Isolation of viable $Mycobacterium$ $bovis$ from faeces of naturally infected free-ranging rural domestic cattle ($Bos$ $taurus$)

Aims: This study investigated the presence of viable Mycobacterium bovis in faecal samples collected from 79 free-ranging domestic cattle in rural KwaZulu-Natal, South Africa. Methods and results: Faecal samples were processed under biosafety level 3 (BSL-3) conditions and analysed using mycobacterial culture followed by molecular speciation, as well as being screened using the GeneXpert® MTB/RIF Ultra (GXU®) assay. Viable M. bovis was isolated from two animals, confirmed through region-of-difference PCR and spoligotyping. These findings provide rare field-based confirmation of natural faecal shedding of viable M. bovis in cattle. The GXU® detected Mycobacterium tuberculosis complex (MTBC) DNA in eight samples (10.1%), including those that were culture positive, supporting its utility as a rapid screening tool. However, its inability to confirm bacterial viability or differentiate MTBC members remains a limitation. Additionally, nontuberculous mycobacteria (NTMs), including M. avium and M. litorale, were isolated, highlighting environmental exposure and diagnostic challenges in endemic regions. Conclusions: This study reinforces the need to consider faecal shedding and environmental reservoirs in bTB transmission dynamics, particularly in communal grazing systems. It also emphasizes the importance of integrating culture-based and molecular diagnostics for accurate detection and differentiation of mycobacterial species. These findings have important implications for One Health approaches to bTB surveillance, control, and zoonotic risk mitigation.