Search

Here we investigate the faecal microbiome of wild European badgers Meles meles using samples collected at post-mortem as part of the All Wales Badger Found Dead study. This is the first published characterisation of the badger microbiome. We initially undertook a sex-matched age comparison between the adult and cub microbiomes, based on sequencing the V3–V4 region of the 16S rRNA gene. Analysis used the QIIME 2 pipeline utilising DADA2 and the Silva database for taxonomy assignment. Fusobacteria appeared to be more abundant in the microbiomes of the cubs than the adults although no significant difference was seen in alpha or beta diversity between the adult and cub badger microbiomes. Comparisons were also made against other wild, omnivorous, mammals’ faecal microbiomes using publicly available data. Significant differences were seen in both alpha and beta diversity between the microbiomes from different species. As a wildlife species of interest to the disease bovine tuberculosis, knowledge of the faecal microbiome could assist in identification of infected badgers. Our work here suggests that, if comparisons were made between the faeces of bTB infected and non-infected badgers, age may not have a significant impact on the microbiome.

Introduction The European badger (Meles meles) is a known wildlife reservoir for bovine tuberculosis (bTB) and a better understanding of the epidemiology of bTB in this wildlife species is required for disease control in both wild and farmed animals. Flow infusion electrospray—high-resolution mass spectrometry (FIE-HRMS) may potentially identify novel metabolite biomarkers based on which new, rapid, and sensitive point of care tests for bTB infection could be developed. Objectives In this foundational study, we engaged on assessing the baseline metabolomic variation in the non-bTB infected badger population (“metabotyping”) across Wales. Methods FIE-HRMS was applied on thoracic fluid samples obtained by post-mortem of bTB negative badgers (n = 285) which were part of the Welsh Government ‘All Wales Badger Found Dead’ study. Results Using principal component analysis and partial least squares—discriminant analyses, the major sources of variation were linked to sex, and to a much lesser extent age, as indicated by tooth wear. Within the female population, variation was seen between lactating and non-lactating individuals. No significant variation linked to the presence of bite wounds, obvious lymphatic lesions or geographical region of origin was observed. Conclusion Future metabolomic work when making comparisons between bTB infected and non-infected badger samples will only need be sex-matched and could focus on males only, to avoid lactation bias.

Mycobacterium bovis is the main cause of bovine tuberculosis (BTB) in cattle and can also infect humans. Zebu cattle are considered more resistant to some infectious diseases compared with Holstein-Friesian (HF) cattle, including BTB. However, epidemiological studies may not take into account usage differences of the two types of cattle. HF cattle may suffer greater metabolic stress due to their more or less exclusive dairy use, whereas Zebu cattle are mainly used for beef production. In experiments conducted so far, the number of animals has been too small to draw statistically robust conclusions on the resistance differences between these cattle breeds. Here, we used a BCG challenge model to compare the ability of naïve and vaccinated Zebu and HF cattle to control/kill mycobacteria. Young cattle of both breeds with similar ages were housed in the same accommodation for the duration of the experiment. After correcting for multiple comparisons, we found no difference between naïve HF and Zebu (ρ = 0.862) cattle. However, there was a trend for vaccinated HF cattle to have lower cfu numbers than non-vaccinated HF cattle (ρ = 0.057); no such trend was observed between vaccinated and non-vaccinated Zebu cattle (ρ = 0.560). Evaluation of antigen-specific IFNγ secretion by PBMC indicated that Zebu and HF cattle differed in their response to mycobacteria. Thus, whilst there may be difference in immune responses, our data indicate that with the number of animals included in the study and under the conditions used in this work, we were unable to measure any differences between Zebu and HF cattle in the overall control of mycobacteria. Whilst determination of different susceptibilities between Zebu and HF cattle using the BCG challenge model will require larger numbers of animals than the number of animals used in this experiment, these data should inform future experiments.

ObjectivesImproved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response ofMycobacterium bovis-infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood—an important component of current bTB diagnostics—will provide new information for development of better diagnostics.MethodsRNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (-1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-weekM. bovisinfection time course experiment with ten age-matched Holstein-Friesian cattle.ResultsIn vitro PPD-b stimulation of peripheral blood fromM. bovis-infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters.ConclusionsA transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers ofM. bovisinfection.

Here we investigate the faecal microbiome of wild European badgers Meles meles using samples collected at post-mortem as part of the All Wales Badger Found Dead study based on sequencing the V3-V4 region of the 16S rRNA gene. This is the first published characterisation of the badger microbiome. We initially undertook a sex-matched age comparison between the adult and cub microbiomes. Analysis used the QIIME 2 pipeline utilising DADA2 and the Silva database for taxonomy assignment. Fusobacteria appeared to be more abundant in the microbiomes of the cubs than the adults although no significant difference was seen in alpha or beta diversity between the adult and cub badger microbiomes. Comparisons were also made against other wild, omnivorous, mammals’ faecal microbiomes using publicly available data. Significant differences were seen in both alpha and beta diversity between the microbiomes from different species. As a wildlife species of interest to the disease bovine tuberculosis, knowledge of the faecal microbiome could assist in identification of infected badgers. Our work here suggests that if comparisons were made between the faeces of bTB infected and non-infected badgers, its possible age may not have a significant impact on the microbiome.

Introduction Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB) in cattle, represents a major disease burden to UK cattle farming, with considerable costs associated with its control. The European badger (Meles meles) is a known wildlife reservoir for bTB and better knowledge of the epidemiology of bTB through testing wildlife is required for disease control. Current tests available for the diagnosis of bTB in badgers are limited by cost, processing time or sensitivities. Materials and Methods We assessed the ability of flow infusion electrospray—high-resolution mass spectrometry (FIE-HRMS) to determine potential differences between infected and non-infected badgers based on thoracic blood samples obtained from badgers found dead in Wales. Thoracic blood samples were autoclaved for handling in a containment level 2 (CL2) hazard laboratory. Results Here we show the major differences associated with with M. bovis infection were changes to folate, pyrimidine, histidine, glycerophospholipid and phosphonate metabolism. Conclusions Our studies have indicated differences in the metabolomic signature of badgers found dead in relation to their infection status, suggesting metabolomics could hold potential for developing novel diagnostics for bTB in badgers. As well as highlighting a potential way to handle samples containing a highly pathogenic agent at CL2 for metabolomics studies.

Members of the Mycobacterium tuberculosis complex (MTBC) show distinct host adaptations, preferences and phenotypes despite being >99% identical at the nucleic acid level. Previous studies have explored gene expression changes between the members, however few studies have probed differences in gene essentiality. To better understand the functional impacts of the nucleic acid differences between Mycobacterium bovis and Mycobacterium tuberculosis we used the Mycomar T7 phagemid delivery system to generate whole genome transposon libraries in laboratory strains of both species and compared the essentiality status of genes during growth under identical in vitro conditions. Libraries contained insertions in 54% of possible TA sites in M. bovis and 40% of those present in M. tuberculosis, achieving similar saturation levels to those previously reported for the MTBC. The distributions of essentiality across the functional categories were similar in both species. 527 genes were found to be essential in M. bovis whereas 477 genes were essential in M. tuberculosis and 370 essential genes were common in both species. CRISPRi was successfully utilised in both species to determine the impacts of silencing genes including wag31, a gene involved in peptidoglycan synthesis and Rv2182c/Mb2204c, a gene involved in glycerophospholipid metabolism. We observed species specific differences in the response to gene silencing, with the inhibition of expression of Mb2204c in M. bovis showing significantly less growth impact than silencing its ortholog (Rv2182c) in M. tuberculosis. Given that glycerophospholipid metabolism is a validated pathway for antimicrobials, our observations suggest that target vulnerability in the animal adapted lineages cannot be assumed to be the same as the human counterpart. This is of relevance for zoonotic tuberculosis as it implies that the development of antimicrobials targeting the human adapted lineage might not necessarily be effective against the animal adapted lineage. The generation of a transposon library and the first reported utilisation of CRISPRi in M. bovis will enable the use of these tools to further probe the genetic basis of survival under disease relevant conditions.

In the absence of biomarkers of protective immunity, newly developed vaccines against bovine tuberculosis need to be evaluated in virulent Mycobacterium bovis challenge experiments, which require the use of expensive and highly in demand Biological Safety Level 3 (BSL3) animal facilities. The recently developed bovine BCG challenge model offers a cheaper and faster way to test new vaccine candidates and additionally reduces the severity of the challenge compared to virulent M. bovis challenge in line with the remits of the NC3Rs. In this work we sought to establish the sensitivity of the BCG challenge model by testing a prime boost vaccine regimen that previously increased protection over BCG alone against M. bovis challenge. All animals, except the control group, were vaccinated subcutaneously with BCG Danish, and half of those were then boosted with a recombinant adenoviral vector expressing Antigen 85A, Ad85A. All animals were challenged with BCG Tokyo into the prescapular lymph node and the bacterial load within the lymph nodes was established. All vaccinated animals, independent of the vaccination regimen, cleared BCG significantly faster from the lymph node than control animals, suggesting a protective effect. There was however, no difference between the BCG and the BCG-Ad85A regimens. Additionally, we analysed humoral and cellular immune responses taken prior to challenge for possible predictors of protection. Cultured ELISpot identified significantly higher IFN-ɣ responses in protected vaccinated animals, relative to controls, but not in unprotected vaccinated animals. Furthermore, a trend for protected animals to produce more IFN-ɣ by quantitative PCR and intracellular staining was observed. Thus, this model can also be an attractive alternative to M. bovis challenge models for the discovery of protective biomarkers.

Improved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response of Mycobacterium bovis-infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood-an important component of current bTB diagnostics-will provide new information for development of better diagnostics.RNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (-1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-week M. bovis infection time course experiment with ten age-matched Holstein-Friesian cattle.In vitro PPD-b stimulation of peripheral blood from M. bovis-infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters.A transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers of M. bovis infection.

The "One world, one health" initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirtythree goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG. © 2013 Pérez de Val et al.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. However, the host and pathogen determinants driving host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed both blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated cells (MNCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multi-nucleation. Importantly, we extend our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNCs. Our findings implicate MNC formation in the contrasting pathology between Mtb and Mbv for the bovine host, and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.

Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naive and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naive cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines.

Vaccination has been proposed as a supplementary tool for the control of tuberculosis in livestock. The long-term immunogenicity elicited by bacillus Calmette&ndash, Guerin (BCG) and the efficacy of revaccination were investigated in thirty goat kids distributed into three groups: unvaccinated controls, BCG (vaccinated at week 0) and BCG-BCG (vaccinated at weeks 0 and 56). Sixty-four weeks after the first vaccination, all animals were challenged with Mycobacterium caprae and examined post-mortem (pathology and bacterial load) at week 73. Antigen-specific interferon-gamma (IFN-&gamma, ) release was measured throughout the experiment. At week 59, peripheral blood mononuclear cells were stained for CD4, CD45RO and IFN-&gamma, to determine the presence of antigen-specific cells secreting IFN-&gamma, The BCG-BCG group showed reductions in rectal temperatures, M. caprae DNA load in pulmonary lymph nodes (LN), the volume of lesions in pulmonary LN, mineralization in lungs, and higher weight gains compared to unvaccinated controls. IFN-&gamma, responses were undetectable from 32 weeks after primary vaccination until revaccination, when the BCG-BCG group showed detectable IFN-&gamma, production and a greater percentage of antigen-specific CD4+CD45RO+IFN&gamma, + and CD4-CD45RO+IFN&gamma, + cells compared to the BCG and control groups, which may be an indicator of the mechanisms of protection. Thus, re-vaccination of goats with BCG appears to prolong protection against infection with M. caprae.

Bovine tuberculosis (BTB) caused by Mycobacterium bovis remains a major problem in both the developed and developing countries. Control of BTB in the UK is carried out by test and slaughter of infected animals, based primarily on the tuberculin skin test (PPD). Vaccination with the attenuated strain of the M. bovis pathogen, BCG, is not used to control bovine tuberculosis in cattle at present, due to its variable efficacy and because it interferes with the PPD test. Diagnostic tests capable of Differentiating Infected from Vaccinated Animals (DIVA) have been developed that detect immune responses to M. bovis antigens absent in BCG; but these are too expensive and insufficiently sensitive to be used for BTB control worldwide. To address these problems we aimed to generate a synergistic vaccine and diagnostic approach that would permit the vaccination of cattle without interfering with the conventional PPD-based surveillance. The approach was to widen the pool of M. bovis antigens that could be used as DIVA targets, by identifying antigenic proteins that could be deleted from BCG without affecting the persistence and protective efficacy of the vaccine in cattle. Using transposon mutagenesis we identified genes that were essential and those that were non-essential for persistence in bovine lymph nodes. We then inactivated selected immunogenic, but non-essential genes in BCG Danish to create a diagnostic-compatible triple knock-out ΔBCG TK strain. The protective efficacy of the ΔBCG TK was tested in guinea pigs experimentally infected with M. bovis by aerosol and found to be equivalent to wild-type BCG. A complementary diagnostic skin test was developed with the antigenic proteins encoded by the deleted genes which did not cross-react in vaccinated or in uninfected guinea pigs. This study demonstrates the functionality of a new and improved BCG strain which retains its protective efficacy but is diagnostically compatible with a novel DIVA skin test that could be implemented in control programmes.

Highlights • BCG-primed, Ad5-85A boosted cattle present Ag85A specific mycobacteriocidal T cells. • Along with mycobacteriocidal activity Ad5-85A boosting induces IL-10 transcription. • Ad5-85A boosting does not alter the transcription pattern of pro-inflammatory cytokines., There is a need to improve the efficacy of the BCG vaccine against human and bovine tuberculosis. Previous data showed that boosting bacilli Calmette-Guerin (BCG)-vaccinated cattle with a recombinant attenuated human type 5 adenovirally vectored subunit vaccine (Ad5-85A) increased BCG protection and was associated with increased frequency of Ag85A-specific CD4+ T cells post-boosting. Here, the capacity of Ag85A-specific CD4+ T cell lines – derived before and after viral boosting – to interact with BCG-infected macrophages was evaluated. No difference before and after boosting was found in the capacity of these Ag85A-specific CD4+ T cell lines to restrict mycobacterial growth, but the secretion of IL-10 in vitro post-boost increased significantly. Furthermore, cell lines derived post-boost had no statistically significant difference in the secretion of pro-inflammatory cytokines (IL-1β, IL-12, IFNγ or TNFα) compared to pre-boost lines. In conclusion, the protection associated with the increased number of Ag85A-specific CD4+ T cells restricting mycobacterial growth may be associated with anti-inflammatory properties to limit immune-pathology.

Bovine tuberculosis (bTB), mainly caused by Mycobacterium bovis (M. bovis), is a major economic disease of livestock worldwide. Vaccination is considered as a potentially sustainable adjunct to the current control strategy. Cattle vaccination with the live attenuated M. bovis bacillus Calmette-Guerin (BCG) confers variable protection; the reasons for this variability are not understood. Indoleamine 2, 3-dioxygenase (IDO), through the catalysis of tryptophan, is thought to have an immunoregulatory role in the immune response to Mycobacterium tuberculosis (M. tuberculosis). In this work, we used immunohistochemistry and digital image analysis to evaluate the presence of IDO in granulomas at different stages of development in cattle that had been BCG-vaccinated or not and then challenged with M. bovis. Our results show that the expression of IDO in granulomas from non-vaccinated M. bovis challenged animals is higher than in granulomas from BCG-vaccinated M. bovis challenged animals. Thus, it is possible that vaccination with BCG prevents the induction of what are thought to be host immunosuppressive pathways by M. bovis, which contribute to pathology during the disease.

The Mycobacterium tuberculosis complex (MTBC) is the collective term given to the group of bacteria that cause tuberculosis (TB) in mammals. It has been reported that M. tuberculosis H37Rv, a standard reference MTBC strain, is attenuated in cattle compared to Mycobacterium bovis. However, as M. tuberculosis H37Rv was isolated in the early 1930s, and genetic variants are known to exist, we sought to revisit this question of attenuation of M. tuberculosis for cattle by performing a bovine experimental infection with a recent M. tuberculosis isolate. Here we report infection of cattle using M. bovis AF2122/97, M. tuberculosis H37Rv, and M. tuberculosis BTB1558, the latter isolated in 2008 during a TB surveillance project in Ethiopian cattle. We show that both M. tuberculosis strains caused reduced gross pathology and histopathology in cattle compared to M. bovis. Using M. tuberculosis H37Rv and M. bovis AF2122/97 as the extremes in terms of infection outcome, we used RNA-Seq analysis to explore differences in the peripheral response to infection as a route to identify biomarkers of progressive disease in contrast to a more quiescent, latent infection. Our work shows the attenuation of M. tuberculosis strains for cattle, and emphasizes the potential of the bovine model as a ‘One Health’ approach to inform human TB biomarker development and post-exposure vaccine development.

Human tuberculosis remains a significant cause of mortality and morbidity throughout the world. The global economic impact of bovine TB is considerable. An effective vaccine would be the most cost-effective way to control both epidemics, particularly in emerging economies. TB vaccine research would benefit from the identification of an immune correlate of protection with which vaccines could be gated at both preclinical and clinical levels. In-vitro mycobacterial growth inhibition assays (MGIA) are functional assays that include most aspects of the complex host immune response to mycobacteria, and they may serve as functional immune correlates for vaccine development. We applied to cattle an MGIA that was developed for use with human and murine samples. Several technical difficulties were encountered while transferring it to the cattle model. However, our data demonstrate that the assay was not discriminatory in cattle and further work is needed before using it for bovine TB vaccine development.

There is an urgent need to identify additional diagnostic biomarkers for bovine TB to complement existing read-out systems such as interferon-gamma and for predictive markers of vaccine efficacy to accelerate vaccine development. To evaluate the potential of miRNAs as such biomarkers, we have analysed their expression in bovine PPD stimulated PBMC isolated from unvaccinated and BCG vaccinated cattle before and following Mycobacterium bovis (M. bovis) infection. Using a bovine microRNA microarray, miR-155 was found to show a significant up-regulation in expression in early (week 2) and late (week 11) M. bovis post-infection samples from unvaccinated cattle, while in BCG vaccinated cattle up-regulation was observed only in late post-infection samples. No differential expression of miR-155 was observed in pre-infection samples from unvaccinated and vaccinated cattle. These observations suggest that miR-155 could be exploited as a marker distinguishing vaccinated from infected animals (DIVA). Analysis by TaqMan RT-PCR, verified the up-regulation of miR-155 in unvaccinated cattle post-infection. Significant correlation was found between the degree of pathology and miR-155 induction in the experimentally infected cattle, suggesting miR-155 is a biomarker of disease development and/or severity. Induction of miR155 expression in cattle sourced from farms with confirmed bTB that tested positive in the tuberculin skin or interferon-gamma blood test was found to be significantly higher in cattle presenting with more advanced pathology (defined by the presence of visible TB lesions) compared to infected cattle without visible pathology and thus likely to be of lower infectivity than those with more advanced disease. In conclusion, our data indicate that miR-155 has potential both as a diagnostic and prognostic biomarker that could be used to identify animals with advanced pathology and as a DIVA test read-out. Its role in the immune biology of bovine TB will also be discussed.

This study reports the performance of the single intradermal tuberculin (SIT) test and the interferon-gamma (IFN-γ) assay for Mycobacterium bovis in a cattle herd with high prevalence of paratuberculosis (PTB). A total of 58/350 animals were selected for necropsy based on one or more of the following criteria: positive to SIT, IFN-γ, a breeding cow that seroconverted to PTB and showed signs compatible with a wasting disease. Infection status was determined by post mortem diagnostic tests that included histopathology examination, mycobacterial cultures and PCR identification for M. bovis and Mycobacterium avium subsp. paratuberculosis (MAP). In 7/58 animals primary tuberculosis (TB) lesions, affecting only the retropharyngeal and/or mediastinal lymph nodes, were found; 3/7 animals were found SIT positive. PTB was confirmed in 35/58 animals, of which 30 had seroconverted and 14 had typical clinical signs. 45/58 animals were IFN-γ + using the most stringent criterion (cut-off point ≥ 0.05); however, IFN-γ test was only positive in 33 animals when using a higher threshold (cut-off point ≥ 0.1). Three animals co-infected also showed extensive TB and diffuse PTB lesions. These results show that the combined use of SIT and IFN-γ, as interpreted using official guidelines, detected all confirmed cases of TB. Individually, the sensitivity of the SIT was inadequate to diagnose TB-positive animals with an advanced stage of PTB. The large number of IFN-γ + animals with no visible TB lesion could be due, in part, to some protection conferred by prior infection with MAP.

Mycobacterium bovis BCG vaccination sensitizes cattle to bovine tuberculin, which compromises the use of the current bovine tuberculosis (TB) surveillance tests. Although the performance of a blood test (that utilizes antigens expressed by Mycobacterium bovis but not by BCG) capable of discriminating infected from vaccinated animals (DIVA interferon gamma test [DIT]) has been evaluated in naturally infected TB field reactors, there is a need to perform similar analysis in a BCG-vaccinated M. bovis -infected population. Furthermore, we explored different scenarios under which a DIT may be implemented alongside BCG vaccination: (i) serial testing to resolve potential false-positive skin test results or (ii) a standalone test to replace the single intradermal comparative cervical tuberculin (SICCT) skin test. Our results demonstrated significantly better relative test sensitivity when the DIT was evaluated in a serial test scenario. Direct comparison of pre- and post-skin test blood samples revealed that the SICCT test induced significant boosting of the gamma interferon response in M. bovis -infected animals to both the ESAT-6–CFP-10 and Rv3615c peptide cocktails that comprise the DIT, which persisted for the ESAT-6–CFP-10 reagent for at least 14 days. Importantly, no similar boosting effects were observed in noninfected BCG vaccinates, suggesting that DIVA blood testing after a recent skin test would have minimal impact on test specificity.

Summary Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2+ CD25lo NK cells were the predominant subset of NK cells within the blood. In contrast, CD2− CD25hi NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2− NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2− subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection.

Highlights • Recovery of BCG from bovine tissues after vaccination. • Potential novel routes of inoculation. • Development of a BCG “challenge” model for the testing of vaccine candidates against bovine TB., Vaccination is being considered as part of a sustainable strategy for the control of bovine tuberculosis (BTB) in the UK. The live attenuated Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been used experimentally to vaccinate cattle against BTB. However, BCG confers partial protection against BTB and therefore, there is a need to develop improved vaccines. BTB vaccine efficacy experiments require the use of biosafety level 3 facilities which are expensive to maintain, generally oversubscribed and represent a bottle neck for the testing of vaccine candidates. One indicator of the induction of protective responses would be the ability of the host's immune response to control/kill mycobacteria. In this work we have evaluated an intranodal BCG challenge for the selection of vaccine candidates at biosafety level 2 which are capable of inducing mycobactericidal responses. To our knowledge, this is the first such report. Whilst BCG only confers partial protection, it is still the standard against which other vaccines are judged. Therefore we tested the BCG intranodal challenge in BCG (Danish strain) vaccinated cattle and showed that vaccinated cattle had lower BCG cfu counts than naïve cattle at 14 and 21 days after intranodal challenge with BCG (Tokyo strain). This model could help prioritize competing TB vaccine candidates and exploration of primary and secondary immune responses to mycobacteria.

There is a requirement for vaccines or vaccination strategies that confer better protection against TB than the current live attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine for use in cattle. Boosting with recombinant viral vectors expressing mycobacterial proteins, such as Ag85A, has shown a degree of promise as a strategy for improving on the protection afforded by BCG. Experiments in small animal models have indicated that broadening the immune response to include mycobacterial antigens other than Ag85A, such as Rv0288, induced by boosting with Ad5 constructs has a direct effect on the protection afforded against TB. Here, we compared the immunogenicity and protection against challenge with M. bovis afforded by boosting BCG-vaccinated cattle with a human type 5 (Ad5)-based vaccine expressing the mycobacterial antigens Ag85A (Ad5-85A); or Ag85A, Rv0251, Rv0287 and Rv0288 (Ad5-TBF); or with protein TBF emulsified in adjuvant (Adj-TBF). Boosting with TBF broaden the immune response. The kinetics of Ad5-TBF and Adj-TBF were shown to be different, with effector T cell responses from the latter developing more slowly but being more durable than those induced by Ad5-TBF. No increase in protection compared to BCG alone was afforded by Ad5-TBF or Adj-TBF by gross pathology or bacteriology. Using histopathology, as a novel parameter of protection, we show that boosting BCG vaccinated cattle with Ad5-85A induced significantly better protection than BCG alone. © 2013.

Cattle vaccination against bovine tuberculosis (bTB) has been proposed as a supplementary method to help control the incidences of this disease. Bacillus Calmette-Guérin (BCG) is currently the only viable candidate vaccine for immunisation of cattle against bTB, caused by Mycobacterium bovis (M. bovis). In an attempt to characterise the differences in the immune response following M. bovis infection between BCG vaccinated and non-vaccinated animals, a combination of gross pathology, histopathology and immunohistochemical (IHC) analyses was used. BCG vaccination was found to significantly reduce the number of gross and microscopic lesions present within the lungs and lymph nodes. Additionally, the microscopically visible bacterial load of stage III and IV granulomas was reduced. IHC using cell surfaces markers revealed the number of CD68+ (macrophages), CD3+ (T-lymphocytes) and WC1+ cells (γδ T-cells) to be significantly reduced in lymph node granulomas of BCG vaccinated animals, when compared to non-vaccinated animals. B lymphocytes (CD79a+) were significantly increased in BCG-vaccinated cattle for granulomas at stages II, III and IV. IHC staining for iNOS showed a higher expression in granulomas from BCG vaccinated animals compared to non-vaccinated animals for all stages, being statistically significant in stages I and IV. TGFβ expression decreased alongside the granuloma development in non-vaccinated animals, whereas BCG vaccinated animals showed a slight increase alongside lesion progression. IHC analysis of the cytokines IFN-γ and TNFα demonstrated significantly increased expression within the lymph node granulomas of BCG vaccinated cattle. This is suggestive of a protective role for IFNγ and TNFα in the response to M. bovis infection. Findings shown in this study suggest that the use of BCG vaccine, can reduce the number and severity of lesions , induce a different phenotypic response and increase the local expression of key cytokines related to protection.

Bovine tuberculosis remains a major economic and animal welfare concern worldwide. Cattle vaccination is being considered as part of control strategies. This approach, used alongside conventional control policies, also requires the development of vaccine-compatible diagnostic assays to distinguish vaccinated from infected animals (DIVA). We discuss progress made on optimizing the only potentially available vaccine, bacille Calmette Guérin (BCG), and on strategies to improve BCG efficacy. We also describe recent advances in DIVA development based on the detection of host cellular immune responses by blood-testing or skin-testing approaches. Finally, to accelerate vaccine development, definition of host biomarkers that provide meaningful stage-gating criteria to select vaccine candidates for further testing is highly desirable. Some progress has also been made in this area of research, and we summarize studies that defined either markers predicting vaccine success or markers that correlate with disease stage or severity.

Experiments in small animal models have indicated that intranasal vaccination confers a greater degree of protection against TB than other routes such as intradermal (i.d.) or intramuscular. In this work, using a prime-boost vaccination strategy, we have compared in cattle vaccinated with BCG as a priming vaccine the boosting capabilities of Ad5-85A delivered either via the endobronchial (e.b.) or i.d. route. We show that Ad5-85A delivered through either route induced comparable peripheral blood antigen specific responses, and that both i.d. and e.b. routes induced bronchioalveolar lavage cells (BALC) that produced antigen-specific IFNgamma. We also show that, regardless of the route of boosting, the kinetics of peripheral blood and BALC responses, as assessed by antigen specific IFNgamma production, are different with systemic responses being detectable earlier than mucosal responses. These results contribute to our understanding on how different vaccination strategies may affect different compartments of the immune response and in turn to the development of safer and more effective vaccines.

This is the first efficacy study using the experimental goat model, a natural host of tuberculosis (TB), to evaluate the efficacy of heterologousMycobacterium bovisbacillus Calmette-Guérin (BCG) prime followed by boosting with a replication-deficient adenovirus expressing the antigen Ag85A (AdAg85A). Three experimental groups of 11 goat kids each were used: BCG vaccinated, BCG vaccinated and AdAg85A boosted, and nonvaccinated. Twenty-two goat kids were vaccinated with ∼5 × 105CFU of BCG (week 0), and 11 of them were boosted at week 8 with 109PFU of AdAg85A. At week 14, all goats were challenged by the endobronchial route with ∼1.5 × 103CFU ofMycobacterium caprae. The animals were euthanized at week 28. Cellular and humoral immunity induced by vaccination andM. capraeinfection was measured throughout the study. After challenge BCG-AdAg85A-vaccinated animals exhibited reduced pathology compared to BCG-vaccinated animals in lungs and in pulmonary lymph nodes. There were significant reductions in bacterial load in both groups of vaccinated goats, but the reduction was more pronounced in prime-boosted animals. Antigen-specific gamma interferon (IFN-γ) and humoral responses were identified as prognostic biomarkers of vaccination outcome depending on their correlation with pathological and bacteriological results. As far as we know, this is the first report using multidetector computed tomography (MDCT) to measure vaccine efficacy against pulmonary TB in an animal model. The use in vaccine trials of animals that are natural hosts of TB may improve research into human TB vaccines.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against infection with virulent M. bovis , the causative agent of bovine tuberculosis (bTB). We compared two strains of BCG, Pasteur and Danish, in order to confirm that the current European human vaccine strain (BCG Danish) induced protective immunity in calves, and we assessed immune responses to determine correlates of protection that could assist future vaccine evaluation in cattle. Both vaccine strains induced antigen (purified protein derivate [PPD])-specific gamma interferon (IFN-γ) in whole-blood cultures. These responses were not significantly different for BCG Pasteur and BCG Danish and peaked at week 2 to 4 postvaccination. Vaccination with either BCG Danish or BCG Pasteur induced significant protection against bTB, with reductions in both lesion score and bacteriological burden evident in both groups of vaccinated calves compared with nonvaccinated control calves. Measurement of IFN-γ-expressing T lymphocytes postvaccination and postchallenge revealed both correlates and surrogates of protective efficacy. The frequency of central memory T lymphocytes present at 12 weeks postvaccination (at the time of M. bovis challenge) correlated significantly with protection. Conversely, the number of IFN-γ-expressing effector T cells present after M. bovis challenge was correlated with disease. These results demonstrate that vaccination of neonatal calves with either BCG Pasteur or BCG Danish induces protective immune responses against TB. In addition, we show that measurement of antigen-specific T lymphocyte populations may provide a reliable means for identifying protective vaccine candidates.

EVALUATION OF: Ryan AA, Nambiar JK, Wozniak TM et al. Antigen load governs the differential priming of CD8 T cells in response to the bacille Calmette-Guérin vaccine or Mycobacterium tuberculosis infection. J. Immunol. 182(11), 7172-7177 (2009). Mycobacterium tuberculosis is the causative agent of human TB, which is responsible for 26% of all preventable deaths in the developing world. Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used as a vaccine against TB since 1921, with protection varying from 0 to 80%. Although the reasons for this variability are unclear, protection is thought to be mediated by Th1-type responses. CD8(+) T cells have been shown to play a role in the response to mycobacteria. However, the nature of the CD8(+) response induced by BCG requires further characterization. In the paper being evaluated, it was shown that the antigenic load is important for the induction of CD8(+) T-cell responses and subsequent protective responses. Identification of the factors affecting induction of CD8(+) responses against mycobacteria will facilitate studies to characterize their nature and provide a platform for the development of systems seeking to improve on protection conferred by BCG.

Summary The protective efficacy of Mycobacterium bovis -bacille Calmette Guerin (BCG) against tuberculosis (TB) is variable in both humans and cattle. Exposure to environmental mycobacteria is thought to result in inappropriate priming of host immune responses. To investigate the impact of environmental mycobacteria on BCG efficacy, cattle were infected with M. avium , vaccinated with BCG, challenged with M. bovis and skin tested prior to necropsy. Elevated levels of IFN γ were evident in M. avium -exposed animals before and after BCG vaccination with a bias towards avian purified protein derivative (PPD-A), suggesting that M. avium primed host immune responses. Exposure to M. avium also resulted in a higher frequency of circulatory IFN γ -producing cells in response to PPD antigens at the time of M. bovis challenge. After M. bovis inoculation, the IFN γ response to bovine PPD (PPD-B) increased compared to pre-challenge levels, indicating that all animals had been exposed to M. bovis. Skin test responses indicated 2/6 M. avium -BCG- M. bovis animals as reactors and 2/6 as inconclusive compared with 6/6 BCG- M. bovis animals as reactors. M. avium -exposed animals also had fewer lesions and the number of tissues containing viable M. bovis at post-mortem was significantly lower ( P M. bovis animals), with two of the animals described as skin test negative with no visible lesions or viable bacteria. Thus, exposure of cattle to environmental mycobacteria such as M. avium prior to BCG vaccination did not dampen BCG-specific immune responses and resulted in lower TB pathology. However, the PPD-A bias associated with M. avium exposure is likely to undermine current TB diagnostic tests and the IFN γ test in cattle.

Bovine tuberculosis (bTB) is caused by Mycobacterium bovis. The incidence of bTB is increasing in cattle herds of developed countries that have a wild life reservoir of M. bovis, such as the UK, New Zealand and the USA. The increase in the incidence of bTB is thought to be due, at least in part, to a wildlife reservoir of M. bovis. M. bovis is also capable of infecting humans and on a worldwide basis, M. bovis is thought to account for up to 10% of cases of human TB [Cosivi O, Grange JM, Daborn CJ et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 1998;4(1):59-70]. Thus, the increased incidence of bTB, besides being a major economic problem, poses an increased risk to human health. In the UK, the incidence of bTB continues to rise despite the use of the tuberculin test and slaughter control policy, highlighting the need for improved control strategies. Vaccination of cattle, in combination with more specific and sensitive diagnostic tests, is suggested as the most effective strategy for bovine TB control. The only vaccine currently available for human and bovine TB is the live attenuated Bacille Calmette Guerin (BCG). BCG is thought to confer protection through the induction of Th1 responses against mycobacteria. However, protection against TB conferred by BCG is variable and to this date the reasons for the successes and failures of BCG are not clear. Therefore, there is a need to develop vaccines that confer greater and more consistent protection against bTB than that afforded by BCG. Given that BCG is currently the only licensed vaccine against human TB, it is likely that any new vaccine or vaccination strategy will be based around BCG. In this review we discuss immune responses elicited by mycobacteria in cattle and the novel approaches emerging for the control of bovine TB based on our increasing knowledge of protective immune responses.

The effector mechanisms used by CD4+ T cells to control mycobacteria differ between humans and rodent models of TB and should be investigated in additional animal models. In these studies, the bovine model was used to characterize the mycobactericidal CD4+ T cell response induced by vaccination with the attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG). Antigenic stimulation of peripheral blood CD4+ T cells from BCG-vaccinated cattle enhanced expression of perforin and IFNgamma in cells expressing a CD45RA-CD45RO+CD62L+ cell surface phenotype, enhanced transcription of granulysin, IFNgamma, perforin, IL-4, IL-13, and IL-21, and enhanced anti-mycobacterial activity of CD4+ T cells against BCG-infected macrophages.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB) and of a proportion of human TB. Protection against TB requires Th1 responses and worsening of disease is associated with Th2 responses. To help clarify the nature of the response to mycobacteria, the responses from M. bovis-BCG vaccinated cattle boosted with live mycobacteria (BCG), bacterial soluble antigens (PPD) or PBS were evaluated. The results indicated that macrophages may be the major cell population ingesting and presenting mycobacteria in BCG boosted animals, while B-cells seem able to ingest and present PPD to T-cells in PPD boosted animals.