<p>Unifrac Results.</p>

Anthrax, caused by Bacillus anthracis (BA), is a prominent neglected zoonosis with major impacts on human, livestock, and wildlife health. Despite this, limited genomic investigation at the One Health interface constrains current understanding of BA transmission and of the ecological and host factors shaping its diversity and population structure. This includes the possibility of host-specific BA lineages, given that anthrax outbreaks often disproportionally affect individual species. This study characterises the genomic diversity of BA in an endemic area, the Ngorongoro Conservation Area (NCA), in northern Tanzania. We analysed 213 BA genomes from livestock, wildlife and humans from cultured isolates combined with a culture-free targeted capture (TC) approach. NCA sequences formed a distinct genetic cluster compared with those from surrounding areas, and we observed surprisingly high levels of strain diversity within apparent epidemiological clusters, as well as within single animals, though strain diversity was lowest at the within host scale. We found limited evidence for seasonal clustering of cases as well as for BA lineages clustering by host species. This indicates that disproportional impacts on certain species during outbreaks are more likely driven by host ecology factors or, hypothetically, by accessory parts of the bacterial genome not represented in our data. TC-derived data significantly expanded the range of host species and geographic locations for genomic analysis, demonstrating the value of this approach. Although TC data may contain artefactual variation, shared SNP profiles between isolate- and TC-derived genomes gave confidence in its use for genotyping. Our analysis demonstrates unexpectedly high BA strain diversity and limited population structure in this endemic area across a range of spatial scales, including within-host. It further highlights the need for high density sampling and adaptable sequencing strategies to generate adequate BA genomic datasets that can enable informative molecular epidemiological studies of anthrax at the One Health interface.