Mycobacterium bovis and M. caprae in Bulgaria: insight into transmission and phylogeography gained through whole-genome sequencing.

BACKGROUND: This study aimed to characterize recent Mycobacterium bovis/M. caprae isolates from Bulgaria by whole-genome sequencing (WGS) to gain a first insight into their molecular diversity, transmission, and position within the global phylogeography of this important zoonotic species. RESULTS: The isolates were obtained from cattle in diverse locations of Bulgaria in 2015-2020 and were identified by microbiological and PCR assays. WGS data were used for phylogenetic analysis that also included M. bovis global dataset. Thirty-seven M. bovis/caprae isolates from Bulgaria were studied and 34 of them were SNP genotyped. The isolates were subdivided into 3 major phylogenetic groups. Type Mbovis-13 (Eu2 complex [western Europe and northern Africa]) included one isolate. Mbovis-37 type included 5 isolates outside of known clonal complexes. The Bulgarian M. caprae isolates formed a sub-group within the Mcaprae-27B cluster which also included 22 M. caprae isolates from Poland, Spain, Germany, and the Republic of Congo. The Bulgarian M. caprae isolates share their latest common ancestors with Spanish isolates. The Mbovis-37 group shares a distant common ancestor (pairwise distance 22-29 SNPs) with an isolate from Poland but was very distant (> 200 SNPs) from the rest of the tree. The Mbovis-13 group shares a common ancestor with two human isolates from Germany. Phylogeographically, both M. bovis clades had limited circulation in northeastern Bulgaria while the majority of the studied isolates (M. caprae) were from central and western provinces. A phylogenetic network-based analysis demonstrated that 11 Bulgarian isolates were separated by 1 to 6 SNPs within four clusters, mostly forming pairs of isolates. CONCLUSION: The obtained WGS analysis positioned the Bulgarian isolates within the global phylogeography of M. bovis/M. caprae. Hypothetically, the observed phylogenetic diversity may not have resulted from livestock trade routes, but instead may reflect the deeply rooted M. bovis/M. caprae phylogeography of Europe. A high level of genetic divergence between the majority of the studied isolates suggests limited active transmission of bTB in Bulgaria during the survey period. At the same time, a possibility of the endemic presence of circulating bTB strains in the form of the latent persistent disease cannot be ruled out.

First Insight into Diversity of Minisatellite Loci in Mycobacterium bovis/M. caprae in Bulgaria.

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.

First insight into phylogeography of Mycobacterium bovis and M. caprae from cattle in Bulgaria.

Bovine tuberculosis (bTB) represents a significant economic burden to the agriculture. In spite of decades of the control program, Mycobacterium bovis infection levels in cattle in Bulgaria continued to rise over recent years. In order to gain a better understanding of the M. bovis diversity, we used spoligotyping for strain differentiation and the data were compared to the international databases Mbovis.org and SITVIT2 for shared type and clade assignment. Study sample included 30 M. tuberculosis complex isolates from cattle originating from different regions of Bulgaria. The isolates were subdivided by spoligotyping into 4 spoligotypes: 2 types shared by 20 and 8 isolates and 2 singletons. SITVIT2-defined types SIT645 and SIT647 belonged to the common and classical bovine ecotype M. bovis (9 isolates) while types SIT120 and SIT339 belonged to the M. caprae ecotype (21 isolates). A certain phylogeographic gradient of the spoligotypes and clades at the within-country level was observed: M. caprae was prevalent in the central/southwestern, while classical M. bovis in the northeastern Bulgaria. Whereas all four types have global or European circulation, neither was described in the neighboring Balkan countries. M. caprae isolates identified in this study mostly belong to the Central/Eastern European cluster. In summary, this study provided a first insight into phylogeography of M. bovis in Bulgaria and described, for the first time, M. caprae as an important infectious agent of bTB in this country.