Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp.holarctica from strains of other F. tularensissubspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulentF. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.

Background Francisella tularensis causes tularaemia, a life-threatening zoonosis, and has potential as a biowarfare agent. F. tularensis subsp. tularensis, which causes the most severe form of tularaemia, is usually confined to North America. However, a handful of isolates from this subspecies was obtained in the 1980s from ticks and mites from Slovakia and Austria. Our aim was to uncover the origins of these enigmatic European isolates.Methodology/Principal FindingsWe determined the complete genome sequence of FSC198, a European isolate of F. tularensis subsp. tularensis, by whole-genome shotgun sequencing and compared it to that of the North American laboratory strain Schu S4. Apparent differences between the two genomes were resolved by re-sequencing discrepant loci in both strains. We found that the genome of FSC198 is almost identical to that of Schu S4, with only eight SNPs and three VNTR differences between the two sequences. Sequencing of these loci in two other European isolates of F. tularensis subsp. tularensis confirmed that all three European isolates are also closely related to, but distinct from Schu S4.Conclusions/SignificanceThe data presented here suggest that the Schu S4 laboratory strain is the most likely source of the European isolates of F. tularensis subsp. tularensis and indicate that anthropogenic activities, such as movement of strains or animal vectors, account for the presence of these isolates in Europe. Given the highly pathogenic nature of this subspecies, the possibility that it has become established wild in the heartland of Europe carries significant public health implications.

The prevalence of ticks infected with F. tularensis was followed during a systematic surveillance in endemic area of tularemia in western Slovakia over the years 1984-93. Ticks were collected from vegetation in localities of Podunajské Biskupice, in the vicinity of the capital of Slovakia, Bratislava, near the river Danube. In total 6033 ticks, mostly adults of Dermacentor reticulatus and Ixodes ricinus (4994 and 1004, respectively) and 35 nymphs of Haemaphysalis concinna, were examined for the presence of F. tularensis. Out of 4542 starving ticks, 34 F. tularensis strains were isolated predominantly from D. reticulatus (30), and to a smaller extent also from I. ricinus (3) and H. concinna (1). Natural infection with F. tularensis was further proved from 1491 adults of D. reticulatus fed on laboratory animals, rabbits and white mice, together in 27 cases. From that, 21 times it was by positive isolation either from suspensions of partly or fully engorged ticks and their feaces, or from spleens of animals dead after the feeding of ticks. In addition, solely the development of antibodies against the agent was confirmed in 6 rabbit hosts. The presence of F. tularensis in all the above mentioned tick species and namely the relatively high and permanent infestation of D. reticulatus adults, ranging between 0.5-2% during the followed time period, demonstrated the maintenance of active natural focus of tularemia in the area under study. The present paper also emphasizes the epidemiologic consequence of various species of ticks in endemic foci of tularemia and the aspect of possible ways of transmission of the agent to humans.

Francisella tularensis subsp. tularensis is the common causal agent of tularemia in the USA and Canada, while F. tularensis subsp. palaearctica (holarctica) occurs in Europe, Asia, and to a minor extent in North America. F. tularensis subsp. mediaasiatica was found only in central Asia in a part of the former Soviet Union. Of the total of 155 F. tularensis strains isolated over the years 1978-1996 during the surveillance of tularemia in Slovakia, 65 were from small mammals, 68 from ticks and 22 from mites and fleas. They were characterized and classified by basic markers of infraspecific taxonomy in tests in vitro and compared with type strains of three subspecies and biovars of F. tularensis. Comparative studies have revealed biological properties characteristic of F. tularensis subsp. tularensis in 17 strains isolated from fleas and mites parasiting on small terrestrial mammals, collected in the Danube region, near Bratislava. These strains fermented glycerol, glucose, were positive for citrulline ureidase and sensitive to erythromycin, in contrast to the other 138 isolates classified as F. tularensis subsp. palaearctica (holarctica), biovar II, which fermented only glucose, were negative for citrulline ureidase and resistant to erythromycin. Two selected pairs of isolates with properties characteristic of F. tularensis subsp. palaearctica (holarctica), biovar II (SE-210, SE-234) and of F. tularensis subsp. tularensis (SE-219, SE-221), as shown in tests in vitro, were further examined for their pathogenicity on white mice, guinea pigs and domestic rabbits. In tests of virulence on domestic rabbits, the isolates SE-210 and SE-234 had low pathogenicity, while the isolates SE-219 and SE-221 exhibited high pathogenicity, which along with their biochemical properties confirmed their identification as strains of F. tularensis subsp. tularensis. The first findings of the highly virulent strains of F. tularensis subsp. tularensis in Europe indicate a serious event from epidemiologic and epiozootologic aspects, requiring systematic surveillance.

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