Amplification products were visualized in a 1% agarose gel stained with ethidium bromide on a UV illuminator and subsequently purified using the PureLink PCR Purification Kit, following the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA). Products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit and purified using the BigDye XTerminator Purification Kit following the manufacturer’s protocols on an AB 3500xL Genetic Analyzer (Applied Biosystems, Grand Island, NY, USA). Due to highly conserved 16S rRNA gene sequences between Francisellaceae, DNA extracts of the ticks were also tested specifically for the presence of Francisella tularensis using a multi-target real-time PCR test employing primers ISFtu 2, iglC and tul4 that are specific for F. tularensis as described in Kugeler et al. (2005) and Versage et al. (2003). All reactions were performed in a final volume of 20 μl and contained LightCycler FastStart DNA Master HybProbe mix (Roche, Mannheim, Germany) at a 1× final concentration, 500 nM forward and reverse primers, 100 nM probes, and 1.25 U of uracil-DNA glycosylase per reaction. For the iglC and tul4 the final MgCl2 concentration was 4 mM, and for the ISFtu2 assay, the final concentration was 5 mM. Real-time PCR cycling conditions were: 50 °C for 2 min; 95 °C for 10 min; 45 cycles of: 95 °C for 10 s, 60 °C for 30 s; and 45 °C for 5 min.