From innate to adaptive immunity by axx49266


									         Plenary Session 1

From innate to adaptive immunity

Chairs: John Hopkins, Bernard Charley

              Room 242

         Tuesday, September 5th

         Ruminant TLRs: what we know and what we do not know

         Werling D.
         Royal Veterinary College, London, UK
         Host immune responses strongly control the outcome of infectious disease, and the resistance to
         viral and bacterial infections in mammals is in part genetically determined. The innate immune
         system senses pathogens largely through signals initiated by a collection of phylogenetically rela-
         ted proteins known as pathogen recognition receptors (PRR) such as "Toll-like receptors" (TLRs),
         of which ten representatives are encoded in the bovine genome, and C-type lectin receptors.
         Cloned receptors can in the meantime be studied for ligand-TLR interaction using specific cell
         systems and analysing for example NF-kB activation. In addition, several genetic variations
         within PRR genes in mouse and men are known to be associated with a variety of inflammatory
         and infectious diseases, such as endotoxic shock syndrome, atherosclerosis and Crohn's diseases.
         Interestingly, TLR4 haplotypes have also been identified in different chicken and cattle breeds.
         Whereas a clear evidence for disease resistance was associated with such alterations in chicken,
         these have not yet been attributed to specific diseases resistance in cattle. In this presentation, we
         will summarise our knowledge about bovine pathogen recognition receptors, present examples
         regarding the analysis of a specific ligand with bovine TLRs and show example of differential
         expression of PRR in cells and in different cattle breeds.

         The avian innate immune response – A similar outcome achieved by a

         different repertoire of molecules and cells to those used in mammals
         Kaiser P.1*, Smith A.1, Wu Z.1, Rothwell L.1, Soulier A.1, Poh T.Y.1, Swaggerty C.2, He
         H.2, Kogut M.2
             Institute for Animal Health, Compton, UK
             USDA-ARS, College Station, Texas, USA
         Chickens mount an effective innate immune response, and this innate response presumably also
         drives appropriate adaptive immune responses, as infections with intracellular pathogens are
         controlled by Th1 responses and those with extracellular pathogens by Th2 responses. However,
         the details of the chicken's innate immune response differ from those of the more commonly stu-
         died biomedical species. The chicken has a different repertoire of Toll-like receptors, defensins,
         pro-inflammatory cytokines and chemokines compared to mammals, and these will be discussed.
         Further, the main effector cell of the induced innate response in mammals, the neutrophil, is repla-
         ced in the chicken by a different polymorphonuclear cell, the heterophil, which shares some but
         by no means all of the characteristics of the mammalian neutrophil. Our knowledge of dendritic
         cells (DC) in the chicken is in its infancy. However, we have recently been able to grow out DC
         from bone marrow with recombinant chicken IL-4 and GM-CSF, and to differentially mature these
          DC with LPS or CD40L to give a Th1- or Th2-promoting phenotype respectively.

             Involvement of FcγRII in plasmacytoid dendritic cell activation by viruses

             Guzylack-Piriou L., Balmelli C., Bergamin F., Mc Cullough K.C., Summerfield A.
             Institute of Virology and immunoprophylaxis, Mittelhausern, Switzerland
          Natural interferon-producing cells (NIPC), also called plasmacytoid dendritic cells, are the most
         potent producers of IFN-alpha in response to viral and bacterial components, serving an important
         function in innate immune defences. Here, we demonstrate the NIPC are also participating in
         adaptive immune responses using two distinct ways of interacting with specific immunoglobulin
         (Ig). The first way is exemplified by the non-enveloped foot-and-mouth disease virus (FMDV),
         which cannot stimulate interferon-α (IFN-α) responses in NIPC, unless complexed with FMDV-
         specific immunoglobulins. Stimulation of NIPC with such immune complexes employs FcgRII

ligation, leading to strong secretion of IFN-α. This required the initiation of the viral replicative
cycle leading to an abortive replication, as witnessed by the decrease of dsRNA levels and viral
titres with time post-infection. Sensitivity of the NIPC stimulation to wortmannin and chloroquin,
but not leupeptin, indicated an essential role for the pre-lysosomal stage endosomal compartment.
The second way of NIPC to interact with specific Ig is exemplified by its interaction with classi-
cal swine fever virus (CSFV). In this model, NIPC responsiveness can be primed by immunisa-
tion, increasing their capacity to produce IFN-alpha after viral infection. NIPC isolated from pigs
immunised were more receptive to viral infection and produced higher levels of IFN-α than NIPC
from immunologically naive animals. This sensitisation of NIPC was maintained for at least 8
months after immunisation. IFN-alpha production was dependent on live virus and required repli-
cation, and the "immune" NIPC responded to lower infectious doses of virus. Like with FMDV-
Ig complexes, co-localisation of the CSFV with FcgRII in polarised structures was observed with
"immune" NIPC only. This FcgRII -dependent virus capture and sensitisation of NIPC, evidently
mediated through cytophilic CSFV-specific antibodies, was inhibited by non-specifically aggre-
gated immunoglobulin as well as by pre-formed virus-antibody complexes. In conclusion, these
results demonstrate that NIPC not only represent a major player in innate immunity but are also
functionally linked to the immunological memory of the adaptive immune system.

Supersialylated collectins: multipotent innate defence proteins with

broad-spectrum activity against influenza A virus
van Eijk M.1, Hartshorn K.L.2, White M.R.2, Haagsman H.P.1*
  Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands
  Department of Medicine, Section of Hematology/Oncology, Boston University School of
Medicine, Boston, USA
Collectins are mammalian collagenous glycoproteins that belong to the C-type lectin superfamily.
These multimeric calcium-dependent lectins can bind to characteristic patterns of carbohydrates
expressed on the surface of a vast range of pathogens including bacteria, yeasts, fungi and viruses.
Previous studies, both in vitro and in vivo, have shown that the lung collectins SP-A and SP-D
contribute to the containment of influenza A virus (IAV). Porcine lung collectins show substan-
tially enhanced inhibitory activity against IAV as compared to those from other species. This
results from interactions mediated by the Asn-linked sugar and terminal sialic acid present in the
lectin domain of both porcine lung collectins. Given the unique anti-IAV properties of porcine
lung collectins due to their extraordinary glycosylation profile, recombinant analogues based on
the human SP-D (hSP-D) sequence are being developed. These recombinant collectins have novel
glycosylation sites engineered into the pathogen recognition domains via site-directed mutagene-
sis. Development of ‘glyco-modified’ collectins may result in new products to prevent and com-
bat infections in humans more effectively. It is envisaged that inhaled supersialylated collectins
may be used as a broad-spectrum prophylaxis to protect against IAV infections.

Cytokine secretion by bovine polymorphonuclear neutrophil leukocytes

Paape M.J.*, Sohn E.J., Bannerman D.D., Fetterer R.H., Connor E.E., Peters R.R.
United States Department of Agriculture, Beltsville, MD 20705, USA
University of Maryland, College Park, MD 20742, USA
Rapid recruitment and bacterial phagocytosis and killing by PMN are the most effective defenses
against establishment of infection. In addition, PMN may play a key supportive role through
secretion of cytokines during the inflammatory response. We sought to determine whether bovine

         PMN secrete cytokines in response to stimulation with lipopolysaccharide (LPS). PMN were expo-
         sed to increasing concentrations of LPS (0, 1, 10 and 100 µg/mL) for 18 h at 37 °C, and cell super-
         natants analyzed for expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-12, IL-8
         and interferon (IFN)-γ by ELISA. TNF-α, IL-1β, and INF-γ increased in a dose-dependent manner,
         while IL-12 decreased with increasing concentrations of LPS. To further study the secretion of IL-
         8, PMN were cultured with either RPMI, 0.1 µg LPS, 10 ηg/mL human TNF-α, or 10 ηg/mL bovi-
         ne IL-1β. PMN were also incubated with 0.1 µg LPS plus 10 ηg/mL TNF-α or 10 ηg/mL IL-1β.
         Incubation with either LPS or LPS together with either TNF-α or IL-1β increased secretion of IL-
         8 when compared to RPMI. It was concluded that LPS stimulation can up-regulate the secretion of
         cytokines by bovine PMN, and that co-incubation of LPS with TNF-α or IL-1β had an additive
         effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic
         and bactericidal properties, may play a supportive role in the innate immune response to infection
         by Gram-negative bacteria by producing cytokines that mediate inflammation.

         Specific innate immune responses to replication of PRRSV in alveolar

         Ait-Ali T.1*, Wilson A.1, Wescott D.2, Clapperton M.1, Mellencamp M.3, Drew T.2, Bishop
         S.1, Archibald A.1
             Roslin institute, Edinburgh, UK; 2 VLA, Weybridge; 3 Sygen International, Franklyn, USA
         Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of an infec-
         tious disease characterized by respiratory disorders and abortion in pregnant sows. First identified
         in the US in 1987 and subsequently in Europe, PRRSV is a positive RNA virus. It replicates in
         lung macrophages and elicits only a minimal interferon response. Infected pigs tend to recover but
         are still able to shed the virus for prolonged periods of time. It is increasingly recognized that the
         differences in virulence of viruses have been attributed to numerous factors including, virus strain
         heterogeneity and management practices. Recent reports show that host genetics also play a pro-
         minent role in resistance to infection in several species. Identifying genetic components involved
         in host susceptibility represent an important step forward for the development of a disease control
         programs for PRRS. The present study was undertaken to evaluate the response of the alveolar
         macrophage from five genetic lines of commercial pigs to PRRSV infection. We used alveolar
         macrophage preparations because macrophages represent the initial and the most susceptible host
         cells targeted by PRRSV. Here we report a comparison of the in vitro replication of PRRSV in
         alveolar macrophages derived from five commercial genetic lines including Largewhite, Pietrain,
         Landrace and two synthetic lines selected for dam line robustness and efficient lean growth. First,
         we showed that, in vitro, PRRSV replication and growth in alveolar macrophages from the
         Landrace animals was significantly and reproducibly reduced when compared with the other four
         lines. Finally we demonstrated that the Landrace phenotype was associated with extensive trans-
         criptional modulations early during infection such as high levels of TNF-α, IL-8 or type-I inter-
         feron pathway mRNA expression. Our data indicate the possibility that apparent genetic factors
         are likely to be responsible for the in vitro attenuation of PRRSV replication.

         Effects of interferon-α on the inflammatory response to bacterial

         endotoxin in swine peripheral blood mononuclear cells
         Amadori M.1*, Begni B.1, Ritelli M.2, Podavini2
           Department of Animal Welfare and Immunoprophylaxis, Istituto Zooprofilattico Sperimentale,
         Brescia, Italy
           Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnologies,
         University of Brescia, Italy

We had previously reported on a role of interferon (IFN)-α in the regulation of inflammatory cyto-
kine genes in swine pulmonary alveolar macrophages (PAM). In this model, IFN-α at a very low
concentration (0.5 U/mL) could reduce the expression of the TNF-α gene, as opposed to a 100-
fold higher concentration. No such effect had been observed with regard to both IL-1β and IL-6
genes. Owing to the above, we wondered if IFN-α could exert a similar regulation on functional-
ly immature cells like blood monocytes. Thus, swine peripheral blood mononuclear cells were
supplemented with IFN-α at different final concentrations (0.5 and 50 U/mL), and then stimula-
ted with LPS. The expression of the TNF-α, IL-1β and IL-6 genes was investigated by semi-quan-
titative PCR. As opposed to the PAM model, IFN-α at both concentrations could significantly
reduce the expression of the IL-6 and not of the TNF-α gene. The results of TNF-α bioassays on
cell culture medium were in agreement with this result. In addition, a significant inhibiting effect
on the IL-1β gene was exerted at 50 U/mL IFN-α, only. The above findings confirm a role of IFN-
α as a homeostatic agent in the inflammatory response. In particular, the preferential regulation of
the IL-6 gene in peripheral blood cells could be related to the role of this cytokine in the acute
phase response. As opposed to the lung environment, the down-regulation in blood of some
inflammatory cytokine genes could be exerted by IFN-α at moderate concentrations (50 U/mL),
during innate immune response to viral and bacterial infections.

Staphylococcus aureus intramammary infection elicits increased

production of transforming growth factor-alpha, beta1, and beta2
Bannerman D., Paape M.*, Chockalingam A.
U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD, USA
Pennsylvania State University, University Park, PA USA
Staphylococcus aureus is one of the most highly prevalent contagious pathogens to cause masti-
tis in cattle. In contrast to other mastitis pathogens, the host response evoked during S. aureus
intramammary infection is marked by the absence of the induction of critical cytokines, including
IL-8 and TNF-alpha, which have established roles in mediating host innate immunity. The eluci-
dation of changes in the expression of other mediators with the potential to regulate mammary
inflammatory responses to S. aureus remains lacking. Transforming growth factor (TGF)-alpha,
TGF-beta1, and TGF-beta2 are cytokines that regulate mammary gland development. Because
these cytokines also have a demonstrated role in mediating inflammation, the objective of the cur-
rent study was to determine whether S. aureus intramammary infection influences their expres-
sion. Ten cows were challenged with S. aureus and milk samples collected. Increases in milk
levels of TGF-alpha were evident within 32 h of infection and persisted for 16 h. Infection with
S. aureus also elicited an increase in TGF-beta1 and TGF-beta2 production within 40 h of infec-
tion and this increase was sustained through the end of the study 5 days later. Thus, in contrast to
IL-8 and TNF-alpha, S. aureus is able to elicit host production of TGF-alpha, TGF-beta1, and
TGF-beta2. This finding may suggest a role for these molecules in mediating mammary gland host
innate immune responses to infection by S. aureus.

Lentivirus-mediated siRNA delivery to porcine dendritic cells to assess

pattern recognition receptor-mediated virus recognition
Alves M.*, Neuhaus V., McCullough K., Summerfield A.
Institute of Virology and Immunoprophylaxis, CH-3147 Mittelhausern, Switzerland
Dendritic cells (DC) play a major role in linking innate to adaptive immunity through the capabi-
lity to sense invading pathogens using pathogen-recognition receptors (PRR). The complexity of
this system is reflected by a cell-type-specific presence of various receptors which can sense defi-
ned microbial components, and have an appropriate cellular localization. In order to investigate
         the PRR's involved in the recognition of viruses by the porcine innate immune system, we have
         established RNA interference-based knockdown of various PRR currently including dsRNA-
         dependent protein kinase (PKR), toll-like receptor (TLR) 7 and the major adaptator protein of
         TLR signalling, MyD88. As delivery method an amphotrophic lentiviral system was used, which
         efficiently transduced monocyte-derived DC (MoDC), Flt3 Ligand induced bone marrow-derived
         DC (Flt3L-DC), as well as their precursors, monocytes and haematopoietic stem cells, respecti-
         vely. Lentivirus-mediated siRNA delivery was efficient in silencing PKR, MyD88 and TLR7
         transcription. Functional tests confirmed that the MyD88 and TLR7 mRNA loss resulted in a redu-
         ced responsiveness to TLR4 and TLR7 ligands. Infection of MoDC and Flt3L-DC by the lentivi-
         rus did neither induce CD80/86 and MHC class II upregulation nor cytokine responses. However,
         studies on the interaction of the lentivirus with natural interferon producing cells (nIPC) demons-
         trated the induction of high levels of INF-α. Taken together, these studies demonstrate the suita-
         bility of lentivirus-mediated siRNA delivery for various porcine antigen presenting cell subsets,
         and enable the study of their interaction with microorganisms.

         Do Digenean parasites use different or common strategies to escape

         intermediate and definitive host's immune response?
         Sibille P.*, Coustau C.
         INRA UR86 Centre de Tours, 37380 Nouzilly, France
         Several organisms are able to parasite two or more successive hosts along their developmental
         stages. In the case of several hosts, whether the parasite should display different subversion stra-
         tegies or cope with one prototypic arsenal should be assessed. Trematode Fasciola hepatica pro-
         duces two infective stages (sporocyst, SP and adult fluke) that settle in a molluscan Lymnaea trun-
         catula and in a grass feeding mammal, respectively. Both stages were used for the collection of
         excretory/secretory (ES) products that were assayed for reactive nitrogen/oxygen intermediates in
         rat or sheep mononuclear cell cultures. Stimulation of resting peritoneal cells by high doses of sp
         ES led to increased NO production, while O2- synthesis was reduced by a broad range of sp ES
         dilutions. Experimental infections with F. hepatica led to reversion of NO production by sp ES-
         stimulated peritoneal cells. Similar experiments performed on F. hepatica infected sheep showed
         that (i) sheep do not produce detectable NO; (ii) peripheral blood leukocytes of control and infec-
         ted sheep produce significantly less O2- when stimulated by either adult or sp ES. These results
         suggest that F. hepatica may use identical mechanisms to down regulate immune response of both
         its intermediate and definitive host’s immune response.

         Innate immunity factors involved in resistance to intestinal carriage of

         Salmonella in birds
         Sadeyen J.-R., Velge P., Derache C., Beaumont C., Lalmanach A.-C.*
         INRA, Nouzilly, France
         Salmonellosis is a major cause of food borne poisoning mainly due to the consumption of conta-
         minated poultry products (meat, eggs…). In birds, Salmonella is able to persist in the digestive
         tract (caecum) for weeks without triggering any symptom. Genetic selection of resistant animals
         as well as immuno-stimulation (recombinant vaccine) represents prophylactic measures that need
         a better knowledge of host resistance factors. In this context, two inbred lines of birds orally ino-
         culated with Salmonella Enteritidis at one week or thirty weeks of age were compared for
         Salmonella colonisation level and for innate immunity candidate gene expression in the caecum.
         The level and duration of the intestinal bacterial load was different between lines showing either
         a resistant or susceptible phenotype which was age-related. A marked differential expression of
         defensin genes was observed between lines. The high level of defensin was linked to the resistan-
ce to Salmonella carriage in adult but not in young birds. The results suggest a microbicidal and/or
a chemotactic activity of defensins towards immune cells in the adult host against Salmonella per-
sistence. This activity could not be functional in young birds and need to be further investigated.

Female sexual steroids modulate the indirect killing of bovine

neutrophilic granulocytes after stimulation of in vitro generated
macrophages with bacterial molecular patterns
Schuberth H.-J.1*, Petz W.2, Schuler G.3, Seyfert H.-M.3, Zerbe H.2
    Institute of Immunology, University of Veterinary Medicine, Hannover, Germany
    Clinic for ruminants, Ludwig-Maximilians-University, Munich, Germany
    Clinic for Obstetrics, Gynecology and Andrology, University of Giessen, Germany
    Research Institute for the Biology of Farm Animals, Dummerstorf, Germany
Mastitis occurs rather frequently during the periparturient period, often due to grampositive or
gramnegative bacterial infection. Thus, it was tested whether pathogen associated molecular pat-
terns (PAMPs) (CpG-DNA, LPS) influence the function of in vitro generated macrophages
(MdM) in the context of relevant sexual steroid hormones. PMN, blood mononuclear cells
[PBMC], purified monocytes and MdM contained progesterone and oestrogen (α, β) receptors as
proven by immunohistology and PCR. Progesterone (P4) and estradiol-17β (E2), at high concen-
trations, significantly inhibited the induced killing of PMN in CpG-stimulated leukocyte popula-
tions (P4: p < 0.05; E2: p < 0.001). P4 had no effect on the numbers of viable PMN after PAMP
stimulation, neither when stimulated alone nor in co-culture with PBMC. Estradiol-17β signifi-
cantly enhanced the LPS-induced, MdM-dependent killing of PMN (p < 0.01), but not the killing
induced by LPS or LPS+CpG in pure PMN cultures. MdM expressed the pattern recognition
receptors Toll like receptor-2, and -4. Quantitative RT-PCR showed no significantly altered
expression for these two gene products after exposition to P4 and E2. In summary, it could be
shown that the tested hormones are able to modulate the response of immune cells to PAMPs but
not at the level of TLR expression.

Expression of toll-like receptor 2 in porcine tissues

Alvarez B.1*, Revilla C.1, Domenech N.2, Perez C.1, Martínez P.1, Alonso F.1, Ezquerra
A.1, Domínguez J.1
  Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria (INIA), Ctra de la Coruña, 28040 Madrid, Spain
  Unidad de Investigacion, Complejo Hospitalario Juan Canalejo, 15006 A Coruna, Spain
Toll-like receptors (TLR) are a group of pattern recognition receptors that play a crucial role in
innate immunity. TLR2 binds a variety of cell wall components of Gram-positive bacteria and
mycobacteria such as peptidoglycan, lipoteichoic acid, lipoproteins and lipoarabinomanan, lea-
ding to cell activation and production of proinflammatory cytokines. We developed a monoclonal
antibody (1H11) to porcine TLR2 after immunising mice with poTLR2-expressing 3T3 transfec-
tants and analysed the distribution of this molecule in porcine tissues. The specificity of this anti-
body was confirmed by its reactivity with CHO cells transfected with poTLR2 but not with non-
transfected cells. By flow cytometry, TLR2 was found on cells of the innate immune system,
including monocytes, macrophages and granulocytes, but not on peripheral blood lymphocytes.
By immunohistochemistry, TLR2 expression was also detected on tracheobronchial and intestinal
epithelial cells and on the basal layer of the epidermis. This distribution is consistent with a sur-
veillance function at entry sites allowing for early detection of microbial invasion.

         In silico B cell epitope mapping of the HN of the porcine rubulavirus and

         its expression in OmpC from S. typhi
         Zenteno-Cuevas R.1*, Huerta-Yepes S.2, Suarez-Güemes F.3, González-Bonilla C.2,
         Ramírez H.4, Agundis C.5, Zenteno E.5
           Instituto de Salud Pública, Universidad Veracruzana, Xalapa, Veracruz 91000 Mexico
           Hospital de Infectología, Centro Medico La Raza, IMSS, 02990, Mexico
           Departamento de Microbiología e Inmunología, Facultad de Medicina Veterinaria y Zootecnia,
         UNAM, 04510 Mexico
           Laboratorio de Producción Porcina, Facultad de Medicina Veterinaria y Zootecnia, UNAM, 04510
           Departamento de Bioquímica Facultad de Medicina UNAM, 04510 Mexico
         Based on the amino acid sequence of the Hemagglutinin-Neuraminidase (HN) from the porcine
         rubulavirus from La Piedad Michoacan (RvpLPM), we applied a combination of physicochemi-
         cal and structural restrictions and prediction algorithms, in order to map the HN protein and iden-
         tify B cell epitopes. From 18 initial candidates only two of them fulfill all the requirements; one
         from residues 533 to 544 (TTTCFRDTDTGK; HN-A) and the second from 251 to 267 (YVATR-
         SETDYYAGNSPPQ; HN-B). Antibodies from RvpLPM-infected pigs were able to recognize
         these peptides by the ELISA assay. HNA and HNB coupled to BSA and administrated to rabbits
         induce the production of antibodies with viral recognition capability. The DNA sequence for the
         HNA peptide was used to replace the loop 5 of the membrane protein ompC from Salmonella
         typhi. The fusion protein was expressed in E. coli UH302 ompC- and OmpF-, and the bacterium
         was used to immunize Balb-c mice by oral and intra-peritoneal via. The antibodies obtained by
         oral administration showed the highest hemagglutinating inhibitory capability (p > 0.001). The
         results of this work lead us to suggest that our in silico prediction system is able to identify pep-
         tides with antigenic relevance, and the HNA peptide is a good sequence target for the study of the
         carbohydrate recognition domain of HN from RvpLPM.

         Porcine β-defensin 2 is upregulated by Salmonella typhimurium in the

         intestinal cell line IPI-2I
         Veldhuizen E.J.A.*, Hendriks H., Kalkhove S., Vo A., Gaastra W., Tooten P., Haagsman
             Department of Infectious Diseases and Immunology, Utrecht University, The Netherlands
             Department of Pathobiology, Utrecht University, The Netherlands
         Introduction: Beta defensins are small antimicrobial peptides that are part of the innate immune
         system of animals and plants. In the intestine, they are produced and secreted by epithelial cells,
         and for some defensins, the expression can be induced upon a bacterial trigger. In this study a por-
         cine Ileal epithelial cell line (IPI) was used to detect whether porcine defensins 1 and 2 (pBD1&2)
         are inducible upon bacterial infection.
         Results: A defensin gene expression increase during growth in DMEM medium/10% FCS was
         observed in IPI cells. To optimize the infection experiments, cells were grown in synthetic serum-
         free medium where the natural pBD production remained constant over the time course of the
         experiment. Salmonella typhimurium infection of IPI cells resulted in elevated levels of pBD2
         mRNA after 6 h and longer while PBD1 mRNA was elevated after 24 h. In addition, elevated
         levels of IL-8 were observed upon infection. Similar infection of IPI cells using Staphylococcus
         aureus, arcobacter and Salmonella enteritidis had no effect on the pBD production. This indicates
         that there is a S. typhimurium specific effect on porcine defensin regulation in this cell line.

Chicken β-defensin gallinacin-6 is expressed in the digestive tract and has

antimicrobial activity against food-borne pathogens
van Dijk A.*, Veldhuizen E.J.A., Kalkhove S.I.C., Tjeerdsma-van Bokhoven J.L.M.,
Haagsman H.P.
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands
In contrast to mammalian β-defensins, little is known about the role of β-defensins in the avian
digestive tract and their efficacy. In this study, the expression of chicken β-defensin gallinacin-6
(Gal-6) and its antimicrobial activity against food borne pathogens were investigated. RT-PCR
analysis showed high expression of Gal-6 mRNA in the oesophagus and crop, moderate expres-
sion in the proventriculus, and low expression throughout the intestinal tract. Crop tissue Gal-6
mRNA levels were breed-independent, but varied greatly in young animals. The presence of puta-
tive transcription factor binding sites for nuclear factor kappa beta, activator protein-1 and nuclear
factor interleukin-6 in the Gal-6 gene upstream region suggests a possible inducible nature. In
colony counting assays, strong bactericidal and fungicidal activity was observed for synthetic Gal-
6 (sGal-6), including bactericidal activity against food-borne pathogens Campylobacter jejuni,
Salmonella typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16
µg/mL sGal-6, resulted in a 3 LOG unit reduction in C. perfringens survival within 60 min, indi-
cating fast killing kinetics. Transmission electron microscopy examination of sGal-6 treated C.
perfringens cells showed dose-dependent changes in morphology after 30 min, including intra-
cellular granulation, cytoplasm retraction, irregular septum formation in dividing cells and cell
lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest
that chicken β-defensin gallinacin-6 plays an important role in chicken innate host defense.

Avian collectins: protectors against infections in birds?

Hogenkamp A.1, van Eijk M.1, van Dijk A.1, van Asten A.J.A.M.2, Veldhuizen E.J.A.1,
Haagsman H.P.1
  Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands
  Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The
Collectins play a key role in innate immunity. These pattern-recognition molecules have been
shown capable of neutralizing various pathogens, including influenza A virus. In avians, the only
collectin described thus far is the chicken mannan binding lectin. In this study, we sought to iden-
tify other collectins in chickens by searching the EST database using the amino acid sequences of
known collectins, and relating the results to the chicken genome. Three chicken collectins were
found and designated chicken Collectin 1 (cCL-1), chicken Collectin 2 (cCL-2), and chicken
Collectin 3 (cCL-3), which resemble the mammalian proteins Collectin Liver 1, Collectin 11 and
Collectin Placenta 1, respectively. Additionally, a lectin resembling Surfactant Protein A (SP-A)
was found. Lacking the collagen-like domain characteristic for collectins, it was named chicken
Lung Lectin (cLL). An extensive tissue distribution analysis showed that cCL-1, cCL-2 and cCL-
3 are expressed in a wide range of tissues throughout the digestive, reproductive and lymphatic
system. Similar to SP-A, cLL is mainly localized in tissues associated with the respiratory system.
The newly found chicken collectins could have a significant role in avian immunity. Work is cur-
rently in progress to produce these lectins in order to study their characteristics. A better unders-
tanding of the role of these proteins may ultimately lead to strategies to reduce infectious diseases
in poultry and zoonotic diseases.

         Porcine circovirus type 2 (PCV2) CpG sequences immunomodulate porcine

         dendritic cells in recall antigen responses
         Montoya M.*, Kekarainen T., Domínguez J., Mateu E., Segalés J.
         Centro de Investigación en Sanidad Animal, UAB, Barcelona 08193, Spain
         Porcine circovirus type 2 (PCV2) is considered the necessary agent for postweaning multisyste-
         mic wasting syndrome (PMWS) in pigs. Interaction of this virus with porcine dendritic cells
         (poDC) induces a degree of immunomodulation that may favour the establishment of other conco-
         mitant infections in the animals (1). In fact, it has been suggested that PMWS affected pigs are
         immunosuppressed, and therefore, more prone to develop concomitant infections (2). The immu-
         nomodulatory effects of oligodeoxyribonucleotides (ODN) containing unmethylated CpG dinu-
         cleotides (CpG-ODN) have also been proven to modulate IFNα secretion in poDC (3). A set of
         21 CpG-ODNs were selected from PCV2 genome with the aim to unravel the stimulatory/inhibi-
         tory effects of PCV2 on in vitro recall antigen responses of pigs. This selection was performed by
         comparing porcine circovirus type 1 and PCV2 full genome sequences. Stimulation of peripheral
         blood mononuclear cells of PCV2-naïve animals with the selected CpGs was performed and cell
         culture supernatants were used to determine IFNα and IL10. The same CpG were used to stimu-
         late bone marrow derived poDC. Surface activation markers and cytokines were also analysed
         after stimulation by themselves and in antigen recall responses. The results indicate that certain
         CpG sequences were able to induce similar immunomodulatory effects to the PCV2 by cytokine
         secretion. The implication of those CpG sequences in PCV2 infection with other pathogens will
         be discussed. This work was partly funded by the Project No. 513928 from the Sixth Framework
         Programme of the European Commission.
         (1) Immunology 115:388-398. (2) Anim. Health. Res. Rev. 6:119-142. (3) Vet. Immunol.
         Immunopathol. 101:87-102.

         ELA-DQA*1301 phenotype frequencies in equidae

         Brown J.J., Carter S., Clegg P., Thomson W., Ollier W.E.R.
         Background: Major histocompatibilty complex (MHC) class II molecules are important regulato-
         ry elements controlling the adaptive immune response. Such genes are highly polymorphic in
         most higher species. These molecules have been implicated in a wide range of immunological dri-
         ven disease. As with most other mammalian species, the equine MHC (ELA) is poorly defined in
         terms of its structure and the number and order of the genes it contains. However, as more resear-
         ch is being carried out into equine immunogenetics, similarities between the equine MHC and that
         of other species have been demonstrated along with several significant differences. The horse
         MHC class II loci are unusual in that the DRA locus is polymorphic and DQA loci exist on two
         separate chromosomes. Objectives: To characterise the ELA-DQA*1301 specificity on horse
         chromosome 5, devise a specific genotyping assay and determine the ELA-DQA*1301 frequency
         in equidae. Methods: An Allele specific PCR was developed which only amplified ELA-
         DQA*1301 on horse chromosome 5, and did not amplify other DQA loci on horse chromosome
         20. Amplification of ELA-DQA*1301 was achieved using the previously published exonic primer
         DQA2e and an ELA-DQA*1301 allele specific primer. DNA from 295 equidae comprising
         Arabians n = 10, Warmbloods n = 21, Thoroughbred crossbreds n = 46, Thoroughbred n = 121,
         Irish draught horse n = 42, one three generation Thoroughbred pedigree, donkeys n = 36, and
         zebras n = 2, were analysed. Results: A range of ELA-DQA*1301 phenotype frequencies were
         observed in the animals tested, Arabians 70%, Warmbloods 43%, Thoroughbred crossbreds 39%,
         Thoroughbred 27%, Irish draught horse 26%, donkeys 53% and zebras 0%. Conclusions: The
         ELA-DQA*1301 phenotype frequencies varied considerably across equidae. This may have
         implications for equine disease susceptibility.

Toll-like receptor mRNA expression in LPS-stimulated porcine alveolar

Ramírez-Boo M., Pérez E., Lucena C., Moreno A., Garrido J.J.
Unidad de Marcadores Genéticos Moleculares, Departamento de Genética, Universidad de
Córdoba, Campus Rabanales, Córdoba, Spain
The immune system in vertebrates consists of two related functional arms: the innate and the adap-
tative immune systems, which function in a coordinated way to protect against infection. Innate
immunity acts as the first line of host defence against microbial pathogens and is conserved over
a wide variety of species from flies to mammals. Pathogens consist of a complicated cocktail of
pathogen-associated molecular patterns (PAMPs) that stimulate a battery of receptors at the sur-
face of the immune cells. These cellular receptors that interact with PAMPs are known as pattern
recognition receptors (PRRs). Members of the Toll-like receptor (TLR) family constitute a class
of PRRs with the ability to recognize pathogens and to initiate signalling events leading to acti-
vation of innate host defences. The main goal of this study was to evaluate the TLR mRNA
expression pattern in porcine alveolar macrophages treated with LPS from Salmonella typhimu-
rium. To achieve our objective, the expression level of TLR-1, TLR-2, TLR-4, TLR-5, TLR-6,
TLR-9 and TLR-10 were examined after 2 and 4 h of incubation in unstimulated control and LPS-
stimulated cells. Our results showed significant up-regulation of TLR-2 and TLR-4 transcript
expression in alveolar macrophages upon LPS challenge whereas the expression levels relative to
the rest of TLRs were similar in both unstimulated and LPS-stimulated cells.

Cell-mediated cytotoxicity and homing in DNA-vaccinated rainbow trout

Oncorhynchus mykiss infected with viral haemorrhagic septicaemia virus
Utke K.1, Sunyer O.2, Bergmann S.1, Lorenzen N.3, Koellner B.1*, Ototake M.4, Fischer U.1
    Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany
    University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA, USA
    Danish Institute for Food and Veterinary Research, Aarhus, Denmark
    National Research Institute of Aquaculture, Tamaki, Japan
Cytotoxic T cells are part of the adaptive immune system and recognize virus-infected target cells
by binding of their T cell receptors to classical MHC class I molecules loaded with viral peptides.
Our previous studies have shown that the allele of the classical MHC class I locus Onmy-
UBA*501 is identical in the rainbow trout clone C25 and in the rainbow trout cell line RTG-2.
This enabled us to develop a cytotoxicity assay with MHC class I-matched effector and target
cells. Peripheral blood leukocytes (PBL) from VHSV infected donors killed MHC class I mat-
ching and later also MHC class I mismatching xenogeneic VHSV infected targets. PBL from
infected fish showed a higher transcriptional level of the CD8α gene than PBL from uninfected
control fish. Concurrently, the expression levels of the natural killer cell enhancement factor
(NKEF)-like gene and of the NK cell marker CD56 were enhanced. This suggests an activation of
both NK and cytotoxic T cells during VHSV infection. The later appearance of PBLs able to kill
VHSV-infected MHC class I mismatching cells is contradictory to the generally accepted rule that
innate immune mechanisms represent the first line of defense after viral infections. One possible
explanation for this discrepancy could be that VHSV-specific antibodies are involved in the obser-
ved NK-like target cell killing.

         The activation of TLR and MHC II+ leukocytes decides the fate of trout

         after infection with aeromonas salmonicida
         Kotterba G.1, Rebl A.2, Seyfert H.M.2, Burr S.3, Koellner B.1*
             Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany
             Research Institute for the Biology of Farm Animals, Dummerstorf, Germany
             Institute for Veterinary Bacteriology, University Bern, Switzerland
         In order to study the host-pathogen bacterial interaction (Aeromonas salmonicida salmonicida,
         A.s.) the immune response after intraperitoneal (i.p.) immunization was compared with those after
         i.p. infection using a model system where about 50% of A.s. infected trout died. The kinetics of
         different leukocyte populations from lymphatic organs was monitored by flowcytometry using
         monoclonal antibodies against surface markers. After i.p. immunization with A.s. a stimulation of
         predominantly MHC II positive leukocytes (B-cells, monocytes) in the spleen followed by acti-
         vation in the blood was found. In infected fish two different kinetics were seen: a loss of MHC II
         positive leukocytes in moribund fish and a strong activation in surviving fish. The immune mecha-
         nisms that are responsible for survival were studied in a time course experiment. The first results
         reveal a clear correlation between the severity of clinical signs with the expression level of MHC
         II and TLR. In moribund trout this level is markedly decreased compared to uninfected control
         trout. In infected trout without clinical signs and in surviving trout the expression of both MHC II
         and TLR was strongly increased. This reactivity was also found in trout that were infected with a
         non-pathogenic knock-out A.s. mutant (type III secretion system knocked out).

         In-vitro activation of porcine natural killer cells: influence of IL-2, IL-12

         and IL-18 on cytolytic activity and cytokine production
         Pintaric M.*, Gerner W., Saalmueller A.
         Clinical Immunology, University of Veterinary Medicine Vienna, Vienna, Austria
         Natural killer (NK) cells are described as large granular lymphocytes that play an important role
         in the innate immune response against infections and tumor cells. NK cells exert their effector
         functions by cytolytic activity and by producing immune regulatory cytokines and chemokines.
         The existence of NK cells in the swine is well known, but their response following activation by
         cytokines is still poorly understood. Therefore, we tested the influence of the immune regulatory
         cytokines IL-2, IL-12 and IL-18 on the cytolytic activity of peripheral blood mononuclear cells
         (PBMC) as effectors and the human tumor cell line K562 as targets in a flow cytometry based
         assay. Combinations of IL-2/IL-12 and IL-12/IL-18 showed a clear dose-dependent increase of the
         cytolytic response compared to the influence of single cytokines. For phenotypical analyses of the
         cytolytic effector cell population, PBMC were separated by magnetic cell separation (MACS)
         using a cocktail of monoclonal antibodies against porcine CD21, CD3 and SWC3. After stimula-
         tion with the cytokine combinations described above, the enriched CD3-CD21-SWC3- cell frac-
         tion showed an evident increase in the cytolytic activity compared to PBMC. This enhanced cyto-
         lytic activity was accompanied by an enrichment of IFN-γ producing cells in the respective cell
         fraction, which was determined with ELISPOT assays. The experiments performed clearly indi-
         cate a stimulatory role of the investigated cytokines in the activation of porcine NK cells in-vitro.
         Also, strong synergistic effects of the different cytokine combinations were obvious. Further stu-
         dies will address the role of NK cells and lymphokine activated killer cells in viral infections.

Pattern recognition receptor expression in normal ovine tissues, DC and

PBL subsets
Nalubamba K.S.1,2, Gossner A.1, Dalziel R.1, Hopkins J.1
    University of Edinburgh, Edinburgh, United Kingdom
    The University of Zambia, Lusaka, Zambia
The innate immune system senses pathogens via germ-line encoded pattern recognition receptors
(PRRs). These include Toll-like receptors (TLRs) and C-type lectins. Each is able to distinguish a
range of microbial signatures or pathogen associated molecular patterns (PAMPs) and can induce
both adaptive and innate, pathogen-tailored responses. The repertoire of different PRRs expressed
by each cell type has an important bearing on the ability to recognize a specific array of PAMPs
on a pathogen and mount a response against such challenge. The cellular content of a tissue thus
has a bearing on its ability to mount a competent response against pathogens. A discernment of
the expression patterns of PRRs in normal cells/tissues will enable us to understand the signifi-
cance of these receptors in the development of the response and form a basis for understanding
disease pathogenesis. Baseline data also allows deviations from normal to be identified. Ovine
PRRs were cloned and sequenced and qRT-PCR assays developed for each of the 10 TLRs as well
as dectin-1 and 2, CARD15 (NOD2) and the adaptor molecule MyD88. Different peripheral blood
leukocytes subsets (CD4+, CD8+ and γ δ T cells, B cells and monocytes) were purified by FACS.
The two DC subsets, obtained by pseudoafferent lymphatic cannulation, were FACS-purified
based on expression of CD172a. The spleen, lung and lymph nodes express all TLRs; the kidney
expresses high levels of TLR1, 2, 3, 4, 5, 6, but very low levels of TLR8, 9 and 10. The skin
expresses low levels of TLR5, 6, 9 and 10 mRNA. CD172a+ DC express TLR3, 4 and 9 while
CD172a- DC are TLR3-.

Pattern recognition receptor mRNA expression in ovine paratberculosis

(Johne’s Disease)
Nalubamba K.S.1,2, Smeed J.1, Gossner A.1, Dalziel R.1, Hopkins J.1
    University of Edinburgh, Edinburgh, United Kingdom
    The University of Zambia, Lusaka, Zambia
Johne's Disease is a chronic fulminating mycobacteriosis of ruminants caused by Mycobacterium
avium paratuberculosis (Map) characterized by weight loss and diarrhoea. It leads to severe eco-
nomic loss worldwide, and treatment is generally ineffective. There are three pathological forms
of disease, asymptomatic, paucibacillary and multibacillary. The innate immune system has a
pivotal role in the control and dissemination in mycobacterial diseases. In the progression of Map
infection in ruminants, the immune response begins with a cytotoxic and pro-inflammatory Th1
activity that is suitable for control of intracellular pathogens such as mycobacteria. However, this
gradually changes to an ineffective Th2-like response, characterized by IgG1 production that
allows for the proliferation of the infection. The precise mechanisms responsible for this shift in
immune responses to mycobacteria are not well defined. We hypothesized that mycobacterium
antigen recognition and processing has a role in the development of the pathological form of
disease. RNA was obtained from RNAlater® stabilized ileum - the target organ for Map infection.
PRR specific mRNA expression was evaluated using qRT-PCR. Our results show the importance
of differential expression of PRRs in Johne's disease pathology. TLR2, TLR8 and CD14 are signi-
ficantly higher in multibacillary form, CARD15 (NOD2), dectin 1 and dectin 2 expression levels
are augmented in both paucibacillary and multibacillary disease forms, with the expression of the
adaptor molecule MyD88 mRNA being significantly higher in paucibacillary form of disease.

         The cDNA and amino acid sequence of equine C1-inhibitor: a key

         inhibitor of inflammation
         Senior M.*, Carter S., Leuwer M., Proudman C.
         University of Liverpool, UK
         Complement system activation occurs in human patients with septic shock and is linked with poor
         outcome. C1-inhibitor (C1-inh), a plasma protein, is a major inhibitor of several pathways invol-
         ved in complement driven inflammation. Colic is a relatively common disease of horses and
         ranges from mild, transient episodes to a life-threatening syndrome that requires surgical inter-
         vention. Many horses with colic have a compromised intestinal mucosal barrier, which results in
         endotoxaemia, a life-threatening disease similar to septic shock. We aim to characterise the role
         of equine C1-inh in colic-derived endotoxaemia. The currently available antibodies against human
         C1-inh show little or no cross reactivity to equine C1-inh. Purified equine C1-inh has yet to be
         extracted from plasma. We aimed to determine the cDNA and amino acid (a.a.) sequence of equi-
         ne C1-inh. We searched unclassified equine RNA sequences (Extended Sequence Tags- ESTs) for
         highly homologous sections to human C1-inh RNA sequence; and from these we designed ‘pri-
         mers’ to use in Polymerase Chain Reaction (PCR) experiments to amplify most of the cDNA
         sequence for equine C1-inh. RNA Ligase Mediated Rapid Amplification of cDNA Ends PCR
         (RLM RACE PCR) was used to determine the 3’ and 5’ ends of the sequence. The full equine C1-
         inh cDNA sequence was analysed to determine the likely a.a. sequence of equine C1-inh. This was
         then compared to the known a.a. sequence of human C1-inh to search for peptide sequences that
         are most likely to be antigenic. The a.a. sequences LIFHVDQPF & LCRMTEDPDLQV have been
         used to custom generate protein fragments of equine C1-inh and antibodies generated. These anti-
         bodies will be tested against the natural protein and if they react will be used to develop an assay
         for equine C1-inh detection in clinical cases.

         Recognition of lipopolysaccharide (LPS) by bovine cells as assessed by

         cells stably expressing bovine TLR4 and MD-2
         Sauter K.-S., Brcic M., Franchini M., Jungi T.W.*
         Institute of Veterinary Virology, University of Bern, Bern, Switzerland
         The reaction of the innate immune system to Gram-negative bacteria is a cardinal host defense
         mechanism of mammals. However, Gram-negative bacteria or lipopolysaccharide (LPS) derived
         from them may result in septic shock and death. In either case, this is due to interaction of LPS
         and the innate immune system, e.g. macrophages. LPS interacts with TLR4 and MD-2, assisted
         by cellular or soluble CD14 and LPS binding protein (LBP). We previously showed that the res-
         ponse of human and bovine macrophages to LPS is serum-independent. Antibodies blocking the
         binding of LPS-FITC to bovine monocytes blocked the activation by LPS of human but not bovi-
         ne macrophages. To unravel the role of TLR4, MD-2, CD14, and LBP in LPS recognition by bovi-
         ne macrophages, stable HEK293 cells generated overexpressing bovine TLR4 with or without
         bovine MD-2. Such lines were assessed for LPS recognition, using interleukin-8 (IL-8) produc-
         tion and NFkB activation as readouts. This was compared with bovine macrophages, using TNF
         production and NO synthesis as readouts. The results of these experiments will be presented. (1)
         They led to the identification of monoclonal antibodies reacting with bovine TLR4 and blocking
         the interaction of LPS with HEK293 cells overexpressing bovine TLR4 and MD-2. (2) The role
         and origin of bovine TLR4, bovine MD-2, CD14 and LBP can now be appreciated for LPS-sti-
         mulated bovine macrophages. (3) Certain LPS types and other molecules are agonists and anta-
         gonists of human and mouse TLR4/MD-2 complexes, respectively; the specificity pattern of catt-
         le TLR4 and MD-2 is delineated here. Collectively, the study is a step forward in understanding
         the molecular mechanisms of LPS recognition by bovine mononuclear phagocytes.

Early protection (5 days) against PRV infection in swine by DNA vaccines

Dory D.*, Torché A.M., Béven V., Jestin A.
French Food Safety Agency (Afssa), Unit of Viral Genetics and Biosafety, 22440, Ploufragan,
In order to measure the efficacy of a DNA vaccine against Pseudorabies virus (PRV) infection,
which is responsible for the Aujeszky's disease, pigs are usually challenged 21 days after injection
of plasmids in a standard protocol. At this time point, the adaptative immune response is assessed
in a challenge model. One potential application of DNA vaccination is to induce a protective immu-
nity in animals earlier. These potentialities could be used in case of infectious disease outbreak.
Aim: The aim of the present study was to analyse the immune responses and to measure the pro-
tection potential achieved 5 days after a single injection of the DNA vaccine.
Method: Pigs were intramuscularly vaccinated with the DNA vaccine composed of plasmids enco-
ding for PRV-gB, -gC and -gD 5 days before an intranasal challenge with a virulent PRV strain,
and the results were compared to those vaccinated 21 days before challenge.
Results: - No pig survived PRV-infection in the non-vaccinated control groups. Two out of 5 pigs
and 3 out of 6 pigs survived PRV infection in the groups vaccinated 5 days and 21 days before
challenge, respectively. - In the group vaccinated 5 days before challenge no reduction of nasal
viral excretion was observed, whereas a 1.5 log reduction of nasal viral excretion was observed in
the other vaccinated group. - No antibodies against PRV were detected in the serum 5 days after
the injection of the plasmids; PRV ab were detected in animals vaccinated 21 days before the ana-
lyses. - Isolated PBMC incubated with PRV produced IFN-γ at 5 and 21 days after injection of the
Conclusion: Cellular-mediated protection against PRV-infection was achieved 5 days after DNA

Comparison of local and systemic cellular responses of cattle

experimentaly infected or vaccinated with Mycobacterium avium
subspecies paratuberculosis
Marché S.1, Govaerts M.1, Godfroid J.2, Rosseels V.3, Huygen K.3, Walravens K.1*
    CERVA-CODA, Brussels, Belgium
    University of Pretoria, Onderstepoort, South Africa
    Wetenschappelijk Instituut voor Volksgezondheid- Pasteur Institute, Brussels, Belgium
The control of cattle paratuberculosis by vaccination is presently hampered by the lack of perfor-
mance of the vaccines for the control of Mycobacterium avium subsp. paratuberculosis (Map)
infection. Therefore, there is a need to better understand local immune parameters that should be
induced to improve the control of bacterial growth at the site of infection. In this study, we com-
pared the mucosal cellular responses induced after Map vaccination or experimental infection.
Lymphocytes prepared from peripheral blood, mesenteric lymph nodes (MLN), lamina propria
(LP) and intraepithelial lymphocytes (IEL) were stimulated in vitro with different mycobacterial
antigens and IFNγ responses were measured in 20-h and 5-day cultures. Avian Purified Protein
Derivative (PPD-A) specific systemic IFNγ responses were recorded at 20 h in immunized and
infected animals. In the same stimulation conditions, no or weak responses were observed in
MLN, LP and IEL preparations. In 5-day cultures from LP, IEL, similar IFNγ responses were
detected in some vaccinated and infected animals following PPD-A stimulation. Strong local IFNγ
responses were recorded in negative control animals as well as in vaccinated or infected animals
after stimulation with other mycobacterial antigens, suggesting that strong local sensitization of
the immune system occurs probably through the contact with environmental mycobacteria. In
conclusion, no difference in the local IFNγ responses was observed between animals experimen-
tally infected with Map or vaccinated with Map by the SC route.
         A role for bovine NK cells in defence against infection

         Storset A.K.1*, Boysen P.1, Olsen I.2, Klevar S.2
             Norwegian School of Veterinary Science, Oslo, Norway
             National Veterinary Institute, Oslo, Norway
         NK cells have been assigned a critical role in the defence against intracellular pathogens in
         humans and rodents. Following infection, APCs recognize pathogens through pattern recognition
         receptors, leading to IL-12 and IL-18 secretion, which potently trigger IFN gamma production
         from NK cells. The IFN gamma both serves to stimulate pathogen elimination by macrophages,
         but also to direct a TH1-differentiation of CD4+ T-cells. These mechanisms have directed the
         focus on NK cells as regulators and effectors of immunity to infection. The characterization of
         bovine NK cells and the identification of a marker that defines most or all bovine NK cells have
         lead to the possibility to study the role of these cells in infections. Through studies of IL-2 expan-
         ded bovine NK cells in vitro, we have found a direct stimulation of NK cells by Neospora cani-
         num tachyzoites which was not dependent of IL-12 but was increased by the addition of this cyto-
         kine. The stimulated NK cells produced IFN gamma and killed N. caninum infected fibroblasts.
         This shows that in addition to the mechanisms described above, bovine NK cells may also be
         directly stimulated by pathogens. In an experimental infection of calves with N. caninum, the per-
         centage of NK cells in blood dropped at day 4-6 after i.v. inoculation of N. caninum tachyzoites.
         This drop was followed by an increase of NK cells in blood until about day 12 post infection. For
         the first time we demonstrate NK-cells to be primary responders in N. caninum infection of calves.

         Ex vivo biological activity of different types of CpG ODN sequences on

         different swine subset leucocytes
         El Garch H.1, Guzylack L.2, Summerfield A.2, Bautista E., Golde W.3, Audonnet J.C.1,
         Andreoni C.1, Juillard V.1
           Merial SAS, Lyon, France, Discovery Research, 254 rue Marcel Mérieux, 69342 Cedex 07, France
           Institute of Virology and Immunoprophylaxis (IVI), Postfach, CH-3147 Mittelhausern,
           Plum Island Animal Disease Center, Foot-and-Mouth Disease Unit PIADC, ARS, USDA P.O. Box
         848 Greenport, NY 11944, USA
         CpG oligonucleotides (ODNs) stimulate cells that express Toll-like receptor 9 (TLR-9), initiating
         an immunomodulatory cascade resulting in the activation of B and T lymphocytes, natural killer
         cells, monocytes, macrophages and dendritic cells. Different types of CpGs have been characteri-
         zed depending on their ex vivo activity and referred to as A-, B- and C-class ODNs. These diffe-
         rent classes of ODNs induce different post-TLR-9 signaling pathways that result in different bio-
         logical activities. We compared the activity of three distinct CpG ODN sequences on different
         subsets of swine leucocytes. One well-known A-class (2216) and two others (2142 and 2143),
         which sequences were predicting a B-class ODN activity, were selected. We observed that the A-
         class ODN 2216 sequence was a strong inducer of IFNa by pig PBMCs, as well as by freshly iso-
         lated plasmacytoid dendritic cells (pDCs). Both predicted B-class CpG ODNs were shown to effi-
         ciently activate B cells. Interestingly, the 2142 sequence also induced a strong IFNα secretion by
         pDCs. These results indicated that this predicted B-class ODN had a broad biological activity,
         similar to the one described for the palindrome bearing C-class ODNs which combine the effects
         of A- and B-class CpG ODNs. Interestingly, the 2142 CpG ODN was the only ODN sequence with
         the capacity to activate skin DCs. Altogether, these data highlight the interest of the 2142 sequen-
         ce as a potential CpG ODN candidate for swine.


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