Tracking Mycobacterium bovis Intra-host Microevolution and Transmission among Vervet Monkeys (Chlorocebus pygerythrus) using Nanopore-Based Genomics

Mycobacterium bovis infection in vervet monkeys (Chlorocebus pygerythrus) was investigated using whole-genome sequencing (WGS) to resolve transmission dynamics and assess genetic diversity within and between outbreaks in the Skukuza section and Sabie Game Reserve regions of the Greater Kruger Conservation Area (GKCA), South Africa. Five cases from Skukuza exhibited extremely low genomic diversity, with six isolates from three individuals being genetically identical and the remaining seven isolates differing by a single nucleotide variant (SNV). These findings indicate recent and localized transmission within this population. In contrast, the Sabie monkey was infected by a highly divergent strain, differing by 68 SNVs from the Skukuza cluster, confirming two epidemiologically unrelated outbreaks. Although all isolates shared the same spoligotype (SB0121), WGS provided substantially higher resolution and demonstrated the limited discriminatory power of conventional genotyping. Within-host microevolution was detected in one individual, where a urinary bladder isolate differed by one SNV from isolates recovered from other organs, suggesting localized diversification and potential urinary shedding. Comparative genomic analysis with regional isolates collected since 1996 revealed no identical matches, although the Skukuza cluster showed close relatedness to a banded mongoose (Mungos mungo) isolate from 2022, differing by six SNVs. The Sabie isolate was most closely related to an African buffalo (Syncerus caffer) isolate from GKCA, differing by 43 SNVs. These findings highlight concurrent independent outbreaks, within-host evolution, and the importance of WGS for resolving M. bovis transmission in wildlife.