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.

Introduction and persistence of tularemia in Bulgaria.

INTRODUCTION: Outbreaks of the zoonotic disease tularemia occurred in north-east Bulgaria in the 1960s. Then came 30 years of epidemiological silence until new outbreaks occurred in west Bulgaria in the 1990s. To investigate how bacterial strains of Francisella tularensis causing tularemia in wildlife and humans in the 1960s and the 1990s were related, we explored their genetic diversity. MATERIAL AND METHODS: Ten F. tularensis genomes from the 1960s (n=3) and the 1990s (n=7) were sequenced, assigned to canonical single-nucleotide polymorphism (canSNP) clades, and compared to reference genomes. We developed four new canSNP polymerase chain reaction (PCR) assays based on the genome sequence information. RESULTS AND DISCUSSION: The genetic analysis showed that the outbreaks in the 1960s as well as in the 1990s involved multiple clones and new genetic diversity. The smallest genetic difference found between any of the Bulgarian strains was five SNPs between the strains L2 and 81 isolated 43 years apart, indicating that F. tularensis may persist locally over long time periods without causing outbreaks. The existence of genetically highly similar strain-pairs isolated the same year in the same area from different hosts supports a hypothesis of local expansion of clones during outbreaks. Close relationship (two SNPs) was found between one strain isolated 1961 in northeast Bulgaria and one strain isolated 5 years before in USSR. Historical data coinciding with the actual time point describe the introduction of water rats from USSR into the Bulgarian outbreak area, which may explain the close genetic relationship and the origin of the outbreak. CONCLUSION: Genome analysis of strains from two outbreaks in the 1960s and the 1990s provided valuable information on the genetic diversity and persistence of F. tularensis in Bulgaria.

Characterization and genotyping of strains of Francisella tularensis isolated in Bulgaria.

A tularaemia focus was detected in 1998 in Bulgaria, in an area where tularaemia had never been reported. The properties of Francisella tularensis subsp. holarctica strains isolated from 1998 to 2005 were studied. The strains showed heterogeneity, based on acid production from glycerol and erythromycin susceptibility. Genotyping by analysis of seven loci containing variable-number tandem repeats showed four genotypes among eight strains.