Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163.

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

Absence of effects of widespread badger culling on tuberculosis in cattle.

Government policy in England aims for the elimination of bovine tuberculosis (bTB). This policy includes culling of European badger (Meles meles) to reduce cattle TB incidence. The rationale is based on a field trial, the Randomised Badger Culling Trial (RBCT) 1998-2005, which reported a substantial decrease in bTB herd incidence where badger culling had been implemented, in comparison to untreated control areas. The RBCT was undertaken because previous studies of reductions in badgers by culling, reported a possible association between bTB in badger and cattle, but none could directly show causation. The effect of intensive widespread (proactive) culling in the RBCT was reported in 2006 in the journal Nature. Analysis of an extensive badger removal programme in England since 2013 has raised concerns that culling has not reduced bTB herd incidence. The present study re-examined RBCT data using a range of statistical models. Most analytical options showed no evidence to support an effect of badger culling on bTB herd incidence 'confirmed' by visible lesions and/or bacterial culture post mortem following a comparative intradermal skin test (SICCT). However, the statistical model chosen by the RBCT study was one of the few models that showed an effect. Various criteria suggest that this was not an optimal model, compared to other analytical options available. The most likely explanation is that the RBCT proactive cull analysis over-fitted the data with a non-standard method to control for exposure giving it a poor predictive value. Fresh appraisal shows that there was insufficient evidence to conclude RBCT proactive badger culling affected bTB breakdown incidence. The RBCT found no evidence of an effect of culling on 'total' herd incidence rates. Total herd incidences include those confirmed as bTB at necropsy and those herds where there was at least one animal animal positive to the comparative intradermal skin test, the standard diagnostic test used for routine surveillance, but not confirmed at necropsy. This was also the case using the more suitable statistical models. Use only of 'confirmed' herd incidence data, together with a more recent (2013) published perception that RBCT data presented 'a strong evidence base….with appropriate detailed statistical or other quantitative analysis' should be reconsidered. The results of the present report are consistent with other analyses that were unable to detect any disease control benefits from badger culling in England (2013-2019). This study demonstrates one form of potential driver to the reproducibility crisis, in this case with disease control management in an increasingly intensified livestock industry.

Assessing the feasibility of test-and-cull and test-and-segregation approaches for the control of high-prevalence bovine tuberculosis in Ethiopian intensive dairy farms.

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.

Expression and field evaluation of new Mycobacterium bovis antigens.

Bovine tuberculosis (bTB) represents a threat to livestock production. Mycobacterium bovis is the main causative agent of bTB and a pathogen capable of infecting wildlife and humans. Eradication programs based on surveillance in slaughterhouses with mandatory testing and culling of reactive cattle have failed to eradicate bTB in many regions worldwide. Therefore, developing effective tools to control this disease is crucial. Using a computational tool, we identified proteins in the M. bovis proteome that carry predictive binding peptides to BoLADRB3.2 and selected Mb0309, Mb1090, Mb1810 and Mb3810 from all the identified proteins. The expression of these proteins in a baculovirus-insect cell expression system was successful only for Mb0309 and Mb3810. In parallel, we expressed the ESAT-6 family proteins EsxG and EsxH in this system. Among the recombinant proteins, Mb0309 and EsxG exhibited moderate performance in distinguishing between cattle that test positive and negative to bTB using the official test, the intradermal tuberculin test (IDT), when used to stimulate interferon-gamma production in blood samples from cattle. However, when combined as a protein cocktail, Mb0309 and EsxG were reactive in 50 % of positive cattle. Further assessments in cattle that evade the IDT (false negative) and cattle infected with Mycobacterium avium paratuberculosis are necessary to determine the potential utility of this cocktail as an additional tool to assist the accurate diagnosis of bTB.

Genome-Wide Association Study Reveals Quantitative Trait Loci and Candidate Genes Associated with High Interferon-gamma Production in Holstein Cattle Naturally Infected with Mycobacterium Bovis.

Mycobacterium bovis (Mb) is the causative agent of bovine tuberculosis (bTb). Genetic selection aiming to identify less susceptible animals has been proposed as a complementary measure in ongoing programs toward controlling Mb infection. However, individual animal phenotypes for bTb based on interferon-gamma (IFNÉ£) and its use in bovine selective breeding programs have not been explored. In the current study, IFNÉ£ production was measured using a specific IFNÉ£ ELISA kit in bovine purified protein derivative (bPPD)-stimulated blood samples collected from Holstein cattle. DNA isolated from the peripheral blood samples collected from the animals included in the study was genotyped with the EuroG Medium Density bead Chip, and the genotypes were imputed to whole-genome sequences. A genome-wide association analysis (GWAS) revealed that the IFNÉ£ in response to bPPD was associated with a specific genetic profile (heritability = 0.23) and allowed the identification of 163 SNPs, 72 quantitative trait loci (QTLs), 197 candidate genes, and 8 microRNAs (miRNAs) associated with this phenotype. No negative correlations between this phenotype and other phenotypes and traits included in the Spanish breeding program were observed. Taken together, our results define a heritable and distinct immunogenetic profile associated with strong production of IFNÉ£ in response to Mb.

Computing metrics to inform selection of candidate areas for a regionalized approach to bovine tuberculosis eradication in Ireland.

We describe the computation of metrics to inform the selection of areas for a regionalised approach to bovine tuberculosis eradication in Ireland. Our aim was not to recommend suitable regions but to elucidate the criteria used in metric selection and comment on the diversity of metric values amongst regions. The 26 counties of Ireland were compared using 20 metrics, grouped into five categories: region size and cattle population, herd fragmentation, cattle movement, bovine TB testing, badger population and control. Fragmentation metrics, measuring the proportion of herds with land in at least two counties, varied considerably by county, from 1% to 24 %.  Between 25 % and 92 % of moves into herds came from a different county, illustrating the likely disruption in trade that a regionalized approach could entail. Cattle movement networks were combined with a risk model to calculate the proportion of moves which would be deemed risky under a risk-based trading regime and these results were compared to a more traditional approach based on the herd type and test history of each herd, with many fewer moves potentially restricted using the latter approach. We show how correlation between region size and some of the metrics complicates their interpretation.

A scoping review on bovine tuberculosis highlights the need for novel data streams and analytical approaches to curb zoonotic diseases.

Zoonotic diseases represent a significant societal challenge in terms of their health and economic impacts. One Health approaches to managing zoonotic diseases are becoming more prevalent, but require novel thinking, tools and cross-disciplinary collaboration. Bovine tuberculosis (bTB) is one example of a costly One Health challenge with a complex epidemiology involving humans, domestic animals, wildlife and environmental factors, which require sophisticated collaborative approaches. We undertook a scoping review of multi-host bTB epidemiology to identify trends in species publication focus, methodologies, and One Health approaches. We aimed to identify knowledge gaps where novel research could provide insights to inform control policy, for bTB and other zoonoses. The review included 532 articles. We found different levels of research attention across episystems, with a significant proportion of the literature focusing on the badger-cattle-TB episystem, with far less attention given to tropical multi-host episystems. We found a limited number of studies focusing on management solutions and their efficacy, with very few studies looking at modelling exit strategies. Only a small number of studies looked at the effect of human disturbances on the spread of bTB involving wildlife hosts. Most of the studies we reviewed focused on the effect of badger vaccination and culling on bTB dynamics with few looking at how roads, human perturbations and habitat change may affect wildlife movement and disease spread. Finally, we observed a lack of studies considering the effect of weather variables on bTB spread, which is particularly relevant when studying zoonoses under climate change scenarios. Significant technological and methodological advances have been applied to bTB episystems, providing explicit insights into its spread and maintenance across populations. We identified a prominent bias towards certain species and locations. Generating more high-quality empirical data on wildlife host distribution and abundance, high-resolution individual behaviours and greater use of mathematical models and simulations are key areas for future research. Integrating data sources across disciplines, and a "virtuous cycle" of well-designed empirical data collection linked with mathematical and simulation modelling could provide additional gains for policy-makers and managers, enabling optimised bTB management with broader insights for other zoonoses.

First time whole genome sequencing of Mycobacterium bovis from the environment supports transmission at the animal-environment interface.

Spreading of Mycobacterium bovis causing animal tuberculosis (TB) at livestock-wildlife-environment interfaces remains a significant problem. Recently, we provided evidence of widespread environmental contamination of an endemic animal TB setting with viable and dormant M. bovis cells able to recover metabolic activity, making indirect transmission via environmental contamination plausible. We now report the first whole genome sequences of M. bovis recovered from the environment. We establish epidemiological links at the environment-animal interface by phylogenomic comparison of these M. bovis genomes with those isolated from livestock and wild ungulates from the same area. Environmental and animal genomes are highly intertwined and distribute similarly into the same M. bovis lineages, supporting several instances of environmental contamination. This study provides compelling evidence of M. bovis excretion into the environment and viability maintenance, supporting the environment as a potential source of new infection. These insights have clear implications for policy formulation, advocating environmental surveillance and an ecosystem perspective in TB control programs. ENVIRONMENTAL IMPLICATION: We report the first whole genome sequences of M. bovis from the environment and establish epidemiological links at the environment-animal interface, demonstrating close phylogenomic relatedness of animal and environmental M. bovis. Definitive evidence of M. bovis excretion into the environment with viability maintenance is provided, supporting the environment as a potential source of new infection. Implications of this work include methodological innovations offering a tool to resolve indirect transmission chains and support customized biosecurity measures. Policy formulation aiming at the control of animal tuberculosis and cost mitigation should consider these findings, encouraging environmental surveillance in official eradication programmes.

'It is impossible to meet our officially bTB-free targets with the current testing policy'.

Neil J Watt and Keith Cutler argue that Defra's aim of achieving officially bovine tuberculosis (bTB)-free status for England by 2038 is unlikely to be met without a drastic change to testing and policy.

Is badger culling associated with risk compensation behaviour among cattle farmers?

BACKGROUND: Risk compensation theory suggests that behaviours are modified in response to interventions that remove risks by substituting them with other risky behaviours to maintain a 'risk equilibrium'. Alternatively, risk reduction interventions may result in spill-over behaviours that seek to minimise risks further. This paper assessed evidence for these behavioural risk responses among farmers in response to badger culling that seeks to remove the risk of bovine tuberculosis in cattle. METHODS: Data from the UK's randomised badger culling trial were re-analysed, comparing farmers' cattle movement practices in proactive and reactive culling areas and control areas. Analysis compared cattle movements during and after the trial using zero-inflated negative binomial regression. RESULTS: The analysis found no strong evidence of risk compensation behaviours among farmers who experienced proactive culling. However, strong evidence for a reduction in cattle movements in reactive culling areas was found. The results indicate high levels of inertia within farming systems in relation to cattle purchasing. LIMITATIONS: Data do not account for the risk of cattle purchases and reflect previous policy regimens. Evidence from recent badger culling interventions should be analysed. CONCLUSION: Proactive badger culling was not associated with risk compensation behaviours, while reactive badger culling was associated with decreased risk taking among farmers.

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

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

Seroprevalence and risk factors associated with bovine tuberculosis in cattle in Eastern Bhutan.

Bovine tuberculosis (bTB) is a chronic zoonotic disease affecting cattle of all age groups including wild animals. It poses a significant threat to public health and high economic losses to dairy farmers. While the disease has been eradicated from most of the developed countries through extensive surveillance, testing and culling strategy, it is endemic in Africa, Asia, and the Middle East countries. Currently, there is limited research regarding the prevalence of bTB in cattle in Bhutan. This study aimed to determine the seroprevalence of bTB in cattle in six districts of eastern Bhutan. A two-stage probability proportional to size (PPS) sampling strategy was used to determine the number of animals from which serum samples needed to be collected in each district and sub-district. All farms and cattle for sampling were randomly selected from the data in the annual livestock census of 2020. The samples were tested using bTB ELISA test kit. The seroprevalence and their 95% confidence intervals were calculated. Logistic regression models were constructed to assess the influence of various individual animal and environmental risk factors (breed, age, sex, source of animal, body condition scores of animals, respiratory system status) associated with sero-positivity in animals. The study revealed an apparent seroprevalence of 2.57% (25/971 cattle; 95% CI:1.58-3.57), with an estimated true seroprevalence of 0.91% (95% CI: 0.0-2.81). However, none of the variables were found to be significantly associated with bTB seroprevalence in cattle. We recommend, further sampling and employment of confirmatory testing to fully ascertain the extent of bTB in the cattle herds in eastern Bhutan for prevention and control.

Population structure and history of Mycobacterium bovis European 3 clonal complex reveal transmission across ecological corridors of unrecognized importance in Portugal.

Mycobacterium bovis causes animal tuberculosis in livestock and wildlife, with an impact on animal health and production, wildlife management, and public health. In this work, we sampled a multi-host tuberculosis community from the official hotspot risk area of Portugal over 16 years, generating the largest available data set in the country. Using phylogenetic and ecological modeling, we aimed to reconstruct the history of circulating lineages across the livestock-wildlife interface to inform intervention and the implementation of genomic surveillance within the official eradication plan. We find evidence for the co-circulation of M. bovis European 1 (Eu1), Eu2, and Eu3 clonal complexes, with Eu3 providing sufficient temporal signal for further phylogenetic investigation. The Eu3 most recent common ancestor (bovine) was dated in the 1990s, subsequently transitioning to wildlife (red deer and wild boar). Isolate clustering based on sample metadata was used to inform phylogenetic inference, unravelng frequent transmission between two clusters that represent an ecological corridor of previously unrecognized importance in Portugal. The latter was associated with transmission at the livestock-wildlife interface toward locations with higher temperature and precipitation, lower agriculture and road density, and lower host densities. This is the first analysis of M. bovis Eu3 complex in Iberia, shedding light on background ecological factors underlying long-term transmission and informing where efforts could be focused within the larger hotspot risk area of Portugal. IMPORTANCE: Efforts to strengthen surveillance and control of animal tuberculosis (TB) are ongoing worlwide. Here, we developed an eco-phylodynamic framework based on discrete phylogenetic approaches informed by M. bovis whole-genome sequence data representing a multi-host transmission system at the livestock-wildlife interface, within a rich ecological landscape in Portugal, to understand transmission processes and translate this knowledge into disease management benefits. We find evidence for the co-circulation of several M. bovis clades, with frequent transmission of the Eu3 lineage among cattle and wildlife populations. Most transition events between different ecological settings took place toward host, climate and land use gradients, underscoring animal TB expansion and a potential corridor of unrecognized importance for M. bovis maintenance. Results stress that animal TB is an established wildlife disease without ecological barriers, showing that control measures in place are insufficient to prevent long-distance transmission and spillover across multi-host communities, demanding new interventions targeting livestock-wildlife interactions.