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Summary: The pathognomonic characteristic of tuberculosis (TB) is the formation of a tuberculous granuloma. The objective of this study was to classify lymph node granulomas from experimentally infected calves into different histopathological stages and characterize them further by studying cell types and markers of fibrosis associated with each of the stages. Four stages of granuloma were identified and mRNA and protein expression for cell markers, cytokines and pro-fibrotic markers were studied by immunohistochemistry (IHC) and in-situ hybridization (ISH). In advanced stage granulomas, there was an increase in the expression of TGF-β, and of type I procollagen as demonstrated by IHC and ISH. As the granulomas advanced, there were fewer CD3+T cells and they tended to be more prominent towards the periphery of the lesions, with a steady increase in the number of CD68+ cells and γδ (WC1+) T cells. Granuloma classification and application of cell cytokine markers will assist in improving understanding of the pathogenesis of bovine TB and may help to identify the immunopathology of active disease versus contained or inactive disease. Such disease correlates may help to inform the development of improved diagnostic methods and support vaccine development programmes. [Copyright &y& Elsevier]

Background: Bovine tuberculosis (bTB) is the most important animal health concern in Wales. Annual testing across all cattle herds in Wales commenced in 2010. In 2017, a new geographic division of Wales was conceived, with bespoke cattle controls and eradication milestones reflecting the geographical heterogeneity of bTB distribution., Methods: This observational study uses descriptive analysis and Poisson regression modelling to analyse bTB surveillance data from all herds in Wales., Results: Since 2010, there has been a significant decrease (p < 0.0001) in bTB incidence (8.6%‒6.3%) and plateauing prevalence across Wales. Conversely, there has been an increase in bTB incidence and prevalence in discrete areas. Recurrence and persistence remain important drivers of bTB infection. One of the sharpest declines in bTB incidence was observed in an intensive action area where enhanced cattle control and wildlife vaccination were implemented in an area of high bTB prevalence. Increased herd size, dairy herd type and herd location are important risk factors affecting the rate of bTB incidents in Wales., Limitations: This study includes data from Wales only., Conclusions: Improvements in trends of bTB occurred from 2010 to 2021, but the spatial variations described in this paper support the continued need for regionally adapted surveillance and control measures., (© 2024 The Author(s). Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

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., (© 2022. The Author(s).)

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne's disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein-Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

After birth, as effectively monogastric animals, calves undergo substantial physiological changes to become ruminants by 3 months of age and reach sexual maturity at approximately 15 months of age. Herein, we assess longitudinal metabolomic changes in Holstein-Friesian (HF) heifers from birth until sexual maturity during this developmental process. Sera from 20 healthy, HF heifers were sampled biweekly from 2 weeks of age until 13 months of age and then monthly until 19 months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares discriminant analysis (PLS-DA) and unsupervised hierarchical clustering analysis (HCA) of the derived metabolomes indicated changes detectable in heifers' sera over time. Time series analyses identified 30 metabolites that could be related to rumen development and weaning at ~3 months of age. Further time series analysis identified 40 metabolites that could be correlated with growth. These findings highlight the role of acetic acid and 3-phenylpropionate (3-PP) in rumen development and growth, suggest that weaning induces elevated levels of fatty acyls in response to a post-weaning stress-induced innate immune response and demonstrate the utilization of fatty acyls in growth. The identified metabolites offer serum metabolites which could inform the nutrition and healthy development of heifers.

Introduction: Paratuberculosis, commonly known as Johne's disease, is a chronic granulomatous infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Clinical signs, including reduced milk yields, weight loss and diarrhoea, are typically absent until 2 to 6 years post exposure., Objectives: To identify metabolomic changes profiles of MAP challenged Holstein-Friesian (HF) cattle and correlate identified metabolites to haematological and immunological parameters., Methods: At approximately 6 weeks of age, calves (n = 9) were challenged with 3.8 × 10 9 cells of MAP (clinical isolate CIT003) on 2 consecutive days. Additional unchallenged calves (n = 9) formed the control group. The study used biobanked serum from cattle sampled periodically from 3- to 33-months post challenge. The assessment of sera using flow infusion electrospray high resolution mass spectrometry (FIE-HRMS) for high throughput, sensitive, non-targeted metabolite fingerprinting highlighted differences in metabolite levels between the two groups., Results: In total, 25 metabolites which were differentially accumulated in MAP challenged cattle were identified, including 20 which displayed correlation to haematology parameters, particularly monocyte levels., Conclusion: The targeted metabolites suggest shifts in amino acid metabolism that could reflect immune system activation linked to MAP and as well as differences in phosphocholine levels which could reflect activation of the Th1 (tending towards pro-inflammatory) immune response. If verified by future work, selected metabolites could be used as biomarkers to diagnose and manage MAP infected cattle., (© 2022. The Author(s).)

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes weight loss, diarrhoea, and reduced milk yields in clinically infected cattle. Asymptomatic, subclinically infected cattle shed MAP bacteria but are frequently not detected by diagnostic tests. Herein, we compare the metabolite profiles of sera from subclinically infected Holstein-Friesian heifers and antibody binding to selected MAP antigens. The study used biobanked serum samples from 10 naturally MAP-infected and 10 control heifers, sampled monthly from ~1 to 19 months of age. Sera were assessed using flow infusion electrospray-high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least-squares discriminant analyses (PLS-DA) and hierarchical cluster analysis (HCA) of the data discriminated between naturally MAP-infected and control heifers. In total, 33 metabolites that differentially accumulated in naturally MAP-infected heifers compared to controls were identified. Five were significantly elevated within MAP-infected heifers throughout the study, i.e., leukotriene B4, bicyclo prostaglandin E2 (bicyclo PGE2), itaconic acid, 2-hydroxyglutaric acid and N6-acetyl-L-lysine. These findings highlight the potential of metabolomics in the identification of novel MAP diagnostic markers and particular biochemical pathways, which may provide insights into the bovine immune response to MAP.

In order to better understand the spatial spread of bovine tuberculosis (bTB) in Wales, an All Wales Badgers Found Dead (AWBFD) survey was carried out from 2014-2016. For Wales, as a whole, there was a significant decrease (p < 0.001) in prevalence of bTB in badgers since a similar survey was carried out in 2005-2006, with a drop from 13.3% to 7.3%. The highest prevalence was observed for the High TB Area East (18.6%), which shares its border with England, and differed significantly (p < 0.001) from the High TB Area West (7.4%). The lowest proportion of carcases diagnosed with the disease (0.7%) was in the Low TB Area, followed by the two Intermediate TB Areas of Wales (2.7%). The M. bovis isolates from badgers tended to be similar to the genotypes of cattle in the same area, except in the Low TB Area. The direction of any cross species transmission and the drivers for this cannot be determined from this study. The spatial variations described here support the need for regionally adapted surveillance and control measures for bovine tuberculosis in Wales.

Quantifying pathogen transmission in multi-host systems is difficult, as exemplified in bovine tuberculosis (bTB) systems, but is crucial for control. The agent of bTB, Mycobacterium bovis , persists in cattle populations worldwide, often where potential wildlife reservoirs exist. However, the relative contribution of different host species to bTB persistence is generally unknown. In Britain, the role of badgers in infection persistence in cattle is highly contentious, despite decades of research and control efforts. We applied Bayesian phylogenetic and machine-learning approaches to bacterial genome data to quantify the roles of badgers and cattle in M. bovis infection dynamics in the presence of data biases. Our results suggest that transmission occurs more frequently from badgers to cattle than vice versa (10.4x in the most likely model) and that within-species transmission occurs at higher rates than between-species transmission for both. If representative, our results suggest that control operations should target both cattle and badgers., Competing Interests: JC, CB, DB, ND, AA, AA, RB, EP, JD, GH, SL, JN, RS, HT, DW, RZ, RD, RK No competing interests declared, (© 2019, Crispell et al.)

Background: Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extrapulmonary TB in Ethiopia., Methods: Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis., Results: No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3., Conclusions: The study suggests a complex role for multiple interacting factors in the epidemiology of extrapulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia.

Background: Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia., Methodology/principal Findings: A 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 (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by "Vordermeier et al. (2002)" was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively., Conclusions/significance: The 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.

Molecular typing of 964 specimens from patients in Ethiopia with lymph node or pulmonary tuberculosis showed a similar distribution of Mycobacterium tuberculosis strains between the 2 disease manifestations and a minimal role for M. bovis. We report a novel phylogenetic lineage of M. tuberculosis strongly associated with the Horn of Africa.

A preliminary study to characterise mycobacteria infecting tuberculous cattle from two different management systems in central Ethiopia was carried out. Approximately 27% of isolates from grazing cattle were Mycobacterium tuberculosis, while cattle in a more intensive-production system were exclusively infected with M. bovis. The practice of local farmers discharging chewed tobacco directly into the mouths of pastured cattle was identified as a potential route of human-to-cattle transmission of M. tuberculosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Rapid, simple, and accurate antemortem tests for tuberculosis (TB) in cattle need to be developed in order to augment the existing screening methods. In particular, as cattle vaccines are developed, such tests would allow the continuation of test-and-slaughter policies alongside vaccination. Therefore, the development of an assay that distinguishes infected from vaccinated animals (a DIVA test) is an urgent research requirement. In this study, we assessed the performance of a novel multiplex serological test with sera collected from 96 skin-tested animals with bovine tuberculosis, 93 TB-free animals, and 39 cattle vaccinated with Mycobacterium bovis BCG. Our results indicate that the test has a relative sensitivity range of 77.0% to 86.5% at corresponding specificity levels of 100.0% to 77.6%. Comparison with the Bovigam gamma interferon antemortem test revealed that this serology test was significantly more sensitive at specificities above 97.9%, while the Bovigam test was, on average, about 10% more sensitive when the test specificity was set below 97%. Importantly, this serological multiplex assay does not react with sera from BCG-vaccinated calves and is therefore suitable as a DIVA test alongside BCG-based vaccine strategies.

Host immune responses to Mycobacterium bovis (M. bovis) infection are variable at the different severity stages of pathology of the disease. In countries like Ethiopia, where routine screening of bovine TB is not undertaken, the use of tests which measure cellular and antibody responses may help for the maximum detection of infection. In the present study, 701 cattle were tested for bovine tuberculosis (BTB) using comparative intradermal tuberculin (CIDT) test, interferon (IFN)-gamma test, and lateral flow assay. The apparent prevalence was 32% when all the three tests were used, but varied from 23 to 25% when a pair of tests was used and from 9% to 15% when a single test was used. Agreement was observed between CIDT and IFN-gamma tests both at a cut-off >2 mm (Kappa +/- standard Error, k +/- SE, 0.129 +/- 0.045; 95%CI = 0.041,0.216) and a cut-off >4 mm (k +/- SE, 0.094 +/- 0.044, 95%CI = 0.008,0.179) while no agreement was observed either between CIDT test and lateral flow assay (k +/- SE, -0.04 +/- 0.033; 95%CI = -0.104,0.024) or between IFN-gamma tests and lateral flow assay (k +/- SE, -0.031 +/- 0.032; 95% CI = -0.093,0.031). Thus, the use of more than one test leads to the detection of the maximum number of infected animals.

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.

Accurate detection and removal of infected cattle, using immunodiagnostic tests such as the comparative intradermal tuberculin (CIDT) test, are the basis of control strategies for bovine tuberculosis (TB). According to the Office des Internationale Epizooties recommendation, the cutoff point for positivity of the CIDT test, calculated as the difference between skin thicknesses after bovine tuberculin (B) and avian tuberculin (A) injections (B - A), is >4 mm. This cutoff point is used worldwide, although it is likely that local conditions influence test performance. Thus, this study was formulated to determine CIDT test cutoff points applicable to cattle in central Ethiopia. Receiver operating characteristic analysis was performed for the CIDT test, using data from 186 Bos indicus (zebu) and Bos taurus (Holstein) cattle. Detailed postmortem examination for the presence of TB lesions was used to define disease status. At a cutoff of >2 mm, CIDT test sensitivity was 69% (95% confidence interval [95% CI], 58.5 to 79%), while it was 59% (95% CI, 49 to 69%) at a cutoff of >4 mm. In contrast, specificities of the CIDT test at these two cutoff values were identical, at 97% (95% CI, 89 to 100%). Thus, the maximum sensitivity of the CIDT test can be realized using a >2-mm cutoff without affecting specificity. The apparent prevalence was significantly (chi(2) = 13.56; P < 0.001) higher at a cutoff of >2 mm (16.0%; n = 5,424) than at a >4-mm cutoff (13.5%; n = 5,424). Nonetheless, no significant difference (chi(2) = 2.15; P = 0.14) in true prevalence was observed at a cutoff of >2 mm (19.6%) and at a cutoff of >4 mm (18.5%). Thus, our study demonstrates the importance of defining local, relevant cutoff values to maximize test sensitivity, and we suggest the application of the >2-mm cutoff for testing of cattle in central Ethiopia.

A comparative study on the prevalence and pathology of bovine tuberculosis (TB) was conducted on 5,424 cattle (2,578 zebus, 1,921 crosses, and 925 Holsteins), which were kept on pasture in the central highlands of Ethiopia, using a comparative intradermal tuberculin test, postmortem examination, and bacteriology. The overall prevalence of bovine TB was 13.5%; prevalence was higher in Holsteins than either zebus (22.2% versus 11.6%, chi(2) = 61.8; P < 0.001) or crosses (22.2% versus 11.9%, chi(2) = 50.7; P < 0.001). Moreover, the severity of pathology in Holsteins (mean +/- standard error of the mean [SEM], 6.84 +/- 0.79) was significantly higher (P = 0.018) than the severity of pathology in zebus (5.21 +/- 0.30). In addition, the risk of TB in Holsteins was more than twice (odds ratio [OR] = 2.32; 95% confidence interval [CI] = 1.89, 2.85) that in zebus. Animals between 5 and 9 years of age were at higher (OR = 2.37; 95% CI = 1.80, 3.12) risk of bovine TB than those 2 years of age or below. A significant difference (chi(2) = 351; P < 0.001) in the occurrence of TB lesions in lymph nodes was recorded; the mesenteric lymph node (mean pathology score +/- SEM, 1.95 +/- 0.08) was most severely affected, followed by the retropharyngeal (0.80 +/- 0.05) and caudal mediastinal (0.8 +/- 0.06) lymph nodes. Fifty-six percent (n = 145) of the animals with gross TB lesions were culture positive; the lowest culture positivity was recorded in the skin lesions (27.3%) and the lesions of the mesenteric lymph node (31.5%). Both the skin test response and the postmortem findings suggested a higher susceptibility to bovine TB in Holsteins than zebus under identical field husbandry conditions (on pasture). In the light of increased numbers of Holstein cattle introduced into this area to raise milk production to satisfy the needs of Addis Ababa's growing population, these findings highlight the need for a control program in these herds.

To understand the relevance of aerogenic transmission for bovine tuberculosis, it is important to study cattle experimentally infected with low doses of Mycobacterium bovis that result in pathology of the lower respiratory tract resembling that of most naturally infected cattle. In this study, we have compared and contrasted granuloma distribution and formation from cattle infected with low doses (1-1000 colony-forming units (cfu)) of M. bovis over 24 weeks. We also studied the expression of the cytokine IFN-gamma in lymph nodes by immunohistochemistry (IHC) as well as by ELISA from in vitro-stimulated peripheral blood cells. Our results confirmed that 50% of cattle infected with only 1cfu of field strain (AF2122/97) M. bovis developed advanced granulomas in thoracic lymph nodes. The degree of lesion advancement and granuloma distribution was similar between the lowest dose group (1cfu) and the highest of the 4 groups (1000cfu). The number of acid-fast bacilli identified within the granulomas was also similar among all groups. IFN-gamma expression was not significantly correlated with the infective dosage in either granuloma IHC or ante-mortem ELISA. These studies confirm that within the same time period, the smallest possible infective dose, properly administered, can produce pathology indistinguishable from a dose 1000-fold greater.

Bovine tuberculosis is a major economic problem and a potential public health risk. Improved diagnostics like the gamma interferon (IFN-gamma) test with ESAT6 and/or CFP10 could contribute to the control program. We assessed IFN-gamma responses in zebu (Ethiopian Arsi breed) and Holstein cattle kept indoors or in a pasture to tuberculin purified protein derivative (PPD) and an ESAT6-CFP10 protein cocktail. Furthermore, the intensity and distribution of pathology of bovine tuberculosis were compared between the two breeds. Our data demonstrated significantly (all P < 0.02) higher IFN-gamma responses to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail in Holstein than in zebu cattle, while lesion severities in infected animals and tuberculin skin test responses did not differ significantly (P > 0.05) between the two breeds. Holstein cattle that were kept indoors produced significantly (all P < 0.01) higher IFN-gamma levels in response to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail than did Holstein cattle kept in a pasture. Moreover, lesion severity was significantly higher in Holstein cattle kept indoors (P = 0.001) than in those kept in the pasture. Lesions were localized predominantly in the digestive tract in cattle kept in a pasture, while they were localized in the respiratory tract in cattle kept indoors. In conclusion, in Holstein cattle, husbandry was a dominant factor influencing the severity of tuberculosis lesions and IFN-gamma responses to mycobacterial antigens compared to breed. A difference in the cellular immune response between zebu and Holstein cattle was observed, while tuberculosis lesion severities were identical in the two breeds, when both were kept in a pasture.

Development of necrotic granulomas in response to Mycobacterium bovis infection in cattle is pathognomonic for bovine tuberculosis. Previously our laboratory reported on M. bovis granuloma classification by stage of lesion advancement within bovine lymph nodes and developed immunohistochemical markers to further characterize these granulomas. In this study of bovine lymph node granulomas we applied this classification system to assess the dynamics of vaccination challenge. Lymph nodes collected from cattle vaccinated with M. bovis bacillus Calmette-Guerin (BCG) and subsequently challenged with virulent M. bovis were compared to lymph nodes from unvaccinated, challenged cattle. Expression of interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), type I procollagen and cell marker identification of T cells, B cells, macrophages and WC1(+)gammadelta TCR+ cells were assessed. Granulomas formed in vaccinated cattle were greatly reduced in number, area, degree of necrosis and peripheral fibrosis and contained fewer Langhans' giant cells, acid fast bacilli, WC1(+)gammadelta TCR+ cells and less TGF-beta expression in comparison to controls. B cells clustered intensely along the outer granuloma margins within vaccinated calves, with significantly more IFN-gamma producing cells identified in the medullary regions of lymph nodes from BCG-vaccinated animals compared to unvaccinated controls. This may be indicative of immune activation and surveillance in regions not directly associated with ongoing disease. Lymph node evaluation using light microscopy and immunohistochemical markers is useful to assess the immune response and discriminate granulomas to determine vaccine efficacy and disease severity.

This slaughterhouse study in Chad shows higher proportions of Mycobacterium bovis isolates among Mbororo than Arabe zebu cattle. Spoligotyping shows a homogenetic population structure for M. bovis and lack of spacer 30, as were found in neighboring Cameroon and Nigeria. This finding suggests transborder and ongoing transmission between cattle.

Bovine tuberculosis (bTB) is an important disease of cattle and an emerging infectious disease of humans. Cow- and badger-based control strategies have failed to eradicate bTB from the British cattle herd, and the incidence is rising by about 18%per year. The annual cost to taxpayers in Britain is currently 74 million UK pounds. Research has focused on the badger as a potential bTB reservoir, with little attention being paid to other mammals common on farmland. We have conducted a systematic survey of wild mammals (n=4393 individuals) present on dairy farms to explore the role of species other than badgers in the epidemiology of bTB. Cultures were prepared from 10397 samples (primarily faeces, urine and tracheal aspirates). One of the 1307 bank voles (Clethrionomys glareolus) live-sampled, and three of the 43 badgers (Meles meles), yielded positive isolates of Mycobacterium bovis. This is the first time the bacterium has been isolated from the bank vole. The strain type was the same as that found in cattle and badgers on the same farm. However, our work indicates that the mean prevalence of infectious individuals among common farmland wildlife is extremely low (the upper 95% confidence interval is < or =2.0 for all of the abundant species). Mathematical models illustrate that it is highly unlikely the disease could be maintained at such low levels. Our results suggest that these animals are relatively unimportant as reservoirs of bTB, having insufficient within-species (or within-group) transmission to sustain the infection, though occasional spill-overs from cattle or badgers may occur.

The definition of antigens for the diagnosis of human and bovine tuberculosis is a research priority. If diagnosis is to be used alongside Mycobacterium bovis BCG-based vaccination regimens, it will be necessary to have reagents that allow the discrimination of infected and vaccinated animals. A list of 42 potential M. bovis-specific antigens was prepared by comparative analysis of the genomes of M. bovis, M. avium subsp. avium, M. avium subsp. paratuberculosis, and Streptomyces coelicolor. Potential antigens were tested by applying them in a high-throughput peptide-based screening system to M. bovis-infected and BCG-vaccinated cattle and to cattle without prior exposure to M. bovis. A response hierarchy of antigens was established by comparing responses in infected animals. Three antigens (Mb2555, Mb2890, and Mb3895) were selected for further study, as they were strongly recognized in experimentally infected animals but with low or no frequency in BCG-vaccinated and naïve cows. Interestingly, all three antigens were recognized in animals vaccinated against Johne's disease, suggesting the presences of epitopes cross-reacting with M. avium subsp. paratuberculosis antigens. Eight peptides from the three antigens studied in detail were identified as immunodominant and were characterized in terms of major histocompatibility complex class II restriction element usage and shown to be restricted through both DR and DQ molecules. Reasons for antigenic cross-reactivity with M. avium subsp. paratuberculosis and refinement of the in silico strategy to predict such cross-reactivity from the primary protein sequence will be discussed. Evaluation of the peptides identified from the three dominant antigens by use of larger field studies is now a priority.

We have previously demonstrated that vaccination of mice with plasmid DNA vectors expressing immunodominant mycobacterial genes induced cellular immune responses and significant protection against challenge with Mycobacterium tuberculosis. We demonstrate here, using in vitro-synthesized RNA, that vaccination with DNA or RNA constructs expressing the M. tuberculosis MPT83 antigen are capable of inducing specific humoral and T-cell immune responses and confer modest but significant protection against M. tuberculosis challenge in mice. This is the first report of protective immunity conferred against intracellular bacteria by an RNA vaccine. This novel approach avoids some of the drawbacks of DNA vaccines and illustrates the potential for developing new antimycobacterial immunization strategies.