Macrophage polarization in lymph node granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex.

Tuberculosis in animals is caused by members of the Mycobacterium tuberculosis complex (MTC), with the tuberculous granuloma being the main characteristic lesion. The macrophage is the main cell type involved in the development of the granuloma and presents a wide plasticity ranging from polarization to classically activated or pro-inflammatory macrophages (M1) or to alternatively activated or anti-inflammatory macrophages (M2). Thus, this study aimed to analyze macrophage polarization in granulomas from cattle and pig lymph nodes naturally infected with MTC. Tuberculous granulomas were microscopically categorized into four stages and a panel of myeloid cells (CD172a/calprotectin), M1 macrophage polarization (iNOS/CD68/CD107a), and M2 macrophage polarization (Arg1/CD163) markers were analyzed by immunohistochemistry. CD172a and calprotectin followed the same kinetics, having greater expression in late-stage granulomas in pigs. iNOS and CD68 had higher expression in cattle compared with pigs, and the expression was higher in early-stage granulomas. CD107a immunolabeling was only observed in porcine granulomas, with a higher expression in stage I granulomas. Arg1+ cells were significantly higher in pigs than in cattle, particularly in late-stage granulomas. Quantitative analysis of CD163+ cells showed similar kinetics in both species with a consistent frequency of immunolabeled cells throughout the different stages of the granuloma. Our results indicate that M1 macrophage polarization prevails in cattle during early-stage granulomas (stages I and II), whereas M2 phenotype is observed in later stages. Contrary, and mainly due to the expression of Arg1, M2 macrophage polarization is predominant in pigs in all granuloma stages.

Bacteriological and molecular characterization of Mycobacterium bovis isolates from tuberculous lesions collected among slaughtered cattle, Northwest Ethiopia.

BACKGROUND: In Ethiopia, the distribution of bovine tuberculosis (BTB) has long been known and documented as a major problem of animal health. However, the burden of circulating M. bovis strains is poorly understood in the country. Therefore; this study aimed to identify and characterize the mycobacterial isolates responsible for BTB in Northwest Ethiopia. METHODS: A cross-sectional study was conducted on tuberculous lesions that had been collected from slaughtered cattle between September 2018 to June 2019. Collected lesions were cultured and tested for tuberculous bacilli. The MPT64 assay and Genotype line probe assay (LPA) were used for identification of mycobacterial isolates, and region of deletion 4 (RD4) typing and spoligotyping were used to characterize the M. bovis strains. RESULTS: Of the total 1458 examined slaughtered cattle, only 62 (4.3, 95%CI; 0.0328-0.0542) had tuberculous lesions. The highest number of gross tuberculous lesions were observed from the lymph nodes of the thoracic cavity; at the mediastinal (40.3%, 25/62) and bronchial (22.6%, 14/62) lymph nodes. Of the 62 collected tuberculous lesions; 18 (29.0%) were culture positive for mycobacterium isolates, and only five isolates were confirmed for M. tuberculosis complex (MTBc) by the MPT64 assay and LPA. All the five MTBc isolates were positive for RD4 typing of M. bovis with a PCR product size of 446 bp, and no isolate was noticed to have M. tuberculosis. The detected M. bovis strains displayed five spoligotypes; with the common SB1176 and SB0133 M. bovis strains, although the two spoligotypes had not been previously reported. CONCLUSION: The present study shows that BTB in North Gondar, Ethiopia, is caused by M. bovis strains SB1176 and SB0033, with low frequency. Thus, the finding highlights the importance of continuous surveillance for mycobacterial strains in cattle populations.

Spoligotype-specific risk of finding lesions in tissues from cattle infected by Mycobacterium bovis.

BACKGROUND: Although the pathogenic effect of members of the Mycobacterium tuberculosis complex in susceptible hosts is well known, differences in clinical signs and pathological findings observed in infected animals have been reported, likely due to a combination of host and pathogen-related factors. Here, we investigated whether Mycobacterium bovis strains belonging to different spoligotypes were associated with a higher risk of occurrence of visible/more severe lesions in target organs (lungs and/or lymph nodes) from infected animals. A large collection of 8889 samples belonging to cattle were classified depending on the presence/absence of tuberculosis-like lesions and its degree of severity. All samples were subjected to culture irrespective of the presence of lesions, and isolates retrieved were identified and subjected to spoligotyping. The association between the presence/severity of the lesions and the isolation of strains from a given spoligotype was assessed using non-parametric tests and Bayesian mixed multivariable logistic regression models that accounted for origin (region and herd) effects. RESULTS: Results suggested a difference in severity in lesioned samples depending on the strain's spoligotype. An association between specific spoligotypes and presence of lesions was observed, with a higher risk of finding lesions in animals infected with strains with spoligotypes SB0120, SB0295 and SB1142 compared with SB0121, and in those coming from certain regions in Spain. CONCLUSIONS: Our results suggest that strains belonging to certain spoligotypes may be associated with a higher probability in the occurrence of gross/macroscopic lesions in infected cattle, although these observational findings should be confirmed in further studies that allow accounting for the effect of other possible confounders not considered here, and ultimately through experimental studies.

Ocular tuberculosis in a calf.

BACKGROUND: Bovine tuberculosis is a chronic inflammatory disease that causes granuloma formation mainly in retropharyngeal, tracheobronchial, mediastinal lymph nodes and lungs of bovines. The presence of these lesions in other tissues such as the eyeball is very rare and difficult to diagnose. This study describes macroscopic and microscopic pathological findings in a calf with ocular and meningeal tuberculosis. CASE PRESENTATION: March 2019, an eight-month-old Holstein Friesian calf was identified in a dairy farm located in central Mexico with a clinical cough, anorexia, incoordination, corneal opacity and vision loss. At necropsy, pneumonia, lymphadenitis, meningitis, and granulomatous iridocyclitis were observed. The histopathological examination revealed granulomatous lesions in lung tissue, lymph nodes, meninges and eyes with the presence of acid-fast bacilli associated with Mycobacterium spp. CONCLUSION: To the best of our knowledge, this is the first report that describes macroscopic and microscopic pathological findings of ocular tuberculosis in cattle. This report highlights the importance of considering bovine tuberculosis in the differential diagnosis of corneal opacity and loss of vision in cattle.

Cellular and Cytokine Responses in the Granulomas of Asymptomatic Cattle Naturally Infected with Mycobacterium bovis in Ethiopia.

Cell (CD3+ T cell and CD68+ macrophages), cytokine (interferon gamma-positive [IFN-γ+] and tumor necrosis factor alpha-positive [TNF-α+]), and effector molecule (inducible nitric oxide synthase-positive [iNOS+]) responses were evaluated in the lymph nodes and tissues of cattle naturally infected with Mycobacterium bovis Detailed postmortem and immunohistochemical examinations of lesions were performed on 16 cows that were positive by the single intradermal cervical comparative tuberculin (SICCT) test and that were identified from dairy farms located around the city of Addis Ababa, Ethiopia. The severity of the gross lesion was significantly higher (P = 0.003) in M. bovis culture-positive cows (n = 12) than in culture-negative cows (n = 4). Immunohistochemical techniques showed that in culture-positive cows, the mean immunolabeling fraction of CD3+ T cells decreased as the stage of granuloma increased from stage I to stage IV (P < 0.001). In contrast, the CD68+ macrophage, IFN-γ+, TNF-α+, and iNOS+ immunolabeling fractions increased from stage I to stage IV (P < 0.001). In the early stages, culture-negative cows showed a significantly higher fraction of CD68+ macrophage (P = 0.03) and iNOS+ (P = 0.007) immunolabeling fractions than culture-positive cows. Similarly, at advanced granuloma stages, culture-negative cows demonstrated significantly higher mean proportions of CD3+ T cells (P < 0.001) than culture-positive cows. Thus, this study demonstrates that, following natural infection of cows with M. bovis, as the stage of granuloma increases from stage I to stage IV, the immunolabeling fraction of CD3+ cells decreases, while the CD68+ macrophage, IFN-γ+, TNF-α+, and iNOS+ immunolabeling fractions increases.

Test characteristics of the tuberculin skin test and post-mortem examination for bovine tuberculosis diagnosis in cattle in Northern Ireland estimated by Bayesian latent class analysis with adjustments for covariates.

The single intradermal comparative cervical tuberculin (SICCT) test and post-mortem examination are the main diagnostic tools for bovine tuberculosis (bTB) in cattle in the British Isles. Latent class modelling is often used to estimate the bTB test characteristics due to the absence of a gold standard. However, the reported sensitivity of especially the SICCT test has shown a lot of variation. We applied both the Hui-Walter latent class model under the Bayesian framework and the Bayesian model specified at the animal level, including various risk factors as predictors, to estimate the SICCT test and post-mortem test characteristics. Data were collected from all cattle slaughtered in abattoirs in Northern Ireland in 2015. Both models showed comparable posterior median estimation for the sensitivity of the SICCT test (88.61% and 90.56%, respectively) using standard interpretation and for post-mortem examination (53.65% and 53.79%, respectively). Both models showed almost identical posterior median estimates for the specificity (99.99% vs. 99.80% for SICCT test at standard interpretation and 99.66% vs. 99.86% for post-mortem examination). The animal-level model showed slightly narrower posterior 95% credible intervals. Notably, this study was carried out in slaughtered cattle which may not be representative for the general cattle population.

Early Pulmonary Lesions in Cattle Infected via Aerosolized Mycobacterium bovis.

Mycobacterium bovis is a serious zoonotic pathogen and the cause of tuberculosis in many mammalian species, most notably, cattle. The hallmark lesion of tuberculosis is the granuloma. It is within the developing granuloma where host and pathogen interact; therefore, it is critical to understand host-pathogen interactions at the granuloma level. Cytokines and chemokines drive cell recruitment, activity, and function and ultimately determine the success or failure of the host to control infection. In calves, early lesions (ie, 15 and 30 days) after experimental aerosol infection were examined microscopically using in situ hybridization and immunohistochemistry to demonstrate early infiltrates of CD68+ macrophages within alveoli and alveolar interstitium, as well as the presence of CD4, CD8, and γδ T cells. Unlike lesions at 15 days, lesions at 30 days after infection contained small foci of necrosis among infiltrates of macrophages, lymphocytes, neutrophils, and multinucleated giant cells and extracellular acid-fast bacilli within necrotic areas. At both time points, there was abundant expression of the chemokines CXCL9, MCP-1/CCL2, and the cytokine transforming growth factor (TGF)-ß. The proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, as well as the anti-inflammatory cytokine IL-10, were expressed at moderate levels at both time points, while expression of IFN-γ was limited. These findings document the early pulmonary lesions after M. bovis infection in calves and are in general agreement with the proposed pathogenesis of tuberculosis described in laboratory animal and nonhuman primate models of tuberculosis.

Different lesion distribution in calves orally or intratracheally challenged with Mycobacterium bovis: implications for diagnosis.

Animal tuberculosis (TB) remains a major problem in some countries despite the existence of control programmes focused mainly on cattle. In this species, aerogenous transmission is accepted as the most frequent infection route, affecting mainly the respiratory system. Under the hypothesis that the oral route could be playing a more relevant role in transmission, diagnosis and disease persistence than previously thought, this study was performed to assess the course of TB infection in cattle and its effects on diagnosis depending on the route of entry of Mycobacterium bovis. Two groups of five calves each were either endotracheally (EC) or orally (OC) challenged. Necropsies were carried out 12 weeks after challenge except for three OC calves slaughtered 8 weeks later. All animals reacted to the tuberculin skin test and the entire EC group was positive to the interferon-gamma release assay (IGRA) 2 weeks after challenge and thereafter. The first positive IGRA results for OC calves (3/5) were recorded 4 weeks after challenge. Group comparison revealed significant differences in lesion and positive culture location and scoring. TB-compatible gross lesions and positive cultures were more frequently found in the thorax (p < 0.001) and lung (p < 0.05) of EC animals, whereas OC animals presented lesions (p = 0.23) and positive cultures (p < 0.05) mainly located in the abdomen. These results indicate that the infection route seems to be a determining factor for both the distribution and the time needed for the development of visible lesions. Our study suggests that confirmation of TB infection in some skin reactor animals can be problematic if current post-mortem examination and diagnostics are not improved.

The C3HeB/FeJ mouse model recapitulates the hallmark of bovine tuberculosis lung lesions following Mycobacterium bovis aerogenous infection.

Achieving the control of bovine tuberculosis (bTB) would require the discovery of an efficient combined immunodiagnostic and vaccine strategy. Since in vivo experiments on cattle are not ethically and economically acceptable there is a need for a cost-effective animal model capable of reproducing, as closely as possible, the physiopathology of bTB to (i) better characterize the cellular and molecular features of bTB immunopathogenesis and (ii) screen preclinical vaccine candidates. To develop such a model, we focused on the C3HeB/FeJ Kramnik's mouse forming hypoxic, encapsulated granulomas with a caseous necrotic center following Mycobacterium tuberculosis infection. Our work represents the first investigation on C3HeB/FeJ interaction with M. bovis, the main agent of bTB. Detailed histopathological analysis of C3HeB/FeJ lung lesions development following aerogenous M. bovis infection unraveled a bimodal evolution of the pathology. The C3HeB/FeJ recapitulated all the hallmarks of classical bovine lung granulomas but also developed, to some extend, lethal necrotic large lesions characterized by high mycobacterial and neutrophil load, and an inefficient collagen-driven lesion encapsulation. Interestingly these rapidly invasive pneumonia lesions, occurring in a constant percentage of the mice, shared all features with some exacerbated lung lesions that we and others have observed in lungs of cattle naturally or experimentally infected with M. bovis. Together, our findings demonstrate the relevance of the C3HeB/FeJ mouse as a comprehensive model to study bTB immunopathology that could be used for further vaccine therapies in the future.

Bovine tuberculosis visible lesions in cattle culled during herd breakdowns: the effects of individual characteristics, trade movement and co-infection.

BACKGROUND: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains a significant problem for livestock industries in many countries worldwide including Northern Ireland, where a test and slaughter regime has utilised the Single Intradermal Comparative Cervical Tuberculin (SICCT) test since 1959. We investigated the variation in post-mortem confirmation based on bTB visible lesion (VL) presence during herd breakdowns using two model suites. We investigated animal-level characteristics, while controlling for herd-level factors and clustering. We were interested in potential impacts of concurrent infection, and therefore we assessed whether animals with evidence of liver fluke infection (Fasciola hepatica; post-mortem inspection), M. avium reactors (animals with negative M. bovis-avium (b-a) tuberculin reactions) or Bovine Viral Diarrhoea Virus (BVDV; RT-PCR tested) were associated with bTB confirmation. RESULTS: The dataset included 6242 animals removed during the 14 month study period (2013-2015). bTB-VL presence was significantly increased in animals with greater b-a reaction size at the disclosing SICCT test (e.g. b-a = 5-9 mm vs. b-a = 0 mm, adjusted Odds ratio (aOR): 14.57; p < 0.001). M. avium reactor animals (b-a < 0) were also significantly more likely to disclose VL than non-reactor animals (b-a = 0; aOR: 2.29; p = 0.023). Animals had a greater probability of exhibiting lesions with the increasing number of herds it had resided within (movement; log-herds: aOR: 2.27-2.42; p < 0.001), if it had an inconclusive penultimate test result (aOR: 2.84-3.89; p < 0.001), and with increasing time between tests (log-time; aOR: 1.23; p = 0.003). Animals were less likely to have VL if they were a dairy breed (aOR: 0.79; p = 0.015) or in an older age-class (e.g. age-quartile 2 vs. 4; aOR: 0.65; p < 0.001). Liver fluke or BVDV variables were not retained in either multivariable model as they were non-significantly associated with bTB-VL status (p > 0.1). CONCLUSIONS: Our results suggest that neither co-infection of liver fluke nor BVDV had a significant effect on the presence of VLs in this high-risk cohort. M. avium tuberculin reactors had a significantly increased risk of disclosing with a bTB lesion, which could be related to the impact of co-infection with M. avium subsp. paratuberculosis (MAP) affecting the performance of the SICCT however further research in this area is required. Movements, test history, breed and age were important factors influencing confirmation in high-risk animals.

Molecular identification of Mycobacterium bovis from cattle and human host in Mali: expanded genetic diversity.

BACKGROUND: Bovine tuberculosis (BTB) is a contagious, debilitating human and animal disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. The study objective were to estimate the frequency of BTB, examine genetic diversity of the M. bovis population in cattle from five regions in Mali and to determine whether M. bovis is involved in active tuberculosis (TB) in humans. Samples from suspected lesions on cattle at the slaughterhouses were collected. Mycobacterial smear, culture confirmation, and spoligotyping were used for diagnosis and species identification. Mycobacterium DNA from TB patients was spoligotyped to identify M. bovis. RESULTS: In total, 675 cattle have been examined for lesions in the five regions of Mali. Out of 675 cattle, 79 specimens presented lesions and then examined for the presence of M. bovis. Thus, 19 (24.1 %) were identified as M. bovis; eight (10.1 %) were non-tuberculous Mycobacterium (NTM). Nineteen spoligotype patterns were identified among 79 samples with five novel patterns. One case of M. bovis (spoligotype pattern SB0300) was identified among 67 TB patients. CONCLUSION: This study estimates a relatively true proportion of BTB in the regions of Mali and reveals new spoligotype patterns.

Comparative assessment of postmortem inspection and immunochromatographic techniques for the detection of bovine tuberculosis in slaughter cattle in Nigeria.

Animals with tuberculosis pose some risks to humans, especially in developing countries of the world. In this study, postmortem inspection (PMI) and immunochromatographic assay (ICA) techniques were compared for the detection of bovine tuberculosis (BTB) in slaughter cattle in Enugu State, Nigeria using culture as the gold standard. A cross-sectional study was conducted from January-June, 2011 on animals presented at four purposively selected slaughterhouses in the study area, involving a total of 500 randomly selected animals. Blood samples were collected from the jugular veins of selected animals and serum samples harvested for ICA. Thorough PMI was carried out and tissue samples from the lung, liver, intestine, and lymph nodes were collected, with or without lesions for culture; from the animals examined, culture detected 11 positive cases giving a prevalence rate of 2.2 %, whereas PMI detected 22 positive cases including 7 (out of the 11) positive cases detected by culture, giving a prevalence rate of 4.4 %. Fifteen of the cases detected as positive by PMI were negative by culture. Therefore, the sensitivity and specificity of PMI were 64 and 97 %, respectively. ICA detected 59 positive cases including 10 of the 11 positive cases detected by culture, hence, a prevalence rate of 11.8 %. Forty-nine of the cases detected as positive by ICA were negative by culture. Hence, the sensitivity and specificity of ICA were 91 and 90 %, respectively. In conclusion, the performance of ICA was found sufficiently high to support its use in BTB surveillance and control in cattle in Enugu State, Nigeria.

Necrosis plays a role in the concentration of mycobacterial antigens in granulomas from Mycobacterium bovis naturally infected cattle.

The dynamics that develop between cells and molecules in the host against infection by Mycobacterium bovis, leads to the formation of granulomas mainly present in the lungs and regional lymph nodes in cattle. Cell death is one of the main features in granuloma organization, however, it has not been characterized in granulomatous lesions caused by M. bovis. In this study we aimed to identify the profiles of cell death in the granuloma stages and its relationship with the accumulation of bacteria. We identified necrosis, activated caspase-3, LC3B/p62 using immunohistochemistry and digital pathology analysis on 484 granulomatous lesions in mediastinal lymph nodes from 23 naturally infected cattle. Conclusions: greater amounts of mycobacterial antigens were identified in granulomas from calves compared with adult cattle. The highest percentage of necrosis and quantity of mycobacterial antigens were identified in granuloma stages (III/IV) from adults. The LC3B/p62 profile was heterogeneous in granulomas between adults and calves. Our data suggest that necrosis is associated with a higher amount of mycobacterial antigens in the late stages of granuloma and the development of autophagy appears to play an heterogeneous effector response against infection in adults and calves. These results represent one of the first approaches in the identification of cell death in the four stages of granulomas in bovine tuberculosis.

Genome-level analyses of Mycobacterium bovis lineages reveal the role of SNPs and antisense transcription in differential gene expression.

BACKGROUND: Bovine tuberculosis (bTB) is a disease with major implications for animal welfare and productivity, as well as having the potential for zoonotic transmission. In Great Britain (GB) alone, controlling bTB costs in the region of £ 100 million annually, with the current control scheme seemingly unable to stop the inexorable spread of infection. One aspect that may be driving the epidemic is evolution of the causative pathogen, Mycobacterium bovis. To understand the underlying genetic changes that may be responsible for this evolution, we performed a comprehensive genome-level analyses of 4 M. bovis strains that encompass the main molecular types of the pathogen circulating in GB. RESULTS: We have used a combination of genome sequencing, transcriptome analyses, and recombinant DNA technology to define genetic differences across the major M. bovis lineages circulating in GB that may give rise to phenotypic differences of practical importance. The genomes of three M. bovis field isolates were sequenced using Illumina sequencing technology and strain specific differences in gene expression were measured during in vitro growth and in ex vivo bovine alveolar macrophages using a whole genome amplicon microarray and a whole genome tiled oligonucleotide microarray. SNP/small base pair insertion and deletions and gene expression data were overlaid onto the genomic sequence of the fully sequenced strain of M. bovis 2122/97 to link observed strain specific genomic differences with differences in RNA expression. CONCLUSIONS: We show that while these strains show extensive similarities in their genetic make-up and gene expression profiles, they exhibit distinct expression of a subset of genes. We provide genomic, transcriptomic and functional data to show that synonymous point mutations (sSNPs) on the coding strand can lead to the expression of antisense transcripts on the opposing strand, a finding with implications for how we define a 'silent' nucleotide change. Furthermore, we show that transcriptomic data based solely on amplicon arrays can generate spurious results in terms of gene expression profiles due to hybridisation of antisense transcripts. Overall our data suggest that subtle genetic differences, such as sSNPS, may have important consequences for gene expression and subsequent phenotype.

Comparison of the MGIT 960, BACTEC 460 TB and solid media for isolation of Mycobacterium bovis in United States veterinary specimens.

BACKGROUND: Bacteriologic culture remains one of the most important methods to diagnose bovine tuberculosis despite the lengthy incubation time, significant decontamination and media expense, and high biocontainment requirements. Media selection is an important determination of culture sensitivity, and the planned discontinuation of the BACTEC 460 TB culture system has challenged veterinary diagnostic laboratories to evaluate alternatives. At the National Veterinary Services Laboratories the BACTEC MGIT 960 and 4 solid media formulations were compared with the BACTEC 460 TB system on 6,795 veterinary diagnostic specimens submitted for Mycobacterium bovis culture. RESULTS: M. bovis was isolated from 2.6% of the samples and atypical mycobacteria from 4.4% of the samples. The BACTEC 12B media isolated significantly more M. bovis (93.1% of positive samples) than MGIT 960 media (81.9%). However, contamination rates were much higher for the MGIT media, 17-24%, compared to 7% for BACTEC, suggesting that contamination was a major cause of MGIT reduced sensitivity. Time to signal positive was 2.37 weeks (95% CI 2.24-2.5) for the MGIT, and 3.2 weeks (95% CI 3.07-3.3) for the BACTEC, both earlier than any solid media. Mycobactosel LJ failed to isolate M. bovis from primary culture. An in-house 7H11 media supplemented with calf sera, hemolyzed blood, malachite green and pyruvate recovered more M. bovis (80.6%) with the least amount of contamination of any other solid media evaluated. CONCLUSION: Decontamination methods may have to be optimized and or MGIT media may have to be altered to reduce contamination in veterinary samples. Despite these issues, the MGIT 960 system is still favored over the use of solid media due to decreased time to recovery and the potential for higher sensitivity.

Prevalence study on bovine tuberculosis and molecular characterization of its causative agents in cattle slaughtered at Addis Ababa municipal abattoir, Central Ethiopia.

A cross-sectional study was conducted on 500 cattle slaughtered at Addis Ababa abattoir to determine the prevalence of bovine tuberculosis (BTB) and characterize its causative agents. Postmortem examination, mycobacteriological culturing, region of difference-4 (RD4)-based PCR and spoligotyping were applied. The prevalence of BTB was 5 % on the basis of postmortem inspection alone but 1.2 % based on molecular confirmation. Factors including age, sex, and breed showed statistically significant association with BTB (p < 0.05). Gross lesions were observed most frequently (68 %) in the lungs and lung-associated lymph nodes compared to other organs and lymph nodes. Of the 25 grossly suspicious TB lesions processed and cultured, only six (24 %) were culture-positive, yielding Mycobacterium bovis confirmed by RD4 deletion typing. Further characterization of the six M. bovis isolates at the strain level by using spoligotyping revealed that one did not belong to any previously known type, while the others belonged to types SB1176 (two), SB1477 (two), and SB0133 (one). The new strain was submitted to the international M. Bovis.org database for international code designation. The study confirms the considerable prevalence of bovine tuberculosis in cattle slaughtered at Addis Ababa abattoir and highlights the need for control of bovine tuberculosis in the country.

Molecular genotyping of Mycobacterium bovis isolated from cattle tissues in the North West Region of Cameroon.

An epidemiological study was carried out to determine the Mycobacterium bovis strains causing bovine tuberculosis (TB) in cattle in North West Cameroon. Suspected TB lesions from slaughtered cattle were cultured on Lowenstein-Jensen and Middlebrook 7 H9 media to isolate mycobacteria agents for molecular genotyping using deletion analysis and spoligotyping. PCR-based genomic deletion typing showed that 54 of 103 tubercle bacilli isolated from cattle tissue were M. bovis strains and the African 1 clonal complex was widespread in affected cattle. Spoligotyping analysis revealed a closely related group of five M. bovis strains. SB0953, the dominant spoligotype pattern, and four new patterns identified as SB2161, SB2162, SB2663 and SB2664 according to the www.Mbovis.org international spoligotype database were identified. These spoligotypes were similar to other M. bovis strains recovered from bordering regions and other parts of Africa. The findings provided useful facts on the zoonotic risks of bovine TB and overwhelming evidence of the significance of M. bovis infection to human TB in the North West Region of Cameroon. The study revealed that bovine TB was widespread in cattle destined for human consumption and also has important implications for the control of TB in animals and humans in Cameroon.

Bovine tuberculosis: prevalence and diagnostic efficacy of routine meat inspection procedure in Woldiya municipality abattoir north Wollo zone, Ethiopia.

Bovine tuberculosis (BTB) is a widespread and endemic disease of cattle in Ethiopia posing a significant threat to public health. Regular surveillance by skin test, bacteriology, and molecular methods is not feasible due to lack of resources. Thus, routine abattoir (RA) inspection will continue to play a key role for national surveillance. A cross-sectional study was conducted at Woldiya municipal abattoir from April 1, 2009 to April 5, 2010 to estimate the prevalence of BTB in slaughtered cattle on the basis of detailed abattoir inspection and to compare efficacy of RA inspection with respect to detailed abattoir inspection and isolation and identification of Mycobacterium. Diagnostic accuracies (with corresponding measures of statistical uncertainty) were determined by computing test property statistics (sensitivity and specificity). Agreement between RA and detailed abattoir inspections was measured using kappa statistics. Out of 1,029 slaughtered heads of cattle examined during the study period, 63 (6.12 %) and 15 (1.45 %) were diagnosed with gross tuberculous lesions by detailed abattoir meat inspections and RA meat inspections, respectively, making a prevalence of 6.12 % (95 % CI: 5.2-7.1) on the basis of detailed abattoir inspection. About 59.45 % of tuberculous lesions were observed in the lungs and associated lymph nodes, whereas 35.13 % lesions were from the lymph nodes of the head. From 63 cattle suspected with tuberculosis (TB) based on detailed abattoir meat inspection, nine (19.05 %) were identified as Mycobacterium bovis, while three (4.8 %) as Mycobacterium tuberculosis. The sensitivity of RA meat inspection was 23.8 % in comparison to the detailed abattoir meat inspection and 25 % in comparison to culture, respectively. Poor agreement (k = 0.37) was seen between RA meat examination and detailed abattoir meat examination methods. Similarly, poor agreement (k = 0.013) was seen between RA meat examination and culture results. In conclusion, relatively higher prevalence (6.12 %) was recorded in Woldiya municipal abattoir on the basis of detailed Abattoir inspection and RA meat inspection protocols currently utilized in Ethiopia which are insufficient to detect the majority (76.19 %) of TB lesions at the gross level, which indicates the magnitude of meat borne zoonotic TB as an ongoing risk to public health. Detailed abattoir inspection protocols were demonstrated to improve the detection level by approximately fourfold. In conclusion, routine meat inspections have limitations in detecting BTB-suggestive lesions which indicate the magnitude of meat-borne zoonotic TB as an ongoing risk to public health.

Factors associated with severity of bovine tuberculosis in Ethiopian cattle.

Bovine TB is a disease of high economic and public health importance particularly in resource poor countries. Many aspects of pathogenesis of bovine TB in cattle have not been well understood. We carried out an investigation on 337 Ethiopian cattle with characteristic TB-like lesions to describe severity of pathology and factors associated with it. Severity of pathology was determined based upon gross lesion characteristics, distribution and presence/absence of viable mycobacteria. Molecular speciation of mycobacteria was performed using Gene-Probe's Accu-Probe method. Mycobacterium bovis was identified by genomic deletion analysis and spoligotyping. Data were analysed using descriptive statistics and regression model. The results showed that TB-like lesions and M. bovis were more frequently observed in lungs and respiratory lymph nodes. Mammary lesions yielded significant proportion of M. bovis upon culturing. Intestinal lesions were the second most frequently encountered pathology; upon culturing, however, the tissue specimens yielded the lowest proportion of M. bovis isolates. Sex, breed and management system were found to significantly affect TB manifestation. Female (ß ± SE = 4.1 ± 1.0; P = 0.00) and exotic breed (ß ± SE = 1.7 ± 0.9; P = 0.045) were at a relatively higher risk of developing severe tuberculosis. TB pathology was more severe in cattle raised under large-scale farming (ß ± SE = 2.3 ± 0.5; P = 0.00). The fact that severe tuberculosis is linked to high degree of disease transmission potential warrants implementation of proper disease surveillance programs in large-scale farms. Isolation of M. bovis from mammary and muscle tissues implies a potential threat of zoonotic transmission, where raw milk and raw beef constitute a customary dietary regimen in Ethiopia.