title: Introduction and persistence of tularemia in Bulgaria.
abstract: BACKGROUND
  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 ofFrancisella tularensiscausing tularemia in wildlife and humans in the 1960s and the 1990s were related, we explored their genetic diversity.
  
  
  METHODS
  TenF. tularensisgenomes 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.
  
  
  CONCLUSIONS
  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 thatF. tularensismay 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.
  
  
  CONCLUSIONS
  Genome analysis of strains from two outbreaks in the 1960s and the 1990s provided valuable information on the genetic diversity and persistence ofF. tularensisin Bulgaria.
authors: Department of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden; kerstin.myrtennas@foi.se.
authors: Kerstin
authors: K
authors: Myrtennäs
authors: Department of Microbiology, Military Medical Academy (MMA), Sofia, Bulgaria.
authors: Krustyu
authors: K
authors: Marinov
authors: Departments of Clinical Microbiology, Clinical Bacteriology, and Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.
authors: Anders
authors: A
authors: Johansson
authors: Biological Threat Identification Center, Military Institute of Hygiene and Epidemiology (MIHE), Pulawy, Poland.
authors: Marcin
authors: M
authors: Niemcewicz
authors: Department of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden.
authors: Edvin
authors: E
authors: Karlsson
authors: Department of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden.
authors: Mona
authors: M
authors: Byström
authors: Department of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden.
authors: Mats
authors: M
authors: Forsman
keywords: Francisella; Illumina; assay; canSNP; genome; outbreak; phylogeography; sequencing; subtyping; zoonosis