Abstract
Mycobacterium bovis infection poses a significant threat to the biodiversity and conservation of South African wildlife. Despite this, few studies have explored transmission dynamics within these complex multi-host systems. This study used whole-genome sequencing to investigate the genetic diversity and relatedness of M. bovis strains across various wildlife species and regions in South Africa to explore transmission patterns. A total of 112 M. bovis isolates from 106 individuals representing 12 species underwent short-read sequencing. Two animal-adapted sub-lineages, La1.7.1 (clonal complex Eu2) and La1.8.1 (Eu1), exhibited geographic clustering and notable genomic diversity. Closely related isolates (≤5 SNP differences) were primarily found within single host species, particularly African buffalo (Syncerus caffer), indicating intra-species transmission and potential source identification. In contrast, other genetically similar isolates (≤12 SNP differences), collected over 25 years, suggest historical inter-species transmission. Understanding these transmission patterns is essential for developing effective strategies to control the spread of M. bovis and protect vulnerable wildlife populations.