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.
['Myrtennäs|Kerstin|K|', 'Marinov|Krustyu|K|', 'Johansson|Anders|A|', 'Niemcewicz|Marcin|M|', 'Karlsson|Edvin|E|', 'Byström|Mona|M|', 'Forsman|Mats|M|']
outbreak Francisella genome sequencing canSNP assay Illumina phylogeography subtyping zoonosis