Seroconversion against antigen MPB83 in badgers (Meles meles) vaccinated with multiple doses of BCG strain Sofia.

Serological diagnosis of Mycobacterium bovis infection in badgers (Meles meles) has relied primarily on antibody recognition of MPB83, a sero-dominant antigen of M. bovis. Most vaccine studies in badgers to date have used the Bacille Calmette-Guerin (BCG) Danish strain, a low producer of MPB83. Due to a supply shortage of the BCG Danish strain, the BCG Sofia SL222 strain has been considered as an alternative vaccine. This strain is a high producer of MPB83 raising the possibility that vaccinated animals will test sero-positive in diagnostic assays that use this antigen. In this study we vaccinated a group of eleven badgers with BCG Sofia SL222 by injection via the intramuscular route and a booster vaccine dose was similarly delivered at 12 weeks and 64 weeks. Primary vaccination did not result in measured detection of antibodies against MPB83 in any badger during the first twelve weeks using serum or whole blood tested by the Dual Path Platform (DPP) VetTB, however, MPB83 antibodies were detected in a semi-quantitative ELISA assay. Following delivery of booster BCG at 12 weeks and 64 weeks, antibody responses against MPB83 were recorded in badgers using whole blood and serum on DPP VetTB and by ELISA. At all time points, vaccination was also associated with the in vitro production of gamma interferon (IFN-γ) following stimulation of lymphocytes with bovine and avian tuberculin (PPD) but not with MPB83 or M. bovis specific antigen CFP-10. The results indicate that serological diagnosis of tuberculosis using tests that target MPB83 may be compromised if badgers are repeatedly vaccinated with BCG Sofia.

Protective immunity against tuberculosis in a free-living badger population vaccinated orally with Mycobacterium bovis Bacille Calmette-Guérin.

Vaccination of badgers with Mycobacterium bovis Bacille Calmette-Guérin (BCG) has been shown to protect badgers against tuberculosis in experimental trials. During the 3-year County Kilkenny BCG vaccine field study, badgers were treated orally with placebo (100% in Zone A), BCG (100% in Zone C) or randomly assigned 50%: 50% treatment with BCG or placebo (Zone B). At the end of the study, 275 badgers were removed from the trial area and subjected to detailed post-mortem examination followed by histology and culture for M. bovis. Among these badgers, 83 (30.2%) were captured for the first time across the three zones, representing a non-treated proportion of the population. Analysis of the data based on the infection status of treated animals showed a prevalence of 52% (95% CI: 40%-63%) infection in Zone A (placebo), 39% (95% CI: 17%-64%) in Zone B (placebo) and 44% (95% CI: 20%-70%) in Zone B (BCG vaccinated) and 24% (95% CI: 14%-36%) in Zone C (BCG vaccinated). There were no statistically significant differences in the proportion of animals with infection involving the lung and thoracic lymph nodes, extra-thoracic infection or in the distribution and severity scores of histological lesions. Among the 83 non-treated badgers removed at the end of the study, the infection prevalence of animals in Zone A (prevalence = 46%, 95% CI: 32%-61%) and Zone B (prevalence = 44%, 95% CI: 23%-67%) was similar to the treated animals in these zones. However, in Zone C, no evidence of infection was found in any of the untreated badgers (prevalence = 0%, 95% CI: 0%-14%). This is consistent with an indirect protective effect in the non-vaccinated badgers leading to a high level of population immunity. The results suggest that BCG vaccination of badgers could be a highly effective means of reducing the incidence of tuberculosis in badger populations.

IL-10 suppression of IFN-γ responses in tuberculin-stimulated whole blood from Mycobacterium bovis infected cattle.

The measurement of bovine interferon-gamma (IFN-γ) forms the basis of a diagnostic test for bovine tuberculosis where Mycobacterium bovis sensitised effector T cells produce IFN-γ following in vitro stimulation with tuberculin antigens. In cattle infected with M. bovis it is also known that the anti-inflammatory IL-10 cytokine can inhibit in vitro production of IFN-γ leading to a reduced response in the IFN-γ diagnostic test. In order to investigate this in greater detail, whole blood samples from tuberculin skin test positive and negative cattle were stimulated with bovine and avian tuberculin antigens and in parallel with a neutralising anti-IL-10 monoclonal antibody. The results showed that IFN-γ protein levels increased when IL-10 activity was suppressed by Anti - IL-10. By using a standard diagnostic interpretation, the elevated levels of IFN-γ were shown to change the level of agreement between the performance of the single intradermal comparative tuberculin test (SICTT) and IFN-γ assay, depending on the tuberculin treatment. A transcriptomic analysis using RT-qPCR investigated the influence of IL-10 activity on expression of a suite of cytokine genes (IFNG, IL12B, IL10 and CXCL10) associated with antigen-stimulated production of IFN-γ. The IFNG and IL12B genes both experienced significant increases in expression in the presence of Anti-IL-10, while the expression of IL10 and CXCL10 remained unaffected.

Oral Vaccination of Free-Living Badgers (Meles meles) with Bacille Calmette Guérin (BCG) Vaccine Confers Protection against Tuberculosis.

A field trial was conducted to investigate the impact of oral vaccination of free-living badgers against natural-transmitted Mycobacterium bovis infection. For a period of three years badgers were captured over seven sweeps in three zones and assigned for oral vaccination with a lipid-encapsulated BCG vaccine (Liporale-BCG) or with placebo. Badgers enrolled in Zone A were administered placebo while all badgers enrolled in Zone C were vaccinated with BCG. Badgers enrolled in the middle area, Zone B, were randomly assigned 50:50 for treatment with vaccine or placebo. Treatment in each zone remained blinded until the end of the study period. The outcome of interest was incident cases of tuberculosis measured as time to seroconversion events using the BrockTB Stat-Pak lateral flow serology test, supplemented with post-mortem examination. Among the vaccinated badgers that seroconverted, the median time to seroconversion (413 days) was significantly longer (p = 0.04) when compared with non-vaccinated animals (230 days). Survival analysis (modelling time to seroconversion) revealed that there was a significant difference in the rate of seroconversion between vaccinated and non-vaccinated badgers in Zones A and C throughout the trial period (p = 0.015). For badgers enrolled during sweeps 1-2 the Vaccine Efficacy (VE) determined from hazard rate ratios was 36% (95% CI: -62%- 75%). For badgers enrolled in these zones during sweeps 3-6, the VE was 84% (95% CI: 29%- 97%). This indicated that VE increased with the level of vaccine coverage. Post-mortem examination of badgers at the end of the trial also revealed a significant difference in the proportion of animals presenting with M. bovis culture confirmed lesions in vaccinated Zone C (9%) compared with non-vaccinated Zone A (26%). These results demonstrate that oral BCG vaccination confers protection to badgers and could be used to reduce incident rates in tuberculosis-infected populations of badgers.

Oral vaccination of badgers (Meles meles) against tuberculosis: comparison of the protection generated by BCG vaccine strains Pasteur and Danish.

Vaccination of badgers by the subcutaneous, mucosal and oral routes with the Pasteur strain of Mycobacterium bovis bacille Calmette-Guérin (BCG) has resulted in significant protection against experimental infection with virulent M. bovis. However, as the BCG Danish strain is the only commercially licensed BCG vaccine for use in humans in the European Union it is the vaccine of choice for delivery to badger populations. As all oral vaccination studies in badgers were previously conducted using the BCG Pasteur strain, this study compared protection in badgers following oral vaccination with the Pasteur and the Danish strains. Groups of badgers were vaccinated orally with 10(8) colony forming units (CFU) BCG Danish 1331 (n = 7 badgers) or 10(8) CFU BCG Pasteur 1173P2 (n = 6). Another group (n = 8) served as non-vaccinated controls. At 12 weeks post-vaccination, the animals were challenged by the endobronchial route with 6 × 10(3) CFU M. bovis, and at 15 weeks post-infection, all of the badgers were euthanased. Vaccination with either BCG strain provided protection against challenge compared with controls. The vaccinated badgers had significantly fewer sites with gross pathology and significantly lower gross pathological severity scores, fewer sites with histological lesions and fewer sites of infection, significantly lower bacterial counts in the thoracic lymph node, and lower bacterial counts in the lungs than the control group. No differences were observed between either of the vaccine groups by any of the pathology and bacteriology measures. The ELISPOT analysis, measuring production of badger interferon - gamma (IFN-γ), was also similar across the vaccinated groups.

Trophic enrichment factors for blood serum in the European badger (Meles meles).

Ecologists undertaking stable isotopic analyses of animal diets require trophic enrichment factors (TEFs) for the specific animal tissues that they are studying. Such basic data are available for a small number of species, so values from trophically or phylogenetically similar species are often substituted for missing values. By feeding a controlled diet to captive European badgers (Meles meles) we determined TEFs for carbon and nitrogen in blood serum. TEFs for nitrogen and carbon in blood serum were +3.0 ± 0.4‰ and +0.4 ± 0.1‰ respectively. The TEFs for serum in badgers are notably different from those published for the red fox (Vulpes vulpes). There is currently no data for TEFs in the serum of other mustelid species. Our data show that species sharing similar niches (red fox) do not provide adequate proxy values for TEFs of badgers. Our findings emphasise the importance of having species-specific data when undertaking trophic studies using stable isotope analysis.

Innate gene repression associated with Mycobacterium bovis infection in cattle: toward a gene signature of disease.

BACKGROUND: Bovine tuberculosis is an enduring disease of cattle that has significant repercussions for human health. The advent of high-throughput functional genomics technologies has facilitated large-scale analyses of the immune response to this disease that may ultimately lead to novel diagnostics and therapeutic targets. Analysis of mRNA abundance in peripheral blood mononuclear cells (PBMC) from six Mycobacterium bovis infected cattle and six non-infected controls was performed. A targeted immunospecific bovine cDNA microarray with duplicated spot features representing 1,391 genes was used to test the hypothesis that a distinct gene expression profile may exist in M. bovis infected animals in vivo. RESULTS: In total, 378 gene features were differentially expressed at the P < or = 0.05 level in bovine tuberculosis (BTB)-infected and control animals, of which 244 were expressed at lower levels (65%) in the infected group. Lower relative expression of key innate immune genes, including the Toll-like receptor 2 (TLR2) and TLR4 genes, lack of differential expression of indicator adaptive immune gene transcripts (IFNG, IL2, IL4), and lower BOLA major histocompatibility complex - class I (BOLA) and class II (BOLA-DRA) gene expression was consistent with innate immune gene repression in the BTB-infected animals. Supervised hierarchical cluster analysis and class prediction validation identified a panel of 15 genes predictive of disease status and selected gene transcripts were validated (n = 8 per group) by real time quantitative reverse transcription PCR. CONCLUSION: These results suggest that large-scale expression profiling can identify gene signatures of disease in peripheral blood that can be used to classify animals on the basis of in vivo infection, in the absence of exogenous antigenic stimulation.

Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD).

Microarray analysis of messenger RNA (mRNA) abundance was used to investigate the gene expression program of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis. An immunospecific bovine microarray platform (BOTL-4) with spot features representing 1336 genes was used for transcriptional profiling of PBMC from six M. bovis-infected cattle stimulated in vitro with bovine purified protein derivative of tuberculin (PPD-bovine). Cells were harvested at four time points (3 h, 6 h, 12 h and 24 h post-stimulation) and a split-plot design with pooled samples was used for the microarray experiment to compare gene expression between PPD-bovine stimulated PBMC and unstimulated controls for each time point. Statistical analyses of these data revealed 224 genes (approximately 17% of transcripts on the array) differentially expressed between stimulated and unstimulated PBMC across the 24 h time course (P<0.05). Of the 224 genes, 87 genes were significantly upregulated and 137 genes were significantly downregulated in M. bovis-infected PBMC stimulated with PPD-bovine across the 24 h time course. However, perturbation of the PBMC transcriptome was most apparent at time points 3 h and 12 h post-stimulation, with 81 and 84 genes differentially expressed, respectively. In addition, a more stringent statistical threshold (P<0.01) revealed 35 genes (approximately 3%) that were differentially expressed across the time course. Real-time quantitative reverse transcription PCR (qRT-PCR) of selected genes validated the microarray results and demonstrated a wide range of differentially expressed genes in PPD-bovine-, PPD-avian- and Concanavalin A (ConA) stimulated PBMC, including the interferon-gamma gene (IFNG), which was upregulated in PBMC stimulated with PPD-bovine (40-fold), PPD-avian (10-fold) and ConA (8-fold) after in vitro culture for 12 h. The pattern of expression of these genes in PPD-bovine stimulated PBMC provides the first description of an M. bovis-specific signature of infection that may provide insights into the molecular basis of the host response to infection. Although the present study was carried out with mixed PBMC cell populations, it will guide future studies to dissect immune cell-specific gene expression patterns in response to M. bovis infection.