Molecular diversity of cox1 and LSU rDNA sequences of Sarcocystis bertrami (syn. S. fayeri) (Apicomplexa: Eucoccidiorida: Sarcocystidae) in horses

Food poisoning caused by consuming raw horsemeat contaminated with Sarcocystis is a significant public health concern. Two morphotypes of sarcocysts in horsemeat, characterized by upright and folded villar protrusions, are typically identified as Sarcocystis fayeri and S. bertrami, respectively. However, recent molecular studies focusing on the ribosomal RNA gene (rDNA) and mitochondrial cytochrome c oxidase subunit I gene (cox1) have indicated a conspecific relationship between these two morphotypes using a limited number of specimens. To explore further genetic diversity in equid sarcocysts, cox1 and large-subunit (LSU) rDNA sequences were analyzed in sarcocysts extracted from horsemeat inspected from 150 horses (76 and 41 horses imported from Canada and France, respectively, and 33 horses reared in Japan). Sarcocysts were detected in the muscles of 71, 2, and 3 horses from Canada, France, and Japan, respectively. Fifty-eight sarcocysts underwent cox1 and the LSU rDNA sequencing. Newly obtained cox1 sequences (n = 53) and sequences labeled as equid S. bertrami, S. fayeri and S. asinus retrieved from GenBank (n = 53) exhibited conspecific relationships. Inter-individual variation in cox1 sequences was observed among various sarcocysts, even within a single host animal, although no intra-individual variation was observed. However, nuclear-embedded mitochondrial DNA (NUMT: cox1 pseudogene) sequences were obtained using inappropriate techniques using certain primers. The LSU rDNA of sarcocysts (211 cloned sequences from 54 sarcocysts) exhibited inter-individual and robust intra-individual variations, indicating significant intragenomic rRNA array mosaicism in S. bertrami. These findings confirmed the conspecificity of classically defined species without geographical subpopulations.