: Ophidiomycosis is an emerging infectious disease caused by the fungus Ophidiomyces ophidiicola (Oo). To date, Oo presence or associated disease condition has been recorded in wild and/or captive snakes from North America, Europe, Asia and Australia, but the data is still scarce outside the Nearctic. Although Italy is a country with a high snake biodiversity in the European panorama, and animals with clinical signs compatible with Oo infection have been documented, to date no investigations have reported the disease in the wild. Therefore, a pilot survey for the Italian territory was performed in conjunction with setting up a complete diagnostic workflow including SYBR Green-based real-time PCR assay for the detection of Oo genomic and mitochondrial DNA combined with histopathology of scale clips. Oo presence was investigated in 17 wild snake specimens from four different species. Four snakes were sampled in a targeted location where the mycosis was suspected via citizen science communications (i.e. North of the Lake Garda), whereas other ophidians were collected following opportunistic sampling. Oo genomic and mitochondrial DNA were detected and sequenced from all four Lake Garda Natrix tessellata, including three juveniles with macroscopic signs such as discolouration and skin crusts. From histopathological examination of scale clips, the three young positive individuals exhibited ulceration, inflammation and intralesional hyphae consistent with Oo infection, and two of them also showed the presence of arthroconidial tufts and solitary cylindrical arthrospores, allowing "Ophidiomycosis and Oo shedder" categorisation. For the remaining snake samples, the real-time PCR tested negative for Oo. This pilot survey permitted to localise for the first time Oo infection in free-ranging ophidians from Italy. Ophidiomycosis from Lake Garda highlights the need to increase sampling efforts in this area as well as in other northern Italian lakes to assess the occurrence of the pathogen, possible risk factors of the infection, its impact on host population fitness and the disease ecology of Oo in European snakes.
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