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Open Access Research article

German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity

Wolfgang Müller1, Helmut Hotzel1, Peter Otto1, Axel Karger2, Barbara Bettin2, Herbert Bocklisch3, Silke Braune4, Ulrich Eskens5, Stefan Hörmansdorfer6, Regina Konrad6, Anne Nesseler5, Martin Peters7, Martin Runge4, Gernot Schmoock1, Bernd-Andreas Schwarz8, Reinhard Sting9, Kerstin Myrtennäs10, Edvin Karlsson10, Mats Forsman10 and Herbert Tomaso1*

Author Affiliations

1 Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96A, Jena D-07743, Germany

2 Institute of Molecular Biology, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Südufer 10, Greifswald-Insel Riems D-17493, Germany

3 Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz, Tennstedter Str. 9, Bad Langensalza D-99947, Germany

4 Lower Saxony State Office for Consumer Protection and Food Safety, Eintrachtweg 17, Hannover D-30173, Germany

5 Landesbetrieb Hessisches Landeslabor, Schubertstr. 60, Gießen D-35393, Germany

6 Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstr. 2, Oberschleißheim D-85764, Germany

7 Staatliches Veterinäruntersuchungsamt Arnsberg, Zur Taubeneiche 10-12, Arnsberg D-59821, Germany

8 Official Laboratory for Public and Veterinary Health Saxony, Leipzig, Bahnhofstr. 58-60, Leipzig D-04158, Germany

9 Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/3, Fellbach D-70763, Germany

10 CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå SE-90182, Sweden

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BMC Microbiology 2013, 13:61  doi:10.1186/1471-2180-13-61

Published: 21 March 2013

Abstract

Background

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). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.

Results

Cultivation 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.

Conclusions

F. 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.