High prevalence of Sarcocystis spp. in the Eurasian wolf (Canis lupus lupus): Third-generation sequencing resolves mixed infections

<i>Sarcocystis</i> 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 <i>Sarcocystis</i> spp. at the same time, which complicates species identification. We aimed to determine the prevalence and species diversity of <i>Sarcocystis</i> 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. <i>Sarcocystis</i> oocysts/sporocysts were detected in 76 % (66/87). DNA was obtained from 57/66 positive samples and 55/57 resulted positive in a <i>Sarcocystis 18S</i> rRNA screening PCR. Additionally, mitochondrial cytochrome <i>c</i> oxidase subunit I (<i>COI</i>) gene PCR and a real-time PCR targeting <i>S. cruzi</i> 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 <i>18S</i> rRNA full-length and <i>COI</i> fragment PCR products. BLAST and phylogenetic analysis were used to validate taxonomic classification. Molecular analysis identified nine known <i>Sarcocystis</i> species: <i>S. tenella, S. arieticanis, S. capreolicanis, S. linearis, S. gracilis, S. cruzi, S. capracanis, S. iberica,</i> and <i>S. venatoria</i>. 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 <i>Sarcocystis</i> species for the first time, including <i>S. linearis</i>, <i>S. iberica</i>, and <i>S. venatoria</i>. Further complementary studies on prey species are needed to clarify host-parasite dynamics.