Morphological and Molecular Characterization of Sarcocystis chenggongensis n. sp. (Apicomplexa: Sarcocystidae) in Domestic Sheep (Ovis aries) from China

Background Sarcocystis infections in sheep (Ovis aries ) are prevalent worldwide, posing significant health and economic challenges. To date, a total of nine Sarcocystis taxa, including seven identified species and two unclassified ones, have been found to form sarcocysts in sheep. Among these, macrocysts with long villar protrusions have been reported only twice: once as S. mihoensis in Japan and once as S. mihoensis -like in Spain. In this study, this distinct morphological type is reported for the first time in China. Methods Between March and September 2025, muscle tissue samples were obtained from 83 domestic sheep in Kunming City, China. The sarcocysts detected within these tissue samples were subjected to morphological characterization using both light microscopy (LM) and transmission electron microscopy (TEM). For molecular analysis, genomic DNA was extracted from individual sarcocysts that were isolated from different sheep. Subsequently, five genetic markers were amplified, sequenced and analyzed. These markers included the nuclear 18S rDNA, 28S rDNA, and ITS - 1 regions, as well as the mitochondrial cox 1 gene and the apicoplast rpoB gene. Results Novel sarcocysts were detected via LM in six out of 83 (7.2%) domestic sheep. These macroscopic sarcocysts measured up to 6230 μm in length and 341 μm in width, possessing a thick wall with numerous sloping villar protrusions (VPs) measuring 6.9–11.9 μm long. Ultrastructural examination showed VPs that were 5.8–9.3 μm long and 0.8–1.1 μm in wide, lined by an electron-dense layer and containing scattered microtubules extending from the apex to the base of these VPs. Sequence comparisons with GenBank entries revealed the highest similarity to Sarcocystis buffalonis for 18S rDNA (97.6–98.0% identity), S. miescheriana for 28S rDNA (92.1–92.3% identity), S. japonica for cox1 (81.2–81.8% identity), and S. arctica for rpoB (88.5–88.8% identity). No significant matches were found for the ITS-1 region. Genetic divergence analysis against other sheep-infecting species indicated the smallest distances with S. medusiformis at 18S rDNA (0.0582), S. gigantea at 28S rDNA (0.0556), and S. gigantea at cox1 (0.2922). Given the distinct morphological features and the unique molecular characteristics, the Sarcocystis organism infecting these Chinese domestic sheep is proposed as a new species, named Sarcocystis chenggongensis n. sp. Conclusions This study marks the third documented instance of macroscopic sarcocysts featuring elongated villar protrusions, succeeding the previous reports of S. mihoensis in Japan and an S. mihoensis -like species in Spain. These results further substantiate the widespread yet sporadic geographical distribution of this distinct morphological group. Nevertheless, taxonomic uncertainties persist, stemming from inconsistent nomenclature and a lack of comprehensive molecular and life-cycle data. Future research should prioritize resolving evolutionary relationships and elucidating the complete life cycles of these morphologically similar species.