Cytokine responses to EHV-1 infection in immune and non-immune ponies

Protecting equids against equine herpesvirus-1 (EHV-1) infection remains an elusive goal. Repeated infection with EHV-1 leads to protective immunity against clinical respiratory disease, and a study was conducted to measure the regulatory cytokine response (IFN-gamma and IL-4) in repeatedly infected immune ponies compared to non-immune ponies. Two groups of four ponies were established. Group 1 ponies had previously been infected on two occasions, and most recently 7 months before this study. Group 2 ponies had no history no vaccination or challenge infection prior to this study. Both groups were subjected to an intranasal challenge infection with EHV-1, and blood samples were collected pre-infection, and at 7 and 21 days post-infection for preparation of PBMCs. At each time point, the in vitro responses of PBMCs to stimulation with EHV-1 were measured, including IFN-gamma and IL-4 mRNA production, and lymphoproliferation. Group 1 ponies showed no signs of clinical disease or viral shedding after challenge infection. Group 2 ponies experienced a biphasic pyrexia, mucopurulent nasal discharge, and nasal shedding of virus after infection. Group 1 ponies had an immune response characterized both before and subsequent to challenge infection by an IFN-gamma response to EHV-1 in the absence of an IL-4 response, and demonstrated increased EHV-1-specific lymphoproliferation post-infection. Group 2 ponies had limited cytokine or lymphoproliferative responses to EHV-1 pre-challenge, and demonstrated increases in both IFN-gamma and IL-4 responses post-challenge, but without any lymphoproliferative response. Protective immunity to EHV-1 infection was therefore characterized by a polarized IFN-gamma dependent immunoregulatory cytokine response.     

Equine herpesvirus-1 infected peripheral blood mononuclear cell subpopulations during viremia

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for β-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.     

Determination of equid herpesvirus 1-specific, CD8+, cytotoxic T lymphocyte precursor frequencies in ponies.

The frequency of antigen-specific, genetically restricted cytotoxic T lymphocyte precursors (CTLp) was measured in peripheral blood mononuclear cells (PBMC) of ponies before and after infection with equid herpesvirus 1 (EHV1). Split-well limiting dilution analysis (LDA) was developed to measure CTLp frequency using EHV1-infected 51Cr-labelled lymphoblasts as targets. Extensive characterisation showed that recombinant human interleukin-2, autologous antigen presenting cells and equine serum containing virus neutralising antibody were necessary for maturation of CTLp into effector CTL in vitro. CTLs were not induced when the equine serum (containing VN antibody) was replaced with either foetal calf serum or foetal equine serum (without VN antibody), or seronegative equine serum. CTLp frequency decreased significantly when CD8+ lymphocytes were depleted from the induction cultures. There was good inter- and intra-assay reproducibility using both fresh and recovered cryopreserved PBMC. Both EHV1 and EHV4 could be used to induce effector CTL which lysed EHV1-infected target cells. CTLp frequencies were measured in 2 groups of ponies: Group 1 consisted of two ponies (approx. 9 years old), which had multiple previous experimental infections with EHV1; Group 2 comprised five young (1-2 years) and two older (7 years) ponies which had presumed natural exposure to EHV1/EHV4 but no previous experimental infections. The results showed that CTLp frequencies were higher in the ponies of Group 1 compared with the others. Moreover, ponies with the higher CTLp frequencies were better protected against re-challenge infection with EHV1, showing reduced or absent clinical and virological signs. Consequently, measurement of EHV1-specific CTLp frequency is a potential in vitro correlate of immunity which may be useful for screening new vaccines in horses before embarking upon challenge protection studies to confirm efficacy.     

Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses

Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in supernatants of mitogen stimulated equine peripheral blood mononuclear cells (PBMC). The sensitivity of the ELISA for detecting secreted IL-10 was found to be around 200pg/ml. The production of intracellular IL-10 was measured in equine PBMC by flow cytometry. PBMC were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of the secretion blocker Brefeldin A. All three anti-IL-10 mAbs detected a positive population in PMA stimulated lymphocytes which was absent in the medium controls. Around 80% of the IL-10(+) cells were CD4(+). Another 15% were CD8(+) cells. Double staining with IL-4 or interferon-gamma (IFN-gamma) indicated that PMA and ionomycin stimulation induced 80% IL-10(+)/IFN-gamma(+) lymphocytes, while only 5% IL-10(+)/IL-4(+) cells were observed. By calculation, at least 60% of the IL-10(+)/IFN-gamma(+) cells were CD4(+) lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T(R)1) cell phenotype. In summary, the new mAbs to equine IL-10 detected native equine IL-10 by ELISA and flow cytometry and can be used for further characterization of this important regulatory cytokine in horses.     

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.     

Immunity to equine herpesvirus type 1 (rhinopneumonitis): in vitro lymphocyte response.

Twenty-two ponies were examined for serum-neutralizing (SN) antibody to equine herpesvirus type 1 and for in vitro lymphocyte transformation in the presence of viral antigen. Six ponies had undetectable levels of neutralizing antibody (titer less than 1:2) and had lymphocytes which did not respond in culture with viral antigen (stimulation index less than 2.0). Four ponies which had SN antibody to equine herpesvirus type 1 did not manifest lymphocyte transformation in vitro. The 12 remaining seropositive ponies had lymphocyte transformation with viral antigen in vitro (stimulation indexes from 2.0 to 23.5). Lymphocyte transformation was specifically suppressed when cells were grown in medium containing autologous serum. The temporal development of in vitro lymphocyte responsiveness and SN antibody in 3 specific-pathogenfree ponies after experimentally induced infection with equine herpesvirus type 1 is described.     

The mucosal humoral immune response of the horse to infective challenge and vaccination with equine herpesvirus-1 antigens.

Equine herpesvirus-1 (EHV-1) remains a frequent cause of upper respiratory tract infection and abortion in horses worldwide. However, little is known about the local antibody response elicited in the upper airways of horses following exposure to EHV-1. This study analysed the mucosal humoral immune response of weanling foals following experimental infection with virulent EHV-1, or vaccination with either of 2 commercial vaccines. Twenty weanlings were assigned to 5 groups and were inoculated with, or vaccinated against, EHV-1 following different regimens. Finally, all weanlings were simultaneously challenged intranasally with virulent EHV-1 Army 183 (A183). Nasal wash and serum samples were collected at regular intervals until 13 weeks after final challenge. Nasal washes were assayed for EHV-1-specific equine IgGa, IgGb, IgG(T), IgA, IgM and total virus-specific antibody using an indirect, quantitative ELISA. Total serum antibody responses were also monitored, and clinical signs of EHV-disease were recorded for each individual. Virus-specific IgA dominated the mucosal antibody response elicited in weanlings inoculated with A183, being detectable at up to 3.1 microg/mg total IgA 13 weeks after challenge. Neither inactivated EHV-1 administered i.m., nor attenuated EHV-1 administered intranasally induced detectable mucosal antibodies. EHV-1-specific mucosal antibodies impeded EHV-1 plaque formation in vitro. Such virus-neutralising antibody probably contributes to a reduction of shedding of EHV-1 from the respiratory tract of virus-infected horses.     

The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV)

Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.     

Antibody and cellular immune responses following DNA vaccination and EHV-1 infection of ponies

Equine herpesvirus-1 (EHV-1) is the cause of serious disease with high economic impact on the horse industry, as outbreaks of EHV-1 disease occur every year despite the frequent use of vaccines. Cytotoxic T-lymphocytes (CTLs) are important for protection from primary and reactivating latent EHV-1 infection. DNA vaccination is a powerful technique for stimulating CTLs, and the aim of this study was to assess antibody and cellular immune responses and protection resulting from DNA vaccination of ponies with combinations of EHV-1 genes. Fifteen ponies were divided into three groups of five ponies each. Two vaccination groups were DNA vaccinated on four different occasions with combinations of plasmids encoding the gB, gC, and gD glycoproteins or plasmids encoding the immediate early (IE) and early proteins (UL5) of EHV-1, using the PowderJect XR research device. Total dose of DNA/plasmid/vaccination were 25 microg. A third group comprised unvaccinated control ponies. All ponies were challenge infected with EHV-1 6 weeks after the last vaccination, and protection from clinical disease, viral shedding, and viremia was determined. Virus neutralizing antibodies and isotype specific antibody responses against whole EHV-1 did not increase in either vaccination group in response to vaccination. However, glycoprotein gene vaccinated ponies showed gD and gC specific antibody responses. Vaccination did not affect EHV-1 specific lymphoproliferative or CTL responses. Following challenge infection with EHV-1, ponies in all three groups showed clinical signs of disease. EHV-1 specific CTLs, proliferative responses, and antibody responses increased significantly in all three groups following challenge infection. In summary, particle-mediated EHV-1 DNA vaccination induced limited immune responses and protection. Future vaccination strategies must focus on generating stronger CTL responses.     

Use of DNA and recombinant canarypox viral (ALVAC) vectors for equine herpes virus vaccination

In this study, experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly with one dose of the appropriate test vaccine at an interval of 5 weeks. Two to 3 weeks after the second vaccination, ponies were infected intra-nasally with the virulent Ab4 strain of EHV-1 after which they were observed clinically and sampled for virological investigations. The results demonstrated that DNA and ALVAC vaccination markedly reduced virus excretion after challenge in terms of duration and magnitude, but failed to protect against cell-associated viremia. Noteworthy was the almost complete absence of virus excretion in the group of ponies vaccinated with ALVAC-EHV in the presence of Carbopol adjuvant or DNA plasmid formulated with aluminium phosphate. The administration of the DNA vaccine in the presence of GM-CSF and formulated in DMRIE-DOPE and of the ALVAC vaccine in the presence of Carbopol adjuvant significantly improved virus neutralising antibody responses to EHV-1. These findings indicate that DNA and ALVAC vaccination is a promising approach for the immunological control of EHV-1 infection, but that more research is needed to identify the immunodominant protective antigens of EHV-1 and their interaction with the equine immune system.     

Absence of viral envelope proteins in equine herpesvirus 1-infected blood mononuclear cells during cell-associated viremia

In vitro studies demonstrated that most equine herpesvirus 1 (EHV-1)-infected peripheral blood mononuclear cells (PBMC) do not expose viral envelope proteins on their surface. This protects them against antibody-dependent lysis. We examined whether viral envelope proteins are also undetectable on infected PBMC during cell-associated viremia. Further, surface expression of major histocompatibility complex (MHC)-I was examined, since MHC-I assists in making infected cells recognizable for cytotoxic T-lymphocytes (CTL). Four ponies, previously exposed to EHV, and two ponies that had no contact with EHV before, were inoculated with EHV-1. PBMC were collected at different time points up to 28 days post inoculation. Ninety-eight percent of the infected PBMC did not show viral envelope proteins on their surface. Moreover, infected PBMC without surface expression only produced immediate early and, at least, one early protein, ICP22, but not late envelope proteins gB and gM. This indicates that surface expression of viral envelope proteins is absent, simply because the PBMC are in an early phase of infection. The percentage of infected PBMC showing surface expression of MHC-I was similar as observed in non-infected PBMC from the same ponies (80-100%). Therefore, inefficient recognition of EHV-1-infected PBMC by CTLs does not arise from absent surface expression of MHC-I.     

A monoclonal antibody specific for bovine T cell surface antigen associated with the E-rosette receptor

From mice immunized with T lymphocyte-enriched bovine peripheral blood mononuclear cells (PBMC), a monoclonal antibody termed BLMo-12 was obtained. BLMo-12 reacted with the antigen of Mr 56,000 in lysate of T lymphocytes. This mAb was found to inhibit spontaneous rosette formation by T-bovine lymphocytes with sheep red blood cells but it did not react with B lymphocytes, monocytes, neutrophils or eosinophils. In frozen section of the thymus, BLMo-12 showed a positive staining both the cortex and the medulla. In lymph nodes, the mAb stained the T-dependent paracortex. BLMo-12 reacted with 49.9% of PBMC and 82.5% of thymocytes. Recognition of the bovine homologue of CD2 on the T lymphocyte surface by this mAb was discussed.     

Cytotoxic T lymphocytes in protection against equine infectious anemia virus

Cytotoxic T lymphocytes (CTL) are associated with virus control in horses infected with equine infectious anemia virus (EIAV). Early in infection, control of the initial viremia coincides with the appearance of CTL and occurs before the appearance of neutralizing antibody. In carrier horses, treatment with immunosuppressive drugs results in viremia before a change in serum neutralizing antibody occurs. Clearance of initial viremia caused by other lentiviruses, including human immunodeficiency virus-1 and simian immunodeficiency virus, is also associated with CTL and not neutralizing antibody. In addition, depletion of CD8+ cells prior to infection of rhesus monkeys with simian immunodeficiency prevents clearance of virus and the same treatment of persistently infected monkeys results in viremia. Cats given adoptive transfers of lymphocytes from vaccinated cats were protected and the protection was MHC-restricted, occurred in the absence of antiviral humoral immunity, and correlated with the transfer of cells with feline immunodeficiency virus-specific CTL and T-helper lymphocyte activities. Therefore, a lentiviral vaccine, including one for EIAV, needs to induce CTL. Based on initial failures to induce CTL to EIAV proteins by any means other than infection, we attempted to define an experimental system for the evaluation of methods for CTL induction. CTL epitopes restricted by the ELA-A1 haplotype were identified and the MHC class I molecule presenting these peptides was identified. This was done by expressing individual MHC class I molecules from cDNA clones in target cells. The target cells were then pulsed with peptides and used with effector CTL stimulated with the same peptides. In a preliminary experiment, immunization of three ELA-A1 haplotype horses with an Env peptide restricted by this haplotype resulted in CTL in peripheral blood mononuclear cells (PBMC) which recognized the Env peptide and virus-infected cells, but the CTL response was transient. Nevertheless there was significant protection against clinical disease following EIAV challenge of these immunized horses when compared with three control horses given the same virus challenge. These data indicated that responses to peptides in immunized horses needed to be enhanced. Optimal CTL responses require help from CD4+ T lymphocytes, and experiments were done to identify EIAV peptides which stimulated CD4+ T lymphocytes in PBMC from infected horses with different MHC class II types. Two broadly cross-reactive Gag peptides were identified which stimulated only an interferon gamma response by CD4+ T lymphocytes, which indicated a T helper 1 response is needed for CTL stimulation. Such peptides should facilitate CTL responses; however, other problems in inducing protection against lentiviruses remain, the most significant of them being EIAV variants that can escape both CTL and neutralizing antibody. A possible solution to CTL escape variants is the induction of high-avidity CTL to multiple EIAV epitopes.     

CD4(+) T-cell responses against the VP1-unique region in individuals with recent and persistent parvovirus B19 infection

To date cellular immune responses against parvovirus B19 (B19) have not been studied extensively. The aim of this study was to examine the T-cell response against the VP1-unique region as the immunodominant part of the viral structural protein VP1 in individuals with different courses of B19 infection. Therefore, a group of 13 parvovirus-positive probands was separated into subgroups characterized for recent or acute, past or persistent infection by means of the presence of specific immunoglobulin (Ig)M and IgG isotypes and of viral DNA in blood and tissue. Transiently transfected B-cells expressing VP1-unique region were used in ELISpot assays to investigate T-cell responses directed against the VP1-unique region in peripheral blood mononuclear cells (PBMC) of individual donors. Significant numbers of interferon-gamma (IFN-gamma) secreting lymphocytes were detectable in PBMC of all individuals with recent, acute or persistent B19 infection, but not in PBMC of donors with past B19 infection and seronegative individuals. A more detailed analysis of IFN-gamma producing cells by intracellular cytokine staining by flow cytometry revealed, that CD4(+) T cells but not CD8(+) cytotoxic lymphocytes (CTL) were the major subpopulation of IFN-gamma producing cells. These data strongly suggest the need of virus protein production for the maintenance of VP1-unique region-specific CD4(+) T-helper cell responses in B19-infected individuals.     

Infection of central nervous system endothelial cells by cell-associated EHV-1

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late-term abortions and equine herpesvirus myeloencephalitis (EHM). Our understanding of EHM pathogenesis is limited except for the knowledge that EHV-1 infected, circulating peripheral blood mononuclear cells (PBMC) transport virus to the central nervous system vasculature causing endothelial cell infection leading to development of EHM. Our objective was to develop a model of CNS endothelial cell infection using EHV-1 infected, autologous PBMC. PBMCs, carotid artery and brain endothelial cells (EC) from 14 horses were harvested and grown to confluency. PBMC or ConA-stimulated PBMCs (ConA-PBMCs) were infected with EHV-1, and sedimented directly onto EC monolayers ('contact'), or placed in inserts on a porous membrane above the EC monolayer ('no contact'). Cells were cultured in medium with or without EHV-1 virus neutralizing antibody. Viral infection of ECs was detected by cytopathic effect. Both brain and carotid artery ECs became infected when cultured with EHV-1 infected PBMCs or ConA-PBMCs, either in direct contact or no contact: infection was higher in carotid artery than in brain ECs, and when using ConA-PBMCs compared to PBMCs. Virus neutralizing antibody eliminated infection of ECs in the no contact model only. This was consistent with cell-to-cell spread of EHV-1 infection from leucocytes to ECs, demonstrating the importance of this mode of infection in the presence of antibody, and the utility of this model for study of cellular interactions in EHV-1 infection of ECs.     

Equine herpesvirus-1-specific interferon gamma (IFNgamma) synthesis by peripheral blood mononuclear cells in thoroughbred horses

REASONS FOR PERFORMING STUDY: An assay has been developed that measures EHV-1 specific interferon gamma synthesis (IFNgamma), a cytokine produced following the activation of memory T lymphocytes and therefore a measure of cell mediated immunity. The method requires validation in the field. OBJECTIVES: To measure the frequency of EHV-1 specific, IFNgamma synthesising peripheral blood mononuclear cells (PBMC) in a population of Thoroughbred horses, and examine its relationship with age, gender, premises and history of vaccination or field infection with EHV-1. METHODS: Lymphocytes from 200 Thoroughbred horses were stimulated with EHV-1 in vitro, and IFNgamma detected using a monoclonal antibody and indirect immunofluorescence. Percent positive cells were enumerated by flow cytometric analysis and the results described and compared statistically between groups. RESULTS: The frequency of IFNgamma+ PBMC was significantly higher in animals age >5 years compared with 2-4 years, in females vs. males, on stud farms vs. training yards and following vaccination of 2-year-olds with inactivated virus compared with nonvaccinates. Age strongly confounded all these associations and care must therefore be taken interpreting these results. Mares exposed to a field infection with EHV-1 also had higher frequencies of IFNgamma+ PBMC than other vaccinated horses. CONCLUSIONS: The frequency of EHV-1 specific, IFNgama+ PBMC among the sample Thoroughbred population was diverse but lowest in young, unvaccinated horses-in-training. POTENTIAL RELEVANCE: The frequency of EHV-1 specific lymphocytes synthesising IFNgamma in this population may be associated with its susceptibility to infection with this virus. This easy technique may be applied to monitor the antigenicity of vaccines and their effectiveness at stimulating cellular immunity.     

Antibody and IFN-gamma responses induced by a recombinant canarypox vaccine and challenge infection with equine influenza virus

In horses, equine influenza virus (EIV) is a leading cause of respiratory disease. Conventional inactivated vaccines induce a short-lived immune response. By comparison, natural infection confers a long-term immunity to re-infection. An aim of new equine influenza vaccines is to more closely mimic natural infection in order to achieve a better quality of immunity. A new live recombinant vaccine derived from the canarypox virus vector and expressing haemagglutinin genes of EIV (subtype H3N8) has been developed. Stimulation of the immune system was studied after immunisation with this canarypox-based vaccine and challenge infection by exposure to a nebulised aerosol of EIV. The humoral immune response was evaluated by measuring serum antibody levels using the single radial haemolysis (SRH) assay. The cellular immune response was assessed by the measurement of interferon gamma (IFN-gamma) synthesis in peripheral blood mononuclear cells (PBMC). Clinical signs of the disease (temperature, coughing, nasal discharge, dyspnoea, depression and anorexia) and virus excretion were monitored after challenge infection. Clinical signs and virus shedding were significantly reduced in vaccinates compared with unvaccinated controls. EIV-specific immunity was stimulated by vaccination with a recombinant vaccine as serological responses were detected after immunisation. This study also provided the first evidence for increased IFN-gamma protein synthesis in vaccinated ponies following challenge infection with EIV compared with control ponies.     

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.     

Decreased expression of L-selectin (CD62L) on lymphocytes in enzootic bovine leukaemia

Expression of L-selectin was determined by single- and two-colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L-selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV-infected animals comprised lymphocytotic and non-lymphocytotic cows. L-selectin was expressed on 90-98% of granulocytes in all tested animals. The percentage of PBMC expressing L-selectin was lower in cattle with persistent lymphocytosis than in non-lymphocytotic or BLV-free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L-selectin was significantly decreased from 60.2 +/- 1.9% in BLV-free cattle to 43.8 +/- 3.6 and 22.5 +/- 5.7% in non-lymphocytotic and lymphocytotic cattle, respectively. B-lymphocytes stained for L-selectin exhibited about 50% reduction in L-selectin expression in BLV-infected cattle compared with BLV-free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L-selectin-positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV-free and BLV-infected cattle. However, L-selectin expression on T lymphocytes was reduced (about 50%) in BLV-infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L-selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.