Red fox (Vulpes vulpes) as wild reservoir of Opisthorchis felineus (Digenea: Opisthorchiidae) in Italy

AIM. Opisthorchiasis by Opisthorchis felineus (Digenea: Opisthorchiidae) is gaining an increasing importance in Italy for the recent occurrence of several human cases due to the consumption of raw or undercooked/marinated tench (Tinca tinca), so far known as the more suitable second intermediate host of the parasite in Italian country. The "classic" life cycle involves the domestic cat (Felis catus) as definitive host assuming the infective stage (metacercaria) of O. felineus by the consumption of infected fish remains, a freshwater snail belonging to Bithynia genus as first intermediate host and cyprinid fish as second intermediate host. Man is a suitable definitive host, such as dog and other fish eating mammals. Recent surveys (Crotti D et al, 2007, G It Microbiol Med Odont Clin, 11: 20-23; De Liberato C et al, 2010, Vet Parasitol, 177: 67-71) have shown, in endemic areas of Central Italy, O. felineus eggs prevalence values in cat feces of 31.8%, 36.6% and 73.3% around Trasimeno, Bolsena and Bracciano Lakes respectively. Although in European and former U.S.S.R. countries the occurrence of a life cycle of O. felineus linked to wild environment is reported (World Health Organization, 1995, Control of Foodborne Trematode Infection. WHO Technical Report Series: 125-126; Schuster R et al, 1999, Parasitol Res, 85: 142-146; Adams AM, 2006, in: Foodborne parasites, Ortega Ed, Springer Science, New York, USA), in Italy only two cases of opisthorchiasis in European polecat Mustela putorius in Pisa province, Tuscany, have been reported (Macchioni G, 1963, Ann Fac Med Vet Pisa, 16: 238-247), in the same area where O. felineus was reported for the first time by Rivolta in XIX century in cats and dogs (Rivolta S, 1884, G Anat Fisiol Patol Anim, 16: 20-28). With the aim of defining the possible role of wild mammals in O. felineus epidemiology in Italy, a parasitological survey has been undertaken on fecal samples collected from foxes in Tuscany. MATERIALS AND METHODS. Five fecal fox samples collected during 2007 in the framework of a research project on epidemiology of Trichinellosis in red foxes of Tuscany (Magi M et al, 2008, Hystrix, 19: 31-38) in Cascina and Bientina areas in Pisa province and stored in 10% buffered formalin were subjected to coprological examination by sedimentation and flotation with zinc sulphate ZnSO4 (s.g. 1.350), searching for Opisthorchiid eggs. Microscopically positive fecal samples were subjected to DNA extraction with QIAamp DNA Stool Mini Kit (Qiagen) and to nested PCR, amplifying the ITS rRNA (Luton K et al, 1992, Mol Biochem Parasitol, 56: 323-328). The PCR product was sequenced in both direction by ABI 3730 DNA Analyzer at StarSEQ GmbH (Mainz, Germany). RESULTS. One out of five fecal fox samples was positive for the presence of digenean eggs, showing measures and morphological features consistent with eggs of Opistorchiidae trematodes. The sequence obtained showed 100% identity with O. felineus. CONCLUSIONS. The possible role played by red fox as wild reservoir of Opisthorchiasis in Italy needs further investigations, already undertaken by our research groups in different Italian regions. The presence of a "wild" life cycle in addition to the "classic" one linked to the cat as main reservoir could complicate the epidemiology and, as a consequence, the control of this parasitic zoonosis in Italy.