Determination of intracellular cytokines IFN-γ and IL-4 in canine T lymphocytes by flow cytometry following whole-blood culture

This report describes a whole-blood flow cytometric method for the determination of intracellular cytokines IFN-γ and IL-4 in canine T lymphocyte subpopulations. Conjugated anti-cytokine antibodies and commercially available reagents for cell fixation and permeabilization were used. Canine peripheral blood was cultured with a combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to promote cytokine synthesis in each cell, along with monensin to increase the sensitivity of the method by retaining IFN-γ and IL-4 within the cell to detectable levels. The optimum concentrations of PMA and ionomycin were determined. Maximum IFN-γ expression from both CD4+ and CD8+ T lymphocytes was detected after 6 h of incubation of cell culture, while maximum IL-4 production took 6 h from CD4+ cells and 4 h from CD8+ cells. This method is a simple immunologic technique for measuring intracellular cytokines which could be of value in the investigation of canine immunological response mainly in various intracellular and extracellular infections, since IFN-γ and IL-4 are considered key cytokines activating the cellular and humoral immunity, respectively.     

Magnitude and kinetics of multifunctional CD4+ and CD8β+ T cells in pigs infected with swine influenza A virus

Although swine are natural hosts for influenza A viruses, the porcine T-cell response to swine influenza A virus (FLUAVsw) infection has been poorly characterized so far. We have studied Ki-67 expression and FLUAVsw-specific production of IFN-γ, TNF-α and IL-2 in CD4⁺ and CD8β⁺ T cells isolated from piglets that had been intratracheally infected with a H1N2 FLUAVsw isolate. IFN-γ⁺TNF-α⁺IL-2⁺ multifunctional CD4⁺ T cells were present in the blood of all infected animals at one or two weeks after primary infection and their frequency increased in four out of six animals after homologous secondary infection. These cells produced higher amounts of IFN-γ, TNF-α and IL-2 than did CD4⁺ T cells that only produced a single cytokine. The vast majority of cytokine-producing CD4⁺ T cells expressed CD8α, a marker associated with activation and memory formation in porcine CD4⁺ T cells. Analysis of CD27 expression suggested that FLUAVsw-specific CD4⁺ T cells included both central memory and effector memory populations. Three out of six animals showed a strong increase of Ki-67⁺perforin⁺ CD8β⁺ T cells in blood one week post infection. Blood-derived FLUAVsw-specific CD8β⁺ T cells could be identified after an in vitro expansion phase and were multifunctional in terms of CD107a expression and co-production of IFN-γ and TNF-α. These data show that multifunctional T cells are generated in response to FLUAVsw infection of pigs, supporting the idea that T cells contribute to the efficient control of infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0182-3) contains supplementary material, which is available to authorized users.     

Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαβ⁺CD8⁺IFN-γ⁺ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαβ⁺CD4⁺ T cells were also found to increase in the infected mice, however later than CD8⁺ T cells. Interestingly, splenic and MLN CD4⁺CD25⁺ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαβ⁺CD8⁺ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.     

Immunity,safety and protection of an Adenovirus 5 prime - Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves

Vaccination is the most cost effective control measure for Johne’s disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4⁺, CD8⁺ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1β and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0112-9) contains supplementary material, which is available to authorized users.     

Memory T cell proliferative responses and IFN-γ productivity sustain long-lasting efficacy of a Cap-based PCV2 vaccine upon PCV2 natural infection and associated disease

Porcine circovirus type 2 (PCV2) vaccination represents an important measure to cope with PCV2 infection; however, data regarding the modulation of the immune cell compartment are still limited, especially under field conditions. This study is aimed at investigating the features of the cellular immune response in conventional piglets induced by vaccination using a capsid (Cap) protein-based PCV2 vaccine compared to unvaccinated animals when exposed to PCV2 natural infection. Immune reactivity was evaluated by quantifying peripheral cell subsets involved in the anti-viral response and characterizing the interferon-gamma (IFN-γ) secreting cell (SC) responsiveness both in vivo and upon in vitro whole PCV2 recall. The vaccination triggered an early and intense IFN-γ secreting cell response and induced the activation of peripheral lymphocytes. The early increase of IFN-γ SC frequencies resulted in a remarkable and transient tendency to increased IFN-γ productivity in vaccinated pigs. In vaccinated animals, soon before the onset of infection occurred 15-16 weeks post-vaccination, the recalled PCV2-specific immune response was characterized by moderate PCV2-specific IFN-γ secreting cell frequencies and augmented productivity together with reactive CD4⁺CD8⁺ memory T cells. Conversely, upon infection, unvaccinated animals showed very high frequencies of IFN-γ secreting cells and a tendency to lower productivity, which paralleled with effector CD4^–CD8⁺ cytotoxic cell responsiveness. The study shows that PCV2 vaccination induces a long-lasting immunity sustained by memory T cells and IFN-γ secreting cells that potentially played a role in preventing the onset of infection; the extent and duration of this reactivity can be an important feature for evaluating the protective immunity induced by vaccination.     

CD27 expression discriminates porcine T helper cells with functionally distinct properties

Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we identified a mAb specific for porcine CD27 and showed that CD27 is expressed by all naïve CD8α^- T helper cells but divides CD8α⁺ T helper cells into a CD27⁺ and a CD27^- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were performed. Naïve CD8α^-CD27⁺ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8α⁺CD27⁺ and CD8α⁺CD27^- T helper cells were found in blood, spleen and liver. Both CD8α⁺CD27⁺ and CD8α⁺CD27^- T helper cells were capable of producing IFN-γ upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8α^-CD27⁺, CD8α⁺CD27⁺ and CD8α⁺CD27^- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN-γ and TNF-α production in the CD8α⁺CD27^- subset. Therefore, these cells resembled terminally differentiated effector memory cells as described in human. This was supported by analyses of CCR7 and CD62L expression. CD8α⁺CD27^- T helper cells were mostly CCR7^- and had considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8α⁺CD27⁺ T helper cells, which also had a proliferation rate similar to naïve CD8α^-CD27⁺ T helper cells and showed intermediate levels of cytokine production. Therefore, similar to human, CD8α⁺CD27⁺ T helper cells displayed a phenotype and functional properties of central memory cells.     

Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L)

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 10^7.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.     

Impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infections are characterized by prolonged viremia and viral shedding consistent with incomplete immunity. Type I interferons (IFN) are essential for mounting efficient antiviral innate and adaptive immune responses, but in a recent study, North American PRRSV genotype 2 isolates did not induce, or even strongly inhibited, IFN-α in plasmacytoid dendritic cells (pDC), representing “professional IFN-α-producing cells”. Since inhibition of IFN-α expression might initiate PRRSV pathogenesis, we further characterized PRRSV effects and host modifying factors on IFN-α responses of pDC. Surprisingly, a variety of type 1 and type 2 PRRSV directly stimulated IFN-α secretion by pDC. The effect did not require live virus and was mediated through the TLR7 pathway. Furthermore, both IFN-γ and IL-4 significantly enhanced the pDC production of IFN-α in response to PRRSV exposure. PRRSV inhibition of IFN-α responses from enriched pDC stimulated by CpG oligodeoxynucleotides was weak or absent. VR-2332, the prototype genotype 2 PRRSV, only suppressed the responses by 34%, and the highest level of suppression (51%) was induced by a Chinese highly pathogenic PRRSV isolate. Taken together, these findings demonstrate that pDC respond to PRRSV and suggest that suppressive activities on pDC, if any, are moderate and strain-dependent. Thus, pDC may be a source of systemic IFN-α responses reported in PRRSV-infected animals, further contributing to the puzzling immunopathogenesis of PRRS.     

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining

Background

The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49–96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-β], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed.

Results

Eleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-β] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-β] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies.

Conclusion

The identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.     

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.     

The effects of protein level on cytokines and chemokines in the uterine environment of beef heifers during development

The development of replacement heifers is crucial for breeding success and herd efficiency. Nutritional management can affect not only reproductive development but also the inflammatory status of the uterine environment, which may impact reproductive functions such as pregnancy establishment and development. The study herein evaluated the concentration of cytokines and chemokines in the uterus of heifers supplemented with different levels of protein. Angus heifers (n = 60) were blocked by body weight (BW) and randomly assigned to 1 of 3 treatments based on protein supplementation level: control of 10% crude protein (CON), 20% crude protein (P20), or 40% crude protein (P40). BW, body condition score, and blood samples were taken every 2 wk for 140 d to monitor development. Uterine flushes were performed monthly and concentrations of cytokines (IL-1α, IL-1β, TNF-α, IFN-γ, IL-10, VEGF-α, IL-17A, and IL-36RA) and chemokines (IL-8, MCP-1, MIP-1α, and MIP-1β) were quantified via ELISA multiplex. To test if there were mean differences in cytokines between the treatment groups or over time, PROC GLIMMIX (SAS v 9.4) was utilized. Concentrations of all cytokines and chemokines, except IL-1α, changed throughout heifer development (P < 0.05). Heifers in the P40 treatment group displayed reduced concentrations of MCP-1 (P = 0.007) and tended to have decreased concentrations of IFN-γ (P = 0.06). Cytokine IL-36RA tended (P = 0.06) to be affected by protein level, with the lowest concentrations observed in CON heifers. Most cytokines and chemokines increased following the initial month of supplementation (P < 0.05). The increase in concentrations after 1 mo may indicate an adaptive response in the uterus to diet change. Cytokines and chemokines fluctuated due to physiological changes occurring during development. Further research is needed to determine the influence of nutrition on uterine inflammation and long-term impacts on reproductive function.     

In vitro effects of meloxicam on the number,Foxp3 expression,production of selected cytokines,and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

The purpose of this study was to evaluate the effect of meloxicam (MEL) on selected immune parameters of bovine CD25^highCD4+, CD25^lowCD4+, and CD25-CD4+ cells. Peripheral blood mononuclear cells (PBMCs) collected from 12-month-old heifers were treated with MEL at a concentration corresponding to the serum level of this medication following administration at the recommended dose (MEL 5 × 10^-6 M) and at a concentration 10 times lower (MEL 5 × 10^-7 M). After 12 and 24 h of incubation with the drug, the percentage of CD25^highCD4+ cells decreased; however, this disturbance was quickly reversed. Furthermore, the absolute number of CD25^highCD4+ cells in the PBMC populations treated with MEL 5 × 10^-6 M for 48 and 168 h was increased. Prolonged (168 h) exposure to the drug increased the percentage of Foxp3+ cells in the CD25^highCD4+ cell subpopulation. The higher dose of MEL was found to significantly increase the percentage of IFN-γ+ cells among the CD25-CD4+ cells. These results indicated that MEL does not exert an immunosuppressive effect by depleting CD4+ cells and suppression of IFN-γ+ production by these cells. Furthermore, IL-10 and TGF-β production was not changed following exposure to MEL.     

Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein N^pro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE^-, N^pro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE^- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE^- vaccine virus in pigs, the IRF3-degrading function of N^pro was not recovered. Therefore, we examined whether restoring the ability of N^pro to block IFN-α/β induction of both the avirulent and moderately virulent GPE^--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in N^pro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional N^pro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional N^pro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.     

Immune Efficacy of different immunization doses of divalent combination DNA vaccine pOPRL+pOPRF of Pseudomonas aeruginosa

At present, there is no vaccine available against Pseudomonas aeruginosa, a common zoonotic pathogenic bacterium. In a previous study, the authors prepared a divalent combination DNA vaccine, pOPRL+pOPRF, which exhibited good protective efficacy. To explore the optimal immunization dose of this divalent combination DNA vaccine, in the present study, chickens were vaccinated with 25, 50, 100, and 200 µg doses. The levels of serum antibody, interferon-γ (IFN-γ), and interleukin-2 (IL-2) were determined, and lymphocyte proliferation assays were performed. After challenge with virulent P. aeruginosa, the protective efficacy was evaluated. Following vaccination, the serum antibodies, stimulation index values, and concentrations of IFN-γ and IL-2 were significantly higher in chickens vaccinated with 100 and 200 µg vaccines than in those vaccinated with 25 and 50 µg doses (P<0.05). IFN-γ and IL-2 concentrations in chickens immunized with 100 µg vaccine were slightly higher than those in chickens immunized with 200 µg vaccine, although the difference was not statistically significant. The protective rates were 55%, 65%, 85%, and 85% with 25, 50, 100, and 200 µg of the pOPRL+pOPRF DNA vaccine, respectively. Thus, the immune efficacy of the pOPRL+pOPRF DNA vaccine increased with an increase in immunization dose, but this does not imply that a higher dose necessarily achieves a better outcome. The optimal immunization dose of pOPRL+pOPRF DNA vaccine in chickens was 100 µg.     

Blood concentrations of the cytokines IL-1beta,IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery

Background

Knowledge of the cytokine response at infection with Brachyspira hyodysenteriae can help understanding disease mechanisme involved during swine dysentery. Since this knowledge is still limited the aim of the present study was to induce dysentery experimentally in pigs and to monitor the development of important immunoregulatory cytokines in blood collected at various stages of the disease.

Methods

Ten conventional pigs (~23 kg) were orally inoculated with Brachyspira hyodysenteriae B204^T. Eight animals developed muco-haemorrhagic diarrhoea with impaired general body condition. Blood was sampled before inoculation and repeatedly during acute dysentery and recovery periods and cytokine levels of IL-1β, IL-6, Il-10, TNF-α and IFN-γ were measured by ELISA.

Results

IL-1β was increased at the beginning of the dysentery period and coincided with the appearance of Serum amyloid A and clinical signs of disease. TNF-α increased in all animals after inoculation, with a peak during dysentery, and IL-6 was found in 3 animals during dysentery and in the 2 animals that did not develop clinical signs of disease. IL-10 was found in all sick animals during the recovery period. IFN-γ was not detected on any occasion.

Conclusion

B. hyodysenteriae inoculation induced production of systemic levels of IL-1β during the dysentery period and increased levels of IL-10 coincided with recovery from dysentery.     

Ovine and murine T cell epitopes from the non-structural protein 1 (NS1) of bluetongue virus serotype 8 (BTV-8) are shared among viral serotypes

Bluetongue virus (BTV) is a non-enveloped dsRNA virus that causes a haemorrhagic disease mainly in sheep. It is an economically important Orbivirus of the Reoviridae family. In order to estimate the importance of T cell responses during BTV infection, it is essential to identify the epitopes targeted by the immune system. In the present work, we selected potential T cell epitopes (3 MHC-class II-binding and 8 MHC-class I binding peptides) for the C57BL/6 mouse strain from the BTV-8 non-structural protein NS1, using H2^b-binding predictive algorithms. Peptide binding assays confirmed all MHC-class I predicted peptides bound MHC-class I molecules. The immunogenicity of these 11 predicted peptides was then determined using splenocytes from BTV-8-inoculated C57BL/6 mice. Four MHC-class I binding peptides elicited specific IFN-γ production and generated cytotoxic T lymphocytes (CTL) in BTV-8 infected mice. CTL specific for 2 of these peptides were also able to recognise target cells infected with different BTV serotypes. Similarly, using a combination of IFN-γ ELISPOT, intracellular cytokine staining and proliferation assays, two MHC-class II peptides were identified as CD4⁺ T cell epitopes in BTV-8 infected mice. Importantly, two peptides were also consistently immunogenic in sheep infected with BTV-8 using IFN-γ ELISPOT assays. Both of these peptides stimulated CD4⁺ T cells that cross-reacted with other BTV serotypes. The characterisation of these T cell epitopes can help develop vaccines protecting against a broad spectrum of BTV serotypes and differentiate infected from vaccinated animals.     

Study of the local immune response to Fasciola hepatica in the liver and hepatic lymph nodes of goats immunised with a peptide of the Sm14 antigen

The nature of the local immune response was assessed studying the distribution of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes, IgM⁺ B cells, IL-4⁺ and IFN-γ⁺ cells in the liver and hepatic lymph nodes (HLN) of goats immunised with a synthetic peptide of the Sm14 antigen from Schistosoma mansoni and challenged with Fasciola hepatica. A morphometric study of HLN was also carried out in order to evaluate the hyperplasia of lymphoid follicles. Despite the decrease in fluke burdens found in the immunised group (45.9%) respect to the infected control group, this difference was not statistically significant due to the high individual variability. In liver, a significant increase of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes was found in the infected groups respect to the uninfected control and in the infected control respect to the immunised group. HLN showed a significant enlargement due to the hyperplasia of lymphoid follicles and infiltration of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes in both infected groups respect to the uninfected control, with no significant differences between the infected control and immunised group. IFN-γ⁺ lymphoid cells was absent or very occasional in HLN where the number of IL-4⁺ cells was higher than that of IFN-γ, suggesting a polarized Th2 response in immunised and in infected control group.     

The innate immune response of equine bronchial epithelial cells is altered by training

Respiratory diseases, including inflammatory airway disease (IAD), viral and bacterial infections, are common problems in exercising horses. The airway epithelium constitutes a major physical barrier against airborne infections and plays an essential role in the lung innate immune response mainly through toll-like receptor (TLR) activation. The aim of this study was to develop a model for the culture of equine bronchial epithelial cells (EBEC) in vitro and to explore EBEC innate immune responses in trained horses. Bronchial epithelial biopsies were taken from 6 adult horses during lower airway endoscopy. EBEC were grown in vitro by an explant method. The innate immune response of EBEC was evaluated in vitro by treatment with TLR ligands. TLR3 is the most strongly expressed TLR at the mRNA level in EBEC and stimulation of EBEC with Poly(I:C), an analog of viral dsRNA, triggers a strong secretion of IFN-β, TNF-α, IL-6 and CXCL8. We further evaluated the EBEC innate immune response in horses that underwent a 4-month-training program. While training had no effect on TLR mRNA expression in EBEC as well as in bronchial biopsies, it increased the production of IFN-β after stimulation with a TLR3 ligand and decreased the secretion of TNF-α and IL-6 after stimulation with a TLR2 and TLR3 ligand. These findings may be implicated in the increased risk for viral and bacterial infections observed in sport horses. Altogether, we report a successful model for the culture of EBEC that can be applied to the investigation of pathophysiologic conditions in longitudinal studies.