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Ophidiomyces ophidiicola, the causative agent of ophidiomycosis, poses a potential threat to wild snakes worldwide. This study aimed to retrospectively investigate the prevalence of O. ophidiicola in archived snake moults collected from the San River Valley in the Bieszczady Mountains, Poland, from 2010 to 2012. Using qPCR for O. ophidiicola detection and conventional PCR for clade characterisation, we analysed 58 moults and one road-killed specimen of Zamenis longissimus and Natrix natrix. A novel combination of primers (ITS2L) was used to simultaneously confirm SYBR Green-based qPCR results and perform genotyping. O. ophidiicola has been detected from two Z. longissimus and one N. natrix specimens. The identified clade (I-B) is consistent with those found in wild snakes of eastern Europe and San River Valley, indicating that O. ophidiicola has been present in this region for at least a decade. This study underscores the value of historical samples in understanding the long-term presence of pathogens and highlights the potential role of environmental reservoirs in the persistence of O. ophidiicola. Our findings are crucial for informing conservation strategies for the endangered Aesculapian snake populations in Poland, emphasising the need for ongoing monitoring and habitat management to mitigate the potential impact of ophidiomycosis.

In the Abruzzi village of Cocullo (Italy), each year, on May 1st, local snake hunters (known as Serpari) display colubrids, captured in the wild, to commemorate the ancient ritual of San Domenico. The ascomycete Ophidiomyces ophidiicola (Oo) is the causative agent of ophidiomycosis, an emerging disease with sublethal effects. Skin lesions, such as dysecdysis, edematous, crusty or necrotic scales, swellings, nodules, and ulcers, are the most common clinical manifestation of the disease. The pathogen and its associated disease are well characterized in wild snakes in North America, whereas broad screenings of free ranging wild ophidians in Europe are rare. In 2019, as part of a multi-year snake health monitoring project, all the Cocullo ophidians were carefully examined for integumentary affections and those showing signs consistent with ophidiomycosis were dry swabbed on the skin and on any visible cutaneous lesions with a single applicator. The extracted DNA underwent a broad-range panfungal PCR targeting the D1-D2 region, as well as two conventional PCRs specific to the ITS2 and IGS regions of Oo DNA. Twenty-three out of 129 snakes (13/82 Elaphe quatuorlineata; 7/31 Hierophis viridiflavus; 3/15 Zamenis longissumus; 0/1 Natrix helvetica) resulted clinically affected, but no specific Oo genomic DNA was detected by PCR. The Cocullo ritual celebration provided a unique opportunity for the first systematic testing of a large sample size of a local snake community for the monitoring of this pathogen in Italy.