DIGITALNA ARHIVA ŠUMARSKOG LISTA
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of the bacterium in the environment. Thereby, these animals could serve as potential reservoirs of bacterium, providing important source of infection for humans (Maurin and Gyuranecz, 2016; Ozanic et al., 2015; Tarnvik, 2007). In the last twenty years, epizootic screening studies of prevalence of F. tularensis among small mammals have been described in different European countries including Hungary (Gyuranecz et al., 2011), Kosovo (Reintjes et al., 2002), Switzerland (Origgiet al., 2015), Slovakia and Austria (Gurycova et al., 2001). Hence, last epizootic reports on prevalence of F. tularensis in Croatia date from the middle seventies of the last century (Borcic et al., 1976). Recently, Tadin et al. (2016) reported the presence of multiple zoonotic pathogens in rodent species in Croatia with high abundance of Leptospira and Hantaviruses and low rate of Francisella tularensis (0.8 %) infection in A. agrarius. The purpose of this study is to screen for the frequency of Francisella spp. among small mammals in distinct areas in Croatia, where the high estincidence of human tularemia was observed. The current study is also part of the program of Croatian Ministry of Agriculture for prevalence of F. tularensis among animals (2016). Samples of small rodents and insectivores were trapped during the two years period in eight different localities in continental Croatia: VelikaGorica, Lipovljani, Nova Subocka, Stara Gradiška, Sunja, Županja, Koprivnica and Čakovec. In this study, three mice were found to be positivefor F. tularensis. MATERIALS AND METHODS MATERIJALI I METODE During a two-year long survey (2014-2016), small rodents and insectivores were collected in eight different localities in Croatia: Lipovljani, Nova Subocka, Velika Gorica, StaraGradiška, Županja, Sunja, Koprivnica and Čakovec, using snap traps, according to the previously described guidelines (Gannon and Sikes, 2007). Total of 444 animals were trapped, 69 in Lipovljani, 6 in Nova Subocka, 88 in Velika Gorica, 24 in Sunja, 2 in Stara Gradiška, 158 in Županja, 74 in Koprivnica and 23 in Čakovec. Species were morphologically determined. Collected species of small rodent sincluded: Apodemus agrarius (197), Apodemus sylvaticus (78), Apodemus flavicollis (92), Myodes glareolus (17), Microtus agrestis (27) and Microtus arvalis (20). Collected insectivores included one species, Sorex araneus (13). Animals were labelled, and spleens were aseptically dissected. The genomic DNA was extracted from spleen using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin, USA). The presence of F. tularensis in collected samples was confirmed using commercial PCRmax Ltd TM qPCR tests (PCR max, Beacon Road, Staffordshire, UK) for F. tularensis succinate dehydrogenase (sdhA) gene, according to manufacturer instruction. RESULTS REZULTATI In this study, 444 spleen samples of small rodents and insectivores were tested for the presence of Francisella spp., using qRT-PCR method. Dead rodents and insectivores were collected in eight different localities in continental Croatia: Lipovljani, Nova Subocka, Velika Gorica, Sunja, Županja, Stara Gradiška, Koprivnica and Čakovec (Figure 1). Number of collected species in each locality and results of testing are summarized in Table 1. Our results show that from total of 444 animals collected, only 3 mice (0.67%) of |