Genotyping of Francisella tularensis subsp. holarctica from Hares in Germany.

Jörg Linde,Mats Forsman,David Sundell,Caroline Öhrman,Julia M. Riehm,Alexandra Dangel,Timo Homeier-Bachmann,Herbert Tomaso

doi:10.3390/microorganisms8121932

Abstract

Francisella tularensis is the causative agent of the zoonotic disease tularemia. In Germany, most human infections are caused by contact with infected hares. The aim of this study was to characterize Francisella tularensis subsp. holarctica strains isolated from hares in Germany and to develop bioinformatics tools to analyze their genetic relatedness. In total, 257 German isolates—obtained mainly from hares (n = 233), other vertebrate animals, and ticks, but also from humans (n = 3)—were analyzed within this study. Publically available sequence data from 49 isolates were used to put our isolates into an epidemiological context and to compare isolates from natural foci and humans. Whole-genome sequences were analyzed using core-genome Multi-Locus-Sequence-Typing, canonical Single Nucleotide Polymorphism (SNP) typing and whole-genome SNP typing. An overall conformity of genotype clustering between the typing methods was found, albeit with a lower resolution for canonical single SNP typing. The subclade distribution, both on local and national levels, among strains from humans and hares was similar, suggesting circulation of the same genotypes both in animals and humans. Whilst close to identical isolates of the same subclade were found distributed over large areas, small geographical foci often harbored members of different subclades. In conclusion, although genomic high-resolution typing was shown to be robust, reproducible and allowed the identification of highly closely related strains, genetic profiling alone is not always conclusive for epidemiological linkage of F. tularensis strains.

Highlights

Tularemia is a zoonotic disease that occurs mainly in the northern hemisphere and is caused by Franciella (F.) tularensis
The F. tularensis subsp. holarctica strains used in the present study were cultivated on cysteine heart agar (CHA, Becton Dickinson, BD Heidelberg, Germany) at 37 ◦C with 5% CO2 for 48 h from human specimens, animal samples, or ticks harvested from these carcasses
Tularemia in hares (Lepus europaeus) is a reportable disease in Germany, so the vast majority of isolates at FLI was obtained from these animals

Summary

Introduction

Tularemia is a zoonotic disease that occurs mainly in the northern hemisphere and is caused by Franciella (F.) tularensis. In Germany, only the subspecies F. tularensis holarctica occurs in natural foci and humans usually acquire the disease due to contact with infected hares (Lepus europaeus) and less frequently from ticks, mosquitoes, contaminated dust, food, or water [4,5,6,7]. Wild boars (Sus scrofa) and foxes (Vulpes vulpes) proved to be useful bioindicators for the presence of the pathogen. They feed upon reservoir animals of F. tularensis such as small mammals and may be exposed to contaminated water, which can be an important source of infection [11,13,14]. In 2019, seventy-two human cases (SurvStat@RKI 2.0, https://survstat.rki.de; accessed 2020/09/11) and 207 cases among animals were reported in Germany (TSN online; accessed 2020/05/13)

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