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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-1β
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-γproduction 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. Igl 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
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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.