Smartphone-based immunochemical sensor exploiting peroxidase-like activity of ligand-capped gold nanostars: A proof-of-concept detection of Mycobacterium bovis.

Bacterial pathogens represent a safety concern in the food industry, and this is amplified by the lack of sensing devices that can be applied on-site by non-trained personnel. In this study, peroxidase-mimicking activity of gold nanostars was exploited to develop a user-friendly colourimetric sensor. A smartphone was exploited as an image reader and analyser, empowered with a novel App developed in-house. The mobile App was evaluated and compared with a commercial smartphone App for its capability to quantify generated colourimetric signals. A major obstacle found with sensors relying on gold nanozymes is the fact that modification of the surface of gold nanoparticles with biorecognition elements generally lead to a suppression of their nanozyme activity. This drawback was overcome by introducing an autocatalytic growth step, which successfully restored the peroxidase-mimicking activity through generation of new gold nanoseeds acting as catalytic centres. A proof-of-concept using this sensing mechanism was developed targeting Mycobacterium bovis, a zoonotic pathogen primarily found in cattle but that can be transmitted to humans by consumption of contaminated food and cause tuberculosis disease. The resulting smartphone-based immunological sensor has shown promising results with a linear response between 104 - 106 CFU/mL, enabling detection of M. bovis at concentrations as low as 7.2·103 CFU/mL in buffer conditions. It is anticipated that the concept of the developed approach will have applicability in many fields relying on smartphone-based biosensing.

Evaluation of the discriminatory power of spoligotyping and 19-locus mycobacterial interspersed repetitive unit-variable number of tandem repeat analysis (MIRU-VNTR) of Mycobacterium bovis strains isolated from cattle in Algeria.

Bovine tuberculosis (bTB) caused by Mycobacterium (M.) bovis and M. caprae is a transmissible disease of livestock, notifiable to the World Organization for Animal Health (OIE). BTB particularly affects cattle and small ruminants and can be transmitted to humans thereby posing a significant threat to veterinary and public health worldwide. M. bovis is the principal cause of bTB in Algeria. In order to better understand the route of spreading and elaborate an eradication program, isolation and characterization of mycobacteria from Algerian cattle was performed. Sixty strains belonging to the M. tuberculosis complex were analyzed by spoligotyping, thereof 42 by 19-locus-MIRU-VNTR-typing. Spoligotyping revealed 16 distinguishable patterns (Hunter-Gaston discriminatory index [HGDI] of 0.8294), with types SB0120 (n = 20) and SB0121 (n = 13) being the most frequent patterns, representing 55% of the strains. Analyses based on 19-locus-MIRU-VNTR yielded 32 different profiles, five clusters and one orphan pattern, showing higher discriminatory power (HGDI = 0.9779) than spoligotyping. Seven VNTR-loci [VNTR 577 (alias ETR C), 2163b (QU11b), 2165 (ETR A), 2461 (ETR B), 3007 (MIRU 27), 2163a (QUB11a) and 3232 (QUB 3232)] were the most discriminative loci (HGDI ˃ 0.50). In conclusion, 19-locus-MIRU-VNTR yielded more information than spoligotyping concerning molecular differentiation of strains and better supports the elucidation of transmission routes of M. bovis between Algerian cattle herds.

Prevalence of Mycobacterium bovis in milk on dairy cattle farms: An international systematic literature review and meta-analysis.

Bovine tuberculosis, caused by Mycobacterium bovis (M. bovis), is a globally distributed chronic disease of animals. The bacteria can be transmitted to humans via the consumption of unpasteurised (raw) milk, thus representing an important public health risk. To investigate the risk of zoonotic transmission of M. bovis via raw milk, this study systematically reviewed published studies to estimate the prevalence of M. bovis in on-farm bulk-tank milk (BTM) and individual cow's milk (IM) by meta-analysis. In total, 1,339 articles were identified through seven electronic databases and initially screened using titles and abstracts. The quality of 108 potentially relevant articles was assessed using full texts, and 67 articles comprising 83 studies (76 IM and 7 BTM), were included in the meta-analysis. The prevalence of M. bovis in IM and BTM was summarised according to the diagnostic test used, and the tuberculin skin test (TST) infection status of the individual cows (for IM) or herds (for BTM). Heterogeneity was quantified using the I-squared statistic. Prediction intervals (95% PIs) were also estimated. For IM, the overall prevalence was summarised at 5% (95%CI: 3%-7%). In TST positive cows, prevalence was summarised at 8% (95%CI: 4%-13%). For BTM, the overall prevalence independent of individual herd TST infection status was summarised at 5% (95%CI: 0%-21%). There was considerable heterogeneity evident among the included studies, while PIs were also wide. Inconsistency in the quality of reporting was also observed resulting in missing information, such as the TST infection status of the individual animal/herd. No study reported the number of M. bovis bacteria in test-positive milk samples. Several studies reported the detection of M. tuberculosis and M. africanum in milk. Despite international efforts to control tuberculosis, this study highlights the risk of zoonotic transmission of M. bovis via unpasteurised milk and dairy products made using raw milk.

Inferring Mycobacterium bovis transmission between cattle and badgers using isolates from the Randomised Badger Culling Trial.

Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.

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

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

Serological detection of Mycobacterium Tuberculosis complex infection in multiple hosts by One Universal ELISA.

Tuberculosis (TB), a contagious disease mainly caused by Mycobacterium tuberculosis (M. tb), Mycobacterium bovis (M. bovis), and Mycobacterium caprae (M. caprae), poses a major global threat to the health of humans and many species of animals. Developing an ante-mortem detection technique for different species would be of significance in improving the surveillance employing a One Health strategy. To achieve this goal, a universal indirect ELISA was established for serologically detecting Mycobacterium tuberculosis complex infection for multiple live hosts by using a fusion protein of MPB70, MPB83, ESAT6, and CFP10 common in M. tb, M. bovis, and M. caprae as the coating antigen (MMEC) and HRP-labeled fusion protein A and G as a secondary antibody. After testing the known positive and negative sera, the receiver operating characteristic curves were constructed to decide the cut-off values. Then, the diagnostic sensitivity and specificity of MMEC/AG-iELISA were determined as 100.00% (95% CI: 96.90%, 100.00%) and 100.00% (95% CI: 98.44%, 100.00%) for M. bovis infection of cattle, 100.00% (95% CI: 95.00%, 100.00%) and 100.0% (95% CI: 96.80%, 100.00%) for M. bovis infection of sheep, 90.74% (95% CI: 80.09%, 95.98%) and 98.63% (95% CI: 95.14%, 99.76%) for M. bovis infection of cervids, 100.00% (95% CI: 15.81%, 100.00%) and 98.81% (95% CI: 93.54%, 99.97%) for M. bovis infection of monkeys, 100.00% (95% CI: 86.82%, 100.00%) and 94.85% (95% CI: 91.22%, 97.03%) for M. tb infection of humans. Furthermore, this MMEC/AG-iELISA likely detects M. caprae infection in roe deer. Thus this method has a promising application in serological TB surveillance for multiple animal species thereby providing evidence for taking further action in TB control.

Development of a Multiplex Real-Time PCR Assay for Mycobacterium bovis BCG and Validation in a Clinical Laboratory.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live attenuated vaccine which can result in local or disseminated infection, most commonly in immunocompromised individuals. Differentiation of BCG from other members of the Mycobacterium tuberculosis complex (MTBC) is required to diagnose BCG disease, which requires specific management. Current methods for BCG diagnosis are based on mycobacterial culture and conventional PCR; the former is time-consuming and the latter often unavailable. Further, there are reports that certain BCG strains may be associated with a higher rate of adverse events. This study describes the development of a two-step multiplex real-time PCR assay which uses single nucleotide polymorphisms to detect BCG and identify early or late BCG strains. The assay has a limit of detection of 1 pg BCG boiled lysate DNA and was shown to detect BCG in both pure cultures and experimentally infected tissue. Its performance was assessed on 19 suspected BCG clinical isolates at Christian Medical College in Vellore, India, taken from January 2018 to August 2020. Of these 19 isolates, 10 were identified as BCG (6 early and 4 late strains), and 9 were identified as other MTBC members. Taken together, the results demonstrate the ability of this assay to identify and characterize BCG disease from cultures and infected tissue. The capacity to identify BCG may improve patient management, and the ability to discriminate between BCG strains may enable BCG vaccine pharmacovigilance. IMPORTANCE Vaccination against tuberculosis with bacillus Calmette-Guérin (BCG) can lead to adverse events, including a rare but life-threatening complication of disseminated BCG. This complication often occurs in young children with immunodeficiencies and is associated with an ∼60% mortality rate. A rapid method of reliably identifying BCG infection is important because BCG requires treatment unique to tuberculosis. BCG is resistant to the first-line antituberculosis drug pyrazinamide. Additionally, diagnosis of BCG disease would lead to further investigation of a possible underlying immune condition. We have developed a diagnostic assay to identify BCG which improves upon previously published methods and can reliably identify BCG from bacterial culture or directly from infected tissue. This assay can also differentiate between strains of BCG, which have been suggested to be associated with different rates of adverse events. This assay was validated on 19 clinical isolates collected at Christian Medical College in Vellore, India.

A new phylodynamic model of Mycobacterium bovis transmission in a multi-host system uncovers the role of the unobserved reservoir.

Multi-host pathogens are particularly difficult to control, especially when at least one of the hosts acts as a hidden reservoir. Deep sequencing of densely sampled pathogens has the potential to transform this understanding, but requires analytical approaches that jointly consider epidemiological and genetic data to best address this problem. While there has been considerable success in analyses of single species systems, the hidden reservoir problem is relatively under-studied. A well-known exemplar of this problem is bovine Tuberculosis, a disease found in British and Irish cattle caused by Mycobacterium bovis, where the Eurasian badger has long been believed to act as a reservoir but remains of poorly quantified importance except in very specific locations. As a result, the effort that should be directed at controlling disease in badgers is unclear. Here, we analyse densely collected epidemiological and genetic data from a cattle population but do not explicitly consider any data from badgers. We use a simulation modelling approach to show that, in our system, a model that exploits available cattle demographic and herd-to-herd movement data, but only considers the ability of a hidden reservoir to generate pathogen diversity, can be used to choose between different epidemiological scenarios. In our analysis, a model where the reservoir does not generate any diversity but contributes to new infections at a local farm scale are significantly preferred over models which generate diversity and/or spread disease at broader spatial scales. While we cannot directly attribute the role of the reservoir to badgers based on this analysis alone, the result supports the hypothesis that under current cattle control regimes, infected cattle alone cannot sustain M. bovis circulation. Given the observed close phylogenetic relationship for the bacteria taken from cattle and badgers sampled near to each other, the most parsimonious hypothesis is that the reservoir is the infected badger population. More broadly, our approach demonstrates that carefully constructed bespoke models can exploit the combination of genetic and epidemiological data to overcome issues of extreme data bias, and uncover important general characteristics of transmission in multi-host pathogen systems.

Prevalence of bovine tuberculosis in dairy cattle in China during 2010-2019: A systematic review and meta-analysis.

BACKGROUND: Bovine tuberculosis (bTB), caused by members of the Mycobacterium tuberculosis complex bacteria, mainly Mycobacterium bovis (M. bovis), is a major threat to public health and economic development. There has been no systematic epidemiological assessment concerning bTB in dairy cattle in China. METHODOLOGY/PRINCIPAL FINDINGS: Literature related to bTB in China was retrieved from China National Knowledge Infrastructure (CNKI), PubMed, ScienceDirect, VIP Chinese Journals Database, and Wan Fang Database to build the first meta-analysis for estimating the prevalence and infection moderators of bTB in dairy cattle in China. A total of 100 relevant studies published from 2010 to 2019 were included. We estimated the overall prevalence of bTB was 2.4% (95% CI: 2.1-2.8) during this decade. In the sampling year subgroup, the prevalence was lowest in 2017 or later at 0.8% (95% CI: 0.3-1.5). The lowest prevalence was 0.7% (95% CI: 0.5-1.0) in Northwestern China. The lowest prevalence was 2.1% (95% CI: 1.8-2.5) using SIT test. Heifer cows had the highest prevalence, which was 27.1% (95% CI: 9.7-49.2). The prevalence in scale farming was 3.7% (95% CI: 3.1-4.3), significantly higher than that in free-range farming (1.7%, 95% CI: 1.1-2.4). The prevalence of bTB was highest in summer at 4.0% (95% CI: 1.7-7.0). In addition, the influence of different geographical factors (altitude, longitude, latitude, precipitation, temperature, humidity) on the prevalence was analyzed. CONCLUSIONS/SIGNIFICANCE: The results showed that bTB was widespread in China but has been gradually reduced through concerted national intervention. It is suggested that different countries should formulate corresponding prevention and control measures according to the epidemic situation in its cattle industry. Enhanced monitoring of warm and humid areas may play an important role in reducing the incidence of bTB. In addition, when large-scale breeding is promoted, attention should be paid to standardizing breeding management and improving animal welfare to reduce the prevalence of bTB in cattle.

Bovine tuberculosis due to Mycobacterium bovis and other mycobacteria among water buffalo (Bubalus bubalis) from the Brazilian Amazon.

INTRODUCTION: Zoonotic tuberculosis is a disease of public health importance worldwide, especially in developing countries. The present study aimed to investigate the role played by Mycobacterium bovis and other mycobacteria as etiologic agents of bubaline tuberculosis (TB) in the Brazilian Amazon region. METHODOLOGY: Granulomatous lesions suggestive of TB obtained from 109 buffaloes (n =109) during sanitary inspection at slaughter were subjected to histopathological evaluation, immunohistochemical (IHC) detection of Mycobacterium antigens, and to molecular tests (PCR) to detect hsp65, IS6110 and RD4 genes, which are specific to Mycobacterium spp., Mycobacterium tuberculosis Complex (MTBC) and M. bovis, respectively. RESULTS: PCR results indicated Mycobacterium infection in 87.2% of the cases, of which 69.5% were positive for M. bovis, 27.4% belonged to MTBC, and 3.1% were probably non-TB mycobacteria. There was good agreement between the genus-specific molecular technique and the histopathological analysis. This high frequency of TB cases caused by non-M. bovis suggests a diversified scenario of mycobacteria associated with bubaline TB in the Brazilian Amazon region. CONCLUSIONS: The results reinforce the need of discussing the inclusion of more accurate techniques in examinations carried out by Inspection Services in Brazil.

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.

Population structure and transmission of Mycobacterium bovis in Ethiopia.

Bovine tuberculosis (bTB) is endemic in cattle in Ethiopia, a country that hosts the largest national cattle herd in Africa. The intensive dairy sector, most of which is peri-urban, has the highest prevalence of disease. Previous studies in Ethiopia have demonstrated that the main cause is Mycobacterium bovis, which has been investigated using conventional molecular tools including deletion typing, spoligotyping and Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Here we use whole-genome sequencing to examine the population structure of M. bovis in Ethiopia. A total of 134 M. bovis isolates were sequenced including 128 genomes from 85 mainly dairy cattle and six genomes isolated from humans, originating from 12 study sites across Ethiopia. These genomes provided a good representation of the previously described population structure of M. bovis, based on spoligotyping and demonstrated that the population is dominated by the clonal complexes African 2 (Af2) and European 3 (Eu3). A range of within-host diversity was observed amongst the isolates and evidence was found for both short- and long-distance transmission. Detailed analysis of available genomes from the Eu3 clonal complex combined with previously published genomes revealed two distinct introductions of this clonal complex into Ethiopia between 1950 and 1987, likely from Europe. This work is important to help better understand bTB transmission in cattle in Ethiopia and can potentially inform national strategies for bTB control in Ethiopia and beyond.

Interferon-Gamma Release Assays Differentiate between Mycobacterium avium Complex and Tuberculous Lymphadenitis in Children.

OBJECTIVES: To assess the performance of interferon-gamma release assays (IGRAs) in the differential diagnosis between Mycobacterium avium complex (MAC) and tuberculosis (TB) in children affected with subacute/chronic submandibular/cervical lymphadenitis. STUDY DESIGN: Multicenter observational study comparing children with microbiologically confirmed MAC lymphadenitis from the European NontuberculouS MycoBacterial Lymphadenitis in childrEn study with children with TB lymphadenitis from the Spanish Network for the Study of Pediatric TB database. RESULTS: Overall, 78 patients with MAC and 34 with TB lymphadenitis were included. Among MAC cases, 44 out of 74 (59.5%) had positive tuberculin skin test (TST) results at the 5-mm cut-off, compared with 32 out of 33 (97%) TB cases (P < .001); at the 10-mm cut-off TST results were positive in 23 out of 74 (31.1%) vs 26 out of 31 (83.9%), respectively (P < .001). IGRA results were positive in only 1 out of 32 (3.1%) patients with MAC who had undergone IGRA testing, compared with 21 out of 23 (91.3%) TB cases (P < .001). Agreement between TST and IGRA results was poor in MAC (23.3%; κ = 0.017), but good in TB cases (95.6%; κ = 0.646). IGRAs had a specificity of 96.9% (95% CI 84.3%-99.8%), positive predictive value of 95.4% (95% CI 78.2%-99.8%), and negative predictive value of 93.9% (95% CI 80.4%-98.9%) for TB lymphadenitis. CONCLUSIONS: In contrast to TST, IGRAs have high specificity, negative predictive value, and positive predictive value for TB lymphadenitis in children with subacute/chronic lymphadenopathy, and consequently can help to discriminate between TB and MAC disease. Therefore, IGRAs are useful tools in the diagnostic work-up of children with lymphadenopathy, particularly when culture and polymerase chain reaction results are negative.

Genomic and temporal analyses of Mycobacterium bovis in southern Brazil.

Mycobacterium bovis is a causal agent of bovine tuberculosis (bTB), one of the most important diseases currently facing the cattle industry worldwide. Tracing the source of M. bovis infections of livestock is an important tool for understanding the epidemiology of bTB and defining control/eradication strategies. In this study, whole genome sequencing (WGS) of 74 M. bovis isolates sourced from naturally infected cattle in the State of Rio Grande do Sul (RS), southern Brazil, was used to evaluate the population structure of M. bovis in the region, identify potential transmission events and date the introduction of clonal complex (CC) European 2 (Eu2). In silico spoligotyping identified 11 distinct patterns including four new profiles and two CCs, European 1 (Eu1) and Eu2. The analyses revealed a high level of genetic diversity in the majority of herds and identified putative transmission clusters that suggested that within- and between-herd transmission is occurring in RS. In addition, a comparison with other published M. bovis isolates from Argentina, Brazil, Paraguay and Uruguay demonstrated some evidence for a possible cross-border transmission of CC Eu1 into RS from Uruguay or Argentina. An estimated date for the introduction of CC Eu2 into RS in the middle of the 19th century correlated with the historical introduction of cattle into RS to improve existing local breeds. These findings contribute to the understanding of the population structure of M. bovis in southern Brazil and highlight the potential of WGS in surveillance and helping to identify bTB transmission.

Antigen-Specific Tissue-Resident Memory T Cells in the Respiratory System Were Generated following Intranasal Vaccination of Mice with BCG.

Tissue-resident memory T cells (TRM) are different from effector memory T cells (TEM) and central memory T cells (TCM) and contribute to the protective immunity against local challenges. Currently, we found that CD4+ and CD8+ TRM cells in the nasal mucosa, trachea, lungs, and lavage fluids were heterogeneous on the expression of CD69 and CD103 as well as the production of cytokines including IFN-γ, IL-2, and TNF-α. After intranasal vaccination of mice with BCG, respiratory tissues expressed higher levels of the chemokine CXCL16 and TRM cells expressed CXCR6 to CXCL16. In addition, antigen-specific CD4+ and CD8+ TRM cells expressed cytokines following the stimulation with BCG and persisted in the nasal mucosa, trachea, and lungs for more than a hundred days. At the same time, mice were infected intranasally with live BCG and the results showed that vaccinated mice cleared up live BCG faster than nonvaccinated mice in the respiratory system. Taken together, our data demonstrated that intranasal vaccination of mice with BCG could induce antigen-specific CD4+ and CD8+ TRM cells in the respiratory system and have the ability to provide protection against pulmonary reinfection.

Serological test performance for bovine tuberculosis in cattle from herds with evidence of on-going infection in Northern Ireland.

The ability to accurately identify infected hosts is the cornerstone of effective disease control and eradication programs. In the case of bovine tuberculosis, accurately identifying infected individual animals has been challenging as all available tests exhibit limited discriminatory ability. Here we assess the utility of two serological tests (IDEXX Mycobacterium bovis Ab test and Enfer multiplex antibody assay) and assess their performance relative to skin test (Single Intradermal Comparative Cervical Tuberculin; SICCT), gamma-interferon (IFNγ) and post-mortem results in a Northern Ireland setting. Furthermore, we describe a case-study where one test was used in conjunction with statutory testing. Serological tests using samples taken prior to SICCT disclosed low proportions of animals as test positive (mean 3% positive), despite the cohort having high proportions with positive SICCT test under standard interpretation (121/921; 13%) or IFNγ (365/922; 40%) results. Furthermore, for animals with a post-mortem record (n = 286), there was a high proportion with TB visible lesions (27%) or with laboratory confirmed infection (25%). As a result, apparent sensitivities within this cohort was very low (≤15%), however the tests succeeded in achieving very high specificities (96-100%). During the case-study, 7/670 (1.04%) samples from SICCT negative animals from a large chronically infected herd were serology positive, with a further 17 animals being borderline positive (17/670; 2.54%). Nine of the borderline animals were voluntarily removed, none of which were found to be infected post-mortem (no lesions/bacteriology negative). One serology test negative animal was subsequently found to have lesions at slaughter with M. bovis confirmed in the laboratory.

Single-nucleotide polymorphism-based epidemiological analysis of Korean Mycobacterium bovis isolates.

BACKGROUND: Bovine tuberculosis (TB) is caused by Mycobacterium bovis, a well-known cause of zoonotic tuberculosis in cattle and deer, and has been investigated in many physiological and molecular studies. However, detailed genome-level studies of M. bovis have not been performed in Korea. OBJECTIVES: To survey whole genome-wide single-nucleotide polymorphism (SNP) variants in Korean M. bovis field isolates and to define M. bovis groups in Korea by comparing SNP typing with spoligotyping and variable number tandem repeat typing. METHODS: A total of 46 M. bovis field isolates, isolated from laryngopharyngeal lymph nodes and lungs of Korean cattle, wild boar, and Korean water deer, were used to identify SNPs by performing whole-genome sequencing. SNP sites were confirmed via polymerase chain reaction using 87 primer pairs. RESULTS: We identified 34 SNP sites with different frequencies across M. bovis isolates, and performed SNP typing and epidemiological analysis, which divided the 46 field isolates into 16 subtypes. CONCLUSIONS: Through SNP analysis, detailed differences in samples with identical spoligotypes could be detected. SNP analysis is, therefore, a useful epidemiological tracing tool that could enable better management of bovine TB, thus preventing further outbreaks and reducing the impact of this disease.

Novel molecular transport medium used in combination with Xpert MTB/RIF ultra provides rapid detection of Mycobacterium bovis in African buffaloes.

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore MTM at ambient temperature until Xpert MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis-unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis-positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis-unexposed animals, showing the potentially high specificity of Ultra with PrimeStore swabs. PrimeStore MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.

Simulating the next steps in badger control for bovine tuberculosis in England.

Industry-led culling of badgers has occurred in England to reduce the incidence of bovine tuberculosis in cattle for a number of years. Badger vaccination is also possible, and a move away from culling was "highly desirable" in a recent report to the UK government. Here we used an established simulation model to examine badger control option in a post-cull environment in England. These options included no control, various intermittent culling, badger vaccination and use of a vaccine combined with fertility control. The initial simulated cull led to a dramatic reduction in the number of infected badgers present, which increased slowly if there was no further badger management. All three approaches led to a further reduction in the number of infected badgers, with little to choose between the strategies. We do note that of the management strategies only vaccination on its own leads to a recovery of the badger population, but also an increase in the number of badgers that need to be vaccinated. We conclude that vaccination post-cull, appears to be particularly effective, compared to vaccination when the host population is at carrying capacity.