Extensive differential DNA methylation between tuberculosis skin test positive and skin test negative cattle.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), represents a significant problem for the agriculture industry as well as posing a risk for human health. Current diagnostic tests for bTB target the cell-mediated immune (CMI) response to infection with M. bovis, primarily through screening of animals with the tuberculin skin test. Epigenetic modifications have been shown to alter the course of the immune response and differentially methylated regions (DMRs) might also influence the outcome of the skin test in cattle. Whole Genome Bisulphite Sequencing (WGBS) was used to profile DNA methylation levels from peripheral blood of a group of cattle identified as test positive for M. bovis (positive for the single intradermal comparative tuberculin test (SICTT) and/or the interferon-γ release assay compared to a test negative control group [n = 8/group, total of 16 WGBS libraries]. Although global methylation profiles were similar for both groups across the genome, 223 DMRs and 159 Differentially Promoter Methylated Genes (DPMGs) were identified between groups with an excess of hypermethylated sites in SICTT positive cattle (threshold > 15% differential methylation). Genes located within these DMRs included the Interleukin 1 receptor (IL1R1) and MHC related genes (BOLA and BOLA-DQB). KEGG pathway analysis identified enrichment of genes involved in Calcium and MAPK signalling, as well as metabolism pathways. Analysis of DMRs in a subset of SICTT negative cattle that were IFN-γ positive showed differential methylation of genes including Interleukin 10 Receptor, alpha (IL10RA), Interleukin 17 F (IL17F) and host defence peptides (DEFB and BDEF109). This study has identified a number of immune gene loci at which differential methylation is associated with SICTT test results and the degree of methylation could influence effective host immune responses.

Bovine tuberculosis in Central Ethiopian slaughterhouses and the identification of causative mycobacteria by multiplex real-time PCR.

BACKGROUND: Bovine tuberculosis (bTB) is a chronic disease caused by members of the Mycobacterium tuberculosis complex (MTBC) that ultimately leads to the development of progressive granulomatous lesions. Although the disease is widespread, especially in crossbred cattle in Ethiopia, routine investigations and surveillance are lacking. Thus, the aim of this study was to determine the prevalence, associated risk factors, and species of mycobacteria causing bTB in slaughtered cattle at four slaughterhouses in Central Ethiopia. METHODS: Postmortem examination of 7,640 cattle was conducted using a cross-sectional slaughterhouse survey. A total of 388 tuberculous-like lesions (TBLs) were collected from 173 animals and cultured. Six target genes were used to differentiate mycobacterial species using multiplex real-time PCR (mRT-PCR). Multivariate logistic regression analyses and related odds ratios (ORs) were used to gauge the strength of the associations between risk factors, TBL incidence and culture growth. RESULTS: The prevalence of TBL was 2.3% (95% CI = 2.0-2.6). Logistic regression analysis indicated an increased risk of TBL in crossbred cattle (OR = 11.8, 95% CI: 6.4, 21.2, p < 0.001). Animals slaughtered at Adama (OR = 3.2, 95% CI: 1.2, 7.3, p = 0.009) or Burayu (OR = 5.8, 95% CI: 3.9, 8.9, p < 0.001) had a greater risk of TBL than those slaughtered at Sululta. There were significantly more TBL-positive lesions in the lungs and lymph nodes related to the lung (OR = 7.1; 95% CI: 2.7, 24.5, p < 0.001) and the head lymph node (OR = 5.6; 95% CI: 1.8, 21.7; p = 0.006) compared to gut associated lymph nodes. Among the 173 TBL-positive animals, 36% (95% CI = 28.8, 43.2), and among the 388 TBL-positive tissues, 24.2% (95% CI = 20, 29) were culture and mRT-PCR positive. All the culture-generated isolates were positive for M. bovis in mRT-PCR. Among them, two animals had mixed infections including one zebu cattle tested positive for both M. caprae and M. bovis, and a crossbred cow tested positive for both M. tuberculosis and M. bovis in mRT-PCR. This suggests persistent transmission within the cattle population, posing a substantial public health threat. CONCLUSION: This study revealed an eleven-fold greater risk of bTB-related lesions in crossbred cattle compared to local zebu cattle. This finding highlights the necessity for targeted interventions, continuous vigilance, and thorough carcass inspection to mitigate public health risks.

A systematic review and meta-analysis of bovine tuberculosis occurrence and burden in Bangladesh, 1970-2023.

We aimed to estimate the overall apparent prevalence, true prevalence, and the spatial, temporal, and test-specific burden of bovine tuberculosis in Bangladesh. PubMed, Web of Science, Scopus, Google Scholar, and BanglaJOL were searched for bovine tuberculosis publications in Bangladesh from 1 January 1970 to 23 June 2023. Of 142 articles screened, systematic review and meta-analysis were performed on 22 (15.5%) articles. The apparent estimated bovine tuberculosis prevalence was 7%. The apparent Bayesian pooled mean bovine tuberculosis prevalences based on caudal fold test and single intradermal comparative tuberculin test were 7.83% and 9.89%, respectively, and the true pooled mean prevalences were 10.39% and 10.48%, respectively. Targeted interventions are recommended for districts with higher prevalence to effectively reduce the bovine tuberculosis burden in those areas. Current diagnostic practices employed in Bangladesh may not accurately reflect the bovine tuberculosis burden. Our findings highlight the need for better diagnostic tools and supplemental testing methods to ensure accurate diagnosis and surveillance. Efforts should prioritize obtaining 'true' prevalence estimates corrected for misclassification bias, rather than relying solely on apparent prevalence. Underestimating the bovine tuberculosis burden could result in inadequate resource allocation and hinder the implementation of effective control measures.

Bacteriological culture and direct PCR for detecting the Mycobacterium tuberculosis complex in the Italian eradication campaign: a decade of experience at the National Reference Laboratory.

AIMS: Our study evaluates the capacity of direct real-time PCR for detecting Mycobacterium tuberculosis complex (MTBC), with a focus on diagnostic performances and the feasibility of implementing this protocol in an eradication campaign. Specifically, we compare the effectiveness of the direct PCR method to various culture systems used by the Italian National Reference Laboratory over the last decade to detect MTBC. METHODS AND RESULTS: Bovine tissue samples were routinely tested and analyzed for bovine tuberculosis (bTB) confirmation using microbiological culture (solid and liquid media), histopathological analysis, and a direct PCR assay targeting IS6110, an insertion sequence specific to the MTBC that is widely used for tuberculosis diagnosis. The direct real-time PCR demonstrated a high concordance (K = 0.871) with microbiological culture, as well as good sensitivity (91.84%) and specificity (95.24%). In contrast, histopathology demonstrated lower concordance (K = 0.746) and performance levels (sensitivity 91.41%, specificity 82.88%). Liquid media promoted faster and more efficient growth of MTBC than solid media. M. bovis and M. caprae had the comparable ability to respond to the direct real-time PCR test and grow on the microbiological medium. CONCLUSIONS: This study confirms that direct real-time PCR can detect MTBC with high diagnostic accuracy within a few days. This study found no significant differences in performance between culture media and direct PCR for M. bovis and M. caprae.

Comparative analysis of tuberculin and defined antigen skin tests for detection of bovine tuberculosis in buffaloes (Bubalus bubalis) in Haryana state, India.

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

Challenges in diagnosing bovine tuberculosis through surveillance and characterization of Mycobacterium species in slaughtered cattle in Kolkata.

BACKGROUND: Tuberculosis in cattle is caused by Mycobacterium tuberculosis complex (MTBC) species. Apart from MTBC, different Nontuberculous Mycobacteria (NTM) species have also been isolated from cattle. The presence of NTM infection in bovines makes the diagnosis of bovine tuberculosis (bTB) a cumbersome task. Therefore, a cross sectional study was conducted to isolate and characterize different Mycobacterium spp. from a slaughterhouse situated in Kolkata, a city in the eastern part of India. RESULTS: Out of 258 morbid samples, 98 isolates were found to be positive for bacterial growth, and 35% (n = 34) were positive for Mycobacterium. 94% of Mycobacterial cultural isolates were NTM (n = 32), and the rest (n = 2) were found to be MTBC. Species-level identification of the isolates by hsp65 sequencing revealed that out of 32 isolates, 24 were M. fortuitum, three were M. abscessus, two each were M. chelonae and M. parascrofulaceum, and one was M. novocastrense. A phylogenetic tree with partial hsp65 gene sequences was also constructed to determine the relatedness of the unknown isolates to the reference strains. CONCLUSION: Both NTM species and MTBCs were identified from TB-like lesions in cattle that were slaughtered at the Kolkata abattoir. This discovery may indicate that NTM contributes to the development of lesions in cattle. Also, we recommend implication of more specific diagnostic tests for bTB.

Prevalence and molecular characterization of Mycobacterium tuberculosis complex in cattle and humans, Maiduguri, Borno state, Nigeria: a cross-sectional study.

INTRODUCTION: Globally, the highest burden of bovine and human tuberculosis resides in Africa and Asia. Tuberculosis (TB) is the second leading single infectious killer after severe acute respiratory syndrome corona virus-2 (SARSCOV-2). Bovine TB remains a treat to wild and domesticated animals, humans and hinders international trade in endemic countries like Nigeria. We aimed at determining the prevalence of bovine and human tuberculosis, and the spoligotypes of Mycobacterium tuberculosis complex in cattle and humans in Maiduguri. METHODS: We conducted a cross sectional study on bovine and human tuberculosis in Maiduguri, Borno state. We calculated sample size using the method of Thrusfield. Lesions suggestive of TB from 160 slaughtered cattle were obtained from Maiduguri Central Abattoir. Sputum samples from humans; 82 abattoir workers and 147 suspected TB patients from hospitals/clinics were obtained. Lesions and sputum samples were cultured for the isolation of Mycobacterium spp. Positive cultures were subjected genus typing, deletion analysis and selected isolates were spoligotyped. Data was analysed using SPSS VERSION 16.0. RESULTS: Prevalence of 32.5% (52/160) was obtained in cattle. Damboa local government area (LGA), where majority of the infected animals were obtained from had 35.5% bTB prevalence. All categories analysed (breed, age, sex, body conformation and score) had P-values that were not significant (P > 0.05). Sputum culture revealed a prevalence of 3.7% (3/82) from abattoir workers and 12.2% from hospitals/clinics. A significant P-value (0.03) was obtained when positive culture from abattoir and that of hospitals/clinics were compared. Out of the 52 culture positive isolates obtained from cattle, 26 (50%) belonged to M. tuberculosis complex (MTC) and 17/26 (65.4%) were characterized as M. bovis. In humans, 7/12 (58.3%) MTC obtained were characterized as M. tuberculosis. Spoligotyping revealed SB0944 and SB1025 in cattle, while SIT838, SIT61 of LAM10_CAM and SIT1054, SIT46 of Haarlem (H) families were obtained from humans. CONCLUSIONS: Cattle in Damboa LGA need to be screened for bTB as majority of the infected animals were brought from there. Our findings revealed the presence of SB0944 and SB1025 spoligotypes from cattle in Borno state. We isolated M. tuberculosis strain of the H family mainly domiciled in Europe from humans.

Evaluation of the performance of the IFN-γ release assay in bovine tuberculosis free herds from five European countries.

The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1-26.3% (Bovigam: ODbovis-ODPBS ≥ 0.1 and ODbovis-ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98-99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status.

Development and validation of a one-tube, nested real-time PCR method suitable for routine detection of Mycobacterium bovis in animal tissue.

AIMS: Development and validation of a real-time PCR test for high-throughput routine screening of animal tissue for Mycobacterium bovis and other Mycobacterium tuberculosis complex (MTBC) members. METHODS AND RESULTS: A preliminary study compared the results of a combination of five tissue preparation/DNA extraction methods and nine PCR assays on a panel of 92 cattle tissue samples of known M. bovis culture status (55 positive and 37 negative). The combination of DNA extraction and PCR was found to be important in achieving optimal detection of M. bovis. The optimal combination of a simple tissue preparation/DNA extraction method and a one-tube, nested real-time PCR to maximize the sensitivity of detection of an M. bovis-specific RD4 deletion and an IS1081 MTBC-specific target was selected for further evaluation. In total, tissue samples collected from 981 cattle and 366 non-bovine animals and submitted for routine TB culture were parallel tested with the selected method, as well as tissue samples obtained from 156 animals in certified TB-free cattle herds. CONCLUSION: For cattle, the optimized RD4-IS1081 PCR test exhibited a diagnostic sensitivity of 96% (95% CI: 94-97%) and specificity of 97% (95% CI: 95-98%) compared to culture. Specificity was 100% when testing the 156 samples from known TB-free cattle. For non-bovine species, the PCR had a diagnostic sensitivity of 93% (95% CI: 83-98%) and a specificity of 99% (95% CI: 97-100%).

[An Infant with Zoonotic Pulmonary Tuberculosis due to Mycobacterium bovis]. / Mycobacterium bovis'e Bagli Zoonotik Pulmoner Tüberküloz Tanili Bir Infant.

Bovine tuberculosis might be seen in low-income countries, especially in children fed with raw milk. The most common transmission route is fecal-oral way, and it is most likely through unpasteurized dairy products. Although clinical and radiological findings are like non-zoonotic tuberculosis, treatment approaches may differ in individuals with zoonotic tuberculosis. Prevention of zoonotic diseases requires multidisciplinary approaches. These approaches include the development of veterinary and surveillance studies for the detection of communicable diseases in farm animals, as well as informing the public about raw milk consumption. In this case report, a patient with zoonotic pulmonary tuberculosis related to Mycobacterium bovis because of consumption of raw milk was presented. A five-month-old male was admitted to the hospital due to a persistent, feverless, non-productive cough since birth. Empirical antibiotic treatment was started with a preliminary diagnosis of pneumonia because of left upper lobe and right pericardial infiltration on chest X-ray. However, after two weeks of antimicrobial therapy, the patient's clinical and laboratory findings did not improve. This led to the referral for a computed tomography imaging, which revealed tracheomalacia, consolidation on the right upper lobe, an indistinguishable mass or consolidation on the left middle lobe of the lung, peribronchial thickening on the basal segment of the lower lobe, and mediastinal lymphadenopathy. Three consecutive days of fasting gastric lavage fluid was sent to the reference laboratory for acid-resistant bacillus examination, polymerase chain reaction (PCR) and culture studies. As the clinical findings were compatible and PCR was positive, the patient was started on quadruple antituberculous therapy. After initiation of anti-tuberculosis drugs, the patient's findings radiologically and clinically were improved. Mycobacterium bovis was grown in the culture. In the meantime, it was discovered that the patient was fed with raw milk. Due to the patient's clinical symptoms and the growth of Mycobacterium bovis in the gastric lavage fluid culture, the diagnosis of bovine tuberculosis was made. The culprit was that the milk of the cow belonging to the patient's family, which was later found to be infected with M.bovis, was milked and given to the patient without boiling. Today, unpasteurized dairy products continue to be consumed, especially in rural areas. One of the most important steps to prevent zoonotic diseases is to raise awareness about not consuming raw milk and undercooked meat. To elucidate the epidemiological link in childhood, taking a good anamnesis, including questioning raw milk consumption, is essential in the diagnosis of tuberculosis.

Real-time PCR using atpE, conventional PCR targeting different regions of difference, and flow cytometry for confirmation of Mycobacterium bovis in buffaloes and cattle from the Delta area of Egypt.

BACKGROUND: Mycobacterium bovis notoriously causes detrimental infections in bovines and humans. In this study, 1500 buffaloes and 2200 cattle were tested by single intradermal comparative cervical tuberculin test and compared with the detection rates of M. bovis isolation, real-time and simplex PCR, and flow Cytometry. RESULTS: The tuberculin test is the reference test in Egypt, the positive rate was 54/3700 (1.5%) composed of 18/1500 (1.2%) buffaloes and 36/2200 (1.6%) cattle which were mandatorily slaughtered under the Egyptian legislation, after postmortem examination the non-visible-lesion proportion was 39/54 (72.2%) which surpassed the visible-lesion rate 15/54 (27.8%) with (p < 0.0001). The samples from each case were pooled into one sample representing the case, and the isolation rate of M. bovis was 25/54 (46.3%). Real-time PCR using atpE was positive for mycobacteria on the genus level in 18/18 (100%) and 5/5 (100%) of tissue samples and isolates, respectively; simplex PCR detected M. bovis in 44/54 (81.5%) and 25/25 (100%) of tissue samples and isolates, respectively. Flow Cytometry evaluation of the CD4+, CD8+, WC1+δγ, and CD2+ cell phenotypes showed increased counts in the tuberculin-positive cases compared with negative cases (p < 0.0001), and these phenotypes in the tuberculin-positive cases increased after antigen stimulation than in the negative cases (p < 0.0001). Detection rates of PCR techniques and flow Cytometry exceeded that of bacterial isolation (p < 0.0001) and exhibited a strong correlation. CONCLUSIONS: The skin test suffers from interference from non-tuberculous mycobacteria able to cause false-positive reactions in cattle and other species. Real-time PCR using atpE, conventional PCR targeting RDs, and flow Cytometry are rapid and accurate methods that correlate with the isolation and can be promising for detection and confirmation of infected live and slaughtered cases.

Untargeted metabolomic analysis of thoracic blood from badgers indicate changes linked to infection with bovine tuberculosis (Mycobacterium bovis): a pilot study.

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.

Development and evaluation of a Mycobacterium bovis interferon-γ enzyme-linked immunospot (ELISpot) assay for detection of bovine tuberculosis.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important zoonotic disease. This infection is difficult to control because of the limited ability of the tuberculin skin test (TST) and ancillary IFN-γ release assay to detect all infected animals. In this study, we aimed to develop an efficient assay based on the enzyme-linked immunospot (ELISpot) technique for the diagnosis of bTB, with IFN-γ monoclonal antibodies 3E9 and Bio-labeled 6F8 used as capture and detection antibodies, respectively. As expected, there were significantly more M. bovis-specific spot-forming units (SFU) in bTB-infected cattle than in healthy cattle when an M. bovis-specific antigen, CFP-10-ESAT-6 fusion protein (CE protein), was used. The M. bovis IFN-γ ELISpot assay demonstrated a high level of agreement (90.83%) with the BOVIGAM ELISA test (Thermo Fisher Scientific) for detecting bTB. Furthermore, 3 of 109 cattle tested negative by both the TST and the BOVIGAM ELISA tests, but positive by the ELISpot assay (TST- ELISA- ELISpot+). During subsequent long-term monitoring, these 3 cattle became TST+ ELISA+ ELISpot+. These results suggest that the M. bovis IFN-γ ELISpot assay we established could detect infected cattle earlier than the BOVIGAM ELISA test.

KLK12 Regulates MMP-1 and MMP-9 via Bradykinin Receptors: Biomarkers for Differentiating Latent and Active Bovine Tuberculosis.

It has been established that kallikrein12 (KLK12) expression is closely related to bovine tuberculosis (bTB) development. Herein, we sought to clarify the regulatory mechanism of KLK12 and its application in tuberculosis diagnosis. KLK12 knockdown macrophages were produced by siRNA transfection. Bradykinin receptors (BR, including B1R and B2R) were blocked with specific inhibitors. Mannose-capped lipoarabinomannan (ManLAM) was extracted from Mycobacterium bovis (M. bovis) and used to study the mechanism of KLK12 activation. In addition, we constructed different mouse models representing the latent and active stages of M. bovis infection. Mouse models and clinical serum samples were used to assess the diagnostic value of biomarkers. Through the above methods, we confirmed that KLK12 regulates MMP-1 and MMP-9 via BR. KLK12 upregulation is mediated by the M. bovis-specific antigen ManLAM. KLK12, MMP-1, and MMP-9 harbor significant value as serological markers for differentiating between latent and active bTB, especially KLK12. In conclusion, we identified a novel signaling pathway, KLK12/BR/ERK/MMPs, in M. bovis-infected macrophages, which is activated by ManLAM. From this signaling pathway, KLK12 can be used as a serological marker to differentiate between latent and active bTB. Importantly, KLK12 also has enormous potential for the clinical diagnosis of human tuberculosis (TB).

Evaluation of using comparative intradermal tuberculin test to diagnose bovine tuberculosis in dairy cattle in Taiwan.

The National Bovine Tuberculosis (bTB) Eradication Program for dairy cattle has been operating in Taiwan for many years and has allowed the prevalence of bTB to decrease gradually. However, 29% of intradermal tuberculin test (ITT)-positive dairy cows were later found to be TB negative based on necropsy, histopathological examination, and mycobacterial isolation results. Studies in Taiwan have indicated that Mycobacterium avium subsp. paratuberculosis (MAP) may lead to false-positive ITT. Due to the high prevalence (over 90%) of paratuberculosis (PTB) serum antibody among Taiwan's farms, comparative ITT (CITT) has been recommended to differentiate between bTB and PTB infections. In this study, we used ITT, CITT, and enzyme-linked immunosorbent assay (ELISA) to evaluate the prevalence of bTB from 2012 to 2018. We also used pathological and bacterial examination from ITT-positive dairy cows to evaluate CITT's diagnostic ability and adjust its cutoff point accordingly. After careful selection, 36 cows (including 31 cows from 11 ITT-positive farms and 5 from 2 ITT-negative farms) were examined by CITT. The cutoff point was adjusted using a receiver operating characteristic (ROC) analysis. Overall, our results identified the ITT-positive prevalence in Taiwan as 0.03-0.22%, and PTB-positive prevalence as 54.55-73.53%. The results of sensitivity, specificity, kappa, and ROC analyses have identified the optimal cutoff point for the CITT in Taiwan as ≥ 3 mm. At this cutoff point value, the sensitivity and specificity were 62.5% and 96.43%, respectively. Our findings can be used to reduce the false-positive response rate caused by PTB cross-reaction and accelerate the eradication of bTB in Taiwan.

Apparent prevalence and risk factors for bovine tuberculosis in the state of Paraná, Brazil: an assessment after 18 years since the beginning of the Brazilian program.

Bovine tuberculosis (bTB) impacts considerably animal production and one health worldwide. To describe the prevalence, risk factors, and spatial pattern of the disease in the state of Paraná, Brazil, a cross-sectional study was conducted from September 2018 to February 2019. The area was divided into seven regions. Within each region, farms were randomly selected, and a predetermined number of cows was selected and tested by a comparative cervical tuberculin test. 17,210 animals were tested across 1757 farms. Herd prevalence of bTB-infected herds in Paraná was 2.5% [1.87-3.00%]. It has varied from 0.8 to 3.98% among seven regions, with clustering being detected in the west, central, and northeast areas. Animal prevalence was 0.35% [0.21-0.59%] and has varied from 0.08 to 0.6% among the pre-set regions. No major shifts in the prevalence of bTB were detected since 2007. Large-sized herds, dairy production, and feeding with whey were detected to be correlated with the presence of bTB. Exclusively among dairy herds, veterinary assistance from cooperatives, possession of self-owned equipment to cool milk, and feeding with whey were correlated with the disease. Considering these results, it is recommended that the state of Paraná seek to implement a surveillance system for the detection of bTB-infected herds transforming them into free ones, if possible, incorporating elements of risk-based surveillance. Health education is also recommended to inform farmers about the risks of introducing animals without testing and of feeding raw whey to calves.

Effect of selective removal of badgers (Meles meles) on ranging behaviour during a 'Test and Vaccinate or Remove' intervention in Northern Ireland.

The role of the Eurasian badger (Meles meles) as a wildlife host has complicated the management of bovine tuberculosis (bTB) in cattle. Badger ranging behaviour has previously been found to be altered by culling of badgers and has been suggested to increase the transmission of bTB either among badgers or between badgers and cattle. In 2014, a five-year bTB intervention research project in a 100 km2 area in Northern Ireland was initiated involving selective removal of dual path platform (DPP) VetTB (immunoassay) test positive badgers and vaccination followed by release of DPP test negative badgers ('Test and Vaccinate or Remove'). Home range sizes, based on position data obtained from global positioning system collared badgers, were compared between the first year of the project, where no DPP test positive badgers were removed, and follow-up years 2-4 when DPP test positive badgers were removed. A total of 105 individual badgers were followed over 21 200 collar tracking nights. Using multivariable analyses, neither annual nor monthly home ranges differed significantly in size between years, suggesting they were not significantly altered by the bTB intervention that was applied in the study area.

Study on supplemental test to improve the detection of bovine tuberculosis in individual animals and herds.

BACKGROUND: Bovine tuberculosis (bTB), is a worldwide disease caused by Mycobacterium bovis (M. bovis). The success of bTB eradication and control programs is based on early detection and the removal of reactors from a herd thus routine testing and cull strategy have been applied globally. Since the late nineteenth century, the Tuberculin Skin Test (TST) has been the primary antemortem test available to support bTB eradication campaigns. Due to the TST limitations in terms of Se and Sp, the credibility of the diagnosis is frequently questioned given the occurrence of false-positive and false-negative reactions, therefore, it is necessary to confirm reactive animals using other methods, ensuring the reliability of the diagnosis. The aim of this study was to evaluate the sensitivity and specificity of a multiplex enzyme-linked immunosorbent assay (ELISA) relative to the tuberculin test used for the diagnosis of tuberculosis in cattle in Brazil. RESULTS: Lack of agreement between comparative cervical tuberculin test and ELISA IDEXX TM was observed. The 2 animals positive on the comparative cervical tuberculin test did not react at the ELISA IDEXX TM and 22 negative reactors by comparative cervical tuberculin test were positive by the ELISA IDEXX TM. The ELISA IDEXX TM showed sensitivity that is significantly lower than the official screening test the single cervical tuberculin. ELISA IDEXX TM also detected infected animals and herds undetected by the comparative cervical tuberculin test. The parallel use of comparative cervical tuberculin test and ELISA IDEXX TM increased sensitivity and the feasibility bTB screening. CONCLUSIONS: The results obtained here suggest that the ELISA IDEXX TM may be a supplemental test for the detection of Mycobacterium bovis infection in regions without routine testing and slaughter, where the disease generally progresses to more advanced stages and antibody responses are likely to be more prevalent. Evidence to support the validation of the ELISA IDEXX™ as a supplemental test for bTB eradication programs was provided.

Evaluation of ELISA and PCR in parallel to single intradermal cervical tuberculin test (SICT) for diagnosis of tuberculosis in buffaloes.

Bovine tuberculosis is an economically important disease with very high zoonotic potential. Single intradermal cervical tuberculin test (SICT) is considered a gold standard assay for the diagnosis of bovine tuberculosis. However, bovines especially buffaloes may produce a false negative result when the animal becomes cell-mediated immune (CMI) anergic in the advanced stage of the disease. In the present study, ELISA and PCR assays were successfully demonstrated to be useful in diagnosing tuberculosis especially in the CMI anergic buffaloes infected with Mycobacterium bovis. ELISA and PCR assays are able to detect 8.94% and 8.13%, respectively, more animals as positive in comparison to standard SICT assay in a selected population of 123 buffaloes. The moderate agreement between SICT and ELISA (k: 0.528; 0.249-0.807), a substantial agreement between SICT and PCR (k: 0.648; 0.364-0.931), and high agreement between ELISA and PCR (k: 0.856; 0.697-1.0) highlight that ELISA and PCR, if used in parallel with SICT, will provide better sensitivity over single assay. Reduction of false negative reactors may help in minimizing the zoonotic threat from bovine tuberculosis especially in disease endemic region where human and livestock interface is quite high.

Effect of co-positivity for brucellosis and tuberculosis on milk yield and fertility of Holstein cows.

This study aimed to determine whether cows detected as tuberculosis (bTB) reactors and seropositive to brucellosis (bBR), as well as co-positive to bBR and bTB (bBR-bTB) and with a complete lactation before slaughter, were associated with reduced milk production and fertility. A total of 8068 productive and reproductive records of high-yielding Holstein cows from a single large dairy herd with a high prevalence of bTB and bBR were collected from 2012 to 2015. Lactation derived either from calving (n = 6019) or hormonally induced lactation (n = 2049), and all cows received growth hormone throughout lactation. For cows not induced into lactation, pregnancy rate to first service for healthy cows (C; 26.6%) was higher (P < 0.01) than bBR (15.2%), bTB (15.8%), and bBR-bTB (1.3%) cows. For induced cows, pregnancy rate to first service did not differ significantly among C, bBR, and bTB (14.5-17.3%) cows, but the percentage success of first service was extremely low (1.3%; P < 0.01) in bBR-bTB cows. Services per pregnancy (only pregnant cows) were lowest for C (3.3 ± 2.9; P < 0.01) and highest (6.4 ± 3.4) for bBR-bTB non-induced cows. This variable was lowest for C (2.9 ± 2.5; P < 0.01) and highest for bBR-bTB non-induced cows (6.3 ± 3.1). Pregnancy rate to all services did not differed for C (79.5%), bBR (76.7%), and bTB (75.9%) but was lower (58.9%; P < 0.01) for bBR-bTB non-induced cows. For induced cows this variable was highest for bBR (53.3%) and lowest for bBR-bTB (34.1%; P < 0.01) non-induced cows. 305-d milk production was increased by 4%, and total milk yield by 7% in TB-positive cows compared to that of the negative cows non-induced hormonally into lactation. This study showed the negative impact of the co-positivity for bTB and bBR on the reproductive efficiency of Holstein cows, although positive bTB and bBR tests enhanced milk yield.