Molecular characterization of Sarcocystis spp. in intestines of coyotes and foxes from Pennsylvania, USA

Background Wild canids serve as definitive hosts for numerous Sarcocystis species infecting livestock and wildlife. However, species-level identification in free-ranging carnivores remains difficult, impeded by the morphological resemblance among the sporocysts of various parasite species. Methods Intestinal mucosal scrapings from 40 wild canids: red fox ( Vulpes vulpes , n = 8), gray fox ( Urocyon cinereoargenteus , n = 6) and coyote ( Canis latrans , n = 26) in Pennsylvania, USA, sampled in 2024, were tested for Sarcocystis sporocysts. Results Sporocysts were detected microscopically in intestinal homogenates digested in Chlorox in 3/6 (50%) gray foxes, 3/8 (37.5%) red foxes and 18/26 (69.2%) coyotes. PCR amplification was successful on 8/18 (44.4%) coyotes, 0/6 Gy foxes and 2/3 (66.6%) red foxes. Multi-locus genotyping was performed for the 18S rRNA, 28S rRNA, COI and ITS1 genetic markers, supporting identification of multiple species of Sarcocystis , including Sarcocystis albifronsi -like, S. cruzi , S. gjerdei -like, S. cristata / S. wenzeli -like and an additional undescribed, ungulate-associated Sarcocystis spp. Conclusions Phylogenetic analysis confirmed clustering with reference sequences with strong support. This study demonstrates that wild foxes and coyotes serve as definitive hosts for multiple species of Sarcocystis , including Sarcocystis cruzi, which uses cattle as intermediate hosts and imposes significant economic burdens on cattle production. Moreover, these findings demonstrate that wild canids harbor diverse Sarcocystis spp., supporting their role in environmental dissemination and potential transmission at the wildlife–livestock interface.