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Abstract Background Bovine tuberculosis (bTB) is a chronic disease that results from infection with any member of the Mycobacterium tuberculosis complex. Infected animals are typically diagnosed with tuberculin-based intradermal skin tests according to World Organization of Animal Health which are presently in use. However, tuberculin is not suitable for use in BCG-vaccinated animals due to a high rate of false-positive reactions. Peptide-based defined skin test (DST) antigens have been identified using antigens (ESAT-6, CFP-10 and Rv3615c) which are absent from BCG, but their performance in buffaloes remains unknown. To assess the comparative performance of DST with the tuberculin-based single intradermal test (SIT) and the single intradermal comparative cervical test (SICCT), we screened 543 female buffaloes from 49 organized dairy farms in two districts of Haryana state in India. Results We found that 37 (7%), 4 (1%) and 18 (3%) buffaloes were reactors with the SIT, SICCT and DST tests, respectively. Of the 37 SIT reactors, four were positive with SICCT and 12 were positive with the DST. The results show that none of the animals tested positive with all three tests, and 6 DST positive animals were SIT negative. Together, a total of 43 animals were reactors with SIT, DST, or both, and the two assays showed moderate agreement (Cohen’s Kappa 0.41; 95% Confidence Interval (CI): 0.23, 0.59). In contrast, only slight agreement (Cohen’s Kappa 0.18; 95% CI: 0.02, 0.34) was observed between SIT and SICCT. Using a Bayesian latent class model, we estimated test specificities of 96.5% (95% CI, 92–99%), 99.7% (95% CI: 98–100%) and 99.0% (95% CI: 97–100%) for SIT, SICCT and DST, respectively, but considerably lower sensitivities of 58% (95% CI: 35–87%), 9% (95% CI: 3–21%), and 34% (95% CI: 18–55%) albeit with broad and overlapping credible intervals. Conclusion Taken together, our investigation suggests that DST has a test specificity comparable with SICCT, and sensitivity intermediate between SIT and SICCT for the identification of buffaloes suspected of tuberculosis. Our study highlights an urgent need for future well-powered trials with detailed necropsy, with immunological and microbiological profiling of reactor and non-reactor animals to better define the underlying factors for the large observed discrepancies in assay performance, particularly between SIT and SICCT.

BackgroundTuberculosis (TB) is a major cause of ill health and one of the leading causes of death worldwide, caused by species of the Mycobacterium tuberculosis complex (MTBC), with Mycobacterium tuberculosis being the dominant pathogen in humans and Mycobacterium bovis in cattle. Zoonotic transmission of TB (zTB) to humans is frequent particularly where TB prevalence is high in cattle. In this study, we explored the prevalence of zTB in central Ethiopia, an area highly affected by bovine TB (bTB) in cattle.MethodA convenient sample of 385 patients with pulmonary tuberculosis (PTB, N = 287) and tuberculous lymphadenitis (TBLN, N = 98) were included in this cross-sectional study in central Ethiopia. Sputum and fine needle aspirate (FNA) samples were obtained from patients with PTB and TBLN, respectively, and cultures were performed using BACTEC™ MGIT™ 960. All culture positive samples were subjected to quantitative PCR (qPCR) assays, targeting IS1081, RD9 and RD4 genomic regions for detection of MTBC, M. tuberculosis and M. bovis, respectively.ResultsTwo hundred and fifty-five out of 385 sampled patients were culture positive and all were isolates identified as MTBC by being positive for the IS1081 assay. Among them, 249 (97.6%) samples had also a positive RD9 result (intact RD9 locus) and were consequently classified as M. tuberculosis. The remaining six (2.4%) isolates were RD4 deficient and thereby classified as M. bovis. Five out of these six M. bovis strains originated from PTB patients whereas one was isolated from a TBLN patient. Occupational risk and the widespread consumption of raw animal products were identified as potential sources of M. bovis infection in humans, and the isolation of M. bovis from PTB patients suggests the possibility of human-to-human transmission, particularly in patients with no known contact history with animals.ConclusionThe detected proportion of culture positive cases of 2.4% being M. bovis from this region was higher zTB rate than previously reported for the general population of Ethiopia. Patients with M. bovis infection are more likely to get less efficient TB treatment because M. bovis is inherently resistant to pyrazinamide. MTBC species identification should be performed where M. bovis is common in cattle, especially in patients who have a history of recurrence or treatment failure.

Abstract The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.

Abstract Bovine tuberculosis (bTB) is a disease of livestock with severe and worldwide economic, animal welfare and zoonotic consequences. Application of test-and-slaughter-based control polices reliant on tuberculin skin testing has been the mainstay of bTB control in cattle. However, little is known about the temporal development of the bovine tuberculin skin test response at the dermal sites of antigen injection. To fill this knowledge gap, we applied minimally-invasive sampling microneedles (SMNs) for intradermal sampling of interstitial fluid at the tuberculin skin test sites in Mycobacterium bovis BCG-vaccinated calves and determined the temporal dynamics of a panel of 15 cytokines and chemokines in situ and in the peripheral blood. The results reveal an orchestrated and coordinated cytokine and local chemokine response, identified IL-1RA as a potential soluble biomarker of a positive tuberculin skin response, and confirmed the utility of IFN-γ and IP-10 for bTB detection in blood-based assays. Together, the results highlight the utility of SMNs to identify novel biomarkers and provide mechanistic insights on the intradermal cytokine and chemokine responses associated with the tuberculin skin test in BCG-sensitized cattle.

The Bacillus Calmette–Guérin (BCG) vaccination provides partial protection against, and reduces severity of pathological lesions associated with bovine tuberculosis (bTB) in cattle. Accumulating evidence also suggests that revaccination with BCG may be needed to enhance the duration of immune protection. Since BCG vaccine cross-reacts with traditional tuberculin-based diagnostic tests, a peptide-based defined antigen skin test (DST) comprising of ESAT-6, CFP-10, and Rv3615c to detect the infected among the BCG-vaccinated animals (DIVA) was recently described. The DST reliably identifies bTB-infected animals in experimental challenge models and in natural infection settings, and differentiated these from animals immunized with a single dose of BCG in both skin tests and interferon-gamma release assay (IGRA). The current investigation sought to assess the diagnostic specificity of DST in calves (Bos taurus ssp. taurus × B. t. ssp. indicus; n = 15) revaccinated with BCG 6 months after primary immunization. The results show that none of the 15 BCG-revaccinated calves exhibited a delayed hypersensitivity response when skin tested with DST 61 days post-revaccination, suggesting 100% diagnostic specificity (one-tailed lower 95% CI: 82). In contrast, 8 of 15 (diagnostic specificity = 47%; 95% CI: 21, 73) BCG-revaccinated calves were positive per the single cervical tuberculin (SCT) test using bovine tuberculin. Together, these results show that the DST retains its specificity even after revaccination with BCG and confirms the potential for implementation of BCG-based interventions in settings where test-and-slaughter are not economically or culturally feasible.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections.

More than 50 million cattle are likely exposed to bovine tuberculosis (bTB) worldwide, highlighting an urgent need for bTB control strategies in low- and middle-income countries (LMICs) and other regions where the disease remains endemic and test-and-slaughter approaches are unfeasible. While Bacillus Calmette-Guérin (BCG) was first developed as a vaccine for use in cattle even before its widespread use in humans, its efficacy against bTB remains poorly understood. To address this important knowledge gap, we conducted a systematic review and meta-analysis to determine the direct efficacy of BCG against bTB challenge in cattle, and performed scenario analyses with transmission dynamic models incorporating direct and indirect vaccinal effects (“herd-immunity”) to assess potential impact on herd level disease control. The analysis shows a relative risk of infection of 0.75 (95% CI: 0.68, 0.82) in 1,902 vaccinates as compared with 1,667 controls, corresponding to a direct vaccine efficacy of 25% (95% CI: 18, 32). Importantly, scenario analyses considering both direct and indirect effects suggest that disease prevalence could be driven down close to Officially TB-Free (OTF) status (

Bovine tuberculosis (bTB) is a zoonotic disease caused mainly by Mycobacterium bovis, which is associated with major economic losses for milk and meat producers. The objective of this trial was to assess the efficacy of the BCG Russia strain in a cohort study performed under field conditions, with the vaccination of calves in seven dairy farms from a high prevalence area in central Chile. The trial was performed with 501 animals, subcutaneously vaccinated with 2–8 × 105 colony-forming units of BCG, whilst 441 matched control animals received a saline placebo. Peripheral blood was collected at 6, 12 and 18 months post-vaccination, and infection status was determined using the IFNγ release assay in conjunction with the DIVA (Detecting Infected amongst Vaccinated Animals) antigens ESAT-6, CFP-10 and Rv3615c. The BCG vaccine showed a low but significant level of protection of 22.4% (95% CI 4.0 to 36.4) at the end of the trial. However, diverse levels of protection and a variable duration of immunity were observed between trial herds. This diverse outcome could be influenced by the general health condition of calves and their exposition to non-tuberculous mycobacteria. These results suggest that BCG vaccination of dairy calves in a natural transmission setting confers variable protection to animals against bTB in a high prevalence area.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic disease of cattle with a detrimental impact on food quality and production. Research on bTB vaccines has predominantly been focused on proteinaceous antigens. However, mycobacteria have a thick and intricate lipid outer layer and lipids as well as lipopeptides are important for immune-evasion and virulence. In humans, lipid extracts of M. tuberculosis have been shown to elicit immune responses effective against M. tuberculosisin vitro. Chloroform-methanol extraction (CME) was applied to M. bovis BCG to obtain a hydrophobic antigen extract (CMEbcg) containing lipids and lipopeptides. CMEbcg stimulated IFN-γ+IL-2+ and IL-17A+IL-22+ polyfunctional T cells and elicited T cell responses with a Th1 and Th17 cytokine release profile in both M. bovis BCG vaccinated and M. bovis challenged calves. Lipopeptides were shown to be the immunodominant antigens in CMEbcg, stimulating CD4 T cells via MHC class II. CMEbcg expanded T cells killed CMEbcg loaded monocytes and the CMEbcg-specific CD3 T cell proliferative response following M. bovis BCG vaccination was the best predictor for reduced pathology following challenge with M. bovis. Although the high predictive value of CMEbcg-specific immune responses does not confirm a causal relationship with protection against M. bovis challenge, when taking into account the in vitro antimycobacterial phenotype of CMEbcg-specific T cells (e.g. Th1/Th17 cytokine profile), it is indicative that CMEbcg-specific immune responses could play a functional role in immunity against M. bovis. Based on these findings we conclude that lipopeptides of M. bovis are potential novel subunit vaccine candidates and that further studies into the functional characterization of lipopeptide-specific immune responses together with their role in protection against bovine tuberculosis are warranted.

In most low- and middle-income countries (LMICs), bovine tuberculosis (bTB) remains endemic due to the absence of control programs. This is because successful bTB control and eradication programs have relied on test-and-slaughter strategies that are socioeconomically unfeasible in LMICs. While Bacillus Calmette–Guérin (BCG) vaccine-induced protection for cattle has long been documented in experimental and field trials, its use in control programs has been precluded by the inability to differentiate BCG-vaccinated from naturally infected animals using the OIE-prescribed purified protein derivative (PPD)-based tuberculin skin tests. In the current study, the diagnostic specificity and capability for differentiating infected from vaccinated animals (DIVA) of a novel defined antigen skin test (DST) in BCG-vaccinated (Bos taurus ssp. taurus x B. t. ssp. indicus) calves were compared with the performance of traditional PPD-tuberculin in both the skin test and in vitro interferon-gamma release assay (IGRA). The IFN-γ production from whole blood cells stimulated with both PPDs increased significantly from the 0 week baseline levels, while DST induced no measurable IFN-γ production in BCG-vaccinated calves. None of the 15 BCG-vaccinated calves were reactive with the DST skin test (100% specificity; one-tailed lower 95% CI: 82). In contrast, 10 of 15 BCG-vaccinated calves were classified as reactors with the PPD-based single intradermal test (SIT) (specificity in vaccinated animals = 33%; 95% CI: 12, 62). Taken together, the results provide strong evidence that the DST is highly specific and enables DIVA capability in both skin and IGRA assay format, thereby enabling the implementation of BCG vaccine-based bTB control, particularly in settings where test and slaughter remain unfeasible.

Vaccination of cattle against bovine Tuberculosis (bTB) has been a long-term policy objective for countries where disease continues to persist despite costly test-and-slaughter programs. The potential use of vaccination within the European Union has been linked to a need for field evaluation of any prospective vaccine and the impact of vaccination on the rate of transmission of bTB. We calculate that estimation of the direct protection of BCG could be achieved with 100 herds, but over 500 herds would be necessary to demonstrate an economic benefit for farmers whose costs are dominated by testing and associated herd restrictions. However, the low and variable attack rate in GB herds means field trials are unlikely to be able to discern any impact of vaccination on transmission. In contrast, experimental natural transmission studies could provide robust evaluation of both the efficacy and mode of action of vaccination using as few as 200 animals.

For several complex intracellular pathogens, we have an urgent need for effective vaccines and yet there are common barriers to vaccine development. These diseases, including tuberculosis, leishmaniasis, leprosy and melioidosis, cause a huge burden of disease and disproportionately affect low and middle income countries. They are therefore often neglected due to the marginalisation of affected populations and the poor predicted commercial return on investment. Barriers to vaccine development include an incomplete understanding of protective immunity and translation from the bench into clinical vaccine trials. The current linear approach to vaccine research and development for these pathogens, which involves basic research, vaccine design, and vaccine evaluation in preclinical challenge models and clinical trials, is inefficient for these complex intracellular pathogens. We have established a Global Challenges Research Fund Network for VAccine deveLopment for complex Intracellular neglecteD pAThogEns, “VALIDATE”, where we aim to adopt a more flexible, integrated cross-pathogen approach to accelerate vaccine research and clinical development for these four pathogens, by cross-pathogen analyses, cross-discipline collaborations, and repeated integration of data from human and animal studies. This network provides a unique opportunity to bring together individuals working on four exemplar complex intracellular neglected pathogens (M.tb, Leishmania spp., B. pseudomallei and M.leprae), which share a common lifestyle as pathogens of macrophages, induce similar end-stage pathologies and alter host immune and metabolic responses. The horizontal collaborations established throughout this network, together with the provision of a protected environment for early data sharing, will exploit these biological synergies. By interrogating mechanisms that lead from infection to disease, we will be able to develop common vaccine development strategies for these and other complex intracellular pathogens.

TBVAC2020 is a research project supported by the Horizon 2020 program of the European Commission (EC). It aims at the discovery and development of novel tuberculosis (TB) vaccines from preclinical research projects to early clinical assessment. The project builds on previous collaborations from 1998 onwards funded through the EC framework programs FP5, FP6, and FP7. It has succeeded in attracting new partners from outstanding laboratories from all over the world, now totaling 40 institutions. Next to the development of novel vaccines, TB biomarker development is also considered an important asset to facilitate rational vaccine selection and development. In addition, TBVAC2020 offers portfolio management that provides selection criteria for entry, gating, and priority settings of novel vaccines at an early developmental stage. The TBVAC2020 consortium coordinated by TBVI facilitates collaboration and early data sharing between partners with the common aim of working toward the development of an effective TB vaccine. Close links with funders and other consortia with shared interests further contribute to this goal.

Paratuberculosis vaccination in cattle has been restricted due to its possible interference with the official diagnostic methods used in tuberculosis eradication programs. To overcome this drawback, new possibilities to detect Mycobacterium bovis infected cattle in paratuberculosis vaccinated animals were studied under experimental conditions. Three groups of 5 calves each were included in the experiment: one paratuberculosis vaccinated group, one paratuberculosis vaccinated and M. bovis infected group and one M. bovis infected group. The performance of the IFN-gamma release assay (IGRA) and the skin test using conventional avian and bovine tuberculins (A- and B-PPD) but also other more specific antigens (ESAT-6/CFP10 and Rv3615c) was studied under official and new diagnostic criteria. Regarding the IGRA of vaccinated groups, when A- and B-PPD were used the sensitivity reached 100% at the first post-challenge sampling, dropping down to 40-80% in subsequent samplings. The sensitivity for the specific antigens was 80-100% and the specificity was also improved. After adapting the diagnostic criteria for the conventional antigens in the skin test, the ability to differentiate between M. bovis infected and non-infected animals included in paratuberculosis vaccinated groups was enhanced. Taking for positive a relative skin thickness increase of at least 100%, the single intradermal test specificity and sensitivity yielded 100%. The comparative intradermal test was equally accurate considering a B-PPD relative skin increase of at least 100% and greater than or equal to that produced by A-PPD. Using the specific antigens as a proteic cocktail, the specificity and sensitivity reached 100% considering the new relative and absolute cut-offs in all experimental groups (Δ≥30% and Δmm ≥ 2, respectively). Results suggest that the interference caused by paratuberculosis vaccination in cattle could be completely overcome by applying new approaches to the official tuberculosis diagnostic tests.

Vaccination for the control of bovine tuberculosis (bTB) in cattle is not currently used within any international control program, and is illegal within the EU. Candidate vaccines, based upon Mycobacterium bovis bacillus Calmette-Guérin (BCG) all interfere with the action of the tuberculin skin test, which is used to determine if animals, herds and countries are officially bTB-free. New diagnostic tests that Differentiate Infected from Vaccinated Animals (DIVA) offer the potential to introduce vaccination within existing eradication programs. We use within-herd transmission models estimated from historical data from Great Britain (GB) to explore the feasibility of such supplemental use of vaccination. The economic impact of bovine Tuberculosis for farmers is dominated by the costs associated with testing, and associated restrictions on animal movements. Farmers' willingness to adopt vaccination will require vaccination to not only reduce the burden of infection, but also the risk of restrictions being imposed. We find that, under the intensive sequence of testing in GB, it is the specificity of the DIVA test, rather than the sensitivity, that is the greatest barrier to see a herd level benefit of vaccination. The potential negative effects of vaccination could be mitigated through relaxation of testing. However, this could potentially increase the hidden burden of infection within Officially TB Free herds. Using our models, we explore the range of the DIVA test characteristics necessary to see a protective herd level benefit of vaccination. We estimate that a DIVA specificity of at least 99.85% and sensitivity of >40% is required to see a protective benefit of vaccination with no increase in the risk of missed infection. Data from experimentally infected animals suggest that this target specificity could be achieved in vaccinates using a cocktail of three DIVA antigens while maintaining a sensitivity of 73.3% (95%CI: 61.9, 82.9%) relative to post-mortem detection.

BackgroundTransmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia.Methodology/principal findingsA study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (pConclusions/significanceThe results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis.

Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.

Ethiopia has the largest cattle population in Africa. The vast majority of the national herd is of indigenous zebu cattle maintained in rural areas under extensive husbandry systems. However, in response to the increasing demand for milk products and the Ethiopian government's efforts to improve productivity in the livestock sector, recent years have seen increased intensive husbandry settings holding exotic and cross breeds. This drive for increased productivity is however threatened by animal diseases that thrive under intensive settings, such as bovine tuberculosis (BTB), a disease that is already endemic in Ethiopia.An extensive study was conducted to: estimate the prevalence of BTB in intensive dairy farms in central Ethiopia; identify associated risk factors; and characterize circulating strains of the causative agent, Mycobacterium bovis. The comparative intradermal tuberculin test (CIDT), questionnaire survey, post-mortem examination, bacteriology, and molecular typing were used to get a better understanding of the BTB prevalence among dairy farms in the study area. Based on the CIDT, our findings showed that around 30% of 2956 tested dairy cattle from 88 herds were positive for BTB while the herd prevalence was over 50%. Post-mortem examination revealed gross tuberculous lesions in 34/36 CIDT positive cattle and acid-fast bacilli were recovered from 31 animals. Molecular typing identified all isolates as M. bovis and further characterization by spoligotyping and MIRU-VNTR typing indicated low strain diversity within the study area.This study showed an overall BTB herd prevalence of 50% in intensive dairy farms in Addis Ababa and surroundings, signalling an urgent need for intervention to control the disease and prevent zoonotic transmission of M. bovis to human populations consuming dairy products coming from these farms. It is suggested that government and policy makers should work together with stakeholders to design methods for the control of BTB in intensive farms in Ethiopia.

Higher IFN-γ responses to mycobacterial antigens were observed in Bos taurus (Holsteins) than in Bos indicus (Zebu) cattle which could due to differences in antigen recognition profiles between the two breeds. The present study was conducted to evaluate mycobacterial antigen recognition profiles of the two breeds. Twenty-three mycobacterial antigens were tested on 46 skin test positive (24 Zebu and 22 Holstein) using enzyme-linked immunospot assay (ELISPOT) and multiple antigen print immunoassay (MAPIA). Herds from which the study cattle obtained were tested for Fasciola antibody. The T cells from both breeds recognized most of the mycobacterial antigens at lower and comparable frequencies. However, antigens such as CFP-10, ESAT-6, Rv0287, Rv0288, MPB87, Acr-2, Rv3616c, and Rv3879c were recognized at higher frequencies in zebu while higher frequencies of T cell responses were observed to Hsp65 in both breeds. Furthermore, comparable antibody responses were observed in both breeds; MPB83 being the sero-dominant antigen in both breeds. The prevalence of Fasciola antibody was 81% and similar in both breeds. This piece of work could not lead to a definitive conclusion if there are differences in mycobacterial recognition profiles between the two breeds warranting for further similar studies using sound sample size from the two breeds.

Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 10(6) CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-gamma and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97-infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis-infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis of host preference, and in particular the molecular basis of virulence in M. bovis.

Toll-like receptors (TLRs) are a potent trigger for inflammatory immune responses. Without tight regulation their activation could lead to pathology, so it is imperative to extend our understanding of the regulatory mechanisms that govern TLR expression and function. One family of immunoregulatory proteins which can provide a balancing effect on TLR activity are the Leukocyte Ig-like receptors (LILRs), which act as innate immune receptors for self-proteins. Here we describe the LILR family, their inhibitory effect on TLR activity in cells of the monocytic lineage, their signalling pathway, and their antimicrobial effects during bacterial infection. Agents have already been identified which enhances or inhibits LILR activity raising the future possibility that modulation of LILR function could be used as a means to modulate TLR activity.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a debilitating disease of cattle. Ethiopia has one of the largest cattle populations in the world, with an economy highly dependent on its livestock. Furthermore, Ethiopia has one of the highest incidence rates of human extrapulmonary TB in the world, a clinical presentation that is often associated with transmission of M. bovis from cattle to humans.Here we present a comprehensive investigation of the prevalence of bTB in Ethiopia based on cases identified at slaughterhouses. Out of approximately 32,800 inspected cattle, approximately 4.7% showed suspect tuberculous lesions. Culture of suspect lesions yielded acid-fast bacilli in approximately 11% of cases, with M. bovis accounting for 58 of 171 acid-fast cultures, while 53 isolates were non-tuberculous mycobacteria. Strikingly, M. tuberculosis was isolated from eight cattle, an unusual finding that suggests human to animal transmission.Our analysis has revealed that bTB is widely spread throughout Ethiopia, albeit at a low prevalence, and provides underpinning evidence for public health policy formulation.

For several complex intracellular pathogens, we have an urgent need for effective vaccines and yet there are common barriers to vaccine development. These diseases, including tuberculosis, leishmaniasis, leprosy and melioidosis, cause a huge burden of disease and disproportionately affect low and middle income countries. They are therefore often neglected due to the marginalisation of affected populations and the poor predicted commercial return on investment. Barriers to vaccine development include an incomplete understanding of protective immunity and translation from the bench into clinical vaccine trials. The current linear approach to vaccine research and development for these pathogens, which involves basic research, vaccine design, and vaccine evaluation in preclinical challenge models and clinical trials, is inefficient for these complex intracellular pathogens. We have established a Global Challenges Research Fund Network for VAccine deveLopment for complex Intracellular neglecteD pAThogEns, “VALIDATE”, where we aim to adopt a more flexible, integrated cross-pathogen approach to accelerate vaccine research and clinical development for these four pathogens, by cross-pathogen analyses, cross-discipline collaborations, and repeated integration of data from human and animal studies. This network provides a unique opportunity to bring together individuals working on four exemplar complex intracellular neglected pathogens ( M.tb, Leishmania spp., B. pseudomallei and M.leprae), which share a common lifestyle as pathogens of macrophages, induce similar end-stage pathologies and alter host immune and metabolic responses. The horizontal collaborations established throughout this network, together with the provision of a protected environment for early data sharing, will exploit these biological synergies. By interrogating mechanisms that lead from infection to disease, we will be able to develop common vaccine development strategies for these and other complex intracellular pathogens.