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Mycobacterium bovis causes tuberculosis (TB) at the human-wildlife-livestock interface. Environmental persistence of M. bovis excreted by infected hosts may cause indirect transmission to other animals. However, methodological constrains hamper assessment of M. bovis viability and molecular signature in environmental matrices. In this work, an innovative, modular, and highly efficient single-cell workflow combining flow cytometry (FLOW), fluorescence in situ hybridization (FISH), and fluorescence-activated cell sorting (FACS) was developed, allowing detection, quantification, and sorting of viable and dormant M. bovis cells from environmental matrices. Validation with spiked water and sediments showed high efficiency (90%) of cell recovery, with high linearity between expected and observed results, both in cell viability evaluation (r2 =0.93) and FISH-labelled M. bovis cells quantification (r2 ≥0.96). The limit of detection was established at 105 cells/g of soil in the cell viability step and 102 cells/g of soil in the taxonomical labelling stage. Moreover, FACS efficiency attained noteworthy recovery yield (50%) and purity (60% viable cells; 70% taxonomically labelled M. bovis). This new methodology represents a huge step for M. bovis assessment outside the mammal host, offering the rapid quantification of M. bovis cell load and cell viability, including viable but non-culturable cells, and further downstream cell analyses after FACS. Subsequent environmental data integration with the clinical component will expand knowledge on transmission routes, promising new paths in TB research and an intervention tool to mitigate the underlying biohazard. [Display omitted] • Environmental contamination with Mycobacterium bovis perpetrates TB in livestock and wildlife. • Mechanistic understanding of M. bovis transmission via environment is key to control TB. • A new high-performance workflow based on FLOW-FISH & FACS was developed to assess M. bovis burden in environmental matrices. • This single cell framework enables detection, quantification, and sorting of metabolically active and quiescent M. bovis. • This tool applicable to the total environment opens new avenues in TB research and biohazard control. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a widespread disease that crosses the human and animal health boundaries, with infection being reported in wildlife, from temperate and subtropical to arctic regions. Often, TB in wild species is closely associated with disease occurrence in livestock but the TB burden in wildlife remains poorly quantified on a global level. Through meta‐regression and systematic review, this study aimed to summarize global information on TB prevalence in commonly infected wildlife species and to draw a global picture of the scientific knowledge accumulated in wildlife TB. For these purposes, a literature search was conducted through the Web of Science and Google Scholar. The 223 articles retrieved, concerning a 39‐year period, were submitted to bibliometric analysis and 54 publications regarding three wildlife hosts fulfilled the criteria for meta‐regression. Using a random‐effects model, the worldwide pooled TB prevalence in wild boar is higher than for any other species and estimated as 21.98%, peaking in Spain (31.68%), Italy (23.84%) and Hungary (18.12%). The pooled prevalence of TB in red deer is estimated at 13.71%, with Austria (31.58%), Portugal (27.75%), New Zealand (19.26%) and Spain (12.08%) positioning on the top, while for European badger it was computed 11.75%, peaking in the UK (16.43%) and Ireland (22.87%). Despite these hard numbers, a declining trend in wildlife TB prevalence is apparent over the last decades. The overall heterogeneity calculated by multivariable regression ranged from 28.61% (wild boar) to 60.92% (red deer), indicating that other unexplored moderators could explain disease burden. The systematic review shows that the most prolific countries contributing to knowledge related with wildlife TB are settled in Europe and Mycobacterium bovis is the most reported pathogen (89.5%). This study provides insight into the global epidemiology of wildlife TB, ascertaining research gaps that need to be explored and informing how should surveillance be refined. [ABSTRACT FROM AUTHOR]