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 M. 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, non-tuberculous 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.