Search

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

This contribution describes the development of a simple, fast, cost-effective, and sensitive impedimetric immunosensor for quantifying bovine tuberculosis (TB) in bovine serum samples. The construction of the immunosensor involved immobilizing the purified protein derivative (PPD) of M. bovis onto a screen-printed electrode that was modified with gold nanoparticles (AuNPs) and a polypyrrole (pPy) film synthesized electrochemically. The immunosensor exhibited a linear range from 0.5 μg mL -1 to 100 μg mL -1 and achieved a limit of detection (LD) of 100 ng mL -1 for the detection of anti-M. bovis antibody. The recovery percentages obtained in bovine serum samples were excellent, ranging between 98 % and 103 %. This device presents several advantages over alternative methods for determining TB in bovine serum samples. These include direct, in situ measurement without the need for pre-treatment, utilization of small volumes, thus avoiding harmful solvents and expensive reagents, and portability. In addition, the immunosensor exhibits both physical and chemical stability, retaining effectiveness even after 30 days of modification. This allows simultaneous incubations and facilitates large-scale detection. Hence, this immunosensor presents itself as a promising diagnostic tool for detecting anti-M. bovis antibodies in bovine serum. It serves as a viable alternative to tuberculin and ELISA tests., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

Bovine tuberculosis (bTB) is endemic and has a substantial impact on the livestock sector in Ethiopia and other low and middle-income countries (LMICs). With a national emphasis on dairy farm intensification to boost milk production and spur economic growth, the incidence of bTB is anticipated to rise. However, Ethiopia, like other LMICs, lacks a comprehensive national bTB control strategy due to the economic and social infeasibility of traditional test-and-cull (TC) approaches. To inform the development of such a strategy, we evaluated the effectiveness and feasibility of TC and test-and-segregation (TSg) strategies for bTB control on Ethiopian dairy farms. A TC approach was used at Farm A [N = 62; comparative cervical test (CCT) > 4 mm, starting prevalence 11.3%] while TSg was implemented at Farm B (N = 45; CCT > 4 mm, prevalence 22.2%), with testing intervals of 2-4 months. Both strategies achieved a reduction in bTB prevalence to 0%, requiring seven rounds of TC over 18 months at Farm A, and five rounds of TSg over 12 months at Farm B's negative herd. The results show that adopting more sensitive thresholds [CCT > 0 mm or single cervical test (SCT) > 2 mm] during later rounds was pivotal in identifying and managing previously undetected infections, emphasizing the critical need for optimized diagnostic thresholds. Cost analysis revealed that TC was approximately twice as expensive as TSg, primarily due to testing, labor, and cow losses in TC, versus construction of new facilities and additional labor for TSg. This underscores the economic and logistical challenges of bTB management in resource-limited settings. Taken together, our study highlights an urgent need for the exploration of alternative approaches including TSg and or vaccination to mitigate within herd transmission and enable implementation of bTB control in regions where TC is not feasible., (© 2024. The Author(s).)

The frequent zoonotic disease known as "bovine tuberculosis" is brought on by the Mycobacterium bovis bacteria, which can infect both people and animals. The aim of this review article is to provide an explanation of the etiology, history, epidemiology, pathogenesis, clinical symptoms, diagnosis, transmission, risk factors, public health importance, economic impact, treatment, and control of bovine tuberculosis. Primarily, bovine tuberculosis affects cattle, but other animals may also be affected. Bovine tuberculosis is present throughout the world, with the exception of Antarctica. Cattle that contract bovine tuberculosis might suffer from a persistent, crippling illness. In the early stages of the disease, there are no symptoms. The tuberculin test is the primary method for detecting bovine tuberculosis in cows. Depending on its localized site in the infected animal, M. bovis can be found in respiratory secretions, milk, urine, feces, vaginal secretions, semen, feces, and exudates from lesions (such as lymph node drainage and some skin lesions). This illness generally lowers cattle productivity and could have a negative financial impact on the livestock business, particularly the dairy industry. The most effective first-line anti-tuberculosis chemotherapy consists of isoniazid, ethambutol, rifampin, and streptomycin. Second-line drugs used against bovine tuberculosis include ethionamide, capreomycin, thioacetazone, and cycloserine. To successfully control and eradicate bovine tuberculosis, developed nations have implemented routine testing and culling of infected animals under national mandatory programs., Competing Interests: The authors declare that there is no conflict of interest.

Bovine tuberculosis (bovine TB) is a chronic wasting disease of cattle caused primarily by Mycobacterium bovis. Controlling bovine TB requires highly sensitive, specific, quick, and reliable diagnostic methods. This systematic review and meta-analysis evaluated molecular diagnostic tests for M. bovis detection to inform the selection of the most viable assay. On a per-test basis, loop-mediated isothermal amplification (LAMP) showed the highest overall sensitivity of 99.0% [95% CI: 86.2%-99.9%] and specificity of 99.8% [95% CI: 96.2%-100.00%]. Quantitative real-time polymerase chain reaction (qPCR) outperformed conventional PCR and nested PCR (nPCR) with a diagnostic specificity of 96.6% [95% CI: 88.9%-99.0%], while the diagnostic sensitivity of 70.8% [95% CI: 58.6-80.5%] was comparable to that of nPCR at 71.4% [95% CI: 60.7-80.2%]. Test sensitivity was higher with the input of milk samples (90.9% [95% CI: 56.0%-98.7%]), while specificity improved with tests based on major M. bovis antigens (97.8% [95% CI: 92.3%-99.4%]), the IS6110 insertion sequence (95.4% [95% CI: 87.6%-98.4%]), and the RD4 gene (90.7% [95% CI: 52.2%-98.9%]). The design of the currently available molecular diagnostic assays, while mostly based on nonspecific gene targets, prevents them from being accurate enough to diagnose M. bovis infections in cattle, despite their promise. Future assay development should focus on the RD4 region since it is the only target identified by genome sequence data as being distinctive for detecting M. bovis. The availability of a sufficiently accurate diagnostic test combined with the routine screening of milk samples can decrease the risk of zoonotic transmissions of M. bovis., Competing Interests: Declaration of Competing Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome, (Copyright © 2024. Published by Elsevier B.V.)

Bovine tuberculosis is usually diagnosed using tuberculin skin tests or at post-mortem. Recently, we have developed a serological test for bovine tuberculosis in cattle which shows a high degree of accuracy using serum samples. Here, we have assessed the performance of the test using individual bovine milk samples. The diagnostic specificity estimate using the high sensitivity setting of the test was 99.7% (95% CI: 99.2-99.9). This estimate was not altered significantly by tuberculin boosting. The relative sensitivity estimates of the test using the high sensitivity setting in milk samples from comparative skin test positive animals was 90.8% (95% CI: 87.1-93.6) with boosting. In animals with lesions, the relative sensitivity was 96.0% (95% CI: 89.6-98.7). Analysis of paired serum and milk samples from skin test positive animals showed correlation coefficients ranging from 0.756-0.955 for individual antigens used in the test. Kappa analysis indicated almost perfect agreement between serum and milk results, while McNemar marginal homogeneity analysis showed no statistically significant differences between the two media. The positive and negative likelihood ratio were 347.8 (95% CI: 112.3-1077.5) and 0.092 (95% CI: 0.07-0.13) respectively for boosted samples from skin test positive animals. The results show that the test has high sensitivity and specificity in individual milk samples and thus milk samples could be used for the diagnosis of bovine tuberculosis., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests:Amanda O’Brien, John Clarke, Alastair Hayton, Keith Cutler, Gordon Harkiss and Neil Watt declare commercial interests in the Enferplex Bovine TB antibody test. Darren Shaw and Andy Adler declare no competing interests., (Copyright: © 2024 O’Brien et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although several brands of tuberculin purified protein derivatives (PPDs) are available for diagnosing bovine tuberculosis (bTB), comparative studies to determine their diagnostic accuracy are infrequent. In Ecuador we compared two different PPD brands for bTB diagnosis using skin testing and measuring skin thickness increase. Additionally, we evaluated four PPD brands, including those used for skin testing, in the Bovine Tuberculosis Interferon Gamma Test (IFN-γ test) measuring IFN-γ induction in whole blood. The study included 17 naturally tuberculosis-infected PPD and IFN-γ test positive bovines. Both the field and laboratory results showed significant differences in classifying the 17 bovines as bTB positive or negative. We hypothesize that several factors, such as the genetic background of the cows, sensitization to environmental mycobacteria, M. bovis strains involved in the bTB infection, and the manufacturing procedures of the PPDs, could have influenced the immune reaction toward the different tuberculin PPD brands. Our study emphasizes the necessity for comparative studies aimed at determining the diagnostic accuracy of PPD brands for bTB diagnosis as well as the development of standardized methods for PPD production and potency determination., (© 2024. The Author(s).)

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8 M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In Ireland, the interferon-gamma (IFN-γ) assay is routinely used as an ancillary test interpreted in parallel with the single intradermal comparative tuberculin test (SICTT) to maximize the detection of bovine tuberculosis (bTB) infected animals. Up until 2018, a positive test result was recorded in the IFN-γ ELISA assay following whole blood stimulation with purified protein derivative (PPD)-bovine (B), PPD-avian (A) and nil sample (N), using the interpretation criteria, B-N > 50 optical density units (OD), B > 100 and B-A > 0. Following a review of available data, the threshold of the B-A component changed to B-A > 80. As predicting the impact of changing the cut-off thresholds for the IFN-γ test de novo is challenging, the aims of this study were to follow animals that initially tested negative using the new IFN-γ assay interpretation criteria and investigate their future risk of disclosure with bTB, with a focus on animals that otherwise would have been removed when using the older interpretation criteria (0 < B-A ≤ 80). Enrolled animals (n = 28,669 cattle from 527 herds) were followed up for two years (2019-2021), or to point of bTB detection or death. At the end of follow-up, 1151 (4.0%) of enrolled animals were bTB cases. The majority of these cases were diagnosed using SICTT (80.5%). The cumulative number of positive animals that would have been removed if the old cut-off (0 < B-A ≤ 80) was used amounted to 1680 cattle (5.9% of the enrolled cohort). Of these, 127 (7.5%) were diagnosed with bTB during follow-up. In contrast, 1024 of the 1151 cattle which subsequently tested positive during the study period following a negative IFN-γ test would not have been identified with the old or new IFN-γ cut-off criteria. Survival analysis showed that animals that would have been removed under the old interpretation criteria were at increased risk of a positive diagnosis with bTB during follow-up compared to other test negative animals. A newly developed risk prediction model (using a Cox proportional hazard model) showed that age, animal number of SICTT tests, number of inconclusive SICTT tests, B-A (IFN-γ assay), B-N (IFN-γ assay), animals from store herds and the percentage of the rest of the herd that were positive during the breakdown were statistically significantly associated with bTB detection. However, inclusion of the IFN-γ OD variables did not show added value in terms of prediction performance of the model., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

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., (© 2024. The Author(s).)

Bovine tuberculosis is usually diagnosed using tuberculin skin and interferon gamma tests. However, it is clear these tests miss infected animals due to poor sensitivity. The Enferplex Bovine TB antibody test has been validated by the World Organisation for Animal Health as fit for purpose in diagnosing bovine TB. A recent paper by Madden and colleagues (Veterinary Research Communications published online 17 August 2023) presented data on the future risk of Enferplex test antibody positive animals developing bovine TB. We argue in this communication that this does not make sense. Also, the study design did not include measuring antibodies at the point of censure of the animals and hence the survival analysis performed was meaningless. Most significantly, the study misses the point that skin and interferon gamma tests fail to detect a significant proportion of infected animals identified by the Enferplex test., (© 2023. The Author(s).)

The Single Intradermal Comparative Tuberculin Test (SICTT) and the interferon-gamma (IFN-γ) assay are the approved diagnostic tests for bovine tuberculosis (bTB) in Ireland. The aim of this pilot study was to explore if there was any added diagnostic benefit from applying the Enferplex bTB test (an antibody test) in severe bTB herd breakdowns after the removal of cattle that had tested positive to the SICTT and the IFN-γ test. In addition to the normal bTB testing and management protocols, the animals in these herds that tested negative to SICTT and the IFN-γ test were followed forward for a period of two years. All animals were tested by Enferplex at enrolment. The time to subsequent bTB detection (diagnosed with SICTT/IFN-γ tests or detection of visible lesions at routine slaughter) for animals that tested positive or negative to the Enferplex bTB test at the start of the study was compared using Kaplan-Meier survival curves and Cox based survival models. Of the 484 enrolled animals (from 11 herds), 171 (35.3%) and 151 (31.1%) initially tested positive in the Enferplex assay under the high sensitivity and high specificity interpretation settings respectively. The results of the survival analysis showed that there was no difference in the survival time to a positive diagnosis with bTB during the follow-up period between animals initially classified as positive and negative by the Enferplex test. Further research is warranted to explore the potential benefit of using the Enferplex test in other scenarios., (© 2023. The Author(s).)

Bovine tuberculosis is an infectious disease of global significance that remains endemic in many countries. Mycobacterium bovis infection in cattle is characterized by a cell-mediated immune response (CMI) that precedes humoral responses, however the timing and trajectories of CMI and antibody responses determined by newer generation assays remain undefined. Here we used defined-antigen interferon-gamma release assays (IGRA) and an eleven-antigen multiplex ELISA (Enferplex TB test) alongside traditional tuberculin-based IGRA and IDEXX M. bovis antibody tests to assess immune trajectories following experimental M. bovis infection of cattle. The results show CMI responses developed as early as two-weeks post-infection, with all infected cattle testing positive three weeks post-infection. Interestingly, 6 of 8 infected animals were serologically positive with the Enferplex TB assay as early as 4 weeks post-infection. As expected, application of the tuberculin skin test enhanced subsequent serological reactivity. Infrequent M. bovis faecal shedding was observed but was uncorrelated with observed immune trajectories. Together, the results show that early antibody responses to M. bovis infection are detectable in some individuals and highlight an urgent need to identify biomarkers that better predict infection outcomes, particularly for application in low-and-middle income countries where test-and-slaughter based control methods are largely unfeasible., (© 2024. Crown.)

Background: Enzyme-linked immunosorbent assays (ELISAs) are the most widely used diagnostic tools in bovine paratuberculosis (bPTB) control. However, their diagnostic accuracy may be compromised by bovine tuberculosis (bTB) infection, as both diseases share diagnostic targets., Methods: The bPTB and bTB infection status of 228 animals was determined using microbiological tissue culture as a reference test. The diagnostic performance (sensitivity, specificity, likelihood ratios and predictive values) of the bPTB-ELISA on blood serum samples, taking into account the bPTB animal-level prevalence of the area and the bTB status of the animals, was evaluated., Results: A sensitivity of 40.7% (95% confidence interval [CI]: 27.5%-53.9%) and a specificity of 94.7% (95% CI: 91.4%-98.0%) were obtained for bPTB-ELISA in all animals. A bPTB-ELISA-positive animal would have a post-test probability of 70% or more of being infected in areas with a bPTB prevalence of 23% or more. A negative bPTB-ELISA result, in areas with a bPTB prevalence of 41% or less, would rule out the disease with more than 70% certainty. In bTB-positive animals, sensitivity increased (94.4% [95% CI: 81.4%-100%] vs. 25.1% [95% CI: 11.8%-38.4%]) and specificity decreased (82.6% [95% CI: 71.8%-93.4%] vs. 99.4% [95% CI: 98.0%-99.9%]). The bPTB-ELISA is a good tool to rule out bPTB co-infection in bTB-positive animals, while in bTB-negative animals, it allows confirmation of disease with more than 70% probability if disease prevalence is 6% or more., Limitations: The observed differences could be enhanced by the effect of frequent application of the intradermal tuberculin test, which was unknown in the animals studied., Conclusions: These results provide useful guidance for the application and interpretation of ELISA as a tool for bPTB disease control., (© 2023 British Veterinary Association.)

Tuberculin skin test (TST) is the standard method for screening of bovine tuberculosis (bTB). However, gamma interferon blood test has been introduced in the bTB control program as an ancillary testing with TST in many countries of the world. The objective of this study was to recommend this screening test as an ancillary testing with TST for field application in Bangladesh. In this study 577 cattle of different age, sex and breeds from twenty nine (29) cattle herds were examined to determine skin response against bTB through single intradermal comparative tuberculin test (SICTT) that comprised of positive (n = 81), inconclusive (n = 44) and negative (n = 452) animals. Of which 74 animals that included positive (n = 63), inconclusive (n = 8) and negative (n = 3) animals were taken under this study. Blood samples were collected in heparinized tube and stimulated overnight with bovine and avian purified protein derivatives (PPDs) for the secretion of gamma interferon, and measured via sandwich ELISA. Cohen's kappa statistics was performed for the evaluation of agreement between the two tests. The agreement obtained between two tests was fair (Kappa agreement, K = 24.0%, 95% CI = 16.9-30.5%, P = 0.037). Of positive (n = 63), inconclusive (n = 8) and negative (n = 3) status of animals at SICTT, 82.54% (n = 52), 62.50% (n = 5), and 33.33% (n = 1) were found to be bTB positive respectively through this ancillary test. This test notably corroborates to TST result. A considerable number of inconclusive TB status animals were found to be positive through this gamma interferon assay. Therefore, this test could be used as an ancillary test with TST to maximize the proportion of bTB estimation in the infected cattle herd for early detection of zoonotic tuberculosis in Bangladesh before transmission at the animal-human interface., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Detecting bovine tuberculosis (bTB) primarily relies on the tuberculin skin test, requiring two separate animal handling events with a period of incubation time (normally 3 days) between them. Here, we present the use of liquid atmospheric pressure (LAP)-MALDI for the identification of bTB infection, employing a three-class prediction model that was obtained by supervised linear discriminant analysis (LDA) and tested with bovine mastitis samples as disease-positive controls. Noninvasive collection of nasal swabs was used to collect samples, which were subsequently subjected to a short (<4 h) sample preparation method. Cross-validation of the three-class LDA model from the processed nasal swabs provided a sensitivity of 75.0% and specificity of 90.1%, with an overall classification accuracy of 85.7%. These values are comparable to those for the skin test, showing that LAP-MALDI MS has the potential to provide an alternative single-visit diagnostic platform that can detect bTB within the same day of sampling.

Background: Tuberculosis (TB) is a major cause of ill health and one of the leading causes of death worldwide, caused by species of the Mycobacterium tuberculosis complex (MTBC), with Mycobacterium tuberculosis being the dominant pathogen in humans and Mycobacterium bovis in cattle. Zoonotic transmission of TB (zTB) to humans is frequent particularly where TB prevalence is high in cattle. In this study, we explored the prevalence of zTB in central Ethiopia, an area highly affected by bovine TB (bTB) in cattle., Method: A convenient sample of 385 patients with pulmonary tuberculosis (PTB, N  = 287) and tuberculous lymphadenitis (TBLN, N  = 98) were included in this cross-sectional study in central Ethiopia. Sputum and fine needle aspirate (FNA) samples were obtained from patients with PTB and TBLN, respectively, and cultures were performed using BACTEC ™ MGIT ™ 960. All culture positive samples were subjected to quantitative PCR (qPCR) assays, targeting IS 1081 , RD9 and RD4 genomic regions for detection of MTBC, M. tuberculosis and M. bovis , respectively., Results: Two hundred and fifty-five out of 385 sampled patients were culture positive and all were isolates identified as MTBC by being positive for the IS 1081 assay. Among them, 249 (97.6%) samples had also a positive RD9 result (intact RD9 locus) and were consequently classified as M. tuberculosis . The remaining six (2.4%) isolates were RD4 deficient and thereby classified as M. bovis . Five out of these six M. bovis strains originated from PTB patients whereas one was isolated from a TBLN patient. Occupational risk and the widespread consumption of raw animal products were identified as potential sources of M. bovis infection in humans, and the isolation of M. bovis from PTB patients suggests the possibility of human-to-human transmission, particularly in patients with no known contact history with animals., Conclusion: The detected proportion of culture positive cases of 2.4% being M. bovis from this region was higher zTB rate than previously reported for the general population of Ethiopia. Patients with M. bovis infection are more likely to get less efficient TB treatment because M. bovis is inherently resistant to pyrazinamide. MTBC species identification should be performed where M. bovis is common in cattle, especially in patients who have a history of recurrence or treatment failure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ayalew, Habtamu, Melese, Tessema, Ashford, Chothe, Aseffa, Wood, Berg, Mihret and The ETHICOBOTS Consortium.)

In the bovine tuberculosis diagnosis, the use of plasma samples (already available for IFNɣ assays) in serological tests might facilitate the work in the field. Here, the performance of two commercial serological tests (ELISA IDEXX M. bovis Ab test and Enferplex Bovine TB antibody test) were evaluated using plasma samples from cattle in Belgium. Specificity values estimated from 567 plasma samples collected from bTB-free cattle were 98.4% when using the ELISA IDEXX M. bovis Ab test, and were 96.5% and 93.3% when using the high specificity and high sensitivity settings of the Enferplex Bovine TB antibody test, respectively. Sensitivity values were calculated relative to SICCT-positive (N = 117) and IFNɣ-positive (N = 132) animals originating from M. bovis-infected herds. Overall, the multiplexed Enferplex Bovine TB antibody test had better sensitivity (mean: 32.5% and 43.4% for the high specificity and sensitivity settings, respectively) compared to the ELISA IDEXX M. bovis Ab test (mean: 12%). Data obtained from plasma samples in the current study were compared to a previous study using both serological tests with sera. In conclusion, both serological tests showed comparable performance with both matrix; although overall specificity values with the Enferplex Bovine TB antibody test were lower when using plasma samples than sera., Competing Interests: Declaration of Competing Interest None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine tuberculosis (bTB) is a disease of significant economic and zoonotic importance, therefore, optimising tests for the identification of Mycobacterium bovis infected cattle is essential. The Interferon Gamma (IFN-γ) Release Assay (IGRA) can diagnose M. bovis infected cattle at an early stage, is easy to perform and can be used alongside skin tests for confirmatory purposes or to increase diagnostic sensitivity. It is known that IGRA performance is sensitive to environmental conditions under which samples are taken and transported. In this study, the association between the ambient temperature on the day of bleeding and the subsequent IGRA result for bTB was quantified using field samples from Northern Ireland (NI). Results of 106,434 IGRA results (2013-2018) were associated with temperature data extracted from weather stations near tested cattle herds. Model dependent variables were the levels of IFN-γ triggered by avian purified protein derivative (PPDa), M. bovis PPD (PPDb), their difference (PPD(b-a)) as well as the final binary outcome (positive or negative for M. bovis infection). IFN-γ levels after both PPDa and PPDb stimulation were lowest at the extremes of the temperature distribution for NI. The highest IGRA positive probability (above 6%) was found on days with moderate maximum temperatures (6-16 °C) or moderate minimum temperatures (4-7 °C). Adjustment for covariates did not lead to major changes in the model estimates. These data suggest that IGRA performance can be affected when samples are taken at high or low temperatures. Whilst it is difficult to exclude physiological factors, the data nonetheless supports the temperature control of samples from bleeding through to laboratory to help mitigate post-collection confounders., Competing Interests: Declaration of Competing Interest The authors declare to have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Bovine tuberculosis is the leading cause of death in cattle and other species worldwide. Quick and precise identification of mycobacteria is critical to control the occurrence of tuberculosis in cattle., Methods: We developed a fluorescent peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) approach to detect Mycobacterium bovis and Mycobacterium avium in cytological smears and tissue sections of bovines suspected of having tuberculosis. PNA-FISH was conducted on smears of lung and lymph node tissues. Standard bovine mycobacterial cultures were used to standardize the probes using 50 % formamide for M. bovis and 30 % formamide for M. avium. M. bovis probe (MTBCcy3), which was standardized at hybridization conditions of (55 °C and 40 % formamide) concentrations, was positive in all cytological smears., Results: Four out of twenty five samples tested positive in tissue sections observed as a bright red fluorescence with a cy3 filter (MTBC probe). No results were observed with (MAV TAMRA ) probe for M. avium which was standardized at hybridization conditions of (55 °C and 30 % formamide). No fluorescence was observed in the control tissue sections. Additionally, the results were juxtaposed with those of other commonly used detection methods such as immunohistochemistry and Polymerase Chain Reaction (PCR) by targeting the esxA gene. None of the samples tested positive for M. avium infection., Conclusion: PNA-FISH can be used to obtain cytological impression smears and tissue sections. When compared to PCR it consumes less time in the diagnosis of bovine tuberculosis in post mortem cases., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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: OD bovis -OD PBS  ≥ 0.1 and OD bovis -OD avium  ≥ 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., (© 2023. The Author(s).)

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.

Bovine tuberculosis (bTB), a chronic disease of cattle, is caused by the Mycobacterium bovis infection. Despite having a serious social and economic impact in the United Kingdom and Ireland, there is no antemortem gold standard diagnostic test. Tuberculin skin tests (CICT) are commonly used as a control measure with the interferon gamma (IFN-γ) assay being applied in certain circumstances. This paper utilizes data gathered describing tuberculin regression in reactors (test positive cattle) following the CICT at 72 ± 4 h post injection in herds with large bTB outbreaks. The work then applies machine learning techniques (Decision Trees, Bagging Trees and Random Forests, alongside several balancing approaches) to predict which cattle were likely to be truly infected with tuberculosis, enabling identification of atypical breakdowns that require extra investigation and providing a mechanism for quality assurance of the existing CICT bTB surveillance scheme. The analysis showed that Random Forests (RF) trained using SMOTE balancing had the joint best performance and accuracy (0.90). The importance of the two components of the interferon gamma assay within the RF model also indicated that varying the assay threshold for large outbreaks would be beneficial. Furthermore, the combined use of the RF and IFN- γ models could lead to the improved detection of infection within breakdown herds, reducing the scale and duration of outbreaks. An additional use of these models would be for quality assuring the current bTB surveillance based on CICT and post mortem inspection. Quality control is well recognized as an essential component of a disease surveillance/eradication programme.Clinical Relevance- Bovine tuberculosis remains a disease that is hard to control on a national level. The use of the machine learning model could lead to significant improved detection of infection within breakdown herds, reducing the scale and duration of outbreaks. Advanced modelling, such as this, has the potential to strengthen the efficacy of disease surveillance and the eradication strategy and can meaningfully contribute to animal disease national control plans.

Persistence of bovine tuberculosis (bTB) in cattle herd remains a major challenge in disease elimination due to the ineffectual removal of all infected animals in a bTB breakdown. Characterization of herds with a higher probability of experiencing further bTB breakdowns can help to implement specific risk-based policies for disease control and eradication. Here, our aim was to identify herd- and breakdown-level risk factors in bTB infected herds in Castilla y Leon, Spain, associated with a decreased time to recurrence and an increased risk of recurrence using a mixed effects Cox proportional hazards model and a multivariable logistic regression model, respectively. Results revealed that location (province), herd size and number of incoming animals/contacts were good predictors of a decreased time to bTB recurrence and an increased risk of becoming a recurrent herd. Additionally, the duration of the previous outbreak and the number of IFN-γ herd-tests applied in it were associated with increased odds of (an early) recurrence. Risk factors identified here can be used for early identification of herds in which bTB eradication may be more challenging and that should thus be subjected to increased control efforts. The characterization of high-risk herds may help to minimize the risk of reinfection and emphasize early detection and removal of bTB positive animals in the herd., Competing Interests: Declaration of Competing Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Ethical review and approval were not required for the animal study because all data was collected as part of authorized regulatory surveillance in the framework of the Spanish National Eradication Program for Bovine Tuberculosis. Written informed consent for participation was not obtained from the owners because approval from premises owners was not required for this study., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis , the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.

Bovine tuberculosis (TB) is a chronically evolving zoonotic infectious disease caused by Mycobacterium bovis. Anatomopathological examination during post mortem inspection in bovines is the main resource engaged in sanitary slaughter; however, it is very troublesome since many granulomatous inflammatory processes have similar morphological characteristics. Thus, this study aims to use complementary diagnosis methods (histopathological and polymerase chain reaction - PCR assays) to confirm the macroscopic assessment of lymphadenopathies indicative of tuberculosis in bovines slaughtered in a refrigerated slaughterhouse in Tailândia city, PA, Brazil. Fifty‑one samples were collected from lesions indicative of tuberculosis in pre‑scapular and pre‑pectoral lymph nodes (or different lymphadenitis) in condemned carcasses. Histological processing employed routine techniques carried out at the Laboratory of Animal Pathology of the Federal Rural University of the Amazon, while the PCR assay was performed at the Bacteriology Laboratory of the Evandro Chagas Institute. Results showed that 1.96% of the histopathology samples corresponded to inflammatory processes typical of TB and that, in PCR, 4.25% of the samples had the amplification profile of the M. bovis species. These results indicate the importance of adding complementary methods to assist the sanitary inspection line and make inspection more efficient in its decisions.

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%)., (© Crown copyright 2023.)

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity., (© 2023. The Author(s).)

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A 202-347 -SOD 1-72 -Ag85B 173-330 -74F 1-148+669-786 ), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum ® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum ® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4 , TLR2 , PTX3 , PTGS2 , PD-L1 , IL1B , IL2 , IL6 , IL12B , IL17A , IL22 , IFNG , CD40 , and CD86 . Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum ® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test., Competing Interests: Authors SG, TW, PM, AH, BB and DW were employed by company AgResearch. The remaining author declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gupta, Wilson, Maclean, Rehm, Heiser, Buddle and Wedlock.)

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&#95;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., (© 2022. The Author(s).)

Recent studies have suggested the potential of innovative serologic tests for accurate and rapid detection of bovine tuberculosis (bTB). Dual Path Platform (DPP) technology has been used to develop rapid animal-side antibody tests for Mycobacterium bovis infection in a range of livestock and wildlife host species. The present study evaluated diagnostic performance of DPP BovidTB IgM/IgG assay designed for differential detection of bovine IgM and IgG antibodies against two chimeric antigens, DID38 and TBf2, respectively, using 662 well-characterized serum samples from M. bovis-infected and bTB-free cattle collected in the United States, Great Britain, France, and South Africa. Test sensitivity and specificity ranged from 71% to 100% and from 95% to 100%, respectively, depending on the country, with overall accuracy of 83%. No significant risk of cross-reactivity with serum samples from cattle infected with most relevant species of mycobacteria other than M. bovis was found. The DPP BovidTB IgM/IgG assay may be suitable for use in multi-test algorithms to improve current strategies for bTB surveillance., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bovine tuberculosis (bTB) is an important zoonotic disease in developing countries including China. Lack of rapid and simple detection methods for surveillance affects efficient control and eradication of this disease. The objective of this study was to determine the feasibility of using an individual and bulk milk sample ELISA based on the fusion protein of four specific antigens (CFP10/ESAT-6/MPB70/MPB83) of Mycobacterium bovis for the rapid detection and surveillance of bTB in dairy cattle herds and to identify individual animal factors that affect its results. The study population consisted of 388 dairy cows from four herds in Henan province, China. The serum sample ELISA (cut-off value 30%) and milk sample ELISA (cut-off value 10%) were shown to have good agreement, while, there was fair agreement between a blood sample IFN-γ in vitro release assay and the milk sample ELISA (cut-off value 10%) (Cohen's kappa statistic of 0.492 and 0.306, respectively). The sensitivity and specificity for the milk sample ELISA using a S/P% cut-off value of 10%, as estimated using a Bayesian latent class analysis model, was 48.4% (95%CI: 31.0 - 85.2) and 97.1% (95%CI: 92.2 - 99.7), respectively. Although both ELISAs had comparable sensitivities and specificities, the milk sample ELISA had a greater discriminatory ability, as measured by the area under the curve (ROC), than the serum sample ELISA with the difference of their AUCs being - 0.069 (95%PCI: -0.121 to -0.021). Further, it was estimated that the lower limit of detection at an animal level lacto-prevalence might be 1.1% with this milk ELISA using bulk milk samples. Additionally, a mixed linear regression model, incorporating herd as a random effect variable, was constructed to investigate the association between natural log-transformed bTB sample-to-positive ratio (LnS/P%) and milk data. Across the model, natural log-transformed Somatic Cell Count (SCC) and parity had a positive effect on the LnS/P% of bTB antibody response, and milk urea nitrogen had a curvilinear association with LnS/P%. In conclusion, a milk ELISA for bTB antibodies, especially bulk tank milk antibodies, could be used as an efficient tool for rapid screening in surveillance, which might be followed by confirmatory testing with IFN-γ assay in dairy cattle, during the implementation of bTB control programmes. Relevant milk factors should be considered to adjust the S/P% with the aim of classifying the infection status of animals and herds as accurately as possible., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Continual tailoring of control programmes of endemic pathogens during long-term eradication campaigns requires detailed analysis of surveillance data to inform evidence-based policy. Bovine tuberculosis is a disease where long-term control and eradication programs are in train in several countries. The primary diagnostic tool, the intradermal tuberculin test, used to identify infected animals can be interpreted using different criteria and cut-offs, facilitating flexibility in its use as a basis to inform interventions. We investigated the comparative risk of animals failing a single intradermal comparative tuberculin test (SICTT) based on their previous tuberculin test result following a higher risk test-type (reactor retest of an infected herd). The study was a retrospective cohort design, and the primary exposure was the test status following a reactor retest classified as mutually exclusive categories based on bovine and avian tuberculin reactions: standard interpretation inconclusive (Sdi), severe interpretation inconclusive (Svi), single intradermal test (SIT) reactors (SITr), SIT inconclusive (SITi), avian tuberculin reactors (Ar), and test negative animals. Random effects multivariable logistic regression was used to investigate future risk. Cross-validation and downscaling was used to explore model performance. Alternate models with differing outcome test types were also explored. The models were trained on 844,207 observations from June 2018 to June 2021. Sdi, Svi, SITr, SITi and Ar were associated with the following odds ratios 12.242 (95 %CIs: 5.236-28.625; p < 0.001), 4.101 (95 %CIs: 3.423-4.913; p < 0.001), 2.503 (95 %CIs: 1.878-3.338; p < 0.001), 1.741 (95 %CIs: 1.195-2.538; p = 0.004) and 1.065 (95 %CIs: 0.833-1.361; p = 0.616) for failing the next test, respectively. High model performance was achieved with inclusion of random effects for both training and test evaluation datasets (AUC: 0.94; Balanced accuracy: 0.84), but fixed-effects only predictions exhibited moderate performance (AUC: 0.70; Balanced accuracy: 0.69). This reflects that 55 % of the risk of test failure relates to between herd heterogeneity based on intra-class correlation, while controlling for fixed effects. Other factors that were associated with increasing risk included age (older cohorts were at greater risk than the youngest cohort), breakdown history of the herd (greater number of breakdowns prior to the study period), and the time between exposure test and outcome test. These results provide further evidence to inform risk-based management policies for TB, including the removal of Sdi animals in higher risk situations, supplementary testing of cattle based on tuberculin responses and the provision of risk management advice to herd owners. The results characterise the future animal-level risk posed by Svis and suggest this risk may require policy led interventions., Competing Interests: Conflict of interest statement The authors of the paper “Can more information be extracted from bovine TB skin test outcomes to inform animal risk management? A retrospective observational animal-level study” submitted to Preventive Veterinary Medicine declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (© 2022. The Author(s).)

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).

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., Competing Interests: The authors have declared that no competing interests exist.

In South Africa, animal tuberculosis (TB) control programs predominantly focus on domestic cattle and African buffaloes (Syncerus caffer) despite increasing global reports of tuberculosis in goats (Capra hircus). Left undetected, Mycobacterium tuberculosis complex (MTBC) infected goats may hinder TB eradication efforts in cattle and increase zoonotic risk to humans. Since the publication of animal TB testing guidelines in 2018, prescribing the use of the tuberculin skin test (TST) for goats in South Africa by the Department of Agriculture, Land Reform, and Rural Development (DALRRD), there have been no published reports of any field application of the prescribed test criteria in goat herds. Therefore, this study aimed to evaluate the performance of these DALRRD guidelines using the single intradermal cervical tuberculin test (SICT) and the single intradermal comparative cervical tuberculin test (SICCT). Between October and December 2020, 495 goats from communal pastures of Kwa-Zulu Natal (KZN), where M. bovis infection has been identified in cattle and where cattle and goats cohabitate, were tested using the SICT and SICCT (M. bovis-exposed group). Additionally, 277 goats from a commercial Saanen dairy herd, with no history of M. bovis, were also tested (M. bovis-unexposed group). Estimated apparent prevalence of TST positive goats was determined based on published test interpretation criteria as described by DALRRD. When proportions of test-positive goats were compared between different DALRRD criteria, the ≥ 4 mm cut-off criterion for the SICCT resulted in the lowest proportion of positive results in the presumably uninfected group (1/277 positive in the unexposed group). The apparent prevalence of TB in the exposed group was estimated at 3.0% (95% CI: 1.7-4.9%), which is similar to previous reports of M. bovis prevalence in cattle from this area (6%). The detection of a significantly greater proportion of SICCT positive goats in the M. bovis-exposed group compared to the unexposed group suggests that MTBC infection is present in this population. Further investigations should be undertaken, in conjunction with confirmatory molecular tests, mycobacterial culture, and advanced pathogen sequencing to establish whether MTBC infection in domestic goats is a true under-recognized threat to the eradication of animal TB in South Africa., Competing Interests: Conflicts of Interest All authors declare no conflict of interest regarding the content of this article., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bovine tuberculosis (BTB) in sheep (Ovis aries) is caused by Mycobacterium bovis and Mycobacterium caprae. Even though sheep have been considered less sensitive to BTB than other ruminants, they have been subject to increasing numbers of tuberculosis cases and it has been suggested that they may act as a disease reservoir in some regions. Aim of the study: Following a report of tuberculous-like gross lesions (repeated cases of purulent or caseous lymphadenitis and a single case of serosal tubercles on the peritoneum) from veterinarians working in a slaughterhouse in the Małopolskie Voivodeship, southern Poland, the aim of this study was to conduct ante-mortem BTB diagnostics in three flocks with suspected BTB. The animals for testing were selected randomly from the flocks; a blood sample for interferon-γ release assay (IGRA) and a single tuberculin skin test (TST) was performed on each sheep. All TST results were negative. The IGRA result was positive in two ewes from the same flock (four and five years of age); these two sheep were tested microbiologically using Stonebrink and Löwenstein-Jensen media. No gross lesions suggesting BTB were observed, and the culture results were negative. Based on the positive IGRA results, together with its high specificity in sheep, and the potential risk to humans posed by consuming local unpasteurized dairy products, we recommend introducing serological BTB screening in sheep from this area, and subjecting the positive results to confirmation by culture., (Copyright© by the Polish Academy of Sciences.)

Objectives: Improved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response of Mycobacterium bovis-infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood-an important component of current bTB diagnostics-will provide new information for development of better diagnostics., Methods: RNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (-1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-week M. bovis infection time course experiment with ten age-matched Holstein-Friesian cattle., Results: In vitro PPD-b stimulation of peripheral blood from M. bovis-infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters., Conclusions: A transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers of M. bovis infection., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB) in cattle, represents a major disease burden to UK cattle farming, with considerable costs associated with its control. The European badger (Meles meles) is a known wildlife reservoir for bTB and better knowledge of the epidemiology of bTB through testing wildlife is required for disease control. Current tests available for the diagnosis of bTB in badgers are limited by cost, processing time or sensitivities., Materials and Methods: We assessed the ability of flow infusion electrospray-high-resolution mass spectrometry (FIE-HRMS) to determine potential differences between infected and non-infected badgers based on thoracic blood samples obtained from badgers found dead in Wales. Thoracic blood samples were autoclaved for handling in a containment level 2 (CL2) hazard laboratory., Results: Here we show the major differences associated with with M. bovis infection were changes to folate, pyrimidine, histidine, glycerophospholipid and phosphonate metabolism., Conclusions: Our studies have indicated differences in the metabolomic signature of badgers found dead in relation to their infection status, suggesting metabolomics could hold potential for developing novel diagnostics for bTB in badgers. As well as highlighting a potential way to handle samples containing a highly pathogenic agent at CL2 for metabolomics studies., (© 2022. The Author(s).)

Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis (M. bovis), is the lead candidate vaccine for control of bovine tuberculosis (TB) in cattle. However, BCG vaccination sensitises cattle to bovine tuberculin, thus compromising the use of the current bovine TB surveillance tests. To address this, we have developed a diagnostic skin test that is not compromised by BCG vaccination and is able to detect BCG vaccinated animals that subsequently develop bovine TB following exposure to M. bovis. Building on previous work using 'in house' formulated protein cocktail reagents, we herein present test performance data for a single fusion protein (DST-F) containing the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c formulated as a 'ready to use' reagent by a commercial manufacturer. Our results demonstrate that, unlike tuberculin reagents, a diagnostic skin test using DST-F maintained high specificity in BCG vaccinated animals. Furthermore, the DST-F skin test demonstrated a high relative sensitivity in identifying M. bovis infected animals, including those where BCG vaccination failed to prevent bovine TB pathology following experimental exposure to M. bovis. The DST-F is currently undergoing field trials in Great Britain to support its licensure and commercialisation., (© 2022. Crown.)

Screening of serum and fecal samples from huemul (Hippocamelus bisulcus) and pudu (Pudu puda) from southern Chile for Mycobacterium bovis and Mycobacterium avium paratuberculosis (MAP) found all but four samples Mycobacterium-negative. The positive sequences showed only 92-93% similarity with MAP and were from remote Isla Riesco populations., (© Wildlife Disease Association 2022.)

Even though enormous efforts and control strategies have been implemented, bovine tuberculosis (TB) remains a significant source of health and socioeconomic concern. The standard method used in TB eradication programs for in vivo detection is the tuberculin skin test. However, the specificity of the tuberculin skin test is affected by infection with non-tuberculous mycobacteria or by vaccination. Thus, some animals are not correctly diagnosed. This study aimed first to identify a plasma metabolic TB profile by high-field (HF) nuclear magnetic resonance (NMR) spectroscopy and second measure this characteristic TB metabolic profile using low-field benchtop (LF) NMR as an affordable molecular technology for TB diagnosis. Plasma samples from cattle diagnosed with TB (derivation set, n = 11), diagnosed with paratuberculosis (PTB, n = 10), PTB-vaccinated healthy control (n = 10) and healthy PTB-unvaccinated control (n = 10) were analyzed by NMR. Unsupervised Principal Component Analysis (PCA) was used to identify metabolic differences between groups. We identified 14 metabolites significantly different between TB and control animals. The second group of TB animals was used to validate the results (validation set, n = 14). Predictive models based on metabolic fingerprint acquired by both HF and LF NMR spectroscopy successfully identified TB versus control subjects (Area under the curve of Receiver Operating Characteristic over 0.92, in both models; Confidence Interval 0.77-1). In summary, plasma fingerprinting using HF and LF-NMR differentiated TB subjects from uninfected animals, and PTB and PTB-vaccinated subjects who may provide a TB-false positive, highlighting the use of LF-NMR-based metabolomics as a complementary or alternative diagnostic tool to the current diagnostic methods., (© 2021 Wiley-VCH GmbH.)