The aim of this study was to assess the diversity of minisatellite VNTR loci in Mycobacterium bovis/M. caprae isolates in Bulgaria and view their position within global M. bovis diversity. Forty-three M. bovis/M. caprae isolates from cattle in different farms in Bulgaria were collected in 2015-2021 and typed in 13 VNTR loci. The M. bovis and M. caprae branches were clearly separated on the VNTR phylogenetic tree. The larger and more geographically dispersed M. caprae group was more diverse than M. bovis group was (HGI 0.67 vs. 0.60). Overall, six clusters were identified (from 2 to 19 isolates) and nine orphans (all loci-based HGI 0.79). Locus QUB3232 was the most discriminatory one (HGI 0.64). MIRU4 and MIRU40 were monomorphic, and MIRU26 was almost monomorphic. Four loci (ETRA, ETRB, Mtub21, and MIRU16) discriminated only between M. bovis and M. caprae. The comparison with published VNTR datasets from 11 countries showed both overall heterogeneity between the settings and predominantly local evolution of the clonal complexes. To conclude, six loci may be recommended for primary genotyping of M. bovis/M. caprae isolates in Bulgaria: ETRC, QUB11b, QUB11a, QUB26, QUB3232, and MIRU10 (HGI 0.77). VNTR typing based on a limited number of loci appears to be useful for primary bTB surveillance.
Keywords: Bulgaria; Mycobacterium bovis; Mycobacterium caprae; VNTR; bTB; genotyping; whole-genome sequencing.