,	respectively.	Goats	in	the	treated	group	receved	bi-monthly	subcu-
                          k-hagi@rakuno.ac.jp                                      taneous	injections	of	2000	µg	of	hydroxycobalamin.	We	compared	the	
                                                                                   chemiluminescence	(CL)	response	of	neutrophils	(PMN)	isolated	from	
      IFN-γ	plays	an	important	role	in	cellular	immunity	leading	to	micro-         these	animals	during	in	vitro	phagocytosis		of	the	target	zymosan	and	
organism	elimination.	We	have	reported	that	bovine	colostrum	contains	             utilized	a	WST-8		assay	to	compare	their	T-cell	blastogenic	responses	
high	levels	of	IFN-γ	as	well	as	immunoglobulin	(1).	Lymphocytes	are	               to	the	mitogens,	pokeweed	mitoge	(PWM)	and	phytohaemagultinin	A	
potent	cells	for	IFN-γ	production,	therefore,	clarification	of	the	lympho-         (PHA).	The	control	goats	exhibited	as	early	as	three	weeks	after	the	
cyte	 population	 in	 the	 colostrum	 would	 help	 to	 clarify	 the	 source	 of	   onset	of	this	study	a	significantly	lower	CL	response	to	zymosan	and	
colostral	cytokines.	In	this	study,	we	clarified	the	population	of	lympho-         this	 lowered	 response	 was	 still	 evident	 after	 two	 and	 three	 months.	
cyte	 subsets	 in	 colostrum-namely,	 CD4	 (Th)	 cells,	 CD8	 (cytotoxic	 T)	      Similarly,	control	goats	exhibited	lower	blastogenic	responses	to	both	
cells	and	γd-T	cells	by	flow	cytometric	analysis	(EPICS	XL,	Beckman	               PWM	and	PHA.	These	preliminary	results	suggest	that	low	dietary	lev-
Coulter),	and	quantified	the	concentration	of	colostral	IFN-γ	by	ELISA.	           els	of	cobalt	lead	to	a	reduction	in	both	phagocytic	activity	as	well	as	
IFN-γ	was	detected	in	the	colostrum	from	all	the	96	healthy	Holstein	              in	T	cell	responses	and	are	sensitive	indicators	of	developing	vitamin	
cows,	the	levels	tended	to	decrease	on	the	day	subsequent	to	parturi-              B12	deficiency
tion.	Flow	cytometric	analysis	showed	that	many	γd-T	and	CD8	positive	
cells	were	contained	in	the	colostrum,	and	CD4/CD8	ratio	was	a	low.	               Key words:	 cobalt,	 goats,	 phagocytosis,	 neutrophils,	 lymphocytic	
The	ratio	of	CD8	and	γd-T	positive	cells	decreased	during	the	5	days	              proliferation,	,	hepatic	lipidosis
                                                                                   Species:	ruminants
80


      er081. INFLUENCE OF HORMONES IN THE ExPRESSION                                            er083. THE EFFECT OF PREGNANCy ON MATERNAL
      OF INDOLEAMINE-2,3 DIOxIGENASE IN CULTURED CELLS                                                        IMMUNITy IN SHEEP
                   FROM BOVINE PLACENTA                                                        SEAn	WATTEGEDERA,	MARA	RoCCHI,	JAynE	HoPE,GARy	
      A	R	LIMA,	JM	MonTEIRo,	RV	BoSCH,	RS	IUnES,	ET	FIoRETTo,	                                                   EnTRICAn	
                             LJ	oLIVEIRA,	JR	KFoURy	JR.                                                             gary.entrican@moredun.ac.uk	
            The	 maternal	 immune	 system	 is	 challenged	 to	 tolerate	 the	 con-
     ceptus	 as	 a	 semi-allogeneic	 graft	 during	 pregnancy.	 The	 enzyme	                  Moredun	Research	Institute,	Pentlands	Science	Park,	Bush	Loan,	
     indoleamine	2,3	dioxygenase	(IDO)	has	been	shown	to	play	a	role	in	                      Midlothian,	EH26	0PZ,	UK,	1	Institute	for	Animal	Health,	Compton,	
     the	 maternal	 immune-tolerance,	 mainly	 through	 catabolism	 of	 tryp-                               Newbury,	Berkshire	RG20	7NN,	UK.	
     tophan,	 resulting	 in	 the	 inhibition	 of	 T-cell	 proliferation	 by	 starvation.	         The	 failure	 of	 eutherian	 mammals	 to	 reject	 the	 semi-allogeneic	
     In	 addition	 the	 maternal	 immune	 system	 hormonal	 regulation	 is	 still	          fetus	 is	 an	 immunological	 paradox	 in	 the	 context	 of	 self-nonself	 dis-
     not	completely	understood.	For	example,	progesterone	increases	the	                    crimination.	In	the	fifty	or	so	years	since	Peter	Medawar	commented	
     production	of	interleukines	(IL	4	and	IL-10)	by	T	cells,	and	the	predomi-              on	this	paradox,	multiple	mechanisms	for	maternal	acceptance	of	the	
     nance	of	estrogen	is	associated	to	the	Th2	response.	The	purpose	of	                   fetus	 have	 been	 postulated	 and	 tested.	 One	 of	 the	 original	 theories	
     this	study	was	to	investigate	the	potential	effect	of	progesterone	and	                was	 that	 the	 maternal	 immune	 system	 was	 suppressed.	 There	 has	
     estrogen	 in	 IDO	 expression	 in	 cultured	 bovine	 placental	 cells	 along	          been	very	little	experimental	evidence	to	support	generalised	maternal	
     the	pregnancy.	Explants	of	placentomes	from	four	animals	from	each	                    immunosuppression,	but	there	is	evidence	for	a	more	subtle	modulation	
     trimester	of	pregnancy	were	cultured	using	supplemented	media	with	                    of	maternal	immunity,	notably	the	down-regulation	of	IFN-γ	production.	
     progesterone	(medroxiprogesterone	acetate,	Promone	E®)	or	estrogen	                    This	has	been	observed	in	humans	and	rodents,	and	in	some	cases	
     (Stradiol	 Cipionate,	 E.C.P.	 ®).	 The	 IDO	 expression	 was	 assessed	 at	           linked	to	pregnancy	failure	during	infection.	We	are	particularly	inter-
     day	zero	(D0)	and	day	one	(D1)	of	culture	by	flow	cytometry	using	anti-                ested	in	host-pathogen	interactions	during	chlamydial	abortion	in	sheep	
     IDO	mouse	monoclonal	antibody	(Upstate®,USA).	Analysis	of	the	IDO	                     since	the	bacterial	pathogen	Chlamydophila	abortus	is	the	single	most	
     levels	in	the	control	group	showed	an	increase	from	D0	to	D1	in	the	                   common	diagnosed	cause	of	infectious	abortion	in	sheep	in	the	UK.	In	
     second	and	third	(64	to	78%	and	67	to	82%,	respectively)	but	not	in	the	               vitro	data	indicate	that	host	control	of	C.	abortus	is	mediated	through	
     first	 trimester	 (74	 to	 75%).	The	 IDO	 expression	 in	 placental	 explants	        IFN-γ	production,	induction	of	indoleamine	2,3-dioxygenase	(IDO)	and	
     in	the	presence	of	estrogen	was	similar	to	the	control	group	in	the	first	             subsequent	deprivation	of	tryptophan.	Since	C.	abortus	establishes	a	
     trimester	 (73%	 to	 76%),	 nevertheless	 the	 second	 and	 third	 trimester	          persistent	infection	that	usually	only	manifests	itself	during	pregnancy,	
     revealed	lower	values,	76%	to	67%	and	84%	to	73%,	respectively.	In	a	                  we	were	interested	to	know	if	T	cells	from	pregnant	sheep	exhibited	a	
     presence	of	progesterone	of	IDO	expression	was	lower	when	compar-                      reduced	capacity	of	IFN-γ	that	could	offer	an	explanation	for	recrudes-
     ing	the	D0	to	D1	in	the	first	and	second	trimesters	(72%	to	68%	and	                   cence	 of	 the	 organism	 and	 invasion	 of	 the	 placenta.	 We	 immunised	
     79%	to	73%),	and	higher	values	in	the	third	trimester	(69%	to	80%).	                   sheep	 with	 the	 nominal	 antigen	 ovalbumin	 (Ova)	 prior	 to	 pregnancy	
     The	 results	 suggest	 that	 in	 vitro	 placental	 expression	 of	 IDO	 can	 be	       and	 monitored	 anti-Ova	 T	 cell	 responses	 throughout	 pregnancy	 and	
     affected	by	the	presence	of	progesterone	and	estrogen.                                 compared	these	to	non-pregnant	controls.	We	also	analysed	responses	
     Key words:	indoleamine	2,3	dioxygenase	•	oestrogen	•	progesterone	                     to	the	T	cell	mitogen	concanavalin	A	(Con	A).	We	found	that	pregnancy	
     •	immune	tolerance	                                                                    did	not	significantly	alter	IFN-γ	production	by	peripheral	maternal	ovine	
     Species:	ruminants                                                                     T	cells	in	response	to	in	vitro	restimulation	with	Ova	(P=0.565)	or	Con	
                                                                                            A	(P=0.110).	The	effects	of	IFN-γ	can	be	counteracted	by	IL-4	and	IL-
                                                                                            10,	two	cytokines	that	have	been	reported	to	be	increased	in	pregnant	
              er082. ISOLATION AND PROLIFERATION OF T
                                                                                            mammals.	 Analyses	 revealed	 low	 levels	 of	 both	 these	 cytokines	 in	
                LyMPHOCyTES FROM BOVINE PLACENTA
                                                                                            restimulated	 cultures,	 and	 neither	 were	 elevated	 during	 pregnancy.	
     J	M	MonTEIRo,	R	V	BoSCH,	A	R	LIMA,	M	SAKAI,	K	KIELInG,	G	M	                            These	 data	 indicate	 that	 T	 cell	 responses	 in	 pregnant	 sheep	 do	 not	
           CoSTA,	R	S	IUnES,	CC	ARAúJo,	JR	KFoURy	JR                                        follow	the	Th1/Th2	paradigm	and	that	other	factors	are	likely	to	be	more	
           	J.	R.	University	of	São	Paulo	School	of	Veterinary	Medicine                     important	in	the	pathogenesis	of	ovine	chlamydial	abortion.																										
           Leucocytes	in	the	placenta	behave	distinctively	from	those	of	the	               Key words:	 abortion,	 Chlamydophila	 abortus	 ,•	 indoleamine	 2,3-
     peripheral	blood	and	this	fact	is	directly	related	to	the	immune-response	             dioxygenase	(IDO),	IFN-γ		
     observed	in	the	maternal-fetal	tolerance	process.	This	behavior	includes	              Species:	ruminants
     predominance	 a	Th2	 orientated	 immunoresponse,	 a	 temporary	 toler-
     ance	to	the	embryo	or	fetal	antigens,	involvement	of	immunosupres-                        er084. IN VITRO ExPRESSION OF BOVINE CLASSICAL
     sion	related	lymphocyte	populations,	etc.	The	distribution	and	function	                     AND NON-CLASSICAL MHC CLASS I PROTEINS
     of	 placental	 lymphocytes	 in	 bovines	 are	 still	 unclear,	 therefore,	 as	 a	
                                                                                              P	PARASAR1,	C	SUAREZ2,	DD	nEW1,	WC	DAVIS1,CJ	DAVIES1
     first	step	to	conduct	studies	on	this	subject,	we	attempted	to	establish	
     isolation	 and	 proliferation	 techniques	 for	 lymphocytes	 from	 placenta.	              1Washington	State	University,	Pullman,	WA	USA,	2Agricultural	
     Placentomes	were	sampled	from	cows	in	different	trimesters	of	preg-                               Research	Service,	USDA,	Pullman,	WA	USA
     nancy	 and	 placed	 in	 culture	 by	 explant	 technique.	After	 two	 days	 of	                In	 humans,	 cattle	 and	 other	 viviparous	 species,	 placental	 MHC	
     culture,	cells	were	incubated	with	primary	antibody	anti-bovine-CD4+	                  class	I		(MHC-I)	expression	is	tightly	regulated.	Bovine	interplacento-
     (VRMD,	Inc.	Pullman,	USA),	washed,	and	labeled	with	secondary	anti-                    mal	trophoblast	cells	express	both	classical	and	non-classical	MHC-I	
     body	anti-mouse	IgG	conjugated	with	magnetic	beads	(Miltenyi	Biotec,	                  genes	 during	 the	 third	 trimester	 of	 gestation	 (Davies	 et	 al.	 Placenta	
     Germany).	CD4+	positive	cells	were	obtained	after	magnetic	separa-                     2000,	 21:194-202).	 Classical	 MHC-I	 antigens	 have	 the	 potential	
     tion	(Vario	MACS,	Miltenyi	Biotec,	Germany)	and	incubated	for	2	hours	                 to	 trigger	 immune-mediated	 abortion	 and	 appear	 to	 be	 involved	 in	
     to	allow	monocytes	to	adhere	to	the	culture	microplate.	After	that,	cells	             placental	 release	 during	 parturition	 (Hill	 et	 al.	 Biol.	 Reprod.	 2002,	
     in	 suspension	 (lymphocytes	 CD4+)	 were	 collected	 and	 added	 CFSE	                67:55-63,	 Davies	 et	 al.	 Anim.	 Reprod.	 Sci.	 2004,	 82-83:267-280).	 In	
     (Carboxyfluoescein	 Diacetate	 Succinimidyl	 Ester).	 After	 4	 days	 of	              contrast,	 non-classical	 MHC-I	 antigens,	 such	 as	 HLA-G,	 seem	 to	 be	
     incubation,	cells	were	analyzed	by	flow	cytometry.	Results	show	that	                  immunosuppressive.	 In	 a	 previous	 study,	 we	 analyzed	 expression	 of	
     lymphoproliferation	 were	 13,81	 ±	 9,19,	 28,33	± 6,07,	 3,40	 ± 15,3	 for	          MHC-I	 genes	 in	 PBMC	 and	 interplacentomal	 trophoblast	 cells	 using	
     lymphocytes	from	first,	second	and	third	trimester	pregnancy,	respec-                  cDNA	cloning,	microarray	analysis	and	sequencing	(Davies	et	al.	Am.	
     tively.	 A	 basal	 placental	 T	 lymphocyte	 proliferation	 without	 specific	         J.	Reprod.	Immunol.	2006,	55:188-200).	Interplacentomal	trophoblast	
     stimulation	was	observed	differently	from	that	of	mouse	lymphocytes	                   cells	from	late	pregnancy	expressed	four	non-classical	MHC-I	loci	with	
     from	spleen	or	peripheral	blood.		Our	results	showed	that	it	is	possible	              differential	expression	in	different	haplotypes.	The	level	of	MHC-I	tran-
     to	isolate	lymphocytes	from	bovine	placenta	and	to	conduct	prolifera-                  scripts	 encoded	 at	 non-classical	 MHC-I	 loci	 was	 significantly	 higher	
     tion	assay	by	using	magnetic	cell	separation	and	CFSE	assay.                           in	 trophoblast	 cells	 than	 in	 PBMC.	 Nevertheless,	 all	 of	 the	 classical	
     Key words:	Placenta,	Lymphocyte,	CFSE,	CD4+.                                           MHC-I	genes	expressed	in	PBMC	were	also	expressed	in	trophoblast	
                                                                                            cells.	 We	 are	 now	 studying	 expression	 of	 bovine	 classical	 and	 non-
                                                                                                                                                                        81


classical	MHC-I	proteins	in	mouse	P815	cells.	Two	classical	and	four	                HERnánDEZ	J1*,	SoTo	E,	REZEnDIZ	M1,	PInELLI-AAVEDRA	A1,	
non-classical	 MHC-I	 alleles	 from	 the	AH11	 haplotype	 were	 amplified	                              KIRT	C	KLASInG2
with	Platinum	Pfx	DNA	Polymerase	and	subcloned	into	the	pcDNA	3.1	                   1Laboratorio	de	Inmunología,	CIAD,	A.C.	Hermosillo,	Sonora,	Mexico,	
expression	 vector.	The	 pcDNA	 3.1	 subclones	 were	 sequenced	 using	                       2Department	of	Animal	Science,	UC	Davis,	USA.
the	 T-7	 forward	 and	 BGH	 reverse	 sequencing	 primers	 and	 perfect	
clones	 were	 selected	 for	 expression.	 By	 using	 two	 different	 reverse	               In	addition	of	its	antioxidant	function,	studies	have	showed	that	vita-
primers	both	clones	with	the	normal	stop	codon	and	clones	expressing	                min	E	can	modulate	the	immune	system.	Different	reports	describe	that	
a	 3’	 6xHis	 tag	 were	 produced.	 P815	 mouse	 mastocytoma	 cells	 were	           vitamin	E	could	increase	the	humoral	and	cellular	immune	response,	
transfected	with	either	a	classical	or	non-classical	MHC-I	gene	using	               including	the	cytokine	production.	The	aim	of	this	work	was	to	evaluate	
lipofectamine.	Bovine	MHC-I	transcripts	were	detected	by	RT-PCR	in	                  the	effects	of	vitamin	E	on	Th1	and	Th2	cytokines	production.	PBMC	
all	transfected	cell	lines.	Transfectants	were	screened	for	cell	surface	            were	 isolated	 from	 conventional	 pigs	 (n=	 8)	 and	 supplemented	 with	
expression	of	bovine	MHC-I	proteins	by	flow	cytometry	with	anti-bovine	              different	concentrations	of	vitamin	E	(alpha-tocopheorl-AT	,	0,	10,	50,	
MHC-I	monoclonal	antibodies,	48	hours	after	being	transfected	and/or	                and	100	µM),	and	stimulated	with	PHA	for	either,	24	h	to	determine:	a)	
after	 selection	 with	 geneticin	 (Gentamycin).	The	 two	 classical	 MHC-I	         the	 concentration	 of	 tocopherol	 incorporated	 in	 the	 membrane	 cells,	
proteins	 (N*01802	 and	 N*01701)	 and	 one	 non-classical	 MHC-I	 pro-              b)	cytokine	production	(IL-2,	IL-4,	IL-10	and	IFN-γ)	and	Th1	and	Th2	
tein	 (NC3*50201)	 were	 detected	 on	 the	 cell	 membrane.	 Isolation	 of	          regulators	genes	(TBX21	and	GATA-3),	or	72	h	to	determine	the	prolif-
His	 tagged	 proteins	 and	 Western	 blotting	 are	 being	 used	 to	 identify	       eration	of	PBMCs.	AT	was	quantified	by	HPLC,	cytokine	production	by	
intracellular	and	secreted	classical	and	non-classical	MHC-I	proteins.	              intracellular	staining	using	FACS	analysis,	mRNA	was	semi-quantified	
This	study	provides	the	first	evidence	for	the	expression	of	cell	surface	           by	conventional	RT-PCR,	TBX21	and	GATA	were	analyzed	by	real	time	
and	secreted	bovine	non-classical	MHC-I	proteins.	                                   PCR,	and	proliferation	was	evaluated	with	CFSE	and	FACS	analysis.	
                                                                                     Our	results	showed	that	in	vitro	supplementation	increased	the	content	
Key words:	 Classical	 MHC	 Class	 I,	 Non-classical	 MHC	 Class	 I,	                of	 vitamin	 E	 in	 PBMC	 according	 the	 concentration	AT	 was	 supplied	
transfected	mouse	P815	cells	                                                        in	the	culture.	The	analysis	of	proliferation	did	not	showed	significant	
Species:	ruminants                                                                   differences	in	the	percentage	of	proliferation	(p>0.05),	but	AT	induced	
                                                                                     multiple	 cycles	 of	 proliferation	 in	 comparison	 of	 cells	 without	 supple-
       er085. T CELLS DISTRIBUTION AND FUNCTION                                      ment.	The	 analysis	 of	 cytokines	 showed	 that	 10	 µM	 of	AT	 increased	
        IN PERIPHERAL BLOOD AND UTERUS DURING                                        the	 mRNA	 expression	 and	 the	 percentage	 of	 cells	 producing	 IL-2	
            PERIIMPLANTATIONAL PERIOD IN PIG                                         (p<0.05).	 The	 production	 of	 IFN-γ	 was	 inconsistent	 and	 no	 changes	
   MARIA	F	CUELLo,	MARIA	C	GRoSSo,	RAMIRoA	MARTInEZ,	                                were	observed	with	any	supplement	of	vitamin	E.		The	mRNA	expres-
            ADRIAnA	B	VIVAS,CECILIA	R	GRECo1                                         sion	of	IL-4	was	not	modified	by	vitamin	E,	however	the	percentage	of	
                                                                                     cells	producing	IL-4	was	increased	significantly	(p<0.05)	by	vitamin	E	
Facultad	de	Agronomía	y	Veterinaria.	1Facultad	de	Ciencias	Exactas.	
                                                                                     (10	µM	).	mRNA	expression	of	IL-10	was	reduced	with	10	and	50	µM	of	
  Universidad	Nacional	de	Río	Cuarto.	5800	Río	Cuarto.	Argentina.	
                                                                                     AT,	as	well	as	the	percentage	of	cells	producing	IL-10	with	10,	50,	and	
                     cgreco@exa.unrc.edu.ar
                                                                                     100	µM.	AT	supplementation	in	all	concentrations	increase	the	relative	
      It	 has	 been	 suggested	 that	 successful	 pregnancy	 in	 pig	 depend	        expression	 of	 mRNA	 of	TBX21	 vs	 GATA3.	These	 results	 showed	 for	
on	 adequate	 immuno-endocrine	 modulation	 at	 conceptus-maternal	                  first	 time	 that	 vitamin	 E	 modulate	Th1	 and	Th2	 cytokines	 in	 pig,	 and	
interface	and	systemic	levels.		The	aim		was		to	study	the		T	cell	dis-              suggest	 that	 vitamin	 E	 down-modulate	 the	 expression	 of	 cytokines	
tributions	 in	 peripheral	 blood	 and	 uterus	 and	 its	 functional	 properties	    associated	with	the	phenotype	Th2	or	anti-inflammatory	in	the	case	of	
during	 peri-implantational	 period.	 Blood	 samples	 were	 obtained	 from	          IL-10.	These	functions	appear	to	be	related	with	the	down-modulation	
pregnant	 sows	 in	 10	 days	 (n=15)	 and	 30	 days	 (n=15)	 of	 pregnancy	          of	GATA3,	a	gene	responsible	of	regulate	Th2	cytokine.		
from	a	breeding	farm.		Pregnant	state	was	determined	by	ultrasound.	
                                                                                     Key words:	Vitamin	E,	Th2	cytokines,	
Non-pregnant	 sows	 (n=15)	 in	 the	 luteal	 period	 of	 the	 oestrus	 cycle	
                                                                                     Species:	swine
were	used	as	control.	Uterine	tracts	were	obtained	from	non	pregnant	
and	pregnant	sows	in	10	days	(n=10)	and	30	days	(n=10)	of	pregnancy	
from	a	slaughter	house.	Peripheral	blood	mononuclear	cells	(PMBCs)	                   er087. NOREPINEPHRINE MODULATES PROLIFERATION
and	 uterine	 mononuclear	 cells	 (UMCs)	 were	 isolated	 by	 Histopaque.	             AND IFN-α PRODUCTION By PORCINE IMMUNE CELLS
Phenotypes	of	porcine	T	lymphocyte	were	determinate	by	two-colour	                      ELoDIE	MERLoT1,2,	FRIDA	HASSLUnG	WIKSTRöM1,	SIRJE	
Flow	Cytometric	Analysis	using	specific	monoclonal	antibodies	against	                     TIMMUSK1,	LISBETH	FUxLER1,CARoLInE	FoSSUM1
CD4	and	CD8	surface	antigens.	Four	subpopulations	CD4+CD8-,	CD4-
                                                                                      1Department	of	Biomedical	Sciences	and	Veterinary	Public	Health,	
CD8low+,	 CD4-CD8high+	 T	 cells	 and	 CD4+CD8+double	 positive	 (DP)	 T	
                                                                                     Section	for	Immunology,	Swedish	University	of	Agricultural	Sciences,	
cells	were	determined.	In	addition,	PMBCs	and	UMCs	were	stimulated	
                                                                                     SE-751	23	Uppsala,	Sweden,	2INRA,	UMR1079	Systèmes	d’Elevage	
separately	with	P4	or	with	Con	-A.	In	supernatants	IL-10,	INF-γ	and	TGF-
                                                                                         Nutrition	Animale	et	Humaine,	F-35590	Saint	Gilles,	France.	
β2	concentrations	were	determined	by	ELISA.	At	the		preimplantational	
                                                                                                         elodie.merlot@rennes.inra.fr
period	an	increase	of	CD4+CD8+DP	T	cells	(p<0.05)	in	peripheral	blood	
and	a	very	significant	increase	of	CD4-CD8high+	T	cells	(p<0.01)		were	                    Sympathetic	 system	 has	 been	 shown	 to	 play	 a	 major	 role	 in	
observed.	Before	embryo	implantation	CD4-CD8high+	T	cells	increased	                 neuroendocrine-immune	 communication	 in	 rodents	 and	 humans.	
in	peripheral	blood	(p<0.01)	and	diminished	significantly	at	uterine	level	          Norepinephrine	(NE)	modulates	T	lymphocyte	proliferation,	production	
(p<0.05).	CD4+CD8+DP	T	cells	remained	increased	at	peripheral	blood	                 of	Th1	and	Th2	cytokines	by	T	lymphocytes	and	dendritic	cells	(DC),	
but	decreased	at	uterine	level.	Distribution	changes	of	CD4+CD8+DP	T	                and	stimulates	migration	of	DC	toward	lymph	nodes.	In	this	study,	we	
cells	were	not	observed.	                                                            investigated	the	effect	of	NE	on	the	proliferation	of	porcine	peripheral	
                                                                                     blood	mononuclear	cells	(PBMC)	and	on	their	ability	to	produce	inter-
      The	above	mentioned	changes	are	accompanied	by	a	special	pat-
                                                                                     feron-α	 (IFN-a).	 PBMC	 were	 isolated	 by	 density	 gradient	 centrifuga-
tern	 of	 cytokine	 production	 when	 the	 UMCs	 are	 progesterone-stimu-
                                                                                     tion	from	heparinized	blood	from	conventionally	reared	Yorkshire	pigs	
lated.	At	10	d	of	pregnancy	increased	significantly	IL-10	(p<0.05)	and	
                                                                                     (8-12	 weeks).	The	 dose-effect	 of	 NE	 was	 investigated	 by	 stimulating	
TGF-β2	 (p<0.05)	 and	 decreased	 INF-γ	 (p<0.01).	 In	 conclusion,	 these	
                                                                                     PBMC	with	various	inducers	and	by	adding	NE	simultaneously	(doses	
findings	 suggest	 that	 at	 uterine	 level	 CD4-CD8high+	 T	 cells	 function	 is	
                                                                                     ranging	from	0.01	to	100µM).	Lymphocyte	proliferation	over	96	hours	
modulated	by	progesterone	presenting	a	special	cytokine	pattern	coin-
                                                                                     was	measured	by	incorporation	of	tritiated	thymidine	during	the	last	24	
cidentally	with	a	modulated	cytolytic	activity.
                                                                                     hours.	For	IFN-a,	the	supernatant	of	PBMC	was	collected	after	20h	of	
Key words:	 uterine	 mononuclear	 cells,	 pregnancy,	 IL-10,	 INF-γ	 ,	              incubation	and	porcine	IFN-α	was	quantified	by	dissociation-enhanced	
TGF-	β2                                                                              lanthanide	fluoro-immunoassay	(DELFIA).	When	NE	alone	was	added	
Species:	swine                                                                       to	PBMC,	proliferation	was	stimulated	at	concentrations	ranging	from	
                                                                                     0,1	to	100µM.	When	NE	was	added	in	combination	with	T-lymphocyte	
er086. VITAMIN E MODULATES THE ExPRESSION OF TH2                                     (ConA,	PHA)	or	B-lymphocyte	(LPS)	mitogens,	the	highest	doses	(10	
           CyTOKINES ON PORCINE PBMC                                                 and	100	µM)	of	NE	decreased	the	mitogen-induced	proliferation.	The	
82


     production	of	IFN-α	was	stimulated	when	PBMC	were	exposed	to	NE	                   adult	horse	(n=3).	To	compare	mRNA	expression	levels	to	protein	pro-
     in	 combination	 with	 oligodeoxyribonucleotide	 (ODN)	 2216	 but	 it	 was	        duction,	 we	 quantified	 the	 concentrations	 of	 several	 immunoglobulin	
     not	affected	when	NE	was	co-administered	with	polyriboinosinic-poly-               isotypes	in	pre-suckle	foal	(n=11)	and	adult	horse	(n=6)	sera	using	com-
     ribocytidylic	acid	(poly	I:C).	When	added	in	combination	with	complex	             mercially	available	radial	immunodiffusion	assays	(IgM,	IgA,	total	IgG	
     microbial	inducers,	NE	at	100	µM	inhibited	IFN-a production	by	PBMC	               and	IgG3-5)	and	ELISA	(IgG1,	IgG4-7).	The	RT-PCR	results	revealed	
     in	 response	 to	 heat	 inactivated	Aujeszky’s	 disease	 DNA	 virus	 (ADV)	        that	 the	 B	 cell	 specific	 mRNA	 expression	 patterns	 were	 consistently	
     and	stimulated	it	in	response	to	live	Sendai	Virus	(SV).	Porcine	IFN-a	            similar	among	individuals	within	the	same	age	category	(fetal,	neonate,	
     producing	cells	that	resemble	the	human	plasmacytoid	dendritic	cells	              young	foal	and	adult	horses).		Fetal	lymphoid	tissues	expressed	mRNA	
     (pDC)	are	believed	to	be	the	main	source	of	IFN-a	when	porcine	PBMC	               for	most	genes	assayed,	including	CD20,	CD21,	CD40,	B220,	CD79A,	
     are	stimulated	with	ODN	2216	or	ADV	(via	TLR-9	activation),	while	it	              and	CD79B.		The	expression	of	RAG-1	and	-2,	and	Tdt	was	temporally	
     is	believed	to	be	the	monocytes	or	monocyte-derived	DC	(moDC)	that	                and	 spatially	 restricted.	 Expressions	 of	 particular	 IgG	 isotypes	 were	
     respond	to	stimulation	by	dsRNA	(via	TLR-3)	e.g.,	poly	I:C	or	replicating	         absent	or	limited	in	the	equine	neonate	and	foal	up	to	3	months	of	age.	
     SV.	Thus,	the	regulatory	effect	of	NE	on	IFN-a	seems	to	depend	on	the	             In	summary,	our	data	indicates	an	active	B	cell	program	during	equine	
     cell	type	and	on	the	pathway	by	which	they	are	activated	by	microbial	             gestation,	but	functionally,	the	adaptive	immune	system	remains	lim-
     components.	 In	 order	 to	 investigate	 this	 interaction,	 we	 are	 currently	   ited	in	the	production	of	certain	immunoglobulin	isotypes	and	diversity	
     studying	how	NE	regulates	IFN-a	production	in	porcine	moDC.	                       in	early	life.	
     Key words:	 Norepinephrine,	 T	 lymphocytes,	 Th1	 cytokines,	 Th2	                Key words:	Fetal	immunoglobulins,	RAG-1,	RAG-2,	Tdt,	CD20,	CD21,	
     cytokines                                                                          CD40,	B220,	CD79A,	CD79B
     Species:	swine                                                                     Species:	Equine

      er088. THE INFLUENCE OF SEASONAL STRESS DURING                                        er090. THE CONTRIBUTION OF BODy CONDITION
       GESTATION ON IMMUNE PARAMETERS IN POSTNATAL                                       SCORE AND PERCENT BODy FAT TO THE INFLAMMATORy
                      STRESSED PIGLETS                                                               RESPONSE IN AGED HORSES
       GABRIELA	RoDRIGUEZ	ALonSo,	noRA	MAyER1,	CECILIA	R	                                AMAnDA	A	ADAMS,	MADHU	KATEPALLI,	KATHARInA	KoHLER,	
           GRECo1,	nAnCy	RoDRÍGUEZ1,ADRIAnA	VIVAS                                           STEPHAnIE	REEDy,	JP	STILZ,	MAnDI	M	VICK,	BARRy	
                                                                                                    FITZGERALD,	DAVID	W	HoRoHoV	
     Facultad	de	Agronomía	y	Veterinaria,	1Facultad	de	Ciencias	Exactas.	
       Universidad	Nacional	de	Río	Cuarto.	5800	Río	Cuarto.	Argentina.	                 Maxwell	H.	Gluck	Equine	Research	Center,	Department	of	Veterinary	
                          cgreco@exa.unrc.edu.ar                                                  Science,	University	of	Kentucky,	Lexington,	KY,		
                                                                                                            amanda.adams@uky.edu	
           Numerous	 infertility	 problems	 are	 presented	 in	Argentina	 during	
     summer	 months.	 The	 most	 important	 clinical	 manifestations	 are	 the	               Advanced	age	is	associated	with	a	low-grade,	systemic	inflamma-
     irregular	zeal	and	early	abortions.	Stress	activation	of	Hypothalamic-             tory	response	in	vivo	and	increased	inflammatory	cytokine	production	
     pituitary-Adrenal	axis	generates	changes	in	immune	system.	However,	               in	vitro.		One	possible	source	of	this	increased	inflammatory	cytokine	
     the	effect	of	different	stimuli	on	summer	infertility	on	offspring	viability	      production	 are	 dysfunctional	 white	 blood	 cells	 associated	 with	 the	
     or	its	immune	response	is	not	well	known.	The	aim	was	to	study	the	                aged	 immune	 system.	 	 In	 previous	 studies	 of	 the	 aged	 horse	 (≥20	
     immune	response	of	piglets	to	prenatal	stress	during	summer	time	with	             yrs),	 our	 results	 demonstrated	 a	 significant	 increase	 in	 the	 percent	
     addition	 of	 postnatal	 stress	 like	 weaning.	 Male	 and	 females	 pigs	 of	     of	 lymphocytes	 from	 old	 horses	 compared	 to	 young	 producing	 inter-
     45-60	days	of	age	were	used,	Landrace	x	Yorkshire,	mothers’	offspring	             feron-gamma	(IFNγ)	and	tumor	necrosis	factor	alpha	(TNFa).		It	is	also	
     under	different	environmental	temperatures	during	the	pregnancy:	high	             known	 that	 increased	 white	 adipose	 tissue,	 associated	 with	 obesity,	
     temperatures	 (summer	 season),	 stocking	 temperatures	 (autumn	 and	             leads	to	increased	production	of	inflammatory	cytokines.		To	date,	it	is	
     spring)	 and	 low	 temperatures	 (winter).	 Forty	 five-	 sixty	 days	 of	 age	    unknown	 whether	 increased	 adiposity	 contributes	 to	 the	 age-related	
     offspring	after	weaning	were	separated	from	their	mothers,	identified	             increased	inflammatory	status.	Therefore,	we	tested	the	hypothesis	that	
     and	leave	in	special	housing	during	three	days.	Blood	samples	were	                increased	body	condition	scores	(BCS≥7)	and	percent	body	fat	(≥10)	of	
     taken	from	males	and	females,	before	and	after	weaning.	The	follow-                aged	horses	are	positively	associated	with	increased	in	vitro	produc-
     ing	 determinations	 were	 made:	 Total	 blood	 leukocytes,	 in	 vitro	 lym-       tion	of	inflammatory	cytokines,	IFNγ	and	TNFa.		Further,	we	proposed	
     phocyte	proliferation,	serum	gammaglobulins	and	IgG	concentrations,	               that	 decreasing	 BCS	 (≤7)	 and	 percent	 body	 fat	 (≤10)	 will	 decrease	
     serum	glucose	and	cortisol	concentrations.	The	addition	of	two	stress	             inflammatory	cytokine	production	both	in	vivo	and	in	vitro.		Body	condi-
     changes	immune	responses	through	decreasing	total	leukocytes	num-                  tion	 scores	 were	 determined	 using	 the	 standard	 nine-point	 Henneke	
     ber,	 a	 decrease	 in	 percentage	 of	 lymphocytes	 accompanied	 with	 an	         Scaling	System.		Body	fat	was	determined	by	ultrasound	measurement	
     increase	of	polymorphonuclear	neutrophils.	In	addition,	mitogen-stimu-             of	rump	fat.	PBMC	were	stimulated	with	either	PMA/ionomyocin.	The	
     lated	 proliferative	 lymphocytes	 were	 disminished.	An	 increase	 in	 IgG	       cells	were	fixed,	permeabilized	and	intracellularly	stained	for	IFNγ	and	
     plasmatic	concentration	was	observed.	In	conclusion,	stress	modifies	              TNFa.	 	 Samples	 were	 acquired	 on	 a	 FACSCaliber.	 	 Percent	 positive	
     the	immune	response	trying	to	avoid	possible	infections.                           were	determined	using	lymphocyte	gates.		TNFa	protein	in	the	serum	
                                                                                        was	measured	using	an	ELISA.	There	was	an	increased	percentage	
                                                                                        of	lymphocytes	staining	for	TNFα	in	old	mares	with	a	BCS	>7	and	per-
      er089. ONTOGENy AND ExPRESSION OF EQUINE FETAL                                    cent	body	fat	>15.		Likewise,	IFNγ	production	tended	to	be	increased	
              AND NEONATAL IMMUNOGLOBULINS                                              in	 the	 fat	 mares.	 	 Furthermore,	 decreasing	 adiposity	 and	 BCS	 score	
        REBECCA	L	TALLMADGE,	KRISTIn	E	MCLAUGHLIn,	MARy	B	                              decreased	 the	 percent	 of	 IFNγ	 	 and	 TNFα	 positive	 lymphocytes.	    	
                   MATyCHAK,MJULIA	BF	FLAMInIo                                          Decreasing	 adiposity	 in	 old	 horses	 also	 decreased	 TNFα	 protein	 in	
                                                                                        serum.		These	results	indicate	that	body	condition	score	and	%	rump	
       Cornell	University,	College	of	Veterinary	Medicine,	Ithaca,	NY	USA
                                                                                        fat	are	confounding	factors	when	assessing	age-associated	inflamma-
           Many	aspects	of	the	immune	system	of	the	horse	develop	during	               tory	responses.		Decreasing	body	fat	and	BCS	could	improve	geriatric	
     fetal	life,	yet	foals	are	dependent	on	the	transfer	of	maternally-derived	         health	conditions	related	to	inflammatory	mediators.
     antibodies	 during	 the	 neonatal	 period	 for	 protection	 against	 environ-      Key words:	 aging,	 IFNγ,	 TNFα,	 lymphocytes,	 flow	 cytometry,	 body	
     mental	pathogens.	In	addition,	studies	involving	infectious	organisms	             condition	scores
     have	indicated	delayed	production	in	certain	immunoglobulin	isotypes	              Species:	Equine
     in	the	foal,	which	could	increase	their	susceptibility	to	pathogens.	This	
     study	 explores	 the	 progressive	 expression	 of	 B	 cell	 markers	 in	 lym-
     phoid	tissues	from	fetal	life	to	adulthood.		Developmental,	activation,	             er091. CHARACTERIZATION OF THE IMMUNOLOGICAL
     and	immunoglobulin	markers	were	selected	for	RT-PCR	experiments.	            	       AND PHySIOLOGICAL RESPONSE OF AGED HORSES TO
     We	tested	mRNA	expression	using	a	two	step	RT-PCR	in	liver,	bone	                              EQUINE INFLUENZA INFECTION
     marrow,	 spleen,	 mesenteric	 lymph	 node,	 lung	 and	 peripheral	 blood	                AMAnDA	A	ADAMS,	CoRMAC	C	BREATHnACH,	TRACy	
     mononuclear	cell	samples	from	equine	fetus,	neonate,	young	foal	and	                      STURGILL,	ToM	CHAMBERS,	DAVID	W	HoRoHoV	
                                                                                                                                                                              83


Maxwell	H.	Gluck	Equine	Research	Center,	Department	of	Veterinary	                     from	 Paxgene®	 tubes	 according	 to	 the	 manufacturer’s	 protocol	 and	
          Science,	University	of	Kentucky,	Lexington,	KY.		                            reverse	transcribed	into	cDNA.		RT-PCR	was	performed	for	the	equine	
                    Amanda.Adams@uky.edu                                               cytokines	IFN-γ,	TNF-α,	IL-4,	IL-10,	and	IL-1	with	β-GUS	being	used	
      Influenza	 is	 a	 serious	 health	 problem	 for	 the	 elderly	 population	       as	the	housekeeping	gene.		PBMC	and	BAL	cells	were	cultured	in	vitro	
and	 ranks	 as	 one	 of	 the	 top	 leading	 causes	 of	 death	 in	 the	 elderly.		     and	 intracellular	 staining	 for	 IFN-γ	 was	 performed.	 	 There	 appeared	
There	are	alterations	in	immune	function	that	occur	with	age	that	affect	              to	be	an	increase	in	IFN-γ	production	in	BAL	cells	after	the	first	treat-
the	ability	of	the	elderly	population	to	respond	to	vaccination	and	resist	            ment,	however	this	was	not	statistically	significant.		Additionally,	while	
infection.	 	 In	 the	 horse	 population,	 equine	 influenza	 virus	 (EIV)	 is	 a	     there	was	an	increase	in	IFN-γ	following	the	second	treatment	in	both	
leading	cause	of	respiratory	disease,	however	the	susceptibility	of	old	               PBMC	and	mRNA,	this,	too,	was	not	statistically	significant.	This	lack	
horses	 to	 EIV	 infection	 remains	 unknown.	 	Advanced	 age	 in	 horses	             of	stimulation	may	be	due	in	part	to	impaired	toll-like	receptor	mediated	
(>20	yrs)	is	associated	with	age-related	changes	in	immune	function.	            	     innate	immunity.		While	EqStim®	failed	to	stimulate	IFN-γproduction	in	
Nevertheless,	 there	 are	 no	 specific	 recommendations	 regarding	 the	              the	foals	in	this	study,	the	role	of	other	immune	modulators	for	use	in	
                                                                                       foals	needs	to	be	investigated	further.	
vaccination	 of	 older	 horses	 even	 though	 a	 well	 characterized	 effect	
of	 aging	 in	 horses	 is	 a	 reduced	 antibody	 response	 to	 standard	 vac-          Key words:	 immune	 modulator,	 EqStim®,	 IFN-γ	 Bronchoalveolar	
cination.	 	 Nor	 do	 we	 know	 if	 natural	 exposure	 to	 EIV	 is	 enough	 to	        lavage	cells,	PBMCs
sustain	a	protective	immune	response	in	the	older	horse.		Therefore,	                  Species:	Equine
we	 evaluated	 the	 immunological	 and	 physiological	 response	 of	 aged	
horses	 to	 EIV	 challenge	 infection.	 	 Naïve	 yearlings	 were	 challenged	             er93. THE COMPARATIVE STUDy OF THE EFFECT OF
with	EIV	for	comparison.	EIV-specific	interferon-gamma	(IFN-γ)	synthe-                    VIRGINAMyCINE AND MANNAN- OLIGOSACCHARIDES
sis	by	peripheral	blood	mononuclear	cells	(PBMC)	in	vitro	was	used	a	                    (MOS) ON HUMORAL IMMUNITy OF BROILER CHICKENS
measure	of	cell-mediated	immunity.		Pro-inflammatory	cytokine	mRNA	
                                                                                                         A	ZAKERI*	1,	M	FADAEI	2,S	ZAKERI	3	
production	in	vivo	was	determined	using	RT-PCR.		Clinical	signs	of	the	
disease	 (coughing,	 nasal	 discharge,	 dyspnea,	 depression,	 anorexia)	              1	Postgraduate	student	of	Avian	Diseases,	Department	of	Veterinary	
as	 well	 as	 rectal	 temperature	 were	 monitored	 post	 challenge.	 There	              Science,	Faculty	of	Agriculture,	Islamic	Azad	University	branch	
was	no	significant	difference	in	clinical	signs	between	naïve	yearlings	               Tabriz,	Tabriz-Iran	and	Young	Researchers	Club	(YRD)	,	Tabriz	Iran.	
and	old	horses	post	challenge.		However,	there	was	a	significant	dif-                  E-mail:	azakerii@yahoo.com	,	zakeri@iaut.ac.ir,	2	Student	of	master	
ference	in	the	febrile	response	between	naïve	and	old	horses	following	                 (MS)	course	of	Mathematics,	Tehran-	Iran,	3	Student	of	Veterinary	
the	challenge.		While	there	was	no	significant	difference	in	EIV-specific	                 Medicine,	Islamic	Azad	University	branch	Tabriz,	Tabriz-Iran
IFN-γ	synthesis	between	the	naive	and	old	horses	prior	to	challenge,	                        MOS	 is	 a	 prebiotic	 and	 virginamycine	 is	 an	 antibiotic	 used	 as	
the	percent	of	EIV-specific	IFN-γ+	lymphocytes	was	significantly	higher	               growth	promoter	(AGP).	In	this	study	360	Cobb	500	broiler	 chickens	
in	 old	 horses	 compared	 to	 naïve	 horses	 post	 challenge.	 	 Both	 naïve	         were	divided	in	three	similar	groups	with	120	chickens	in	each	group	
and	older	horses	exhibited	similar	increases	in	the	expression	of	IL-6,	               (with	four	replicates	of	30	chickens	in	each	group).	One	kg	/	ton	MOS	
IFN-γ	and	IL-10	mRNA	post	challenge.		However,	there	was	significant	                  for	experimental	group	Ι	and	100	g/	ton	virginamycine	for	experimental	
increase	 in	 IL-1β	 and	TNF-α	 expression	 in	 the	 old	 horses	 compared	           group	ΙΙ	were	added	to	the	basic	diets	while	the	control	group	chick-
to	 the	 naïve	 yearlings	 post	 challenge.	 	 In	 summary,	 the	 clinical	 data	      ens	 were	 fed	 only	 with	 basic	 diet.	 On	 days	 9,	 17	 and	 25	 of	 growth	
show	that	old	horses	are	just	as	susceptible	to	EIV	infection	as	naïve	                (1	 day	 before	 and	 7,	 14	 days	 after	 first	 Newcastle	 B1	 vaccination),	
yearlings.	 	 The	 increased	 EIV-specific	 IFN-γ	 production	 may	 be	 an	            from	each	group,	each	time	40	chickens	were	chosen	randomly	and	
indication	 that	 older	 horses	 have	 immunological	 memory	 to	 EIV,	 but	           serum	antibodies	titers	were	measured	against	Newcastle	vaccine	by	
not	enough	to	insure	protection	from	EIV	infection.		                                  HI	 test.	After	 reviewing	 and	 analyzing	 the	 statistic	 data,	 the	 statistic	
                                                                                       results	of	the	serum	antibodies	titers	by	HI	test	(5.40±0.44	in	control	
Key words:	aged,	equine,	influenza
                                                                                       group,	5.87	±	0.47	in	experimental	group	Ι	and	5.61±041	in	experimen-
Species:	equine
                                                                                       tal	group	ΙΙ)	indicated	a	statistical	difference	(P	<	0.05)	between	each	
                                                                                       three	groups.	MOS	is	a	natural	substance	that	does	not	have	any	drug	
  er092. INTERFERON-GAMMA ExPRESSION IN yOUNG                                          residual	 in	 meat	 of	 poultry	 and	 it	 is	 a	 suitable	 alternative	 for	 growth	
  FOALS WHEN TREATED WITH AN IMMUNE MODULATOR                                          promoters	antibiotics.
      DVM	TRACy	L	STURGILL,	KRISTIn	A	HAnDKE,	DAVID	W	                                      Ref:
                        HoRoHoV
                                                                                           Milner	,	JA.	,	M	.	Roberforid	,	1999	.	Nutritional	properties	of	inulin	
  Gluck	Equine	Research	Center,	Department	of	Veterinary	Science,	                     and	oligofructose.	J	Nutr	:	129	,	S	1395	–	502.
         University	of	Kentucky,	Lexington,	KY	40546-0099.	
                                                                                           Rober	foid	,	MB.,	2000	.	Health	benefits	of	non	–	digestible	oligo-
                          tlstur2@uky.edu	
                                                                                       saccharides.	Adv	,Exp,	Med	Bull.	427	,	211	–	219.
      The	 equine	 neonate,	 like	 neonates	 of	 other	 species,	 is	 uniquely	
                                                                                           Salminen.S.	 ,C.	 Bouley	 ,	 MC.	 Boutron	 –	 ruoult	 ,	 1998	 .	 function	
susceptible	 to	 infectious	 disease.	 	 These	 diseases	 are	 a	 significant	
                                                                                       food	science	and	gastroinestinal	physiology	and	function	.	Bry	Nutr	,	
cause	 of	 neonatal	 mortality,	 thereby	 causing	 considerable	 economic	
                                                                                       80(suppl)	,S147	–	71.
loss	to	the	equine	industry.		Neonatal	immune	responses	are	consid-
ered	to	be	immature.		The	mechanisms	involved	in	this	immaturity	are	                       Vegad	,	JL	.	,2004	.Prebiotics	,Probiotic	,Acidfires	and	Antibiotic	
numerous	and	incompletely	understood.		Immune	modulators	that	con-                     growth	Promotors	.	poultry		diseases	a	guide	for	farmers	and	Poultry	
tain	various	pathogen-associated	molecular	patterns	have	been	used	                    Professional	.	First	edn	.	339	-	346.
extensively	 in	 the	 horse	 to	 combat	 respiratory	 and	 other	 infections.	         Key words:	humoral	immunity,	virginamycine	,MOS	,broiler	chickens			
                                                                                                                                                        	
While	the	use	of	immunomodulators	in	adults	results	in	the	upregula-                   Species:	avian
tion	 of	 IFN-γ,	 their	 effect	 on	 IFN-γ	 production	 in	 the	 neonatal	 foal	 is	
unknown.	 	 The	 purpose	 of	 this	 study	 was	 to	 determine	 the	 effect	 of	            er094. EFFECT OF EARLy STIMULATIONS ON SOME
treatment	with	an	immune	modulator	on	cytokine	production	in	foals.	               	       IMMUNE PARAMETERS IN PRENATAL STRESS RATS
Thirteen	 foals	 were	 randomly	 divided	 into	 a	 treatment	 group	 receiv-
                                                                                         AnA	LIAUDAT,	AURELIA	SARAnDón	,nAnCy	RoDRIGUEZ,	
ing	EqStim®	or	the	control	group	receiving	no	treatment.		Treatments	
                                                                                       CECILIA	R	GRECo	,	ADRIAnA	B	VIVAS1	HECToR	FGAUnA,noRA	
were	administered	within	the	first	five	days	of	life	and	repeated	at	30	
                                                                                                               MAyER	
days	of	age.		EqStim®	1ml,	q	48	hrs,	IV,	was	administered	for	a	total	of	
three	 treatments,	 as	 recommended	 by	 the	 manufacturer.	 	 Starting	 at	           1Facultad	de	Agronomía	y	Veterinaria;	Facultad	de	Ciencias	Exactas.	
birth	and	continuing	weekly,	peripheral	blood	was	aseptically	collected	                 Universidad	Nacional	de	Río	Cuarto;	5800	Río	Cuarto.	Argentina.	
via	 jugular	 venipuncture	 from	 each	 foal	 into	 heparinized	 tubes	 and	                                cgreco@exa.unrc.edu.ar
Paxgene®	tubes.		Bronchoalveolar	lavage	(BAL)	was	performed	nine	                            The	application	of	stressors	during	pregnancy	produces	an	altera-
days	post	initiation	of	treatment.		Heparinized	blood	was	used	for	isola-              tion	of	the	hypothalamic-pituitary-adrenal	(HPA)	axis	that	would	induce	
tion	of	peripheral	blood	mononuclear	cells	(PBMC).		RNA	was	isolated	                  a	 long-term	 alteration	 of	 the	 immune	 function	 in	 the	 offspring.	 Early	
84


     postnatal	 stimulations	 produce	 beneficial	 effect	 on	 the	 long-	 term	        els	of	corticosterone.	Thus,	the	spleens	of	all	animals	were	removed	for	
     emotional	response	and	HPA	axis	activity	that	could	revert	the	effect	             lymphocytes	T	culture.	The	profile	of	the	leucocytes,	lymphocytes	and	
     of	prenatal	stress.	The	aim	of	this	study	was	to	investigate	the	effect	           neutrophils	was	similar	in	EP	and	C,	however	response	was	depressed	
     of	early	stimulations	in	prenatal	stressed	animals	through	the	distribu-           in	M	animals	under	postnatal	IMO.	The	in	vitro	proliferations	of	lympho-
     tion	of	the	subpopulations	of	leucocytes	and	the	in	vitro	proliferations	          cytes	T	increased	in	development	of	M	animals.	In	conclusion,	postna-
     of	 lymphocytes	 T	 in	 response	 to	 acute	 stress	 in	 rats.	 For	 the	 study	   tal	stimulation	reverts	the	effects	of	prenatal	stress	on	the	distribution	
     we	used:	three	months	old	male	offsprings	from	immobilization	(IMO)	               of	the	subpopulations	of	leucocytes	and	on	the	in	vitro	development	of	
     stressed	mothers	(EP)		and	offprings	from	non	stressed	mothers.(CP).	              lymphocytes	T	under	the	same	postnatal	stress.
     Half	of	the	EP	animals	were	manipulated	(M)	during	the	first	week	of	              Key words:	prenatal	stress,	immune	parameters,	rats	
     life.	Before	extraction	of	blood	for	basal	determinations,	the	animals	of	         Species:	other	(Rat)
     both	groups	were	under	acute	stress	IMO	(20	minutes).	Then,	blood	
     was	extracted	at	20,	60,	90,	120,	150	and	330	minutes	post-stress	to	
     count	white	blood	cells,	the	subpopulations	of	leucocytes	and	the	lev-
            4. IMMUNOLOGy OF THE MUCOSAE AND SKIN AND OF THE MAMMARy GLAND: POSTERS SM095-SM118



   sm095. ROLE OF BASOPHILS IN THE RESISTANCE OF                                    inflammatory	infiltrates	elicited	by	tick	bites	in	skin	of	resistant	(R)	and	
    SENSITIZED GOATS TO AMBLyoMMA CAJENNENSE                                        susceptible	 (S)	 bovines.	 R	 and	 S	 cattle	 underwent	 three	 successive	
               FABRICIUS (1787) NyMPHS                                              infestations	and	biopsies	were	taken	at	the	feeding	site	of	nymphs	and	
                                                                                    adults,	from	normal	skin	of	the	same	host	or	from	skin	of	naïve	controls.	
                  GER	MonTEIRo1,2,	GH	BECHARA2
                                                                                    Total	and	differential	cells	counts	were	made	on	paraffin-fixed	sections	
1Departamento	de	Paraclinicas,	FV-Universidade	Eduardo	Mondlane,	                   stained	by	May-Grünwald	e	Giemsa,	in	infested	skin	they	were	limited	
  Maputo,	Mozambique,	2Departamento	de	Patologia	Veterinária,	                      to	the	area	of	the	tick	cement	cone.	Bites	with	adult	ticks	recruit	more	
   FCAV-Universidade	Estadual	Paulista,	Jaboticabal-SP,	Brazil.                     inflammatory	cells	than	those	by	nymphs	(P<0.05,	one-way	ANOVA).	
      Lone-star	 ticks	 Amblyomma	 cajennense	 parasite	 primarily	                 Neutrophils	are	more	numerous	in	skin	infested	with	adults	than	with	
horses,	but	it	can	infest	cows,	deers,	dogs,	birds	and	men	also.	It	is	             nymphs	(P<0.05).	Conversely,	mononuclear	cells	are	more	abundant	
the	main	vector	of	horse	babesiosis	and	human	spotted	fever	in	Brazil.	             in	 skin	 infested	 with	 nymphs	 than	 with	 adults	 (P=0.001).	 Mast	 cell	
According	to	preliminary	results	of	the	laboratory	goats	acquire	partial	           numbers	were	equally	diminished	in	adult-infested	skin	of	both	breeds	
resistance	against	A.	cajennense	nymphs	after	repeated	infestations.	               when	compared	with	non-infested	skin	(P<0.05).	Nymph-infested	skin	
The	aim	of	this	study	was	to	characterize	this	tick-host	interaction	by	            had	more	mast	cells	than	R	adult-infested	skin	(P<0.05).	Eosinophils	
examining	morphological	features	of	the	tick	bite	lesion	during	repeated	           were	 absent	 in	 skin	 from	 naïve	 animals,	 but	 were	 present	 in	 normal	
infestations.	 Ten	 naive	 goats	 aged	 six	 months,	 of	 both	 sexes,	 were	       and	infested	skin,	however	they	were	reduced	in	infested	skin	of	both	
distributed	into	two	groups:	test	(n=5),	infested	with	15	nymphs	thrice	            breeds	 (P<0.05)	 and	 more	 significantly	 so	 in	 adult-infested	 skin	 of	 R	
at	30	days	interval	and	control	(n=5).	Skin	fragments	of	tick	bite	sites	           hosts	(P<0.05).	Basophils	were	more	abundant	in	R	than	in	S	adult-
were	collected	24,	48,	72	and	120hours	post	infestation	and	processed	              infested	skin	(P<0.05).	Mast	cells	are	source	of	cytokines	and	inflam-
according	to	routine	histotechnology.	Hematoxylin-eosin	and	Giemsa-                 matory	 mediators	 that	 play	 effectors	 and	 modulator	 roles	 in	 immune	
stained	skin	sections	of	4	µm	thickness	were	observed,	respectively	for	            responses,	their	reduction	possibly	due	to	degranulation	by	inflamma-
general	aspects	and	inflammatory	cells’	count	under	the	cement	cone	                tory	 cytokines.	The	 lower	 amount	 of	 neutrophils	 in	 infested	 skin	 may	
through	 an	 integrated	 eyepiece	 (10x	 magnification)	 and	 an	 objective	        reflect	the	fact	that	only	nymphs	express	RGD-containing	disintegrins,	
100x	 magnification	 (total	 area=	 0.0052mm2).	At	 the	 tick	 feeding	 site,	      which	 are	 possibly	 neutrophil-specific.	 Eosinophils,	 as	 well	 as	 baso-
it	was	observed	epidermal	fracture	suggesting	tick	mouthpart-induced	               phils,	have	been	shown	to	be	important	in	resistance	to	ticks	in	experi-
tissue	destruction,	epidermal	hyperplasia	and	pustule-like	intra-epider-            mental	 models	 and	 their	 skin	 kinetics	 suggests	 a	 systemic	 effect	 of	
mal	 vesicles	 filled	 with	 polymorphonuclear	 cells,	 mainly	 neutrophils.	       tick	infestations.	The	greater	number	of	basophils	in	infested	skin	of	R	
Alimentary	 cavity	 was	 present	 sometimes	 at	 the	 dermal	 layer,	 distal	       hosts	suggests	that	they	are	the	pivotal	cells	that	impair	hematophagy.	
to	the	cement	cone.	In	addition,	it	was	observed	infiltrated	inflamma-              Our	results	reflect	the	fact	that,	while	the	tick	bite	induces	inflammation,	
tory	 cells	 into	 the	 dermis,	 mainly	 neutrophils,	 eosinophils,	 basophils,	    tick	saliva	contains	anti-adhesive	and	immunosuppressive	molecules,	
mononuclear	cells	and	mast	cells.	The	intensity	of	these	cells	varied	              many	of	which	are	stage-specific.	
according	to	the	experimental	situation.	In	fact,	the	first	infestation	was	             Supported:	CNPq,	CAPES	and	FAPESP.	
dominated	 by	 a	 neutrophils	 influx,	 its	 number	 maintained	 high	 in	 the	
subsequent	 infestations.	 On	 the	 other	 hand,	 a	 cutaneous	 basophilia	         Key words:	Ticks,	Bovine,	Basophil,	Eosinophil,	Skin	
was	evidenced	by	a	higher	number	of	infiltrating	basophils	increasing	              Species:	ruminants
significantly	from	the	48th	to	72nd	hour	after	both	the	2nd	and	3rd	infesta-
tions.	Mononuclear	cells	appeared	in	the	inflammatory	focus,	but	their	                 sm097. SEQUENTIAL MORPHOLOGy AND GENE
number	varied	little	among	infestations,	with	only	a	significant	increase	           ExPRESSION PROFILES OF CUTANEOUS REACTIONS TO
120hours	after	2nd	infestation.	Eosinophils	and	mast	cells	were	found	                          TICK ANTIGENS IN BOVINES
occasionally	with	no	statistical	difference	when	compared	the	1st	and	               ARR	ABATEPAULo1,	JoÃo	MoRELLI2,	MATHIAS	P	SZABó2,4,	
subsequent	 infestations.	 It	 is	 concluded	 that	 basophils	 may	 play	 an	         SS	KASHIno3,	CJ	nARDELLI3,	GR	GARCIA1,	E	RAMIRo	DA	
important	 role	 in	 the	 mechanism	 of	 resistance	 of	 goats	 against	 A.	        SILVA	JR3,	oB	REGo	nETo3,	JS	SILVA1,	GH	BECHARA2,	IKF	DE	
cajennense	nymphs.                                                                                     MIRAnDA	SAnToS1
Key words: cutaneous	basophilia,	nymphs,	amblyomma	cajennense,	                       1Ribeirão	Preto	Medical	School,	University	of	São	Paulo,	Ribeirão	
resistance                                                                           Preto,	SP,	BRAZIL;	2Faculty	of	Agricultural	and	Veterinary	Sciences,	
Species: ruminants                                                                    State	University	of	São	Paulo,	Jaboticabal,	SP,	BRAZIL;3	Brazilian	
                                                                                    Enterprise	for	Agricultural	Research,	Brasília,	DF,	BRAZIL;	4Faculty	of	
   sm096. SKIN LESIONS INDUCED By TICKS RECRUIT                                     Veterinary	Medicine,	Universidade	Federal	de	Uberlândia,	Uberlândia,	
  DISTINCT CELLULAR POPULATIONS IN RESISTANT AND                                                                  MG,	BRAZIL
             SUSCEPTIBLE BOVINE HOSTS.                                                   Introduction:	 In	 cattle	 the	 level	 of	 infestation	 with	 ticks	 varies	
   FRAnZIn	AM1,	MoRé	DD1,	CARVALHo	WA1,	ConTI	LHA2,	                                according	to	breed	and	the	different	phenotypes	are	heritable,	geneti-
  JoF	PAULA	2,	AAM	MAIA2,	JS	SILVA1,	BR	FERREIRA1,	IKF	DE	                          cally	 susceptible	 animals	 harbour	 significantly	 more	 parasites	 than	
                    MIRAnDA	SAnToS1	                                                resistant	breeds,	even	after	repeated	infestations.
   1Dept.	of	Biochemistry	and	Immunology,	Ribeirão	Preto	Medical	                       Objectives:	 To	 study	 the	 molecular	 and	 cellular	 components	 of	
 School,	Ribeirão	Preto,	SP,University	of	São	Paulo,	Brazil,	2Dept.	of	             cutaneous	inflammatory	reactions	elicited	with	tick	antigens	in	different	
  Basic	Sciences,	School	of	Animal	Sciences	and	Food	Technology,	                   phenotypes	of	tick	infestations	in	bovines.	
          Pirassununga,	SP,University	of	São	Paulo,	Brazil.	                              Methods:	Animals	 of	 a	 resistant	 (R,	 Bos	 indicus,	 Nelore,	 N	 =	 6)	
                          alefranzin@usp.br                                         and	susceptible	(S,	B.	taurus,	Holstein,	N	=	6)	breed	were	managed	
      Cattle	 present	 variable	 and	 heritable	 levels	 of	 resistance	 to	 the	   in	 a	 pasture	 infested	 with	 the	 cattle	 tick,	 Rhipicephalus	 (Boophilus)	
cattle	tick,	Rhipicephalus	(Boophilus)	microplus.	In	order	to	obtain	some	          microplus.	Status	of	resistance	was	verified	by	counting	female	ticks	
of	 the	 immune	 correlates	 of	 these	 phenotypes,	 we	 characterized	 the	        larger	than	4	mm	on	each	host.	Antigen	(50	µg	in	100	µl	of	an	extract	
86


     of	unfed	larvae	(UFLE)	from	R.	microplus)	was	injected	in	the	dermis	               The	observed	depletion	of	CD3+	T	cells,	B	cells	and	γd+/WC1+	T	cells,	
     of	the	internal	surface	of	one	ear	and	the	same	volume	of	the	dilution	             but	not	of	CD8+	T	cells,	in	tick	bite	lesions	is	possibly	due	to	cell	death,	
     buffer	 was	 injected	 in	 the	 opposite	 ear	 of	 the	 animals.	 Skin	 biopsies	   emigration	and/or	impaired	cellular	migration	to	tick	bite	lesions.	It	sug-
     were	 taken	 one,	 72	 and	 96	 hours	 after	 inoculation	 of	 UFLE	 or	 buff-      gests	that	the	parasite	impairs	the	acquired	immune	response	via	anti-
     ered	 saline	 and	 processed	 for	 staining	 with	 haematoxylin/eosin	 and	         adhesive	 and/or	 immunosuppressive	 molecules.	 On	 the	 other	 hand,	
     May-Grünwald	 and	 Giemsa	 or	 extraction	 of	 total	 RNA.	Total	 and	 dif-         the	greater	reduction	of	inflammatory	γd+	T	cells	seen	in	the	infested	
     ferential	cells	counts	were	made	of	tissue	sections	and	quantification	             skin	of	resistant	bovines	indicates	that	these	cells	may	play	a	role	in	
     of	expression	of	candidate	genes	coding	for	chemokines	and	anti-	and	               resistance	to	ticks.	The	expression	of	chemokine	receptors	associated	
     proinflammatory	 cytokines	 was	 done	 with	 Real	Time	 PCR.	 Student’s	            with	migration	of	γd+	T	lymphocytes	to	inflamed	and	normal	skin	must	
     t-test	was	used	to	evaluate	significance	among	group	medians	and	a	                 be	examined.
     P-value	<	0,05	was	used	to	establish	the	level	of	significance.	
                                                                                              Supported	by	CNPq,	CAPES	and	FAPESP.
           Results	and	Discussion:	Haematoxylin/eosin	and	Giemsa-stained	
     sections	of	skin	biopsies	taken	one	hour	after	injection	revealed	similar	          Key words:	lymphocytes,	tick,	bovine,	skin,	immunohistochemistry
     total	and	differential	cell	counts	at	both	test	and	control	sites	for	both	         Species:	ruminants
     breeds,	except	eosinophils,	which	were	significantly	more	abundant	in	
     the	skin	of	S	animals.	After	72	hours,	in	relation	to	controls	the	global	                  sm099. INFLAMMATORy CELL INFILTRATION
     numbers	of	cellular	infiltrates,	as	well	as	of	eosinophils,	increased	sig-                 AND INFLAMMATORy CyTOKINES: INDICATORS
     nificantly	and	equally	in	S	and	R	bovine	skin	stimulated	with	UFLE,	how-                  OF STREPTOCOCCUS AGALACTIAE INFECTION IN
     ever,	at	this	time	point	the	numbers	of	basophils	increased	significantly	                       ExPERIMENTAL MOUSE MASTITIS
     in	R,	but	not	S	bovine	skin	stimulated	with	UFLE.	In	relation	to	gene	
                                                                                           GABRIELA	TRIGo1,2,	AnGELA	FRAnçA1,	MáRCIA	DInIS1,2,	
     expression,	R	bovines	presented	higher	level,	but	not	significantly,	of	
                                                                                           RUI	GIL	DA	CoSTA1,	ELVA	AnDRADE1,	PAULA	FERREIRA1,2,	
     expression	of		IFN-γ	in	IH	test	reactions	and	significantly	higher	expres-
                                                                                                            DELFInA	TAVARES1,2	
     sion	of	IGF-1,	IDO,	TGFb,	IL-16,	SLURP-1	and	Endotelin-1	in	delayed	
     reactions	significantly.	This	molecular	pattern	suggests	that	R	bovines	                 1Instituto	de	Ciências	Biomédicas	Abel	Salazar	(ICBAS),	
     have	 a	 greater	 capacity	 to	 mount	 inflammatory	 reactions	 comprising	          Universidade	do	Porto,	Portugal;	2Instituto	de	Biologia	Molecular	e	
     basophils	that	are	detrimental	to	the	tick.	                                                           Celular	(IBMC),	Porto,	Portugal	
          Supported	by:	FAPESP	and	CNPq.                                                                        dcb03002@icbas.up.pt

     Key words:	 gene	 expression,	 cutaneous	 reactions,	 tick	 antigens,	                    Streptococcus	 agalactiae	 is	 a	 major	 contagious	 pathogen	 caus-
     bovines                                                                             ing	mastitis	highly	adapted	to	survive	in	bovine	mammary	gland.	This	
     Species:	ruminants                                                                  study	focuses	on	the	use	of	a	mouse	model	of	Streptococcus	agalac-
                                                                                         tiae-induced	mastitis	as	a	practical	approach	for	regarding	the	patho-
                                                                                         genesis	of	the	bacteria.	BALB/c	mice	in	10-15	 th	day	of	lactation	were	
     sm098. BOVINE γδ/WC1+ AND B LyMPHOCyTES, BUT NOT
                                                                                         intramammary	 (I.ma)	 challenge	 on	 both	 L4	 (on	 the	 left)	 and	 R4	 (on	
     CD8+ CELLS, ARE REDUCED IN INFLAMMATION INDUCED
                                                                                         the	 right)	 abdominal	 mammary	 glands	 with	 108	 cells	 of	 S.	agalactiae	
                         By TICKS
                                                                                         isolated	 from	 bovine	 mastitis	 (infected	 animals)	 or	 with	 PBS	 (control	
        ALESSAnDRA	M	FRAnZIn1,	DAnIELA	D	MoRé1,WAnESSA	                                  animals).	Throughout	the	study	the	colonization	was	evaluated	by	bac-
       A	CARVALHo	1,	LUIZ	HA	ConTI2,	PAULA	JoF2,	AnTonIo	AM	                             terial	counts	(CFU)	in	the	mammary	gland,	kidneys,	spleen	and	liver.	
        MAIA2,	MARK	A	JUTILA,	JS	SILVA1,	BR	FERREIRA1,	IKF	DE	                           Mammary	 tissue	 alterations	 were	 evaluated	 by	 haematoxylin-eosin	
                         MIRAnDA	SAnToS1	                                                staining	 of	 mammary	 sections.	 Cytokine	 production	 in	 the	 mammary	
        1Dept.	of	Biochemistry	and	Immunology,	Ribeirão	Preto	Medical	                   gland	 during	 infection	 was	 evaluated	 by	 ELISA.	 S.	 agalactiae	 I.ma	
      School,	Ribeirão	Preto,	SP,University	of	São	Paulo,	Brazil;	2Dept.	of	             infection	 showed	 that	 the	 bacteria	 replicates	 in	 the	 mammary	 gland	
       Basic	Sciences,	School	of	Animal	Sciences	and	Food	Technology,	                   and	peaked	24	h	later.	At	the	same	time,	a	massive	infiltration	of	poly-
               Pirassununga,	SP,University	of	São	Paulo,	Brazil.	                        morphonuclear	cells	(PMNs)	and	an	increase	in	IL-1β,	IL-6	and	TNF-a	
                              	alefranzin@usp.br                                         (inflammatory	cytokines)	levels	were	detected	in	the	mammary	gland.	
           Cattle	 present	 variable	 and	 heritable	 levels	 of	 resistance	 to	 the	   After	this,	a	gradual	decrease	on	bacteria	load	was	observed	which	was	
     cattle	 tick,	 Rhipicephalus	 (Boophilus)	 microplus.	 In	 order	 to	 obtain	       accompanied	by	a	decrease	in	the	number	of	PMNs	and	an	increase	of	
     some	of	the	immune	correlates	of	these	phenotypes,	we	characterized	                macrophages	and	lymphocytes,	indicating	an	evolution	into	a	chronic	
     the	inflammatory	infiltrates	elicited	by	tick	bites	in	skin	of	resistant	(R)	       process	 in	 the	 mammary	 tissue.	A	 decrease	 in	 the	 levels	 of	TNF-	 a,	
     and	susceptible	(S)	bovines.	R	and	S	cattle	underwent	three	succes-                 IL-1β	and	IL-6	were	observed	in	the	mammary	gland	72	h	after	infec-
     sive	infestations	and	biopsies	were	taken	at	the	feeding	site	of	nymphs	            tion,	which	was	accompanied	by	an	increase	in	the	levels	of	IL-12	and	
     and	 adults,	 from	 normal	 skin	 of	 the	 same	 host	 or	 from	 skin	 of	 naïve	   IL-10.	Dissemination	of	the	bacteria	from	the	mammary	glands	to	the	
     controls.	Lymphocyte	surface	antigens	were	detected	with	specific	anti-             kidneys,	spleen	and	liver	was	observed	6	hours	after	infection,	with	a	
     bodies	reacting	with	acetone-fixed	cryostat	sections	of	biopsies	stained	           peak	of	the	CFU	after	12	hours	for	the	kidneys	and	spleen	and	after	24	
     using	 the	 indirect	 immunoperoxidase	 technique	 and	 counter-stained	            hours	for	the	liver.	The	bacterial	load	in	these	organs	was	significantly	
     with	 May-Grünwald	 and	 Giemsa,	 in	 infested	 skin,	 sections	 were	 lim-         lower	 than	 the	 one	 observed	 in	 the	 mammary	 gland,	 throughout	 the	
     ited	to	the	area	of	the	tick	cement	cone.	Bites	by	nymphs	recruit	more	             study.	In	conclusion,	the	mouse	model	of	infectious	mastitis	proposed	
     mononuclear	cells	than	those	of	adults	(P=0.001,	One-way	ANOVA).	                   here	is	suitable	and	less	costly	than	cows	for	the	study	of	pathogenesis	
     The	 numbers	 of	 an	 important	 subset	 of	 mononuclear	 cells,	 CD3+	 T	          of	S.	agalactiae.
     lymphocytes,	were	reduced	in	adult-infested	skin	of	both	breeds	when	
                                                                                             This	work	was	supported	by	the	Fundação	da	Ciência	e	Tecnologia	
     compared	with	those	found	in	skin	of	naïve	and	non-infested	controls	
                                                                                         (FCT)	grant	POCI/CVT/57144/2004	and	FEDER
     (P<0.05).	Numbers	of	proinflammatory	γd	WC1+/CD3+	T	cells	were	also	
     diminished	(P<0.05)	in	nymph	and	adult-infested	skin	of	both	breeds,	               Key words:	 Streptococcus	 agalactiae	 ,	 mastitis,	 inflammation,	
     when	compared	with	those	found	in	control	skin.	Moreover,	numbers	                  cytokines,	
     of	γd+	T	cells	were	even	lower	(P<0.05)	in	nymph-infested	R	skin	when	              Specie:	ruminants
     compared	 with	 adult-infested	 R	 skin.	 B	 lymphocytes	 were	 present	 in	
     skin,	 but	 their	 numbers	 were	 reduced	 (P<0.05)	 in	 nymph	 and	 adult-          sm100. ELUCIDATING THE BIOSyNTHETIC PATHWAy OF
     infested	skin,	regardless	of	the	breed	of	host.	As	the	distance	from	the	            PAF PRODUCTION By MAMMARy ENDOTHELIAL CELLS
     cement	cone	increased,	numbers	of	all	these	cells	reached	the	same	                         FOLLOWING ENDOTOxIN STIMULATION
     as	 normal	 skin.	 Numbers	 of	 another	 CD3+	 T	 cell	 subset,	 CD8+	 cyto-
     toxic	lymphocytes,	were	similar	in	infested,	normal	or	naïve	skin.	Skin	                       JC	GAnDy,	CM	CoRL,	K	BEGIn,LM	SoRDILLo
     immune	responses	involve	resident	and	infiltrating	subsets	of	lympho-                   Department	of	Large	Animal	Clinical	Sciences,	Michigan	State	
     cytes	that	are	sources	of	immune	regulatory	and	effectors	responses.	                                   University,	East	Lansing,	MI
                                                                                                                                                                             87


      Acute	symptoms	associated	with	coliform	mastitis	are	due	to	rapid	                Key words:	ileal	Peyer’s	patch,	jejunal	Peyer’s	patch,	B-cell	repertorie,	
growth	of	the	organism,	the	release	of	endotoxin,	and	the	subsequent	                   prenatal	and	postnatal
development	 of	 an	 exacerbated	 inflammatory	 reaction.	 	 Endotoxin	                 Species:	ruminants
release	promotes	the	production	of	potent	pro-inflammatory	mediators,	
such	 as	 platelet	 activating	 factor	 (PAF),	 that	 lead	 to	 the	 pathophysi-             sm102. STABILITy OF THE RECOMBINANT GM-CSF
ological	 changes	 witnessed	 during	 gram-negative	 infections.	 	 While	                   PRODUCED By BACULOVIRUS GENE ExPRESSION
much	attention	has	been	given	to	the	production	of	PAF	by	neutrophils	
                                                                                                               SySTEM
and	macrophages,	very	little	research	has	focused	on	PAF	production	
by	mammary	endothelial	cells	which	are	vital	to	maintaining	vascular	                   SHIGEKI	InUMARU,	HIDEyUKI	TAKAHASHI,	SAToKo	WATAnABE,	
homeostasis	 during	 the	 acute	 phase	 response.	 	 Previous	 research	                            MASATo	oHTA,	TAKAyUKI	KUBoTA
has	 shown	 increased	 lyso-PAF:acetyl	 coenzyme-A	 acetyltransferase	                                  NARO	National	Institute	of	Animal	Health	
(Lyso-PAF-AcT)	 activity,	 the	 rate	 limiting	 enzyme	 of	 PAF	 production,	                                   inumaru@affrc.go.jp
following	LPS	stimulation	in	bovine	mammary	endothelial	cells	(BMEC)	                         GM-CSF	is	known	as	a	cytokine	that	affects	the	various	haema-
occurs	prior	to	increases	in	the	acute	phase	cytokine	response.		The	                   topoietic	cells.	Bovine	GM-CSF	is	expected	to	use	a	therapeutic	agent	
biosynthetic	pathway	leading	to	this	increased	Lyso-PAF-AcT	activity	                   for	 diseases	 caused	 by	 complex	 and	 opportunistic	 infection	 such	 as	
and	 subsequent	 PAF	 production	 following	 LPS	 stimulation	 has	 not	                mastitis	in	cows.	Since	natural	GM-CSF	is	produced	only	trace	amount	
been	determined.		It	is	hypothesized	that	phosphatidic	acid	(PA)	and	                   by	 particular	 cells,	 we	 established	 the	 efficient	 method	 to	 produce	
phospholipase	D	(PLD)	are	preliminary	mediators	in	the	biosynthesis	                    recombinant	bovine	GM-CSF	(rboGM-CSF)	by	baculovirus/cell	culture	
of	 PAF	 by	 BMEC	 following	 endotoxin	 stimulation.	 	 Utilizing	 primary	
                                                                                        gene	 expression	 system	 and	 reported	 that	 this	 rboGM-CSF	 was	 a	
BMEC	 stimulated	 with	 LPS	 the	 production	 of	 PA	 and	 activity	 of	 PLD	
                                                                                        potential	therapeutic	agent	for	subclinical	mastitis	of	dairy	cows	caused	
was	 measured,	 as	 well	 as	 the	 activity	 of	 the	 PAF	 catabolic	 enzyme	
                                                                                        by	 S.	 aureus	 infection.	 Though	 the	 information	 about	 stability	 of	 the	
PAF-acetylhydrolase	 (PAF-AH).	 	 Preliminary	 data	 suggests	 that	 LPS	
                                                                                        rboGM-CSF	is	important	to	develop	rboGM-CSF	agent,	it	is	not	cleared	
stimulation	causes	an	increase	in	both	PA	production	and	PLD	activity	
                                                                                        up.	Therefore	 we	 are	 studying	 the	 stability	 of	 the	 rboGM-CSF	 under	
in	 BMEC.	 	 Endotoxin	 stimulation	 did	 not	 significantly	 alter	 the	 activ-
                                                                                        several	conditions.	METHODS:	Bovine	GM-CSF	cDNA	sequence	was	
ity	of	PAF-AH,	further	supporting	our	focus	on	the	initial	synthesis	of	
                                                                                        inserted	 to	 a	 baculovirus	 (AcNPV)	 genome.	 The	 rboGM-CSF	 was	
PAF.	 	 Determining	 the	 early	 mediators	 of	 the	 acute	 phase	 response	
                                                                                        prepared	 with	 this	 recombinant	 virus	 infected	 insect	 cell(	 TN5	 cells).	
during	 gram-negative	 infections	 could	 lead	 to	 the	 development	 of	
                                                                                        The	culture	fluid,	containing	rboGM-CSF	was	ultra-filtrated	to	remove	
novel	therapeutic	targets	to	diminish	the	uncontrolled	inflammation	and	
                                                                                        virus	particle	and	diluted	with	PBS	with	10	%	FBS	(PBS+)	or	without	
pathophysiological	effects	of	coliform	mastitis.
                                                                                        FBS(PBS-).	 It	 is	 then	 stored	 at	 4	 C	 or	 –20	 C.	 The	 biological	 activi-
Key words:	 Endothelial	 cell,	 inflammation,	 Platelet	 activating	 factor,	           ties	were	measured	with	rboGM-CSF	adopted	TF-1	cells.	RESULTS	&	
cytokine                                                                                DISCUSSION:	To	study	the	stability	of	rboGM-CSF	at	4	C,	rboGM-CSF	
Species:	ruminants                                                                      in	PBS+	was	stored	at	4	C,	and	the	biological	activity	was	measured.	
                                                                                        The	activity	was	not	reduced	at	least	5	month.	Similarly,	the	activity	of	
  sm101. DyNAMICS OF B-CELL REPERTOIRE IN SHEEP                                         rboGM-CSF	in	PBS-	was	not	reduced	at	least	5	month.	To	study	the	
 JEJUNAL AND ILEAL PEyER’S PATCH SINGLE FOLLICLES                                       stability	of	rboGM-CSF	on	the	freeze	and	thaw,	rboGM-CSF	in	PBS+	
MASAHIRo	yASUDA,	CRAIG	n	JEnnE,	LAURIE	J	KEnnEDy,JoHn	                                  was	frozen	at	–20	C	and	thawed	repeatedly.	At	least	3	times	repetition,	
                    D	REynoLDS                                                          the	biological	activity	was	not	reduced.	The	activity	of	rboGM-CSF	in	
                                                                                        PBS-	was	also	not	decreased	by	the	freeze	and	thaw	at	least	3	times	
   Immunology	Research	Group,	Department	of	Cell	biology	and	
                                                                                        repetition.	 These	 results	 clearly	 showed	 that	 rboGM-CSF	 produced	
 Anatomy,	University	of	Calgary,	Department	of	Veterinary	Anatomy,	
                                                                                        by	 baculovirus/cell	 culture	 gene	 expression	 system	 is	 very	 stable	 at	
                       University	of	Miyazaki
                                                                                        4	C	and	by	freeze	and	thaw.	It	is	an	advantageous	factor	to	develop	
      In	the	ruminant’s	intestine,	there	are	two	types	of	gut-associated	               rboGM-CSF	 therapeutic	 agent	 for	 mastitis	 etc.	 We	 are	 studying	 the	
lymphoid	organ:	jejunal	Peyer’s	patch	(PP)	and	ileal	PP.			Ileal	PP	is	                 stability	 under	 some	 other	 conditions.	 CONCLUSIONS:	 The	 rboGM-
thought	to	be	the	primary	lymphoid	organ	of	B-cell,	whose	repertoire	                   CSF	produced	by	baculovirus/cell	culture	gene	expression	system	is	
is	diversified	by	gene	conversion	and/or	somatic	hypermutation.			On	                   stable	for	storage	at	4C	and	freeze	and	thaw.
the	 other	 hand,	 jejunal	 PP	 is	 thought	 to	 be	 the	 secondary	 lymphoid	
                                                                                        Key words:	GM-CSF,	stability,	therapeutics,	mastitis
organ	 for	 local	 mucosal	 immunity	 and	 the	 functions	 of	 this	 lymphoid	
                                                                                        Species:	ruminants
organ	 keep	 throughout	 the	 animal’s	 life.	 	 	The	 prenatal	 development	
of	 follicles	 in	 the	 PP	 begins	 first	 in	 the	 jejunum	 during	 the	 middle	 of	
gestation	and	then	in	the	ileum	during	late	gestation.			Therefore	it	can	                   sm103. RELATIONSHIP OF T CELL PROLIFERATION
be	considered	that	jejunal	PP	follicle	also	contributes	making	primary	                      AND THE UDDER TRANSCRIPTOME WITH MASTITIS
B-cell	repertoire	as	well	as	ileal	PP	follicle	at	fetal	development.		Then	                    RESISTANCE TO STAPHLoCoCCUS AUREUS
after	birth,	jejunal	PP	may	form	the	character	for	local	mucosal	immu-                   nICoLA	HASTInGS1,	FIonA	yoUnG2,	JoHn	WILLIAMS3,	JULIE	
nity.			We	attempt	to	analyze	B-cell	repertoire	of	ileal	and	jejunal	PP	                           FITZPATRICK4,ELIZABETH	J	GLASS1
single	 follicles	 during	 ontogeny.	 	 	 Both	 PP	 single	 follicles	 at	 prenatal	
                                                                                          1Roslin	Institute,	Roslin,	Midlothian,	EH25	9PS,	U.K.;	2Agriculture	
and	postnatal	development	were	isolated	under	stereoscopic	micros-
                                                                                          Branch,	Agri-Food	and	Biosciences	Institute,	Hillsborough,	County	
copy.	 	 	 Igl	 light	 chain	 is	 dominant	 light	 chain	 in	 sheep.	 Vl-Jl1	 was	
                                                                                        Down,	Northern	Ireland,	BT26	6DR.;	3Parco	Tecnologico	Padano,Via	
amplified	by	PCR	or	PT-PCR.			At	the	postnatal	stage,	ileal	PP	follicles	
                                                                                          Einstein,	Polo	Universitario,	Lodi	26900,	Italy;	4Moredun	Reseach	
contained	 oligoclonal	 B-cell,	 but	 jejunal	 PP	 follicles	 contained	 much	
                                                                                         Institute,	Pentlands	Science	Park,	Bush	Loan,	Penicuik,	Midlothian,	
more	polyclonal	B-cell.			Similar	tendency	observed	at	prenatal	stage.	             	
Hence	clonality	of	both	PP	follicles	was	markedly	different.			At	prenatal	                                          EH26	0PZ,	U.K.
stage,	point	mutation	accumulated	in	CDR	region	in	Vl	gene	of	both	                             Mastitis	remains	one	of	the	most	important	diseases	in	dairy	cattle	
PP	follicular	B-cell	(14-19	point	mutations/kb	were	in	ileal	PP	and	3-13	               particularly	in	the	western	world.	It	costs	the	EU	alone	€100-200	million/
mutations/kb	were	in	jejunal	PP).			B-cell	diversity	is	observed	in	not	                year	with	economic	losses	due	to	reduced	milk	production,	lower	milk	
only	ileal	PP	follicle	but	also	jejunal	PP	follicle.		The	data	show	that	both	          quality,	veterinary	treatment	and	also	culling.		In	addition,	as	mastitis	
PP	follicles	contribute	making	B-cell	repertoire	during	prenatal	develop-               is	an	extremely	painful	disease,	there	are	serious	welfare	implications.	
ment.			At	the	postnatal	development,	much	more	mutations	observed	                     Current	control	measures	are	not	always	effective	and	developing	vac-
in	 CDR	 regions	 in	 Vl	 gene	 of	 both	 PP	 follicular	 B-cell	 (32-64	 point	        cines	targeted	at	mucosal	surfaces	such	as	the	udder	are	proving	dif-
mutations/kb	were	in	ileal	PP	and	39-54	mutations/kb	were	in	jejunal	                   ficult.	Breeding	for	resistance	to	mastitis	could	be	an	alternative	means	
PP).			Especially	many	replacement	mutations	observe	in	CDR3	region	                    of	control	but	is	not	straightforward	for	a	number	of	reasons.	Although	
of	ileal	PP	follicles.			Therefore	ileal	PP	follicles	contribute	making	very	           it	 is	 clear	 that	 there	 is	 a	 genetic	 component	 accounting	 for	 variation	
wide	 diversity	 of	 B-cell	 after	 birth.	 	 	 This	 event	 probably	 causes	 the	     in	mastitis	resistance,	it	is	likely	to	be	multigenic	and	the	trait	has	low	
appearance	many	self-reactive	B-cell.                                                   heritability,	possibly	because	mastitis	scoring	for	genetic	analysis	usu-
88


     ally	does	not	take	into	consideration	that	mastitis	is	caused	by	different	              antigenic	components	(57,	43	and	35	kDa),	thus	representing	potential	
     pathogens	which	elicit	different	host	responses.	Additionally	mastitis	is	               antigenic	markers	to	discriminate	vaccinated	from	infected	cattle.
     becoming	more	of	an	issue	for	dairy	farmers	because	it	has	a	negative	                   Key words:	Brucella	abortus,	Avidity-Immunoblot,	Triton	X-114,	Cattle,	
     correlation	 with	 genetic	 selection	 for	 increased	 productivity.	 We	 pro-           LPS.	
     pose	to	take	a	different	approach	by	taking	advantage	of	the	genomic	                    Specie:	ruminants
     resources	now	available	for	cattle	to	identify	key	genes	and	pathways	
     that	 could	 ultimately	 lead	 to	 new	 genetic	 tests	 and	 selectable	 mark-           sm105. IDENTIFICATION OF ADAPTIVE TRAITS OF BOVINE
     ers	 for	 breeding	 for	 mastitis	 resistance.	 Earlier	 studies	 indicated	 that	               MASTITIS ESCHERICHIA COLI STRAINS
     measurement	of	the	proliferative	response	of	bovine	peripheral	blood	
                                                                                                  S	BLUM1,	S	SELA2,	o	HAMMER-MUnTZ2,	o	KRIFUCKS1,	L	
     T	 cells	 to	 formalin-fixed	 Staphylococcus	 aureus	 might	 be	 used	 as	 a	
                                                                                                         WEISBELITH1,	D	HELLER3,	G	LEITnER1*
     potential	mastitis-resistance	predictor	with	high	responders	potentially	
     predicting	greater	resistance	to	S.	aureus	mastitis	than	those	with	a	low	                 1National	Mastitis	Reference	Center,	Kimron	Veterinary	Institute,	
     proliferative	response.	Our	studies	in	a	cattle	cross	population	showed	                    Ministry	of	Agriculture	&	Rural	Development,	Bet	Dagan,	Israel,	
     that	the	level	of	proliferation	to	S.	aureus	was	at	least	partially	geneti-              2Microbial	Food-Safety	Research	Unit,	Department	of	Food	Sciences,	
     cally	determined	(h2	=	0.2).	We	now	aim	to	directly	investigate	whether	                  Agricultural	Research	Organization	(ARO),	The	Volcani	Center,	Bet	
     young	heifers	selected	on	the	basis	of	their	T	cell	response	to	S.	aureus	                 Dagan,	Israel,	3Faculty	of	Agriculture,	The	Hebrew	University	of	
     into	two	groups	of	high	and	low	responders,	do	differ	in	their	response	                                       Jerusalem,	Rehovot,	Israel.	
     to	experimental	S.	aureus	challenge	in	vivo	at	6-weeks	of	lactation.	In	                                           leitnerg@moag.gov.il	
     addition	to	measuring	clinical	parameters,	we	will	also	transcriptionally	                      Escherichia	coli	is	a	major	agent	of	acute	bovine	mastitis.	In	spite	
     profile	the	cells	entering	the	udder	and	the	udder	tissue	itself	following	              of	extensive	research,	the	bacterial	pathogenic	factors	associated	with	
     infection.	This	project	could	provide	more	direct	evidence	of	the	predic-                E.	coli	bovine	mastitis	are	still	unclear	and,	to	date,	no	E.	coli	strains	
     tive	 ability	 of	 the	T	 cell	 test,	 and	 has	 the	 potential	 to	 reveal	 relevant	   subset	or	virulence	factors	have	been	particularly	associated	with	the	
     gene	and	pathway	targets	as	candidates	for	new	genetic	tests	of	mas-                     disease.	In	this	context,	this	study	aimed	to	assess	if	bovine	mastitis	E.	
     titis	resistance.                                                                        coli	strains	have	unique	adaptive	traits.	For	this	purpose,	E.	coli	isolates	
     Key words:	mastitis,	resistance,	cattle,	microarray                                      from	acute	bovine	mastitis	and	from	the	environment	were	compared	
     Species:	ruminants                                                                       regarding	 phenotypic	 and	 physiological	 traits,	 resistance	 to	 killing	 by	
                                                                                              bovine	 leukocytes	 and	 genetically,	 by	 Pulse-field	 gel-electrophoresis	
                                                                                              (PFGE).	Mastitis	and	environmental	strains	could	not	be	phenotypically	
          sm104. EVALUATION OF BRUCELLA ABoRTUS
                                                                                              distinguished	by	means	of	the	conventionally	studied	traits,	namely	O	
         HyDROPHOBIC AND HyDROPHILIC FRACTIONS By
                                                                                              antigen	and	antibiotic	sensitivity.	The	physiologic	traits	evaluated	were	
      AVIDITy-IMMUNOBLOT FOR SERODIAGNOSIS OF BOVINE                                          bacterial	growth	in	milk,	nutrient	broth	and	lactose	fermentation	rates.	
                       BRUCELLOSIS                                                            While	no	differences	in	bacterial	growth	rates	were	observed	in	nutrient	
        AnA	C	A	M	PAJUABA,	DEISE	A	o	SILVA,	DÂMASo	P	RIBEIRo,	                                broth,	 in	 milk,	 however,	 bacterial	 growth	 rates	 of	 all	 mastitis	 isolates	
                           JoSé	R	MInEo*                                                      were	significantly	higher	than	most	of	environmental	strains.	Similarly,	
      Laboratory	of	Immunoparasitology,	Institute	of	Biomedical	Sciences,	                    the	 rate	 of	 lactose	 fermentation	 was	 higher	 in	 mastitis	 isolates	 than	
         Federal	University	of	Uberlândia,	Av.	Pará	1720,	38400-902	                          environmental	 ones,	 being	 positively	 correlated	 to	 growth	 in	 milk.	 In	
                           Uberlândia,	MG,	Brazil		                                           addition,	the	mean	resistance	to	killing	by	bovine	leukocytes	was	higher	
                               jrmineo@ufu.br                                                 in	 mastitis	 isolates	 than	 in	 environmental	 strains.	 PFGE	 revealed	 a	
                                                                                              higher	 genetic	 variability	 among	 environmental	 strains	 than	 in	 masti-
           Brucellosis	 is	 a	 major	 zoonosis	 and	 has	 been	 considered	 an	
                                                                                              tis	 isolates,	 ~	 40%	 of	 mastitis	 isolates	 formed	 a	 relatively	 genetically	
     emerging	 or	 re-emerging	 disease	 worldwide,	 particularly	 leading	 to	
                                                                                              similar	cluster.	Since	only	part	of	the	environmental	strains	physiologi-
     abortion	 and	 infertility	 in	 domestic	 herds.	 Diagnosis	 of	 brucellosis	 in	
                                                                                              cally	and	genetically	resembled	mastitis	isolates,	our	results	reinforce	
     cattle	 is	 mainly	 based	 on	 serological	 methods	 that	 have	 used	 whole	
                                                                                              the	idea	of	an	E.	coli	subset	of	strains	more	adapted	to	cause	acute	
     cell	 preparations,	 sonicated	 cell	 extracts	 or	 lipopolysaccharide	 (LPS)	
                                                                                              bovine	 mastitis.	 This	 subset	 is	 apparently	 characterized	 by	 adaptive	
     enriched	fractions.	As	specificity	of	these	tests	is	low,	alternative	anti-
                                                                                              traits	related	to	a	higher	ability	to	multiply	and	evade	the	host	immune	
     gens	have	been	characterized	as	potentially	useful	tools	in	diagnostic	
                                                                                              response	within	the	bovine	udder	and	genetic	homogeneity.
     tests	for	brucellosis.	In	this	context,	the	Triton	X-114	nonionic	detergent	
     has	 been	 successfully	 used	 for	 extraction	 of	 membrane-associated	                 Key words:	 Escherichia	 coli,	 mastitis,	 adaptive	 traits,	 lactose	
     proteins,	but	no	data	are	available	on	its	use	for	Brucella	abortus	anti-                fermentation
     gen	 extraction.	 Smooth	 lipopolysaccharide	 (S-LPS)	 and	Triton	 X-114	                Species:	ruminants
     fractions	 from	 B.	 abortus	 were	 analyzed	 in	 immunoblot	 and	 avidity-
     immunoblot	 to	 identify	 differences	 between	 antigenic	 markers	 from	                 sm106. INFLUENCE OF SEMINAL PLASMA ON TGF BETA1
     S19	 vaccinated	 from	 non-vaccinated	 seropositive	 cows.	 Four	 groups	                        MRNA ExPRESSION IN THE PIG OVIDUCT
     with	15	cattle	sera	each	were	analyzed:	(I)	non-vaccinated	seropositive	                     J	JIWAKAnon1,	M	BERG2,	C	FoSSUM2,	E	PERSSon3,	AM	
     cows	 from	 Brucella-endemic	 areas,	 (II)	 non-vaccinated	 seropositive	                                         DALIn1
     cows	 from	 Brucella	 non-endemic	 areas,	 (III)	 S19	 vaccinated	 heifers,	
     and	 (IV)	 non-vaccinated	 seronegative	 cows.	 Classical	 agglutination	                    1Dept.	of	Clinical	Sciences,	2Dept.	of	Biomedical	Science	and	
     tests	were	used	to	select	the	groups	of	cattle	sera.	S-LPS	immunoblot	                        Veterinary	Public	Health,	3Dept.	of	Anatomy,	Physiology	and	
     showed	a	wide	cluster	of	bands	(22	to	105	kDa)	recognized	by	sera	                                        Biochemistry,	SLU,	Uppsala,	Sweden
     from	non-vaccinated	cows	(groups	I	and	II)	while	a	more	restrict	cluster	                       It	has	been	shown	that	semen	induces	uterine	immune	response	
     (43	to	58	kDa)	was	seen	in	sera	from	vaccinated	heifers	(group	III).	In	                 and	 that	 seminal	 plasma	 may	 regulate	 uterine	 cytokine	 expression.	
     avidity-immunoblot	using	S-LPS,	no	distinct	reactivity	profile	could	be	                 The	aim	of	the	present	study	was	to	evaluate	if	seminal	plasma	per	se	
     identified.	Triton	X-114	hydrophobic	fraction	revealed	a	wide	cluster	of	                affects	the	expression	of	the	potent	immunoregulatory	cytokine,	trans-
     antigenic	bands	in	contrast	to	hydrophilic	fraction	that	showed	a	more	                  forming	growth	factor	beta	1	(TGFβ1)	in	the	porcine	oviduct.	Sixteen	
     clearly	defined	reactivity	profile.	Immunoblot	using	the	hydrophilic	frac-               gilts	 were	 inseminated	 once,	 15-20h	 after	 standing	 reflex,	 with	 fresh	
     tion	revealed	some	immunodominant	antigens	(30,	35,	43,	57	and	73	                       semen	diluted	in	BTS	(extender)	(n=4),	seminal	plasma	(n=4),	sperms	
     kDa)	 in	 all	 three	 seropositive	 groups,	 whereas	 a	 significantly	 lower	           isolated	 by	 colloidal	 centrifugation	 and	 diluted	 in	 BTS	 (n=4)	 or	 BTS	
     reactivity	was	seen	for	the	69	and	65	kDa	bands	only	in	vaccinated	heif-                 alone	(n=4).	The	gilts	were	slaughtered	5–6	h	after	insemination	and	
     ers	(group	III).	Avidity-immunoblot	with	the	hydrophilic	fraction	showed	                oviductal	samples	were	taken	immediately,	plunged	into	liquid	nitrogen	
     significant	 reactivity	 impairment	 for	 the	 immunodominant	 antigenic	                and	 stored	 at	 -80	 °C	 until	 analyzed.	 TGFβ1	 mRNA	 expression	 level	
     bands	(57,	43	and	35	kDa)	recognized	by	sera	of	group	III,	reflecting	                   in	oviductal	tissues,	isthmus	and	infundibulum,	was	quantified	by	real	
     in	lower	vaccinal	antibody	avidity	for	such	antigenic	markers.	In	con-                   time	PCR	using	TaqMan-probe	normalized	to	the	geometric	mean	of	
     clusion,	B.	abortus-specific	IgG	reactivity	profile	in	avidity-immunoblot	               two	 housekeeping	 genes,	 hypoxanthine-guanine-phosphoribosyl-
     demonstrated	that	vaccinal	antibodies	showed	a	lower	avidity	for	some	                   transferase	(HPRT)	and	cyclophilin.	The	mRNA	expression	of	TGFβ1	
                                                                                                                                                                           89


was	similar	in	infundibulum	compared	to	in	isthmus,	regardless	if	the	                     ∗Corresponding	author:	Ulf	Magnusson,	Department	of	Clinical	
gilts	were	inseminated	with	fresh	semen,	purified	sperm	or	the	extender	                    Sciences,	Faculty	of	Veterinary	Medicine	and	Animal	Science,	
alone.	 Thus,	 the	 results	 suggest	 that	 seminal	 plasma	 per	 se	 has	 no	             Swedish	University	of	Agricultural	Sciences,	P.O.	Box	7054,	SE-
early	 effect	 on	 the	 level	 of	 TGFβ1	 mRNA	 in	 the	 oviduct	 and	 similar	                               75007	Uppsala,	Sweden	
expression	level	of	TGFβ1	mRNA	were	observed	between	the	upper	                                               ulf.magnusson@kv.slu.se
part	and	the	lower	part	of	the	oviduct.
                                                                                              Twelve	healthy	primiparous	sows	received	intramammary	inocula-
Key words:	gilt,	TGF	beta,	oviduct,	and	seminal	plasma                                  tion	with	Escherichia	coli	(E.	coli,	serotype	O127)	during	the	24-hour	
Species:	swine                                                                          period	 preceding	 parturition.	 Eight	 of	 the	 sows	 remained	 clinically	
                                                                                        healthy,	 whereas	 four	 developed	 clinical	 signs	 of	 mastitis	 such	 as	
       sm107. THE IN VIVO EARLy TRANSCRIPTIONAL                                         fever,	 mammary	 swelling,	 and	 lethargy.	 Jugular	 blood	 samples	 and	
    INTESTINAL RESPONSE TO ROTAVIRUS INFECTION IN                                       biopsy	samples	from	the	inoculated	glands	were	collected	immediately	
                  GERM-FREE PIGLETS                                                     before	inoculation,	 and	24	hours	after	inoculation.	Twenty	four	hours	
  THEo	nIEWoLD1,	MARCEL	HULST,	HInRI	KERSTEnS,	AGnES	                                   after	inoculation,	the	serum	concentrations	of	TNFa	were	higher	(P	<	
       DE	WIT,	MARI	SMITS	AnD	JAn	VAn	DER	MEULEn	                                       0.001)	in	sows	that	developed	clinical	signs	of	mastitis	compared	with	
  Animal	Sciences	Group	of	Wageningen	University	and	Research,	                         those	 that	 remained	 clinically	 healthy.	 In	 the	 mammary	 gland	 biopsy	
 P.O.	Box	65,	Lelystad,	The	Netherlands,	1Nutrition	and	Health	Unit,	                   specimens,	analysis	of	TNFa	by	immunohistochemistry	revealed	that	
 Faculty	of	Bioscience	Engineering,	Katholieke	Universiteit	Leuven,	                    the	production	of	TNFα	24	hours	after	inoculation	was	higher	(p	<	0.05)	
            B-3001	Heverlee,	Belgium	(presenting	author)                                in	sows	that	developed	clinical	signs	of	mastitis	compared	with	those	
                                                                                        that	remained	clinically	healthy.	Reverse	transcription-PCR	(RT-PCR)	
      Rotaviruses	are	a	major	cause	of	severe	diarrhea	in	young	chil-
                                                                                        analysis	of	the	biopsy	specimens	showed	that	TNF-a	mRNA	expres-
dren	 worldwide.	 Most	 studies	 regarding	 the	 molecular	 mechanism	
underlying	rotavirus	induced	disease	focus	on	isolated	enterocytes	or	                  sion	 increased	 in	 the	 inoculated	 mammary	 glands	 of	 both	 sows	 that	
enterocyte	 cell-lines.	 In	 vivo,	 mature	 enterocytes	 lining	 the	 intestinal	       developed	clinical	signs	of	mastitis	(P	<	0.01)	and	those	that	remained	
epithelial	 layer	 are	 the	 primarily	 target	 cells	 for	 rotavirus	 replication,	    clinically	healthy	(P	<	0.05)	24	hours	after	inoculation,	however	there	
however,	the	different	types	of	cells	that	compose	the	intestinal	muco-                 were	no	differences	in	mRNA	expression	between	the	two	groups	of	
sal	layer	are	probably	also	involved	in	anti-viral	responses.	The	in	vivo	              sows.	 	 Here	 we	 show	 that	 the	 higher	 serum	 concentrations	 of	 TNF-
system	is	also	much	more	complex	for	the	presence	of	the	intestinal	                    α	 in	 sows	 that	 developed	 systemic	 clinical	 signs	 of	 mastitis	 were	 in	
microbiota.	In	order	to	separate	rotavirus	specific	effects	from	micro-                 accordance	 with	 a	 higher	 protein	 production	 in	 the	 mammary	 gland.	
biota	associated	effects,	we	used	germ-free	piglets.	Piglets	housed	in	                 However,	such	a	difference	between	healthy	and	not	healthy	sows	was	
sterile	incubators	were	orally	infected	with	virulent	group	A	rotavirus	at	3	           not	seen	at	the	gene	expression	level.	These	data	illustrates	the	dis-
weeks	and	whole	mucosal	gene	expression	vs	uninfected	controls	was	                     crepancy	between	different	read-outs	when	relating	TNF-a	to	clinical	
analyzed	by	cDNA	microarray	on	12	and	18	hours	post	infection.	IFN-γ	                   disease.
mRNA	levels	were	10	to	50-fold	higher	in	infected	piglets.	Microarray	                  Key words:	cytokine,	mammary	gland,	mastitis,	E.	coli
analysis	 identified	 13	 down-,	 and	 19	 up-regulated	 genes	 in	 infected	           Species:	swine
piglets.	 Microarray	 data	 were	 validated	 by	 Northern	 blot	 analysis	 of	
nine	 selected	 genes.	 Regulated	 genes	 were	 functionally	 clustered	 in	
interferon-regulated	 genes,	signal	transduction	and	apoptosis	 genes,	                 sm109. MODULATION OF PERIPHERAL DENDRITIC CELLS
(enterocyte)	metabolism	and	cell	maintenance	genes,	and	genes	with-                       TOWARDS MUCOSA-TyPE DENDRITIC CELLS By ALL-
out	a	known	function.	Up-regulation	was	observed	for	several	genes	                                  TRANS RETINOIC ACID
associated	 with	 the	 innate	 defense	 against	 viral	 infections,	 such	 as	                  LESLIE	SAURER,	KEnnETH	MCCULLoUGH,	ARTUR	
IFN-γ	induced	guanylate	binding	protein	2	(GBP-2),	a	protein	that	was	                                         SUMMERFIELD
described	earlier	to	effectively	inhibit	VSV	and	EMCV	virus	replication	
                                                                                         Institute	of	Virology	and	Immunoprophylaxis,	Sensemattstrasse	293,	
in	vitro.	Furthermore,	a	gene	coding	for	an	uncharacterized	hypothetical	
                                                                                                           CH-3147	Mittelhäusern,	Switzerland.
protein	was	upregulated,	carrying	a	phospholipase	A2	inhibitor	domain,	
suggesting	involvement	in	(anti-)	inflammatory	pathways.	We	hypoth-                           Efficient	induction	of	mucosal	immunity	most	often	employs	nasal	
esize	that	both	these	proteins	participate	in	cellular	mechanism(s)	that	               or	oral	vaccination	while	parenteral	immunization	generally	is	ineffective	
provide	 the	 intestinal	 mucosa	 protection	 to	 the	 effects	 of	 rotavirus	 in	      at	generating	mucosal	immune	responses.	This	relates	to	the	unique	
the	 jejunum.	 Histological	 analysis	 showed	 a	 significant	 reduction	 of	           ability	 of	 resident	 mucosal	 dendritic	 cells	 (DC)	 to	 induce	 IgA	 switch-
villus	length	due	to	rotavirus	infection.	It	is	concluded	that	differential	            ing	and	to	imprint	mucosa-specific	homing	receptors	on	lymphocytes.	
expression	reflects	in	part	a	shift	in	the	relative	contribution	of	certain	            Based	on	the	well-known	plasticity	of	the	DC	system,	this	study	sought	
cell	types	(e.g.	loss	of	mature	epithelial	cells),	and	in	another	part	rep-             to	 investigate	 whether	 peripheral	 DC	 could	 be	 modulated	 towards	
resents	 induction	 by	 rotavirus	 itself.	 Finally,	 a	 most	 striking	 finding	 is	   “mucosa-type”	DC	by	treatment	with	immunomodulatory	and	therefore	
the	limited	number	of	differentially	expressed	genes	in	intact	mucosa	in	               potentially	adjuvant-like	factors.	Here,	we	show	that	monocyte-derived	
vivo	compared	to	those	obtained	in	in	vitro	enterocyte	cultures.	Dilution	              dendritic	cells	(MoDC)	pre-treated	with	the	vitamin	A	derivative	all-trans	
of	expression	may	play	a	role	in	that,	but	our	findings	are	probably	also	              retinoic	acid	(RA)	indeed	acquired	several	attributes	characteristic	of	
consistent	with	modulatory	effects	of	the	heterogenous	cell	population	                 mucosal	 DC:	 secretion	 of	TGFβ	 and	 IL-6	 and	 the	 capacity	 to	 induce	
in	vivo.	                                                                               IgA	responses	and	the	expression	of	mucosal	homing	receptors	in	co-
Key words:	intestinal	genomics,	rotavirus,	piglets                                      cultured	lymphocytes.	Addition	of	a	TGFβ	neutralizing	Ab	significantly	
Species:	swine                                                                          inhibited	 induction	 of	 a4β7	 integrin,	 but	 not	 of	 CCR9	 mRNA	 expres-
                                                                                        sion	 by	 the	 RA-treated	 MoDC.	 Both	 α4β7	 integrin	 and	 CCR9	 mRNA	
  sm108. COLIFORM MASTITIS IN SOWS ENHANCES TNF-                                        expression,	but	not	IgA	production	were	suppressed	in	the	presence	of	
  ALPHA PROTEIN AND MRNA ExPRESSION IN DIFFERENT                                        a	retinoic	acid	receptor	antagonist.	Collectively,	our	findings	identified	
                  COMPARTMENTS                                                          a	novel	role	for	RA	as	mucosal	immune	modulator	targeting	DC.	Such	
    yAoHonG	ZHU1,3,	MIKAEL	BERG2,	CARoLInE	FoSSUM2,	                                    a	role	would	be	consistent	with	the	vicinity	of	mucosal	DC	to	RA-pro-
                 ULF	MAGnUSSon1,3,*                                                     ducing	intestinal	epithelial	cells	and	the	autocrine	production	of	RA	by	
                                                                                        intestinal	DC.	Importantly,	our	results	further	demonstrate	that	DC	can	
1Department	of	Clinical	Sciences,	Faculty	of	Veterinary	Medicine	and	
                                                                                        act	as	efficient	carriers	of	RA	at	least	in	vitro.	DC	targeting	with	RA	may	
  Animal	Science,	Swedish	University	of	Agricultural	Sciences,	SE-
 75007	Uppsala,	Sweden;	2Department	of	Biomedical	Sciences	and	                         thus	 hold	 promise	 for	 promoting	 vaccine-induced	 mucosal	 immune	
Veterinary	Public	Health,	Swedish	University	of	Agricultural	Sciences,	                 responses	via	the	parenteral	route	of	immunization.
  SE-75123	Uppsala,	Sweden;	3Centre	for	Reproductive	Biology	in	                        Key words:	dendritic	cells,	mucosal	imprinting,	all	trans	retinoic	acid
                    Uppsala,	Uppsala,	Sweden	                                           Species:	swine
90


         sm110. THE MUCOSAL IMMUNOGENICITy IN PIGS OF                                       to	non-vaccinated	sows.	These	colostral	leukocytes	were	assessed	by	
         F4(K88) FIMBRIAE IS DECREASED By REDUCING ITS                                      flow	cytometry	for	intra-cellular	cytokines	and	by	IFNγ ELIspots	assay	
                      POLyMERIC STABILITy                                                   for	their	ability	to	produce	IFN	γ	and	TNF a	upon	antigenic	re-stimula-
         F	VERDonCK1,	J	DE	MEyER1,	J	JoEnSUU2,	M	MUILU2,	B	                                 tion.	These	functional	experiments	clearly	showed	that	colostrum	from	
             GoDDEERIS1,3,	V	nIKLAnDER-TEERI2,	E	Cox1                                       vaccinated	sows	contained	IFN	γ-	and	TNF	a-producing	PCV2-specific	
                                                                                            CD4+	and	CD8+	T	cells,	whereas	no	IFNγ	or	TNF	a	production	could	be	
      1Laboratory	of	Immunology,	Faculty	of	Veterinary	Medicine,	Ghent	                     detected	in	the	colostrum	of	non-vaccinated	sows.	Our	data,	together	
      University,	Belgium;	2Department	of	Applied	Biology,	University	of	                   with	 previous	 reports	 demonstrating	 that	 leukocytes	 isolated	 from	
     Helsinki,	Finland;	3Department	of	Biosystems,	Katholieke	Universiteit	                 colostrum	 can	 pass	 through	 the	 intestinal	 barrier	 of	 newborn	 piglets,	
                               Leuven,	Belgium
                                                                                            strongly	 suggest	 that	 maternal	 antigen-specific	 leukocytes	 may	 be	
            ETEC	 are	 an	 important	 cause	 of	 diarrhoea	 in	 man	 and	 animals	          transferred	to	the	piglets	via	the	colostrum	and	constitute	another	line	
     and	 there	 is	 need	 for	 the	 development	 of	 vaccines	 against	 these	             of	active	defense	against	infections	in	neonate	piglets.
     pathogens.	We	have	shown	that	purified	fimbriae	from	F4+ETEC	can	
                                                                                            Key words:	Colostrum,	PCV2,	cellular	immune	response,	inactivated	
     induce	a	protective	F4-specific	intestinal	immune	response	against	an	
                                                                                            vaccine,	pig
     oral	challenge	with	F4+ETEC	when	given	oral	to	F4	receptor-positive	
                                                                                            Species:	swine
     (F4R+)	 piglets.	 Adhesion	 to	 the	 F4R	 is	 necessary	 for	 this	 mucosal	
     immunogenicity.	 Binding	 of	 other	 soluble	 antigens	 such	 as	 F18	 fim-
     briae	 to	 enterocytes	 does	 not	 lead	 to	 a	 mucosal	 immune	 response,	             sm112. CPG ODN ACTIVITy IN SWINE: FROM Ex VIVO TO
     so	 F4	 must	 have	 interesting	 properties	 that	 can	 explain	 its	 mucosal	                          IN VIVO ACTIVITy
     immunogenicity.	 One	 of	 the	 important	 differences	 lays	 in	 their	 struc-         A	GoUBIER1,	F	PIRAS2,	H	EL	GARCH,	S	RICHARD1,L	FoREST1,	
     ture.	F4	fimbriae	are	mainly	composed	of	adhesive	FaeG	subunits	and	                   C	AnDREonI1,	JC	AUDonnET1,	R	noRDGREn3,V	JUILLARD1*
     some	less	frequent	minor	subunits,	whereas	in	F18	the	adhesin	Fed	
                                                                                            1Merial	S.A.S.,	Discovery	Research,	254	rue	Marcel	Mérieux,	69342	
     F	 is	 only	 present	 at	 the	 tip	 of	 the	 fimbriae	 and	 the	 major	 subunit	 is	
                                                                                             Cedex	07	Lyon,	France;	2sanofi	pasteur,	Research,	1541	avenue	
     non-adhesive.		Furthermore,	we	demonstrated	that	F4	forms	a	stable	
                                                                                            Marcel	Mérieux,	69280,	Marcy	l’Etoile,	France;	3Merial	Limited,	3239	
     polymeric	structure,	whereas	FedF	in	F18	has	a	weak	interaction	with	
     the	major	subunit	FedA.	To	analyse	the	importance	of	these	observa-                                      Satellite	Blvd.,	Duluth,	GA,	USA
     tions,	two	point	mutations	were	made	in	FaeG	resulting	in	a	reduced	                         CpG	ODNs	signal	through	TLR9	and	trigger	a	cascade	of	events	
     stability	and	polymeric	nature	of	F4	fimbriae	but	retaining	its	capacity	              that	lead	to	activation	of	innate	and	adaptive	immune	responses.	The	
     to	bind	to	the	F4R.	Comparison	of	wild	type	F4	(wtF4)	and	mutant	F4	                   post-TLR9	 signalling	 pathways	 and	 the	 biological	 activities	 resulting	
     (mF4)	in	an	oral	immunization	 experiment	in	piglets	showed	that	the	                  from	the	activation	depend	on	the	class	of	CpG	ODN	used.	Interestingly,	
     induction	of	an	intestinal	F4-specific	immune	response	is	more	effec-                  it	has	been	shown	that	synthetic	CpG	ODNs	may	enhance	the	quality	
     tive	with	wtF4	than	with	the	mF4.	with	higher	numbers	of	F4-specific	                  of	 the	 immune	 response	 to	 vaccination,	 an	 effect	 that	 could	 be	 rein-
     IgA	 antibody	 secreting	 cells	 in	 the	 jejunal	 and	 ileal	 Peyer’s	 patches,	      forced	by	using	appropriate	formulation	agents.	
     mesenteric	lymph	nodes	and	lamina	propria	of	wtF4	(means	of	20,	9,	                         This	 study	 aimed	 at	 selecting	 an	 effective	 combination	 of	 CpG	
     27	and	9	per	5.106	monomorphonuclear	cells	respectively)	than	of	mF4	                  ODN	and	formulation	agents	which	could	be	used	to	enhance	the	por-
     immunized	animals	(3,	4,	16	and	4	respectively).	These	results	show	                   cine	immune	response	to	vaccination.
     that	the	stable	polymeric	appearance	of	the	F4	adhesines	is	important	
     for	its	mucosal	immunogenicity.                                                              Different	 CpG	 ODN	 sequences	 were	 analyzed	 for	 their	 capacity	
                                                                                            to	activate	porcine	PBMCs	ex	vivo,	especially	their	induction	of	IFN-a	
     Key words:	 enterotoxigenic	 E.	 coli,	 F4	 fimbriae,	 oral	 immunisation,	
                                                                                            secretion	and	promotion	of	B	cell	proliferation.	Two	sequences	showing	
     intestinal	immunity
                                                                                            promising	features	were	retained	and	assessed	in	vivo	in	combination	
     Species:	swine
                                                                                            with	two	different	oil-in-water	emulsions.	Various	systemic	parameters	
                                                                                            were	 followed	 up	 at	 different	 times	 after	 injection,	 and	 the	 results	
        sm111. COLOSTRUM FROM SOWS VACCINATED WITH                                          were	analyzed	as	a	combination	of	linear	parameters	in	a	discrimina-
         AN INACTIVATED PCV2 VACCINE CONTAINS ANTIGEN                                       tory	 analysis.	 The	 results	 of	 that	 analysis	 allowed	 us	 to	 discriminate	
                     SPECIFIC LEUKOCyTES                                                    both	 sequences	 and	 to	 select	 one	 of	 the	 emulsions	 for	 its	 ability	 to	
      A	GoUBIER1,	F	PIRAS2,	M	GnUDI3,	L	CHAPAT1,	H	EL	GARCH1,	                              increase	 the	 biological	 activity	 of	 the	 CpG	 ODNs	 tested.	 The	 more	
         F	JoISEL3,	C	CHARREyRE4,	S	RICHARD1,	L	FoREST1,	C	                                 potent	 sequence,	 in	 combination	 with	 the	 oil	 emulsion	 showing	 best	
                     AnDREonI1	AnD	V	JUILLARD1                                              effects,	was	further	assessed	as	adjuvant	of	a	recombinant	protein	in	
     1Merial	S.A.S.,	Discovery	Research,	254	rue	Marcel	Mérieux,	69342	                     pigs.	 We	 showed	 that	 CpG	 ODN	 enhanced	 IFN-γ+	 antigen-specific	T	
      Cedex	07	Lyon,	France;	2Sanofi	pasteur,	Research,	1541	avenue	                        cell	 responses.	 Interestingly,	 analysis	 of	 cellular	 immune	 responses	
       Marcel	Merieux,	69280,	Marcy	l’Etoile;	3Merial	S.A.S.,	Technical	                    after	re-stimulation	with	an	overlapping	peptide	library	clearly	showed	
      Support,	254	rue	Marcel	Mérieux,	69342	Cedex	07	Lyon,	France,	                        that	CpG	ODN	increased	the	number	of	T	cell	epitopes	recognized	by	
     4Merial	S.A.S.,	R&D,	254	rue	Marcel	Mérieux,	69342	Cedex	07	Lyon,	                     antigen	 specific-T	 cells.	This	 CpG-mediated	T	 cell	 epitope	 spreading	
                                   France                                                   emphasizes	that	CpG	ODN	not	only	improve	the	magnitude	of	the	cel-
                                                                                            lular	immune	response	but	also	impact	on	the	quality	of	this	response.	
           In	 species	 having	 an	 epitheliochorial	 placenta,	 like	 swine,	 it	 has	
                                                                                            Collectively,	our	data	suggest	that	CpG	ODN	could	be	employed	as	an	
     been	 largely	 demonstrated	 that	 colostrum	 from	 vaccinated	 mothers	
                                                                                            effective	immuno-adjuvant	to	optimize	immune	responses	induced	by	
     contains	 antigen-specific	 maternal	 immunoglobulins	 which	 have	 an	
     important	role	in	the	passive	protection	of	newborn	animals.	However,	                 vaccination	in	pigs.	
     it	has	been	demonstrated	that	colostrum	also	contains	T	and	B	lympho-                  Key words:	 CpG,	 swine,	 TLR9,	 IFNγ,	 immunomodulation,	 epitope	
     cytes.	Little	is	known	about	the	functionality	of	these	colostral	leuko-               spreading
     cytes	and	their	importance	in	protection	of	newborns	against	infection.	               Species:	swine
     The	aim	of	this	study	was	to	evaluate	if	sow	vaccination	would	lead	to	
     the	presence	not	only	of	specific	immunoglobulin	but	also	of	specific	                    sm113. A WEST NILE VIRUS (WNV) RECOMBINANT
     immune	cells	in	colostrum.	Two	groups	of	specific	pathogen	free	sows	                    CANARyPOx VIRUS (ALVAC) VACCINE ELICITS WNV
     were	 included	 in	 this	 study.	 	 The	 first	 group	 was	 vaccinated	 with	 an	         SPECIFIC NEUTRALISING ANTIBODIES AND T-CELL
     inactivated	PCV2	vaccine,	whereas	the	other	group	did	not	receive	the	
                                                                                            MEDIATED IMMUNE RESPONSES IN THE HORSE: ABSENCE
     vaccine.	As	expected,	PCV2-specific	IgG1	and	IgG2	antibodies	were	
     detected	in	colostrum	of	vaccinated	sows	by	ELISA.	Interestingly,	the	
                                                                                              OF INHIBITING ANTI-VECTOR IMMUNITy FOLLOWING
     IgG1/IgG2	ratio	differed	between	blood	and	colostrum	immunoglobulins	                                 REPEATED INJECTIONS
     with	a	lower	ratio	in	the	colostrum	than	in	the	blood.	Phenotypic	analysis	                H	EL	GARCH1,	JM	MInKE1,	J	REHDER2,	S	RICHARD1,	
     of	colostral	leukocytes	demonstrated	an	increase	in	CD8+	T	cells	and	a	                 CToULEMonDE3,	SDInIC3,	C	AnDREonI1,	JC	AUDonnET1,	R	
     diminution	of	CD4+	T	cells	in	vaccinated	sows’	colostrum	as	compared	                                noRDGREn4,V	JUILLARD1*
                                                                                                                                                                        91


 1Merial	SAS,	Discovery	Research,	254	rue	Marcel	Mérieux,	69342	                    Key words:	 FIV	 Gut-associated	 lymphoid	 tissue,	 CXCR4,	 CD134,	
Cedex	07	Lyon,	France,	2Merial	Limited,	115	Transtech	Drive,	Athens,	               CD134+	B	cells,	CD4+CD134+	T	cells
  GA,	USA,	3Merial	SAS,	Clinical	Operations	Saint	Vulbas,	France,	                  Species:	Feline
       4Merial	Limited,	3239	Satellite	Blvd.,	Duluth,	GA,	USA
      Successful	vaccination	against	West	Nile	Virus	(WNV)	is	thought	               sm115. CHARACTERIZATION OF INFLAMMATORy CELLS
to	require	induction	of	both	neutralizing	antibodies	and	cell-mediated	               POPULATION IN MAMMARy TUMOR IN FEMALE DOGS
immune	 responses.	 In	 this	 study,	 we	 have	 assessed	 the	 ability	 of	 a	         AnA	CARoLInA	TRoMPIERI	SILVEIRA1,	AnTonIo	CARLoS	
recombinant	 ALVAC®-WNV	 vaccine	 (RECOMBITEK	 WNV)	 to	 elicit	                       ALESSI1,	RoSAnGELA	ZACARIAS	MACHADo1,	FELIPE	
neutralizing	 antibodies	 and	 virus	 specific	 T	 cell-mediated	 immune	                           AUGUSTo	RUIZ	SUEIRo2
responses	in	horses.	In	addition,	we	examined	whether	prior	exposure	
to	ALVAC®-WNV	 vaccine	 would	 inhibit	 B	 and	 T	 cell	 specific	 immune	           1Universidade	Estadual	Paulista	(UNESP),	Jaboticabal,	SP,	Brazil,				
responses	 against	 the	 transgene	 product	 upon	 subsequent	 booster	                  2Universidade	de	Franca	(UNIFRAN),	Franca,	SP,	Brazil	
immunizations	with	the	same	vaccine.	The	results	demonstrated	that	                                    actrompieri@yahoo.com.br
the	 recombinant	 ALVAC-WNV	 vaccine	 induced	 neutralising	 antibod-                      Mammary	neoplasias	are	the	most	frequently	observed	tumor	type	
ies	 and	 prM/E	 insert-specific	 IFNγ+	T	 cell	 responses	 against	 WNV	 in	       in	 the	 female	 dogs.	 The	 incidence	 is	 threefold	 higher	 in	 comparison	
vaccinated	 horses.	 Prior	 exposure	 to	 ALVAC®-WNV	 vaccine	 did	 not	            to	woman	breast	tumors.	Canine	mammary	neoplasias	are	adequate	
impair	 the	 ability	 of	 horses	 to	 respond	 to	 two	 subsequent	 booster	        models	 for	 biological	 and	 therapeutic	 studies.	 Studies	 evaluating	 the	
injections	 with	 the	 same	 vaccine,	 although	 anti-vector-specific	T	 and	       spontaneous	 tumor	 type	 in	 domestic	 animals	 have	 been	 considered	
B	 cell	 responses	 were	 induced	 in	 vaccinated	 horses.	 We	 showed	 in	         a	 good	 choice	 to	 elucidate	 important	 issues	 related	 to	 oncology	 and	
this	study	that	ALVAC	vector	allows	for	a	strong	immunogenicity	of	the	             probably	may	be	useful	on	studies	approaching	human	neoplasia.	Data	
recombinant	antigen	in	the	absence	of	an	inhibitory	anti-vector	immune	             obtained	from	human	tumor	have	suggested	that	relationship	between	
response	even	after	multiple	injections.	In	this	regard,	ALVAC	vectors	             tumor	and	host	cells	may	stimulate	and	also	inhibit	anti-tumor	immu-
successfully	combine	the	safety	of	an	inactivated	vaccine	with	the	effi-            nity,	 according	 to	 heterogeneous	 cell	 infiltrate	 composition,	 and	 then	
cacy	of	a	modified	live	vaccine.                                                    different	tumor	cells	interaction,	which	in	turn	may	lead	to	variable	prog-
                                                                                    nosis.	Specimens	used	in	this	study	were	obtained	from	1995	to	2006	
                                                                                    from		the	Histopathology	Laboratory	files	at	the	Veterinary	Pathology	
sm114. CD134+ T AND B LyMPHOCyTES FROM EFFECTOR                                     Department.	Such	samples	were	divided	into	three	groups:	malignant	
   SITES OF THE UPPER GASTROINTESTINAL TRACT                                        mixed	 type	 tumor,	 adenocarcinoma	 and	 solid	 carcinoma.	 The	 immu-
 CONSTITUTE A SITE OF ACTIVE REPLICATION OF FIV IN                                  nohistochemical	 stainings	 were	 performed	 using	 the	 avidin-biotin-
           CHRONICALLy INFECTED CATS                                                peroxidase	complex	method	(ABC).	Cellular	infiltrate	characterization	
    H	EL	GARCH1,	A	GoUBIER1,	H	PoULET1,	y	MUELLE1,	S	                               was	performed	by	using	monoclonal	antibodies:	T	CD3,	B	CD79a	and	
RICHARD1,	L	FoREST1,	L	CHAPAT1,	C	AnDREonI1,V	JUILLARD1                             HLA-DR,	all	antibodies	were	produced	in	mouse	against	human	anti-
1Merial	S.A.S.,	Discovery	Research,	254	rue	Marcel	Mérieux,	69342	                  gen,	and	also	the	TGF	-β	cytokine	polyclonal	antibody	were	used.	The	
                      Cedex	07	Lyon,	France                                         anti-CD4	and	anti-CD8,	T	helper	and	T	citotoxic	lymphocyte	and	prog-
                                                                                    nostic	markers	Ki-67,	Estrogen	receptor	(ER),	Progesterone	receptor	
      Feline	Immunodeficiency	Virus	(FIV)	is	a	major	pathogen	of	cats,	             (PR)	and	p53	are	being	standardized.	First	results	had	demonstrated	
responsible	 for	 an	 acquired	 immuno-deficiency	 syndrome	 (AIDS),	               that	cellular	inflammatory	infiltrate	had	positive	cells	marked	with	anti-
comparable	 to	 Human	 Immunodeficiency	 Virus	 (HIV)-associated	                   CD3,	anti-CD79a,	anti-HLA-DR	and	anti-TGF-β	at	different	proportions	
AIDS	in	humans.	Therefore,	not	only	is	FIV	a	major	issue	in	veterinary	             on	 different	 tumors	 studied.	 Intense	 staining	 were	 observed	 on	 cells	
medicine,	but	it	also	provides	a	natural	model	to	study	lentivirus	patho-           marked	 by	 CD3	 and	 HLA-DR,	 but	 weak	 staining	 were	 observed	 on	
genesis.	Entry	of	HIV	and	FIV	in	host	cells	follows	a	two-step	model	               cells	 marked	 by	 TGF-β	 and	 CD79a.	 Inflammatory	 cells	 distribution	
in	 which	 binding	 to	 a	 primary	 receptor	 induces	 conformational	 rear-        were	interstitial	predominantly.
rangements	 exposing	 the	 co-receptor	 binding	 site.	 Both	 viruses	 use	
CXCR4	as	a	co-receptor	but	differ	in	their	primary	receptor	(CD4	for	               Key words:	 Immunohistochemistry,	 Mammary	 neoplasias,	 dog,	
HIV	and	CD134	for	FIV)	and	in	their	tropism,	restricted	to	CD4+	T	cells	            antibody
for	HIV	and	broader	for	FIV.	Gut-associated	lymphoid	tissue	(GALT)	is	              Species:	canine
a	reservoir	for	the	virus	and	a	site	of	CD4+	T	cells	depletion	in	chroni-
cally	HIV-infected	humans,	but	its	role	in	FIV	pathogenesis	needs	to	be	            sm116. IMMUNOHISTOCHEMICAL ExPRESSION OF TGF-β,
investigated	further.	                                                               LyMPHOCyTES T, B, MACROPHAGES, AND MHC CLASS II
     Our	 objectives	 were	 to	 characterize	 FIV-induced	 immune	 disor-               OVER CANINE TRANSMISSIBLE VENERAL TUMOR
der	and	FIV	target	population	in	GALT	of	chronically	infected	cats.	We	                  AnA	CARoLInA	TRoMPIERI	SILVEIRA1,	MIRELA	TInUCCI	
therefore	developed	a	method	to	isolate	GALT	immune	cells	and	ana-                           CoSTA1,	DAnIEL	GERARDI1,	JULIAnA	MoRo1
lyzed	 immunological	 changes	 and	 virus	 multiplication	 through	 detec-           1Universidade	Estadual	Paulista	(UNESP),	Jaboticabal,	SP,	Brazil,	
tion	of	p24.	We	first	observed	a	depletion	of	mucosal	CD4+	T	cells	and	                                actrompieri@yahoo.com.br
B	 cells.	As	 in	 HIV	 infection,	 this	 depletion	 is	 preferentially	 observed	
                                                                                          Canine	 transmissible	 veneral	 tumor	 (TVT)	 is	 a	 experimentally	
in	 GALT	 effector	 sites	 (intestinal	 epithelium	 and	 lamina	 propria),	 as	
                                                                                    transplantable	tumor	and	has	been	used	as	an	experimental	model	of	
compared	 to	 the	 inductive	 compartments	 (mesenteric	 lymph	 nodes	
                                                                                    the	relation	tumor	versus	host.	Naturally,	TVT	is	transmitted	by	sexual	
and	Peyer’s	Patches).	By	focusing	on	CXCR4	and	CD134	expressing	
                                                                                    contact,	through	de	deposit	of	tumoral	cells	on	the	mucosa	scared	during	
cells,	we	observed	a	preferential	depletion	of	mucosal	CD134+	B	cells	
                                                                                    coitus.	This	study	had	the	goal	to	evaluate	the	infiltrating	T	lymphocytes	
and	CD4+CD134+	T	cells,	especially	in	those	co-expressing	CD134	and	
                                                                                    (CD3,	CD4,	CD8),	B	(CD79-a),	macrophages	(MAC-3),	to	the	expres-
CXCR4.	Whether	these	depletions	were	associated	with	the	induction	
                                                                                    sion	 of	 the	 major	 histocompatibility	 complex	 class	 II	 (MHC	 II)	 and	 of	
of	apoptosis	was	not	investigated.	
                                                                                    TGF-β	on	TVT,	by	means	of	immunohistochemistry	(ABC	method).	The	
     To	 further	 characterize	 FIV	 replication	 in	 the	 gut	 of	 chronically	    experimental	groups	were	composed	of	tumors	of	natural	occurrence	
infected	cats,	the	expression	of	p24	was	analyzed	by	flow	cytometry	in	             (Group	1)	(n=8),	and	of	others	from	dog	pups	that	developed	TVT	after	
mucosal	immune	cells.	We	observed	that	p24	positive	cells	were	within	              transplantation	in	the	phases	of	tumor	progression	(n=8)	(Group	2a),	
the	CD134+	cell	population	and	were	preferentially	observed	in	GALT	                latency	(n=8)	(Group	2b),	and	regression	(n=7)	(Group	2c).	The	lym-
effector	compartments,	a	T-cell	rich	site.	                                         phocytes	T	CD3+	prevailed	in	the	progression	and	regression	phases	
     Altogether	our	data	showed	that	mucosal	CD4+	T	cells	and	B	cells	              in	comparison	to	latency	phase	and	natural.	The	lymphocytes	T	CD4+,	
constitute	a	site	of	active	replication	of	the	virus	in	chronically	infected	       CD8+	prevailed	in	the	progression	phase	followed	by	natural	TVT	and	
cats.	 Our	 results	 emphasize	 the	 role	 of	 GALT	 in	 FIV	 pathogenesis,	        showed	a	smaller	expressiveness	for	TVT	in	regression	phase.	MHC	
further	 reinforcing	 the	 homologies	 observed	 between	 FIV	 and	 HIV	            II	prevailed	in	natural	TVT	followed	by	TVT	in	progression	phase	and	a	
infection.                                                                          smaller	concentration	in	the	regression	phase.	Lymphocytes	B	were	in	
92


     greater	amount	in	the	regression	phase,	followed	by	natural	TVT,	and	               (macrophages,	B	cells,	CD4+	and	CD8+	T	cells)	in	infiltrating	the	site	
     with	 little	 expression	 in	 the	 progression	 phase.	 TGF-β	 was	 similarly	      of	vaccination.	
     expressed	in	the	phases	of	the	transplanted	TVT	in	any	phases	of	the	               Key words:	Fowlpox	virus,	recombinant	vaccine
     transplanted	TVT	and	of	the	natural	TVT.	TVT	cells	reacted	to	macro-                Species:	avian
     phage	marker,	TGF-β	and	MHC	II,	however,	they	did	not	react	to	any	of	
     the	leucocyte	markers	used.	The	results	from	this	experiment	showed	
                                                                                         sm118. EFFECT OF TRANSFERRINS ON CHLAMyDOPHILA
     that	there	was	no	prevalence	of	a	specific	cell	type	in	any	of	the	trans-
     planted	 TVT	 development	 phases.	 The	 reactivity	 of	 tumoral	 cells	 to	                            PSITTACI
     histiocytic/macrophagic	markers	(MAC-3)	and	to	MHC	II	corroborates	                 DELPHInE	BEECKMAn	CARoLInE	VAn	DRooGEnBRoECK	AnD	
     the	histiocytic	origin	of	TVT.	The	expression	of	TGF-β	cytokine	by	the	                              DAISY	VANROMPAY	
     tumor	cells	and	leucocytes	suggests	that	this	cytokine	may	be	one	of	                Ghent	University,	Department	of	Molecular	Biotechnology,	Coupure	
     the	responsible	elements	for	the	immunoregulation	observed	in	TVT.	                                   Links	653,	9000	Ghent,	Belgium.	
     Apparently,	the	tumoral	tissue	donor	interferes	on	the	tumor	x	host	rela-                               Daisy.Vanrompay@ugent.be
     tion,	at	least	in	terms	of	the	amount	of	inflammatory	cells	infiltrated	in	
                                                                                               Objectives:	 the	 effect	 of	 ovotransferrin	 (ovoTF),	 human	 lacto-
     the	transplated	TVT.
                                                                                         ferrin	 (hLF)	 and	 bovine	 lactoferrin	 (bLF)	 on	 the	 obligate	 intracellular	
     Key words:	 canine	 transmissible	 veneral	 tumor,	 infiltrating	 T	                Gram-negative	bacterium	Chlamydophila	(Cp.)	psittaci	was	evaluated	
     lymphocytes,	macrophages,	immunohistochemistry	                                     using	African	Green	Monkey	(BGM)	kidney	epithelial	cells	and	chicken	
     Species:	canine                                                                     macrophages	 (HD11	 cells).	 Subsequently,	 transferrins	 were	 used	 to	
                                                                                         prevent	 a	 Cp.	 psittaci	 infection	 in	 specified	 pathogen	 free	 (SPF)	 tur-
        sm117. PERSISTENCE OF RECOMBINANT FOWLPOx                                        keys.	Results:	firstly,	the	effect	of	transferrins	on	extracellular	bacteria	
      VIRUSES IN CHICKEN TISSUES AND THE LOCAL IMMUNE                                    was	evaluated.	Pre-incubation	of	Cp.	psittaci	with	0.5	to	5	mg/ml	ovoTF	
                          RESPONSE                                                       prior	to	infecting	BGM	cells	significantly	lowered	the	infection	rate.	For	
          I	ELDAGHAyES1,2,	L	RoTHWELL2,	M	A	SKInnER3	AnD	P	                              both	lactoferrins,	the	infection	rate	could	only	be	reduced	with	5	mg/ml,	
                               KAISER2                                                   albeit	not	significantly	as	compared	to	the	infection	rate	created	by	the	
                                                                                         untreated	 bacteria.	 Secondly,	 transferrins	 were	 tested	 for	 their	 ability	
      1Department	of	Microbiology	and	Parasitology,	Faculty	of	Veterinary	               to	influence	bacterial	adhesion	and	entry	in	HD11	cells.	Maximal	non-
        Medicine,				Al-Fateh	University,	P.	O.	Box	13662,		Tripoli,	Libya.;	            cytotoxic	and	non-bactericidal	concentrations	of	0.05	mg/ml	ovoTF	and	
       2Institute	for	Animal	Health,	Compton,	Berkshire	RG20	7NN,	U.K.;	                 0.5	mg/ml	hLF	and	bLF	were	used.	Overall,	ovoTF	was	more	effective	
      3Dept	of	Virology,	Faculty	of	Medicine,	Imperial	College	London,	St.	              than	human	and	bovine	LF	in	inhibiting	bacterial	attachment	and	cell	
              Mary’s	Campus,	Norfolk	Place,	London,	W2	1PG,	U.K.	                        entry	and	the	latter	was	accompanied	by	a	dose-dependent	reduction	
                            ibrahim1971@hotmail.com                                      of	 actin	 recruitment	 at	 the	 bacterial	 entry	 site.	 However,	 once	 bacte-
           Fowlpox	virus	(FPV)	has	been	under	development	as	a	recombi-                  ria	 had	 entered	 HD11	 cells,	 transferrins	 had	 apparently	 no	 effect	 on	
     nant	vaccine	vector	for	20	years.	To	date,	surprisingly,	very	few	data	             intracellular	replication.	Thirdly,	ovoTF	being	the	most	effective	anti-Cp.	
     exist	on	the	persistence	of	fowlpox	vaccine	virus	in	chicken	tissues,	or	           psittaci	 transferrin	 in	 vitro,	 was	 evaluated	 for	 protecting	 SPF	 turkeys	
     the	immune	cells	that	respond	to	the	vaccination	at	the	site	of	inocula-            against	an	aerogenic	chlamydial	infection.	Results	of	the	in	vivo	study	
     tion.	Although	both	humoral	and	cellular	mediated	immunity	(CMI)	play	              will	be	presented.	Conclusion:	present	findings	suggest	a	possible	role	
     a	 part	 in	 overall	 immunity	 against	 FPV,	 little	 is	 known	 regarding	 the	   for	transferrins	and	especially	ovoTF,	in	preventing	avian	Cp.	psittaci	
     cell-mediated	immune	responses	to	FPV	infection.                                    infections.	
          The	main	aim	of	this	study	was	to	measure	persistence	of	recom-                Key words:	transferrins,	Chlamydophila	psittaci,	respiratory	disease.
     binant	fowlpox	vaccine	virus	in	skin	tissues	following	vaccination.	The	            Species:	avian
     recombinant	FPVs	did	not	persist	for	long.	Virus	was	detected	in	skin	
     tissue	 after	 vaccination	 at	 very	 high	 concentrations	 2	 days	 post-vac-
     cination	(dpv),	to	a	lesser	degree	at	4	dpv	and	was	almost	cleared	from	
     6	dpv.	We	also	investigated	the	kinetics	of	response	of	immune	cells	
                       5. ANTIGEN PRESENTATION AND DENDRITIC CELLS, EFFECTOR CELLS, B AND T CELLS,
                             NK AND NK T CELLS, IMMUNOREGULATORy CELLS: POSTERS AP119-AP140


  AP119. GENERATION OF B CELLS IN BOVINE FETUSES                                     phenotypes:	B-1a	(CD5+/CD11b+)	(13	cases),	B-1b	(CD5-/CD11b+)	(8	
                    AnnA	EKMAn,AnTTI	IIVAnAInEn	                                     cases)	and	B-2	(conventional	B)	(CD5-/CD11b-)	(8	cases).	The	results	
                                                                                     of	TNF-Rs	expression	were	all	tumor	cells	in	EBL	uniformly	expressed	
   Department	of	Basic	Veterinary	Sciences,	University	of	Helsinki,	                 the	TNF-RII,	but	not	TNF-RI.	
                          Helsinki,	Finland	
                    antti.iivanainen@helsinki.fi                                           TNF-a	 activity	 is	 mediated	 by	 two	 functionally	 different	 cell	 sur-
                                                                                     face	 receptors,	 TNF-RI	 (55kDa)	 and	 TNF-RII	 (75kDa).	 Most	 biologic	
      The	 human	 and	 murine	 bone	 marrow	 generates	 naïve	 B	 cells	             responses	of	TNF-α,	such	as	the	induction	and	suppression	of	apop-
throughout	postnatal	life.	In	many	other	species,	however,	B	cell	ontog-             tosis,	are	considered	to	be	mediated	by	these	two	different	receptors.	
eny	is	apparently	limited	to	the	fetal	period,	founder	B	cells	are	few	in	           In	 this	 study,	 EBL	 tumor	 cells	 expressed	 TNF-RII,	 but	 not	 TNF-RI,	
number	and	the	recombinational	repertoire	is	limited.	At	late	fetal	and	             suggesting	that	TNF-RII	carries	out	important	functions	in	BLV-induced	
early	 postnatal	 age,	 the	 preimmune	 repertoire	 is	 diversified	 through	        leukemogenesis.
post-recombinational	 mechanisms.	 The	 B	 cell	 population	 is	 dramati-
cally	expanded	in	various	gut	associated	lymphoid	tissues,	i.e.	in	avian	                 We	 conclude	 that	 TNF-Rs	 play	 the	 most	 important	 role	 in	 the	
bursa,	rabbit	appendix	and	ruminant	ileal	Peyer’s	patch	(IPP).	Despite	              malignant	proliferation	of	B	cells	and	formation	of	lymphomas	in	EBL.	
the	well	established	role	of	ruminant	IPP	in	the	generation	of	B	cell	mass	          However,	 in	 future	 studies,	 the	 observations	 of	 TNF-a expression	 in	
and	preimmune	repertoire,	very	little	specific	information	is	available	on	          neoplastic	 tissues	 in	 EBL	 and	 TNF-Rs	 expression	 in	 aleukemic	 (AL)	
the	 ontogeny	 of	 ruminant	 B	 cells.	 We	 have	 systematically	 assessed	          and	persistent	lymphocyotsis	(PL)	will	be	necessary	to	clarify	the	pro-
hematopoietic	tissues	of	bovine	fetuses	from	different	developmental	                cess	by	which	EBL	arises	from	BLV	infection.
stages	 for	 signs	 of	 B	 lymphopoiesis.	 The	 material	 consisted	 of	 29	         Key words:	Tumor	Necrosis	Factor	Receptor	I,	Tumor	Necrosis	Factor	
fetuses	ranging	from	85	to	280	days	of	embryonic	development.	By	a	                  Receptor	II,	enzootic	bovine	leukosis,	cell	apoptosis
combination	of	immunohistochemistry	and	quantitative	RT-PCR,	a	set	                  Species:	Ruminants
of	markers	characterizing	B	cell	differentiation	was	analysed.	Surface	
IgM	positive	immature	B	cells	were	detected	during	the	third	trimerster	                  AP121. IDENTIFICATION OF BOVINE CyTOTOxIC T
in	 spleen	 and	 in	 lymph	 nodes.	 CD21-positive,	 CD79a-positive	 and	                  LyMPHOCyTE EPITOPES ON THE INTRACELLULAR
surface	 IgM-low	 or	 IgM-negative	 serial	 sections	 in	 spleen	 and	 lymph	                      PARASITE THEILERIA PARVA
nodes	suggest	the	presence	of	pre-B	cells	during	the	second	trimester.	                    SIMon	P	GRAHAM1,	RoGER	PELLé1,	ETIEnnE	P	DE	
Quantitation	 of	 RAG-1	 and	 RAG-2	 expression	 revealed	 low	 levels	 of	           VILLIERS1,	MAT	yAMAGE1,	DUnCAn	M	MWAnGI1,	yoSHIKAZU	
recombination	activation	gene	activity	in	spleen,	lymph	nodes	and	the	                  HonDA1,	SHIRLEy	A	ELLIS2,	nIALL	D	MACHUGH3,	W	IVAn	
bone	marrow	but	not	in	the	terminal	small	intestine.	Even	though	we	                              MoRRISon3,EVAnS	L	n	TARACHA1	
did	not	purify	the	IPP	in	this	work,	the	results	suggest	that	massive	B	
                                                                                     1International	Livestock	Research	Institute,	P.	O.	Box	30709,	Nairobi	
lymphoid	differentiation	characterized	by	RAG	mediated	immunoglobu-
                                                                                       00100,	Kenya;2Immunology	Division,	Institute	for	Animal	Health,	
lin	 gene	 recombination	 does	 not	 take	 place	 in	 fetal	 bovine	 IPP.	 It	 is	
                                                                                        Compton,	RG20	7NN,	UK;	3Department	for	Animal	Health	and	
possible,	however,	that	low	levels	of	B	lymphoid	differentiation	occurs	
                                                                                       Welfare,	Royal	(Dick)	School	of	Veterinary	Studies,	University	of	
in	fetal	spleen,	bone	marrow	and	lymph	nodes.	
                                                                                                Edinburgh,	Easter	Bush,	Roslin,	EH25	9RG,	UK	
Key words:	B	cells,	ontogeny,	CD79a,	CD21,	RAG-1,	RAG-2                                                 s.graham@vla.defra.gsi.gov.uk
Species:	ruminants
                                                                                            Immunity	 against	 the	 bovine	 intracellular	 protozoan	 parasite	
    AP120. TUMOR NECROSIS FACTOR RECEPTORS ON                                        Theileria	parva	has	been	shown	to	be	mediated	through	the	destruc-
                                                                                     tion	of	parasite	infected	lymphocytes	by	MHC	class	I	restricted	CD8+	
    LyMPHOMA CELLS IN ENZOOTIC BOVINE LEUKOSIS
                                                                                     CTL.	Six	parasite	proteins	targeted	by	CTL	from	T.	parva	immune	cattle	
   KoSUKE	oKADA1,2,MAnABU	IKEDA1,2,	SAToRU	KonnAI3,	                                 have	been	identified	and	raised	the	prospect	of	a	subunit	vaccine.	In	
 MISAo	onUMA3,	nAoTAKA	ISHIGURo1,MASAnoBU	GoRyo1,2	                                  this	study,	we	identified	nine	CTL	antigenic	peptides	on	these	six	pro-
1Department	of	Pathogenic	Veterinary	Science,	The	United	Graduate	                   teins	with	minimal	peptide	lengths	of	between	9	to11	amino	acids	long.	
   School	of	Veterinary	Sciences,	Gifu	University;2Department	of	                    The	bovine	MHC	(BoLA)	class	I	alleles	that	restrict	these	epitopes	were	
  Veterinary	Pathology,	Faculty	of	Agriculture,	Iwate	University,	and	               identified	 by	 functional	 screening	 of	 BoLA	 class	 I	 cDNA	 clones	 and	
 3Laboratory	of	Infectious	Diseases,	Department	of	Disease	Control,	                 showed	that	individual	alleles	restricted	five	of	these	peptides	and	two	
    Graduate	School	of	Veterinary	Medicine,	Hokkaido	University                      additional	alleles	were	each	found	to	restrict	two	peptides.	These	data	
      Enzootic	 bovine	 leukosis	 (EBL)	 is	 a	 complex	 disease	 of	 cattle	        were	used	to	evaluate	the	ability	of	available	bioinformatics	software	
associated	 with	 B	 lymphocytotropic	 retrovirus,	 bovine	 leukemia	 virus	         to	successfully	identify	bovine	CTL	epitopes.	A	high	degree	of	agree-
(BLV).	Since	the	disease	takes	a	long	period	to	develop,	it	is	believed	             ment	was	observed	between	the	positions	of	epitopes	predicted	to	bind	
that	BLV	and	the	host	immune	responses	are	closely	related.	A	recent	                human	or	mouse	MHC	class	I	molecules	and	those	actually	recognized	
study	on	the	relationship	between	tumor	necrosis	factor	(TNF)	and	BLV	               by	 bovine	 T	 cells.	 These	 results	 support	 the	 application	 of	 available	
infection	suggested	that	TNF	is	a	key	factor	in	the	disease	progression.	            software	 to	 assist	 in	 the	 screening	 of	 CD8+	 T	 cell	 epitopes	 recog-
The	purpose	of	this	study	was	to	report	the	expressions	of	TNF	recep-                nised	 by	 veterinary	 species,	 although	 the	 efficiency	 of	 this	 process	
tors	(TNF-Rs)	on	tumor	cells	obtained	from	cattle	with	EBL,	discuss	the	             will	 undoubtedly	 be	 improved	 as	 more	 species-specific	 MHC-binding	
relationship	among	TNF-Rs	expression,	phenotype	of	tumor	cells	and	                  motifs	are	developed.	
cellular	morphology,	and	attempt	to	clarify	the	pathogenesis	of	EBL.                 Key words:	Theileria	parva	,	CTL	antigenic	peptides
      Obtained	 lymphomas	 in	 29	 animals	 with	 EBL	 were	 histopatho-             Species:	ruminants
logically	 classified	 into	 three	 types:	 Diffuse	 mixed	 type	 (10	 cases)	 ,	
diffuse	large	type	(9	cases)	,	and	diffuse	large	cleaved	type	(10	cases).	               AP122. IDENTIFICATION OF CIRCULATING LINEAGE-
Immunohistochemically	using	a	monoclonal	antibody	to	a	bovine	lym-                        NEGATIVE TyPE-I IFN PRODUCING PLASMACyTOID
phocyte	 surface	 antigen,	 the	 lymphomas	 were	 classified	 into	 three	               DENDRITIC CELL-LIKE CELLS IN THE BOVINE BLOOD
94


     A	STALKER1,	J	BRoWnLIE1,	S	MIAH3,	P	GRIEBEL2,	D	WERLInG1                               AP124. CHARACTERIZATION OF BOVINE REGULATORy
          1Royal	Veterinary	College,	Dept.	of	Pathology	and	Infectious	                                        CELLS
        Diseases,	Hawkshead	Lane,	Hatfield,	UK;	2Veterinary	Infectious	                    AAD	HoEK*1,	VICToR	RUTTEn1,	JoLAnDA	KooL1,	ILDIKo	VAn	
            Disease	Organization	(VIDO),	120-Veterinary	Road,	U.	of	                                        RHIJn1,AnD	KoETS2
       Saskatchewan,	Saskatoon,	Canada;	3University	College	London,	                         1Departments	of	Infectious	Diseases	and	Immunology	and	Farm	
      Institute	of	Orthopaedics	&	Musculoskeletal	Science,	Stanmore,	UK                     Animal	Health;	2Faculty	of	Veterinary	Medicine,	Utrecht	University,	
            The	 clearance	 of	 a	 viral	 infection	 by	 the	 host	 often	 depends	 on	                             The	Netherlands.		
     the	host-ability	to	mount	an	interferon	response.	Thus,	several	viruses	                                       a.hoek@vet.uu.nl
     have	evolved	to	interfere	with	this	early	innate	immune	response.	Over	                     Regulatory	 cells,	 especially	 the	 natural	 regulatory	 T	 cell	 (CD4+/
     the	recent	years,	it	has	become	evident	that	natural	interferon	produc-              CD25+High),	 are	 regarded	 as	 essential	 for	 controlling	 the	 immune	
     ing	cells/plasmacytoid	dendritic	cells	(pDC)	are	in	the	centre	of	inducing	          reactivity.	 Cows	 suffering	 from	 clinical	 paratuberculosis,	 an	 intestinal	
     such	a	type	I	interferon	response.	However,	this	cell	type	has	not	yet	              inflammatory	disease	caused	by	infection	with	Mycobacterium	avium	
     been	defined	in	the	bovine	system.	A	subset	of	immune	cells	potentially	             subspecies	 paratuberculosis	 (MAP)	 suffer	 from	 a	 loss	 of	T	 cell	 func-
     resembling	bovine	pDC	have	been	isolated	through	negative	selection	                 tion,	and	show	increased	IL10	production,	which	may	indicate	a	role	
     from	whole	blood.	These	cells	were	subsequently	characterised	for	sur-               for	 (increased)	 regulatory	 T	 cell	 activity	 	 in	 the	 pathogenesis	 during	
     face	antigen	expression	and	type-I	IFN	production.	These	cells	express	              progressive	stages	of	disease.
     do	not	express	lineage-specific	surface	antigen,	but	do	express	mark-                       To	enable	identification	of	regulatory	(T)	cells,	cell	(sub)populations	
     ers	expressed	by	myeloid	as	well	lymphoid	cells,	as	similarly	described	             were	 isolated	 from	 peripheral	 blood	 and	 lamina	 propria	 cell	 suspen-
     for	pDC	in	the	porcine,	human	and	murine	system.	In	addition,	activa-                sions	 of	 cows	 with	 known	 paratuberculosis	 status	 by	 flowcytometric	
     tion	 of	 these	 cells	 by	 type	A	 CpG	 ODN,	 but	 not	 type	 B	 or	 poly(I:C)	     sorting.	These	subpopulations	were	characterized	by	quantitative	real	
     induced	 a	 cell-specific	 IFNαβ	 response.	 In	 addition,	 these	 cells	 had	       time	 PCR	 (qRT-PCR),	 intracellular	 staining	 (ICS)	 specific	 for	 bovine	
     mRNA	for	TLRs	normally	present	with	pDC	of	other	species.	SEM	and	                   Foxp3,	 IL-10,	 TGF-β	 and	 IFN-γ.	 In	 addition	 IL-10,	 and	 IFN-γ	 Elispot	
     TEM	of	these	cells	revealed	a	size	similar	to	lymphocytes	with	small	                assays	 were	 performed.	 Lymfocyte	 stimulation	 assays	 (LST)	 and	 in	
     dendrites	protruding	from	the	surface.	Thus,	our	data	describe	for	the	              vitro	co-culture	assays	(IVCA)	in	combination	with	PBMC	depleted	for	
     first	time	the	presence	of	a	circulating	cell-type	with	bovine	peripheral	           specific	subpopulations,	blocking	by	mAb	neutralizing	bovine	IL-10	or	
     blood	potential	resembling	plasmacytoid	dendritic	cells.                             γd	 TCR	 and	 CFSE	 staining	 were	 applied	 to	 test	 	 cellular	 functional-
                                                                                          ity	 of	 the	 isolated	 populations.	 Results	 of	 qRT-PCR,	 Elispot	 and	 ICS	
     Key words:	plasmacytoid	dendritic	cells,		natural	interferon
                                                                                          assays	performed	on		purified	bovine	cell	(sub)populations	measuring	
     Species:	ruminants
                                                                                          bovine	Foxp3,				IL-10,	TGF-β	and	IFN-γ	mRNA/protein,	were	used	to	
                                                                                          classify	 potential	 regulatory	 cells.	These	 showed	 to	 be	 CD3+	T	 cells,	
       AP123. TyPE I INTERFERON PRODUCING CELLS (IPC)
                                                                                          CD4+/CD25+High/Low	 T	 cells,	 CD14+	 monocytes	 and	 γd	 T	 cell	 subsets.	
      CAN GAIN LyMPH NODES VIA THE AFFERENT LyMPHATIC                                     Intracellular	 staining	 by	 cross	 reactive	 anti-Foxp3	 mAb	 in	 cow	 CD4+/
                            ROUTE                                                         CD25+High	 cells	 was	 found.	 Cow	 CD4+/CD25+High	 T	 cells	 isolated	 from	
          FLoREnTInA	PASCALE1	,	VAnESSA	ConTRERAS1	,	                                     peripheral	blood	or	lamina	propria	showed	no	suppressive	activity	in	a	
        ALExAnDRE	CoURBET1	,	MICHEL	BonnEAU2	,	STEFAn	                                    bovine	in	vitro	co-culture	assay,	while		human	CD4+/CD25+High	T	cells	
          CHILMonCZyK1	,	MATHIEU	EPARDAUD1	,	CLAUDIA	                                     tested	in	a	parallel	in	vitro	co-culture	assay	displayed	strong	functional	
           BEVILACqUA3	,	AnnE-MARIE	BALAZUC4	,	ARTUR	                                     suppression	in	a	dose	dependent	manner.	
     SUMMERFIELD5,	BéATRICE	RITEAU1,	JAynE	HoPE6	,	BERnARD	                                    Based	 on	 phenotyping	 and	 Foxp3	 measurements	 bovine	 CD4+/
          CHARLEy1*,	AnD	ISABELLE	SCHWARTZ-CoRnIL1*                                       CD25+High	 population	 was	 considered	 comparable	 to	 human	 CD4+/
     1Virologie	et	Immunologie	Moléculaires,	UR892	INRA,	Domaine	de	                      CD25+High	natural	Treg,	however	the	bovine	cells	did	not	display	func-
     Vilvert,	78352	Jouy-en-Josas	Cedex,	France		2Centre	de	Recherche	                    tional	suppression	in	an	in	vitro	co-culture	assay.	Furthermore	based	
      en	Imagerie	Interventionnelle,	Domaine	de	Vilvert,	78352	Jouy-en-                   on	IL-10	measurements	cow	CD14+	(monocytes)	and	γd	T	cell	subsets	
      Josas	Cedex,	France		3PICT,	Domaine	de	Vilvert,	78352	Jouy-en-                      could	be	classified	as	potential	regulatory	cells	and	preliminary	in	vitro	
      Josas	Cedex,	France		4Plate-forme	de	cytométrie,	Institut	Pasteur,	                 co-culture	 assays	 showed	 indications	 for	 functional	 suppression	 by	
      Paris		5Institute	of	Virology	and	Immunoprophylaxis,	Mittelhausern,	                these	cells	indicating	that	immune	regulation	in	cattle	may	be	of	a	dif-
     Switzerland		6Institute	for	Animal	Health,	Compton,	United	Kingdom	                  ferent	nature	as	compared	to	human	and	rodent	systems.
                         *	isabelle.schwartz@jouy.inra.fr	                                    This	 investigation	 received	 financial	 support	 from	 the	 Dutch	
                                                                                          Technology	Foundation	STW/NWO.			
           	While	most	dendritic	cells	(DC)	enter	lymph	nodes	(LN)	by	migrat-
     ing	from	peripheral	tissues,	plasmacytoid	DC	(pDC),	also	referred	as	                Key words:	 T	 regulatory	 cells,	 Foxp3,	 IL-10,	 TGF-β,	 IFN-γ,	 IL-10,	
     interferon	producing	cells	(IPC),	are	know	to	gain	LN	directly	from	the	             IFN-γ
     blood	by	crossing	high	endothelium	venules.	We	demonstrate	here	that	                Species:	ruminants
     bona	fide	IPC	are	migrating	in	afferent	lymph	in	two	large	mammals,	
     sheep	and	mini-pigs.	In	sheep,	lymph	cells	produced	type	I	IFN	/in	vivo/	                 AP125. DRAINING LyMPH NODES APCS IN THE
     after	 type-A	 CpG	 oligonucleotide	 (ODN)	 injection	 and	 /in	 vitro/	 after	        RESISTANCE OF GOATS TO AMBLyOMMA CAJENNENSE
     stimulation	with	type-A	CpG-ODN	and	enveloped	viruses.	Sheep	lymph	                                FABRICIUS 1787 NyMPHS
     IPC	were	found	within	a	non	B	non	classical	DC	(cDC)	minor	subset	(<	                   G	E	R	MonTEIRo1,2,	G	H	BECHARA2,	AM	FRAnZIn3,	IK	DE	
     1%)	expressing	CD45RB.	The	CD11cneg	Bneg	CD45RBpos	cells	showed	                                        MIRAnDA	SAnToS3
     high	levels	of	TLR-3,	7	and	9	mRNA,	they	displayed	constitutive	IRF-7	
                                                                                                  1Departamento	de	Paraclínicas,	FV-UEM,	Maputo,	
     mRNA	 expression,	 they	 presented	 a	 plasmacytoid	 morphology,	 they	
                                                                                          Moçambique;2Departamento	de	Patologia	Veterinária,	FCAV-UNESP,	
     induced	IFN-γ	production	in	allogeneic	CD4	T	cells	and	they	differenti-
                                                                                            Jaboticabal-SP,	Brazil;	3Laboratório	de	Imunologia,	FMRP-USP,	
     ated	into	DC-like	cells	under	viral	stimulation.	Furthermore	in	mini-pig,	                                Ribeirão	Preto-SP,	Brazil.
     a	CD4pos	SIRPpos	subset	in	afferent	lymph	cells,	corresponding	to	pDC	
     homologues,	produced	type	I	IFN	after	type-A	CpG-ODN	triggering.                           Preliminary	results	of	the	laboratory	showed	that	goats	acquire	par-
                                                                                          tial	resistance	against	nymphs	of	the	lone-star	tick	Amblyomma	cajen-
          Altogether,	this	work	demonstrates	that	cells	with	IPC/pDC	char-                nense	 after	 repeated	 infestations.	 On	 the	 other	 hand	 it	 is	 well-known	
     acteristics	migrate	in	afferent	lymph,	thus	being	ideally	located	to	pos-            that	antigen-presenting	cells	develop	an	important	role	in	the	immune	
     sibly	 transport	 antigen	 from	 tissue	 and	 rapidly	 interact	 with	 incoming	     reaction	 to	 tick	 infestation.	 The	 most	 potent	 well-defined	 antigen-pre-
     cDC	in	lymph	nodes.                                                                  senting	cells	for	Th	cells	are	dendritic	cells,	mononuclear	phagocytes	
     Key words:	 plasmacytoid	 dendritic	 cells;	 CpG	 oligonucleotide;	 type	            and	B-lymphocytes.	Langerhan	cells	are	one	of	the	first	to	be	exposed	to	
     I	IFN                                                                                tick	immunogens	in	skin	for	which	they	migrate	to	draining	lymph	nodes.	
     Species:	ruminants                                                                   In	the	paracortical	area	of	lymph	nodes	Langerhan	cells	transform	into	
                                                                                                                                                                           95


dendritic	cells	and	function	there	as	antigen-presenting	cells	for	T-cells.	          autologous	T.	annulata-infected	lymphocytes	every	10-14	days.	These	
Immunohistochemistry	analysis	of	draining	lymph	nodes	of	goats	repeat-                cells	have	a	CD2+,	CD3-,	CD8lo,	NKp46+,	CD16+	phenotype	consistent	
edly	infested	with	the	ixodid	tick	A.	cajennense	was	performed	to	search	             with	previous	descriptions	of	bovine	NK	cells.	Initial	observations	indi-
for	antigen-presenting	cells.	Three	goats	aged	six	month	of	both	sexes	               cate	 that	 by	 day	 10	 post-stimulation	 there	 is	 a	 4-10	 fold	 increase	 in	
were	used	throughout	the	experiment.	Two	of	them	were	infested	thrice	                NK	 cell	 numbers.	 Studies	 to	 examine	 the	 mechanisms	 by	 which	 co-
with	 A.	 cajennense	 nymphs	 at	 30	 days	 intervals.	 The	 third	 goat	 was	        culture	with	Theileria-infected	cells	maintains	bovine	NK	proliferation	in	
used	as	control	with	no	infestation.	Pre-scapular	lymph	nodes	draining	               vitro	and	to	define	the	function	of	the	NK	clones	obtained	are	currently	
the	tick	infestation	sites	were	collected	15	days	after	both	the	first	and	           being	undertaken.	The	ability	to	propagate	bovine	NK	clones	in	vitro	for	
the	third	infestations.	Lymph	nodes	of	the	non-infested	goat	were	used	               prolonged	periods	and	achieve	significant	expansion	of	cell	numbers	
as	control.	The	immunohistochemical	analysis	was	performed	in	lymph	                  provides	a	valuable	method	which	will	facilitate	in	depth	characterisa-
nodes	cryostat	sections	by	using	the	ABC	method.	It	was	used	three	                   tion	of	bovine	NK	cells	at	the	clonal	level.
cellular	 surface	 markers,	 Cd11b	 (CC126),	 CD11c	 (CC21)	 and	 CD21	               Key words:	NK,	bovine,	culture
(BAC153A)	for	macrophages,	dendritic	cells	and	B	cells,	respectively.	                Species:	ruminants
Pre-scapular	lymph	nodes	from	tick	infested	goats	showed	increased	
CD11b,	CD11c	and	CD21	cellular	infiltration	when	compared	to	those	
                                                                                      AP128. ExPRESSION OF TOLL-LIKE RECEPTOR 2 (TLR2) IN
from	non-infested	animals.	However,	there	were	no	significant	changes	
in	the	number	of	cells	infiltrating	lymph	nodes	of	goats	infested	once	or	
                                                                                          PORCINE LEUKOCyTE SUBSETS AND TISSUES
thrice.	It	is	concluded	that	antigen-presenting	cells	may	play	an	impor-                    BELEn	ALVAREZ1,	ConCEPCIón	REVILLA1,	nIEVES	
tant	role	in	acquired	resistance	mechanism	of	goats	against	nymphs	of	                   DoMEnECH2,	CARLoS	PEREZ1,	PALoMA	MARTÍnEZ	DE	LA	
the	lone-star	tick	A.	cajennense.                                                        RIVA1,	FERnAnDo	ALonSo1,	AnGEL	EZqUERRA1,	JAVIER	
                                                                                                            DoMÍnGUEZ1
Key words:	 Amblyomma	 cajennense,	 goats,	 lymph	 nodes,	
immunohistochemistry,	APCs.                                                                  1Dpto	de	Biotecnología,	INIA,	Madrid,	Spain;	2Unidad	de	
Species:	ruminants                                                                            Investigación,	Hospital	Juan	Canalejo,	A	Coruña,	Spain	
                                                                                                                  juncal@inia.es
AP126. QUANTIFyING THE CONTRIBUTION OF BOLA-DRβ3                                            Toll-like	receptors	(TLR)	are	a	group	of	pattern	recognition	recep-
TO IMMUNE RESPONSES IN A CATTLE CROSS-POPULATION                                      tors	that	play	a	crucial	role	in	innate	immunity.	TLR2	recognizes	a	variety	
                                                                                      of	 microbial	 components	 including	 bacterial	 lipopeptides,	 lipoteichoic	
REBECCA	BAxTER1,	nICoLA	HASTInGS1,	y	LAW1,ELIZABETH	J	
                                                                                      acid,	lipoarabinomannan,	peptidoglycan	and	zymosan,	leading	to	the	
                      GLASS1
                                                                                      development	of	inflammatory	and	immune	responses.	To	characterize	
             1Roslin	Institute,	Roslin,	Midlothian,	EH54	9PS                          the	 expression	 and	 functional	 properties	 of	 porcine	 TLR2,	 we	 raised	
      One	approach	to	reducing	the	impact	that	pathogens	have	on	live-                a	 panel	 of	 mAb	 against	 this	 molecule.	 Mouse	 3T3	 cell	 transfectants	
stock	health	and	welfare	is	to	breed	for	enhanced	disease	resistance.	            	   expressing	pTLR2	were	used	for	immunization	of	mice.	The	specificity	
Suitable	 selectable	 markers	 may	 be	 found	 in	 genes	 within	 the	 Major	         of	 these	 antibodies	 was	 confirmed	 by	 their	 reactivity	 with	 CHO	 cells	
Histocompatibility	Complex	(MHC),	in	particular	the	highly	polymorphic	               transfected	 with	 pTLR2	 but	 not	 with	 pTLR4	 or	 with	 non-transfected	
MHC	class	II	DR	genes.		The	polymorphisms	reside	in	exon	2	of	the	                    cells.	Using	one	of	these	mAbs,	named	1H11,	TLR2	was	found	on	cells	
drβ3	gene	which	encodes	the	peptide	binding	cleft.		As	the	polymor-                   of	the	innate	immune	system,	including	monocytes,	macrophages	and	
phisms	can	alter	the	conformation	of	the	PBC,	they	affect	the	binding	                granulocytes,	 but	 not	 on	 peripheral	 blood	 lymphocytes.	 	 Staining	 of	
efficacy	of	the	pathogen-derived	peptides,	and	consequently	the	level	of	             tissue	sections	showed	that	TLR2	is	also	expressed	on	epithelial	cells	
the	ensuing	immune	response.		However	other	regions	of	the	genome	                    lining	the	tracheobronchial	and	intestinal	tracts,	bile	ducts	in	the	liver	
are	likely	to	account	for	a	significant	proportion	of	the	variability	seen	           and	renal	tubules	and	on	the	basal	layer	of	the	epidermis.	This	distribu-
in	 disease	 resistance.	 	 In	 order	 to	 assess	 the	 relative	 contribution	 of	   tion	is	consistent	with	a	surveillance	function	at	entry	sites	allowing	for	
the	MHC	to	disease	resistance	and	immune	responsiveness	in	cattle,	                   early	detection	of	microbial	invasion.
we	 have	 measured	 a	 range	 of	 immune	 traits	 in	 a	 500	 F2	 Charolais-          Key words:	Toll-like	receptors,	monoclonal	antibodies
Holstein	cross	population.	We	have	improved	a	sequence	based	typing	                  Species:	swine
(SBT)	method	to	reliably	determine	bovine	MHC	(BoLa)	class	II	drβ3	
exon	2	polymorphisms,	 and	have	typed	all	of	 these	cattle.		Although	
                                                                                       AP129. CD2 AND CD21 ExPRESSION CAN BE USED TO
104	alleles	have	been	identified	for	BoLA,	only	22	different	alleles	were	
present	 in	 this	 population.	 	 Preliminary	 evidence	 from	 117	 animals	
                                                                                      DESCRIBE MATURATION PATHWAy FOR PORCINE B CELLS
has	 identified	 significant	 associations	 between	 some	 of	 the	 immune	               MAREK	ŠInKoRA1,	JAnA	ŠInKoRoVá2,JoHn	E	BUTLER3
traits	and	BoLa	class	II	drβ3	types,	and	further	analysis	is	on-going.	               1Academy	of	Science	of	the	Czech	Republic,	Institute	of	Microbiology,	
This	population	has	also	been	genotyped	for	139	microsatellite	mark-                     Sector	of	Immunology	and	Gnotobiology,	Nový	Hrádek,	Czech	
ers	across	the	genome.		This	information	will	enable	us	to	determine	                 Republic;	2A.R.T.,	Nový	Hrádek,	Czech	Republic;	3University	of	Iowa,	
the	 relative	 importance	 of	 the	 BoLa	 drβ3	 genes	 compared	 to	 other	                      Department	of	Microbiology,	Iowa	City,	IA,	USA,		
regions	of	the	genome	in	determining	immune	responsiveness.                                              marek.sinkora@worldonline.cz
Key words:	BoLA,	disease	resistance                                                          Analysis	 of	 CD2	 and	 CD21	 expression	 on	 porcine	 B	 cells	 dur-
Species:	ruminants                                                                    ing	 ontogeny	 showed	 that	 the	 vast	 majority	 of	 µHC+	 B	 cells	 in	 the	
                                                                                      fetus	are	CD2+CD21+.	This	contrasts	with	the	postnatal	period	where	
AP127. IN VITRo MAINTENANCE OF BOVINE NK CELLS By                                     many	B	cells	from	different	lymphoid	organs	are	CD2—	and/or	CD21—.	
  CULTURE WITH THEILERIA-INFECTED LyMPHOCyTES                                         Moreover,	in	vivo	studies	of	germ-free	piglets	infected	with	PRRSV,	SIV	
                                                                                      or	PCV-2	developed	various	proportions	of	CD2—	and/or	CD21—	B	cells	
  T	ConnELLEy,	n	MACHUGH1,	A	SToRSET2,	WI	MoRRISon1
                                                                                      always	greater	than	sham-infected	controls.	These	findings	prompted	
  1Division	of	Clinical	Veterinary	Sciences,	University	of	Edinburgh,	                in	 vitro	 studies	 using	 different	 CD2/CD21	 subpopulations	 of	 B	 cells	
  Edinburgh,	United-Kingdom;	2Dept.	of	Food	Safety	and	Infection	                     sorted	as	pure	populations	by	flow	cytometry.	These	studies	suggested	
   Biology,	Norwegian	School	of	Veterinary	Science,	Oslo,	Norway.                     that	CD2+CD21+	B	cells	represent	inexperienced	mature	B	cells.	These	
     We	report	a	method	of	generating	and	maintaining	in	vitro	bovine	                may	 proliferate	 and	 down-regulate	 CD21	 expression	 spontaneously	
NK	clones	using	co-culture	of	PBMC	from	naïve	cattle	with	autologous	                 during	culture	or	after	in	vitro	activation.	The	resultant	CD2+CD21—	B	
Theileria-infected	lymphocytes.	Following	depletion	of	CD4+	and	γd T-                 cells	can	subsequently	generate	large	proliferating	μHC—	plasmablasts	
cells	 from	 cultures	 of	 naïve	 PBMC	 stimulated	 with	 either	 T.	 parva	 or	      and	small	non-dividing	µHC—	plasma	cells.	Sorting	experiment	revealed	
T.	annulata-infected	lymphocytes,	NK-enriched	populations	have	been	                  that	once	down-regulated,	CD21	molecule	cannot	be	re-expressed	and	
identified	from	which	individual	NK	clones	could	be	derived.	Nine	NK	                 therefore	 is	 valuable	 as	 a	 developmental	 marker	 for	 B	 cell	 in	 swine.	
clones	generated	from	one	animal	have	been	maintained	in	culture	in	                  On	the	other	hand,	CD2,	which	acts	as	functional	adhesin	during	inter-
IL-2	supplemented	media	for	a	period	of	2	months	by	re-stimulation	with	              cellular	 B	 cells	 interactions,	 is	 down-regulated	 by	 cell-to-cell	 contact.	
96


     Once	 B	 cells	 recover	 from	 such	 intereactions,	 CD2	 expression	 on	 B	          we	identified	a	population	of	CD79a+CD21–	cells	which	accounted	for	
     cells	is	re-established	in	a	short	period	of	time.	During	the	CD2—CD21+	              3	to	8%	of	total	lymphocytes.	With	this	phenotype,	the	cells	resemble	
     stage,	 B	 cells	 cannot	 be	 activated	 and	 cannot	 generate	 proliferating	        early	transitional	(T1)	B	cells	which	should	be	in	regard	to	their	immu-
     µHC—	plasmablast	or	non-dividing	µHC—	plasma	cells.	However,	once	                    noglobulin	class	IgD–	and	IgMhigh.	For	further	studies	on	this	minor	B-cell	
     they	re-express	CD2	and	become	CD2+CD21+	B	cells,	at	least	a	sub-                     subset	we	isolated	CD79a+CD21–	cells	by	fluorescence	activated	cell	
     population	 can	 progress	 to	 the	 CD2+	 CD21—stage	 of	 differentiation.	           sorting	and	performed	reverse	transcription	PCR,	specifically	targeting	
     This	work	was	supported	Grant	524/07/0087	and	523/07/0088	from	the	                   porcine	 IgD	 transcripts.	 Interestingly,	 the	 CD79a+CD21–	 subset	 con-
     Grant	Agency	of	the	Czech	Republic	and	by	Grant	A5020303	from	the	                    tained	cells	positive	for	IgD	transcripts.	Also,	analyses	of	IgM	surface	
     Grant	Agency	of	the	Academy	of	Sciences	of	the	Czech	Republic.                        expression	by	flow	cytometry	indicated	IgM-expressing	B	cells	within	
     Key words:	 B	 cells,	 development,	 maturation,	 differentiation,	                   that	 subpopulation	 with	 moderate	 IgM	 surface	 expression.	 However,	
     ontogeny,	                                                                            there	 was	 no	 difference	 in	 the	 IgM	 expression	 pattern	 between	 the	
     Species:	swine                                                                        two	 CD21-defined	 B	 cell	 subpopulations.	 Further	 analyses	 of	 the	
                                                                                           CD79a+CD21–	 subpopulation	 within	 PBMC	 showed	 that	 these	 cells	
     AP130. THE FINAL CUT: CD1+CD2+CD8— γδ THyMOCyTES                                      were	mainly	negative	for	CD5	but	the	majority	expressed	CD2	(∼	70%)	
       ARE PRECURSORS OF ALL PERIPHERAL γδ T CELLS                                         with	a	high	antigen	density	comparable	to	the	CD2	antigen	expression	
             MAREK	ŠInKoRA1,	JAnA	ŠInKoRoVá2,WoLFGAnG	                                     on	T	lymphocytes.
                            HoLTMEIER3                                                          By	 analysing	 further	 lymphatic	 organs	 of	 adult	 and	 six	 month	
     1Academy	of	Science	of	the	Czech	Republic,	Institute	of	Microbiology,	                old	 swine	 we	 detected	 only	 low	 frequencies	 (3%	 and	 below)	 of	
        Sector	of	Immunology	and	Gnotobiology,	Nový	Hrádek,	Czech	                         CD79+CD21–	cells	in	spleen,	lymph	nodes	and	bone	marrow.	
     Republic;	2A.R.T.,	Nový	Hrádek,	Czech	Republic;	3Johann-Wolfgang	                          Taken	together,	these	data	indicate	that	the	identified	population	
         Goethe	Universität	Frankfurt,	Frankfurt	am	Main,	Germany,		                       of	CD79+CD21–	cells	in	swine	despite	this	phenotype,	which	points	to	
                        marek.sinkora@worldonline.cz                                       an	immature	developmental	stage,	show	a	number	of	phenotypical	dif-
           Using	flow	cytometry	sorting,	in	vitro	cultures,	analysis	of	cell	cycle	        ferences	to	transitional-1	B	cells	described	for	mice.	This	clearly	raises	
     and	lymphocyte-specific	transcripts	and	in	vivo	studies	of	individual	γd	             the	question	of	the	function	of	these	cells	which	will	be	addressed	in	
     thymocyte	 subsets	 during	 ontogeny,	 we	 have	 shown	 that	 CD1	 and	               further	studies.	
     CD45RC	 expression	 on	 porcine	 γd	 thymocytes	 define	 subpopula-                   Key words:	B	cells,	swine,	CD79a,	IgD,	IgM
     tions	 with	 developmentally	 dependent	 changes	 in	 their	 phenotype.	              Species:	swine
     Proliferating	 CD2+CD8—CD1+CD45RC—	 γd	 thymocytes	 are	 the	 com-
     mon	precursor	giving	rise	to	all	γd	T	cells	found	in	the	periphery.	These	
     precursors	differentiate	into	CD2+CD8aa+,	CD2+CD8—	and	CD2—CD8—
                                                                                           AP132. SyNERGISTIC EFFECTS OF IL-2, IL-12 AND IL-18 ON
     γd	 thymocytes,	 which	 subsequently	 mature	 by	 loss	 of	 CD1	 and	 by	             CyTOLITIC ACTIVITy AND IFN-γ PRODUCTION OF PORCINE
     eventual	 gain	 of	 CD45RC	 expression.	 γd	 thymocytes	 that	 lose	 CD1	                            NATURAL KILLER CELLS
     expression	 can	 be	 exported	 to	 the	 periphery	 so	 the	 final	 maturation	           MaŠa	PIntarIČ,	WILHELM	GERnER,	ARMIn	SAALMÜLLER
     step	from	CD1—CD45RC—	into	CD1—CD45RC+	cells	may	occur	there.	
                                                                                               Clinical	Immunology,	University	of	Veterinary	Medicine	Vienna,	
     Extrathymic	maturation	of	CD2+CD8—	into	CD2+CD8aa+T	cell	was	also	
                                                                                                                      Vienna,	Austria	
     shown	to	occur	upon	activation.	However,	the	origin	of	CD2+CD8αα+T-
                                                                                                             armin.saalmueller@vu-wien.ac.at
     cells	can	be	monitored	by	MHC	class	II	expression	since	those	which	
     are	 MHC-II—	 appear	 to	 originate	 in	 the	 thymus,	 while	 MHC-II+	 cells	              Natural	killer	(NK)	cells	are	one	of	the	main	cellular	components	of	
     arise	in	the	periphery	after	stimulation	of	their	CD2+CD8—	counterparts.	             the	innate	immune	system.	They	play	an	important	role	in	the	immune	
     These	 results	 indicate	 that	 the	 maturation	 of	 γd	 thymocytes	 occurs	          response	against	infections	as	well	as	tumour	cells	and	therefore	have	
     after	successful	expression	of	TCRγd	and	that	although	γd	T	cells	do	                 two	major	properties:	production	of	immune	regulatory	cytokines	and	
     not	follow	αβT	cell	progenitors	through	MHC	dependent	positive	and	                   chemokines	as	well	as	cytolytic	destruction	of	particular	target	cells.
     negative	selection,	αβ	and	γdcell	lineages	require	about	the	same	time	                     The	existence	of	NK	cells	in	swine	is	well	known	as	well	as	the	
     for	full	maturation.	Apart	from	the	above	mentioned	CD4—	γd	T	cells,	                 phenotype	 of	 resting	 NK	 cells,	 but	 their	 response	 following	 activa-
     a	small	fraction	of	γd	thymocytes	are	CD4+CD8αβ+CD1+.	These	cells	                    tion	 by	 cytokines	 is	 still	 poorly	 understood.	 Therefore,	 we	 tested	 the	
     have	no	counterpart	in	the	periphery	and	were	shown	to	be	a	transient	                influence	of	the	immune	regulatory	cytokines	IL-2,	IL-12	and	IL-18	on	
     and	independent	population	of	thymocytes	that	are	able	to	extinguish	                 cytolytic	activity,	phenotype,	IFN-γ	production	and	the	accumulation	of	
     their	 TCRγd	 expression	 and	 differentiate	 along	 the	 aβ	 T	 cell	 lineage	
                                                                                           perforin	in	cytoplasm	of	peripheral	blood	mononuclear	cells	(PBMC)	as	
     program.	
                                                                                           well	as	purified	NK	cells.	NK	cells	were	enriched	from	PBMC	using	a	
         This	 work	 was	 supported	 Grant	 524/07/0087	 and	 523/07/0088	                 magnetic	cell	separation	(MACS)	strategy	with	monoclonal	antibodies	
     from	the	Grant	Agency	of	the	Czech	Republic	and	by	Grant	A5020303	                    against	CD3,	CD21	and	SWC3,	thereby	removing	T-,	B-	and	myeloid	
     from	 the	 Grant	 Agency	 of	 the	 Academy	 of	 Sciences	 of	 the	 Czech	             cells.	Respective	fractions	were	used	in	flow	cytometry	(FCM)	based	
     Republic.                                                                             cytolytic	assays	with	the	human	tumour	cell	line	K562	as	target.	After	
     Key words:	 γd	 T	 cells,	 development,	 maturation,	 differentiation,	               stimulation	 with	 the	 cytokines	 described	 above,	 the	 NK	 cell	 enriched	
     ontogeny,	                                                                            CD3–CD21–SWC3–	 fraction	 showed	 an	 evident	 increase	 in	 the	 cyto-
     Species:	swine                                                                        lytic	activity	compared	to	PBMC.	This	enhanced	cytolytic	activity	was	
                                                                                           accompanied	by	a	strong	enrichment	of	IFN-γ	producing	cells	when	a	
     AP131. CHARACTERIZATION OF CD79α+CD21– B CELLS IN                                     combination	of	all	three	cytokines	(IL-2/IL-12/IL-18)	was	used,	as	deter-
                         SWINE                                                             mined	in	ELISPOT	assays	and	intracellular	staining	of	IFN-γ	in	FCM.	
      WILHELM	GERnER,	SABInE	HAMMER,	ToBIAS	KäSER,	nICoLA	                                 Also,	the	combination	of	these	three	cytokines	led	to	an	accumulation	
             DIny,	MAŠA	PInTARIC,	ARMIn	SAALMÜLLER                                         of	perforin	in	the	cytoplasm	and	an	up-regulation	of	CD25	compared	to	
                                                                                           control	cultures	incubated	in	medium	without	cytokines.
         Clinical	Immunology,	University	of	Veterinary	Medicine	Vienna,	
                                Vienna,	Austria	                                                 The	 experiments	 performed	 so	 far	 clearly	 indicate	 a	 stimulatory	
                        wilhelm.gerner@vu-wien.ac.at	                                      role	and	strong	synergistic	effects	of	the	investigated	cytokines	in	the	
                                                                                           activation	of	porcine	NK	cells	in	vitro,	inducing	IFN-γ,	perforin	produc-
          The	identification	of	B	cells	by	their	expression	of	CD21	is	a	com-
                                                                                           tion	 and	 cytotoxicity	 against	 target	 cells.	 Further	 studies	 will	 address	
     mon	approach	in	swine	immunology.	However,	in	mice	the	expression	
     of	 CD21	 is	 restricted	 to	 mature	 and	 late	 transitional	 (T2)	 B	 cells.	 In	   the	role	of	NK	cells	and	lymphokine	activated	killer	cells	against	virus-
     contrast,	the	expression	of	CD79a	and	CD79β	occurs	already	from	the	                  infected	target	cells	and	in	vivo	during	viral	infections.
     pro-B	cell	stage	onwards.	By	combining	antibodies	against	CD79a	and	                  Key words:	Natural	Killer	cells,	IFN-γ,	perforin,	IL-2,	IL-12,	IL-18
     CD21	in	flow	cytometry	with	PBMC	from	adult	and	six	month	old	swine	                  Species:	swine
                                                                                                                                                                            97


   AP133. CLASSICAL SWINE FEVER VIRUS INDUCES                                            sion	 of	 C80/86,	 MHC-II,	 CD-172a,	 CD1	 y	 CD14	 was	 analyzed	 by	
DENDRITIC CELL ACTIVATION IN BLOOD AND SECONDARy                                         FACS.	DCs	were	infected	for	1	h	at	m.o.i.	of	0.1,	washed	and	cultur-
                LyMPHOID ORGANS                                                          ing	 for	 24	 h.	 The	 expression	 of	 mRNA	 IFN-a,	 IL-10	 and	 IL-12	 were	
                                                                                         analyzed	 by	 qPCR.	 Apoptosis	 was	 evaluated	 with	 FITC-conjugated	
   AGnèS	JAMIn1,	STéPHAnE	GoRIn1,	RoLAnD	CARIoLET2,	
                                                                                         Annexin	 V	 and	 propidium	 iodide,	 and	 endocytosis	 and	 phagocytosis	
   MARIE-FRéDéRIqUE	LE	PoTIER1,	GAëLLE	KUnTZ-SIMon1
                                                                                         was	 evaluated	 with	 FITC-latex	 particles	 and	 –dextran,	 respectively.	
  1AFSSA-LERAPP,	Swine	Virology	Immunology	Unit,	Ploufragan,	                            The	homologous	and	heterologous	stimulation	of	T	cell	was	analyzed	
  France;	2AFSSA-LERAPP,	Section	of	Healthy	Pig	Production	and	                          with	CFSE.	T	regulators	cells	(Tregs)	was	analyzed	by	the	expression	
              Experimentation,	Ploufragan,	France	                                       of	CD25+	and	Foxp3+.	Our	results	showed	that	iDCs	and	skin-derive	
                    g.kuntz.simon@afssa.fr                                               DCs	were	infected	with	PRRSv.	Infected	skin-derived	DCs	expressed	
      Classical	 swine	 fever	 virus	 (CSFV)	 causes	 indirect	 leucopoenia	             levels	of	mRNA	INF-α	and	low	expression	of	mRNA	IL-10.	Infection	of	
and	 disruption	 of	 in	 vitro	 T	 cell	 stimulation	 capacity.	 It	 can	 efficiently	   iDCs	revealed	an	mRNA	increment	of	1,000	fold	compared	with	mock	
replicate	in	monocyte-derived	dendritic	cells	and	blood-isolated	natural	                treated	iDCs,	and	IFN-a	not	was	expressed.	Apoptosis	was	detected	in	
interferon	 producing	 cells	 without	 interfering	 with	 their	 immune	 reac-           iDCs	at	24	h	post-infection,	increasing	at	48	h	and	72	h	post-infection.	
tivity	 (1,	 2),	 but	 no	 evidence	 has	 been	 found	 of	 a	 role	 of	 stimulated	      The	endocytosis	and	phagocytosis	was	reduced	on	infected	iDCs,	and	
dendritic	 cells	 (DC)	 in	 immunosuppression.	 Intending	 to	 understand	               the	capacity	to	stimulate	heterologous	was	reduced	30%	compared	to	
interactions	between	CSFV	and	DC	in	infected	pigs,	we	investigated	                      mock-treated	cells.	The	analysis	of	homologous	stimulation	was	also	
activation	of	conventional	DC	(cDC)	and	plasmacytoid	DC	(pDC),	two	                      reduced,	and	showed	the	presence	of	CD25+Foxp3+	cells	(9±4%)	in	
DC	substets	we	previously	characterized	in	swine	secondary	lymphoid	                     no-adherent	 cells	 stimulated	 with	 infected	 iDCs,	 this	 phenotype	 was	
organs	 and	 blood	 at	 the	 steady	 state	 (3).	 Changes	 in	 these	 popula-            due	to	virus	replication,	because	heat	inactivated	virus	did	not	produce	
tions	were	studied	in	the	early	time	course	post-inoculation,	together	                  CD25+Foxp3+	cells.	Also,	there	was	not	change	of	CD25+Foxp3+	yield	
with	viral	components	dissemination	in	host	and	cytokine	production	in	                  when	infected	DCs	were	treated	with	IFN-a.	Preliminary	results	test-
serum.	 Whereas	 CD11R1+CD172a+	 cDC	 frequencies	 were	 markedly	                       ing	 porcine	 circovirus	 showed	 that	 CD25+Foxp3+	 was	 not	 produced.	
reduced	in	blood	and	spleen	after	infection,	analysis	of	CD4+CD172a+	                    In	conclusion,	the	modulation	of	cytokine	production	and	the	induction	
pDC	 numbers	 revealed	 a	 rapid	 turn-over	 of	 this	 DC	 type	 in	 tissues.	           of	cells	with	phenotype	of	Tregs	cells	could	be	a	mechanism	used	by	
Both	 DC	 subsets	 matured	 and	 were	 activated,	 as	 demonstrated	 by	                 PRRSv	to	evade	the	immune	response.
down-regulation	of	CD1a,	up-regulation	of	the	co-stimulation	molecule	                   Key words:	Porcine	reproductive	and	respiratory	syndrome,	dendritic	
CD80/86	 and	 expression	 of	 cytokines.	 cDC	 mainly	 expressed	 tumor	                 cells,	CD25+Foxp3+	T	regulatory	cells
necrosis	 factor	 alpha	 (TNF-α)	 and	 interleukin	 (IL)-10,	 whereas	 pDC	              Species:	swine
produced	alpha	interferon	(IFN-α)	and	IL-12.	IFN-α	and	TNF-α	produc-
tions	revealed	enhancement	of	innate	anti-viral	immune	responses.	IL-
                                                                                            AP135. DOG LEUKOCyTE ANTIGENS IDENTIFIED By A
10	expression	indicated	initiation	of	humoral	response,	also	evidenced	
by	detection	of	antigen	activated	B	lymphocytes	in	tonsil	T-cell	areas	at	                                 CDNA LIBRARy
72	h,	subsequently	to	the	transient	translocation	of	the	viral	E2	protein	                                          PAULo	H	P	AGUIAR
within	germinal	centres	at	48	h.	IL-12	expression,	as	well	as	transient	                    Escola	de	Medicina	Veterinária	–	Universidade	Federal	da	Bahia
detection	of	IL-18	and	IFN-γ	in	serum,	would	reflect	initiation	of	cellular	
                                                                                               The	identification	of	antigens	recognized	by	monoclonal	antibod-
responses.	However,	the	uncommonly	high	levels	of	IFN-α	and	TNF-α	
                                                                                         ies	 (MoAbs)	 directed	 against	 dog’s	 Peripheral	 Blood	 Mononuclear	
produced	by	DC	and	measured	in	serum	would	play	a	role	in	disrup-
                                                                                         Cells	(PBMCs)	was	performed	by	using	a	complementary	DNA	(cDNA)	
tion	of	immune	system	cells,	either	inducing	apoptosis	or	impairing	DC	
                                                                                         library,	 produced	 by	 following	 the	 manufacturer	 protocol	 (picoBlue	
functionalities.	
                                                                                         Immunoscreening	 kit,	 Stratagene	 Cat.).	This	 procedure	 was	 used	 as	
     (1)	 Carrasco	C.P.,	Rigden	R.C.,	Vincent	I.E.,	Balmelli	C.,	Ceppi	                  an	 alternative	 to	 immunoprecipitation	 because	 it	 has	 the	 advantage	
M.,	Bauhofer	O.,	Tache	V.,	Hjertner	B.,	McNeilly	F.,	van	Gennip	H.G.,	                   of	 allowing	 the	 subcloning	 of	 the	 genes	 identified	 and	 the	 produc-
McCullough	K.C.,	Summerfield	A.	(2004)	Interaction	of	classical	swine	                   tion	 of	 recombinant	 proteins	 in	 the	 amount	 necessary	 for	 further	
fever	virus	with	dendritic	cells.		J.	Gen.	Virol.	85,	1633-1641.                         assays.	Twelve	 monoclonal	 antibodies	 were	 produced	 and	 tested	 by	
      (2)	Balmelli	C.,	Vincent	I.E.,	Rau	H.,	Guzylack-Piriou	L.,	McCullough	             (1)	 recognizing	 different	 dog’s	 leukocytes	 populations	 in	 an	 Indirect	
K.,	 Summerfield	A.	 (2005)	 Fc	 gamma	 RII-dependent	 sensitisation	 of	                Immunofluorescence	(IIF)	using	cryopreserved	tissue	sections	(lymph	
natural	interferon-producing	cells	for	viral	infection	and	interferon-alpha	             node,	liver,	skin	and	kidney)	and/or	healthy	dog’s	PBMCs	and	(2)	by	
responses.	Eur.	J.	Immunol.	35,	2406-2415.                                               recognizing	antigens	with	distinct	molecular	weights	on	western	blot-
     (3)	 Jamin	A.,	 Gorin	 S.,	 Le	 Potier	 M.-F.,	 Kuntz-Simon	 G.	 (2006)	            ting	assays.	Only	one	MoAb,	called	as	AB9	(IgG2a,	34	KDa	identifying	
Characterization	 of	 conventional	 and	 plasmacytoid	 dendritic	 cells	                 membrane	and/or	cytoplasmic	proteins),	recognized	a	phage	clone	on	
in	 swine	 secondary	 lymphoid	 organs	 and	 blood.	 Vet.	 Immunol.	                     the	library.	This	positive	clone	was	further	bi-directionally	sequenced,	
Immunopathol.	114,	224-237.                                                              twice.	 The	 evaluation	 of	 the	 other	 eleven	 MoAbs	 resulted	 negative,	
                                                                                         possibly	by	inherent	peculiarities	associated	to	the	use	of	the	genetic	
Key words:	Classical	swine	fever	virus,	dendritic	cells,	plasmacytoid	                   material	for	in	vitro	protein	production.	The	production	of	a	cDNA	library	
dendritic	cells,	TNF-α,	IFN-a,	IL-10,	IL-12                                              starts	by	the	isolation	of	total	RNA	from	the	target	tissue	and	the	further	
Species:	swine                                                                           selection	 of	 the	 poliA+	 RNA,	 which	 accounts	 for	 the	 majority	 of	 the	
                                                                                         messages	 on	 a	 given	 cell.	 The	 isolation	 of	 good	 quality	 poliA+	 RNA	
    AP134. PRRSV MODULATE CyTOKINE RESPONSE OF                                           is	 essential,	 as	 any	 degradation	 will	 affect	 the	 library	 sequences	 as	
    PORCINE DENDRITIC CELLS AND COMPROMISE THE                                           representative.	 Although	 cDNA	 libraries	 are	 standard	 tools	 for	 gene	
               ACTIVATION OF T CELLS                                                     expression	 studies	 and	 theoretically	 have	 complete	 copies	 of	 each	
E	SILVA-CAMPA,	L	FLoRES-MEnDoZA,	M	RESénDIZ-SAnDoVA,	                                    messenger	 RNA	 obtained	 from	 the	 initial	 sample,	 losses	 may	 occur	
                     J	HERnánDEZ*                                                        during	the	procedure	of	cloning	techniques.	Hence,	libraries	may	con-
                                                                                         tain	 gene	 fragments	 which	 would	 be	 different	 than	 the	 real	 genome,	
1Laboratorio	de	Inmunología,	CIAD,	A.C.	Hermosillo,	Sonora,	Mexico.                      what	would	interfere	with	the	further	analysis.
      Porcine	reproductive	and	respiratory	syndrome	(PRRS)	infection	                    Key words:	DLA,	cDNA	libraries
is	characterized	by	an	unconventional	immune	response,	our	hypoth-                       Species:	canine
esis	 is	 that	 the	 PRRS	 vs.	 dendritic	 cells	 (DCs)	 interaction	 contribute	
to	 this	 type	 of	 response.	 Previous	 reports	 have	 showed	 that	 PRRSv	
infect	and	replicate	on	immature	 (iDCs)	 and	mature	 DCs	(mDCs).	In	                        AP136. IGM SELECTIVE DEFICIENCy AND TOLL-LIKE
this	work	we	used	iDC	and	skin-derived	DCs	to	investigate	the	modula-                                RECEPTORS – A BRAZILIAN STUDy
tory	proprieties	of	PRRSv.	iDCs	were	generated	by	culturing	adherent	                        C	MASSoCo,	M	Z	MELEIRo,	Ay	A	HoGE,	M	M	CAMARGo	
cells	 with	 IL-4	 and	 GM-CSF	 for	 5	 days,	 and	 skin-derived	 DCs	 were	                  Selective	 IgM	 deficiency	 (SIgMD)	 is	 a	 rare	 primary	 immunodefi-
obtained	from	the	supernatant	of	culturing	skin	for	24	h.	The	expres-                    ciency	whose	molecular	mechanisms	remain	unclear.	Herein	we	pres-
98


     ent	ten	adult	horses	of	several	breeds	whose	SIgMD	was	diagnosed	                    1Department	of	Biochemistry	and	Immunology,	School	of	Medicine	of	
     on	 the	 basis	 of	 clinical	 history	 of	 recurrent	 bacterial	 infections	 and	    Ribeirão	Preto,	USP,	SP,	Brazil;		2Division	of	Molecular	Immunology,	
     reduced	serum	IgM	concentrations	(more	than	2	standard	deviations	                      Cincinnati	Children’s	Hospital	Medical	Center	and	University	of	
     below	the	normal	mean).	All	ten	horses	had	a	history	of	late	develop-                 Cincinnati	College	of	Medicine,	Cincinnati,	OH,	USA;		3Department	
     ment	 and	 respiratory,	 gastrointestinal	 and/or	 reproductive	 disorders.	          of	Maternal-Child	Nursing	and	Public	Health	of	the	Ribeirao	Preto,	
     Immunoglobulin	 quantification	 was	 carried	 by	 radial	 immunodifusion	                      School	of	Nursing	of	the	University	of	Sao	Paulo		
     and	 revealed	 normal	 IgG,	 IgG(T)	 and	 IgA	 concentrations,	 but	 very	                                      carlo@usp.br	
     low	IgM	concentration.	In	vitro	proliferation	studies	revealed	a	hetero-                   Ticks	are	blood-feeding	arthropods	of	vast	medical	and	veterinary	
     geneous	 inter-individual	 profile	 of	 proliferation	 in	 response	 to	 PHA,	       public	health	importance	due	to	direct	damage	caused	by	feeding	and	
     flagelin	and	Pam3Cys.	In	contrast,	all	of	these	individuals	had	skewed	              their	roles	in	transmitting	well	known	and	emerging	infectious	agents.	
     responses	 to	 LPS,	 while	 proliferative	 responses	 to	 ConA	 and	 polyI:          Many	studies	have	shown	that	tick	saliva	contains	a	variety	of	active	
     C	 were	 comparable	 to	 healthy	 controls.	 TLR	 expression	 patterns	 of	          molecules	that	antagonize	host	inflammatory	and	immune	responses.	
     several	individuals	will	be	shown.	As	these	specific	defects	leading	to	             As	dendritic	cells	(DCs)	play	a	major	role	in	host	immune	responses,	
     failures	in	the	immune	response	are	identified	and	correlated	with	the	              we	studied	the	effects	of	Rhipicephalus	sanguineus	tick	saliva	on	DC	
     pathogenesis	of	this	disease	we	will	extend	our	understanding	of	the	                migration	 and	 function.	 To	 evaluate	 the	 effect	 of	 tick	 saliva	 on	 DC	
     working	of	the	equine	immune	system.                                                 migration,	 a	 Boyden	 microchamber	 migration	 assay	 was	 performed.	
     Key words:	IgM	deficiency,	Toll-like	receptors                                       The	 percentage	 of	 cells	 expressing	 chemokine	 receptors	 was	 mea-
     Species:	Equine                                                                      sured	 by	 flow	 cytometry	 analysis.	 To	 investigate	 the	 effect	 of	 saliva	
                                                                                          on	 the	 function	 of	 DCs	 a	 cell	 adoptive	 transfer	 assay	 was	 done	 and	
                                                                                          the	 T	 cell	 proliferation	 and	 cytokine	 production	 was	 evaluated.	 Bone	
         AP137. GENERATION AND CHARACTERISATION OF
                                                                                          marrow-derived	 immature	 DCs	 pre-exposed	 to	 tick	 saliva	 showed	
         EQUINE DENDRITIC CELLS FROM HIGHLy ENRICHED
                                                                                          reduced	 migration	 towards	 Macrophage	 Inflammatory	 Protein	 (MIP)-
                PERIPHERAL BLOOD MONOCyTES                                                1α,	 MIP-1β	 and	 Regulated	 upon	Activation,	 Normal	T-cell	 Expressed	
                      E	C	SIFFRIn,	J	RoHWER,	W	LEIBoLD                                    and	Secreted	(RANTES)	by	approximately	60%.	This	inhibition	medi-
          Dendritic	 cells	 play	 a	 pivotal	 role	 in	 initiating	 a	 wide	 range	 of	   ated	 by	 saliva	 significantly	 reduced	 the	 percentage	 (59.8%)	 and	 the	
     immune	reactions	against	most	antigens.	Peripheral	blood	monocytes	                  average	cell-surface	expression	of	CC	chemokine	receptor	CCR5.	In	
     are	 a	 suitable	 source	 to	 generate	 dendritic	 cells	 (DC)	 in	 vitro	 under	    contrast,	saliva	did	not	alter	migration	of	DCs	towards	MIP-3β		not	even	
     appropriate	 conditions.	 Aiming	 for	 a	 high	 yield	 of	 peripheral	 blood	        if	the	cells	were	induced	for	maturation.	When	evaluated	the	effect	of	
     monocytes	 which	 have	 neither	 been	 preselected	 nor	 preactivated	 by	           tick	 saliva	 on	 the	 activity	 of	 chemokines	 related	 to	 DC	 migration,	 we	
     the	harvesting	procedure	we	scrutinized	different	published	methods.	                showed,	in	a	Boyden	microchamber	assay,	that	tick	saliva	per	se	inhib-
     Subsequently,	the	enriched	monocytes	were	used	to	generate	mono-                     its	the	chemotactic	function	of	MIP-1α	(40.7	%),	while	it	did	not	affect	
     cytes-derived-DC	(MoDC)	which	were	characterised	phenotypically	by	                  RANTES,	MIP-1β	and	MIP-3β.	These	saliva	effects	could	reduce	the	
     means	 of	 their	 CD83	 and	 CD86	 expression	 and	 functionally	 by	 their	         pool	of	immature	DCs	present	at	the	tick-feeding	site.	Finally,	to	test	the	
     antigen	presenting	capacity.	                                                        biological	activity	of	the	saliva-exposed	DCs,	we	transferred	DCs	pre-
           Enrichment	of	equine	monocytes	due	to	their	adherence	on	plastic	              cultured	with	saliva	and	loaded	with	the	Keyhole	Limpet	Hemocyanin	
     surfaces	turned	out	to	be	ineffective,	because	it	comprised	a	consider-              (KLH)	antigen	to	mice	and	measured	their	capacity	to	induce	specific	
     able	loss	of	monocytes	and	an	insufficient	purity	of	maximally	30%	of	               T-cell	 cytokines.	 Data	 showed	 that	 saliva	 reduced	 the	 synthesis	 of	T	
     the	mononuclear	cell	(MNC)	fraction.	Depleting	MNC	from	CD4-	and	                    helper	 (Th)1	 and	 Th2	 cytokines,	 suggesting	 the	 induction	 of	 a	 “non-
                                                                                          responsive”	 T-cell.	 These	 findings	 propose	 that	 the	 inhibition	 of	 DCs	
     CD8-positive	cells	by	magnetic	cell	sorting	(MACS)	resulted	in	a	higher	
                                                                                          migratory	ability	and	function	may	be	potent	mechanisms	used	by	ticks	
     purity	of	monocytes	(up	to	40%	of	MNC)	but	with	an	unacceptable	low	
                                                                                          to	paralyze	the	immune	response	of	the	host.	
     gain	of	only	less	than	7	%	of	the	MNC	to	start	with,	plus	a	selective	
     loss	 of	 CD4+	 and	 CD8+	 monocytes.	 The	 enrichment	 of	 IgE	 positive	                Financial	Support:	FAPESP,	PRODETAB	and	CNPq.
     monocytes	 via	 MACS	 resulted	 in	 an	 impressive	 purity	 of	 monocytes	           Key words:	 Ticks,	 Dendritic	 cells,	 Chemokines,	 Rhipicephalus	
     (up	 to	 70%	 of	 MNC),	 but	 led	 to	 a	 monocyte	 subset	 selection	 (IgE+	        sanguineus.	
     monocytes)	 and	 a	 very	 low	 yield	 of	 monocytes	 (less	 than	 2%	 of	 the	       Species:	other
     monocytes	 available).	 The	 best	 method	 to	 enrich	 equine	 monocytes	
     from	whole	blood	proved	to	be	a	centrifugation	on	a	hyperosmotic	den-                AP139. RAPID IMMUNIZATION AGAINST SCORPION TOxIN
     sity	gradient,	resulting	in	a	purity	of	up	to	90%	monocytes	and	a	yield	of	              By IN VIVO TARGETING MICE DENDRITIC CELLS
     70%	of	the	monocytes	available.	By	this	method	neither	a	preselection	
     nor	a	detectable	preactivation	of	monocytes	took	place.	                                 GP	ESPIno-SoLIS1,	AF	LICEA-nAVARRo2,	GA	GURRoLA-
                                                                                                            BRIonES,LD	PoSSAnI
          To	examine	whether	these	monocytes	are	a	reasonable	source	for	
     the	generation	of	DC,	they	were	cultured	for	7	days	in	medium	supple-                     1Universidad	Nacional	Autónoma	de	México	–	Instituto	de	
                                                                                          Biotecnología	–	Departamento	de	Medicina	Molecular	y	Bioprocesos,	
     mented	 with	 rec.eq.IL4	 and	 rec.hu.GM-CSF.	 Phenotypical	 charac-
                                                                                            Avenida	Universidad,	2001,	Apartado	Postal	510-3.	Cuernavaca,	
     terisation	with	monoclonal	antibodies	against	human	CD83	and	CD86	
                                                                                             Morelos	62210	MEXICO;	2Centro	de	Investigación	y	Educacion	
     revealed	 up	 to	 30%	 CD83+	 and	 up	 to	 60%	 CD86+	 DC.	The	 antigen	
                                                                                             Superior	de	Ensenada.	Departamento	de	Biotecnologia	Marina,	
     presenting	capacity	of	the	generated	MoDCs	were	tested	in	allogeneic	
                                                                                                          Esenada,	Baja	California,	MEXICO.
     and	autologous	mixed	leucocyte	reactions	(MLR).	Unpulsed	allogeneic	
     DC	(without	exogenous	antigen	loading)	led	to	a	significantly	increased	                  Scorpionism	 (accidents	 caused	 by	 scorpion	 stings)	 in	 Mexico	 is	
     T-cell	proliferation	but	did	not	stimulate	autologous	T-cells	to	a	signifi-          a	 public	 health	 problem	 with	 an	 excess	 of	 200,000	 cases,	 annually.	
     cant	 proliferation.	 Exogenous	 loading	 of	 DC	 with	 antigen	 24h	 before	        The	need	of	anti-venom	production	is	mandatory.	Here	we	describe	a	
     application	of	the	DC	in	the	MLR	caused		clear	stimulation	of	allogeneic	            rapid	protocol	to	produce	neutralizing	antibodies.	Toxin	Cn2,	which	is	
     as	well	as	autologous	T-cells.	                                                      most	abundant	component	(6.8%)	of	the	most	dangerous	scorpion	of	
                                                                                          Mexico	(Centruroides	noxius)	was	covalently	coupled	to	a	monoclonal	
     Key words:	Dendritic	cells,		IgE	positive	monocytes,	mixed	leucocyte	
                                                                                          antibody	 capable	 of	 binding	 directly	 to	 the	 integrin	 CD11c,	 a	 specific	
     reactions
                                                                                          marker	of	the	dendritic	cells	of	mice.	The	antibody	used	was	N418	and	
     Species:	Equine
                                                                                          the	toxin	was	attached	by	a	Schiff’s	base	to	the	carbohydrate
                                                                                               moiety	of	this	antibody,	via	NaIO4	oxidation.	A	group	of	five	mice	
        AP138. TICK SALIVA SUPPRESSES DENDRITIC CELL
                                                                                          was	immunized	once	by	intradermic	injection	of	the	complex	N418-Cn2	
       MIGRATION By REGULATING CHEMOKINE ACTIVITy AND                                     dissolved	in	phosphate	saline	buffer,	pH	7.2.	An	antisera	capable	of	rec-
              CHEMOKINE RECEPTOR ExPRESSION                                               ognizing	Cn2	(titer	1:6000)	was	obtained	after	seven	days.	This	serum	
     CJF	oLIVEIRA1,	KA	CAVASSAnI1,	JCS	ALIBERTI2,	JS	SILVA1,	BR	                          was	shown	to	neutralize	toxin	Cn2	with	a	50%	efficiency	compared	to	
                            FERREIRA3                                                     100%	obtained	with	traditional	immunization	protocols,	which	include	
                                                                                                                                                                        99


several	immunizations	during	a	90	days	period	of	time.	This	strategy	              BW.	At	sacrifice,	the	spleen	was	removed	to	prepare	cell	suspension.	
opens	the	way	for	a	quick	production	of	antitoxin	sera	in	mice.                    From	the	cells	obtained	in	each	mouse,	1x107	cells/mL	were	incubated	
    Acknowledgements:	 This	 work	 was	 supported	 in	 part	 by	 grants	           with	1	µL	of	CFSE	for	20	min	in	the	dark.	After	this,	cells	were	washed	
from:	DGAPA-UNAM	No.	IN227507	and	Instituto	Bioclón	S.A.	de	C.V.	                  and	 suspended	 at	 2x106	 cells/mL	 in	 RPMI	 and	 100	 µL	 aliquots	 were	
GPES	have	a	fellowship	from	CONACYT	No.	169946.                                    added	 to	 a	 96-well	 microtiter	 plate.	 	 Untreated	 and	 PHA	 (10µL/well)	
                                                                                   treated	 were	 set	 up	 in	 triplicates	 and	 incubated	 for	 96h	 at	 37°	 C	 in	
Key words:	Scorpion	toxin,	anti-venom,	dendritic	cells                             a	humidified	atmosphere	at	5%	CO2.	The	samples	were	analysed	by	
Species:	other                                                                     flow	cytometry.	In	another	study,	for	the	analysis	of	macrophages,	eight	
                                                                                   C57BL/6	male	mice	were	used	per	group	and	the	same	protocol	was	
   AP140. EFFECTS OF PTERIDIUM AqUILINUM ON MICE                                   utilized	for	treatment,	but	on	10th	day	all	mice	were	injected	ip	with	1	mL	
  ARE NOT ASSOCIATED WITH THE ELICITATION STAGE IN                                 of	thioglycollate	10%.		At	sacrifice,	the	peritoneal	macrophages	were	
     DELAyED-TyPE HyPERSENSITIVITy RESPONSE                                        collected	 with	 PBS.	 	 For	 the	 oxidative	 burst	 analysis,	 from	 the	 cells	
AnDREIA	o	LAToRRE1,	MonICA	SAKAI1,	MITSUE	HARAGUCHI2,	                             obtained	in	each	mouse,	1x106	cells/tube	were	incubated	during	30min	
                 SILVAnA	L	GóRnIAK1                                                at	37ºC	with	either	DCFH-DA	or	DCFH-DA	and	PMA	or	DCFH-DA	and	
                                                                                   Staphylococcus	aureus	conjugated	with	propidium	iodide	(SAPI).	For	
1Department	of	Pathology,	Faculty	of	Veterinary	Medicine	and	Animal	
                                                                                   the	 phagocytosis	 analysis,	 from	 the	 cells	 obtained	 in	 each	 mouse,	
 Sciences,	University	of	São	Paulo,	Brazil;	2Biological	Institute,	São	
                                                                                   1x106	 cells/tube	 were	 incubated	 30min	 at	 37ºC	 with	 either	 SAPI	 or	
                            Paulo,	Brazil	
                                                                                   DCFH-DA	and	SAPI.	The	samples	were	analysed	by	flow	cytometry.	
                          alatorre@usp.br
                                                                                   No	change	in	the	proliferation	of	T	lymphocytes	and	macrophage	activi-
      Pteridium	aquilinum	(Pa)	is	one	of	the	five	most	common	plants	on	           ties	was	observed	after	Pa	treatment,	suggesting	that	the	reduction	in	
the	planet	and	is	known	to	cause	cancer	in	animals	and	humans.	Our	                DTH	response	is	probably	due	to	inefficiency	on	the	sensitization	stage	
earlier	studies	have	shown	that	Pa	causes	injury	in	lymphoid	organs,	              (activation	 and	 expansion	 of	 antigen-specific	 memory	Th1	 cells)	 and	
decreases	bone	marrow	cellularity,	reduces	delayed-type	hypersensi-                not	on	the	elicitation	stage	(Th1	and	macrophage	activation).	Further	
tivity	 (DTH)	 response	 and	 NK	 cytotoxicity	 in	 mice.	 In	 this	 study,	 we	   experiments	are	in	progress	to	see	the	immunomodulatory	effects	of	
have	evaluated	the	activity	of	T	lymphocytes	of	spleen	and	peritoneal	             Pa.	
macrophages	in	order	to	see	whether	there	is	any	correlation	of	these	
                                                                                   Key words:	toxic	plants,	immunomodulation
effects	with	reduction	in	DTH	response.	For	T	lymphocytes	proliferation	
                                                                                   Species:	other
assay,	nine	C57BL/6	male	mice	were	used	per	group	and	treated	by	
gavage	up	to	14	days	as	follows:	control	(Co)0.0,	Pa	(P)30.0	gPa/Kg	
                          6. IMMUNOPARASITOLOGy: IMMUNE RESPONSES TO PROTOZOA, HELMINTHS AND
                             ECTOPARASITES; CANINE VISCERAL LEISHMANIASIS: POSTERS PR141-PR196

  Pr141. DEVELOPMENT OF AN ANTI-TICK VACCINE TO                                                                    Sapporo,	Japan	
 PROTECT CATTLE AGAINST TICK-BORNE TRANSMISSION                                                               luisfparizi@cbiot.ufrgs.br
               OF THEILERIA PARVA                                                           The	ticks	Riphicephalus	(Boophilus)	microplus	and	Haemaphysalis	
   SAIKI	IMAMURA,	SAToRU	KonnAI,	CHIE	nAKAJIMA,	SHInJI	                              longicornis	are	blood-sucking	ectoparasities	of	bovines,	causing	seri-
    yAMADA,yUKo	ITo,	KAZUHIKo	oHASHI,	MISAo	onUMA                                    ous	damages	to	the	livestock	production.	The	main	method	of	control	
                                                                                     is	 based	 on	 the	 acaricides.	 However,	 the	 use	 of	 vaccines	 has	 been	
Graduate	School	of	Veterinary	Medicine;	Hokkaido	University,	Kita	18,	               studied	 as	 a	 promising	 control	 method.	 The	 calreticulin	 (CRT)	 is	 a	
           Nishi	9,	Sapporo	060-0818,	Hokkaido,	Japan.	                              multifunctional	protein	present	in	almost	all	cells	of	animals.	The	secre-
                    saiki-i@vetmed.hokudai.ac.jp                                     tion	 of	 CRT	 during	 feeding	 might	 be	 linked	 to	 the	 modulation	 of	 the	
      Ticks	are	facultative	blood	sucking	ectoparasites	found	in	all	ter-            parasite-host	 interaction.	 In	 the	 present	 study,	 recombinant	 CRTs	 of	
restrial	regions	of	the	world,	where	they	are	the	major	vectors	transmit-            R.	microplus	(rBmCRT)	cloned	in	pET-5b	and	H.	longicornis	(rHlCRT)	
ting	quite	a	large	number	of	pathogens.		Therefore,	suppression	of	the	              cloned	in	pET-43a	were	expressed	in	Escherichia	coli	and	purified	by	
tick	vector	population	is	considered	to	control	specific	diseases	trans-             ion	exchange	chromatography	and	used	for	immunization	of	bovines.	
mitted	by	ticks.		The	application	of	anti-tick	vaccine	has	been	shown	               The	fraction	used	for	tests	in	vitro	was	purified	by	ion	exchange	and	
to	be	the	most	promising	alternative	tick	control	strategy	compared	to	              gel	filtration	chromatography.	By	ELISA,	it	was	demonstrated	that	both	
the	current	use	of	acaricides,	because	acaricides	suffer	from	a	number	              CRTs	 are	 recognized	 by	 immunized	 bovines.	 The	 immunogenic	 and	
of	disadvantages	such	as	chemical	pollution	of	the	food	chain	and	the	               antigenic	 capacities	 of	 rBmCRT	 and	 rHlCRT	 were	 analyzed	 by	 two	
environment	 as	 well	 as	 the	 rapid	 development	 of	 resistance	 against	         methods.	 In	 silico,	 despite	 the	 difference	 in	 amino	 acid	 sequences,	
acaricides	in	ticks.		                                                               antigenic	index	analysis	of	rHlCRT	and	rBmCRT	with	the	Jameson-Wolf	
      Rhipicephalus	appendiculutus	serpin-3	(RAS-3),	R.	appendiculu-                 algorithm	indicated	that	both	proteins	were	very	similar	in	the	antigenic-
tus	serpin	-4	(RAS-4)	and	RIM36,	a	36	kDa	immuno-dominant	protein	                   ity	 index.	Although	 six	 different	 regions	 between	 the	 tick	 CRTs	 have	
of	 R.	 appendiculatus,	 were	 reported	 as	 anti-tick	 vaccine	 candidates	         been	determined.	In	vitro,	this	data	were	corroborated	by	competitive	
for	the	ixodid	tick.		Among	many	candidate	antigens	considered	so	far,	              ELISA	that	suggests	the	presence	of	different	epitopes	between	pro-
serpins	 (e.g.	 RAS-3	 and	 -4)	 and	 cements	 (e.g.	 RIM36)	 may	 be	 the	          teins.	 By	 Western	 blot,	 anti-native	 rBmCRT	 and	 rHlCRT	 bovine	 sera	
most	interesting	antigens	for	the	development	of	an	anti-tick	vaccine,	              also	 recognized	 the	 native	 proteins	 in	 larvae	 extracts.	 These	 results	
because	of	their	important	roles	in	tick	physiology.			                              demonstrate	 the	 presence	 of	 shared	 epitopes	 between	 recombinant	
      In	the	current	study,	we	generated	recombinant	proteins	of	RAS-3,	             and	native	proteins.	In	conclusion,	the	results	suggest	that	the	rBmCRT	
-4,	 and	 RIM36	 and	 assessed	 their	 potency	 as	 an	 anti-tick	 cocktail	         and	rHlCRT	could	have	a	similar	immunogenicity	for	bovines.
vaccine	 in	 cattle	 model.	 	 Immunization	 of	 cattle	 with	 a	 combination	          Supported	 by	 CNPq,	 FAPERGS,	 FINEP,	 PRONEX	 (Brazil)	 and	
of	 rRAS-3,	 -4	 and	 rRIM36	 raised	 antibody	 against	 all	 recombinants	          JSPS	(Japan)
and	tick	whole	extract	as	determined	by	Western	blot.		Tick	challenge	               Key words:	calreticulin,	vaccine,	Riphicephalus	(Boophilus)	microplus	
infestation	demonstrated	significant	protective	immunity	against	ticks,	             e	Haemaphysalis	longicornis
resulting	 in	 41.3	 and	 12.8	 %	 of	 mortality	 rate	 for	 the	 vaccinated	 and	   Species:	ruminants
control	group,	respectively.		In	order	to	evaluate	the	levels	of	pathogen	
transmission	 capacity	 by	 Theileria	 parva-infected	 ticks	 fed	 on	 immu-
                                                                                         Pr143. EVALUATION OF DNA IMMUNIZATION WITH
nized	animal,	an	appearance	of	T.	parva	in	the	parotid	lymph	node	and	
                                                                                       PLASMIDS ExPRESSING RByC (BOOPHILUS yOLK PRO-
peripheral	blood	was	also	determined	and	quantified	by	real-time	PCR.	           	
T.	parva	DNA	load	in	the	lymph	node	was	lower	in	the	immunized	group	
                                                                                                         CATHEPSIN)
than	in	the	control	group.		An	appearance	of	the	pathogen	in	the	blood	                MARIA	LúCIA	S	MEDEIRoS1,	ALExAnDRE	T	LEAL1,	CARLoS	
was	delayed	1-	to	2-day	post	tick	challenge	in	the	vaccinated	group.	                 LoGULLo5,	SAnDRA	E	FARIAS1,4,	AoI	MASUDA1,3,	ITABAJARA	
      Our	findings	suggested	a	beneficial	effect	of	the	anti-tick	vaccine	                              DA	SILVA	VAZ	JR1,2
for	the	prevention	of	both	tick	and	infectious	diseases	transmitted	by	               1C.	Biotecnologia-UFRGS;	2Fac.	Veterinária-UFRGS;	3Depto	Biol.	
ticks.		However,	it	is	necessary	to	identify	other	tick	antigens	that	can	              Molecular	e	Biotecnologia-UFRGS;	4Depto	Fisiologia-UFRGS;	
be	 used	 in	 a	 cocktail	 to	 induce	 anti-tick	 immunity	 more	 potently	 pre-              5LQFPP-CBB-UENF,	Campos	dos	Goytacazes,RJ.		
venting	the	transmission	of	pathogens	of	tick-borne	diseases.                                               malu@cbiot.ufrgs.br
Key words:	 anti-tick	 vaccine,	 Theileria	 parva,	 Rhipicephalus	                        The	Boophilus	microplus	tick	is	the	major	bovine	ectoparasite	and	
appendiculatus                                                                       causes	important	economical	losses	on	cattle	breeding.	The	immuno-
Species:	ruminants                                                                   logic	 control	 has	 been	 studied	 as	 an	 alternative	 method	 for	 the	 tick	
                                                                                     control.	BYC	(Boophilus	Yolk	Pro	Cathepsin)	is	an	aspartic	proteinase	
  Pr142. EVALUATION OF IMMUNOGENICITy IN BOVINES                                     found	 in	 eggs	 that	 is	 involved	 in	 the	 embryogenesis	 of	 B.	 microplus,	
   OF RIPHICEPHALUS (BOOPHILUS) MICROPLUS AND                                        and	it	has	been	proposed	as	a	probable	antigen	in	vaccine	develop-
    HAEMAPHySALIS LONGICORNIS RECOMBINANT                                            ment.	 The	 purpose	 of	 this	 study	 was	 to	 evaluate	 whether	 the	 DNA	
                                                                                     immunization	containing	BYC	cDNA	could	elicit	the	specific	anti-BYC	
                  CALRETICULINS
                                                                                     immune	 response	 in	 vivo.	 The	 cDNA	 of	 BYC	 was	 amplified	 by	 PCR	
     LUÍS	FERnAnDo	PARIZI1,2,	HERBERT	RECH1,2,	SAIKI	                                and	 it	 was	 cloned	 into	 two	 eukaryotic	 expression	 vectors	 (pcDNA3	
  IMAMURA4	MISAo	onUMA4,	AoI	MASUDA1,3,ITABAJARA	DA	                                 and	 pME18-Neo).	 XL1-Blue	 E.coli	 were	 transformed	 with	 clones,	
                    SILVA	VAZ	JR1,2                                                  BYC-pcDNA3	 or	 BYC-pME18-Neo,	 and	 the	 plasmids	 were	 purified	
 1Centro	de	Biotecnologia,	Universidade	Federal	do	Rio	Grande	do	                    by	alkaline	lysis	method.	In	order	to	evaluate	immunogenicity	of	BYC,	
 Sul,	Avenida	Bento	Gonçalves,	9500,	Porto	Alegre-RS	91501-970,	                     BALB/c	 mice	 were	 immunized	 with	 DNA	 vaccines	 by	 intramuscular	
   Brazil;	2Faculdade	de	Veterinária,	Universidade	Federal	do	Rio	                   injection.	The	mice	received	two	intramuscular	inoculations	of	100μg	
 Grande	do	Sul,	Porto	Alegre-RS,	Brazil;	3Departamento	de	Biologia	                  plasmids	 DNA	 (BYC-pcDNA3	 or	 BYC-pME18-Neo)	 and	 the	 negative	
   Molecular	e	Biotecnologia,	UFRGS,	RS;	4Hokkaido	University,	                      controls	received	only	PBS,	pcDNA3	or	pME-18-Neo.	The	production	
of	 antibody	 after	 the	 immunizations	 was	 evaluated	 by	 Western	 Blot	          vector	and	nine	strains	of	E.	coli	were	transformed	with	resultant	plas-
and	ELISA.	Antibodies	against	BYC	were	detected	in	mice	inoculated	                  mid.	The	best	conditions	established	for	production	of	the	recombinant	
with	BYC-pcDNA3.	These	results	show	that	DNA	vaccination	produces	                   protein	 (rTHAP	 with	 fusion	 protein	 NusTag)	 in	 the	 soluble	 form	 was	
specific	anti-BYC	antibodies	and	suggest	that	DNA	could	prove	useful	                the	expression	in	E.coli	BL21	(DE3)	RIL	at	23º	C	and	1	mM	of	IPTG	
for	vaccine	development.                                                             for	4	hours.	Analysis	of	the	expression	was	performed	by	SDS-PAGE	
     Supported	by		FAPERGS,	FINEP,	PRONEX	                                           and	 immunoblotting	 with	 a	 rabbit	 anti-THAP	 serum.	 rTHAP-NusTag	
                                                                                     was	 purified	 by	 affinity	 chromatography	 with	 sepharose-Ni2+	 resin.	A	
Key words: B.	microplus,	vaccine	DNA,	Boophilus	Yolk	Pro	Cathepsin,	
                                                                                     partially	purified	fraction	was	obtained	and	submitted	to	hydrolysis	for	
embryogenesis
                                                                                     removal	 the	 fusion	 protein	 (NusTag).	 The	 rTHAP	 enzymatic	 activity	
Species: ruminants
                                                                                     was	 assayed	 with	 fluorogenic	 substrate	Abz-AIAFFSRQ-EDDnp	 and	
                                                                                     was	monitored	for	1	hour	with	F-MAX	fluorometer.	The	specific	activity	
     Pr144. CLONING AND ExPRESSION OF THE
                                                                                     obtained	was	9.55	RFU/	min/	mg	of	protein	and	the	activity	was	blocked	
RECOMBINANT TRIOSEPHOSPHATE ISOMERASE (TIM), AN
                                                                                     by	 20	 µM	 of	 pepstatin	A.	 In	 order	 to	 characterize	 the	 imunogenicity	
ENZyME INVOLVED IN METABOLISM OF TICK BOOPHILUS                                      of	the	protein,	two	fragments	of	the	cDNA	encoding	the	protein	were	
                   MICROPLUS                                                         cloned.	 One	 cDNA	 fragment	 encoding	 the	 first	 170	 amino	 acids	 and	
   FERnAnDA	E	K	SILVA1,	JoRGE	L	DA	C	MoRAES3,	CARLoS	                                another	 encoding	 the	 last	 185	 amino	 acids.	The	 encoding	 sequence	
  LoGULLo3,	AoI	MASUDA1,4,	ITABAJARA	DA	SILVA	VAZ	JR1,2                              of	amino	acids	of	the	THAP	were	obtained	by	PCR	and	cloned	in	the	
    1Centro	de	Biotecnologia-UFRGS;	2Fac.	Veterinária-UFRGS;	                        pET23a	expression	vector.	The	recombinant	proteins	were	produced	
    3Universidade	Estadual	do	Norte	Fluminense;	4Depto	Biologia	                     in	insoluble	form	by	the	expression	at	37º	C	and	1	mM	of	IPTG	for	4	
                 Molecular	e	Biotecnologia-UFRGS	                                    hours.	The	full	protein	and	carboxi-end	fragment	were	immuno-reactive	
                       feklein@cbiot.ufrgs.br                                        against	sera	from	rabbit	and	bovine	previously	immunized	with	native	
                                                                                     THAP.	Further	studies	on	the	THAP	enzymatic	activity	and	its	potential	
      	      Cattle	 raising	 is	 a	 very	 expressive	 economical	 activity	 in	
                                                                                     immunoprotective	role	are	in	progress	to	define	the	importance	of	this	
Brazil.	Nevertheless,	B.	microplus	is	responsible	for	losses	in	the	order	
                                                                                     protein	to	tick	control.	
of	 one	 billion	 dollars	 a	 year.	 Consequently,	 it	 is	 necessary	 to	 design	
new	 control	 strategies	 that	 are	 technically	 and	 economically	 viable.	           This	work	was	supported	by	grants	from	CNPq-	PIBIC,	CAPES,	
The	homodimeric	enzyme	triosephosphate	isomerase	(TIM)	converts	                     PRONEX-	FAPERJ.
glyceraldehyde-3-phosphate	 to	 dehydroxyacetone	 phosphate,	 a	 key	                Key words:	thap,	tick,	vaccine,	embryogenesis
reaction	in	glycolysis.	Previous	studies	of	this	enzyme	in	other	parasite-           Species:	ruminants
host	model	have	indicated	that	TIM	is	a	promising	anti-parasite	vaccine	
antigen.	 Therefore,	 the	 study	 of	 the	 enzyme	 B.	 microplus	 TIM	 could	           Pr146. CLONING AND CHARACTERIZATION OF THE
contribute	to	develop	a	new	vaccine	for	immune	protection	against	this	               RECOMBINANT GLyCOGEN SyNTHASE KINASE (GSK-3β)
parasite.	In	order	to	study	this	enzyme	at	the	molecular	level,	we	have	               FROM BooPHILUS MICRoPLUS ExPRESSED IN E. CoLI
isolated,	cloned,	sequenced	and	expressed	one	cDNA	clone	encoding	
                                                                                         CARoLInE	P	DE	AnDRADE1,	JoSIAnA	G	DE	AnDRADE3,	
TIM	 from	 tick	 eggs	 using	 RT-PCR	 with	 specific	 primers.	 The	 ampli-
                                                                                       SAIKI	IMAMURA2,	MISAo	onUMA2,	AoI	MASUDA1,5,	CARLoS	
con	with	750	pb	was	digested	with	restriction	enzymes	and	ligated	in	
                                                                                              LoGULLo3,	ITABAJARA	DA	SILVA	VAZ	JUnIoR1,4
pET-43a	 expression	 vector.	 The	 cloning	 was	 confirmed	 for	 cleavage	
with	restriction	enzymes,	PCR	and	DNA	sequencing.	Escherichia	coli	                    1C.	Biotecnologia-UFRGS;	2Hokkaido	University;	3LQFPP-CBB-
AD494	 (DE3)	 pLysS	 was	 transformed	 with	 TIM-pET-43a	 vector	 for	                  UENF;	4Fac.	Veterinária-UFRGS;	5Depto	Biologia	Molecular	e	
expression	 of	 recombinant	 protein.	 Optimal	 rTIM-Bm	 (a	 protein	 with	                                 Biotecnologia-UFRGS	
two	subunits	of	the	27	kDa	and	with	His-	tag)	production	was	achieved	                                   carolandrade@cbiot.ufrgs.br
by	 testing	 different	 growth	 temperatures,	 times	 and	 IPTG	 concentra-                Boophilus	 microplus	 is	 a	 hematophagous	 parasite	 that	 infests	
tions.	The	expression	was	analyzed	for	SDS-PAGE	12%	and	the	pres-                    animals	 of	 economic	 importance,	 causing	 losses	 in	 cattle	 production	
ence	of	the	recombinant	protein	was	confirmed	for	Western	blot,	using	               world-wide.	The	 conventional	 method	 for	 the	 control	 is	 based	 on	 the	
monoclonal	antibody	anti-histidine.	The	purification	of	the	protein	has	             use	 of	 synthetic	 chemical	 products.	 However,	 these	 products	 cause	
been	done	by	Ni2+	affinity	chromatography.	Immunization	will	be	done	                the	selection	of	resistant	tick	to	the	acaricides	being	used	and	cause	
in	mice	and	cattles	to	assess	its	capacity	to	induce	immune	response	                pollution	 in	 the	 environment.	 A	 vaccine	 is	 a	 reasonable	 alternative	
in	the	animals	and	immune	protection	in	the	host.                                    approach	and	several	steps	towards	its	development	have	been	taken.	
     Supported	by:	CNPq,	FAPERGS,	FINEP,	PRONEX,	CAPES.                              The	success	of	this	strategy	is	dependent	on	the	cloning	and	charac-
                                                                                     terization	of	the	physiological	function	of	tick	molecules.	A	combination	
Key words:	triosephosphate	isomerase,	boophilus	microplus,	cloning,	
                                                                                     of	antigens	may	be	required	both	to	improve	vaccine	efficacy	and	to	cir-
vaccine
                                                                                     cumvent	possible	parasite	evasion	methods.	The	Glycogen	Synthase	
Species:	ruminants
                                                                                     Kinase	 (GSK-3β)	 is	 a	 serine/threonine	 kinase	 that	 is	 involved	 in	 gly-
                                                                                     cogen	 synthase	 phosphorylation	 in	 glycogen	 metabolism,	 during	 tick	
  Pr145. RECOMBINANT ExPRESSION AND PARTIAL                                          embryogenesis.	Degenerate	PCR	primers	were	designed	to	anneal	to	
CHARACTERIZATION OF AN ASPARTIC PROTEINASE FROM                                      highly	conserved	nucleotide	sequences	based	on	the	sea	urchin	GSK-
             BOOPHILUS MICROPLUS                                                     3β	cDNA	sequence	and	utilized	in	PCR	reactions	to	amplify	a	partial	
PAULA	C	PoHL1,	MARCoS	SoRGInE2,	ITABAJARA	DA	SILVA	VAZ	                              GSK-3β	sequence	from	the	B.	microplus	cDNA.	The	PCR	produced	a	
               JUnIoR1,	3,	AoI	MASUDA1,	4	                                           fragment	 of	 approximately	 600bp,	 corresponding	 to	 the	 partial	 GSK-
   1Centro	de	Biotecnologia-UFRGS;	2Depto	Bioquímica	Médica-                         3β	 cDNA.	To	 obtain	 the	 full-length	 cDNA	 encoding	 GSK-3β,	 5´RACE	
  UFRJ;		3	Depto	Biologia	Molecular	e	Biotecnologia-UFRGS;	4Fac.	                    and	3´RACE	of	cDNA	ends	were	produced	using	the	sequence	of	the	
                        Veterinaria-UFRGS		                                          600bp	amplified	fragment.	The	products	of	5´RACE	and	3´RACE	were	
                       paula@cbiot.ufrgs.br                                          cloned	 in	 pGEM-TEasy	 vector	 and	 sequenced.	 To	 confirm	 that	 the	
                                                                                     5´RACE	and	3´RACE	products	correspond	to	the	authentic	transcripts,	
      The	tick	B.	microplus	is	a	hematophagous	ectoparasite	of	bovine.	              the	 full-length	 cDNA	 was	 amplified	 by	 PCR,	 from	 total	 RNA	 isolated	
The	actual	method	for	the	control	of	cattle	tick	is	the	use	of	chemical	             from	egg.	The	amplified	of	1,230bp	(full-length	cDNA)	was	cloned	into	
pesticides,	 which	 causes	 damage	 to	 environment	 and	 contaminates	              pGEM-TEasy	 and	 sequenced.	 Thereafter	 to	 confirm	 the	 full-length	
the	meat	and	milk.	An	alternative	and	promising	method	for	control	is	               cDNA	sequence,	the	fragment	was	cloned	into	expression	vector	pET-
the	use	of	vaccines.	The	researches	are	guided	towards	the	identifica-               5b,	to	expression	and	purification	of	the	recombinant	protein	and	evalu-
tion	of	new	proteins	that	have	immunoprotective	potential.	THAP	(tick	               ation	of	immunogenicity.
heme-binding	aspartic	proteinase)	is	an	aspartic	proteinase	present	in	
eggs	of	B.	microplus	involved	in	the	embryogenesis,	by	degrading	vitel-                  Supported	 by	 CNPq	 FAPERGS,	 FINEP,	 PRONEX,	 FAPERJ	
lin	according	with	the	necessity	of	embryonic	development.	The	cDNA	                 (Brazil)	and	JSPS	(Japan).
encoding	full	sequence	of	THAP	was	cloned	in	the	pET43a	expression	                  Key words:	Boophilus	microplus,	glycogen	synthase	kinase,	cloning,	
102


      embryogenesis                                                                          tory	 showed	 that	 goats	 acquire	 partial	 resistance	 against	 nymphs	 of	
      Species:	ruminants                                                                     the	lone-star	tick	Amblyomma	cajennense	after	repeated	infestations.	
                                                                                             With	 the	 aim	 to	 search	 for	 antigens	 from	A.	 cajennense	 recognized	
          Pr147. GSK ACTIVITy IS INVOLVED TO PEPCK GENE                                      by	sera	from	repeatedly	infested	goats,	five	animals	aged	six	months,	
          ExPRESSION DURING TICK EMBRyO DEVELOPMENT                                          of	 both	 sexes,	 were	 infested	 thrice	 with	 100	A.	 cajennense	 nymphs	
          CARLoS	LoGULLo1,2,	WILLIAn	WIToLA2,	CARoLInE	                                      at	30	days	interval.	Sera	from	these	goats	were	collected	before	the	
       AnDRADE3,	JoSIAnA	GoMES1,	ITABAJARA	DA	SILVA	VAZ	JR3,	                                infestation	 and	 30	 days	 after	 both	 the	 1st	 and	 3rd	 infestations	 and	
      SAToRo	KonnAI2,	KAZUHIKo	oHASH2,	SAIKI	IMAMURA2,MISAo	                                 used	in	western	blot	analysis	to	identify	potential	antigens	from	unfed	
                             onUMA2                                                          nymphal	 extract	 homogenate.	 Sera	 collected	 from	 goats	 infested	 at	
                                                                                             same	conditions	with	A.	hebraeum	ticks	were	used	to	search	for	cross-
      1Laboratório	de	química	e	Função	de	Proteínas	e	Peptídeos	-	CBB	-	
                                                                                             reactivity	 between	 tick	 antigens	 from	 the	 ixodid	 species.	 These	 later	
      UEnF,	Campos	dos	Goytacazes,	RJ,	Brazil;	2Laboratory	of	Infectious	
                                                                                             sera	revealed	nine	polypeptides	of	12,	19,	24,	34,	46,	52,	66,	83	and	
         Diseases,	Department	of	Disease	Control,	Graduate	School	of	
                                                                                             160	 kDa	 while	 those	 	 collected	 from	 goats	 infested	 once	 and	 thrice	
      Veterinary	Medicine,	Hokkaido	University,	Sapporo,	Hokkaido,	Japan;	
                                                                                             with	A.	cajennense	nymphs	revealed	eight	common	polypeptides	of	26,	
          3Centro	de	Biotecnologia	-	UFRGS,	Porto	Alegre,	RS,	Brazil.		
                                                                                             36,5,	37,5,	55,	72,	86,	98,	and	170	kDa.	At	non-infested	animals’	blots,	
                               logullo@uenf.br
                                                                                             it	was	observed	reactive	polypeptides	as	well,	revealing	the	existence	
            Ticks	 are	 the	 major	 ectoparasite	 that	 causes	 vast	 economical	            of	 cross	 reactivity	 with	 antigens	 from	 ectoparasites	 other	 than	 ticks.	
      losses	in	cattle	production	on	the	world.		The	study	of	the	embryogen-                 However,	it	should	be	stressed	that	three	polypeptides	of	14,	11	and	7	
      esis	of	ticks	has	sees	fundamental	paper	that,	in	agreement	with	the	                  kDa	related	to	A.	cajennense	nymphs	observed	only	when	used	sera	
      drawing	 of	 the	 new	 methodologies	 proposed	 for	 the	 control	 of	 these	          were	from	animals	infested	thrice	could	play	an	important	role	in	induc-
      vectors.		In	many	cases,	genes	that	participate	in	different	metabolic	                ing	 resistance	 in	 goats	 to	 such	 tick	 species.	 Finally,	 the	 presence	 of	
      pathways	or	genes	regulators	of	different	aspects	of	the	adult	organism	               close	 polypeptides	 reactive	 to	 A.	 hebraeum	 serum	 probably	 reveals	
      are	 also	 expressed	 during	 the	 embryogenesis	 and	 your	 modification	             the	 existence	 of	 cross	 reactivity	 between	 the	 two	 ixodid	 species,	 but	
      can	 result	 in	 decrease	 of	 the	 viability	 or	 even	 in	 precocious	 lethality.	   forward	studies	are	needed	to	confirm	it.	
      In	our	previous	work	we	demonstrated	that	the	embryogenesis	of	the	
      hard	tick	Boophilus	microplus	was	metabolically	separated	in	two	spe-                  Key words:	Immunoblotting,	Amblyomma	cajennense,	A.	hebraeum,	
      cial	phases:		An	initial	phase,	until	the	cellular	blastoderm	formation,	              goats
      characterized	by	the	consumption	of	the	maternal	glycogen	and	a	sec-                   Species: ruminants
      ond	phase	characterized	by	an	intense	amino	acids	degradation	that	
      promotes	in	accumulation	of	glycogen	and	glucose	in	this	stage.		We	                    Pr149. TRANSCRIPTOMES OF TICKS FED ON RESISTANT
      also	observed	that	the	specific	activity	of	PEPCK,	a	key	gluconeogen-                   AND SUSCEPTIBLE CATTLE: GENES AFFECTED By HOST
      esis	enzyme,	is	surprisingly	increasing	after	the	embryo	cellularization	                             IMMUNE RESPONSES
      and	its	activity	is	majority	in	the	mitochondrial	compartment.	Glycogen	               SRC	MARUyAMA1,	GR	GARCIA1,	LG	BRAnDÃo1,	JMC	RIBEIRo2,	
      synthase	kinase	3	(GSK3)	has	been	classically	described	as	involved	                           BR	FERREIRA1,	IKF	DE	MIRAnDA	SAnToS1	
      to	phosphorylate	glycogen	synthase.		The	activity	of	GSK3	is	depen-
      dent	 of	 insulin	 cascade	 involving	 a	 protein	 kinase	 B,	 called	Akt.	 	Akt	       1	Departamento	de	Bioquímica	e	Imunologia,	FMRP-USP,	Ribeirão	
      mediates	the	inhibition	of	GSK3	and	contributes	to	activation	of	glyco-                          Preto,	Brasil.	2	NIAID-NIH,	Rockville-MD,	USA
      gen	and	protein	synthesis.		GSK3β	has	been	shown	by	homologue	of	                            Introduction	 and	 Objectives:	 Rhipicephalus	 microplus	 causes	
      the	shaggy	(or	zestewhite	3),	in	Drosophila	melanogaster	and	GSK3	                     enormous	 losses	 for	 animal	 production	 and	 health.	 Ticks	 induce	
      in	 xenopus.	 	 In	 both	 cases	 this	 gene	 is	 implicated	 in	 dorsoventral	         immune	 response	 in	 their	 hosts,	 indicating	 that	 its	 immunobiological	
      patterning	of	the	early	embryos.		GSK3	activity	was	required	for	both	                 control	 is	 possible.	 Bovines	 present	 different	 phenotypes	 related	 to	
      PEPCK	and	G6Pase	promoter	activity	and	which	selective	inhibitors	of	                  intensity	 of	 tick	 infestations	 and	 those	 phenotypes	 are	 mediated	 by	
      reduce	the	gene	transcription	of	these	enzymes	in	mammalians	cells.	               	   qualitatively	distinct	immune	response.	Ticks	fed	on	resistant	bovines	
      However,	there	are	evidences	that	insulin	regulates	a	gene	expression	                 do	not	feed	well	and	display	low	reproductive	efficiency.	Our	hypothesis	
      of	 G6Pase	 and	 PEPCK	 in	 mammals’	 cells.	 In	 the	 present	 study,	 we	            is	that	different	levels	of	host	anti-tick	immunity	affect	gene	expression	
      have	cloned	and	studied	the	expression	of	GSK3	and	PEPCK	during	                       in	 cattle	 ticks.	 The	 objective	 is	 to	 discover	 larvae	 and	 salivary	 gland	
      tick	 embryo	 development.	 	 On	 the	 other	 hand	 we	 have	 investigated	            genes	whose	expression	is	affected	by	immune	responses	of	suscep-
      the	 influence	 of	 GSK	 activity	 in	 the	 PEPCK	 expression	 during	 tick	           tible	and	resistant	hosts.	
      embryogenesis.	 	 We	 also	 demonstrated	 that	 lithium	 chloride,	 insulin	                 Methods	and	Results:	The	cDNA	libraries	were	constructed	with	
      and	SB216763,	the	inhibitors	of	GSK3	activity,	can	reduce	drastically	                 SMART	 (Clontech-BD)	 technology	 and	 mRNA	 from	 unfed	 larvae	 of	
      an	expression	of	PEPCK	in	embryonic	tick	cells	culture.		We	attributed	                unfed	 larvae	 proceeding	 by	 female	 fed	 on	 susceptible	 or	 resistant	
      that	regulation	activity	of	GSK	probably	occurs	by	the	insulin	cascade	                bovines;	salivary	glands	of	nymphs,	males	and	females	fed	on	suscep-
      during	this	development	process.                                                       tible	or	resistant	bovines.	The	clones	were	randomly	selected	for	PCR	
           Supported	by	CNPq,	FAPERJ,	JSPS	and	CAPES.	                                       amplification	and	the	DNA	inserts	were	sequenced	and	analyzed	with	
                                                                                             bioinformatic	tools.	We	generated	7,923	ESTs,		which	were	trimmed	of	
                                                                                             primer	and	vector	sequences,	clusterized	into	3,342	contigs	and	com-
      Pr148. IMMUNOBLOTTING OF AMBLyOMMA CAJENNENSE
                                                                                             pared	with	public	databases	such	as	NR,	GO,	KOG,	P-fam,	SMART,	
      ANTIGENS USING SERA FROM SENSITIZED GOATS. CROSS
                                                                                             rRNA,	MIT-PLA,	and	private	ones	containing	sequences	for	Acari	and	
                 REACTIVITy WITH A. HEBRAEUM                                                 ESTs	 of	 R.	 microplus	 and	 submitted	 in	 batch	 to	 the	 SignalP	 server.	
             GER	MonTEIRo1,	2,	R	Z	MACHADo2,	G	H	BECHARA2                                    Many	clusters	presented	differential	expression	according	to	the	origin	
       1Departamento	de	Paraclínicas,	FV-UEM,	Maputo,	Moçambique;2	                          of	the	ticks	blood	meal	–	susceptible	(RmS)	and	resistant	(RmR)	hosts.	
       Departamento	de	Patologia	Veterinária,	FCAV-UNESP,	Jaboticabal-                       The	 most	 of	 high	 expression	 clusters	 are	 in	 RmH,	 like	 clusters	 with	
                                  SP,	Brazil                                                 similarity	histamine	binding	proteins	and	oxidant	metabolism	proteins.	
                                                                                             Noteworthy,	clusters	presenting	with	similarities	to	anticoagulant	proteins	
           The	control	of	ticks	has	been	a	major	concern	because	of	the	very	
      high	 costs	 of	 the	 chemical	 methods	 of	 control	 usually	 practiced,	 the	        (ACs)	 that	 are	 essential	 success	 blood	 feed	 and	 metalloproteinases	
      environmental	contamination	they	produce,	and	the	development	of	tick	                 (MMPs)	can	be	related	with	scape	immune	response	and	extracellu-
      resistance	problems.	Antigenic	extracts	derived	from	whole	tick	body	                  lar	matrix	degeneration.	RmS	presents	63	ESTs	(expected	43),	while	
      homogenates	of	both	adult	and	immature	instars	and	of	internal	organs	                 RmR	exhibits	14	ESTs	(expected	33)	similar	to	ACs	(P<0.001),	being	
      as	 salivary	 gland	 and	 gut	 of	 partially	 fed	 female	 ticks	 were	 already	       that	expression	was	more	notable	in	nymphs	and	males	stages.	Similar	
      tested	 and	 demonstrated	 to	 induce	 various	 degrees	 of	 resistance	 to	           to	MMPs,	RmH	presents	26	ESTs	(expected	18),	while	RmR	exhibits	7	
      ticks	 in	 several	 hosts.	 This	 approach	 is	 promising	 as	 an	 alternative	        ESTs	(expected	14)	P=0.016	and	this	case	the	differential	expression	
      method	 for	 tick	 control.	 In	 addition,	 preliminary	 results	 of	 the	 labora-     appearance	only	between	males	(RmSxRmN).
                                                                                                                                                                          103


     Conclusion:	 Our	 data	 suggest	 that	 the	 host	 immune	 response	            artificial	infestations	with	15.000	larvae	of	R.	microplus.	Skin	biopsies,	
affects	gene	expression	in	R.	microplus	and	the	study	of	tick	transcrip-            with	 or	 without	 a	 feeding	 tick	 attached,	 were	 collected	 with	 an	 8mm	
tomes	can	be	a	relevant	source	of	antigens	for	vaccines	anti-ticks.                 punch.	 Blood	 from	 the	 biopsy	 lesion	 were	 collect	 immediately	 with	 a	
     Financial	Support:	CNPq,	FAPESP	and	Valée	SA.	                                 capillary	for	counting	CT.	These	experiments	were	performed	with	ticks	
                                                                                    at	different	times	in	their	life	cycle.	Peripheral	blood	was	also	collected	
Key words:		Rhipicephalus	microplus,	Transcriptome,	salivary	glands,	
                                                                                    from	infested	animals	to	compare	the	local	and	systemic	CT.	Results	
host	immune	response
                                                                                    were	analyzed	with	One	Way	ANOVA	test.	The	results	showed	that	in	
Species:	ruminants
                                                                                    susceptible,	 but	 not	 in	 resistant	 bovines	 the	 local	 CT	 is	 significantly	
                                                                                    (P	<	0.05)	increased	in	skin	infested	with	larvae	or	adults	when	com-
Pr150. COMPARATIVE IGG RECOGNITION OF ADULT TICK                                    pared	to		the	CT	of	bleeding	from	uninfested	skin.	There	was	no	dif-
  ExTRACTS By SERA OF ExPERIMENTALLy INFESTED                                       ference	 in	 the	 systemic	 CTs	 of	 resistant	 and	 susceptible	 bovines	 or	
                    BOVINES                                                         between	infested	bovines	and	uninfested	animals.	This	data	suggests	
 APR	CRUZ1,2,	RT	MATToS1,2,	PRM	DE	LISA3,	SS	SILVA3,	I	DA	                          that	tick	saliva	inhibits	the	coagulation	cascade	only	at	the	site	of	tick	
       SILVA	VAZ	JR2,	A	MASUDA2,	CAS	FERREIRA1                                      attachment	 and	 the	 mechanism	 involved	 does	 not	 affect	 the	 animal	
                                                                                    systemically.	The	 fact	 that	 the	 local	 CT	 time	 is	 not	 increased	 in	 tick-
       	1Laboratório	de	Imunologia,	Fabio,	PUCRS;	2Centro	de	
                                                                                    infested	skin	of	resistant	bovines	indicates	that	their	immune	response	
  Biotecnologia,	UFRGS;	3Departamento	de	Veterinária	Preventiva,	
                                                                                    may	 be	 targeting	 and	 neutralizing	 tick	 anticoagulant	 molecules.	 This	
                               UFPel
                                                                                    possibility	is	further	supported	by	the	poor	feeding	efficiency	displayed	
      The	 tick	 Boophilus	 microplus	 is	 a	 hematophagous	 ectoparasite	          by	females	feeding	on	resistant	bovines.
of	bovines,	widely	distributed	in	herds	from	America,	Asia,	Africa	and	
                                                                                    Key words:	Rhipicephalus	microplus,	clotting	time,	Zebuine,	Taurine
Oceania.	 The	 use	 of	 acaricides	 is	 the	 main	 method	 for	 tick	 control,	
                                                                                    Species:	ruminants
however	it	may	become	unfeasible	due	to	the	cost	of	drugs	and	labor	
required	to	apply	the	treatment,	as	well	as	the	increasingly	appearance	
of	resistant	ticks	to	various	acaricides.	Also,	chemical	residues	in	food	             Pr152. IMMUNOBLOT ANALySIS OF IGG ANTIBODy
and	environmental	pollution	are	major	concerns	nowadays.	The	devel-                      RESPONSES TO RHIPICEPHALUS (BOOPHILUS)
opment	of	an	immunological	control	method	as	an	alternative	for	the	                 MICRoPLUS IN RESISTANT AND SUSCEPTIBLE BOVINES.
chemical	control	depends	on	finding	out	tick	antigenic	molecules	that	                WAnESSA	A	CARVALHo1,	ALExAnDRE	FIRMIno2,	DAnIELA	
induce	a	protective	immune	response	in	the	host.	As	bovines	develop	                  D	MoRé1,	GERVASIo	H	BECHARA3,	BEATRIZ	F	DE	MIRAnDA	
resistance	to	ticks	during	successive	infestations,	the	analysis	of	the	                         SAnToS	RoSSETTI,	KF	ISABEL1
immune	 responses	 developed	 by	 infested	 bovines	 may	 become	 of	
                                                                                    1Dept.	Biochemistry	and	Immunology,	Ribeirão	Preto	Medical	School,	
great	importance	in	the	search	for	protective	antigens.	Enzyme-linked	
                                                                                    University	of	São	Paulo,	Ribeirão	Preto,	Brazil;	2EMBRAPA	Genetics	
immunosorbent	assay	(ELISA)	and	western-blot	analyses	were	used	
                                                                                     Resources,	Brasília,	Brazil;	3Dept.	Veterinary	Pathology,	School	of	
to	 investigate	 the	 antibody	 responses	 of	 six	 bovines	 experimentally	
                                                                                     Veterinary	and	Agronomical	Sciences,	São	Paulo	State	University,	
infested	twelve	times,	during	18	months,	with	B.	microplus against	sali-
                                                                                                             Jaboticabal,	Brazil		
vary	gland,	gut	and	larvae	protein	extracts.	The	levels	of	IgG	against	
                                                                                                          wanac30@hotmail.com
all	three	extracts	presented	an	increase	following	the	initial	infestations	
whereas	a	significant	decrease	was	shown	following	the	final	infesta-                      Bovines	 express	 breed-specific,	 heritable,	 contrasting	 pheno-
tions.	Individual	variations	were	observed	in	ELISA,	as	well	as	and	in	             types	 when	 exposed	 to	 the	 cattle	 tick,	 R.	 microplus.	 Breeds	 of	 Bos	
the	 pattern	 of	 molecules	 recognized	 by	 western-blot,	 which	 showed	          indicus	(R)	are	significantly	more	resistant	than	those	of	B.	Taurus	(S).	
that	a	greater	number	of	antigens	were	recognized	by	the	initial	infesta-           Different	breeds	develop	qualitative	and	quantitative	different	immune	
tions	sera	with	the	presence	of	different	profiles.	Although	the	profiles	          responses	against	ticks	including	production	immunoglobulin	and	anti-
of	the	final	infestations	show	a	scarcer	number	and	less	intense	mol-               bodies	specific	for	tick	saliva.	This	study	was	performed	to	determine	
ecules,	new	molecules	were	recognized	and	some	increased	its	rela-                  the	 frequencies	 and	 specificities	 of	 bovine	 IgG	 antibodies	 binding	 to	
tive	intensity.	Higher	IgG	levels	and	a	major	number	of	molecules	were	             components	 of	 saliva,	 egg	 extracts	 (EE)	 and	 unfed	 larvae	 extracts	
observed	against	salivary	gland	extract,	which	corroborate	that	saliva	is	          (UFLE)	 of	 R.	 microplus	 and	 also	 to	 determine	 whether	 there	 are	 dif-
the	greater	natural	source	of	immunogenic	molecules	of	B.	microplus.                ferent	 immunological	 patterns	 of	 recognition	 of	 proteins	 from	 these	
                                                                                    materials	by	R	(n=5;	B.	indicus,	Nelore	breed)	and	S	(n=5;	B.	Taurus,	
     Support:	CAPES,	FAPERGS,	CNPq	e	CNPq/PRONEX-FAPERJ.
                                                                                    Holstein	breed)	bovines	undergoing	high	and	low	natural	tick	infesta-
Key words:	Boophilus	microplus,	tick	antigens,	immunoglobulin	G                     tions.	In	order	to	determine	the	level	of	infestation	in	each	breed	ticks	
Species:	ruminants                                                                  were	counted	on	one	side	of	S	cattle	at	weekly	intervals	for	the	duration	
                                                                                    of	 the	 experiment	 and	 serum	 was	 collected	 at	 different	 time	 points.	
      Pr151. INFESTATIONS WITH RHIPICEPHALUS                                        Proteins	from	tick	saliva,	EE	and	UFLE	were	separated	by	SDS-PAGE	
  (BOOPHILUS) MICRoPLUS TICKS INCREASE LOCAL AND                                    and	were	electrophoretically	transferred	onto	nitrocellulose	(NC)	paper.	
   SySTEMIC BLOOD CLOTTING TIME IN TICK RESISTANT                                   IgG-binding	proteins	were	detected	in	individual	sera	from	infested	ani-
              AND SUSCEPTIBLE CATTLE                                                mals	and	as	a	control	we	used	the	sera	from	these	animals	before	they	
                                                                                    have	any	contact	with	the	ectoparasite.	A	total	of	fifteen	protein	bands	
    WAnESSA	A	CARVALHo1,	DAnIELA	D	MoRé1,	AnTônIo	
                                                                                    with	molecular	weights	ranging	from	200	to10	KDa	were	recognized	in	
    ABATEPAULo1,	GUSTAVo	R	GARCIA1,	ALESSAnDRA	M	
                                                                                    tick	saliva,	but	resistant	cattle	recognized	less	protein	bands	whether	
   FRAnZIn1,	LUÍS	HEnRIqUE	A	ConTI2,	AnTônIo	A	MAIA2,	
                                                                                    exposed	to	high	or	low	numbers	of	ticks.	EE	and	UFLE	are	recognized	
  GERVASIo	H	BECHARA3,	ISABEL	KF	DE	MIRAnDA	SAnToS1
                                                                                    with	the	same	pattern	by	R	and	S	bovines	and	this	pattern	is	consistent	
1Dept.	Biochemistry	and	Immunology,	Ribeirão	Preto	Medical	School,	                 across	all	levels	of	infestation.	Salivary	proteins	were	separated	in	a	
  University	of	São	Paulo,	Ribeirão	Preto,	Brazil;	2Dept.	Zootenics,	               2-D	 gel	 and	 transferred	 to	 NC	 paper.	A	 spot	 differentially	 recognized	
  Pirassununga	Veterinary	and	Zootecnic	School,	University	of	São	                  by	R	sera	was	eluted	from	a	replicate	gel	and	its	amino	acid	sequence	
 Paulo,	Pirassununga,	Brazil;	3Dept.	Veterinary	Pathology,	School	of	               revealed	100%	homology	with	bovine	hemoglobin,	however	its	pI	was	
  Veterinary	and	Agronomical	Sciences,	São	Paulo	State	University,	                 not	 as	 expected	 for	 the	 native	 protein,	 suggesting	 it	 had	 been	 modi-
                         Jaboticabal,	Brazil		                                      fied	 in	 the	 tick	 salivary	 gland.	 Immunoblots	 of	 saliva	 with	 lectins	 and	
                   E-mail:	wanac30@hotmail.com                                      anti-Lewisx	 antibody	 indicate	 that	 the	 modification	 is	 due	 to	 fucosyl-
      Tick	 saliva	 contains	 many	 molecules	 described	 as	 inhibitors	 of	       ated	 carbohydrates.	 Our	 results	 indicate	 that	 there	 is	 a	 difference	 in	
blood	coagulation	in	vitro.	It	is	believed	that	these	molecules	promote	            the	reactivity	to	antigenic	proteins	in	saliva	from	R.	microplus	and	that	
efficiency	of	blood	feeding	by	ticks,	but	this	effect	has	not	been	evalu-           the	level	of	infestation	is	related	to	the	antibody	response	of	the	host.	
ated	in	vivo.	This	work	was	performed	to	evaluate,	in	vivo,	the	systemic	           In	addition,	antigens	that	are	differentially	recognized	by	resistant	hosts	
and	 local	 clotting	 time	 (CT)	 in	 tick-resistant	 (Bos	 indicus,	 Nelore;	 n	   may	target	components	of	tick	saliva	that	are	essential	for	blood	feed-
=	 4)	 and	 -susceptible	 cattle	 (B.	 taurus,	 Holstein;	 N	 =	 4)	 undergoing	    ing	and	constitute	useful	antigens	for	a	vaccine.
104


      Key words:	 Rhipicephalus	 microplus,	 antibody,	 lectin,	 saliva,	                 great	economical	damages	in	tropical	and	subtropical	countries.	The	
      immunoblot                                                                          transmission	occurs	due	to	the	haematophagy	of	the	arthropods	from	
      Species:	ruminants                                                                  the	 family	 Ixodidae,	 being	 the	 biological	 vector	 in	America	 is	 the	 tick	
                                                                                          Riphicephalus	(Boophilus)	microplus.	In	the	present	work	the	humoral	
         Pr153. KINETICS OF LEUKOCyTE RESPONSES OF                                        immune	 response	 of	 the	 Holstein	 breed	 7/8	 (Holstein	 x	 Gir)	 animals	
       BOVINES IMMUNIZED WITH SBBO23290 (BABESIA BoVIS)                                   was	evaluated.	The	animals	were	immunized	with	the	synthetic	immu-
      AND SBM7462 (RIPHICEPHALUS (BooPHILUS) MICRoPLUS)                                   nogens	SBbo23290	and	SBm7462	for	the	control	of	Babesia	bovis	and	
                                                                                          R.	(B.)	microplus,	respectively.	They	were	divided	in	four	experimental	
      DIoGo	CoELHo	DE	PADUA	oLIVEIRA,	HUGo	GUIEIRo	RIBEIRo	                               groups,	 each	 one	 with	 four	 animals	 that	 received	 three	 immuniza-
      RoCHA,	BRUnA	ALVES	DEVénS,	MARILIA	noGUEIRA	DA	GAMA,	                               tions	 every	 30	 days,	 via	 the	 subcutaneous	 route.	 In	 the	 Group	 I	 the	
       AnnA	PAULA	B	RIBEIRo	FERREIRA,	CARLA	LEITE	MEDEIRoS,	                              inoculation	 were	 applied	 in	 the	 cervical	 area,	 right	 and	 left	 sides,	 for	
        JoAqUÍn	HERnAn	PATARRoyo,	MARLEnE	ISABEL	VARGAS	                                  each	 one	 of	 the	 immunogens	 individually	 (monovalent	 form),	 using	
                              VILoRIA                                                     saponina	 as	 adjuvant.	 In	 the	 Group	 II	 these	 peptides	 were	 prepared	
          Laboratory	of	Biology	and	Control	of	Haematozoa	and	Vectors,	                   in	 association	 doses	 (polyvalent	 form),	 inoculated	 in	 the	 same	 side	
       Institute	of	Biotechnology	Applied	to	Agriculture	and	Animal	Science	              of	 the	 cervical	 area,	 using	 saponina	 as	 adjuvant.	The	 animals	 of	 the	
        (BIOAGRO/Veterinary	Departament),	Federal	University	of	Viçosa,	                  Group	III	received	2mL	of	Milli-Q	water	and	the	animals	of	the	Group	
                           36571-000,	Viçosa,	MG,	Brazil	                                 IV	received	1,5mg	of	saponina.	The	animals	were	challenged	30	and	
                                  jpatarro@ufv.br                                         34	days	after	the	third	inoculation,	being	put	±2000	larvaes	of	R.	(B.)	
            The	tick	Riphicephalus	(Boophilus)	microplus	is	responsible	for	the	          microplus	 (haemoparasites	 free)	 and	 inoculating	 the	 B.	 bovis	 (strain	
      great	economical	damages	in	the	cattle	breeding,	because	it	causes	                 UFV1	-	9th	passage	-	1x106	babesias/mL),	respectively.	The	evaluation	
      reduction	in	the	production	of	milk	and	meat	and	it	provokes	damages	               of	 the	 humoral	 response	 was	 made	 by	 ELISA	 test	 (l=	 492nm)	 and	
      to	the	leather.	This	parasite	is	the	transmitter	of	the	protozoa	Babesia	           the	 data	 were	 submitted	 to	 the	 analysis	 of	 variance	 and	 comparison	
      bovis	and	Babesia	bigemina	and	of	the	rickettsiae	Anaplasma	margi-                  among	 averages	 applying	 the	 Dunnet	Test.	The	 results	 showed	 high	
      nale,	 which	 cause	 the	 Bovine	Tick	 Fever.	 Nowadays	 these	 parasites	          levels	 of	 specific	 total	 IgG	 for	 the	 peptide	 SBbo23290	 since	 the	 first	
      present	high	incidence	and	prevalence	in	Brazil	and	in	others	countries	            week	after	the	inoculation,	with	titles	of	1,14	± 0,2968	for	monovalent	
      of	tropical	climate.	The	present	work	had	as	objective	to	analyze	the	              and	 1,06	 ±0,2486	 for	 polyvalent	 form.	 The	 titles	 of	 specific	 total	 IgG	
      leukocyte	kinetic	of	bovine	immunized	with	the	synthetic	immunogens	                for	SBm7462	were	present	in	high	levels	in	the	eighth	week	with	1,75	
      SBm7462	 and	 SBbo23290,	 in	 the	 monovalent	 and	 polyvalent	 forms.	             ±0,1849	 for	 the	 monovalent	 and	 1,13	 ±0,2819	 for	 polyvalent	 form.	
      These	 peptides	 were	 prepared	 using	 saponina	 as	 adjuvant,	 being	             The	humoral	response	for	the	peptide	SBbo23290	had	high	levels	of	
      applied	 via	 the	 subcutaneous	 route,	 individually	 in	 the	 left	 and	 right	   IgG1	on	IgG2.	It	can	be	concluded	that	crossed	responses	conferred	
      cervical	area	(Treatment	1)	and	in	associated	doses	in	the	same	side	               by	 intraclonal	 competitions	 could	 be	 determining	 the	 immunological	
      of	the	cervical	area	(Treatment	2).	Each	treatment	was	applied	three	               effect	 in	 the	 Groups	 I	 and	 II.	The	 immunological	 protective	 response	
      times	 with	 intervals	 of	 30	 days.	The	 challenge	 was	 made	 30	 and	 34	       conferred	by	the	peptide	SBm7462	in	the	polyvalent	form	did	not	have	
      days	after	the	third	inoculation,	being	put	±2000	larvaes	of	R.	(B.)	micro-         a	direct	effect	in	the	production	of	specific	IgG1	for	this	peptide,	and	
      plus	 (strain	 free	 from	 haemoparasites)	 and	 inoculating	 the	 B.	 bovis	       specific	 IgG1	 for	 the	 peptide	 SBbo23290	 showed	 crossed	 protection	
      (strain	UFV1	-	9th	passage	in	the	concentration	of	1x106	babesias/mL),	             with	the	peptide	SBm7462.
      respectively.	Through	the	analysis	of	the	results,	was	observed	that	all	           Key words:	 Humoral	 immune	 response,	 synthetic	 immunogens,	
      the	 vaccinated	 animals,	 independently	 of	 the	 inoculation	 form	 of	 the	      Babesia	bovis,	Riphicephalus	(Boophilus)	microplus
      immunogen	(monovalent	and	polyvalent),	presented	alterations	in	the	                Species:	ruminants
      population	of	lymphocytes	present	in	the	circulatory	blood.	Among	the	
      sub-populations	of	lymphocytes	analyzed,	there	was	more	prominence	                 Pr155. BIOLOGICAL PARAMETERS OF TICKS COLLECTED
      for	the	B	cells	CD21+	and	T	cells	WC1+,	which	have	important	functions	              FROM ANIMALS IMMUNIZED WITH PEPTIDES SBBO23290
      in	the	control	of	the	babesioses.	It	was	also	verified	by	the	analysis	of	                             AND SBM7462
      the	samples,	accomplished	five	days	after	the	second	vaccination,	a	
                                                                                             JAVIER	AnTônIo	BEnEVIDES	MonTAño,	HUGo	GUIEIRo	
      significant	increase	in	the	number	of	T	cells	CD4+.	Through	the	evalu-
                                                                                             RIBEIRo	RoCHA,	MARILIA	noGUEIRA	DA	GAMA,	VInICIUS	
      ation	of	the	cellular	immune	response	presented	by	the	animals,	it	can	
                                                                                            E	B	CAMPoS,	AnnA	PAULA	B	RIBEIRo	FERREIRA,	GABRIEL	
      be	 concluded	 that	 the	 synthetic	 peptides	 SBbo23290	 and	 SBm7462	
                                                                                           DoMInGoS	CARVALHo,	FABRÍCIo	LUCIAnI	VALEnTE,	JULIAnA	
      in	the	treatments	I	and	II,	using	saponina	as	adjuvant,	were	capable	
                                                                                              DEL	GIúDICE	PAnIAGo,	JoAqUÍn	HERnAn	PATARRoyo
      to	stimulate	the	immune	system	of	the	bovine	and,	besides,	occurred	
      a	high	association	between	the	conferred	protection	by	the	synthetic	                   Laboratory	of	Biology	and	Control	of	Haematozoa	and	Vectors,	
      peptides	and	great	levels	of	T	cells	WC1+	and	CD4+.                                  Institute	of	Biotechnology	Applied	to	Agriculture	and	Animal	Science	
                                                                                            (BIOAGRO/Veterinary	Departament),	Federal	University	of	Viçosa,	
      Key words:	 Leukocyte	 kinetic,	 Babesia	 bovis,	 Riphicephalus	
                                                                                                               36571-000,	Viçosa,	MG,	Brazil
      (Boophilus)	microplus,	Vaccine
      Species:	ruminants                                                                                                  jpatarro@ufv.br
                                                                                                The	 tick	 Riphicephalus	 (Boophilus)	 microplus,	 belonging	 to	 the	
          Pr154. HUMORAL IMMUNE RESPONSES TO THE                                          family	 Ixodidae,	 is	 considered	 the	 main	 ectoparasite	 with	 economi-
       SyNTHETIC IMMUNOGENS SBBO23290 (BABESIA BOVIS)                                     cal	 importance,	 due	 to	 its	 capacity	 to	 cause	 damages	 to	 the	 animal	
      AND SBM7462 (RIPHICEPHALUS (BOOPHILUS) MICROPLUS)                                   host,	 through	 the	 haematophagy,	 inoculation	 of	 toxins,	 depreciation	
         JAVIER	AnTônIo	BEnEVIDES	MonTAño,	HUGo	GUIEIRo	                                  of	the	leather	and	transmission	of	multiple	pathogens	as	the	haema-
          RIBEIRo	RoCHA,	VInICIUS	E	B	CAMPoS,	AnnA	PAULA	B	                               tozoa	 Babesia	 bovis	 and	 Babesia	 bigemina.	 In	 countries	 of	 tropical	
         RIBEIRo	FERREIRA,	CARLoS	HEnRyqUE	SoUZA	E	SILVA,	                                and	 subtropical	 climate	 the	 babesioses	 constitutes	 a	 limitant	 factor	
       GABRIEL	DoMInGoS	CARVALHo,	LARISSA	TAVARES	CyRIno,	                                for	 the	 development	 of	 the	 cattle	 breeding,	 causing	 problems	 to	 the	
       SIDIMAR	SoSSAI,	JoAqUÍn	HERnAn	PATARRoy	o,	MARLEnE	                                animal	 health,	 with	 high	 mortality	 and	 morbosity,	 losses	 in	 the	 meat	
                       ISABEL	VARGAS	VILoRIA                                              and	milk	production,	and	high	costs	with	the	prophylaxia	 and	control	
                                                                                          of	the	diseases.	The	objective	of	the	present	work	was	to	evaluate	the	
          Laboratory	of	Biology	and	Control	of	Haematozoa	and	Vectors,	                   biological	 parameters	 of	 ticks	 collected	 from	 bovines	 of	 the	 Holstein	
       Institute	of	Biotechnology	Applied	to	Agriculture	and	Animal	Science	              breed	 7/8	 (Holstein	 x	 Gir)	 which	 were	 immunized	 with	 the	 synthetic	
        (BIoAGRo/Veterinary	Departament),	Federal	University	of	Viçosa,	                  peptides	 SBbo23290	 and	 SBm7462,	 against	 B.	 bovis	 and	 R.	 (B.)	
                           36571-000,	Viçosa,	MG,	Brazil	                                 microplus,	respectively,	in	the	monovalent	and	polyvalent	forms.	These	
                                  jpatarro@ufv.br                                         peptides	were	prepared	using	saponina	as	adjuvant,	being	applied	via	
           The	 bovine	 babesioses	 caused	 by	 a	 protozoa	 obligatory	 intra-           the	subcutaneous	route,	individually	in	the	left	and	right	cervical	area	
      cellular	 parasite	 from	 the	 generous	 Babesia.	 This	 illness	 occasions	        (Treatment	1)	and	in	associated	doses	in	the	same	side	of	the	cervical	
                                                                                                                                                                            105


area	(Treatment	2).	Each	treatment	was	applied	three	times	with	inter-                  Pr157. TICK INFESTATIONS AFFECT SUBPOPULATIONS
vals	of	30	days.	The	challenge	was	made	30	and	34	days	after	the	third	                   OF PERIPHERAL BLOOD LyMPHOCyTES OF BOTH
inoculation,	 being	 put	 ±2000	 larvaes	 of	 R.	 (B.)	 microplus	 (strain	 free	          SUSCEPTIBLE AND RESISTANT BOVINE HOSTS
from	haemoparasites)	and	inoculating	the	B.	bovis	(strain	UFV1	-	9th	                    D	D	MoRé1,	I	K	DE	MIRAnDA	SAnToS1,	A	M	FRAnZIn1,	W	A	
passage	in	the	concentration	of	1x106	babesias/mL),	respectively.	The	                    CARVALHo1,	A	A	M	MAIA2,	J	S	SILVA1,	A	K	SToRSET3,	M	A	
engorged	females	were	collected	after	the	21st	day	after	the	challenge,	                                 JUTILA	4,	B	R	FERREIRA5
weighed	 individually	 in	 precision	 scale,	 identified	 and	 conditioned	 in	
culture	 plates	 of	 24	 wells.	They	 were	 incubated	 at	 28ºC	 and	 relative	        1Department	of	Biochemistry	and	Immunology,	School	of	Medicine	
                                                                                       of	Ribeirão	Preto,	University	of	São	Paulo,	Brazil;	2Department	of	
humidity	 of	 80%,	 and	 the	 eggs	 were	 collected	 and	 weighed	 until	 15	
                                                                                       Basic	Sciences,	School	of	Animal	Science	and	Food	Engineering,	
days	after	the	laying.	The	data	were	submitted	to	the	analysis	of	vari-
                                                                                        University	of	São	Paulo,	Brazil;	3Department	of	Food	Safety	and	
ance	and	comparison	among	averages	applying	the	Dunnet	Test.	The	
                                                                                        Infection	Biology,	Norwegian	School	of	Veterinary	Science,	Oslo,	
results,	in	relation	to	the	reduction	of	the	eggs	weight	were	2.5%	and	
                                                                                      Norway;	4Department	of	Veterinary	Molecular	Biology,	Montana	State	
8.8%	in	the	Treatments	1	and	2,	respectively.	The	Treatment	2	showed	
                                                                                       University,	Bozeman,	USA;	5Department	of	Maternal-Child	Nursing	
a	 great	 reduction	 in	 the	 number	 of	 the	 engorged	 females	 (32.98%),	
                                                                                      and	Public	Health,	School	of	Nursing	of	Ribeirão	Preto,	University	of	
decrease	 in	 the	 fertility	 (14.24%)	 and	 a	 efficacy	 of	 the	 immunogens	
                                                                                                                São	Paulo,	Brazil	
(46.80%).	 It	 can	 be	 concluded	 that	 the	 associated	 immunogens	 rep-
                                                                                                                ddmore@usp.br
resent	 an	 important	 tool	 for	 the	 control	 of	 the	 populations	 of	 R.	 (B.)	
microplus,	reducing	the	economic	losses	caused	by	this	parasite.                            Success	of	tick	infestation	depends	on	the	outcome	of	host	defense	
                                                                                      responses	 versus	 parasite	 escape	 mechanisms.	 Bovines	 express	
Key words:	Biological	parameters,	tick,	peptides,	vaccine                             breed-specific,	heritable,	contrasting	phenotypes	when	exposed	to	the	
Species:	ruminants                                                                    cattle	tick,	Rhipicephalus	microplus.	Breeds	of	Bos	indicus	(i.e.	Nelore)	
                                                                                      are	significantly	more	resistant	than	those	of	Bos	taurus	(i.e.	Holstein),	
Pr156. ExPRESSION OF PROINFLAMMATORy CyTOKINES                                        while	 animals	 from	 populations	 derived	 from	 crosses	 between	 these	
AND CHEMOKINES IS INCREASED IN TICK-INFESTED SKIN                                     groups	 show	 varying	 levels	 of	 resistance.	 This	 offers	 a	 good	 oppor-
             OF RESISTANT BOVINES                                                     tunity	 to	 identify	 specific	 mechanisms	 of	 response	 associated	 with	
ARR	ABATEPAULo,	WA	CARVALHo,	DD	MoRé,	BR	FERREIRA,	JS	                                resistance	 to	 ticks,	 once	 that	 different	 breeds	 develop	 qualitatively	
           DA	SILVA,IKF	DE	MIRAnDA	SAnToS                                             different	 immune	 responses	 against	 ticks.	In	order	 to	identify	cellular	
                                                                                      differences	 between	 these	 breeds,	 20,000	 larvae	 of	 Rhipicephalus	
    Dept.	of	Biochemistry	and	Immunology,	Ribeirão	Preto	Medical	
                                                                                      microplus	tick	were	placed	on	the	back	of	susceptible	(n=4)	and	resis-
         School,	University	of	São	Paulo,	Ribeirão	Preto,	SP
                                                                                      tant	(n=4)	animals	and	the	tick	infestation	was	accompanied	for	three	
      Introduction	and	Objectives	Ticks	are	hematophagous	arthropods	                 weeks.	The	blood	samples	were	collected	in	each	stage	of	infestation	
that	cause	serious	losses	to	animal	and	public	health	and	demand	new	                 (larvae,	 nymph	 and	 adult),	 at	 one	 week	 interval,	 for	 flow	 cytometer	
control	methods.		Vaccines	are	one	alternative	because	hosts	produce	                 analysis	 for	 CD3,	 CD4,	 CD8,	 NKp46,	 TCR	 γd	 and	 WC1	 molecules	
immune	responses	against	the	parasite.	It	is	necessary,	however,	to	first	            expression	in	two	consecutive	infestations.	At	the	last	infestation,	the	
understand	 the	 mechanisms	 that	 participate	 in	 protective	 responses	            brachial	lymph	nodes	also	were	collected	for	the	same	analysis.	The	
against	ticks.		Bovine	hosts	express	heritable,	contrasting	phenotypes	               results	of	peripheral	blood	showed	that,	during	the	first	infestation,	the	
of	infestations,	there	being	susceptible	(S)	or	resistant	breeds	(R).		This	          percentage	 of	 CD4+	 cells	 in	 adult-infested	 susceptible	 animals	 was	
work	seeks	to	compare	the	expression	profile	of	genes	that	code	for	                  augmented	 two	 fold	 relative	 to	 larvae-	 and	 nymph-infested	 bovines.	   	
molecules	that	are	candidates	to	mediate	the	anti-R.	microplus	immune	                Moreover,	CD8+	cells	were	significantly	increased	during	the	infestation	
responses	that	may	result	in	these	phenotypes.                                        with	nymphs	in	both	breeds	when	compared	with	other	stages	of	the	
      Methods	 and	 Results:	 Genetically	 susceptible	 (Holstein;	 N	=		5)	          life	cycle	(P=0.004	in	Holsteins;	P	<0.001	in	Nelores).	Additionally,	the	
and	 resistant	 (Nelore;	 	 N	 =	 5)	 breeds	 of	 bovines	 were	 managed	             percentage	of	p46NK+	cells	was	higher	in	larvae-infested	susceptible	
in	 a	 pasture	 naturally	 infested	 with	 the	 cattle	 tick,	 Rhipicephalus	         animals	than	in	larvae-infested	resistant	animals	(P=0.004).	During	the	
(Boophilus)	 microplus,	 and	 weekly	 tick	 counts	 were	 done	 during	 16	           larval	 and	 nymph	 stages,	 two	 time	 tick-infested	 Holsteins	 presented	
months.		Biopsies	of	normal	and	infested	(with	an	ingurgitating	female	               a	 significant	 reduction	 (P<0.001)	 on	 the	 percentage	 of	 p46NK+	 cells	
tick)	skin	were	collected	from	each	animal	at	distinct	time	points	and	               when	compared	to	the	first	infestation,	while	during	the	nymph	stage	
                                                                                      two	time	tick-infested	Nelores	presented	a	decrease	in	CD8+	lympho-
RNA	 was	 extracted.	 Expression	 was	 measured	 by	 quantitative	 RT-
                                                                                      cytes	(P=0.004).	No	differences	in	lymph	nodes	cell	populations	were	
PCR	 for	 the	 following	 candidate	 genes:	 	 TGF-β,	 IFN-γ,	 TNF-a,	 IL-4,	
                                                                                      observed.	Taken	together,	these	results	indicate	that	tick	infestations	
IL-8,	IL-10,	IL-18,	iNOS,	IFN-a,	MIP1-a	IP-10,	IGF-1,	MCP-1,	IDO	and	
                                                                                      affect	the	blood-lymphocyte	populations	in	both	resistant	and	suscep-
IL5.	Expression	of	IGF-1,	a	chemokine	that	is	selective	for	basophils,	
                                                                                      tible	cattle	breeds.	Support:	CNPq,	FAPESP,	Valleé.
was	 significantly	 higher	 in	 the	 infested	 skin	 of	 R	 bovines	 when	 com-
pared	 with	 its	 expression	 in	 S	 bovines	 undergoing	 high	 and	 medium	          Key words:	Tick	infestation,	Cattle,	Lymphocytes
tick	infestations	but	not.	Expression	of	IL-8,	and	IDO	was	significantly	             Species:	ruminants
(P	=	0,05,	t	test)	higher	in	the	infested	skin	of	R	bovines	when	com-
pared	with	expression	in	S	bovines	during	periods	of	high	infestations.	                   Pr158. TLR ExPRESSION IN BOVINE MONOCyTES
Expression	 of	 MCP-1,	 MIP1-a	 and	 	 iNOS	 was	 higher	 in	 the	 infested	              DERIVED FROM CATTLE BREEDS WITH DIFFERING
skin	of	R	bovines	when	compared	with	expression	in	S	bovines	dur-                            SUSCEPTIBILITy TO TROPICAL THEILERIOSIS
ing	periods	of	high	infestations	(P=0,06	t	test),	Expression	of	the	other	                  LA	RoWAn,	GD	MAKInS,	L	SMITH,	K	JEnSEn,EJ	GLASS
proinflammatory	cytokines	was	higher	in	the	infested	skin	of	R	bovines	
                                                                                                 Roslin	Institute,	Roslin,	Midlothian,	EH25	9PS,	UK
when	compared	with	normal	skin	of	same	animal	and	with	infested	skin	
of	S	bovines,	although	the	differences	were	not	significant.                               Theileria	annulata	is	an	apicomplexan	parasite	of	global	economic	
                                                                                      importance.	 It	 is	 the	 causative	 agent	 of	 tropical	 theileriosis,	 which	 is	
    Conclusion:	Our	findings	show	that	tick	infestations	modulate	the	
                                                                                      a	debilitating	disease	of	cattle.	It	is	spread	by	ticks	of	the	Hyalomma	
expression	 of	 various	 molecules	 that	 may	 participate	 in	 the	 distinct	
                                                                                      species	 and	 covers	 areas	 from	 the	 Mediterranean	 basin	 to	 China.	
outcomes	of	infestations	in	R	and	S	bovine	hosts.	Taken	together	they	
                                                                                      European	cattle	such	as	Holstein-Friesians	(Bos	taurus)	are	very	sus-
suggest	that	basophils	are	pivotal	effector	cells,	and	that	their	recruit-
                                                                                      ceptible	 to	 tropical	 theileriosis	 and	 the	 mortality	 rates	 amongst	 these	
ment	is	more	efficient	in	R	hosts,	in	which	a	TH1	profile	dominates	the	              breeds	 is	 between	 40	 and	 90%.	 However,	 several	 breeds	 native	 to	
microenvironment	of	the	tick’s	feeding	site.                                          endemic	areas,	such	as	the	Sahiwal	cattle	(Bos	indicus)	from	Pakistan,	
     Supported	by	FAPESP,	CNPq	and	Vallée	SA.                                         are	tolerant	to	the	disease.
Key words:	 Resistence,	 Tick,	 Chemokines,	 PROINFLAMMATORY	                               T.	 annulata	 primarily	 infects	 bovine	 macrophages,	 which	 are	
CYTOKINES                                                                             reversibly	transformed	causing	the	macrophage	and	parasite	to	pro-
Species:	ruminants                                                                    liferate	 in	 synchrony.	As	 a	 result	 of	 this	 transformation	 ex-vivo	 cell	
106


      lines,	derived	from	experimental	infection	of	cattle,	have	been	grown	              1Division	of	Clinical	Veterinary	Sciences,	University	of	Edinburgh,	
      in	 culture.	 Infection	 with	 T.	 annulata	 alters	 the	 function	 and	 phe-        Edinburgh,	United-Kingdom;	2International	Livestock	Research	
      notype	 of	 the	 macrophage,	 for	 example,	 the	 pathogen	 recognition	                    Institute,	P,	O,	Box	30709,	Nairobi	00100,	Kenya
      receptor	 (PRR)	 CD14,	 which	 is	 involved	 in	 macrophage	 activation,	               Theileria	parva	is	an	intracellular	protozoal	parasite	which	causes	
      is	down-regulated.	In	order	to	better	understand	the	roles	of	PRRs	               East	Coast	Fever	(ECF),	a	major	constraint	to	cattle	production	in	east-
      during	 infection	 with	 T.	 annulata	 we	 have	 investigated	 the	 expres-       ern	and	southern	Africa.	Characteristics	of	the	protective	CD8+	T-cell	
      sion	 of	 Toll-like	 receptors	 (TLRs)	 in	 resting,	 activated	 and	 infected	   response	against	T.	parva	suggest	it	is	focused	on	a	limited	number	of	
      monocytes.                                                                        immunodominant	epitopes	that	exhibit	polymorphism	between	parasite	
            Using	 ex-vivo	 cell	 lines	 we	 have	 shown	 that	 all	 10	 TLRs	 are	     strains.	The	 recent	 identification	 of	 antigens	 recognised	 by	 T.	 parva-
      expressed	during	T.	annulata	infection	and	a	correlation	between	pro-             specific	CD8+	T-cells	has	created	new	opportunities	in	the	development	
      inflammatory	cytokines	and	TLR	expression	has	also	been	observed.	                of	a	subunit	vaccine,	and	permitted	the	specificity	of	the	CD8+	response	
      A	bovine	macrophage-specific	cDNA	microarray	has	been	constructed	                to	 be	 examined.	 Cyotoxicity	 assays	 with	 peptide-loaded	 targets,	 and	
      to	investigate	breed-specific	differences	in	Sahiwal	and	Holstein	mono-           analyses	of	TCRBV	gene	usage	have	been	used	to	study	the	antigen	
      cytes	during	infection	with	T.	annulata	and	after	activation	with	lipopoly-       specificity	and	clonal	composition	of	the	CD8+	memory	population	in	T.	
      saccharide	 and	 interferon-γ.	Analysis	 of	 the	 microarray	 data	 showed	       parva	 immune	 animals.	Analyses	 of	 large	 panels	 of	 T.	 parva-specific	
      a	 breed-specific	 difference	 in	 TLR10	 expression	 during	 monocyte	           CD8+	 T-cell	 clones	 from	 memory	 T-cells	 of	 immune	 animals	 homo-
      infection	 and	 activation.	 In	 Sahiwal	 macrophages	 TLR10	 expression	         zygous	 for	 MHC	 haplotypes	 expressing	 either	 the	A10	 or	A18	 class	
      is	up-regulated	after	infection	and	activation,	while	the	Holstein	macro-         I	 specificities,	 revealed	 that	 over	 65%	 of	 the	 response	 was	 directed	
      phages	slightly	down-regulate	TLR10	at	the	same	time	point.	                      against	 a	single	 immunodominant	 epitope	 (Tp1.1	for	A18	and	Tp2.2.	
           	Additional	studies	are	in	progress	to	investigate	further	the	impor-        for	A10).	TCRB-chain	sequence	analysis	showed	that	for	each	immu-
      tance	of	TLRs	and	pro-inflammatory	cytokines	and	to	elucidate	further	            nodominant	 epitope	 the	 antigen-specific	 T-cells	 were	 polyclonal	 but	
      the	roles	they	play	during	T.	annulata	infection.		                               dominated	by	large	oligo-clonal	expansions.	These	clonal	expansions	
      Species:	ruminants                                                                utilized	 different	 VB	 gene	 segments	 in	 different	 animals.	 Importantly,	
      Key words:	macrophage,	protozoan,	toll-like	receptor,	cytokine                    using	 a	 newly	 developed	 bovine	TCRB-CDR3	 heteroduplex	 assay,	 it	
                                                                                        has	been	possible	to	identify	the	same	large	T-cell	clonal	expansions	
                                                                                        in	vivo	following	challenge	with	T.	parva,	indicating	that	the	immuno-
      Pr159. MODULATION OF THEILERIA PARVA POPULATIONS
                                                                                        dominant	populations	of	CD8+	T-cells	observed	in	in	vitro	cultures	are	
               By THE BOVINE IMMUNE RESPONSE
                                                                                        representative	 of	 the	 memory	 population	 present	 in	 vivo.	 	 This	 work	
      FRAnK	KATZER1,2,	DAnIEL	nGUGI2,	ALAn	R	WALKER2,DECLAn	                            provides	 the	 first	 definitive	 demonstration	 of	 immunodominance	 in	 a	
                          J	MCKEEVER1,2                                                 CD8+	T-cell	response	to	a	protozoan	parasite,	providing	an	explanation	
        1The	Moredun	Research	Institute,	Pentlands,	Science	Park,	Bush	                 for	the	incomplete	cross-protection	between	T.	parva	strains	and	has	
         Loan,	Penicuik,	Midlothian,	EH26	0PZ,	UK;	2Centre	for	Tropical	                important	implications	for	the	design	of	a	subunit	vaccine.
        Veterinary	Medicine,	Easter	Bush	Veterinary	Centre,	University	of	              Key words:	Theileria,	Immunodominance,	T-cells
               Edinburgh,	Easter	Bush,	Midlothian,	EH25	9RG,	UK	                        Species:	ruminants
            Theileria	 parva	 is	 a	 tick-borne	 intracellular	 protozoan	 parasite	
      that	 causes	 a	 severe	 and	 often	 fatal	 lymphoproliferative	 disease	           Pr161. TISSUE ESTABLISHMENT OF L3 HAEMONCHUS
      known	 as	 East	 Coast	 fever	 in	 cattle	 in	 sub-Saharan	 Africa.	 The	             CONTORTUS LARVAE IS PREVENTED IN HIGHLy
      protective	 immunity	 of	 the	 bovine	 host	 against	 T.	 parva	 is	 medi-                         SENSITISED SHEEP
      ated	 by	 parasite-specific	 class	 I	 MHC	 restricted	 CD8+	 cytotoxic	 T	
      lymphocytes.		This	response	is	tightly	focused	in	individual	animals	                J	M	KEMP,	n	RoBInSon,	E	n	T	MEEUSEn,	D	M	PIEDRAFITA	
      recognising	 only	 a	 few	 antigenic	 determinants.	 So	 far	 ten	 parasite	        Animal	Biotechnology	Research	Laboratories,	Monash	University,	
      antigens,	 seen	 by	 the	 bovine	 CTL	 response,	 have	 been	 identified	                                 Clayton,	Australia
      and	almost	all	of	these	proteins	are	polymorphic.	The	tight	focus	of	                   Haemonchus	contortus	(Hc)	is	a	blood	sucking	nematode	of	rumi-
      the	immune	response	could	permit	the	parasite	to	evade	recognition	               nants,	residing	in	the	abomasum	(fourth	stomach).	It	is	responsible	for	
      through	 antigenic	 diversity	 and	 by	 shuffling	 of	 antigen	 variants	 via	    significant	 losses	 in	 animal	 production	 and	 death	 of	 stock.	 Immunity	
      recombination.	Here	we	provide	evidence	that	the	immune	response	                 against	Hc	does	establish	over	time,	however	the	immune	mechanisms	
      can	specifically	eliminate	a	predominant	parasite	genotype,	which	it	             behind	nematode	death	and/or	rejection	are	not	yet	fully	elucidated.	In	
      was	 primed	 against,	 during	 an	 infection	 while	 permitting	 minor	 and	
                                                                                        order	 to	 explore	 these	 mechanisms,	 we	 are	 using	 an	 ex	 vivo	 	 larval	
      antigenically	distinct	genotypes	to	establish	infection	and	to	transmit	
                                                                                        establishment	 assay	 (LEA)	 which	 we	 have	 adapted	 for	 Hc.	 Sheep	
      to	 the	 tick	 host.	 Experimental	 evidence	 for	 breakthrough	 parasite	
                                                                                        were	sensitised	with	high	or	low	doses	of	Hc,	given	one	infecting	dose	
      populations	was	obtained	by	immunising	cattle	with	clone	72-01	and	
                                                                                        of	Hc	or	left	naïve.	The	animals	were	euthanized	and	abomasal	folds	
      challenging	them	with	stabilate	72.	This	particular	stabilate	contains	
                                                                                        removed	 and	 used	 in	 the	 LEA.	The	 tissue	 explants	 were	 challenged	
      72-01	 as	 a	 dominant	 parasite	 genotype,	 which	 makes	 up	 75%	 of	
                                                                                        with	L3	larvae	and	the	number	of	larvae	that	penetrated	the	tissue	was	
      parasite	 numbers.	 	 All	 calves	 showed	 mild	 levels	 of	 infection	 and	
                                                                                        counted.	The	ability	to	prevent	larval	establishment	in	the	tissue	was	
      self	 cured	 although	 piroplasms	 could	 be	 detected.	 Characterisation	
                                                                                        dependent	 on	 previous	 exposure	 to	 the	 parasite.	 Thus	 those	 sheep	
      of	breakthrough	parasite	populations	revealed	that	clone	72-01	was	
                                                                                        that	 had	 been	 repeatedly	 sensitised	 (either	 high	 or	 low	 dose)	 had	
      eliminated	during	infection	and	its	genotype	was	no	longer	present	in	
                                                                                        significantly	 lower	 larval	 establishment	 than	 those	 that	 had	 only	 one	
      the	 daughter	 stabilate.	 We	 also	 found	 evidence	 that	 recombination	
                                                                                        challenge.	The	lower	larval	establishment	was	associated	with	a	signifi-
      frequently	occurs	in	the	tick	host,	which	allows	shuffling	of	antigenic	
                                                                                        cantly	greater	number	of	globule	leukocytes	and	mast	cells,	and	their	
      determinants.	 The	 resulting	 recombinant	 parasites,	 with	 reshuffled	
      antigens,	will	have	a	greater	chance	of	survival	in	subsequent	rounds	            activity	is	currently	under	investigation.
      of	infection	of	cattle	which	had	been	infected	previously	with	different	         Key words:	Haemonchus	contortus,	larval	establishment,	mast	cells,	
      T.	parva	genotypes.                                                               globule	leukocytes.
      Key words:	Theileria,	CTL	response,	Antigenicity,	Recombination                   Species:	ruminants
      Species:	ruminants
                                                                                           Pr162. EFFECTS OF HAEMoNCHUS CoNToRTUS On
          Pr160. THE BOVINE CD8+ T-CELL RESPONSE TO                                       THE HUMORAL AND CELLULAR IMMUNE RESPONSE OF
      THEILERIA PARVA DISPLAyS EPITOPE IMMUNODOMINANCE                                             PARASITE-RESISTANT HAIR SHEEP
                     AND OLIGOCLONALITy                                                  KATHRyn	M	MACKInnon,	ISIS	K	MULLARKy,DAVID	R	noTTER
        T	ConnELLEy1,	n	MACHUGH1,	S	GRAHAM2,	E	TARACHA2,	A	                                             Virginia	Tech,	Blacksburg,	Virginia,	USA		
                BURRELLS1,	D	nGUGI1,	WI	MoRRISon1                                                                  kmackinn@vt.edu	
                                                                                                                                                                          107


      Among	 sheep	 producers,	 the	 parasitic	 nematode	 Haemonchus	               and	5	animals	without	infection	(control	group).	Abomasal	lymph	nodes	
contortus	is	a	major	animal	health	concern.		Caribbean	hair	sheep	are	              were	collected	seven	days	after	the	infection,	total	RNA	was	isolated	
known	 to	 be	 more	 resistant	 to	 this	 abomasal	 parasite	 than	 conven-         using	Trizol	reagent	and	cDNA	was	synthesized	by	reverse	transcrip-
tional	wool	sheep.		Our	objective	was	to	determine	differences	in	gene	             tion.	 Cytokine	 quantification	 was	 evaluated	 by	 the	 RT-PCR	 real	 time	
expression	associated	with	parasite	resistance	between	resistant	hair	              technique,	using	the	housekeeping	gene	RPL-19	as	control	and	SYBR	
and	susceptible	wool	lambs.		To	address	this	objective,	12	hair	and	12	             Green	as	florescent	label.	Results	were	analyzed	by	the	software	REST	
wool	 lambs	 were	 each	 infected	 with	 10,000	 H.	 contortus	 larvae	 and	        (Relative	Expression	Software	Tool).	Differences	of	mRNA	abundance	
14	animals	of	each	breed	were	left	as	uninfected	controls.		Fecal	egg	              were	observed	for	the	interleukin	genes	IL-4	and	IL-13	(P<0,05).	IL-4	
counts	(FEC)	were	measured	at	day	0,	16,	21	and	27	in	all	animals	to	               was	15	times	up-regulated	in	challenge	group	in	comparison	to	control	
assess	worm	burden.		Susceptible	wool	lambs	had	higher	FEC	com-                     group	 and	 IL-13	 was	 up-regulated	 in	 challenge	 group	 by	26	 times	in	
pared	 to	 resistant	 hair	 lambs	 throughout	 the	 study.	 	 Total	 RNA	 was	      comparison	to	control	group.	For	the	other	genes,	no	differences	were	
extracted	 from	 abomasum	 and	 abomasal	 lymph	 node	 tissues.	 	After	            observed	between	the	challenge	and	control	groups	analyzed.	It	can	
reverse	transcription,	gene	expression	of	Th1	and	Th2	cytokines,	cyto-              be	concluded	that,	for	the	analyzed	experimental	conditions,	the	first	
kine	 receptors	 and	 antibodies	 were	 evaluated	 by	 real-time	 RT-PCR.	      	   interleukins	 to	 be	 stimulated	 and	 produced	 after	 primary	 infection	 by	
Gene	expression	patterns	were	relatively	consistent	between	aboma-                  Haemonchus	spp	are	IL-4	and	IL-13.	These	results	corroborate	several	
sum	and	lymph	node	tissues,	indicating	potential	coordination	of	local	             studies	that	indicate	cytokine	polarization	Th2	in	infections	caused	by	
and	humoral	immune	responses	during	infection.		Compared	to	unin-                   endoparasites.	In	order	to	obtain	a	broader	view	of	the	bovine	immune	
fected	 control	 animals,	 infected	 sheep	 had	 decreased	 expression	 of	         response	of	this	parasite,	other	genes	related	to	the	immune	system	of	
IFN-γ	 in	 lymph	 node	 tissue	 and	 increased	 expression	 of	 IL-5,	 IL-13	       the	host	will	be	selected	and,	later,	analyzed.
and	 IgE	 in	 both	 tissues.	 	 However,	 even	 though	 Th2-type	 mecha-            Key words:	 Haemonchus	 spp,	 gene	 expression,	 cytokine,	 real	 time	
nisms	 seemed	 to	 be	 in	 place	 by	 this	 time,	 there	 was	 no	 difference	      PCR
in	 expression	 level	 of	 the	 cross-regulatory	 cytokine	 IL-4.	 	At	 3	 days	    Species:	ruminants
post-infection,	resistant	hair	sheep	had	greater	(P	<	0.01)	expression	
of	IgE	in	lymph	node	tissue	when	compared	to	wool	lambs.		By	27	days	
                                                                                       Pr164. EFFECT OF ANTHELMINTHIC COMBINED WITH
post-infection,	resistant	hair	lambs	had	lower	(P	<	0.10)	expression	of	
IFN-γ,	and	higher	(P	<	0.10)	expression	of	IgE	and	Th2	cytokine	IL-13	                  IMMUNOSTIMULANT ON GASTROINTESTINAL SHEEP
in	 both	 tissues	 as	 compared	 to	 susceptible	 wool	 lambs.	 	 The	 mean	                            NEMATODES
level	 of	 expression	 of	 IL-4	 receptor	 α	 did	 not	 differ	 between	 breeds,	        P	DEnAPoLI1,	S	CHEn1,	CF	GARCIA1,	FE	TonIAZZI1,	JS	
tissues	and	days,	but	individual	expression	levels	of	this	receptor	were	                MUSSALEM2,	CC	SqUAIELLA2,	I	LonGo-MAUGERI2,	RJ	
highly	 correlated	 (-0.98)	 with	 FEC	 at	 27	 days	 post-infection.	 	 These	                          SHAW3,LCJ	ABEL1	
results	suggest	that	gastrointestinal	nematode	infection	in	resistant	as	               1Curso	de	Medicina	Veterinária-Universidade	Paulista	-	UNIP.	
compared	 to	 susceptible	 sheep	 elicits	 a	 modified	 Th2-type	 immune	              Rua	Dr	Bacelar	1212,	São	Paulo-SP	05726-100;	2Disciplina	de	
response,	 characterized	 by	 decreased	 IFN-γ,	 steady	 IL-4	 expression	            Imunologia	do	Depto	de	Microbiologia,	Imunologia	e	Parasitologia	
and	increased	IL-13	and	IgE.		Differential	regulation	of	Th2	cytokines	                  da	Universidade	Federal	de	São	Paulo-	São	Paulo	-	Brasil3;	
between	breeds	may	be	partially	responsible	for	differences	in	parasite	             3Wallaceville	Research	Centre	Ward	Street,	PO	Box	40-063,	Upper	
resistance.	                                                                                                 Hutt,	New	Zealand	
KEy WORDS:	 Haemonchus	 contortus,	 cytokine,	 abomasum,	 lymph	                                           luciaabel@uol.com.br				
node                                                                                      							The	failure	of	anthelmintics	to	control	nematode	parasites	in	
Species:	ruminants	(sheep)                                                          sheep	 is	 a	 worldwide	 phenomenon	 of	 rapidly	 increasing	 dimensions.	
                                                                                    Development	 of	 alternative	 strategies	 and	 immunological	 methods	
Pr163. CyTOKINE QUANTIFICATION IN ABOMASAL LyMPH                                    to	 control	 the	 parasites	 is	 needed.	The	 purpose	 of	 this	 study	 was	 to	
  NODE OF BOVINE INFECTED WITH HAEMoNCHUS SPP                                       determine	 the	 effectiveness	 of	 an	 immunostimulant	 combined	 with	
                    PARASITES                                                       anthelmintic	 on	 experimental	 helminthiasis	 through	 IgE	 analysis,	 T	
 ADRIAnA	M	G	IBELLI1,	LILIAnE	C	nAKATA,	RoGéRIo	AnDRéo,	                            cell	 proliferation	 assay	 and	 fecal	 egg	 counts	 (FEC).	 Crossbred	 12-
   MáRCIA	C	S	oLIVEIRA2,	LUIZ	L	CoUTInHo3,	LUCIAnA	C	A	                             month-old	male	sheep	were	grouped	into	4	groups:	I-	animals	treated	
                         REGITAno2                                                  with	LPS	and	Propionibacterium	acnes	and	anthelminthic;	II-	animals	
                                                                                    treated	 with	 Propionibacterium	 acnes	 and	 anthelminthic;	 III-	 animals	
      1Programa	de	Pós-Graduação	em	Genética	e	Evolução,	                           treated	 with	 only	 anthelmintic;	 and	 IV-	 animals	 treated	 with	 a	 sterile	
    Universidade	Federal	de	São	Carlos,	São	Carlos,	SP,	Brasil;2	                   saline	 solution	 0.9%,	 as	 untreated	 controls.	All	 the	 animals	 received	
 Empresa	Brasileira	de	Pesquisa	Agropecuária,	Centro	de	Pesquisa	                   10,000	 infective	 larvae	 (L3)	 orally	 on	 day	 0	 and	 were	 treated	 with	
   Pecuária	do	Sudeste,	São	Carlos,	SP,	Brasil;3	Escola	Superior	                   when	the	fecal	egg	counts	peaked	at	35	days;	that	is,		the	treatment	
Agrícola	Luiz	de	Queiroz,	Universidade	de	São	Paulo,	Piracicaba,	SP,	               was	administered	on	the	35th,	63rd	and	67th	days	after	infection.	Fecal	
                                Brasil		                                            and	blood	samples	were	collected	weekly	for	fecal	egg	counts	(FEC),	
                      adriana.ibelli@gmail.com                                      IgE	 antibodies	 detection	 and	 T	 cell	 proliferation	 assay.	 The	 results	
      Infections	 caused	 by	 gastrointestinal	 parasites	 represent	 great	        showed	that	PBMC	from	group	I,	II,	III	displayed	enhanced	proliferation	
losses	 in	 animal	 production	 all	 over	 the	 world,	 affecting	 both	 bovine	    response	 to	 Concanavalina	 -	A	 with	 maximal	 response	 observed	 on	
and	 ovine.	 In	 the	 Brazilian	 southeast,	 worms	 with	 higher	 prevalence	       day	28	and	35	with	stimulation	indices	significantly	higher	in	group	II	
in	bovine	are	from	Haemonchus	spp	and	Cooperia	spp.	These	endo-                     than	group	IV	(p=0.0121).
parasites	 are	 responsible	 for	 several	 immune	 responses,	 innate	 as	                								During	the	infection,	the	animals	from	group	I	and	II	had	higher	
well	 as	 acquired	 by	 the	 host.	 The	 cytokines	 present	 a	 central	 paper	     IgE	 antibody	 levels	 in	 serum	 than	 other	 groups	 (p<0.05).	 Significant	
in	 the	 modulation	 of	 the	 immune	 response,	 including	 lymphocytes	            reduction	 in	 the	 fecal	 egg	 counts,	 determined	 42	 days	 after	infection,	
activation,	proliferation,	differentiation,	cellular	apoptosis	and	Th1/Th2	         were	 observed	 in	 groups	 I,	 II	 and	 III	 compared	 with	 group	 IV	 (I	 x	 IV	
polarization.	 Some	 of	 them,	 as	 for	 instance,	 IL-12,	 IL-8	 and	 MCP-1	       p=0.0083;II	x	IV	p=0.	0081	and	III	x	IV	p=0.0013).	These	results	suggest	
mediate	 innate	 immunity	 recruiting	 lymphocytes	 to	 the	 inflammation	          that	the	anthelminthic	combined	with	immunostimulant	can	be	effective	
sites.	Another	 are	 more	 related	 with	 the	 acquired	 immunity,	 as	 IL-2,	      for	the	treatment	of	gastrointestinal	sheep	nematodes	by	stimulation	of	
the	principal	factor	of	T	cells	growth	and	IL-4,	that	stimulates	IgE	pro-           IgE	antibodies	and	unspecific	immune	response.
duction.	 The	 objective	 of	 this	 project	 was	 to	 verify	 messenger	 RNA	
(mRNA)	abundance	of	the	cytokines	IL-2,	IL-4,	IL-8,	IL-12,	IL-13,	IFN-γ,	           Key words:	helminths,	sheep,	Propionibacterium	acnes,	anthelminthic
MCP-1	and	TNF-a	in	abomasal	lymph	nodes	of	Bos	indicus		submit-                     Species:	ruminants	(sheep)
ted	 to	 first	 infection	 with	 Haemonchus	 spp.	Ten	 calves	 of	 the	 Nelore	
breed	 were	 maintained	 without	 parasitic	 infections	 since	 they	 were	                 Pr165. ADJUVANT EFFECT OF LPS AND
born.	Those	 animals	 were	 split	 in	 two	 groups:	 5	 calves	 submitted	 to	          PRoPIoNIBACTERIUM ACNES ON ExPERIMENTAL
the	artificial	infection	with	Haemonchus	spp	larvae	(challenge	group)	                GASTROINTESTINAL NEMATODE INFESTATION IN SHEEP
108


             S	CHEn1,	LG	RICCA1,	MF	MARTInS1,	M	GARCIA1,	RZ	                                             Results	and	conclusions:	Proteases	of	H.	contortus	lysate	digest	
            AnAnIAS2,	JS	MUSSALEM2,	CC	SqUAIELLA2,	I	LonGo-                                         completely	 polypeptide	 chain	 of	 goat	 Hb.	 This	 effect	 is	 abrogate	 by	
                           MAUGERI2,LCJ	ABEL                                                        E64,	 which	 indicate	 that	 probably	 the	 involved	 proteolytic	 enzymes	
         1Curso	de	Medicina	Veterinária-Universidade	Paulista	-	UNIP	                               belong	to	the	class	of	cysteine	proteases.	Among	the	tested	plant	prod-
        Rua	Dr	Bacelar	1212,	São	Paulo-SP	05726-100;	2Disciplina	de	                                ucts	only	an	inseparable	mixture	of	N-alkyl	ferulates	from	Maproumea	
      Imunologia	do	Depto	de	Microbiologia,	Imunologia	e	Parasitologia	da	                          Guianensis	(Euphorbiaceae)	showed	an	inhibitory	effect	on	hemoglobi-
            Universidade	Federal	de	São	Paulo-	São	Paulo	-	Brasil	                                  nase	activities,	in	a	dose	dependent	manner,	sugesting	a	specificity	for	
                            luciaabel@uol.com.br                                                    cysteine	proteinases.	Supported	by:	BNB-FUNDECI;	FAPESB-PIBIC;	
                                                                                                    UFBA.		
           	 	 	 	 	 	 	 	 	 	 	 Gastrointestinal	 helminthic	 infection	 is	 one	 of	 the	 most	
      important	 sheep	 diseases	 worldwide	 which	 can	 cause	 anemia,	                            Key words:	Proteases,	Haemonchus,	hemoglobinolytic,	Maproumea,	
      anorexia,	 depression,	 progressive	 weight	 loss	 and	 eventual	 death.	                     n-alkil	ferulates.
      The	 emergence	 of	 anthelmintic	 resistance	 in	 sheep	 has	 led	 to	 seek	                  Speciez:	ruminants
      new	therapeutic	alternatives	to	control	helminthiasis	in	sheep.	Recent	
      reports	 demonstrated	 that	 LPS	 and	 Propionibacterium	 acnes	 have	                           Pr167. PROTECTIVE EFFECTS OF BRAZILIAN NATIVE
      an	adjuvant	effect	in	the	innate	and	acquired	immune	response.	The	                                PLANTS 0N GOATS NATURALLy INFECTED WITH
      overall	aim	of	this	study	was	to	evaluate	the	adjuvant	effect	of	LPS	and	                                  HAEMoNCHUS CoNToRTUS
      Propionibacterium	acnes	in	the	induction	of	experimental	helminthiasis	                          RoBERTo	R	B	DoS	SAnToS2,	JUCEnI	P	DAVID1,	JoRGE	M	
      in	sheep.                                                                                        DAVID4,	MARCUS	V	BAHIA4,	SÍLVIA	C	o	SAnToS1,	FARoUK	
            											Crossbred	male	sheep,	12-18	months	old,	were	divided	                          ZACHARIAS3,	MARIA	TEREZA	B	GUEDES2,	FERnAnDA	W	DE	
      into	two	groups.	Group	I,		received	LPS	and	Propionibacterium	acnes	                                              MEnDonçA	LIMA1
      and	 group	 II	 received	 sterile	 saline	 0.9%.	 Both	 groups	 were	 infected	                 1Faculdade	de	Farmácia	-	UFBA;2	Escola	de	Medicina	Veterinária	
      with	10,000	infective	larvae	(L3)	orally	on	day	0.	Parasitological,	hae-                         -	UFBA;	3Empresa	Baiana	de	Desenvolvimento	Agrícola	-	EBDA;	
      matological	examinations	and	T	cell	proliferation	assay	were	made	on	                                            4Instituto	de	Química	-	UFBA
      days	 0,	 14,	 21,	 28,	 35	 after	 infection.	 The	 results	 showed	 that	 fecal	
      egg	counts	were	significantly	lower	in	group	I	than	group	II	(p<0.05).	                             Introduction	and	Objectives:	Plant	products	are	known	to	exhibit	
      The	mean	fecal	egg	count	reached	a	peak	at	35	days.	The	hemato-                               medicinal	value	against	diseases	(Desai	et	al,	2002).	Our	earlier	stud-
      logical	parameters	showed	an	increase	in	the	leucocytes,	eosinophils,	                        ies	 showed	 that	 crude	 extract	 of	 two	 Leguminosae	 plants	 (Cratylia	
      lymphocytes	at	the	14th,	21th	and	35th	day	in	the	animals	from	Group	II	         	            mollis	 and	 Caesalpinia	 pyramidalis)	were	 effective	on	 inhibition	 of	H.	
      treated	with	immunostimulant	when	compared	to	Group	I	(p<0,05)	and	              	            contortus	 parasitism	 in	 natural	 infected	 goats.	 In	 order	 to	 investigate	
      the	response	of	PBMC	to	mitogen	(Conc-A)	were	higher	in	the	animals	                          a	 possible	 immunomodulator	 role,	 	 the	 humoral	 immune	 response	
      from	Group	II	(	p<0,05).                                                                      was	 evaluated	 in	 treated	 and	 untreated	 animals	 with	 these	 aqueous	
                                                                                                    extracts.	
            	 Our	 findings	 suggest	 that	 immunostimulants	 can	 be	 used	 as	
      a	 strategy	 to	 control	 helminthiasis	 in	 sheep.	A	 significant	 correlation	                     Animals	and	Methods:	Sixty	animals	were	subdivided	in	five	groups	
      between	 the	 LPS	 and	 P	 acnes	 administration	 and	 number	 of	 worms	                     of	twelve	each,	being:	Group	1	(G1)	negative	control;	G2	positive	con-
      was	also	observed.                                                                            trol	 for	 anti-helminthic	 action	 (Doramectin	 1mL	 /50kg,	 Dectomax®,	
                                                                                                    Pfizer);	G3	received	extract	from	C.	mollis	in	the	concentration	of	2.5	
      Key words:	helminthiasis,	lymphocytes,	adjuvant,	sheep                                        mg	 for	 kg/body	 weight,	 G4	 and	 G5	 received	 C.	 pyramidalis	 extracts	
      Species:	ruminants	(sheep)                                                                    (2,5	mg/kg		and	5	mg/kg	body	weight,	respectively).	The	animals	had	
                                                                                                    been	kept	on	the	same	breeding	conditions,	in	an	extensive	regimen	
        Pr166. INHIBITION OF HAEMoNCHUS CoNToRTUS                                                   of	 pasturing.	 Blood	 samples	 of	 5	 mL	 of	 each	 animal	 were	 collected	
      HEMOGLOBINOLyTIC ACTIVITIES By N-ALKyL FERULATES                                              from	the	jugular	vein,	and	their	serum	were	tested	for	concentration	of	
                FROM MAPRoUMEA GUIANENSIS                                                           specific	IgG	anti-H.	contortus.	Total	IgA	and	IgG	was	also	quantified	by	
        SÍLVIA	C	o	SAnToS1,	JUCEnI	P	DAVID1	,	JoRGE	M	DAVID2,	                                      using	a	commercial	Kit	of	sandwich	ELISA	(Bethyl,	Inc,U.S.).	
      JoRGE	A	LóPEZ1,	FARoUK	ZACHARIAS3,	MARIA	T	B	GUEDES3,	                                              Results	 and	 conclusions:	 G5	 animals	 showed	 the	 best	 results	
                   FERnAnDA	W	DE	MEnDonçA	LIMA1                                                     of	 specific	 humoral	 immune	 response,	 with	 a	 significant	 variation	 of	
         1Faculdade	de	Farmácia	-	UFBa;	2Instituto	de	Química	-	UFBA;	                              33.3%	over	the	baseline	concentration	of	anti-H.	contortus	antibodies.	
          3Escola	de	Medicina	Veterinária	-	UFBa	4Empresa	Baiana	de	                                The	highest	concentrations	of	IgA	were	detected	in	samples	from	G4	
                      Desenvolvimento	Agrícola	-	EBDA                                               and	 G5.	 The	 concentrations	 of	 G5	 were	 581	 ng/mL	 on	 the	 baseline	
                                                                                                    moment,	 615	 ng/mL	 on	 30	 days	 and	 575	 ng/mL	 60	 days	 later.	 With	
             Introduction	 and	 objectives:	 Haemonchus	 contortus	 is	 a	 blood-                   the	exception	of	variation	observed	for	G4,	that	showed	a	biggest	IgA	
      sucking	nematode	occurring	in	the	fourth	stomach	of	sheep	and	other	                          concentration	after	60	days	of	treatment,	being	367	ng/mL	before	treat-
      ruminants.	The	H.	contortus	L4	larvae	and	adults	cause	considerable	                          ment,	400	ng/mL	on	30	days	and	411	ng/mL	on	60	days	after	treatment,	
      damage	to	the	mucosal	lining	of	the	infected	sheep	abomasum,	result-                          remaining	groups	had	a	increasing	concentration	values	from	baseline	
      ing	in	extensive	hemorrhages	and	severe	chronic	anemia.	Proteinases	                          until	30	days	and	decreasing	concentrations	on	60	days.	The	observed	
      from	adult	H.	Contortus	degrade	hemoglobin	(Hb)	and	this	degradation	                         IgG	concentrations	were	rising	only	in	G4,	which	values	ranged	from	
      is,	 in	 part,	 due	 to	 cysteine-	 and	 aspartic	 proteinases,	 	 but,	 probably,	           412	 ng/mL	 before	 treatment	 to	 451	 ng/mL	 after	 60	 day	 of	 treatment.	
      also	 by	 metallo-	 and	 serine	 proteinases	 (McKerrow,	 2000;	 Redmond	                     Weight	 gain	 in	 the	 treatment	 groups	 was	 superior	 to	 the	 control	 that	
      &	Windham,	2005).	In	the	present	study,	we	investigated	the	inhibitory	                       received	only	saline,	being	G2	90%,	G3	10%,	G4	50%	and	G5	20%,	
      effect	on	hemoglobinolytic	activity	of	H.	Contortus	proteases	in	different	                   respectively.	 Although	 the	 need	 of	 evaluating	 others	 immunological	
      products	obtained	from	semi-arid	native	plants	of	Bahia,	Brazil.                              aspects	to	explain	the	improved	resistance	to	H.	contortus	on	treated	
           Material	and	methods:	For	standardization	of	the	nematode	hemo-                          goats	of	G4	and	G5,	in	this	work	it	was	demonstrated	some	protective	
      globinolytic	activities,	lysates	(8	mg/mL	of	protein)	were	incubated	with	                    effect	exhibit		by	the	tested	plants	on	the	control	of	this	infection.	
      a	solution	of	goat	Hb	10.5	mg/mL	at	37°C	over	night	in	a	citrate	phos-                           Supported	 by:	 BNB-FUNDECI;	 FAPESB;	 EBDA-SEAGRI-BA;	
      phate	buffer	pH	5.0	with	2-ME	2mM,	in	a	final	volume	of	500	uL.	Control	                      UFBA.	
      of	Hb	was	done	by	incubating	the	protein	without	lysate,	in	the	same	
      conditions.	 The	 products	 of	 hemoglobin	 hydrolysis	 were	 electropho-                     Key words:	 Cratylia	 mollis	 	 e	 Caesalpinia	 piramidalis,	 Haemonchus	
      resed	on	15%	SDS-PAGE	and	gels	were	stained	with	comassie	blue	                               contortus,	goats.
      (Lewis,	1999).	Inhibition	assays	were	performed	by	mixing	the	parasite	                       Specie:	ruminants
      lysate	 (72	 ug/mL	 protein)	 with	 different	 products	 isolated	 from	 native	
      plants.	Positive	control	of	inhibition	was	achieved	using	a	standard	cys-                       Pr168. TRANSFER OF MATERNAL HUMORAL PASSIVE
      teine	protease	inhibitor	E64.	A	lysate	sample	at	the	same	concentration	                      IMMUNITy TO KIDS IN GOAT HERD AGAINST HAEMoNCHUS
      was	also	analysed	by	SDS-PAGE	under	same	conditions.                                                         CoNToRTUS INFECTION
                                                                                                                                                                       109


  MARIA	TEREZA	B	GUEDES2,	FARoUK	ZACHARIAS3,	KELy	C	                                control.	 In	 addition,	 supernatants	 from	 each	 treatment	 were	 placed	
 PEDRoZA1,	SILVIA	CARoLInE	o	SAnToS1,	RoBERTo	R	B	DoS	                              on	monocyte	bovine	cells	(BM)	that	were	later	inoculated	with	live	n.	
       SAnToS2,	FERnAnDA	W	DE	MEnDonçA	LIMA1                                        caninum	 tachyzoites.	 Lysis	 plaque	 formation	 was	 evaluated	 on	 the	
 1Faculdade	De	Farmácia	-	UFBA;	2Escola	De	Medicina	Veterinária	                    BM	cell	monolayer	using	Image	Pro	plus	software.	Significative	differ-
  -	UFBA;	3Empresa	Baiana	De	Desenvolvimento	Agrícola	-	EBDA                        ences	were	observed	among	groups	(x2	p<	0.001).	Supernatant	from	
                                                                                    cells	group	I,	FI	and	SI	had	a	protective	effect	on	BM	cells.	Also,	INF-γ	
      Introduction	and	objectives:	Infection	with	Haemonchus	contortus	             mRNA	expression	was	evaluated	by	RT-PCR,	revealing	INF-γ	expres-
represents	 the	 main	 cause	 of	 economic	 loss	 in	 caprine	 breeding	 in	        sion	in	cells	from	group	I,	as	well	as	in	group	SI.	It	is	concluded	that	
tropical	and	subtropical	areas	of	the	world	(Melo	et	al,	2003).	The	aim	            tachyzoite	lysate	combined	with	an	appropriate	adjuvant	may	provide	a	
of	 this	 study	 was	 to	 evaluate	 the	 humoral	 immune	 response	 of	 goat	       valuable	tool	for	designing	protection	strategies	for	neosporosis.
kids,	before	and	after	natural	colostrums	ingestion,	against	H.	contor-
tus.	The	efficiency	of	maternal	immunity	transfer	and	its	influence	on	             Key words:	 Neospora	 caninum,	 gamma	 interferon,	 antigen,	 spleen	
kids’	health	was	also	investigated.																	                                cells
                                                                                    Specie:	ruminants
      Animals	 and	 methods:	 Thirty	 new	 born	 kids	 were	 weighted	 and	
had	 blood	 sample	 collected	 before	 colostrum	 ingestion	 and	 after	 30,	
60,	120,	180	and	260	days	after	ad	libitum	colostrum	and	milk	inges-
                                                                                    Pr170. BOVINE NATURAL KILLER CELLS ACT AS PRIMARy
tion.	The	twenty	four	dams	were	also	clinical	examined	and	had	sample	                RESPONDERS IN THE EARLy STAGES OF NEOSPORA
of	blood	and	feces	collected.	The	IgG	class	antibodies	to	herd-homolo-                          CANINUM INFECTED CATTLE
gous	 strains	 of	 H.	 contortus	 were	 estimated	 using	 ELISA	 of	 sera	                S	KLEVAR1,	S	KULBERG1,	P	BoySEn2,	A	SToRSET2,	T	
samples	of	dams	and	sera	of	kids.	The	faecal	egg	count	and	culture	for	                          MoLDAL1,	C	BJØRKMAn3,	I	oLSEn1
helminthes	in	kids	and	dams	faecal	sample	were	done.		                                1Department	of	Animal	Health,	National	Veterinary	Institute,	P.	O.	
      Results	and	conclusion:	Here	we	are	showing	for	the	first	time	the	            Box,	8156	Dep.,	N-0033	Oslo,	Norway;	2Department	of	Food	Safety	
nature	 of	 maternal	 transfer	 of	 immunity	 against	 H.	 contortus	 in	 goat.	      and	Infection	Biology,	Norwegian	School	of	Veterinary	Science,	P.	
First	of	all,	every	serum	sample	from	kids	before	colostrums	ingestion	               O.	Box,	8146	Dep.,	N-0033	Oslo,	Norway;	3Department	of	Clinical	
had	negative	results	for	antibodies	to	H.	contortus.		The	ELISA	of	the	                Sciences,	Swedish	University	of	Agricultural	Sciences,	P.	O.	Box	
serum	 samples	 collected	 on	 that	 moment	 gave	 optical	 density	 (DO)	                           7054,	SE-750	07	Uppsala,	Sweden
always	under	0.04	nm.	However,	results	observed	with	serum	sample	                        The	 intracellular	 protozoan	 parasite	 n.	 caninum	 is	 a	 cause	
collected	on	second	and	third	moment	after	colostrum	and	milk	inges-                of	 abortion	 and	 congenital	 diseases	 in	 cattle	 world	 wide.	 We	 have	
tion,	 were	 superior	 to	 0.867	 +	 0.735	 DO.	 None	 of	 sample	 collected	       previously	 shown	 that	 NK	 cells	 can	 produce	 IFN-γ	 in	 response	 to	 n.	
after	collostrum	ingestion	was	inferior	to	0.17	DO.	The	faecal	examina-             caninum	tachyzoites	in	vitro.		This	study	aimed	to	investigate	cellular	
tions	of	the	kids	had	been	presented	negative	for	Strongiloydea,	what	              immune	 responses	 in	 an	 experimental	 infection	 model	 in	 cattle	 and	
it	is	compatible	with	the	moment	of	this	collection,	these	animals	are	             in	particular	the	role	of	NK	cells	in	early	innate	immune	responses.	In	
fed	 only	 suckling	 their	 dams,	 therefore	 had	 not	 had	 contact	 with	 the	    infected	calves,	the	percentage	of	NK	cells	in	blood	dropped	at	day	4-6	
grass.	 The	 results	 of	 the	 maternal	 faecal	 culture	 identified	 1.85%	 of	    after	 inoculation	 followed	 by	 an	 increase	 in	 the	 percentage	 of	 CD8+	
Haemonchus,	 but	 in	 faecal	 sample	 kids	 it	 was	 of	 0	 %.	 On	 average	        T	cells	on	day	7	and	then	an	increase	of	NK	cells	around	day	12.		By	
until	 the	 present	 moment,	 the	 weight	 among	 in	 the	 studied	 animals	        using	a	whole	blood	flow-cytometric	assay	to	identify	IFN-γ	producing	
varied	from	2.7	kg	of	newborn	up	to	7.3	kg.	According	to	the	results	of	            cells	we	observed	that	NK	cells	and	CD8+	T-cells	where	an	important	
the	present	work,	we	can	predict	the	best	moment	for	management	of	                 source	of	IFN-γ	in	the	early	stages	of	infection	while	the	CD4+	T-	cells	
prophylactics	or	anthelmintic	intervention.	Early	studies	have	already	             dominated	 later	 on.	 We	 also	 compared	 the	 ability	 of	 two	 different	 n.	
pointed	the	passive	transfer	of	immunity	trough	the	colostrum	in	goats,	            caninum	 antigen	 preparations,	 i.e.,	 sonicated	 soluble	 antigens	 and	
but	they	did	not	specified	the	immune	response	against	H.	contortus	                intact	heat-inactivated	parasites,	to	induce	proliferation	and	IFN-γ	pro-
infection	 (Simões	 et	 al,	 2005)	 Supported	 by:	 BNB-FUNDECI;	 EBDA-             duction	in	various	cell	types.	The	heat	inactivated	tachyzoites	induced	
SEAGRI-BA;	UFBA.                                                                    a	3.7		times	higher	increase	in	the	number	of	IFN-γ	producing	NK	cells	
Key words:	Haemonchus,	passive	immunity,	colostrums,	antibodies.                    compared	to	the	sonicated	soluble	fraction.	This	indicates	the	presence	
Species:	ruminants                                                                  of	NK	cell	stimulating	antigens	in	the	intact	tachyzoite.	We	also	found	
                                                                                    that	heat	inactivated	whole	tachyzoites	clearly	inhibited	γd	T-cells	prolif-
      Pr169. INTERFERON GAMMA PRODUCTION AND                                        eration	while	the	soluble	antigens	from	n.	caninum	did	not.	For	the	first	
                                                                                    time	we	demonstrate	the	role	of	bovine	NK	cells	as	primary	responders	
    PROTECTIVE EFFECTS AFTER NEoSPoRA CANINUM
                                                                                    in	a	n	caninum	infection	in	calves,	and	additionally	a	distinct	difference	
    TACHyZOITE ACTIVATION OF BOVINE SPLEEN CELLS
                                                                                    in	 the	 effect	 of	 heat-inactivated	 n.	 caninum	 tachyzoites	 and	 soluble	
DIAnA	BACIGALUPE,	MARÍA	C	VEnTURInI,	GASTón	MoRé,JUAn	                              antigens	on	the	IFN-γ	production	from	NK	cells	and	proliferation	of	γd	
   M	UnZAGA,	ALEJAnDRA	LARSEn,	WALTER	BASSo,	LUCILA	                                T-cells.
                      VEnTURInI
                                                                                    Key words:	NEOSPORA	CANINUM,	NK	cells,	gd	T-cells
      Laboratorio	de	Inmunoparasitología,	Facultad	de	Ciencias	                     Species:	ruminants
      Veterinarias,	Universidad	Nacional	de	La	Plata,	Argentina.		
                        dianab@fcv.unlp.edu.ar                                         Pr171. INTERLEUKIN-4 DOWNREGULATES THE GOAT
       neospora	 caninum	 is	 a	 major	 cause	 of	 abortion	 in	 bovines	                BETA-DEFENSIN-2 GENE IN CAPRINE INTESTINAL
throughout	 the	 world.	 Protection	 from	 disease	 is	 primarily	 given	 by	            EPITHELIAL CELLS INFECTED WITH EIMERIA SPP
cellular	 immunity,	 and	 interferon	 gamma	 (IFNγ)	 is	 one	 of	 the	 most	
                                                                                          FRoyLAn	IBARRA-VELARDE,yAZMIn	ALCALA-CAnTo
relevant	 cytokines	 for	 protection.	 In	 order	 to	 detect	 a	 possible	 IFNγ-	
mediated	protective	effect,	bovine	cells	were	stimulated	with	different	             Departamento	de	Parasitología.	Facultad	de	Medicina	Veterinaria	y	
neospora	caninum	antigens.	Bovine	spleen	cells	from	cows	serologi-                         Zootecnia.	Universidad	Nacional	Autónoma	de	México		
cally	negative	to	n.	caninum	were	cultured	and	stimulated	with	live	n.	                                	yazmin@servidor.unam.mx
caninum	tachyzoites	(group	I),	formalin-	heat-	inactivated	tachyzoites	                   Defensins	are	antimicrobial	peptides	produced	by	leukocytes	and	
(group	FI),	and	a	tachyzoite	lysate	obtained	through	sonication	(group	             epithelial	cells.	These	peptides	have	been	shown	to	play	an	important	
SI),	 leaving	 uninfected	 cells	 as	 a	 negative	 control.	An	 ELISA	 test	 for	   role	 in	 innate	 immune	 responses.	 However,	 the	 role	 of	 defensins	 in	
IFNγ		(Bovigamtm)	was	performed	on	supernatants	from	treated	cells	                 goat	 eimeriosis	 remains	 unknown.	 Therefore,	 this	 study	 investigated	
collected	 at	 days	 3,	 7	 and	 10	 after	 inoculation	 with	 negative	 results	   the	 expression	 of	 the	 goat	 beta-defensin	 named	 GBD-2	 in	 caprine	
in	all	treatments.	In	addition,	stimulated	and	control	spleen	cells	were	           intestinal	 epithelial	 cells	 (CIEC)	 stimulated	 with	 recombinant	 bovine	
stained	 with	 Giemsa.	Typical	 activation	 morphology	 was	 observed	 in	          interferon-gamma	(IFN-gamma)	in	the	presence	or	absence	of	recom-
cells	 treated	 with	 live	 tachyzoites	 and	 lysate,	 while	 those	 that	 were	    binant	bovine	interleukin-4	(IL-4)	by	a	reverse	transcriptase-polymerase	
treated	 with	 inactivated	 tachyzoites	 remained	 similar	 to	 the	 negative	      chain	reaction	(RT-PCR)	assay.	GBD-2	mRNA	was	clearly	expressed	
110


      in	 IFN-gamma-stimulated	 CIEC.	 On	 the	 other	 hand,	 the	 direct	 addi-             Pr173. RHIPICEPHALUS SANGUINEUS TICKS FED ON
      tion	of	IL-4	showed	no	significant	effect	on	GBD-2	expression	in	CIEC.	               RESISTANT HOST: HISTOLOGy OF LESIONS ASSOCIATED
      However,	when	supernatants	from	peripheral	blood	mononuclear	cells	                                     WITH IMMUNITy
      (PBMC)	 cultured	 with	 IL-4	 were	 added	 to	 CIEC,	 the	 expression	 of	               VIVIAnE	APARECIDA	VERonEZ1	,	MáRCIo	BoTELHo	DE	
      GBD-2	decreased.	To	elucidate	if	IFN-gamma	functions	as	a	signaling	                   CASTRo2,	GERVáSIo	HEnRIqUE	BECHARA1,	MATIAS	PABLo	
      molecule	that	facilitates	the	generation	of	GBD-2	against	Eimeria	spp.	                                   JUAn	SZABó1,3
      in	 goats,	 anti-	 IL-4	 was	 added	 to	 PBMC	 from	 Eimeria-infected	 goats	
                                                                                              1Departamento	de	Patologia	Veterinária,	Universidade	Estadual	
      and	levels	of	IFN-gamma	in	culture	supernatants	were	determined	by	
                                                                                               Paulista,	Jaboticabal,	SP,	Brasil;	2Faculdade	de	Agronomia	e	
      an	 enzyme-linked	 immunosorbent	 assay	 test.	 Results	 showed	 that	                 Medicina	Veterinária,	Universidade	de	Brasília,	Brasília,	DF,	Brasil;	
      IFN-gamma	secretion	increased	when	anti-IL-4	was	added	to	PBMC.	                         3Faculdade	de	Medicina	Veterinária,	Universidade	Federal	de	
      Hence	 it	 is	 suggested	 that	 IL-4	 may	 be	 a	 further	 factor	 in	 the	 patho-                    Uberlândia,	Uberlândia,	MG,	Brasil		
      genesis	of	goat	coccidiosis	and	its	induction	may	be	part	of	an	evasion	                                      szabo@famev.ufu.br
      strategy	of	the	parasite	to	avoid	pro-inflammatory	responses.
                                                                                                 Dog	 is	 the	 natural,	 non	 resistant	 host,	 of	 the	 tick	 Rhipicephalus	
      Key words:	defensins,	eimerosis,	caprine,	cytokines                                  sanguineus	 whereas	 guinea	 pigs	 develop	 a	 strong	 resistance	 to	 the	
      Species:	ruminants                                                                   same	tick	species	following	repeated	infestations.	In	this	work	histologi-
                                                                                           cal	features	of	ticks	fed	on	dogs	and	on	guinea	pigs	were	compared	to	
           Pr172. FEEDING PROBIOTIC BACTERIA TO SWINE                                      evaluate	caused	by	the	immune	response	of	the	resistant	host	lesions	
              ENHANCES IMMUNITy TO ASCARIS SUUM                                            in	target	tissues.	Additionally	host	complement	fraction	C3	and	IgG1	and	
                                                                                           IgG2	antibodies	were	searched	in	tick	tissues	by	immunohistochemistry	
          GLoRIA	SoLAno-AGUILAR1,	TEREZ	SHEA-DonoHUE2,	                                    to	 associate	 effector	 mechanisms	 with	 lesions.	Ticks	 from	 each	 host	
           KATHLEEn	MADDEn3,	HARRy	DAWSon1,	ETHIoPIA	                                      species	were	collected	during	the	first	and	the	third	infestation	and	pro-
         BESHAH1yoLAnDA	JonES1,	MARTA	RESTREPo1,JoSEPH	                                    cessed	 according	 to	 routine	 histological	 techniques.	 It	 was	 observed	
                           URBAn	JR1                                                       that	 many	 ticks	 from	 guinea	 pigs,	 especially	 during	 third	 infestation	
          1Nutrient	Requirements	and	Functions	Laboratory,	Beltsville	                     where	 unattached,	 dehydrated	 and	 smaller	 when	 compared	 to	 those	
         Human	Nutrition	Research	Center,	ARS-USDA,	Beltsville,	MD	                        from	dogs.	Gut	of	13.6	%	and	38%	of	the	ticks	fed	on	guinea	pigs	during	
       20705,	2Mucosal	Biology	Research	Center,	University	of	Maryland,	                   first	and	third	infestation,	respectively,	had	host	inflammatory	cells	with	
           Baltimore,	3Uniformed	Services	University,	Bethesda,	MD		                       the	 features	 of	 eosinophils	 and	 basophils,	 whereas	 no	 inflammatory	
                       gloria.solanoaguilar@ars.usda.gov                                   cell	was	seen	in	the	gut	of	ticks	fed	on	dogs.	Vacuolization	of	gut	cells	
                                                                                           were	observed	in	all	ticks	except	in	those	fed	on	dogs	for	more	than	
            Probiotic	 bacterial	 species	 are	 included	 in	 the	 diet	 to	 promote	      96	hours.	It	was	also	observed	that	85.7%	of	ticks	fed	on	guinea	pigs	
      health.	Probiotics	purportedly	protect	the	intestine	against	pathogenic	             during	first	infestation	had	vacuolated	tracheae	and	all	displayed	swell-
      microorganisms	and	can	reduce	inflammation,	however,	quantitative	                   ing	of	Malpighian	tubules.	During	third	infestation,	all	ticks	from	guinea	
      measurement	 of	 probiotic	 growth	 and	 related	 effects	 on	 intestinal	           pigs	had	vacuolated	tracheae	and	swelling	of	Malpighian	tubules	and	
      function	are	often	lacking.	Ascaris	suum	commonly	infects	pigs	and	                  in	25%	of	these	ticks	vacuolization	of	oocytes	was	also	observed.	At	
      induces	a	Th2-derived	response	in	the	intestine	that	is	associated	with	             the	same	time	such	alterations	were	lacking	in	ticks	from	dogs.	Another	
      expulsion	of	the	fourth-stage	larvae	(L4)	from	the	jejunum.	The	physi-               interesting	feature	was	the	presence	of	an	increased	number	of	gua-
      ological	aspect	of	expulsion	represent	a	“weep	and	sweep”	response	                  nine	granules	in	ticks	fed	on	guinea	pigs,	especially	during	third	infesta-
      characterized	 by	 smooth	 muscle	 hyper-contractility	 and	 increased	              tion.	No	association	between	host	complement	and	antibodies	with	tick	
      luminal	fluid	associated	with	reduced	sodium-linked	glucose	absorp-                  lesions	could	be	done	because	of	the	diffuse	staining	of	several	tissues	
      tion.	 We	 investigated	 the	 effect	 of	 feeding	 Bifidobacterium	 lactis	          on	ticks	from	both	dogs	and	guinea	pigs	during	the	first	and	third	infes-
      subspecies	animalis	(Bb12)	on	the	immune	and	intestinal	function	of	                 tation.	Overall	results	show	that	the	natural	resistance	of	guinea	pigs	to	
      young	pigs	subsequently	infected	with	A.	suum.	Pregnant	sows	were	                   R.	sanguineus	ticks	is	associated	with	lesions	in	many	different	tissues	
      orally	inoculated	with	a	daily	dose	of	Bb12	(3.5	x	1010	cfu)		or	a	pla-              and	dehydration	of	the	tick.	Eosinophils	and	basophils	in	the	gut	might	
      cebo	during	the	last	trimester	of	pregnancy,	and	to	their	offspring	from	            be	interfering	with	tick	feeding.	Finally	irrespective	of	host	resistance	
      birth	until	weaning.	Six	weeks	after	weaning,	piglets	were	inoculated	               host	 complement	 and	 antibodies	 seem	 to	 reach	 several	 tick	 tissues	
      with	A.	suum	and	the	jejunal	mucosae	was	stripped	and	mounted	in	                    during	infestations	and	thus	functional	activities	of	these	proteins	inside	
                                                                                           the	ticks	should	be	evaluated.	
      Ussing	chambers	to	determine	changes	in	permeability	and	glucose	
      absorption	at	21	days	after	infection.	Pig	jejunum,	mesenteric	lymph	                     Financial	support:	FAPESP	and	FAPEMIG
      nodes,	 and	 proximal	 colon	 were	 collected	 and	 assayed	 for	 gene	              Key words:	 Rhipicephalus	 sanguineus,	 lesions,	 immune	 response,	
      expression	by	real	time	PCR.		Bb12	was	quantitatively	detected	using	                dog,	guinea	pig
      a	single	copy	tuf	gene	and	found	at	the	highest	concentration	in	the	                Species:	dog,	guinea	pig
      colon	 of	 probiotic-treated	 piglets.	 Probiotic	 treatment	 did	 not	 affect	
      intestinal	 permeability,	 but	 significantly	 attenuated	 the	 reduction	 in	            Pr174. SEROLOGICAL, HISTOLOGICAL AND
      glucose	 absorption	 and	 the	 hyper-secretory	 response	 to	 histamine	              IMMUNOHISTOCHEMICAL TESTS FOR CANINE VISCERAL
      induced	by	A.	suum	infection;	suggesting	a	selective	effect	of	the	pro-                          LEISHMANIASIS DIAGNOSIS
      biotic	on	nutrient	absorption	and	mast	cell	responses	against	parasite	                 nInA	MARI	G	P	DE	qUEIRoZ1,	WILMA	A	STARKE-BUZETTI1,	
      infection.	 Probiotic	 treatment	 of	 A.	 suum-infected	 pigs	 significantly	           RITA	DE	CASSIA	S	VIVEIRoS1,	SILVAnA	C	PAULAn1,	KAREn	
      increased	 mRNA	 expression	 of	 genes	 associated	 with	 enhanced	                      I	TASCA1,FLáVIA	L	LIMA1,	MICHELy,	S	TEnoRIo1,	MARIA	
      protection	 against	 parasitic	 infection,	 including	 IL-25,	 RETNLB,	 and	            FRAnCISCA	nEVES1,	RoSAnGELA	ZACARIAS	MACHADo2,	
      SOCS3,	 and	 did	 not	 interfere	 with	 normal	 expulsion	 of	 L4	 from	 the	              TRICIA	MARIA	DE	oLIVEIRA2,	AnTonIo	CARLoS	F	DE	
      jejunum.	 The	 results	 show	 that	 probiotic	 bacteria	 can	 selectively	                                noRonHA	JúnIoR3
      enhance	local	immunity	to	A.	suum	without	affecting	nutrient	absorp-                     1University	of	São	Paulo	State,	FEIS/UNESP-Campus	of	Ilha	
      tion.	This	experimental	system	could	be	used	to	evaluate	the	effect	of	               Solteira,	SP,	Brazil;	2University	of	São	Paulo	State,	FCAVJ/UNESP-
      feeding	Bb12	and	other	probiotics	on	responses	to	different	infectious	               Campus	of	Jaboticabal,	SP,	Brazil;	3Centre	of	Zoonosis	Control,	Ilha	
      agents	to	reduce	antibiotic	use.		It	also	models	the	effect	of	feeding	                                         Solteira,	SP,	Brazil
      probiotics	to	mothers	and	their	newborn	children	on	reduced	expres-
                                                                                                 Canine	visceral	leishmaniasis	(CVL)	is		caused	by	a	parasite	of	
      sion	of	allergic	disease	in	human.	
                                                                                           the	 specie	 Leishmania	 (L.)	 chagasi,	 	 endemic	 for	 humans	 and	 dogs	
      Key words:	 probiotic,	 Ascaris	 suum,	 immunity,	 gastrointestinal	                 in	 many	 regions	 of	 Brazil.	 The	 purpose	 of	 the	 present	 study	 was	 to	
      function                                                                             evaluate	 the	 serological	 testes	 (RIFI	 and	 ELISA)	 in	 asymptomatic,	
      Species:	swine                                                                       oligosymptomatic	 and	 	 symptomatic	 Leishmania-infected	 dogs	 from	
                                                                                                                                                                        111


Solteira,	 SP,	 Brazil.	 These	 methods	 were	 also	 compared	 with	 other	         for	 T.	 cruzi	 and	 Leishmania	 antibodies	 by	 ELISA	 tests.	 Fourteen	 out	
methods	used	for	diagnosis	of this	disease	in	dogs	such	as	histological	            of	31	T.	cruzi	seropositive	dogs	were	tested	for	presence	of	parasite	
(H&E)	and	immunohistochemical	(IMHC)		methods	for	demonstration	                    DNA	by	PCR	technique,	which	resulted	in	50%	of	positivity	(7/14	dogs).	
of	Leishmania	in	skin.		When	serological	tests	were	done,	24	(70.6%)	               Conclusions:	Canine	natural	infection	for	T.	cruzi	was	confirmed	in	the	
dogs	were	positive	by		ELISA	or	IFAT,	but	the	numbers	of	dogs		posi-                studied	area	by	ELISA	and	PCR	techniques.	Due	to	the	high	frequency	
tive	by	ELISA	was	slightly	higher	than	IFAT	(56%	for	IFAT	and	65%	for	              of	 double	 positivity	 for	 T.	 cruzi	 and	 Leishmania	 antibodies	 in	 ELISA	
ELISA),	and	in	7	(21%)	dogs	there	was	no	agreement	between	ELISA	                   tests	for	serological	diagnosis	of	these	infections,	the	use	of	more	spe-
and	 IFAT.	 	 There	 was	 also	 no	 agreement	 between	 serological	 tests	         cific	 and	 accurate	 techniques,	 such	 as	 PCR,	 become	 indispensable	
and	H&E	or	IMHC	in	32.4%,	particularly	in	asymptomatic	(8	dogs)	and	                for	distinguishing	infections	in	endemic	areas.	Since	the	efficiency	and	
oligosymptomatic	(17	dogs)	groups.	In	asymptomatic	group,	5	(62.5%)	                the	 reliability	 of	 diagnostic	 methods	 are	 important	 for	 the	 success	 of	
were	negative	by	both	serological	tests,	but	3	(37.5%)	of	these	dogs	               disease	 control	 measures,	 more	 accurate	 serological	 methods	 need	
were	positives	by	the	presence	of	the	parasite	in	their	skins	confirmed	            to	be	standardized.
by	 H&E	 and	 IMHC	 methods.	 On	 the	 other	 hand,	 one	 asymptomatic	
                                                                                    Key words:	Trypanosoma	cruzi,	ELISA,	PCR
dog	was	positive	in	all	tests	(serological,	H&E	and	IMHC),	indicating	
                                                                                    Species:	canine
that	even	dogs	without	any	clinical	signs	can	be	infected.		In	symptom-
atic	group	(9	dogs),	the	agreement	among	tests	was	100%,	but	1	dog	
(11.1%)	of	this	group	was	negative	and	8	(88.8%)	were	positives.	The	               Pr176. STUDy OF THE CANINE ExPERIMENTAL MODEL OF
skin	 biopsies	 from	 asymptomatic	 dogs	 had	 negligible	 if	 any	 lesions,	        THE INFECTIVITy AND IMMUNOGENICITy OF LEISHMANIA
and	 parasite	 direct	 examination	 showed	 that	 the	 most	 of	 these	 dogs	           INFANTUM NEW VARIANTS ISOLATED FROM HIV-
(62.5%)	were	negative	or	suspect,	but		3	(37.5%)	were	positive	in	skins	                     LEISHMANIA CO INFECTED PATIENTS
without	any	dermatological	alterations.		In	oligosymptomatic	dogs,	14	                  JoRGE	MIRET1,	JAVIER	nIETo1,	CARMEn	CHICHARRo1,	
(82.3%)	showed	discrete	parasite	load,	particularly	in	lesion	skins.		In	               CARMEn	CAñAVATE1,	EUGEnIA	CARRILLo2,	FERnAnDo	
symptomatic	 group,	 100%	 of	 dogs	 had	 several	 forms	 of	 cutaneous	                          GonZáLEZ3,	JAVIER	MoREno2
alterations	with	strong	parasite	loud	in	their	lesion	skins.		In	compari-
son	with	serological	tests	(IFAT	or	ELISA),	the	immunohistochemistry	               1Centro	Colaborador	de	la	OMS	para	Leishmaniasis.	Centro	Nacional	
method	showed	a	sensitivity	of	65%	and	specificity	of	100%	that	was	                de	Microbiología,	Instituto	de	Salud	Carlos	III,	Majadahonda,	Madrid,	
higher	in	lesion	than	in	normal	skins.			In	conclusion,	because	of	the	               Spain;	2Centro	de	Investigaciones	Biológicas,	Consejo	Superior	
complexity	of	this	disease,	the	results	of	the	present	study	support	the	             de	Investigaciones	Científicas,	Madrid,	Spain;	3Departamento	de	
observations	that	only	one	test	(serological	or	parasitological)	may	be	              Toxicología	y	Farmacología,	Facultad	de	Veterinaria,	Universidad	
not	sufficient	for	LVC	diagnosis.	In	addition,	IMHC	was	considered	a	                                   Complutense,	Madrid,	Spain		
valuable	method	to	support	the	diagnosis	of	his	disease	in	addition	to	                                   javier.moreno@cib.csic.es
serological	and	parasitological	tests.                                                    L.	 infantum/L.	 chagasi	 is	 the	 causative	 agent	 of	 zoonotic	 vis-
Key words:	 Dogs;	 Diagnosis,	 Histochemical,	 /Leishmania	 chagasi,/	              ceral	 leishmaniasis	 (ZVL)	 in	 the	 Mediterranean	 basin,	 China	 and	
Leishmaniose	Visceral	Canina.                                                       South	 America.	 Enzymatic	 characterization	 of	 L.	 infantum	 isolates	
Species:	canine                                                                     from	HIV	patients	has	showed	that	some	strains	are	new	variant	that	
                                                                                    have	not	been	described	to	cause	VL	in	the	dog,	the	main	reservoir	of	
                                                                                    the	parasite.	At	the	present	work	we	have	evaluated	on	the	dog,	the	
   Pr175. TRyPANOSOMA CRUZI NATURAL INFECTION
                                                                                    infectivity,	 pathogenicity,	 immune	 response	 and	 the	 possible	 protec-
  OF HOUSEHOLD DOGS DIAGNOSED By PCR AND ELISA
                                                                                    tive	 capacity	 of	 two	 new	 variants	 isolated	 from	 immunocompromised	
  METHODS IN AN ENDEMIC AREA FOR CANINE VISCERAL                                    patients.	The	L.	infantum	strains	LLM-759	(zymodeme	MON-190)	and	
  LEISHMANIOSIS IN BARRA DO POJUCA, BAHIA, BRAZIL                                   LLM-480	(zymodeme	MON-228).	both	isolated	from	immunodeficient	
   C	S	AGUIAR,	A	C	DE	ALCAnTARA,	D	V	V	BITTEnCoURT,	R	o	                            patients,	 and	 the	 highly	 virulent	 strain	 LLM-724	 (zymodeme	 MON-1)	
  G	GAMA,	A	C	C	SILVEIRA,	R	S	LIMA,	C	M	B	GoMES-nETo,	C	R	                          were	 used	 in	 this	 study.	 Dogs	 were	 experimentally	 infected	 with	 108	
         FRAnKE,	SM	BARRoUIn-MELo,	P	H	P	AGUIAR	                                    promastigotes,	 and	 the	 clinical,	 immunological,	 and	 parasitological	
          Universidade	Federal	da	Bahia,	salvador,	BA,	Brazil		                     status	 was	 monitored	 monthly	 during	 1	 year.	 Dogs	 infected	 with	 the	
                       aguiarcs@yahoo.com.br	                                       strains	LLM-759	and	LLM-480	did	not	showed	clinical,	immunological	
      Chagas	 disease	 or	 American	 trypanosomiasis	 is	 restricted	 to	           and	parasitological	status	of	disease	along	the	follow	up.	However	the	
the	 American	 continent	 and	 is	 considered	 a	 major	 tropical	 infection	       dogs	inoculated	with	the	strain	LLM-724	showed	alterations	in	all	the	
of	the	world.	Characterized	by	endemic	nature	and	chronic	evolution,	               parameters	studied	and	a	parasitological	positive	status	one	year	after	
the	disease	is	caused	by	a	flagellate	protozoan	named	Trypanosoma	                  infection.		In	order	to	test	the	protective	capability	of	these	new	vari-
cruzi,	which	 is	transmitted	to	humans	and	other	mammals	mostly	by	                 ants	of	L.	infantum,	those	dogs	infected	with	the	strains	LLM-759	and	
hematophagous	bugs	of	the	subfamily	Triatominae.	Due	to	the	vector’s	               LLM-480	 were	 challenged	 intravenously	 with	 the	 virulent	 strain	 and	
predominantly	 domiciliary	 and	 anthropophilic	 behaviour,	 the	 parasit-          monitored	during	8	months.	Leishmania-specific	serum	antibodies	and	
ism	 was	 transformed	 into	 zoonoses,	 including	 domestic	 animals	 like	         presence	 of	 the	 parasite	 was	 confirmed	 in	 those	 animals	 previously	
cats	and	dogs,	as	well	as	wild	animals	like	rodents,	monkeys,	ground	               infected	with	LLM-480	6	months	after	challenge	with	the	virulent	strain,	
squirrels	and	others,	which	could	also	serve	as	important	parasite	res-             while	dogs	infected	with	LLM-759	remained	parasitologically	negative.	
ervoirs.	 Dogs	 are	 considered	 important	 reservoirs	 of	 T.	 cruzi	 in	 Latin	   These	results	confirm	that	new	L.	infantum	variants	LLM-759	and	LLM-
America	 countries.	 There	 are	 reports	 of	 clinical	 disease	 in	 domestic	      480	are	not	able	to	induce	the	outcome	of	the	disease	in	the	canine	
dogs	in	some	areas	of	the	United	States	and	in	endemic	areas	at	the	                host	and	the	possible	protective	capability	of	the	strain	LLM-759.
Northeast	 region	 of	 Brazil.	 This	 study	 aimed	 to	 investigate	 the	 pres-     Key words:	 canine	 visceral	 leishmaniasis,	 experimental	 infection,	
ence	of	natural	canine	infection	by	T.	cruzi	in	the	District	of	Barra	do	           virulence,	protection
Pojuca,	 Bahia,	 Brazil,	 where	 human	 infection	 by	 T.	 cruzi	 has	 been	        Species:	canine
reported	and	zoonotic	visceral	leishmaniosis	is	endemic.	Material	and	
Methods:	274	randomly	collected	dog	serum	samples	were	analyzed	                           Pr177. CHARACTERIZATION OF CLINICAL,
by	indirect	ELISA	for	seropositivity	to	anti-T.	cruzi	and	anti-Leishmania	
                                                                                     IMMUNOLOGICAL AND PARASITOLOGICAL PARAMETERS
spp.	 antibodies.	 The	 ELISA	 test	 was	 performed	 with	 crude	 soluble	
                                                                                      DURING A STEADy STATE OF IMPROVEMENT OF AFTER
antigens	from	epimastigote	forms	identified	as	being	Colombian	strain	
by	comparison	to	the	reference	strain.	All	seropositive	dogs	for	anti-T	
                                                                                      CHEMOTHERAPy OF LEISHMANIA CHAGASI NATURALLy
cruzi	 antibodies,	 as	 well	 as	 double	 seropositive	 dogs	 for	 anti-T	 cruzi	                     INFECTED DOGS
and	anti-Leishmania	spp.	antibodies	in	ELISA	tests	were	evaluated	by	                 DAnIELA	FARIAS	LARAnGEIRA1,	PAULo	HEnRIqUE	PALIS	
PCR	with	primers	designed	to	amplify	specific	T.	cruzi	minicircle	DNA	              AGUIAR1,2,	GERALDo	GILEno	DE	Sá	oLIVEIRA1,	WASHInGTon	
sequence.	 Results:	 The	 seroprevalence	 for	 T.	 cruzi	 infection	 was	 of	       LUÍS	ConRADo	DoS-SAnToS1,	LAIn	PonTES-DE-CARVALHo1,	
11.31%	(31/274	dogs),	being	29.03%	(9/31	dogs)	double	seropositive	                             STELLA	MARIA	BARRoUIn-MELo1,2
112


        	1Centro	de	Pesquisas	Gonçalo	Moniz	-	FIOCRUZ,	R.	Waldemar	                        LAnA2,	WASHInGTon	LUIZ	TAFURI1,	VAnJA	MARIA	VELoSo1,	
         Falcão,	121,	Salvador,	BA,	CEP:	40295-001;	2Departamento	de	                      CLáUDIA	MARTInS	CARnEIRo2,	ALExAnDRE	BARBoSA	REIS2
        Patologia	e	Clínicas;	Escola	de	Medicina	Veterinária	-	UFBA,	Av.	                   1UFOP-	Laboratório	de	Imunopatolgia,	Núcleo	de	Pesquisas	em	
            Adhemar	de	Barros,	500,	Salvador,	BA,	CEP:	40170-000;                            Ciências	Biológicas;	2UFOP-	Escola	de	Farmácia;	3	CPqRR-	
            Canine	leishmaniosis	(CL)	is	in	an	expanding	zoonosis	in	the	state	           Laboratório	de	Doença	de	Chagas,	Centro	de	Pesquisa	René	Rachou	
                                                                                                                                                         	
      of	Bahia,	Brazil,	despite	the	governmental	programs	for	the	control	of	                                alexreis@nupeb.ufop.com.br
      the	infection.	There	is	a	consensus	about	the	need	for	new	methods	                       Previous	 studies	 in	 our	 laboratory	 have	 demonstrated	 that	 dogs	
      to	 control	 the	 disease,	 like	 vaccines	 and	 immunotherapy,	 since	 the	        experimentally	 infected	 with	 Berenice-78	 T.	 cruzi-strain	 presented	 a	
      host	immune	response	plays	an	important	role	in	the	development	of	                 longer	patent-parasitemia,	and	myocarditis	of	variable	degree.	Despite	
      resistance	to	parasite	infection.	Resistant	dogs	would	be	desirable	for	            these	 parasitological	 and	 pathological	 findings,	 little	 is	 known	 about	
      living	in	endemic	areas,	since	they	would	be	poorly	or	non-parasitized,	            the	humoral	immune	response	during	acute	phase	of	the	infection	in	
      being,	 therefore,	 bad	 reservoirs	 for	 transmitting	 the	 parasite	 to	 phle-    dogs.	 Herein	 we	 have	 performed	 a	 detailed	 follow-up	 investigation	
      botomine	 vectors.	 In	 this	 study,	 we	 aimed	 to	 characterize	 a	 conjunct	     of	 serological	 profile	 by	 ELISA	 using	 specific	 monoclonal	 anti-canine	
      of	 parameters	 of	 infected	 dogs	 before	 and	 after	 treatment	 with	 an	        isotypes	 (IgG,	 IgG1,	 IgG2,	 IgM,	 IgA	 e	 IgE)	 employing	 a	 soluble	 epi-
      anti-leishmania	drug,	in	order	to	make	feasible	the	perceivance	of	any	             mastigotes	 T.	 cruzi	 antigen.	 Twenty-four	 dogs	 were	 subdivided	 in	
      additional	effect	of	immunotherapy	applied	from	a	post-chemotherapy	                six	 experimental	 groups	 with	 four	 animals	 in	 each	 one:	 uninfected	
      steady	 state.	 Material	 and	 method:	 Nine	 dogs	 naturally	 infected	 with	      control,	 infected	 with	 blood	 trypomastigotes,	 infected	 with	 metacyclic	
      Leishmania	 chagasi	 were	 studied	 in	 controlled	 kennels,	 located	 in	 a	       trypomastigotes,	 uninfected	 immunossupressed,	 infected	 with	 blood	
      non-endemical	 area	 for	 CL,	 at	 the	 Research	 Center’s	 premises.	The	          trypomastigotes	immunossupressed	and	infected	with	metacyclic	try-
      animals	were	classified	according	to	the	intensity	of	the	clinical	symp-            pomastigotes	immunossupressed.	Were	used	2,000	metacyclic	trypo-
      toms	as	asymptomatic	(AS),	symptomatic	(SY)	and	oligosymptomatic	                   mastigotes	(MT)	or	blood	trypomastigotes	(BT)	of	BE-78	T.	cruzi	strain,	
      (OL).	Each	animal	was	evaluated	by	means	of	parasitological	diagnosis	              per	Kg/body/weight,	typifying	vectorial	and	transfusional	transmission	
      by	culture	of	spleen	aspirates,	serology	by	ELISA,	in	vitro	Leishmania	             of	human	Chagas	disease,	during	35	days	of	the	acute	phase	of	the	
      antigen-induced	 lymphoproliferation	 using	 PBMC,	 hemogram	 and	                  infection.	The	kinetics	of	immunoglobulin	levels	was	evaluated	in	zero,	
      serum	 biochemistry	 (BUN,	 bilirubin,	 proteins,	 albumin	 and	 globulin)	         seven,	 14,	 21,	 28	 and	 35	 days	 after	 infections	 including	 the	 correla-
      before	 and	 three	 and	 six	 months	 after	 having	 received	 treatment	 by	       tions	with	the	parasitemia	curve.	Dogs	infected	with	BT	presented	an	
      a	short	course	of	meglumine	antimoniate.	The	dogs	were	divided	into	                increase	 of	 IgG	 and	 IgM	 levels,	 maintenance	 of	 IgG1,	 IgG2	 and	 IgA	
      groups	for	3	different	treatments,	having	each	group	one	animal	with	               levels	along	the	time	and	an	increase	of	IgE	followed	by	a	decrease.	
      each	profile,	AS,	SY	and	OL.	The	dogs	of	groups	1	and	2	were	treated	               On	the	other	hand,	dogs	infected	with	forms	MT	presented	an	increase	
      with	100	mg/Kg	of	meglumine	antimoniate	subcutaneously,	twice	daily	                of	 IgG,	 IgG2,	 IgM	 and	 IgA	 and	 maintenance	 of	 IgG1	 and	 IgE	 levels.	
      for	10	days.	The	dogs	of	group	2	received	two	additional	doses	of	imu-              Independent	 of	 the	 infective	 forms,	 the	 profile	 of	 humoral	 response	
      notherapic	preparation	(hidroxide	of	aluminum	+	recombinant	proteins	               presented	reduced	levels	of	IgG,	IgG1,	IgG2,	IgA	and	IgE	in	all	animals	
      of	Leishmania)	after	the	course	of	antimony	and	the	dogs	of	group	3	                immunossupressed.	 However,	 the	 IgM	 showed	 the	 elevations	 levels	
      did	not	suffer	any	intervention.	Results:	Before	treatment,	all	animals	            during	the	kinetics	in	immunossupressed	groups.	Taken	together,	these	
      presented	variable	alterations	in	serum	biochemistry	and	hemograms,	                findings	emphasize	the	importance	of	the	inoculum	source,	suggesting	
      positive	parasitology	and	high	Optical	Density	(OD)	readings	in	ELISA	              that	vectorial	or	transfusional	routes	of	T.	cruzi	infection	may	trigger	dis-
      for	detecting	anti-Leishmania	antibodies	in	sera.	Only	the	symptomatic	             tinct	parasite-host	interaction	during	acute	Chagas	disease.	During	the	
      animals	presented	absence	of	PBMC	proliferation	in	the	presence	of	                 acute	phase	of	the	infection	it	was	not	found	a	correlation	between	the	
      Leishmania	 antigen	 as	 stimulus.	 After	 3	 months	 all	 treated	 animals	        profiles	of	immunoglobulins	and	parasitemia	in	both	immunocompetent	
      (groups	 1	 and	 2)	 presented	 remission	 of	 clinical	 signs,	 improvement	       and	immunossupressed	dogs.	Ours	results	still	suggest	also	that	the	
      of	laboratory	parameters	of	hemogram	and	biochemistry	and	negative	                 early	production	of	IgG	and	IgG2	seems	to	play	a	pivotal	role	control	
      parasitological	tests.	PBMC	from	all	animals	from	these	groups,	includ-             of	 the	 immunopathology	 observed	 in	 acute	 canine	 Chagas	 disease.	
      ing	the	two	symptomatic	dogs,	presented	positive	Leishmania	antigen-                In	 addiction,	 these	 data	 suggested	 that	 the	 route	 of	 infection	 can	 be	
      induced	 proliferation.	 At	 this	 time	 point,	 the	 animals	 of	 group	 3	 had	   an	 important	 element	 driving	 the	 immune	 response	 in	 infected	 dogs.	
      to	 be	 treated	 due	 the	 worsening	 of	 the	 symptoms.	After	 6	 months	 of	      Therefore	the	different	profile	of	class	and	subclass	of	immunoglobu-
      treatment,	3	dogs,	two	from	the	chemotherapy	group	and	one	from	the	                lins	could	be	contribute	for	the	development	of	immunophatogenesis	of	
      chemotherapy	+	immunotherapy	group	presented	positive	parasitology	                 canine	experimental	infections.
      and	begun	to	have	clinical	signs.	All	animals	had	normal	hemograms	
                                                                                             Supported	 by:	 CNPq,	 CAPES,	 FAPEMIG,	 CPqRR-FIOCRUZ	 &	
      and	 serum	 biochemistry,	 as	 well	 as	 positive	 in	 vitro	 lymphoprolifera-
                                                                                          UFOP
      tion.	The	ELISA	tests	of	all	dogs	showed	high	levels	of	OD	during	the	
      whole	time	of	study.	Conclusions:	The	6-month	period	of	remission	of	               Key words:	Chagas	disease,	immunossupressed,	humoral	response,	
      clinical	signs	and	improvement	of	laboratory	parameters,	including	as	              cell	response
      well	the	recovery	of	antigen-specific	lymphoproliferation	and	reduction	            Species:	canine
      of	splenic	parasitism	to	undetectable	levels,	was	achieved	with	a	short	
      course	of	chemotherapy	with	an	anti-leishmania	drug.	These	findings,	               Pr179. KINETICS OF DERMAL CELLULAR INFILTRATES IN
      although	 carried	 out	 in	 controlled	 conditions,	 are	 in	 accordance	 with	     DOGS VACCINATED WITH LEISHMANIA-VACCINE, SAPONIN
      the	 majority	 of	 the	 reported	 results	 of	 CL	 chemotherapy,	 in	 special-           AND SALIVA FROM LUTzoMyIA LoNGIPALPIS
      ized	literature.	After	a	course	of	chemotherapy,	naturally	infected	dogs	                JULIAnA	VIToRIAno	DE	SoUZA1,	náDIA	DAS	DoRES	
      come	 to	 a	 steady	 state	 in	 which	 immunological	 modulators	 could	 be	           MoREIRA1,	HEnRIqUE	GAMA	KER1,	RoDRIGo	CoRRêA-
      introduced	in	experimental	studies	for	the	development	of	efficient	pro-             oLIVEIRA2,	RoDoLFo	CoRDEIRo	GIUnCHETTI1,	ALExAnDRE	
      tocols	for	controlling	the	transmission	from	dogs	to	vectors.	Serology	                    BARBoSA	REIS1,	CLáUDIA	MARTInS	CARnEIRo1
      by	ELISA	was	not	a	good	tool	for	the	evaluation	of	the	animals	during	
      the	studied	time	since	it	did	not	show	any	modification	along	with	the	               1Laboratório	de	Imunopatologia,	NUPEB/UFOP;	2Laboratório	de	
      other	parameters.                                                                          Imunologia	Celular	e	Molecular,	IRR,	FIOCRUZ/MG
      Key words:	canine	leishmaniasis,	immunotherapy	e	chemotherapy.                            Efforts	 have	 focused	 on	 understanding	 the	 early	 events	 that	
      Species:	canine                                                                     influence	 effectiveness	 of	 vaccine	 antigens.	 In	 the	 present	 study,	 we	
                                                                                          investigated	 kinetics	 of	 dermal	 cellular	 infiltrates	 in	 dogs	 vaccinated	
                                                                                          with	 Leishmania-vaccine,	 Saponin	 and	 saliva	 of	 Lutzomyia	 longipal-
          Pr178. TRyPANoSoMA CRUzI: CORRELATION OF                                        pis.	Based	on	cell	migrations,	we	have	developed	a	new	experimental	
        IMMUNOGLOBULINS PROFILES WITH PARASITEMIA IN                                      model	to	study	some	aspects	of	the	inflammatory	immune	response.	
       DOGS IMMUNOSSUPRESSED AND IMMUNOCOMPENTENT                                         In	this	sense,	intradermal	injections	into	the	dorsal	region	of	dogs	were	
      WEnDEL	CoURA	VITAL1,	HELEn	RoDRIGES	MARTInS1,	oLInDo	                               performed	at	different	times	(1,	12,	24,	48,	96	hours).	Stimuli	with	dis-
      ASSIS	MARTInS-FILHo3,	MARIA	TEREZInHA	BAHIA1,	MARTA	DE	                             tinct	 vaccine	 antigen	 components	 were	 evaluated,	 such	 as	 saponin	
                                                                                                                                                                            113


(Sap),	sand	fly	saliva	(Sv),	whole	parasite	antigen	against	VL	(WPA),	                   TRÍCIA	MARIA	FERREIRA	DE	SoUSA	oLIVEIRA1,	MELInA	
and	WPA	associated	with	Sap.	Those	stimuli	were	compared	with	con-                      BASon1,	TIAGo	WILSon	PATRIARCA	MInEo1,	RoSAnGELA	
trols	groups:	salina	inoculum	(Sn)	and	without	salina	(Ns).	Macroscopic	                                ZACARIAS	MACHADo1
observations	 (local	 swelling,	 hyperemia	 and	 necrosis)	 were	 reported	            1Universidade	Estadual	Paulista	(UNESP),	Jaboticabal,	SP,	Brazil		
during	the	course	of	the	experiment.	Skin	samples	were	fixed	in	10%	                                     triciaoliveira@yahoo.com.br
neutral	 buffered	 formalin	 for	 routine	 histopathological	 examination	
of	 sections	 by	 subsequent	 haematoxylin	 and	 eosin	 (HE)	 staining.	                   Visceral	leishmaniasis	is	a	zoonotic	disease,	caused	by	Leishmania	
The	 dermal	 cellular	 infiltrate was	 graded	 in	 absent	 (–);	 discreet	 (+);	     chagasi	and	transmitted	by	the	bite	of	sandflies	from	Lutzomiya	longi-
moderate	(++)	and	intense	(+++).	In	dogs	inoculated	with	saponin	or	                 palpis	 species	 in	 Brazil.	 In	 this	 country,	 it	 is	 an	 emerging	 disease	 in	
saponin	 plus	 WPA,	 we	 observed	 local	 swelling	 and	 hyperemia	 after	           urban	 areas,	 and	 dogs	 are	 the	 main	 domestic	 reservoir.	 The	 aim	 of	
12hs.	 However,	 no	 macroscopic	 alterations	 were	 observed	 in	 dogs	             this	 study	 was	 to	 analyze	 the	 presence	 of	 anti-Leishmania	 IgG	 sub-
inoculated	with	sand	fly	saliva.	All	groups	presented	cellular	infiltrates	          class	antibodies	by	ELISA	in	121	sera	of	seropositive	dogs	from	Belo	
composed	mainly	of	neutrophils.	The	major	findings	occurred	at	12,	24	               Horizonte	(MG)	endemic	area,	and	40	sera	of	vaccinated	animals	with	
and	48	hours	after	inoculation:	increased	cellular	infiltrates	in	Sap	and	           vaccine	Leishmune	(Fort	Dodge®).	This	study	also	aims	to	verify	the	
WPA	plus	Sap	when	compared	to	Sn	and	Ns	groups.	Moreover,	after	                     diference	of	IgG	subclass	among	the	groups.	The	proportion	of	dogs	
48	hours,	the	group	inoculated	with	Sv	presented	an	increased	cellular	              witch	 produced	 detectable	 levels	 of	 anti-Leishmania	 IgG	 subclass	
infiltration	 when	 compared	 with	 Sn.	Additionally,	 after	 96	 hours,	 only	      antibodies	from	Belo	Horizonte	was	80,17%	(97/121)	for	IgG1;	0,83%	
Sap	and	WPA	plus	Sap	inoculated	dogs	showed	enhanced	dermal	cel-                     (1/121)	 for	 IgG2;	 39,67%	 (48/121)	 for	 IgG3	 and	 55,37%	 (67/121)	 for	
lular	infiltrates.	Our	data	point	to	the	potential	early	stimulatory	effects	        IgG4.	The	results	showed	significant	higher	percentage	in	IgG1	anti-
on	innate	immune	responses,	particularly	neutrophil	recruitment,	after	              bodies	 (p<0,01).	 The	 proportion	 of	 vaccinated	 dogs	 which	 produced	
saponin	inoculation.	Furthermore,	we	observed	a	delay	in	cell	infiltra-              detectable	levels	of	anti-Leishmania	IgG	subclass	antibodies	was	22%	
tion	in	the	Sv	group,	which	may	reflect	the	immunomodulatory	proper-                 (9/40)	for	IgG1;	87,5%	(35/40)	for	IgG2;	2,50%	(1/40)	for	IgG3	and	5%	
ties	of	saliva.	Even	though,	edematous	reactions	have	frequently	been	               (2/40)	for	IgG4.	Result	analysis	disclosed	significant	increase	in	IgG2	
observed	after	saponin	injection	or	in	combination	with	WPA,	the	induc-              antibodies	(p<0,01)	in	vaccinated	dogs,	and	that	the	IgG2	response	of	
tion	of	a	strong	cellular	response,	simple	formulation,	safety,	and	the	             vaccinated	dogs	was	significantly	higher	than	in	infected	dog	(p<0,01).	
low	costs,	allow	its	use	as	alternative	adjuvant	in	veterinary	medicine.	            These	results	suggest	that	there	was	great	variation	in	levels	of	IgG1	
Key words:	Leishmania,	dogs	and	Kinetics                                             and	IgG2	between	the	two	groups,	but	it	needs	more	studies	to	use	the	
Species:	canine                                                                      markers	of	resistance	or	susceptibility	of	individual	dogs.	25,4	mm
                                                                                     Key words:	leishmania,	subclass,	IgG	antibodies,	ELISA
  Pr180. SELECTION OF RECOMBINANT PEPTIDES FOR                                       Species:	canine
 CANINE LEISHMANIASIS DETECTION By PHAGE DISPLAy
                                                                                       Pr182. COMPARISON BETWEEN ANTIGEN TOTAL ELISA
 JULIANA	FRANCO	ALMEIDA,	GUILHERME	ROCHA	LINO	SOUZA,	
                                                                                        AND FML ELISA IN DIAGNOSIS OF VISCERAL CANINE
FAUSTO	EMÍLLIO	CAPPARELLI,	CARLOS	ROBERTO	PRUDÊNCIO,	
                                                                                                        LEISHMANIASIS
    ANA	PAULA	PERES	FRESCHI,	RONE	CARDOSO,	FLÁVIA	
        FIGUEIRA	MESSIAS,	LUIZ	RICARDO	GOULART                                           TERESInHA	CRISTInA	CÂnDIDo,	TATIAnA	DE	oLIVEIRA	
                                                                                                       GERZoSCHKWITZ,	MARIA	
    1Instituto	de	Genética	e	Bioquímica	-	Universidade	Federal	de	                      CECÍLIA	RUI	LUVIZoTTo,	VALéRIA	MARçAL	FELIx	DE	LIMA
                           Uberlândia	-	MG		
                       	almeidajf@hotmail.com                                         Departamento	de	Apoio	Produção	e	Saude	Animal,	Faculdade	de	
                                                                                      Odontologia	de	Araçatuba,	Universidade	Estadual	Paulista	Júlio	de	
       Human	 visceral	 leishmaniasis	 (HVL)	 is	 a	 canine	 zoonose	 lead-                                   Mesquita	Filho
ing	to	500,000	new	cases/year,	in	which	no	vaccine	is	available,	and	
chemotherapy	is	highly	toxic.	Canine	leishmaniasis	(CL)	is	a	systemic	                    Visceral	canine	leishmaniasis	or	calazar	is	Brazilian	an	endemic	
disease	 caused	 by	 different	 species	 of	 the	 genus	 Leishmania	 that	 is	       antropozoonosis	 caused	 by	 Leishmania	 L.	 chagasi.	 The	 immunoen-
transmitted	by	blood	sucking	phlebotomine	sandflies.	Dogs	are	consid-                zymatic	assay	in	solid	phase	test	(ELISA)	has	been	used	in	routine	of	
ered	the	main	domestic	reservoir	of	the	parasite,	constituting	part	of	the	          epidemiological	studies	and	diagnostic	of	dogs.	The	ELISA	assay	using	
epidemiological	cycle	of	human	transmission,	accounting	for	more	than	               Leishmania	L.	chagasi	total	antigen	(AgT-ELISA)	and	Fucose	Manose	
90%	 of	 visceral	 leishmaniasis	 cases	 described	 in	 the	 world.	 Phage	          Ligand-ELISA	(FML-ELISA)	has	been	used	to	detected	leishmaniasis	
display	 has	 been	 utilized	 for	 numerous	 purposes,	 including	 mapping	          in	dogs.	The	present	paper	has	the	goal	to	compare	the	efficiency	of	
protein-ligand	interactions,	identifying	binding	antagonists	and	enzyme	             two	antigens	by	ELISA	assay	in	dogs	from	Araçatuba,	SP-	Brazil,	an	
inhibitors	through	the	design	of	mimotopes.	This	technology	was	used	                endemic	 leishmaniasis	 area.	 Thirty	 dogs	 were	 selected	 by	 positive	
to	 identify	 peptides	 against	 HVL	 antibodies	 expanding	 its	 purpose	 to	       imprints	 and	 divided	 into	 two	 groups:	 oligosymptomatic	 (increase	 of	
CL	epitopes	screening	in	the	perspective	of	using	them	as	vaccine	or	                lymphonodes	and	a	good	condition)	and	symptomatic	presence	of	at	
diagnostics’	 antigens.	 IgG	 was	 purified	 from	 sera	 of	 human	 visceral	        least	three	clinical	signs	associated	with	active	visceral	leishmaniasis	
leishmaniasis	patients,	and	used	for	screening	against	a	random	pep-                 (fever,	 dermatitis,	 lymphoadenopathy,	 onychogryphosis,	 weight	 loss,	
tide	phage	library.	The	selection	was	performed	in	liquid	phase	through	             cachexia,	locomotory	difficulty,	conjunctivitis,	epistaxis,	hepatospleno-
agarose	 beads	 (G	 protein	 agarose).	After	 four	 rounds	 of	 biopanning,	         megaly,	 edema,	 and	 apathy).	 Control	 group	 was	 constituted	 of	 dogs	
phage	 clones	 were	 isolated,	 sequenced,	 translated,	 and	 submitted	             from	 Leishmania	 free.	 Dogs	 oligosymptomatics	 presented	 86,7%	 of	
to	 bioinformatic	 analyses.	 Peptide	 analysis	 revealed	 a	 high	 degree	          sensibility	in	AgT-ELISA	and	90%	at	FML-ELISA.	The	specificity	was	
of	sequence	similarities	between	important	parasite	proteins	involved	               100%	at	AgT-ELISA	and	96.7%	for	FML-ELISA.	At	symptomatics	dogs	
in	 the	 disease	 development,	 such	 as	 gp46,	 gp63,	 kinesin	 K39,	 DNA	          the	sensibility	and	specificity	for	AgT-ELISA	were	respectively	90%	and	
topoisomerase	 II,	 LACK	 antigen,	 among	 others.	 Two	 highly	 frequent	           93.3%	and,	the	FML-ELISA	showed	86.7%	and	96.7%.	On	ELISA-AgT	
epitopes	 were	 identified,	 and	 the	 phagotopes	 were	 used	 in	 ELISA	            the	positive	predictive	valor	was	93.1%	at	symptomatic	and	100%	at	
assays	 against	 positive	 and	 negative	 canine	 sera,	 showing	 differen-          oligosypmtomatic,	moreover	in	the	dogs	tested	by	FML-ELISA	the	valor	
tial	 reactivity,	 with	 83%	 of	 sensitivity.	The	 highly	 reactive	 clones	 may	   was	96.3%	for	the	symptomatic	and	96.4%	for	oligosymptomatics.	The	
be	 useful	 in	 diagnostics	 and	 therapeutics	 of	 Canine	 Leishmaniases.	          Kappa	 statistics	 was	 used	 for	 measure	 the	 real	 concordance	 grade	
Financial	support:	FINEP,	CNPq,	UFU,	DiaMed,	ImunoScan.                              between	the	two	ELISA	assay	and	the	direct	parasitological	exam	and	
Key words:	Canine	Leishmaniasis,	epitopes,	Phage	Display.                            showed	a	good	concordance	between	both	tested	methods.	In	conclu-
Species:	canine                                                                      sion,	both	methods	showed	good	sensibility	and	specificity	for	detec-
                                                                                     tion	disease	in	asymptomatic	and	oligosymptomatic	and		can	be	useful	
  Pr181. DETECTION OF ANTI-LEISHMANIA CHAGASI IGG                                    to	diagnosis	of	visceral	canine	leishmaniosis.	
   SUBCLASS ANTIBODy IN INFECTED AND VACCINATED                                      Key words:	Diagnosis,	ELISA,	Visceral	Leishmaniasis
                       DOGS                                                          Species:	canine
114


      Pr183. MyELOPOIESIS AND ERyTHROPOIESIS IN CANINE                                   compared	to	NI	and	HP	groups.	In	both	parasitized	compartments,	we	
                  VISCERAL LEISHMANIASIS                                                 didn’t	 find	 any	 statistically	 significant	 differences	 in	 the	 frequency	 of	
      RAqUEL	TRóPIA	DE	ABREU1,	MARIA	DAS	GRAçAS	CARVALHo2,	                              CD4+,	CD45RA+	and	CD45RB+	neutrophils	between	the	groups.	The	
         RoDoLFo	CoRDEIRo	GIUnCHETTI11,	CLáUDIA	MARTInS	                                 decrease	in	the	frequency	of	CD14+	neutrophils	in	HP	group	and	the	
        CARnEIRo1,	BRUno	MEnDES	RoATT1,	RoDRIGo	DIAn	DE	                                 increase	 in	 the	 frequency	 of	 MHC-II+	 neutrophils	 in	 MP	 group,	 both	
           oLIVEIRA	AGUIAR1,	ALExAnDRE	BARBoSA	REIS1                                     showing	patterns	of	activated	cells,	suggest	the	participation	of	innate	
                                                                                         cells	trying	to	eliminate	the	parasites	through	phagocytic	mechanisms	
        1Laboratório	de	Imunopatologia,	NUPEB/UFOP;	2Laboratório	de	                     and	through	an	oxidative	burst.	Our	next	aim	is	to	evaluate	the	different	
             Hematologia	Clínica	Faculdade	de	Farmácia/UFMG                              phenotypic	markers	by	frequency	and	Medium	Fluorescence	Channel	
            Canine	 visceral	 leishmaniasis	 (CVL)	 manifests	 itself	 as	 a	 broad	     in	eosinophils	and	monocytes	populations	of	dogs	presenting	different	
      clinical	spectrum	ranging	from	asymptomatic	infection	to	patent	severe	            tissue	L.	(L.)	chagasi	densities.				
      disease.	Herein,	bone	marrow	(BM)	smears	stained	by	Giemsa	were	                       Supported	 by:	          FAPEMIG,	       PAPES	       IVb,	   UFOP	      and	
      evaluated	considering	three	clinical	groups	of	dogs	naturally	infected	by	         IRR/FIOCRUZ/MG.
      Leishmania	chagasi	[i.e.	asymptomatic	(AD,	n	=	54),	oligosymptomatic	
      (OD,	n	=	48)	and	symptomatic	(SD,	n	=	73)]	compared	with	non-infected	             Key words:	 canine	 visceral	 leishmaniasis,	 Leishmania	 (Leishmania)	
      dogs	(NID,	n	=	14).	Analysis	using	preliminary	data	on	erythropoiesis	             chagasi,	tissue	parasite	load,	polymorphonuclear	leukocytes
      showed	no	significant	differences	in	proerythroblast,	basophilic,	poly-            Species:	canine
      chromatic	 and	 orthochromatic	 erythroblasts	 when	 all	 different	 clinical	
      groups	 were	 compared	 to	 control	 and	 among	 themselves.	 After	 a	             Pr185. CHARACTERIzATIoN oF PoTENTIAL SECRETED/
      minucious	observation	of	the	bone	marrow	smears	we	have	found	that	                  ExCRETED ANTIGENS oF LEISHMANIA CHAGASI AND
      leucopoiesis	has	presented	some	alterations	in	infected	dogs.	To	date,	                        LEISHMANIA BRAzILIENSIS
      eosinophilic	cells	number	has	shown	a	significant	decrease	in	the	dif-               SoLAnGECU	BUSEK1,	DAnIEL	M	SoUZA1,	AnDREA	R	S	C	
      ferent	 clinical	 groups	 compared	 to	 control.	 Neutrophilic	 cells	 number	     GUIMARÃES1,	SAMUELL	BRAGA2,	BRUno	M	RoATT2,	RoDRIGo	
      show	 slight	 fluctuations	 in	 the	 different	 clinical	 groups,	 however,	 no	    D	o	A	SoARES2,	REnATA	GUERRA	SA3,	RoDRIGo	CoRREA-
      difference	 was	 found	 among	 them.	 Related	 to	 mononuclear	 cells,	 it	                   oLIVEIRA1,	ALExAnDRE	B	REIS	1,2
      was	observed	for	limphocytes	number	a	significant	increase	in	OD	and	
      SD	groups	when	compared	to	AD	group.	Similar	results	were	found	for	                 	1Laboratório	de	Imunologia	Celular	e	Molecular	-	IRR/FIOCRUZ;	
      plasma	 cell	 number	 showing	 a	 clear	 tendence	 to	 a	 gradual	 increase	         2Laboratório	de	Imunopatologia	-	NUPEB/UFOP;	3Laboratório	de	
      according	to	the	severity	of	the	infection.	By	other	hand,	monocytes	cell	                   Bioquímica	e	Biologia	Molecular	-	NUPEB/UFOP		
      number	has	significantly	decreased	in	all	clinical	groups	compared	to	                                 danielsouza@cpqrr.fiocruz.br
      control.		The	bone	marrow	changes	observed	in	infected	dogs,	mainly	                     The	 Leishmaniasis	 ranges	 from	 asymptomatic	 infection	 to	 self-
      those	 in	 mononuclear	 cells,	 suggest	 that	 defence	 mechanisms	 were	          limiting	cutaneous	lesion(s)	or	fatal	visceral	forms,	depending	on	the	
      triggered	in	order	to	combat	the	infectious	agent	by	cellular	response	            Leishmania	 species	 involved	 and	 on	 the	 immunological	 response	 of	
      and	antibody	production.	In	conclusion,	our	study	shows	that	the	clinical	         the	vertebrate	host.	Previous	reports	stressed	that	secreted/excreted	
      evolution	of	CVL	in	naturally	infected	dogs	promotes	clear	alterations	            (SE)	 proteins	 from	 the	 several	 intracellular	 pathogens	 contain	 highly	
      in	 bone	 marrow	 cells.	 Since	 these	 alterations	 are	 directly	 correlated	    immunogenic	and	protective	antigens	in	vaccine	models.	Similarly,	SE	
      with	CVL	clinical	status	would	be	taken	into	account	when	dealing	with	            antigens	of	L.	major	may	constitute	putative	virulence	factors.	In	addi-
      diagnosis	and	prognosis	features.	                                                 tion,	after	its	phagocytosis	by	macrophages,	molecules	released	by	pro-
           Financial	support:	CNPq,	FAPEMIG,	UFOP	and	UFMG.                              mastigotes	of	Leishmania	could	represent	an	immune	evasion	strategy	
                                                                                         to	 avoid	 cellular	 immune	 response.	 To	 characterize	 SE	 proteins,	 we	
      Key words:	leishmania,	bone	marrow,	myelopoiesis,	erythropoiesis                   obtained	 culture	 supernatants	 of	stationary-phase	 promastigotes	 col-
      Species:	canine                                                                    lected	12	h	after	incubation,	with	the	pH	and	temperature	similar	of	the	
                                                                                         phagolysosomal	 vacuole	 conditions.	 Our	 results	 showed	 that	 L.	 cha-
                Pr184. PHENOTyPIC CHANGES IN                                             gasi	incubated	in	normal	culture	conditions	(pH	7.2	and	26ºC)	released	
         POLyMORPHONUCLEAR CELLS OF BRAZILIAN DOGS                                       more	proteins	than	L.	braziliensis.	On	the	other	hand,	in	conditions	that	
         NATURALLy INFECTED By LEISHMANIA (LEISHMANIA)                                   partially	mimic	macrophage	vacuole	environment	(pH	5.5	and	35ºC),	
                           CHAGASI                                                       the	release	of	proteins	in	both	species	was	diminished.	However,	the	
      LUAnDA	L	GUERRA1,	AnDRéA	TEIxEIRA-CARVALHo1,3,	oLInDo	                             profile	of	proteins	of	both	species	was	similar	in	the	two	pH	conditions.	
        A	MARTInS-FILHo3,	RoDoLFo	C	GIUnCHETTI1,2,	RoDRIGo	                              Western	blot	was	carried	out	to	identify	SE	antigens.	The	majority	of	
              CoRRêA-oLIVEIRA1,	ALExAnDRE	B	REIS1,2                                      proteins	from	supernatant	of	L.	chagasi	in	the	both	conditions	of	culture	
      1Laboratório	de	Imunologia	Celular	e	Molecular,	IRR-FIOCRUZ-Belo	                  strongly	reacted	with	serum	of	naturally	infected	dogs	with	L.	chagasi.	
        Horizonte/MG;	2Laboratório	de	Imunopatologia,	ICEBII-NUPEB/                      In	contrast,	only	five	proteins	of	L.	braziliensis	were	identified	as	anti-
       UFOP-Ouro	Preto/MG;	3Laboratório	de	Doença	de	Chagas,	IRR-                        genic	by	serum	of	naturally	infected	dogs	with	L.	braziliensis.	The	time	
                        FIOCRUZ-Belo	Horizonte/MG		                                      course	 of	 the	 study	 showed	 that	 secretion/excretion	 of	 antigens	 was	
                           luanda@cpqrr.fiocruz.br                                       qualitatively	and	quantitatively	different	among	the	species	studied	and	
                                                                                         the	 repertoire	 of	 these	 secreted	 proteins	 will	 help	 us	 select	 relevant	
           Canine	Visceral	Leishmaniasis	(CVL)	is	one	of	the	most	important	             antigens	for	the	development	of	anti-Leishmania	vaccine.
      emerging	diseases	with	high	prevalence	in	Latin	American	countries.	
      Considering	the	putative	role	of	dogs	in	the	transmission	of	the	disease	          Key words:	Leishmania	chagasi;	Leishmania	braziliensis	;	antigens
      and	 the	 importance	 of	 polymorphonuclear	 leukocytes	 in	 Leishmania	           Species:	canine
      infections,	 we	 proposed	 to	 evaluate	 the	 frequency	 of	 different	 cell	
      markers	 (CD4,	 CD14,	 CD45RA,	 CD45RB	 and	 MHC-II)	 in	 systemic	                    Pr186. NEoSPoRA CANINUM MODULATES CANINE
      neutrophils	 of	 dogs	 naturally	 infected	 by	 Leishmania	 (Leishmania)	              SySTEMIC CELLULAR IMMUNE RESPONSES DURING
      chagasi.	 Herein,	 30	 dogs	 were	 subdivided	 into	 three	 groups	 accord-                       ACUTE ORAL INFECTION
      ing	to	spleen	or	bone	marrow	parasitism:	Low	Parasitism	(LP;	n=10),	                  TIAGo	WILSon	PATRIARCA	MInEo,	RoSAnGELA	ZACARIAS	
      Medium	 Parasitism	 (MP;	 n=10)	 and	 High	 Parasitism	 (HP;	 n=10).	                                     MACHADo	
      The	 criteria	 used	 for	 parasite	 load	 was	 the	 LDU	 values	 (“Leishman	
      Donovan	 Units”),	 defined	 such	 as	 number	 of	 amastigotes	 per	 1000	           Department	of	Veterinary	Pathology,	FCAV/UNESP,	Jaboticabal,	SP
      nucleated	cells.	Twenty	non-infected	dogs	(NI),	used	as	control	group,	                 neospora	 caninum	 is	 a	 protozoan	 parasite	 which	 has	 been	
      were	 serologically	 and	 parasitologically	 negative	 for	 L.	 (L.)	 chagasi.	    pointed	out	as	a	major	abortion	inducing	disease	in	cattle.	Dogs,	coy-
      Our	 data	 demonstrated	 a	 decrease	 in	 the	 frequency	 of	 CD14+	 neu-          otes	 and,	 probably,	 other	 canids	 are	 the	 parasite’s	 definitive	 hosts,	
      trophils	in	dogs	with	high	splenic	parasitism	in	comparison	to	the	LP	             responsible	for	the	fecal	elimination	of	resistant	forms	(oocysts),	which	
      group.	The	 results	 of	 bone	 marrow	 parasitism	 showed	 that	 MP	 dogs	         are	 disseminated	 through	 the	 environment.	 Canine	 cellular	 immune	
      presented	an	increase	in	the	frequency	of	MHC-II+	neutrophils	when	                responses	after	n.	caninum	infection	have	not	been	evaluated	to	date.	
                                                                                                                                                                          115


Cattle	 and	 mice	 respond	 to	 infection	 by	 the	 protozoa	 in	 a	 predomi-       higher	susceptibility	of	puppies	to	n.	caninum	may	be	attributed	to	the	
nantly	Th1	manner,	controlling	parasite	replication.	The	known	excep-               ontogeny	of	the	immune	system.
tions	 are	 gestational	 periods,	 when	 female	 hormone	 production	 is	           Key words:	 Neospora	 caninum,	 Antibody	 kinetics,	 Delayed	
shifted	towards	progesterone,	which	possess	potent	anti-inflammatory	               seroconversion,	Ontogeny	of	immune	system
action	at	the	fetal-maternal	interface.	Taken	together,	this	work	aimed	            Species:	canine
to	 observe	 the	 events	 triggered	 in	 canine	 systemic	 cellular	 immunity	
by	 oral	 infection	 with	 n.	 caninum.	 Clinically	 healthy	 dogs,	 serologi-
                                                                                      Pr188. FREQUENCIES OF ANTI-TRyPANOSOMA CRUZI
cally	negative	for	n.	caninum	and	correlated	parasites,	were	selected	
and	divided	into	three	main	groups:	Inoculated	adult	dogs,	inoculated	               ANTIBODIES IN DOGS FROM SOUTHERN OF RIO GRANDE
puppies,	and	control	group.	Inoculated	animals	were	submitted	to	oral	                                 DO SUL STATE
intake	of	500g/day	of	fresh	bovine	meat,	nervous	tissues	and	offal,	for	               CHARLEnE	n	S	TRInDADE,	FABIo	PL	LEITE,nARA	R	FARIAS
three	consecutive	days.	Blood	samples	were	collected	by	vein	punc-                           Departamento	de	Microbiologia	e	Parasitologia-UFPel	
tion	at	close	time	intervals	to	observe	CD4+	and	CD8+	T	cell	kinetics	                                   fabio_leite@ufpel.tche.br
by	FACS	till	200th	days	post-infection	(dpi),	and	popliteal	lymph	nodes	
were	 removed	 surgically	 at	 30	 dpi	 for	 immunohistochemical	 assays.	                Chagas’	 disease	 is	 a	 trypanosomiasis	 found	 in	 the	 Americas	
Additionally,	 acute	 phase	 (90	 dpi)	 cytokine	 mRNA	 expression	 was	            whose	etiological	agent	is	Trypanosoma	cruzi.	The	different	epidemio-
measured	in	PBMC	from	the	puppy	group	by	Real-time	RT-PCR.	The	                     logical	and	clinical	manifestations	of	the	infection	and	the	mechanisms	
results	herein	obtained	indicate	that	dogs	present	a	protracted	acute	              and/or	 factors	 that	 determine	 the	 development	 of	 morbidity	 are	 still	
phase,	with	oocyst	shedding	being	correlated	to	a	drop	in	CD4+	and	                 unclear.	 Several	 important	 points	 remain	 to	 be	 clarified	 with	 regard	
CD8+	T	cell	blood	levels,	and	low	MHC	class	II	expression	in	the	lymph	             to	the	natural	history	of	Chagas’	disease.	One	of	these	is	the	role	of	
nodes.	Cytokine	mRNA	profile	revealed	that	dogs	present	high	modu-                  domestic’s	 animals,	 principally	 the	 dog,	 as	 potential	 element	 for	 the	
latory	cytokine	expression	till	the	60th	dpi,	followed	by	a	sharp	increase	         transmission	 and	 maintenance	 of	 the	 parasite	 in	 the	 human	 popula-
in	 inflammatory	 cytokines,	 as	 IFNγ.	 Based	 on	 these	 results,	 we	 may	       tion.	 The	 prevalence	 of	 infection	 among	 dogs	 from	 endemic	 zones,	
conclude	that	n.	caninum	modulates	the	dog	cellular	immune	response	                the	 close	 relationship	 between	 human	 and	 the	 identity	 of	 parasites	
during	its	sexual	cycle.                                                            infecting	humans	and	dogs,	suggest	that	this	disease	possibly	involves	
                                                                                    these	animals	as	reservoirs.	The	aim	of	this	study	was	to	characterize	
Key words:	Neospora	caninum,	CD4+	and	CD8+	T	cells,	MHC	class	                      the	 frequency	 of	 antibodies	 anti-T.	 cruzi	 	 in	 a	 dog	 population	 from	 a	
II,	Cytokine	mRNA	expression                                                        Chagas’	disease	in	an	endemic	area	located	at	the	south	of	Rio	Grande	
Species:	canine                                                                     do	 Sul	 state.	 Serum	 from	 99	 dogs	 was	 collected,	 in	 rural	 and	 urban	
                                                                                    area,	 and	 analyzed	 by	 a	 commercial	 indirect	 immunofluorescent	 test	
Pr187. CANINE HUMORAL IMMUNE RESPONSES ELICITED
                                                                                    (IFI)	using	an	anti-dog	IgG	labeled	with	FITC.	The	sera	were	diluted	1:	
   IN ORAL INFECTIONS WITH NEoSPoRA CANINUM                                         32	and	analyzed	microscopically	in	duplicate.	From	the	99	sera	studied	
       TIAGo	WILSon	PATRIARCA	MInEo,	MICHAEL	J	DAy*,	                               90.8%		(89)	were	positive	to	T.	cruzi.	The	majority	of	the	positive	dogs	
              RoSAnGELA	ZACARIAS	MACHADo                                            to	T.	cruzi	live	in	houses	with	people,	independently,	of	rural	or	urban	
Department	of	Veterinary	Pathology,	FCAV/UNESP,	Jaboticabal,	SP;	                   area.	These	results	suggest	that	the	dog	might	play	an	important	role	
*	Division	of	Veterinary	Pathology,	Infection	and	Immunity,	School	of	              in	the	transmission	and/or	maintenance	of	T.	cruzi	in	endemic	areas	in	
    Clinical	Veterinary		Science,	University	of	Bristol,	Bristol,	UK                the	south	of	Brazil.	This	work	is	relevant	to	the	question	whether	dogs	
                                                                                    act	 as	 reservoirs,	 disseminating	 the	 infection	 or	 are	 just	 susceptible	
      neospora	caninum	is	an	Apicomplexan	parasite	firstly	described	
                                                                                    to	 T.	 cruzi	 without	 having	 a	 major	 role	 in	 the	 spread	 of	 the	 disease.	
as	 cause	 of	 encephalomyelitis	 in	 puppies	 serologically	 negative	 to	
                                                                                    Thus,	 better	 diagnostics	 and	 epidemiological	 survey	 should	 be	 done	
Toxoplasma	 gondii.	 Previous	 reports	 on	 the	 parasite’s	 definitive	 host	
                                                                                    in	domestic’s	animals	in	order	to	prevent	this	disease.	This	observa-
indicate	a	late	IgG	antibody	response	and	that	clinical	disease	is	difficult	
to	be	induced.	The	aim	of	this	study	was	to	investigate	canine	humoral	             tions	suggest	that	population	in	this	areas	should	be	alerted	as	well	the	
immunity	during	n.	caninum	oral	infection.	For	that	purpose,	clinically	            authorities	responsible	for	the	control	of	this	important	zoonose.				
healthy	 dogs,	 serologically	 negative	 for	 n.	 caninum	 and	 correlated	         Key words:		Trypanosoma	cruzi,	dogs,	antibodies
parasites	were	selected	and	divided	into	three	main	groups:	inoculated	             Species:	canine
adult	dogs,	inoculated	puppies,	and	control	group.	Inoculated	animals	
were	submitted	to	oral	intake	of	500g/day	of	fresh	bovine	meat,	ner-                      Pr189. LEISHIMUNE VACCINE-INDUCED IMMUNE
vous	 tissues	 and	 offal,	 for	 three	 consecutive	 days.	 Blood	 samples	               RESPONSE IN DOGS FROM AN ENDEMIC AREA OF
were	collected	by	vein	punction	at	close	time	intervals	to	observe	total	                           VISCERAL LEIHMANIASIS
and	 specific	 antibody	 kinetics	 for	 250	 days	 post-infection	 (dpi).	 Total	         VMF	LIMA1,FA	IKEDA2,	Cn	RoSSI2,	M	M	FEIToSA1,	Ro	
antibody	 production	 was	 followed	 up	 by	 commercial	 ELISA	 kits	 and	                     VASConCELoS2,	DP	MUnARI,2,	H	GoTo3	
specific	immunoenzimatic	assays	were	standardized	for	IgM,	IgA,	IgE,	
IgG	and	subclasses	(IgG1,	IgG2,	IgG3,	IgG4).	Additionally,	detection	                 1Dept.	of	Clinic,	Surgery	and	Animal	Reproduction-	FO	-	UNESP-	
of	specific	IgM	and	IgG	were	realized	through	indirect	immunofluores-                  Araçatuba-S.P-Brazil;	2UNESP-	Jaboticabal-	Brazil;	3Tropical	
cence	 antibody	 tests	 (IFAT),	 and	 IgG	 antigen	 recognition	 profile	 was	                 Medicine	Institute	-	USP-São	Paulo-	S.P-Brazil
visualized	through	one	dimensional	western	blotting	(WB).	The	results	                    Dogs	with	visceral	leishmaniasis	(VL)	constitutes	a	public	health	
obtained	indicate	that	dogs	present	a	low	specific	antibody	production	             problem	as	they	are	the	potential	sources	of	the	parasites	transmitted	
during	acute	phase	of	infection,	showing	an	unstable	seroconversion	                to	 humans	 through	 the	 vector.	To	 control	 the	 transmission	 a	 vaccine	
pattern,	with	only	IgG1	and	IgG3	being	detected	in	adult	dogs	and	pup-              named	 Leishmune	 (Fort	 Dodge)	 based	 on	 Fucose-mannose	 Ligand	
pies,	respectively,	between	the	second	and	third	months	of	infection.	              (FML)	 of	 L.	 chagasi	 has	 been	 widely	 used,	 but	 the	 immune	 reponse	
Corroborating	with	those	results,	WB	analysis	demonstrated	absence	                 in	 vaccinated	 dogs	 has	 not	 been	 extensively	 studied.	 Twenty	 dogs	
of	antigen	recognition	within	the	first	30	dpi	in	almost	all	animals.	IFAT	         from	Araçatuba,	endemic	area	for	VL	in	the	Southeast	of	Brazil,	with-
showed	similar	pattern,	with	late	IgM	and	IgG	seroconversion.	During	               out	previous	Leishmania	infection	screened	by	serology	and	negative	
the	 experimental	 period,	 five	 IgG2	 and	 IgE	 peaks	 were	 observed	 in	        parasitological	exams	for	leishmaniasis,	with	normal	hemogram,	were	
an	 associated	 manner,	 and	 were	 also	 correlated	 to	 lower	 Th1	 type	         vaccinated	subcutaneously	with	Leishmune	and	cellular	and	humoral	
antibody	 production.	Analyzing	 total	 antibody	 production,	 it	 could	 be	       immune	 responses	 evaluated	 before	 (control)	 and	 ten	 and	 30	 days	
observed	that	younger	animals	present	a	crescent	curve	in	all	analyzed	             after	 vaccination.	After	 vaccination,	 the	 lymphoproliferative	 response	
antibodies,	whereas	adult	animals	demonstrate	stable	serum	antibody	                of	peripheral	blood	mononuclear	cells	(PBMCs)	was	positive	in	80%	to	
concentrations.	After	60	dpi,	specific	antibody	levels	showed	an	incre-             Leishmania	whole	antigen	and	in	85%	to	FML	antigen.	The	expression	
ment,	 especially	 IgG1	 and	 IgG4	 in	 puppies.	Taken	 together,	 we	 may	         of	CD4/CD25+	on	T	cells	(by	cytometry)	decreased	significantly	after	
conclude	that	antibody	production	is	initially	impaired	in	dogs	after	oral	         immunization	(p<	0.05).	The	IFN-γ	level	was	higher	in	supernatant	of	
intake	of	parasite	contaminated	tissues,	and	that	Th1	and	Th2	profile	              Leishmania	 antigen-	 or	 FML-induced	 culture	 cells	 after	 vaccination	
antibodies	 are	 present	 concomitantly	 after	 infection.	 Moreover,	 the	         (P<0,05),	but	IL-4	and	TNF-a	levels	were	similar	in	these	periods,	and	
116


      no	IL-10	was	detected.	The	vaccinated	dogs	produced	high	levels	of	                    antibodies	was	used	protein	A	peroxidase	conjugated,	the	cut-off	was	the	
      antibody	against	Leishmania	whole	antigen	and	FML	antigen	(ELISA)	                     same	used	to	domestic	dog.	The	DNA	was	isolated	following	the	method	
      that	 remainded	 high	 after	 one	 year	 (P	 <0.05)	 although	 the	 animals	           phenol/clorophormio/isoamílic	 (25:24:1)	 and	 PCR	 was	 performed	 with	
      were	parasitologically	negative.		Positive	lymphoproliferative	response	               13	A,	13	B	primer.	In	two	animal	investigated	by	ELISA	positive	reaction	
      to	FML	and	Leishmania	antigen,	decreased	frequency	of	CD4/CD25+	                       was	observed.	The	biopsys	analyzed	by	PCR	showed	positive	in	liver	
      T	 cell,	 an	 increase	 of	 IFN-γ	 level	 and	 no	 detectable	 IL-10	 in	 culture	     and		lymphonodes	samples.		This	is	the	first	report	that	indicate	that	this	
      supernatant	 may	 be	 considered	 a	 counterpart	 of	 protective	 immune	              species	as	a	possible	reservoir	of	Leishmania	sp	
      response	against	Leishmania	parasite.                                                  Key words:	LEISHMANIA,	Speothos	venaticus,	ELISA,	PCR
      Key words:	 vaccine,	 leishmania,	 cellular	 immunity,	 visceral	                      Species:	other
      leishmaniasis
      Species:	canine                                                                        Pr192. NEoSPoRA CANINUM TACHyZOITES, ATTENUATED
                                                                                              By PASSAGE IN TISSUE CULTURE, INDUCE PROTECTIVE
      Pr190. ALTERNATIVE METHODS TO DIFFERENTIATE DOGS                                                            IMMUNITy
       VACCINATED WITH LEISHMMUNE AND INFECTED DOGS IN                                         ELISABETH	A	InnES,	PAUL	M	BARTLEy,	STEPHEn	E	WRIGHT,	
      AN ENDEMIC AREA OF CANINE VISCERAL LEISHMANIASIS                                                  FRAnCESCA	CHIAnInI,DAVID	BUxTon
                        VMF	LIMA,	JP	CoRREA,	KR	FATToRI	                                       Moredun	Research	Institute,	Pentlands	Science	Park,	Bush	Loan,	
        Depto	Clínica,	Cirurgia	e	Reprodução	Animal	–	UNESP	Araçatuba	                                            Midlothian,	EH26	OPZ		
                             vmflima@fmva.unesp.br                                                              lee.innes@moredun.ac.uk
             Zoonotic	 visceral	 leishmaniasis	 is	 caused	 by	 Leishmania	                       neospora	caninum	is	an	apicomplexan	parasite	that	is	recognised	
      (Leishmania)	 chagasi.	 It	 is	 endemic	 in	 America,	 Europe,	 and	 Asian	            as	a	major	cause	of	reproductive	failure	in	cattle	worldwide.	Cell-medi-
      countries	with	new	dissemination	areas	being	identified.	Transmission	                 ated	immune	responses	involving	CD4+	T-cells	and	interferon	gamma	
      between	vertebrate	hosts	is	by	the	phlebotomine	hematophagous	bite	                    (IFNγ)	are	known	to	be	important	in	host	protection.	Vaccine	develop-
      of	Lutzomyia	longipalpis.	Dogs	(Canis	familiares)	were	considered	the	                 ment	to	help	control	bovine	neosporosis	is	likely	to	require	induction	of	
      most	 important	 urban	 reservoirs	 of	 L.	 (L.)	 chagasi,	 as	 show	 a	 larger	       specific	 CMI	 responses	 and	 this	 is	 often	 easier	 to	 achieve	 using	 live	
      prevalence	to	infection	and	from	where	it	gets	transmitted	to	humans	                  preparations.	
      Dogs	 have	 been	 shown	 to	 be	 highly	 susceptible	 to	 infection	 and	 the	               Live	vaccines	using	attenuated	organisms	have	been	successful	
      current	treatments	have	limited	efficiency.	Therefore,	the	use	of	a	pre-               in	inducing	protective	immunity	against	other	protozoan	parasites	such	
      ventive	vaccine	is	fundamental.	Several	studies	have	been	shown	that	                  as	 Leishmania,	 Toxoplasma	 and	 Theileria	 where	 induction	 of	 CMI	 is	
      the	 vaccination	 with	 Leishimmune	 vaccine	 induces	 a	 strong	 humoral	             important.	neospora	caninum	tachyzoites	were	passaged	within	Vero	
      response	with	production	of	antibodies	to	FML	antigen	and	antibodies	                  cells	for	different	lengths	of	time	in	vitro	and	compared	for	their	ability	
      that	 reacts	 with	 total	 antigen	 from	 Leishmania	 sp.	 The	 antigen	 total	        to	cause	disease	following	inoculation	into	mice.	Mice	inoculated	with	
      is	 frequently	 used	 in	 RIFI	 and	 ELISA	 assay	 to	 detect	 disease,	 so	 in	       the	high-passage	parasites	survived	significantly	longer	(P<0.05)	and	
      endemic	areas	the	infection	in	vaccinated	dogs	should	be	monitoring	                   showed	fewer	clinical	symptoms	compared	to	mice	receiving	a	similar	
      by	 other	 methods.	 The	 aims	 of	 this	 study	 was	 investigated	 by	 PCR	           dose	of	the	low-passage	parasites.	The	high	passage	parasites	mul-
      in	 peripherical	 blood	 of	 infected	 dogs	 and	 the	 sororeactivity	 of	 dogs	       tiplied	more	rapidly	(P<0.001)	in	vitro	than	the	low	passage	parasites.	
      vaccinated	to	antigen	k39	as	a	alternative	methods	to	diagnosis	and	                   Similar	results	were	achieved	in	three	separate	experiments	indicating	
      differenciation	of	infected	and	vaccinated	dogs.	To	PCR	one	hundred	                   that	it	is	possible	to	attenuate	the	virulence	of	n.caninum	tachyzoites	in	
      animals	infected	were	used,	DNA	from	peripherical	blood	was	obtained	                  mice	through	prolonged	in	vitro	passage.
      and	 the	 PCR	 with	 primer	 13A	 and	 13B	 was	 performed.	 The	 PCR	                      Mice	inoculated	with	attenuated	n.caninum	tachyzoites	were	fully	
      showed	 91%	 sensibility.	 In	 sororeactivity	 against	 K39	 twenty	 healthy	          protected	against	a	challenge	dose	of	parasites	that	proved	to	be	lethal	
      animals	were	vaccinated	and	ten	days	after	the	sera	was	tested,	only	                  in	naïve	control	mice.	Brain	tissue	was	examined	at	28	days	after	chal-
      10%	 of	 group	 showed	 positive	 reaction	 to	 this	 antigen,	 in	 the	 end	 of	      lenge,	in	all	mice,	using	a	quantitative	PCR	test	for	neospora-specific	
      vaccination	the	bone	marrow	exams	was	negative,	suggesting	that	the	                   DNA.	In	mice	initially	inoculated	with	the	attenuated	parasites	and	then	
      positivity	 observed	 can	 be	 due	 to	 cross	 reaction.	 In	 conclusion,	 both	       challenged,	56%	had	neospora-specific	DNA	in	brain	tissue,	whereas	
      PCR	 and	 serum	 reactivity	 against	 k39	 can	 be	 useful	 to	 diagnosis	 of	         100%	of	the	control	mice	had	neospora-specific	DNA	following	chal-
      canine	 visceral	 leishmaniosis	 with	 a	 security	 of	 90%	 in	 areas	 where	         lenge	 and	 in	 significantly	 higher	 quantity	 (P<0.001)	 compared	 to	 the	
      Leishimmune	vacccine	have	been	used.                                                   immunised	group.
           Financial	support:	FAPESP                                                              Attenuation	of	virulence	was	achieved	through	prolonged	passage	
      Key words:	LEISHIMMUNE	VACCINE,	DIAGNOSIS,	PCR,	K-39                                   of	n.caninum	tachyzoites	in	tissue	culture	and	inoculation	of	the	attenu-
      Species:	canine                                                                        ated	parasites	into	mice	induced	protection	against	a	lethal	challenge.
                                                                                             Key words :	Neospora	caninum,	attenuation,	virulence,	protection
             Pr191. AMERICAN VISCERAL LEISHMANIASIS IN                                       Species:	other
                       SPEoTHoS VENATICUS
                                                                                                  Pr193. PARTICIPATION OF THE MACROPHAGE
      KARInA	REInALDo	FATToRI1,	VALéRIA	MARçAL	FELIx	DE	LIMA2
                                                                                                 INFLAMMATORy PROTEIN-CCL3 IN THE IMMUNE
         1UNESP-FCAV-Campus	Jaboticabal,	Programa	de	Microbiologia	                           RESPONSE INDUCED By FASCIOLA HEPATICA INFECTION
          Agropecuária;	2Depto.	Clínica,	Cirurgia	e	Reprodução	Animal	-	
                                                                                                              IN C57BL/6J MICE
                             UNESP	ARAÇATUBA
                                                                                                 REnATA	CRISTInA	DE	PAULA1,	ADRIAno	LUIS	SoARES	
            Visceral	Leishmaniasis	(VL)	caused	by	Leishmania	L	chagasi,	is	a	
                                                                                                SoUZA2,	GIoVAnI	CASSALI3,	MARCo	PEZZI	GUIMARÃES1,	
      disease	of	human	and	canids.	The	domestic	dog	is	the	main	source	of	
                                                                                               MAURo	MARTInS	TEIxEIRA2,	DéBoRAH	nEGRÃo-CoRRêA1
      human	infection,	with	peridomestic	and	domestic	transmission	effected	
      principally	 by	 sandfly	 vector	 Lutzomya	 longipalpis.	 Control	 measures	            	1Department	of	Parasitology2	2Biochemistry	and	Immunology	and	
      have	 included	 the	 controversial	 extermination	 of	 serologically	 or	 para-        Pathology;	3Biological	Science	Institute	-	UFMG,	Belo	Horizonte,	MG
      sitologically	 positive	 dog,	 but	 the	 existence	 of	 wild	 animal	 host	 clearly	         Fasciola	 hepatica	 is	 a	 trematode	 parasite	 that	 inhabits	 the	 liver	
      complicates	the	situation.	The	“wild”	canids	as	additional	reservoirs	have	            and	 gallbladder	 ducts	 of	 hosts,	 including	 bovine,	 ovine	 and	 human.	
      been	studied.	The	aims	of	the	present	studies	were	to	investigate	the	                 The	 hepatic	 injuries	 are	 associated	 with	 direct	 lesion	 as	 well	 as	 the	
      presence	of	antibodies	anti-Leishmania	by	ELISA	in	sera	and	the	pres-                  intensity	 of	 the	 inflammatory	 reaction	 induced	 by	 the	 parasite	 dur-
      ence	of	DNA	from	Leishmania	by	PCR		in	biopsies	from	lymphonodes,	                     ing	 the	 migration	 of	 the	 immature	 forms	 and	 establishment	 of	 adult	
      spleen,	skin	and	liver	from	two	Speothos	venaticus	(maintained	in	Center	              worms.	 Macrophage	 inflammatory	 protein	 (CCL3),	 produced	 and	
      of	sylvester	animals-CESP-	Ilha	Solteira-S.P-	Brazil).	ELISA	assay	the	                secreted	 mainly	 by	 activated	 macrophages,	 has	 a	 crucial	 role	 in	 the	
      antigen	used	was	a	total	antigen	from	Leishmania	and	to	detection	of	                  recruitment	 of	 pro-inflammatory	 cells	 to	 the	 lesion	 site.	 Therefore,	 in	
                                                                                                                                                                        117


the	 present	 work	 we	 evaluated	 the	 immune	 response	 induced	 by	 F.	              Department	of	Disease	Control,	Graduate	School	of	Veterinary	
hepatica	 infection	 (10	 metacercariae/mice)	 in	 C57Bl/6J	 mice	 geneti-            Medicine,	Hokkaido	University,	Sapporo,	Hokkaido	060-0818,	Japan		
cally	deficient	in	CCL3	production	(CCL3KO)	versus	the	wild	type	mice	                                 konnai@vetmed.hokudai.ac.jp
(WT).	A	significantly	higher	number	of	immature	worms	were	recovered	                      Previously,	a	novel	immunosuppressive	protein	coding	gene	was	
from	CCL3KO	mice	compared	to	WT	mice.	Although	in	higher	number,	                    identified	from	a	cDNA	library	derived	from	salivary	gland	of	partially-
the	liver	lesion	induced	by	F.	hepatica	was	less	extensive	in	CCL3KO	                fed	 Haemaphysalis	 longicornis	 (hard	 tick).	 	 By	 real-time	 PCR	 assay,	
infected	 mice.	 In	 addition,	 antigen-stimulated	 spleen	 cells	 recovered	        the	 gene	 was	 expressed	 during	 blood	 feeding	 and	 suggested	 to	 be	
from	20	day-infected	CCL3KO	mice	produced	significantly	lower	level	                 expressed	 mainly	 in	 the	 salivary	 gland.	 	 In	 order	 to	 investigate	 the	
of	 IL-4,	 IL-13,	 IL-10	 and	 IFN-γ	 cytokines	 and	 the	 liver	 of	 these	 mice	   function	of	this	novel	protein,	we	examined	the	proliferative	response	
showed	 significantly	 lower	 eosinophil	 peroxidase	 and	 mieloperoxi-
                                                                                     and	 cytokine	 expressions	 of	 mitogen-stimulated	 peripheral	 blood	
dase	activity	than	the	WT-infected	mice.	The	diminished	inflammatory	
                                                                                     mononuclear	 cells	 (PBMCs)	 and	 splenocytes	 from	 cattle	 and	 mice	
response	of	CCL3KO	infected-mice	was	followed	by	significantly	lower	
                                                                                     by	addition	with	this	recombinant	protein	(recombinant	H.	longicornis	
F.	hepatica-induced	mortality	rate	between	20	and	23	days	of	infection,	
                                                                                     immunosuppressor:	rHeLIS).		Addition	of	rHeLIS	and	cultivation	for	72	
however,	 the	 mortality	 increases	 in	 CCL3KO	 infected-mice	 after	 25	
                                                                                     h	clearly	showed	the	inhibition	of	the	proliferation	of	mitogen-stimulated	
days	of	infection.	The	results	indicated	that	CCL3	production	is	crucial	
                                                                                     cells	in	a	dose-dependent	manner,	and	down	regulation	of	the	mean	
to	cellular	activation	and	migration	to	infection	site	during	F.	hepatica	
                                                                                     interleukin-2	mRNA	expression	level.		Addition	of	anti-HeLIS	antibody	
in	mice.
                                                                                     with	 rHeLS	 and	 cultivation	 for	 72	 h	 clearly	 enhanced	 proliferation	 of	
Key words:	 chemokines,	 macrophages	 migration,	 CCL3,	 Fasciola	                   cells,	indicating	a	direct	involvement	of	HeLIS	in	the	cell	proliferation.	    	
hepatica                                                                             Furthermore,	 in	 in	 vivo	 experiment,	 proliferative	 response	 of	 spleno-
Species:	other                                                                       cytes	isolated	from	rHeLIS-inoculated	mice	(150µg	x3)	was	significantly	
                                                                                     lower	than	that	of	control	mice.		Interestingly,	microarray	analysis	of	the	
   Pr194. INDUCTION OF ExPERIMENTAL AUTOIMMUNE                                       splenocytes	 derived	 from	 rHeLIS	 inoculated	 mice	 showed	 significant	
    MyOCARDITIS WITH AUTOANTIGEN IMMUNIZATION                                        down	regulation	of	several	immunomodulating	genes,	such	as	MHC,	
   ASSOCIATED WITH LOW NUMBERS OF TRyPANoSoMA                                        CD2	and	CD8.		In	conclusion,	these	results	suggest	that	HeLIS	is	an	
                       CRUzI                                                         immunosuppressor,	which	might	play	an	important	role	in	the	modula-
    L	S	DE	ARAGÃo1,2,	R	R	S	FEIToSA3,2,	R	S	LIMA2,	V	M	G	                            tion	of	host	immune	responses.				
  SILVA1,2,	P	S	oLIVEIRA4,2,	T	BAqUEIRo5,	M	B	P	SoARES2,	R	                          Key Words:	Tick,	salivary	gland,	immunosuppressor,	cell	proliferation
  RIBEIRo	DoS	SAnToS2,	W	L	C	DoS-SAnToS2,	L	PonTES	DE	                               Species:	other
                         CARVALHo2
1Instituto	de	Ciências	da	Saúde,	UFBA,	Salvador,	Brazil;	2Centro	de	                 Pr196. DETECTION OF ANTIBODIES AGAINST EHRLICHIA
 Pesquisa	Gonçalo	Moniz,	FIOCRUZ,	Salvador,	Brazil;	3Escola	de	                      CANIS AND BABESIA CANIS IN BRAZILIAN WILD CAPTIVE
Medicina	Veterinária,	UFBA,	Salvador,	Brazil;	4Instituto	de	Biologia,	                                    FELIDS
 UFBA,	Salvador,	Brazil;	5Instituto	multidisciplinar	de	Saúde,	UFBA,	                  MARCoS	R	AnDRé1,	RoSAnGELA	Z	MACHADo1,	SILMARA	M	
                           Salvador,	Brazil                                             ALLEGRETTI2,	CRISTInA	H	ADAnIA3,	PAULo	A	n	FELIPPE2,	
      Myocarditis	can	be	induced	in	mice	by	immunization	with	myosin-                          KETTy	F	SILVA1,	AnDRéA	C	H	nAKAGHI1
enriched	 heart	 antigen	 (myosin-enriched	 Ag)	 emulsified	 in	 complete	
                                                                                      1Universidade	Estadual	Paulista	(UNESP),	Jaboticabal,	SP,	Brazil;	
Freund´s	adjuvant	(CFA)(1,2).	This	procedure,	however,	did	not	con-
                                                                                      2Universidade	Estadual	de	Campinas	(UNICAMP),	Campinas,	SP,	
sistently	induce	myocaditis	in	different	mouse	strains	in	our	laboratory.	
                                                                                     Brazil;	3Centro	Brasileiro	de	Conservação	de	Felídeos	Neotropicais,	
In	this	work	a	protocol	involving	the	immunization	of	BALB/c	mice	with	
                                                                                                               Jundiaí,	SP,	Brazil		
myosin-enriched	Ag,		emulsified	in	CFA,	followed	by	infection	with	a	low	
                                                                                                            aguaron@fcav.unesp.br
number	of	Y-strain	T.	cruzi	trypomastigotes,	was	evaluated.	The	myo-
sin-enriched	Ag	used	in	this	protocol	was	produced	in	our	laboratory	as	                   Ehrlichiosis	is	an	emergent	zoonosis	caused	by	an	obligate	intra-
described	by	Margossian	et	al	(3).	This	protocol	induced	myocarditis	in	             cellular	bacteria	belonging	to	Anaplasma,	Ehrlichia	and	neorickettsia	
all	treated	mice,	which	was	significantly	more	severe	than	in	mice	only	             genera,	 present	 in	 the	 environment	 through	 complex	 interactions	
infected	with	T.	cruzi.	Immunization	with	myosin-enriched	Ag	alone	did	              among	 invertebrates	 and	 vertebrates	 hosts.	 Babesiosis	 is	 caused	
not	 induced	 myocarditis.	 Preliminary	 results	 indicate	 that	 a	 reduction	      by	 infection	 with	 intraerythrocytic	 parasites	 of	 the	 genus	 Babesia.	
of	 Mycobacterium	 tuberculosis	 concentration	 in	 the	 myosin-enriched	            Both	parasites	are	transmitted	by	ticks.	Brazilian	dogs	are	commonly	
Ag	 emulsion	 increases	 the	 severity	 of	 the	 experimental	 myocarditis.	         infected	by	E.	canis	and	B.	canis,	whose	vector	is	the	dog	brown	tick,	
Experiments	to	investigate	if	the	use	of	the	low	M.	tuberculosis	amount	             the	Rhipicephalus	sanguineus.	Little	is	known	about	the	epidemiology	
improves	the	efficacy	of	the	two-step	protocol	are	underway.                         of	ehrlichiosis	and	babesiosis	in	wild	felids,	which	can	be	seen	as	good	
     1.	Pontes	de	Carvalho	L,	Santana,	C.	C.,	Soares,	M.	B.	P.,	Oliveira,	           sentinels	for	these	diseases,	since	they	are	hosts	for	both	hemopara-
G.	 G.	 S.,	 Cunha-Neto,	 E.,	 Santos	 RR.	 Experimental	 chronic	 Chagas	           sites	and	vectors.	The	present	work	aimed	to	detect	antibodies	against	
disease	myocarditis	is	an	autoimmune	disease	preventable	by	induc-                   E.	canis	and	B.	canis	vogeli	in	72	Brazilian	wild	captive	felids	by	IFA	
tion	of	immunological	tolerance	to	myocardial	antigens.	J.	Autoimmun.,	              (Indirect	Immunofluorescent	Assay),	trying	to	verify	if	these	animals	are	
(2002)	18,	131–138                                                                   exposed	to	these	parasites.	Blood	samples	were	collected	from	Puma	
                                                                                     concolor	 (cougar),	 Leopardus	 pardalis	 (ocelot),	 Puma	 yagouaroundi	
     2.	 Godsel	 L.M.,	 Wang	 K.,	 Schodin	 B.A.,	 Leon	 J.S.,	 Miller	 S.D.,	
                                                                                     (aguarondi),	Leopardus	wiedii	(margay),	Leopardus	tigrinus	(little	spot-
Engman	 D.M.	 Prevention	 of	 autoimmune	 myocarditis	 through	 the	
                                                                                     ted	cat),	oncifelis	colocolo	(pampas	cat)	and	Panthera	onca	(jaguar)	
induction	 of	 antigen-specific	 peripheral	 immune	 tolerance.	 (2001)	
                                                                                     maintained	in	captivity	in	Brasilia,	Ribeirão	Preto,	Pedreira,	Campinas	
Circulation	103:	1709–1714
                                                                                     Zoos	 and	 in	 Associação	 Mata	 Ciliar	 de	 Jundiaí	 (Brazilian	 Center	 of	
    3.	Margossian	SS,	Lowey	S.	Preparation	of	myosin	and	its	sub-                    Neotropic	 Felids	 Conservation).	 Forty-four	 (70.96%)	 of	 62	 animals	
fragments	from	rabbit	skeletal	muscle.	Meth	Enzymol	85:	55-71,	1982                  were	seroreagents	for	B.	canis	and	24	%	for	E.	canis.	Antibodies	titers	
Key words:	autoimmunity	;	Inflammation	;	disease	;	myocarditis	                      found	ranged	from	1:20	(cut	off)	to	1:2,560	for	E.	canis	and	from	1:40	
Species:	other                                                                       (cut	off)	to	1:1,280	for	B.	canis.	Fourteen	felids	were	seropositives	for	
                                                                                     both	 antigens,	 while	 seventeen	 animals	 were	 seronegatives	 for	 both	
Pr195. SUPPRESSION OF PROLIFERATION AND CyTOKINE                                     agents.	To	authors´s	knowledge,	this	is	the	first	report	of	seroreactivity	
ExPRESSION By HELIS, A TICK SALIVARy GLAND-DERIVED                                   of	Brazilian	wild	felids	for	both	E.	canis	and	B.	canis	vogeli,	showing	
     PROTEIN OF HAEMAPHySALIS LoNGICoRNIS                                            that	 these	 animals	 are	 exposed	 to	 vector	 and	 agents	 of	 ehrlichiosis	
    SAToRU	KonnAI,	CHIE	nAKAJIMA,	SAIKI	IMAMURA,	MICHI	                              and	babesiosis.
     KoDAMA,	SHInJI	yAMADA,	HIDETo	nISHIKADo,	WIToLA	                                Key words:	wild	felids,	Ehrlichia	canis,	Babesia	canis,	antibodies
      WILLIAM	HARoLD,	KAZUHIKo	oHASHI,MISAo	onUMA                                    Species:	feline
                                7. COMPARATIVE IMMUNOLOGy; IMMUNOECOLOGy: POSTERS CI197-CI206


Ci197. SIMILARITIES AND DIFFERENCES OF MICROSCOPIC                                    Key words:	Piaractus	mesopotamicus,	anatomy,	histology,	spleen	and	
  STRUCTURE OF SPLEEN IN BELUGA HUSo HUSo WITH                                        thymus.
                TERRESTRIAL ANIMALS                                                   Species:	fish
                         MoHAMMADT	SHEIBAnI	
                                                                                        Ci199. IMUNOLOCALIZATION OF HEAT SHOCK PROTEIN
    Department	of	Basic	Sciences,	Faculty	of	Veterinary	Medicine,	
                                                                                           (HSP) 70 IN THE LIVER OF DIPLOID AND TRIPLOID
                  University	of	Tehran	Tehran,	Iran		
                       tsheibani@yahoo.com
                                                                                                           RAINBOW TROUT
                                                                                      R	S	IUnES1,	J	M	MonTEIRo1,	G	M	CoSTA,	AR	LIMA,	RV	BoSCH1	
      One	of	the	largest	and	the	most	important	sturgeons	in	the	Caspian	
                                                                                                           JR	KFoURy	JR1
Sea	 from	 the	 aspect	 of	 caviar	 production	 is	 beluga.	 For	 this	 investi-
gation	a	total	number	of	three	belugas	fished	from	southern	Caspian	                   1Medical	Surgical	Department	of	Veterinary	Medicine	and	Zootenic	
Sea,	were	subjected	to	study.	By	an	incision	on	the	abdominal	wall	the	                             College	of	the	University	of	São	Paulo
spleen	was	removed	and	after	fixation	in	10%	buffered	formalin,	routine	                    Heat	shock	proteins	(HSPs)	belong	to	a	highly	conserved	family	
histological	processes	were	done,	paraffin	blocks	were	sectioned	at	6	                of	cellular	proteins	present	in	all	organisms.	HSPs	are	expressed	in	dif-
microns	 and	 stained	 with	 Hematoxylin	 and	 Eosin	 and	 studied	 under	            ferent	tissues	under	normal	conditions;	however	it	increases	substan-
light	microscope.	The	results	showed	that	the	spleen,	unlike	to	those	                tially	when	stress	is	present.		Fish	show	two	types	of	stress	response:	
of	mammals,	was	surrounded	with	a	capsule	composed	of	three	layers	                   one	 is	 physiological	 and	 the	 other	 cellular.	 HSPs	 play	 a	 role	 at	 the	
including	an	epithelium	of	cuboidal	to	columnar	cells	with	some	small	                latter,	where	a	fish	may	present	physiological	response	to	a	stressor	
round	secretory	cells	with	eosinophilic	granules.	Under	which	a	waving	               agent	without	showing	any	change	of	the	cellular	HSPs	profile.	Among	
layer	 of	 elastic	 fibers	 and	 then	 a	 condensed	 connective	 tissue	 were	        the	 HSPs,	 HSP	 70	 is	 the	 most	 studied	 in	 fish,	 and	 we	 attempted	 to	
located.	Under	the	capsule	a	dense	lymphatic	tissue	containing	some	                  compare	its	expression	in	the	liver	of	diploid	and	triploid	rainbow	trout	
melanomacrophages	 was	 observed.	 The	 parenchyma	 of	 the	 spleen	                  (oncorhynchus	mykiss).	Liver	from	eight	diploid	and	ten	triploid	trouts	
was	composed	of	white	and	red	pulps	as	in	other	vertebrates.	White	                   were	fixed	in	10%	formalin	and	processed	for	immunohistochemistry,	
pulps	 consisted	 of	 a	 lymphatic	 nodule	 containing	 a	 small	 branch	 of	         using	 a	 monoclonal	 antibody	 specific	 to	 HSP	 70	 (Stressgen®	 mouse	
splenic	artery,	as	the	central	artery,	like	that	of	mammals,	 but	also	 a	            anti-HSP	70	monoclonal	SPA-810).	The	immunohistochemistry	results	
venous	branch,	unlike	mammals	that	differ	from	this	aspect.	The	lym-                  showed	a	cytoplasmatic	expression	of	HSP	70	in	hepatocytes	and	in	
phatic	nodule	is	followed	by	a	lymphatic	cord	consisting	of	an	aggre-                 the	 epithelial	 cells	 of	 the	 biliary	 duct.	 Differences	 on	 the	 intensity	 of	
gation	 of	 lymphocytes	 around	 an	 arteriole,	 as	 periarterial	 lymphatic	         staining	 were	 observed	 in	 both	 ploidy,	 where	 triploid	 fish	 presented	
sheath.	The	red	pulp	contained	too	many	sinusoids	filled	with	red	blood	              a	more	evident	variation,	from	intense	to	negative	staining.	Negative	
cells	 surrounded	 by	 some	 trabeculae	 and	 diffused	 lymphatic	 tissues	           controls	showed	no	staining	in	both	diploid	and	triploid	fish.	Our	results	
throughout	the	red	pulp	of	spleen.			                                                 suggested	triploid	rainbow	trout	show	differences	on	HSP	70	expres-
Key Words:	Spleen,	Beluga,	Terrestrial	animals,	Histology                             sion	characteristics	from	its	diploid	counterpart.
Species:	fish                                                                         Key words:	Trout,	HSP,	stress,	liver
                                                                                      Species:	fish
Ci198. GROSS AND MICROSCOPICAL CHARACTERIZATION
     OF THE THyMUS AND SPLEEN OF PIARACTUS                                                    Co200. CELLULAR FUNCTION EVALUATION OF
              MESOPOTAMICUS (PACU)                                                             SANGUINEOUS LEUKOCyTES IN PHRyNoPS
  GM	CoSTA,	CE	BARRoSo,	AS	oLIVEIRA,	C	BIASI,	RS	IUnES,	                                       GEoFFRoANUS USING FLOW CyTOMETRIC
 AR	LIMA,	J	M	MonTEIRo,	R	V	BoSCH,	P	FAVARon,	M	A	IVo,	JR	                                                METHODOLOGy
                       KFoURy	JR                                                         A	GEnoy-PUERTo1,	S	RoSSI1,	B	o	FERRonATo2,	V	M	Sá-
  Medical	Surgical	Department	of	Veterinary	Medicine	and	Zootenic	                       RoCHA3,	C	S	LISBoA4,	L	C	Sá-RoCHA3,	E	R	MATUSHIMA3	
               College	of	the	University	of	São	Paulo                                   1Academic	Program	of	Experimental	and	Compared	Pathology	-	
       	 Piaractus	 mesopotamicus	 (Pacu)	 is	 a	 fish	 from	 the	 Characidae	        FMVZ/USP;	2Academic	Program		in	Applied	Ecology	-	ESALQ/USP;	
Family,	 intensively	 bred	 in	 Brazil	 because	 of	 its	 rusticity,	 easy	 rais-     3Departament	of	Pathology	-	FMVZ	-	USP;	4Sector	of	Reptiles	of	the	
ing	 and	 adaptation,	 besides	 its	 excellent	 flavor.	 In	 order	 to	 produce	                  Foundation	Zoological	Park	of	São	Paulo		
a	 healthy	 fish,	 information	 on	 its	 immunological	 system	 including	 the	                           alexandergenoy@usp.br	
histology	of	the	lymphoid	organs	is	needed.	Therefore,	this	project	per-                    Although	Phrynops	geoffroanus	displays	ample	geographical	dis-
formed	a	gross	and	histological	analysis	of	the	thymus	and	spleen	of	                 tribution	in	South	America,	few	studies	had	been	developed	in	relation	
five	young	(about	25cm	in	length)	and	five	adult	pacus	(about	38	cm	in	               to	 the	 natural	 history	 of	 this	 freshwater	 turtle.	This	 species	 frequently	
length).	The	thymus	appeared	as	a	paired	organ,	placed	on	the	caudal	                 appears	 in	 contaminated	 water	 sources,	 butthe	 effect	 of	 	 adverse	
portion	 of	 the	 head,	 dorsally	 to	 the	 gills	 in	 the	 opercular	 cavity	 and	   situations	 (anthrophic	 effects)	 on	 the	 animals	 ‘	 cellular	 immune	 activ-
involved	 by	 a	 membranous	 capsule.	 The	 thymus	 was	 more	 evident	               ity	are	unknown.	In	this	work	a	method	employing	flow	cytometry	was	
in	 fish	 older	 than	 five	 months;	 it	 presented	 a	 gelatinous	 consistency	      developed	 in	 order	 to	 evaluate	 the	 cellular	 functions	 of	 phagocytosis	
varying	from	vivid	to	dark	red	color.	Histologically,	it	showed	medullar	             and	spontaneous	and	induced	oxidative	burst	in	leukocytes	from	blood	
and	 cortex	 regions	 consisted	 by	 conjunctive	 tissue	 and	 colagenous	            samples	of	Phrynops	geoffroanus	found	in	contaminated	environments	
fibers	and	the	presence	of	thymocytes	and	lymphocytes.	The	spleen	                    of	 the	 Piracicaba	 river	 and	 Piracicamirim	 brook,	 in	 São	 Paulo	 State.	
was	 located	 caudally	 to	 the	 liver,	 ventrally	 to	 the	 swim	 bladder,	 dor-     Blood	samples	were	obtained	from	8	animals	from	Piracicaba	river,	9	
sally	to	the	medium	caudal	portion	of	the	biliary	vesicle.	It	was	rounded	            from	Piracicamirim	brook,	and	7	from	Zoological	Park	Foundation	of	São	
shaped,	dorsoventrally	flattened	and	it	presented	an	irregular	surface	               Paulo;	the	samples	from	zoological	park	were	used	as	control	group.	
in	young	and	lobed	in	adult	fish.	It	was	delimited	by	a	membrane	and,	                The	samples	had	been	kept	in	culture	in	RPMI	medium	under	refrigera-
histologically,	it	was	composed	by	a	white	pulp,	rich	in	melanomacro-                 tion	and	directed	to	the	Neuro-immunology	Laboratory	of	Department	of	
phages	and	extensive	red	pulp	areas.                                                  Pathology	in	FMVZ/USP	to	be	processed.	The	fact	that	the	red	blood	
cells	are	nucleated	in	these	animals,	made	necessary	the	use	of	Ficoll-              RoBERT	KAMMERER1,2,	TAnJA	PoPP1,	STEFAn	HAERTLE3,	
PaqueTM	PLUS	at	different	densities	(1,085;	1,070	and	1,055	g/mL),	                  BERnHARD	B	SInGER4,	KATHRyn	V	HoLMES5,,WoLFGAnG	
in	order	to	pre-separate	these	cells.	Then	the	sample	was	centrifuged	                                       ZIMMERMAnn1
to	obtain	cells	of	interest,	at	18	°C,	450	x	g,	acceleration	9	and	brake	              1Tumor	 Immunology	 Laboratory,	 LIFE	 Center,	 Klinikum	
0,	resulting	in	a	white	ring	of	leukocytes	located	between	the	superior	          Grosshadern,	 LMU,	 Munich,	 Germany;	 2Institute	 for	 Molecular	
layers	 of	 the	 Ficoll.	The	 samples	 were	 submitted	 to	 two	 washes	 with	    Immunology,	 GSF	 National	 Research	 Center	 for	 the	 Environment	
RPMI	to	remove	the	excesses	of	Ficoll.	After	the	washes,	the	samples	             and	Health,	Munich,	Germany;	3Institute	for	Animal	Physiology,	LMU,	
were	suspended	again	in	500	µL	RPMI	for	making	a	viability	counting	              Munich,	 Germany;	 4Institute	 for	Anatomy,	 University	 Hospital	 Essen,	
with	Trypan	Blue;	samples	with	inferior	percentages	to	94	%	were	not	             Essen,	Germany;	5University	of	Colorado,	Health	Sciences	Center	at	
used	 in	 the	 stimulates:	 Zymosan	A	 (Sacaromyces	 cerevisiae)	 the	 Bio	       Fitzsimons,	Department	of	Microbiology,	Aurora,	USA
Particles®,	Alexa	Fluor®	594	conjugate	to	induce	phagocytosis	and	12-                   The	primordial	CEA	gene	family,	which	is	located	in	the	expanded	
myristate	13-acetic	acid	(PMA)	and	Sacaromyces	cerevisiae	(Zymosan)	              leukocyte	receptor	complex,	was	composed	of	five	genes	(CEACAM1,	
in	the	induction	of	oxidative	burst.	The	animal’s	leukocytes	of	the	zoo	          CEACAM16,	 CEACAM18,	 CEACAM19,	 CEACAM20).	 The	 CEACAM1	
presented	 greater	 phagocytosis	 intensity	 induced	 by	 Zymosan	 A,	 in	        gene	 gave	 rise	 to	 expansions	 of	 the	 CEA	 families	 by	 gene	 duplication	
relation	with	to	the	cells	of	the	animals	of	the	Piracicaba	river.	The	inten-     and	 exon	 shuffling.	 In	 rodents	 and	 primates,	 the	 CEACAM1-related	
sity	of	oxidative	burst	induced	by	PMA	was	smaller	in	the	animals	of	the	         members	 of	 the	 CEA	 family	 can	 be	 divided	 into	 the	 CEA-related	 cell	
Piracicamirim	brook	when	compared	with	turtles	of	Piracicaba	river	and	           adhesion	 molecules	 (CEACAM)	 and	 the	 pregnancy-specific	 glycopro-
the	zoo.	Financial	support:	CNPq,	CAPES	and	FAPESP                                teins	(PSG).	While	various	CEACAM	are	pivotal	for	a	well-coordinated	
                                                                                  immune	 response,	 the	 PSG	 are	 supposed	 to	 be	 instrumental	 for	 suc-
Key words:	Cellular	function	Evaluation,	Leukocytes,	Flow	Cytometric,	            cessful	pregnancies.	The	characterization	of	the	CEA	families	in	primates	
Phrynops	geoffroanus                                                              and	 rodents	 revealed	 profound	 species-specific	 differences.	 In	 the	
Specie:	reptile	(turtle)                                                          mouse	 three	 CEACAM	 (CEACAM1,	 CEACAM2	 and	 CEACAM17)	 are	
                                                                                  transmembrane	 bound	 while	 the	 other	 seven	 are	 secreted.	 In	 humans	
   Ci201. CHARACTERIZATION OF BOVINE ACTIVATION-                                  CEACAM1,	CEACAM3	and	CEACAM4	are	transmembrane	bound	while	
            INDUCED CyTIDINE DEAMINASE                                            the	other	four	are	GPI	anchored.	CEACAM1	and	CEACAM2	harbor	ITIM	
      SUBHASH	VERMA,	ToM	GoLDAMMER1,	RoB	AITKEn2	                                 and	CEACAM3	and	CEACAM4	ITAM	in	their	cytoplasmic	tails.	Since	little	
                                                                                  is	known	about	the	CEA	gene	families	in	other	species,	we	have	analyzed	
  Division	of	Infection	and	Immunity,	Glasgow	Biomedical	Research	
                                                                                  CEA-related	 genes	 in	 cattle,	 dog	 and	 horse.	 We	 identified	 orthologous	
Centre,	University	of	Glasgow,	G12	8TA,	United	Kingdom;	1	Research	
                                                                                  genes	for	CEACAM1	and	the	other	ancestral	members	in	all	three	spe-
  Unit	Molecular	Biology,	Research	Institute	for	the	Biology	of	Farm	
                                                                                  cies.	Expansion	of	CEACAM1-related	genes	occurred	in	these	species,	
 Animals,	Dummerstorf,	D-18296,	Germany;	2	Corresponding	author
                                                                                  leading	to	3,	7	and	11	different	CEACAM1-related	genes	in	cattle,	dog	
      Activation	induced	cytidine	deaminase	(AID)	plays	pivotal	role	in	          and	horse,	respectively.	Most	striking	is	the	complete	absence	of	PSG	in	
the	generation	of	antibody	diversity,	being	essential	for	somatic	hyper-          both	cattle	and	dog.	In	the	horse	five	possibly	secreted	CEACAMs	were	
mutation	(SHM),	gene	conversion	(GC)	and	class	switch	recombination	              identified.	Based	on	three	EST	clones,	one	of	these	genes	is	expressed	
(CSR).	Here,	we	report	the	sequencing,	mapping,	expression	pattern	               in	 placenta.	 Therefore,	 the	 existence	 of	 PSG	 in	 the	 horse	 is	 possible.	
and	mutator	phenotype	of	AID	from	Bos	taurus	(BoAID).	                            The	CEACAM1-related	molecules	in	cattle	and	dogs	are	transmembrane	
      At	the	gene	level,	exon	sequences	were	found	to	be	very	similar	            molecules,	which	harbor	ITAM	ore	ITAM-like	motifs	and	are	expressed	by	
to	their	counterparts	from	humans	and	mice,	implying	a	high	level	of	             immune	cells.	In	the	horse,	there	are	two	genes,	which	may	be	ortholo-
stability	 throughout	 vertebrate	 evolution.	 In	 contrast,	 little	 sequence	   gous	 to	 CEACAM1	 (CEACAM1,	 CEACAM43).	 The	 closest	 relative	 to	
similarity	could	be	detected	between	intronic	sequences	of	bovine	AID	            CEACAM1	is	an	ITAM-bearing	molecule,	similar	to	the	findings	in	cattle	
and	 its	 counterparts	 from	 other	 mammals.	 Bovine	 intron	 sequences	         and	dogs.	The	closest	relative	to	CEACAM43	is	a	possibly	secreted	pro-
were	also	consistently	longer	than	those	of	humans	and	mice.	Mapping	             tein,	similar	to	the	situation	previously	found	in	mice.	Taken	into	consider-
                                                                                  ation	that	CEACAM1	represents	a	virus	receptor	in	mice	and	a	bacterial	
using	a	radiation	hybrid	panel	successfully	placed	the	BoAID	gene	on	
                                                                                  receptor	in	humans	we	hypothesize	that	the	structure	of	the	CEA	family	
Bos	taurus	chromosome	5	(BTA5)	in	a	region	synthenic	with	HSA12.
                                                                                  mirrors	the	history	of	the	arms’	race	between	CEACAM1-binding	patho-
      BoAID	 cDNA	 had	 an	 open	 reading	 frame	 of	 199	 amino	 acid	           gens	and	the	host.	Therefore,	we	generated	anti-bovine	CEACAM1	mAb	
residues.	A	comparison	with	its	counterpart	in	mammals	and	chicken	               and	bovine	CEACAM1-Fc	fusion	proteins	for	the	screening	of	pathogens	
identified	mostly	conserved	amino	acid	residues	especially	within	the	            binding	bovine	CEACAM1.	Indeed,	we	have	recently	 identified	 a	 virus	
cytidine	 deaminase	 motif.	 BoAID	 carried	 an	 extra	 codon	 in	 exon	 3,	      which	infects	cells	by	binding	to	bovine	CEACAM1.
which	codes	for	lysine,	the	significance	of	which	is	unknown.
                                                                                  Key words:	carcinoembryonic	antigen,	pathogen	receptor,	leukocyte	
      The	level	of	transcription	of	BoAID	was	comparable	in	young	and	            receptor	complex,	pathogen-host	coevolution
old	animals.	Interestingly,	BoAID	transcripts	were	present	in	a	day	old	          Species:	other
animal	even	though	germinal	centres	could	not	be	detected	in	the	lym-
phoid	 tissue	 by	 histology.	 BoAID	 mRNA	 in	 these	 animals	 was	 been	             Ci203. MHC POPULATION STRUCTURE IN THE NEW
shown	 to	 be	 present	 in	 both	 lymphoid	 and	 non-lymphoid	 tissues	 but	                    ZEALAND BRUSHTAIL POSSUM
transcripts	were	observed	particularly	abundant	in	the	spleen	and	mes-
                                                                                      oLIVIA	J	HoLLAnD1,2,4,	PHILIP	E	CoWAn3,	LAWREnCE	W	
enteric	lymph	nodes.	Much	lower	abundance	was	evident	in	the	liver,	
                                                                                                 CHAMLEy4,	DIAnnE	M	GLEESon2
and	BoAID	mRNA	was	undetectable	in	muscle	tissue.	
                                                                                     1National	Research	Centre	for	Possum	Biocontrol,	Landcare	
      The	 BoAID	 cDNA	 was	 cloned	 into	 a	 bacterial	 expression	 vector	
                                                                                  Research,	Auckland,	New	Zealand;	2Ecological	Genetics	Laboratory,	
and	when	induced	in	Escherichia	coli,	it	conferred	a	mutator	phenotype.	
                                                                                      Landcare	Research,	Auckland,	New	Zealand;	3Pest	Control	
This	was	evident	through	disruption	of	the	lacZ	reading	frame	and	in	
                                                                                  Technologies,	Palmerston	North,	Landcare	Research,		New	Zealand;	
separate	experiments,	through	the	appearance	of	rifampicin	resistant	             4Obstetrics	and	Gynaecology,	University	of	Auckland,	Auckland,	New	
colonies,	a	result	of	high	frequency	mutation	in	rpoB.                                                         Zealand		
      Cloning	 of	 BoAID	 and	 definition	 of	 its	 chromosomal	 localization	                     hollando@landcareresearch.co.nz
and	functional	properties	will	facilitate	our	understanding	of	the	role	of	
                                                                                         The	brushtail	possum	(Trichosurus	vulpecula)	is	a	major	invasive	
this	enzyme	in	the	immunobiology	of	B.	taurus.                                    pest	in	New	Zealand,	causing	severe	damage	to	native	ecosystems	and	
Key words:	Bovine,	cytidine	deaminase,	BTA5                                       acting	as	a	vector	of	bovine	tuberculosis.		One	option	for	controlling	this	
Specie:	ruminants                                                                 pest	may	be	the	use	of	immunocontraceptive	vaccines,	a	method	of	fer-
                                                                                  tility	control	that	employs	the	immune	system	to	attack	reproductive	cells	
Ci202. THE CARCINOEMBRyONIC ANTIGEN (CEA) FAMILy                                  or	proteins.		Trials	have	shown	there	is	variability	in	immune	responses	
IN PLACENTAL MAMMALS OF THE SUPERORDINAL CLADE                                    among	individual	possums	to	the	various	immunocontraceptive	vaccines	
                 LAURASIATHERIA                                                   currently	undergoing	laboratory	trials.		If	this	variability	is	under	genetic	
120


      control,	 then	 the	 application	 of	 immunocontraception	 as	 a	 population	         	United	States	Department	of	Agriculture,	Agricultural	Research	
      control	mechanism	will	have	to	be	carefully	managed	to	avoid	preferen-                 Service,	Beltsville	Human	Nutrition	Research	Center,	Nutrient	
      tial	selection	of	animals	that	fail	to	mount	a	significant	immune	response	         Requirements	and	Functions	Laboratory,	Beltsville,	Maryland,	20705	
      and	 remain	 fertile.	 	The	 major	 histocompatibility	 complex	 (MHC)	 is	 an	                                   USA
      important	component	of	the	immune	system,	which	influences	the	nature	                    	A	 literature	 and	 laboratory-based	 analysis	 compared	 selected	
      of	 immune	 responses	 and	 has	 been	 extensively	 studied	 in	 eutherian	         features	of	genotype,	phenotype,	and	functional	expression	of	the	por-
      mammals	and	birds,	however	to	date	only	limited	data	is	available	for	              cine,	murine,	and	human	immune	systems.		A	total	of	147	parameters	
      the	 marsupial	 MHC	 loci.	 This	 study	 aims	 to	 characterize	 alleles	 and	      were	examined.		Post-genomic	analysis	found	about	300	unique	mRNA	
      document	genetic	variation	in	the	MHC	loci	of	New	Zealand	possums,	
                                                                                          coding	sequences	between	mice	and	humans	with	approximately	100	
      and	investigate	whether	there	is	a	relationship	between	MHC	haplotypes	
                                                                                          related	to	immunity.		To	date,	we	or	others	have	identified	43	porcine	
      and	individual	immune	responses	to	fertility	control	vaccines.		We	used	
                                                                                          immune-related	 genes	 not	 found	 in	 rodents.		 Further	 analysis	 identi-
      known	marsupial	(possum,	red-necked	wallaby,	tammar	wallaby,	opos-
                                                                                          fied	a	limited	number	of	genes	present	in	rodents	and	pigs	but	not	in	
      sum)	 MHC	 sequences	 to	 design	 PCR	 primers	 for	 possum	 MHC	 loci.	        	
                                                                                          humans,	and	genes	absent	in	pigs	but	found	in	rodents	and	humans.	          	
      The	 variability	 of	 these	 loci	 was	 screened	 in	 populations	 of	 possums	
                                                                                          The	phenotype	of	many	immune	cells,	including	alternatively	activated	
      from	 locations	 throughout	 New	 Zealand,	 as	 well	 as	 between	 individu-
                                                                                          macrophages	and	T	regulatory	cells,	are	more	similar	between	pigs	and	
      als	with	known	responses	to	immunocontraceptive	vaccines.		Alpha	and	
                                                                                          humans	compared	to	rodents.	Pigs	are	naturally	susceptible	to	infection	
      beta	chains	from	two	class	II	families,	which	are	specific	to	marsupials,	
                                                                                          with	species	of	helminths	that	are	closely	related	or	identical	to	those	
      (DA	and	DB)	have	been	confirmed	to	be	present	in	possums	throughout	
                                                                                          infecting	 humans	 (Ascaris,	 Taenia,	 Trichuris,	 Trichinella,	 Shistosoma,	
      New	 Zealand.	 	 Over	 20	 new	 MHC	 alleles	 have	 been	 identified	 in	 the	
                                                                                          Strongyloides)	 indicating	 functionally	 similar	 host	 characteristics.	  	
      possum	 and	 preliminary	 population	 surveys	 have	 shown	 evidence	 for	
                                                                                          Additionally,	 pigs	 are	 excellent	 models	 for	 bacterial	 (Campylobacter,	
      variation	 in	 the	 distribution	 of	 MHC	 alleles	 in	 geographically	 separate	
      New	Zealand	possum	populations.		The	levels	of	variability	in	the	MHC	              E.	coli,	Helicobacter,	neisseria,	Mycoplasma,	Salmonella),	protozoan	
      of	this	marsupial	appear	to	be	comparable	to	those	of	eutherian	species.	       	   (Toxoplasma)	and	viral	infections	(Coronavirus,	Hepatitis	E,	Influenza,	
      In	this	study,	we	have	identified	a	large	number	of	novel	possum	MHC	               nipah,	Reovirus,	Rotavirus)	infections.		Overall,	approximately	80%	of	
      alleles	and	found	evidence	for	MHC	clustering/variability	in	different	pos-         the	 147	 parameters	 examined	 were	 more	 similar	 between	 pigs	 and	
      sum	populations	in	New	Zealand.		Such	extensive	variation	may	greatly	              humans,	suggesting	that	evaluating	immune	function	in	pigs	provides	
      impact	the	effectiveness	of	immunocontraceptive	control	measures.                   data	that	is	more	physiologically	relevant	to	humans.
      Key words:	Immunocontraception,	MHC,	PCR,	marsupial                                 Key words:	review,	human,	mouse
      Species:	other	(marsupial)                                                          Species:	other

       Ci204. ENZOOTIC CALCINOSIS: EFFECTS ON ORGANS                                      Ci206. GENE ExPRESSION OF CHICKEN INTERLEUKINE-4
      AND CELLS OF THE IMMUNE SySTEM IN ExPERIMENTALLy                                                    By BACULOVIRUS
                     INTOxICATED RABBITS                                                    TAKAyUKI	KUBoTA,	MAKIKo	yoKAyAMA,	MASATo	KISHIMA,	
        PAULA	A	FonTAnA1,2,	CARoLInA	n	ZAnUZZI1,2,	LEonARDo	                                SAToKo	WATAnABE,	MASATo	oHoTA,	HIRoKAZU	HIKono,	
          A	CHInCHILLA1	,	CLAUDIo	G	BARBEITo1,2,	EDUARDo	J	                                      CHARLES	o	A	oMWAnDHo,SHIGEKI	InUMARU
                 GIMEno1,2,	EnRIqUE	L	PoRTIAnSKy1,2	                                                         National	Institute	of	Animal	Health		
            1Instituto	de	Patología	.Facultad	de	Ciencias	Veterinarias.	                                            tkubota@affrc.go.jp
       Universidad	Nacional	de	La	Plata.	60	y	118	(1900)	La	Plata.	Buenos	                      INTRODUCTION:	 Mammalian	 T	 cells	 are	 classified	 into	 Th1	
       Aires.	Argentina;		2CONICET	(Consejo	Nacional	de	Investigaciones	                  and	Th2	 according	 to	 the	 cytokine	 production.	Although	 chicken	Th2	
                               Científicas	yTécnicas)	                                    cytokines	have	not	been	identified	until	recent	years,	existence	of	Th2	
                             pfontana@fcv.unlp.edu.ar                                     cytokine	was	suspected	temporarily.	Following	the	cloning	of	Th2	cyto-
            Solanum	glaucophyllum	(Sg)	is	a	calcinogenic	plant	that	causes	               kines	such	as	interleukin	4	(IL-4),	IL-5,	and	IL-13	in	2004,	it	became	
      “Enzootic	calcinosis”	in	cattle.	Its	main	toxic	principle	is	the	1,25-dihy-         clear	that	these	cytokines	and	their	loci	are	conserved	in	chicken.	In	
      droxyvitamin	D.	Vitamin	D	acts	in	calcium	homeostasis	and	has	pro-                  this	study,	we	used	a	baculovirus	gene	expression	system	to	express	
      nounced	immunomodulatory	properties.	The	goal	of	the	present	work	                  chicken	IL-4	and	investigate	its	function.	METHODS:	mRNA	was	iso-
      was	to	test	immune	system	modifications	due	to	VitD3	in	Sg-intoxicated	             lated	from	chicken	peripheral	blood	or	thymus	and	RT-PCR	was	used	
      animals.	 Rabbits	 were	 selected	 as	 experimental	 models	 due	 to	 their	        to	 synthesis	 cDNA.	 IL-4	 cDNA	 was	 inserted	 into	 pFastBac1	 vector	
      high	susceptibility	to	the	Sg	intoxication.	New	Zealand	males	received	             and	 transferred	 into	AcNPV	 baculovirus	 by	 using	 Bac-to-Bac	 system	
      125	mg/kg	of	Sg	leaves	powder	twice	a	week	during	15	and	30	days.	                  (Invitrogen).		Recombinant	virus	was	infected	into	Tn5	insect	cells.	The	
      A	 group	 was	 intoxicated	 during	 15	 days	 but	 sacrificed	 at	 30	 days	 pi	    culture	supernatant	containing	recombinant	chicken	IL-4	(rIL-4)	protein	
      (recovery	 group).	 Control	 animals	 were	 sacrificed	 at	 0	 and	 30	 days.	      was	harvested	and	partially	purified	by	ammonium	sulfate	sedimenta-
      Phagocytosis	tests,	immunohistochemistry	for	CD4,	CD8	and	B	cells	                  tion	 method.	 Biological	 activity	 was	 examined	 by	 3H-tymidne	 uptake.	
      on	 frozen	 isolated	 lymphocytes	 from	 lymph	 nodes,	 and	 histopathol-           RESULT	 &	 DICUSSION:	 After	 the	 recombinant	 baculovirus	 was	
      ogy	 of	 thymus,	 appendix,	 popliteal	 lymph	 nodes	 and	 spleen	 were	            infected	into	Tn5	insect	cells,	the	protein	secreted	in	the	medium	was	
      performed.	 The	 phagocytic	 activity	 in	 peritoneal	 macrophages	 was	            accumulated	into	the	culture	supernatant.	Supernatant	was	collected	
      progressively	reduced	in	the	intoxicated	animals	with	time.	A	significant	          on	 the	 5th	 day	 when	 protein	 concentration	 was	 highest.	 When	 par-
      decrease	 in	 the	 number	 of	 CD4+,	 CD8+	 and	 B	 cells	 were	 observed	          tial	refining	of	this	protein	was	carried	out	with	the	ammonium	sulfate	
      as	 well.	 Histopathology	 showed	 a	 gradual	 increase	 of	 the	 degree	 of	       sedimentation	method,	it	precipitated	between	80%	to	90%	saturation.	
      thymic	 and	 appendix	 atrophy.	 However,	 no	 changes	 were	 observed	             Expression	 was	 checked	 by	 tricine	 polyacrylamide	 gel	 electrophore-
      in	 the	 analyzed	 lymph	 nodes	 and	 in	 the	 spleen.	 Recovery	 group	            sis	(PAGE).	Two	unique	bands	were	found.	The	molecular	masses	of	
      showed	 similar	 results	 than	 controls.	These	 results	 demonstrate	 that	        the	 two	 expressed	 proteins	 were	 about	 18.6kDa	 and	 15.5kDa.	 The	
      Sg	principles	induce	immune	system	alterations	in	this	animal	model.	               molecular	mass	of	mature	chicken	IL-4	calculated	from	the	amino	acid	
      The	changes	observed	in	the	Sg-intoxicated	animals	resembled	those	                 sequence	is	12.4kDa	and	the	precursor	with	signal	peptide	is	14.8kDa.	
      produced	under	physiological	aging	process.                                         We	presume	larger	size	of	these	proteins	on	the	gel	were	due	to	gly-
      Key words:	 Enzootic	 Calcinosis,	 Solanum	                    glaucophyllum,	      cosylation,	because	the	sequence	of	chicken	IL-4	contains	5	N-linked	
      hypervitaminosis	D,	immune	system,	rabbits                                          glycosylation	sites	and	those	diffused	bands	are	typical	of	glycoprotein.	
      Specie:	other	(rabbits)                                                             These	precipitated	proteins	facilitated	proliferation	activity	of	peripheral	
                                                                                          white	 blood	 cell	 in	 dose	 dependent	 manner	 suggesting	 chicken	 pos-
          Ci205. A COMPARATIVE ANALySIS OF THE PORCINE,                                   sibly	have	Th2	function.
               MURINE, AND HUMAN IMMUNE SySTEMS                                           Key words:	“intereukin4”,	“chicken”,	“protein”,	“activity”
        HARRy	D	DAWSon,	JoSHUA	J	REECE,	JoSEPH	F	URBAn	JR                                 Species:	avian
         8. MEDIATORS OF RECRUITMENT AND FUNCTION OF CELLS OF THE IMMUNE SySTEM; FC RECEPTORS AND
         IMMUNOGLOBULINS; SIGNAL TRANSDUCTION AND GENE ExPRESSION IN CELLS OF THE IMMUNE SySTEM:
                                            POSTERS MI207-MI210

mi207. IDENTIFICATION OF A MHC CLASS I-RESTRICTED                                published	bovine	Fc[gamma]RII	sequence	was	99%	and	100%	for	the	
CD8+ IMMUNE RESPONSE TO FOOT-AND-MOUTH DISEASE                                   extracellular	and	intracellular	domains,	respectively.		It	is	hypothesized	
                 VIRUS IN CATTLE                                                 that	the	resulting	protein	is	a	soluble	isoform	of	CD32;	this	phenome-
          EFRAIn	GUZMAn,	GERALDInE	TAyLoR,	BRyAn	                                non	has	been	described	previously	only	in	mice	and	humans.		Analysis	
                 CHARLESTon,SHIRLEy	ELLIS                                        of	the	second	sequence	revealed	a	putative	extracellular	domain	with	
                                                                                 96%	 sequence	 identity	 to	 CD32;	 however,	 the	 predicted	 transmem-
    Institute	for	Animal	Health.	Division	of	Immunology.	Compton,	
                                                                                 brane	and	intracellular	regions	displayed	79%	sequence	identity	to	the	
                  Newbury	RG20	7NN.	United	Kingdom		
                                                                                 Fc[gamma]RIII	(CD16)	molecule.		This	is	of	particular	note	as	CD16,	
                      efrain.guzman@bbsrc.ac.uk
                                                                                 through	non-covalent	association	with	either	the	Fc[epsilon]RI	gamma	
      Foot	and	mouth	disease	virus	(FMDV),	an	apthovirus,	is	a	member	           chain	 or	 the	TCR	 zeta	 chain,	 acts	 to	 cause	 cell	 activation	 by	 means	
of	the	genus	picornaviridae	and	can	be	classified	into	seven	serotypes.	         of	 ITAM	 motifs	 present	 in	 their	 cytoplasmic	 tails.	 	 We	 have	 detected	
It	causes	a	highly	contagious	viral	infection	in	ruminants,	pigs	and	clo-        soluble	 CD32	 transcript	 in	 B	 cells,	 CD4+	 and	 CD8+	 T	 cells,	 CD14+	
ven-hooved	animals	with	very	important	economic	consequences.	The	               monocytes	and	neutrophils	isolated	from	bovine	blood.		Transcript	for	
disease	 is	 characterised	 by	 the	 formation	 of	 vesicles	 on	 the	 mouth,	   the	CD32/CD16	chimeric	sequence	was	found	in	B	cells,	CD4+	T	cells	
tongue,	 nose	 and	 feet.	 Control	 of	 the	 disease	 is	 achieved	 by	 vacci-   and	 CD14+	 monocytes.	 	 Furthermore,	 cultured	 bovine	 B	 cells	 (BL3)	
nation	 with	 a	 chemically	 inactivated	 whole	 virus	 vaccine	 emulsified	     were	found	to	secrete	a	protein	reactive	with	an	anti-CD32	monoclonal	
with	adjuvant,	providing	only	short-term,	serotype	specific	protection.	
                                                                                 antibody	(CCG32,	IAH	Compton).		This	is	the	first	time	a	soluble	FcR	
A	 better	 understanding	 of	 protective	 immune	 mechanisms	 may	 help	
                                                                                 has	been	reported	in	a	species	other	than	murine	or	human.		The	exis-
in	 development	 of	 novel	 vaccines.	 While	 much	 attention	 has	 been	
                                                                                 tence	of	these	novel	receptors	adds	a	new	level	of	complexity	to	cell	
devoted	to	humoral	responses	to	FMDV,	less	is	known	about	the	role	
                                                                                 regulatory	functions	involving	IgG	immune	complexes.	
of	cell-mediated	responses	in	protective	immunity.	An	in	vitro	assay	for	
the	detection	of	antigen-specific	interferon-gamma	(IFN-γ)	release	by	           Key words:	soluble	Fc	receptor;	CD16;	CD32
CD8+	T	cells	was	used	to	determine	the	level	of	CD8+	T	cell	activity	            Species:	ruminants
in	vaccinated	and	infected	cattle	of	known	MHC	type.	MHC-restricted	
CD8+	T	 cell	 recognition	 of	 the	 structural	 proteins	 (P1)	 of	 FMDV	 was	      mi209. VARIATION IN ExPRESSION OF FC GAMMA
detected	in	vaccinated	cattle.	A	significant	cellular	immune	response	to	        RECEPTOR IIB AND CD21 ON BOVINE LyMPHOCyTES WITH
both	live	and	UV-inactivated	FMDV	and	to	the	P1	region	of	FMDV	was	                                      AGE
also	detected	in	FMDV-infected	animals.	Using	mouse	cells	expressing	
individual	cattle	MHC	class	I	genes,	we	have	identified	specific	alleles	                                       KS	CHATTHA
responsible	for	the	presentation	of	FMDV	P1	peptides	to	CD8+	T	cells.	                      Pathobiology,	OVC,	University	of	Guelph,	Canada	
We	are	currently	mapping	MHC	class	I-restricted	CD8+	epitopes	which	                   It	is	difficult	to	induce	active	immune	responses	in	neonates,	partly	
will	enable	us	to	carry	out	a	detailed	study	of	the	role	of	CD8+	T	cells	        due	 to	 the	 limited	 functional	 ability	 of	 neonate’s	 immune	 system	 and	
following	vaccination	or	infection	in	cattle.                                    partly	 due	 to	 inhibition	 mediated	 by	 maternal	 antibodies.	 Fc	 gamma	
Key words:	Foot	and	mouth	disease	virus;	CD8+	T	cells;	aβ	CD4+	T	                receptor	II	B	(CD32)	 is	a	major	receptor	responsible	 for	suppression	
cells;	interferon-γ;	MHC	class	I-restricted	CD8+	epitopes                        of	antibody	responses,	through	interaction	with	immune	complexes	of	
Species:	ruminants                                                               maternal	antibodies	(primarily	IgG)	and	antigens,	by	virtue	of	its	intra-
                                                                                 cellular	 immunoreceptor	 tyrosine-based	 inhibitory	 motif	 (ITIM).	 CD21	
 mi208. IDENTIFICATION OF NOVEL RECEPTORS FOR IGG                                (CR2),	 a	 receptor	 for	 complement	 component	 C3d,	 is	 expressed	 by	
             FC IN BOVINE LyMPHOCyTES                                            B	 lymphocytes	 and	 binding	 results	 in	 lowering	 of	 the	 threshold	 for	 B	
 MATT	A	FIRTH,	KULDEEP	S	CHATTHA,	DoUGLAS	C	HoDGInS,	                            cell	 activation.	Thus	 it	 plays	 an	 important	 role	 in	 enhancing	 antibody	
                   PATRICIA	E	SHEWEn                                             responses	to	complement-associated	antigens.	CD21	(activating	com-
                                                                                 ponent),	B	cell	receptor	(membrane	IgM)	and	CD32	(inhibitory	compo-
University	of	Guelph,	Department	of	Pathobiology,	Ontario	Veterinary	
                                                                                 nent)	form	an	intricate	triad	for	determining	the	threshold	of	activation	
                 College.		Guelph,	Ontario,	Canada		
                                                                                 for	B	cells.	Because	cellular	distribution	of	CD21	and	CD32	of	cattle	has	
                        mfirth@uoguelph.ca
                                                                                 not	been	well	documented,	this	study	aimed	to	determine	the	variation	
      Receptors	 for	 the	 Fc	 portion	 of	 immunoglobin	 molecules	 (FcR)	      in	 expression	 of	 CD21	 and	 CD32	 on	 bovine	 lymphocytes	 with	 age,	
provide	 an	 important	 and	 vital	 link	 between	 circulating	 antibody	 and	   particularly	emphasizing	differences	between	neonates	and	adults.	
cellular	effector	functions.		Cell	surface	FcR	for	IgG,	IgE,	IgA	and	IgM	
have	been	identified	on	many	cell	types.		Much	of	the	current	literature	              Blood	was	collected	from	20	healthy	Holstein	calves,	1	to	90	days	
focuses	on	FcR	sequence	and	function	in	the	murine	and	human	spe-                of	 age,	 and	 8	 healthy	 adults.	 The	 percentage,	 absolute	 number	 and	
cies.		To	date,	only	a	handful	of	these	FcRs	have	been	sequenced	and	            mean	 fluorescence	 intensity	 of	 CD21	 and	 CD32	 positive	 cells	 was	
characterized	 in	 livestock	 species.	 	 Fc[gamma]RII	 (CD32)	 is	 an	 FcR	     determined	 using	 fluorochrome	 labelled	 monoclonal	 antibodies	 and	
present	in	several	isoforms	on	a	wide	variety	of	cells	including	B	cells,	       flow	 cytometry.	 The	 percentage	 and	 absolute	 number	 of	 CD21	 and	
monocytes	 and	 neutrophils.	 	 On	 B	 cells,	 the	 Fc[gamma]RIIb	 isoform	      CD32	positive	lymphocytes	increased	with	age	from	birth	until	about	60	
acts	to	down	regulate	IgG	production	in	direct	opposition	to	the	B	cell	         days	of	age,	with	CD21	showing	a	greater	percentage	increase	com-
co-receptor	complex	consisting	of	CD19,	CD21	and	CD81	by	means	of	               pared	to	CD32.	The	proportion	of	CD32	positive	lymphocytes	express-
an	intracellular	ITIM	motif.		A	recent	examination	of	the	expression	pro-        ing	 CD21	 also	 increased	 with	 age.	 In	 both	 calves	 and	 adults,	 the	
file	of	CD32	in	bovine	blood	mononuclear	cells	revealed	the	presence	            number	of	cells	expressing	CD32	was	greater	than	those	expressing	
of	two	previously	uncharacterized	CD32-like	sequences.		Sequencing	              CD21.	Mean	fluorescence	intensity	for	CD32	was	significantly	higher	
of	the	first	uncharacterized	transcript	indicated	it	to	be	a	CD32	mRNA	          for	adults	(P<0.01),	indicating	a	greater	number	of	CD32	receptors	per	
splice	variant,	lacking	a	120	bp	region	corresponding	to	the	transmem-           lymphocyte	compared	to	neonates.	RT-PCR	using	RNA	extracted	from	
brane	region	in	the	full-length	molecule.		Sequence	homology	with	the	           PBMCs	of	neonatal	calves	suggested	production	of	a	soluble	version	of	
122


      Fc	gamma	RIIB	(sCD32),	lacking	transmembrane	region,	which	might	                     most	of	the	IgG	transcribed	during	fetal	life.	IgG2,	also	with	two	alleles,	
      contribute	to	lower	membrane	CD32	expression	in	neonates.                             is	transcribed	at	much	lower	levels	as	are	IgG4,	IgG5	and	IgG6.	IgG3	
             Lower	 expression	 of	 CD21	 in	 neonates	 combined	 with	 reduced	            is	a	unique	long-hinged	variant	which	accounts	for	>50%	of	IgG	tran-
      levels	of	C3d	in	serum	may	be	a	limiting	factor	for	activation	of	neo-                scripts	in	the	ileal	Peyers	patches	(IPP)	and	MLN	of	newborns	but	rela-
      natal	B	cells.	An	age	related	increase	in	the	percentage	and	absolute	                tive	transcription	declines	as	the	IPP	involute	(7).		Sequence	analyses	
      number	 of	 CD21	 and	 CD32	 positive	 lymphocytes,	 along	 with	 reduc-              confirm	that	speciation	preceded	subclass	evolution	so	that	a	subclass	
      tion	 in	 the	 level	 of	 maternal	 antibodies	 might	 help	 explain	 the	 ability	   with	 a	 certain	 name,	 e.g.	 IgG1,	 does	 not	 imply	 homology	 with	 those	
      of	older	calves	to	produce	effective	antibody	responses	compared	to	                  of	the	same	name	in	other	species.		Therefore	establishing	structure-
      neonates.                                                                             function	 relationships	 must	 be	 done	 for	 each	 species	 individually.	
                                                                                            Porcine	 IgG	 subclass	 proteins	 cannot	 be	 biochemically	 purified	 so	
      Key words:                                                                            porcine-camelid	 chimeric	 heavy	 chain	 antibodies	 (HCAb)	 have	 been	
      Species:	ruminants                                                                    constructed	and	expressed.	Specificity	tests	failed	to	identify	any	sub-
                                                                                            class-specific	monoclonal	or	polyclonal	antibodies	and	showed	most	to	
            mi210. ELEVEN PORCINE C GAMMA (Cγ) GENES:                                       be	biased	to	the	IgG1b	allelic	variant.
          PHyLOGENy, ExPRESSION AND ALLOTyPIC VARIANTS                                          1.	 Metzger,	 J.J.	 and	 M.	 Fougereau,	 1967.	 C.R.	 Hebd.	 Seances	
           JoHn	E	BUTLER1,	nAnCy	WERTZ1,	nICK	DESCHACHT2,3,	                                Acad.	Sci.	Ser	D.	Sci	nat.		      265:724
                SERGE	MUyLDERMAnS2,3,JoAn	K	LUnnEy4                                              2.	Kaltreider,	H.B.	and	J.S.	Johnson.	1972.	J.	Immunol.	109:992
      	               1Dept.	Microbiology,	Univ.	of	Iowa,	USA;	2Cellular	and	                     3.	 Rapacz,	 J	 and	 J.	 Hasler-Rapacz.	 1982.	 Proc.	 2nd	 World	
            Molecular	Immunology,Vrije	Universiteit	Brussel,,	BELGIUM;	                     Congress	on	Genetics	Applied		            to	 Livestock	 Production.	 Vol	
            3Department	of	Molecular	and	Cellular	interaction,	VIB,	Vrije	                  VIII.	Editorial	Garsi,	Madrid
          Universiteit		Brussel	BELGIUM;	4	ANRI,ARS,USDA	Beltsville,	MD,	
                                           USA		                                               4.	Bokhout,	B.A.,	J.J.	van	Asten-Noordijk	and	W.	Stock.	1986.	Mol.	
                                 john-butler@uiowa.edu                                      Immunol.	23:675.
            Subclass	heterogeneity	of	swine	IgG	was	recognized	>40	years	                       5.	Van	Zaane,	D.	and	M.M.	Hulst.	1987.	Vet.	Immunol.	Immunopath.	
      ago	 (1,2),	 allotypic	 variation	 was	 described	 in	 1982	 (3),	 differential	      16:23.
      specificity	of	anti-swine	IgG	antibodies	has	been	recognized	(4,5	)	and	                  6.	Kacskovics,	I.,	J.	Sun	and	J.E.	Butler.	1994.	J.	Immunol.	153:	
      five	 Cγ	 gene	 sequences	 have	 been	 reported	 (6).	The	 full	 complexity	          3536.
      of	 the	 system	 remains	 unresolved	 but	 we	 present	 here	 incremental	                 7.	Butler,	J.E.	and	n.	Wertz.	2006.	J.	Immunol.	177:	5480.
      data	 on	 eleven	 expressed	 Cγ	 genes	 which	 appear	 to	 comprise	 six	
                                                                                            Key Words:	IgG	subclasses;	phylogeny;	fetus;	IPP
      subclasses.	The	allelic	variants	of	IgG1	(IgG1a	and	IgG1b)	account	for	
                                                                                            Species:	swine
      a	very	large	proportion	of	the	IgG	transcribed	in	swine.		This	includes	
           9. INNATE IMMUNITy, INFLAMMATION AND ADJUVANTS; MEMORy, ACQUIRED IMMUNITy AND VACCINES:
                                              POSTERS VA211-VA250

   VA211. NATURAL KILLER CELLS IN LyMPH NODES OF                                 inhibitor	 of	 phagosomal	 acidification)	 prior	 to	 stimulation	 with	 ORN	
    HEALTHy CALVES ExPRESS CD16 AND SHOW BOTH                                    abolished	 the	 cytokine	 responses,	 confirming	 that	 the	 receptor(s)	
   CyTOTOxIC AND CyTOKINE-PRODUCING PROPERTIES                                   which	 mediate	 the	 ORN-induced	 responses	 are	 intracellular.	 These	
 PREBEn	BoySEn1*,	GJERMUnD	GUnnES2,	DAnIELA	PEnDE3,	                             results	 demonstrate	 for	 the	 first	 time	 that	 ORN	 have	 strong	 immune	
          METTE	VALHEIM4,AnnE	K	SToRSET1                                         stimulatory	 effects	in	 cattle,	and	 suggest	 that	further	investigation	 on	
                                                                                 the	potential	of	TLR7/8	ligands	to	activate	innate	and	adaptive	immune	
  1Dept.	of	Food	Safety	and	Infection	Biology	and	2Dept.	of	Basic	               responses	in	domestic	animals	are	warranted.	
  Sciences	and	Aquatic;	Medicine,	Norwegian	School	of	Veterinary	
Science,	Oslo,	Norway;	3Istituto	Nazionale	per	la	Ricerca	sul	Cancro,	           Key words:	Innate	immunity,	CD14+,	TLR7,	TLR8,	ssRNA,	CpG,	IL-
   Genova,	Italy;	4Dept.	for	Animal	Health,	Section	for	Pathology,	              12,	IFN-a,	IFN-γ,	TNF-a,	ELISA,	ELISPOT,	bafilomycin,	cattle
             National	Veterinary	Institute,	Oslo,	Norway		                       Species:	ruminants
                     preben.boysen@veths.no
      Natural	killer	(NK)	cells	were	recently	shown	to	play	an	important	         VA213. PROMISCUOUS EPITOPES OF MyCoBACTERIUM
immunomodulatory	role	in	lymph	nodes.	We	here	report	the	presence,	                 PARATUBERCULoSIS 70 KD HEATSHOCK PROTEIN
phenotype	 and	 function	 of	 NK	 cells	 resident	 in	 lymph	 nodes	 of	 sev-               ACTIVATING BOVINE T CELLS
eral	 anatomical	 sites	 of	 healthy	 calves.	 NK	 cells,	 defined	 by	 NKp46	     AAD	HoEK*1,	VICToR	RUTTEn1,	CHRIS	DAVIES3,AD	KoETS2
expression,	were	present	in	the	paracortex	and	the	medulla	of	bovine	              Departments	of	Infectious	Diseases	and	Immunology1	and	Farm	
lymph	nodes.	Most	lymph	node-derived	NK	cells	expressed	CD16	and	
                                                                                  Animal	Health2,	Faculty	of	Veterinary	Medicine,	Utrecht	University,	
perforin,	and	a	lytic	capacity	was	demonstrated,	while	a	well-developed	
                                                                                 The	Netherlands.	Veterinary	Microbiology	&	Pathology3,	Washington	
interferon-gamma	response	to	interleukin-2	and	interleukin-12	stimula-
                                                                                                        State	University,	USA	
tion	was	also	seen.	Lymph	node-derived	NK	cells	differed	from	those	
                                                                                                          a.hoek@vet.uu.nl
in	 blood	 by	 a	 higher	 expression	 of	 the	 activation	 markers	 CD44	 and	
CD25,	as	well	as	CD8.	Lselectin                                                        Bovine	paratuberculosis,	an	intestinal	inflammatory	disease	caused	
                                                                                 by	 infection	 of	 calves	 with	 the	 intracellular	 pathogen	 Mycobacterium	
     (CD62L)	 was	 expressed	 by	 the	 majority	 of	 lymph	 node-derived	
                                                                                 avium	subspecies	paratuberculosis	(MAP),	constitutes	a	threat	to	cat-
NK	cells,	consistent	with	a	dependency	of	this	molecule	for	migration	
                                                                                 tle	worldwide.	In		earlier	work	the	recombinant	MAP	70	kD	heat	shock	
to	lymph	nodes.	Unlike	in	blood,	the	majority	of	lymph	node	NK	cells	
                                                                                 protein	(MAP	Hsp70)	showed	to	be	an	immunodominant	antigen	and	
had	little	or	no	CD2	expression.	Compared	to	available	literature,	calf	
                                                                                 a	vaccine	candidate	for	paratuberculosis*.	In	the	present	study	peptide	
lymph	 nodes	 contained	 NK	 cells	 in	 numbers	 equal	 to	 or	 higher	 than	
                                                                                 epitopes	 of	 MAP	 Hsp70	 were	 identified	 by	 the	 capacity	 of	 overlap-
reported	 in	 humans,	 and	 clearly	 higher	 than	 in	 mice.	 These	 findings	
suggest	a	cytotoxic	role	of	lymph	node	residing	NK	cells,	beyond	the	            ping	MAP	Hsp70	14-mer	peptides	to	elicit	cell-mediated	responses	of	
predominantly	cytokine-producing	role	previously	inferred	from	studies	          PBMC	of	a	MAP	Hsp70	immunized	high	responder	cow.	Subsequently	
on	human	NK	cells.                                                               immune	responsiveness	to	selected	MAP	Hsp70	peptides	was	tested	
                                                                                 in	bovine	MHC	typed	control	animals	(n=5)	and	cows	that	were	MAP	
Key words:	 natural	 killer	 cells,	 lymphoid	 organs,	 CD16,	 cytotoxicity,	    challenged	(n=28)	and	later	on	immunized	with	MAP	Hsp70.
interferon-gamma,	
Species:	ruminants                                                                     Nine	out	of	124	MAP	Hsp70	peptides	induced	proliferation	of	PBMC	
                                                                                 and	6	T	cell	lines	/clones	were	generated	by	regular	restimulations	with	
                                                                                 3	out	of	the	5	most	immunogenic	Hsp70	peptides	and	peptide	cores	
VA212. CD14+ CELLS ARE REQUIRED FOR TLR7/8 LIGAND-
                                                                                 were	mapped	by	Pepscan	techniques.	PBMC	of	MAP	challenged	and	
 INDUCED IL-12 AND IFN-γ RESPONSES IN BOVINE BLOOD
                                                                                 MAP	Hsp70	immunized	cows	responded	p