Revisiting the relative effectiveness of slaughterhouses in Ireland to detect tuberculosis lesions in cattle (2014-2018).

Slaughterhouse or meat factory surveillance to detect factory lesions (FL) at slaughter is an important part of the bovine tuberculosis (bTB) eradication program in Ireland. The objective of this study was to quantify the effectiveness of Irish slaughterhouses or factories in submitting FL and the proportion of those submitted FL confirmed as being due to bTB in slaughtered cattle, and to identify and quantify the association of risk factors at animal, herd, and factory level with FL submission and confirmation. The data consisted of 6,611,854 animals slaughtered in Irish factories from 2014 to 2018 obtained from the Department of Agriculture, Food and Marine (DAFM), Ireland. Selected risk factors for this study included factory, year and month of slaughter, age, sex, breed, animal movement, ever inconclusive in the standard or severe skin test, herd type, herd size, and bTB history. The association of each risk factor on the FL submission and confirmation risk were analysed with univariable followed by a multivariable logistic regression with herd as random effect. Factories were ranked and compared based on the odds ratio (OR) obtained from the univariable (crude OR) and multivariable (adjusted OR) analysis. The average submission risk of all factories was 20 per 10,000 animals slaughtered, ranging from 1 to 42 per 10,000 animals slaughtered, and the average confirmation risk over all factories was 40.72%, ranging from 0.00 to 61.84%. The odds of submitting and confirming FL as bTB positive were higher in animals over eight years old compared to animals 1-2 years old (OR = 1.91, 95 CI 95% 1.77-2.06 and OR = 4.05, 95% CI 3.17-5.18, respectively), and were higher in animals that ever had inconclusive skin result based on severe interpretation (OR = 2.83, 95% CI 2.44-3.27 and OR = 4.48, 95% CI 2.66-7.54, respectively), animals originating from sucklers herds (OR = 1.08, 95% CI 1.02-1.14 and OR = 1.31, 95% CI 1.11-1.55, respectively), or herds with bTB history in the past three years (OR = 4.46, 95% CI 4.28-4.66 and OR = 319.90, 95% CI 237.98-430.04, respectively). The odds of FL submission and confirmation decreased as the herd size increased (OR = 0.95, 95% CI 0.93-0.96 and OR = 0.82, 95% CI 0.78-0.86, respectively). An inverse relationship of FL submission and confirmation was present for variable sex and inconclusive skin result with standard interpretation, where submission odds were higher in males (OR = 1.05, 95% CI 1.00-1.10) and ever inconclusive animals (OR = 74.24, 95% CI 69.39-79.43), although the confirmation odds were lower (males OR = 0.66, 95% CI 0.56-0.76; ever inconclusive OR = 0.44, 95% CI 0.36-0.54). The crude and adjusted ranking of factories did not differ greatly for FL submission, indicating that factory-related factors may contribute significantly to the submission variation between factories. However, a substantial difference between crude and adjusted confirmation ranking was present which may indicate that animal and herd-related factors were associated to variation in confirmation risk between factories.

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.

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.

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.

Bayesian hierarchical models for disease mapping applied to contagious pathologies.

Disease mapping aims to determine the underlying disease risk from scattered epidemiological data and to represent it on a smoothed colored map. This methodology is based on Bayesian inference and is classically dedicated to non-infectious diseases whose incidence is low and whose cases distribution is spatially (and eventually temporally) structured. Over the last decades, disease mapping has received many major improvements to extend its scope of application: integrating the temporal dimension, dealing with missing data, taking into account various a prioris (environmental and population covariates, assumptions concerning the repartition and the evolution of the risk), dealing with overdispersion, etc. We aim to adapt this approach to model rare infectious diseases proposing specific and generic variants of this methodology. In the context of a contagious disease, the outcome of a primary case can in addition generate secondary occurrences of the pathology in a close spatial and temporal neighborhood; this can result in local overdispersion and in higher spatial and temporal dependencies due to direct and/or indirect transmission. In consequence, we test models including a Negative Binomial distribution (instead of the usual Poisson distribution) to deal with local overdispersion. We also use a specific spatio-temporal link in order to better model the stronger spatial and temporal dependencies due to the transmission of the disease. We have proposed and tested 60 Bayesian hierarchical models on 400 simulated datasets and bovine tuberculosis real data. This analysis shows the relevance of the CAR (Conditional AutoRegressive) processes to deal with the structure of the risk. We can also conclude that the negative binomial models outperform the Poisson models with a Gaussian noise to handle overdispersion. In addition our study provided relevant maps which are congruent with the real risk (simulated data) and with the knowledge concerning bovine tuberculosis (real data).

Severity of bovine tuberculosis is associated with innate immune-biased transcriptional signatures of whole blood in early weeks after experimental Mycobacterium bovis infection.

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a pathogen that impacts both animal and human health. Consequently, there is a need to improve understanding of disease dynamics, identification of infected animals, and characterization of the basis of immune protection. This study assessed the transcriptional changes occurring in cattle during the early weeks following a M. bovis infection. RNA-seq analysis of whole blood-cell transcriptomes revealed two distinct transcriptional clusters of infected cattle at both 4- and 10-weeks post-infection that correlated with disease severity. Cattle exhibiting more severe disease were transcriptionally divergent from uninfected animals. At 4-weeks post-infection, 25 genes had commonly increased expression in infected cattle compared to uninfected cattle regardless of disease severity. Ten weeks post-infection, differential gene expression was only observed when severely-affected cattle were compared to uninfected cattle. This indicates a transcriptional divergence based on clinical status following infection. In cattle with more severe disease, biological processes and cell type enrichment analyses revealed overrepresentation of innate immune-related processes and cell types in infected animals. Collectively, our findings demonstrate two distinct transcriptional profiles occur in cattle following M. bovis infection, which correlate to clinical status.

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.

BoLA-DRB3 exon2 polymorphisms among tuberculous cattle: Nucleotide and functional variability and their association with bovine tuberculosis pathology.

Bovine tuberculosis (bTB) is caused by Mycobacterium bovis and disseminated worldwide. In Argentina, the highest prevalence occurs in dairy areas. BoLA DRB3.2 is related to the adaptive immunity in mycobacterial infections. Genetic polymorphisms of this marker have been associated with resistance or susceptibility to bovine diseases. We evaluated the association between BoLA DRB3.2 polymorphisms and bTB pathology scores in dairy and beef cattle breeds of Argentina. Most bovines exhibited visible lesions compatible with tuberculosis and, furthermore, 150 (85.7%) were also positive by bacteriology. A pathology index showed a variable degree of disease, from 3 to 76 (median pathology score = 9 (IQR: 7-15)). Thirty-five BoLA DRB3.2 alleles were identified with an associated frequency from 16% to 0.3%, distributed 73% (n = 128) in heterozygosis and 27% (n = 47) in homozygosis, with 12 BoLA DRB3.2 alleles (*0101, *1101, *1501, *0201, *2707 *1001, *1002, *1201, *14011, *0501 *0902 and *0701) representing the 74.7% of the population variability. A functional analysis grouped them in 4 out of 5 clusters (A-D), suggesting a functional overlapping. Among the 90 identified genotypes, *1101/*1101, *1101/*1501 and *0101/*0101 were the most frequent (10%, 8.9% and 8.9%, respectively). No association was detected between the pathology scores and a specific DRB3.2 allele (p > .05). Animals infected with M. bovis spoligotype SB0153 showed a significantly higher pathology score than those affected by the spoligotype SB0145 (p = .018). Furthermore, the Aberdeen Angus breed exhibited highest pathological scores (p < .0001), which were associated with disseminated lesion, thus suggesting that the host component could be important to the disease progression.

Epidemiological analysis of bovine tuberculosis in the southern region of Rio Grande do Sul from 2000 to 2015 / Análise epidemiológica da tuberculose bovina na região sul do Rio Grande do Sul no período de 2000 a 2015

Bovine tuberculosis (BTB) is an infectious and zoonotic disease with socioeconomic importance, responsible for economic losses in livestock due to the decrease in animal productivity, mortality, bovine carcass condemnation and restriction on exportations. The objective of the present study was to evaluate the epidemiological aspects of this disease through a descriptive analysis and time series of cases of bovine tuberculosis diagnosed at the Laboratório Regional de Diagnóstico (LRD) of the Faculdade de Medicina Veterinária, Universidade Federal de Pelotas (UFPel) from 2000 to 2015. A survey was carried out in the LRD database of cases diagnosed as tuberculosis in cattle during this period. The incidence rate of BTB ([cases/population] x 100000) was calculated. Qualitative variables (sex, age, race, origin, type of material and presumptive diagnosis) were analyzed in a descriptive way, aiming to establish the profile of affected animals, characterize the municipalities in which the disease is originated and also fix the main referral organs for diagnosis. The time series analysis was performed using the Gretl statistical software 1.9.12 (GNU Regression, Econometric and Time-series Library). To verify the existence of the stationary distribution, the test of Dickey-Fuller was used, considering a value of p<0.05. During this period, 331 cases of BTB were diagnosed in the LRD. The present study identified that the predominant profile of the bovines affected by tuberculosis was female bovine aged more than two years old and crossbreed; the main organ sent for histopathological examination was lymph nodes. The microregion of Pelotas presented the highest incidence of the disease. There was also a tendency of a decrease in the incidence rate of this disease over the years, absence of seasonal influence in the occurrence of the disease and no cases of epidemics occurred from 2002 to 2015 in the area covered by the LRD-UFPel.(AU)

Tuberculose bovina é uma enfermidade infectocontagiosa e zoonótica, de importância socioeconômica, responsável por prejuízos econômicos na pecuária em decorrência da diminuição na produtividade animal, mortalidades, condenações de carcaças e restrições nas exportações. O objetivo do presente estudo foi avaliar os aspectos epidemiológicos, por meio de uma análise descritiva e de séries temporais, dos casos de tuberculose bovina diagnosticados no Laboratório Regional de Diagnóstico da Faculdade de Veterinária, Universidade Federal de Pelotas (LRD-UFPel), no período de 2000 a 2015. Foi realizado um levantamento no banco de dados do LRD dos casos diagnosticados como tuberculose em bovinos, entre os anos de 2000 e 2015. Foi calculada a taxa de incidência da tuberculose bovina ([casos/população] x 100000). As variáveis qualitativas (sexo, idade, raça, procedência, tipo de material e diagnóstico presuntivo) foram analisadas de forma descritiva, tendo como finalidade estabelecer o perfil dos animais acometidos, caracterizar os municípios de origem, e estabelecer os principais órgãos remetidos para diagnóstico. A análise de série temporal foi feita através do software estatístico Gretl 1.9.12 (GNU Regression, Econometricand Time-series Library) e para verificar a existência da estacionariedade utilizou-se o teste de Dickey-Fuller aumentado, considerando um valor de p<0,05. Durante este período foram diagnosticados 331 casos de tuberculose bovina no LRD. O presente estudo permitiu concluir que o perfil predominante dos bovinos acometidos por tuberculose foi de fêmeas, com mais de dois anos de idade e mestiças; o principal órgão encaminhado para exame histopatológico foram linfonodos e a microrregião de Pelotas apresentou a maior incidência da doença. Foi constatada, ainda, uma tendência de queda na taxa de incidência da doença ao longo dos anos analisados, ausência de influência sazonal na ocorrência da enfermidade e, não foram registrados casos de epidemias nos anos de 2002 a 2015, na área de abrangência do LRD-UFPel.(AU)

Bovine tuberculosis: diagnosis in dairy cattle through the association of analyzes / Tuberculose bovina: diagnóstico em bovinos leiteiros através da associação de análises

Tuberculosis is a chronic anthropozoonosis of worldwide occurrence, caused by the bacterium Mycobacterium tuberculosis and its variants. In Brazil, the National Program for the Control and Eradication of Brucellosis and Tuberculosis in cattle, is responsible for diagnosing and the correctly allocate positive animals, but there is still a lack of definitive diagnosis of the disease. This study described the use of five diagnostic tools that can be used, preferably together, for the confirmation of suspected cases. These tools included the clinical examination comparative cervical tuberculin test, macroscopic findings during the slaughtering and histopathology of the damaged tissues followed by histochemistry. We evaluated a total of 211 dairy cattle, where 15.1% (32/211) had classic clinical signs of bovine tuberculosis, 74 (35%) showed reactivity in the comparative cervical tuberculin test. Of the total number of animals, 141 (66.8%) were referred for sanitary slaughter due to legal and control issues in the outbreaks of the disease. In the follow-up of slaughtering and inspection of viscera and carcasses, 74 (52.5%) had macroscopic lesions compatible with bovine tuberculosis, while 67 (47.5%) showed no visible changes. During the inspection, fragments of lymph nodes and liver and lung parenchyma were collected from five cattle with macroscopic lesions and five with no lesions. The histopathological analysis showed numerous areas of caseous necrosis with or without central calcification and granulomatous inflammatory infiltrate. In the special staining of Ziehl-Neelsen, numerous acid-fast bacilli were evidenced in all cases.(AU)

A tuberculose é uma antropozoonose crônica de ocorrência mundial, causada pela bactéria Mycobacterium tuberculosis e suas variantes. No Brasil existe o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose em bovinos que viabiliza o diagnóstico e a destinação correta dos animais positivos, porém ainda há carência quanto ao diagnóstico da doença. Assim, este trabalho descreve a utilização de cinco ferramentas diagnósticas para a confirmação de casos suspeitos de tuberculose. As ferramentas utilizadas compreenderam o exame clínico, teste tuberculínico cervical comparativo, os achados macroscópicos durante o abate sanitário e a histopatologia dos tecidos lesados seguido de histoquímica. O estudo avaliou um total de 211 bovinos leiteiros, dos quais 15,1% (32/211) apresentaram sinais clínicos clássicos de tuberculose bovina, 35,1% (74/211) apresentaram reatividade no teste tuberculínico cervical comparativo, e 143 animais (67,8%) foram encaminhados para abate sanitário devido a questões legais e de controle nos focos da doença. No acompanhamento do abate e inspeção sanitária de vísceras e carcaças verificou-se que 51,8% (74/143) dos bovinos abatidos apresentavam lesões macroscópicas compatíveis com tuberculose bovina, enquanto 48,2% (69/143) não apresentavam alterações visíveis. Durante a inspeção foram coletados fragmentos de linfonodos e parênquima de fígado e pulmão de cinco bovinos com lesões macroscópicas e de cinco sem lesões, que na análise histopatológica apresentaram numerosas áreas de necrose caseosa com ou sem calcificação central e infiltrado inflamatório granulomatoso. Na coloração de Ziehl-Neelsen foram evidenciados numerosos bacilos álcool-ácido resistentes em todos os casos. Assim, diante dos resultados obtidos verifica-se que as análises empregadas no presente estudo foram de extrema importância para o diagnóstico acurado de tuberculose em bovinos.(AU)

Isolation and comparative genomics of Mycobacterium tuberculosis isolates from cattle and their attendants in South India.

The major human pathogen Mycobacterium tuberculosis is rarely reported to cause disease in other animals. Cases in livestock are thought to occur through contact with infected handlers, but previous studies evaluating putative livestock-human transmission used typing techniques with limited resolution. Here, we undertook cross-sectional surveillance for tuberculosis in 271 livestock handlers and 167 cattle on three farms in Chennai, India and defined the relatedness of cultured isolates using whole genome sequencing. Humans and livestock were screened for active mycobacterial infection, and opportunistic post-mortem examination was performed on comparative intradermal test-positive cattle that died. Four cattle and 6 handlers on two farms were culture-positive for M. tuberculosis; M. bovis was not isolated. All 10 isolates (one from each case) belonged to Lineage 1. Pairwise genome comparisons of single nucleotide polymorphism (SNP) differences ranged from 1 to 600 SNPs, but 3 isolate pairs were less than 5 SNPs different. Two pairs were from handlers and the third pair were from two cattle on the same farm. The minimum pairwise SNP difference between a cattle and human isolate was >250 SNPs. Our study confirms the presence of M. tuberculosis infection in cattle in India, sequencing of which characterised relatedness between human and cattle-derived isolates.

Assessing effects from four years of industry-led badger culling in England on the incidence of bovine tuberculosis in cattle, 2013-2017.

The objective was to measure the association between badger culling and bovine tuberculosis (TB) incidents in cattle herds in three areas of England between 2013-2017 (Gloucestershire and Somerset) and 2015-2017 (Dorset). Farming industry-selected licensed culling areas were matched to comparison areas. A TB incident was detection of new Mycobacterium bovis infection (post-mortem confirmed) in at least one animal in a herd. Intervention and comparison area incidence rates were compared in central zones where culling was conducted and surrounding buffer zones, through multivariable Poisson regression analyses. Central zone incidence rates in Gloucestershire (Incidence rate ratio (IRR) 0.34 (95% CI 0.29 to 0.39, p < 0.001) and Somerset (IRR 0.63 (95% CI 0.58 to 0.69, p < 0.001) were lower and no different in Dorset (IRR 1.10, 95% CI 0.96 to 1.27, p = 0.168) than comparison central zone rates. The buffer zone incidence rate was lower for Gloucestershire (IRR 0.64, 95% CI 0.58 to 0.70, p < 0.001), no different for Somerset (IRR 0.97, 95% CI 0.80 to 1.16, p = 0.767) and lower for Dorset (IRR 0.45, 95% CI 0.37 to 0.54, p < 0.001) than comparison buffer zone rates. Industry-led culling was associated with reductions in cattle TB incidence rates after four years but there were variations in effects between areas.

A natural gene drive system influences bovine tuberculosis susceptibility in African buffalo: Possible implications for disease management.

Bovine tuberculosis (BTB) is endemic to the African buffalo (Syncerus caffer) of Hluhluwe-iMfolozi Park (HiP) and Kruger National Park, South Africa. In HiP, the disease has been actively managed since 1999 through a test-and-cull procedure targeting BTB-positive buffalo. Prior studies in Kruger showed associations between microsatellite alleles, BTB and body condition. A sex chromosomal meiotic drive, a form of natural gene drive, was hypothesized to be ultimately responsible. These associations indicate high-frequency occurrence of two types of male-deleterious alleles (or multiple-allele haplotypes). One type negatively affects body condition and BTB resistance in both sexes. The other type has sexually antagonistic effects: negative in males but positive in females. Here, we investigate whether a similar gene drive system is present in HiP buffalo, using 17 autosomal microsatellites and microsatellite-derived Y-chromosomal haplotypes from 401 individuals, culled in 2002-2004. We show that the association between autosomal microsatellite alleles and BTB susceptibility detected in Kruger, is also present in HiP. Further, Y-haplotype frequency dynamics indicated that a sex chromosomal meiotic drive also occurred in HiP. BTB was associated with negative selection of male-deleterious alleles in HiP, unlike positive selection in Kruger. Birth sex ratios were female-biased. We attribute negative selection and female-biased sex ratios in HiP to the absence of a Y-chromosomal sex-ratio distorter. This distorter has been hypothesized to contribute to positive selection of male-deleterious alleles and male-biased birth sex ratios in Kruger. As previously shown in Kruger, microsatellite alleles were only associated with male-deleterious effects in individuals born after wet pre-birth years; a phenomenon attributed to epigenetic modification. We identified two additional allele types: male-specific deleterious and beneficial alleles, with no discernible effect on females. Finally, we discuss how our findings may be used for breeding disease-free buffalo and implementing BTB test-and-cull programs.

Enzyme-linked immunosorbent assay as complement of intradermal skin test for the detection of mycobacterium bovis infection in cattle.

Diagnostic tests based on cell-mediated immunity are used in programs for the control and eradication of bovine tuberculosis (bTB), which is mainly caused by Mycobacterium bovis. Additional serological assays could be performed as an ancillary method to detect an infected animal that fails to produce an immune response against the intradermal reaction (IDR), the official bTB test. In this study, we evaluated the effectiveness of an enzyme-linked immunosorbent assay (ELISA) that uses bovine PPD as a capture antigen as a complement to the IDR in herds with confirmed cases of bTB. The study was conducted in two stages. First, a panel of 200 serum samples was analyzed by ELISA. The sensitivity and specificity obtained were 60% and 99%, respectively. The subsequent stage consisted of evaluating 7,494 bovines from 14 selected dairy farms. The number of animals yielding a IDR negative/ELISA positive result were 200. A necropsy analysis of 33 of these IDR negative/ELISA positive animals revealed that 30 (91%) presented granulomatous lesions and positive M. bovis isolation. This finding confirmed bTB in most cases. Altogether, the results obtained in the present study suggest that the combined use of IDR and ELISA is an effective strategy to improve the control of bTB in endemic herds.

Impact of Mycobacterium bovis-induced pathology on interpretation of QuantiFERON®-TB Gold assay results in African buffaloes (Syncerus caffer).

The cytokine interferon gamma-inducible protein 10 (IP-10) is a sensitive biomarker of Mycobacterium bovis (M. bovis) infection in African buffaloes (Syncerus caffer). However, elevated levels of IP-10 in QuantiFERON®-TB Gold (QFT) unstimulated whole blood compromises the utility of this biomarker. In this study, IP-10 and interferon gamma (IFN-γ) concentrations in whole blood samples from M. bovis culture-confirmed buffaloes with varying degrees of pathological changes (n = 72) and uninfected controls (n = 70) were measured in the IP-10 release assay (IPRA) and IFN-γ release assay (IGRA), respectively. Findings suggest that concentrations of both cytokines in QFT Nil tubes were higher in infected buffaloes with macroscopic pathological changes consistent with bovine tuberculosis compared to uninfected controls, and IGRA values increased with more severe pathological changes in infected buffaloes (p < 0.05). Finally, in culture-confirmed buffaloes with IPRA-negative and IGRA-positive test results, most animals were also those with the most advanced pathology. We conclude that IP-10 and IFN-γ concentrations measured in QFT Nil tubes may provide insight into the presence of M. bovis pathology in infected buffaloes. Furthermore, this study highlights the value in evaluating cytokine production in both antigen-stimulated and unstimulated samples when interpreting cytokine release assay results.

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.

Atypical granuloma formation in Mycobacterium bovis-infected calves.

Bovine tuberculosis is a chronic inflammatory disease that causes granuloma formation. Characterization of granulomatous lesions of Mycobacterium bovis (M. bovis) experimentally infected cattle has helped to better understand the pathogenesis of this disease. However, few studies have described granulomas found in M. bovis naturally infected cattle. The aim of this work was to examine granulomas from Holstein-Friesian cattle naturally infected with M. bovis from a dairy basin located in the central region of Mexico. Tissue samples from thirty-two cattle with lesions suggestive of tuberculosis were collected post-mortem. Fifteen of the 32 sampled animals (46.8%) were 4 months of age or younger (calves), whereas the rest (53.2%, 17/32) were over one year old (adults). Macroscopic lesions suggestive of tuberculosis were found in the mediastinal lymph node chain of all animals (32/32). From the 1,143 granulomatous lesions that were microscopically analyzed, 34.6% (396/1143) were collected from adult animals and subsequently classified according to the nomenclature suggested by Wangoo et al., 2005. Surprisingly, lesions from calf tissues showed an atypical pattern which could not be fitted into the established developmental stages of this classification. Granulomatous lesions found in calves covered most of the affected organ, histologically showed large necrotic areas with central calcification, absence of a connective tissue capsule, and few giant cells. Also, there was a higher percentage of lesions with acid-fast bacilli (AFB) when compared to studied granulomas in adults. Growth of Mycobacterium spp was detected in 11 bacteriological tissue cultures. Genotypic identification of M. bovis was performed by DNA extraction from bacterial isolates, formalin-fixed and paraffin-embedded (FFPE) tissues and samples without bacterial isolation. M. bovis was detected by PCR in 84.3% (27/32) of the studied cases; whereas other AFB were observed in tissues of the remaining sampled animals (5/32). Our results describe atypical granuloma formation in calves 4 months of age or younger, naturally infected with M. bovis. These findings contribute to better understanding the physiopathology of M. bovis infection in cattle.

Kallikrein 12 Regulates Innate Resistance of Murine Macrophages against Mycobacterium bovis Infection by Modulating Autophagy and Apoptosis.

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1ß, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-ß expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.

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.