Sarcocystis spp. (Apicomplexa: Coccidia) are obligate heteroxenous protozoa that infect a wide range of host species. Transmission follows a predator-prey cycle involving an intermediate host (IH) and a definitive host (DH). For many species, only IHs have been identified, while DHs remain unknown. DHs can be infected with multiple Sarcocystis spp. at the same time, which complicates species identification. We aimed to determine the prevalence and species diversity of Sarcocystis infections in free-ranging wolves in Switzerland using both coprological and molecular methods. A further goal was to evaluate the utility of Third-generation sequencing for resolving mixed infections. A total of 87 wolf intestinal content samples were collected between 2017 and 2023 and analyzed coproscopically by a sedimentation-flotation method. Sarcocystis oocysts/sporocysts were detected in 76 % (66/87). DNA was obtained from 57/66 positive samples and 55/57 resulted positive in a Sarcocystis 18S rRNA screening PCR. Additionally, mitochondrial cytochrome c oxidase subunit I (COI) gene PCR and a real-time PCR targeting S. cruzi were performed. PCR products from conventional PCRs were submitted for Sanger sequencing. Monoinfections were identified in 16 % (9/55) and mixed infections in 84 % (46/55) of the samples. A subset of five samples was analyzed by Third-generation sequencing (Pacific Biosciences) of the 18S rRNA full-length and COI fragment PCR products. BLAST and phylogenetic analysis were used to validate taxonomic classification. Molecular analysis identified nine known Sarcocystis species: S. tenella, S. arieticanis, S. capreolicanis, S. linearis, S. gracilis, S. cruzi, S. capracanis, S. iberica, and S. venatoria. Newly developed pipelines for the Third-generation sequencing data provided high-resolution species-level identification in samples with mixed infections. These findings confirm the Eurasian wolf as natural DH for multiple Sarcocystis species for the first time, including S. linearis, S. iberica, and S. venatoria. Further complementary studies on prey species are needed to clarify host-parasite dynamics.
