Antigen-specific CD4+ and CD8+ T-cell responses in PBMC from pony mares immunized with either native or recombinant zona pellucida vaccines.

Few studies have investigated the cell mediated immune response during zona pellucida-based immunocontraception, despite hypothesized cytotoxic T-cell involvement in ovarian dysfunction associated with these vaccines. This study aimed to investigate antigen-specific anamnestic responses of helper (CD4+) and cytotoxic (CD8+) T-lymphocytes in peripheral blood mononuclear cells (PBMC) isolated from pony mares before and after their treatment with native porcine zona pellucida (pZP), recombinant pZP3 and pZP4 antigens (reZP) or adjuvanted saline. Mares were randomly assigned to pZP, reZP and control groups (n = 7 per group). Treatments consisted of a primary vaccination or saline (V1; Day 0) incorporating Freund's modified complete adjuvant, followed by a single booster (V2; Day 35) incorporating Freund's incomplete adjuvant. Peripheral blood mononuclear cells were isolated and cryopreserved immediately prior to V1 (Day 0) and five weeks post V2 (Day 70). Relative proliferation of T-lymphocytes in response to pZP antigen was assessed using carboxyfluorescein diacetate succinimidyl ester dilution with immunophenotyping, analysed via flow cytometry. Significant pZP-specific CD4+ and CD8+ T-lymphocyte responses were detected in PBMC isolated from mares treated with either pZP or reZP, in comparison to pre-treatment samples. In the pZP group, but not the reZP group, CD8+ T-cell proliferation showed significant negative correlations to circulating progesterone, oestradiol and anti-Müllerian hormone levels. Results suggest that antigen-specific CD8+ T-cells may play a role in ovarian suppression observed during pZP immunocontraception in this species.

Immune response profiles of calves following vaccination with live BCG and inactivated Mycobacterium bovis vaccine candidates.

Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlife-livestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4-6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferon-γ responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG. The animals in the heat-killed M. bovis group demonstrated strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heat-killed M. bovis group. In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.

Use of the mice passive protection test to evaluate the humoral response in goats vaccinated with Sterne 34F2 live spore vaccine.

The Sterne live spore vaccine (34F2) is the most widely used veterinary vaccine against anthrax in animals. Antibody responses to several antigens of Bacillus anthracis have been described with a large focus on those against protective antigen (PA). The focus of this study was to evaluate the protective humoral immune response induced by the live spore anthrax vaccine in goats. Boer goats vaccinated twice (week 0 and week 12) with the Sterne live spore vaccine and naive goats were used to monitor the anti-PA and toxin neutralizing antibodies at week 4 and week 17 (after the second vaccine dose) post vaccination. A/J mice were passively immunized with different dilutions of sera from immune and naive goats and then challenged with spores of B. anthracis strain 34F2 to determine the protective capacity of the goat sera. The goat anti-PA ELISA titres indicated significant sero-conversion at week 17 after the second doses of vaccine (p = 0.009). Mice receiving undiluted sera from goats given two doses of vaccine (twice immunized) showed the highest protection (86%) with only 20% of mice receiving 1:1000 diluted sera surviving lethal challenge. The in vitro toxin neutralization assay (TNA) titres correlated to protection of passively immunized A/J mice against lethal infection with the vaccine strain Sterne 34F2 spores using immune goat sera up to a 1:10 dilution (rs ≥ 0.522, p = 0.046). This study suggests that the passive mouse protection model could be potentially used to evaluate the protective immune response in livestock animals vaccinated with the current live vaccine and new vaccines.

Serological response of foals to polyvalent and monovalent live-attenuated African horse sickness virus vaccines.

African horse sickness (AHS) is typically a highly fatal disease in susceptible horses and vaccination is currently used to prevent the occurrence of disease in endemic areas. Similarly, vaccination has been central to the control of incursions of African horse sickness virus (AHSV) into previously unaffected areas and will likely play a significant role in any future incursions. Horses in the AHSV-infected area in South Africa are vaccinated annually with a live-attenuated (modified-live virus [MLV]) vaccine, which includes a cocktail of serotypes 1, 3, 4 (bottle 1) and 2, 6-8 (bottle 2) delivered in two separate doses at least 21 days apart. In this study, the neutralising antibody response of foals immunized with this polyvalent MLV AHSV vaccine was evaluated and compared to the response elicited to monovalent MLV AHSV serotypes. Naïve foals were immunized with either the polyvalent MLV AHSV vaccine, or a combination of monovalent MLV vaccines containing individual AHSV serotypes 1, 4, 7 or 8. There was a marked and consistent difference in the immunogenicity of individual virus serotypes contained in the MLV vaccines. Specifically, foals most consistently seroconverted to AHSV-1 and responses to other serotypes were highly variable, and often weak or not detected. The serotype-specific responses of foals given the monovalent MLV vaccines were similar to those of foals given the polyvalent MLV preparation suggesting that there is no obvious enhanced immune response through the administration of a monovalent vaccine as opposed to the polyvalent vaccine.

Passive transfer and rate of decay of maternal antibody against African horse sickness virus in South African Thoroughbred foals.

REASONS FOR PERFORMING STUDY: African horse sickness is an insect-transmitted, noncontagious disease of equids caused by African horse sickness virus (AHSV). Mortality can exceed 90% in fully susceptible horse populations. A live-attenuated (modified live) cell-culture-adapted (MLV) polyvalent AHSV vaccine is widely used to control African horse sickness in endemic areas in southern Africa. Field studies detailing antibody responses of vaccinated horses are lacking. OBJECTIVES: To determine antibody titres to the 9 known serotypes of AHSV in a cohort of broodmares that were regularly vaccinated with the MLV AHSV vaccine and to measure the passive transfer and rate of decay of maternal antibody to the individual virus serotypes in foals. METHODS: Serum was collected from 15 mares before foaling and from their foals after foaling and monthly thereafter for 6 months. Antibody titres to each of the 9 AHSV serotypes were determined by serum virus neutralisation assay. RESULTS: There was marked variation in the antibody response of the mares to individual AHSV serotypes even after repeated vaccination, with consistently higher titre responses to some virus serotypes. Likewise, the duration of maternally derived antibodies in foals differed among serotypes. CONCLUSIONS: Data from this study confirm variation of the neutralising antibody response of individual mares to repeated vaccination with polyvalent AHSV vaccine. Virus strains of individual AHSV serotypes included in the vaccine may vary in their inherent immunogenicity. Passively acquired maternal antibodies to AHSV vary markedly among foals born to vaccinated mares, with further variation in the duration of passive immunity to individual AHSV serotypes. POTENTIAL RELEVANCE: These data are relevant to the effective utilisation of live-attenuated AHSV vaccines in endemic regions, and potentially to the use of vaccines in response to future incursions of AHSV into previously free regions. Further studies involving a larger population will be required to determine the optimal time for vaccinating foals.

An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses.

A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from <10 to 40) 8 weeks after vaccination leading us to hypothesize that there could be a participation of cell mediated immunity (CMI) in protection against AHSV4. The present study aimed at characterizing the CMI induced by the experimental ALVAC(®)-AHSV4 vaccine. Six horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(®)-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(®)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-γ responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4.

A competitive ELISA for the detection of group-specific antibody to equine encephalosis virus.

A polyclonal antibody-based, group-specific, competitive ELISA (C-ELISA) for the detection of antibodies to equine encephalosis virus (EEV) was developed. The assay measures the competition between a specific guinea pig antiserum and a test serum, for a pre-titrated EEV antigen. The C-ELISA detected antibodies to the seven known EEV serotypes. Reference antisera raised against other arboviruses did not cross react with EEV antigen. Negative sera from horses in the United Kingdom were used to establish the baseline for a negative population. Negative and positive populations of South African horses, selected on the basis of virus neutralisation were assayed subsequently. Optimal test parameters, where sensitivity≅specificity≅100%, were calculated by two-graph receiver operator characteristic (TG-ROC) analysis to be at a cut-off value of 29.5% inhibition. Results show the EEV C-ELISA described to be sensitive, specific and reliable. Used in conjunction with ELISAs available for African horse sickness virus (AHSV), differential serological diagnosis between EEV and AHSV can be achieved.

Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay.

The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra) and domestic animal (cattle, sheep and goat) populations was studied in wildlife/livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA), using a recombinant antigen (MSP-5) from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA), as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers.

Experimental reproduction of severe bluetongue in sheep.

Sheep inoculated with a virulent South African strain of bluetongue (BT) virus serotype 4 developed severe clinical signs and lesions characteristic of fulminant BT, including coronitis, hemorrhage and ulceration of the mucosal lining of the oral cavity and forestomaches, hemorrhage in the wall of the pulmonary artery, and focally extensive necrosis of skeletal muscle, especially of the neck. At necropsy, up to 14 days after infection, the infected sheep exhibited striking pulmonary edema, edema of the subcutaneous tissues and fascial planes of the head and neck, and pleural and pericardial effusion of varying severity. A reliable model for experimental reproduction of fulminant BT in sheep will facilitate future studies to better characterize the pathogenesis of this disease, particularly as it regards the mechanisms responsible for the increased vascular permeability that characterizes BT and related orbiviral diseases such as African horse sickness.

A potential krimpsiekte vaccine.

Krimpsiekte, a chronic form of cardiac glycoside poisoning, is an important plant-induced intoxication of small stock in South Africa. It is caused by cumulative, neurotoxic bufadienolides, such as cotyledoside. A cotyledoside-bovine serum albumin conjugate was synthesized to immunize animals. The efficacy of the cotyledoside-conjugate in inducing an immunological response was ascertained in rabbits (n = 4) and sheep (n = 4) by determining cotyledoside antibody titres with an ELISA using cotyledoside-hen ovalbumin as antigen. The formation of anticotyledoside antibodies was induced in both rabbits and sheep following immunization with the cotyledoside-protein conjugate. Protection provided by the vaccine was demonstrated by challenging sheep (n = 4) with repeated, daily doses of cotyledoside (0.015 mg/kg) administered intravenously, commencing 45 days after the initial vaccination. One control animal died on Day 3 of the challenge period and the other was severely affected after administration of the third cotyledoside dose. The immunized ewes (n = 2) remained clinically unaffected and the challenge was suspended following six daily injections. Vaccination as a means of preventing krimpsiekte seems to be quite feasible and deserves further investigation.

A group-specific, indirect sandwich ELISA for the detection of equine encephalosis virus antigen.

A polyclonal antibody-based, group-specific, indirect, sandwich ELISA (S-ELISA) for the detection of equine encephalosis virus (EEV) antigen was developed. Purified EEV particles were titrated in the S-ELISA and the limit of detection was determined to be approximately 9.0 ng of antigen/ml (0.45 ng/well). Positive S-ELISA reactions were recorded with seven serologically distinct EEV serotypes. No cross-reactions were recorded with other arboviruses including African horse sickness virus (AHSV) serotypes 1-9, bluetongue serotypes 1-24, epizootic haemorrhagic disease serotypes 1-8 and isolate 318, and selected isolates of Palyam, Eubenangee, Corriparta, Warrego, Akabane and bovine ephemeral fever viruses. The assay proved to be sensitive and specific for the rapid detection of EEV in cell cultures and in homogenated suckling mouse brain (MB). The data generated in this study suggest that the ELISA will be valuable for epidemiological studies of EE and will assist in making a reliable differential diagnosis between EEV and AHSV infections.