Abstract
BackgroundTularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.ResultsCultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level.ConclusionsF. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.
Highlights
Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates
F. tularensis subsp. holarctica was identified in all 52 cases
variable-number of tandem repeats (VNTRs) typing In a pilot study, six loci (Ft-M3, Ft-M6, Ft-M20, Ft-M21, Ft-M22, and Ft-M24) were amplified and sequenced, but only the loci Ft-M3, Ft-M6, and Ft-M24 were discriminatory
Summary
Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). Most infections in animals and humans are caused by two F. tularensis subspecies, F. tularensis subsp. After years without reported cases in animals the re-emergence of tularemia started in 2004 with an outbreak of tularemia in a semi-free living group of marmosets (Callithrix jacchus) in Lower Saxony [7], and in December 2005 an outbreak with 15 human cases due to contact with infected hares was reported from Hesse [8]. Holarctica in organ samples of these hares using PCR assays was the beginning of our investigations of tularemia in European brown hares (Lepus europaeus) in Germany The detection of F. tularensis subsp. holarctica in organ samples of these hares using PCR assays was the beginning of our investigations of tularemia in European brown hares (Lepus europaeus) in Germany
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