Epizootic investigations of tularemia and the comparative characterization of Francisella tularensis strains
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Francisella tularensis is the etiological agent of tularemia, a lethal zoonotic disease and a potential biological warfare agent. In the last decades, several emergences or re-emergences of tularemia were seen all over the world which has focused the attention on this disease. The first human tularemia cases were observed in Hungary in 1951 and the disease has been diagnosed every year ever since. In most cases tick bite, close contact with European brown hares (Lepus europaeus), hamsters (Cricetus cricetus) or rats (Rattus spp.) were found in the anamnesis. Furthermore, in efforts to replenish game populations for sporting purposes, thousands of brown hares are annually translocated from Hungary to France and Italy which is a significant income for the country. The tularemia free status of the exported hares is crucial to maintain this export. Through retrospective data collection it was shown that the number of human cases in Hungary ranged between 20 and 148 per year during the past two decades (1984-2009). At the same time the prevalence of tularemia among hares, captured for live animal export (2.8- 40 thousand exported hares/year) ranged between 0.31% and 20.2%. A one-year study of the ecological cycle of F. tularensis was performed in an enzootic area during an inter-epizootic period. The study was based on multiple sampling of all major elements of the disease cycle. Seroprevalence of tularemia in the European brown hare population was 5.1% (10/197) with low titers (1/10 and 1/20) and F. tularensis ssp. holarctica was isolated from four hares. Based on these results the modification of the diagnostic tube agglutination titer 1/80 was presumed. F. tularensis was not detected by real-time polymerase chain reaction in any of the trapped 38 common voles (Microtus arvalis), 110 yellow-necked mice (Apodemus flavicollis), 15 stripped field mice (Apodemus agrarius) and a by-catch of 8 Eurasian pygmy shrews (Sorex minutus) and 6 common shrews (Sorex araneus). A total of 1106 Ixodes ricinus and 476 Haemaphysalis concinna ticks were collected from vegetation and 404 I. ricinus, 28 H. concinna ticks and 15 Ctenophtalmus assimilis and 10 Nosopsyllus fasciatus fleas were combed off small mammals. One H. concinna female and one nymph collected from the vegetation were infected with F. tularensis ssp. holarctica thus resulting a 0.42% (2/476) prevalence. F. tularensis was not detected in environmental water samples and the examined 100 sheep, 50 cows and 50 buffaloes, grazed in the study area, were all found seronegative. It can be hypothesized that during interepizootic periods F. tularensis ssp. holarctica persists only in the European brown hare – H. concinna cycle. H. concinna may not serve exclusively as an arthropod vector but it might also harbor bacteria for three to four years through multiple life stages and act as an important reservoir of F. tularensis. Rodent species probably do not play as true reservoir hosts of F. tularensis. 7 The role of the common hamster in the natural cycle of F. tularensis was examined using serologic methods on 900 hamsters and real-time polymerase chain reaction on 100 hamsters in an endemic agricultural area. 374 Ixodes acuminatus ticks were collected from the hamsters and tested by real-time polymerase chain reaction. The results of all tests were negative. To examine clinical signs, pathology and histopathology of acute tularemia infection similar to the natural infection, two hamsters were infected with a large dose of a wild strain of F. tularensis ssp. holarctica. After a short period of apathy, the animals died on the 8th and 9th days postinfection. The pathological, histopathological and immunohistochemical examination contributed to the diagnosis of septicemia in both cases. The results confirmed previous findings that common hamsters are highly sensitive to F. tularensis. It was concluded that although septicemic hamsters could pose substantial risk to humans during tularemia outbreaks, hamsters in interepizootic periods do not act as a significant reservoir of F. tularensis. Lesions induced by F. tularensis were examined in 50 cases of naturally infected, seropositive European brown hares. Gross pathological examination revealed scant to numerous, grayishwhite foci with a diameter of 0.1-1 cm in single (24 cases) or multiple organs (20 cases) in a total of 44/50 (88%) cases. These lesions were proven to be areas of granulomatous inflammation, frequently encompassing necrosis. F. tularensis antigen was detected with immunohistochemistry in a total of 46/50 (92%) cases, while F. tularensis ssp. holarctica was isolated by culture and identified by polymerase chain reaction from 35/50 cases (70%). Infection by respiratory route was presumed by the presence of tissue lesions in the thoracic organs in 44/50 (88%) cases. These results emphasize the importance of the European brown hare as a reservoir of F. tularensis. Generalized tularemia were diagnosed in a patas monkey (Erythrocebus patas) and a vervet monkey (Chlorocebus aethiops), which both died suddenly in Szeged Zoo, Hungary. Macroscopic lesions in each animal included disseminated, grayish-white foci in the lungs, lymph nodes, spleen, liver, and kidney. All focal lesions were characterized microscopically as purulent to pyogranulomatous to granulomatous inflammation with necrosis. F. tularensis ssp. holarctica strains were isolated from tissue samples and identified by a commercial carbonsource utilization test and polymerase chain reaction. A F. tularensis ssp. holarctica strain collection was established in Hungary. Sixty-three strains were isolated from European brown hares originating from different parts of Hungary and two strains from Austria. Two further strains were isolated from the patas monkey and the vervet monkey from Szeged Zoo. Utilisation of carbon sources of 15 F. tularensis strains was characterised with the Biolog system. The system was already able to identify the strains after 4 hours of incubation, instead of the standard 24 hours. After the analysis and comparison of the metabolic profiles 8 of our strains with the Biolog database, it was concluded that not all carbon sources indicated in the database were utilized by our isolates. The Biolog software failed to distinguish the highly virulent F. tularensis ssp. tularensis and the moderately virulent F. tularensis ssp. holarctica. Still the Biolog microplates could be manually read to differentiate the two subspecies based on glycerol source utilisation. As none of the studied strains was able to use glycerol they could be identified as F. tularensis ssp. holarctica. The dendrogram based on the metabolic relationship of the strains showed that the isolates are very similar to each other, which correlates with the conservative genetic character of F. tularensis ssp. holarctica. The whole genome of a Hungarian F. tularensis ssp. holarctica isolate was sequenced and was compared to 5 other complete genomes. The phylogenetic characterization of 19 F. tularensis isolates from Hungary and Italy was also performed. F. tularensis isolates from Hungary belonged to the B.Br.013 lineage and descended from a diverse set of minor subclades comprised of strains found throughout Central Europe, Scandinavia and Russia. F. tularensis isolates native to Italy belonged to the B.Br.FTNF002-00 subclade, a distinct genetic group comprising isolates from France, Spain, Switzerland and parts of Germany. The results on the genetic differences of the strains enabled us to contradict the hypothesis that Central Europe is the direct source of Western European (e.g. France, Italy) F. tularensis strains through hare importation. Important additions to the European phylogeographic model of F. tularensis were also provided and a set of powerful molecular tools for investigating F. tularensis dispersal throughout Europe was presented.