Development and validation of species-specific nested PCRs for diagnosis of acute sarcocystiosis in sheep.

Sheep may be infected by four species of Sarcocystis. Two of these species, Sarcocystis tenella and Sarcocystis arieticanis, are pathogenic. They may cause abortion or acute disease during the early phase of infection, and chronic disease during the late phase of infection. Thus far, diagnosis of sarcocystiosis in sheep has been limited, because traditional diagnostic tests based on the detection of Sarcocystis-specific antibodies are only genus-specific and, thus, cannot differentiate between pathogenic and non-pathogenic species. In addition, most of these tests show a reasonable sensitivity only for the late phase of infection. Therefore, diagnosis of acute sarcocystiosis has been based mainly on post-mortem examination, i.e. after the animal had succumbed to the disease. Here we established species-specific nested PCR assays based on unique small subunit ribosomal RNA gene sequences of S. tenella and S. arieticanis. These PCR assays specifically detect DNA of the homologous species in blood samples of sheep. No cross-reactions were observed with the heterologous pathogenic species, the non-pathogenic species Sarcocystis gigantea, or the closely related coccidia Toxoplasma gondii and Neospora caninum. In sheep experimentally infected with S. tenella or S. arieticanis, positive PCR results were correlated with the early phases of multiplication (endopolygeny) of the parasites. By contrast, Sarcocystis-specific antibodies were detected by an enzyme-linked immunosorbent assay only during the terminal phase of endopolygeny or thereafter. Thus, the nested PCR assays developed here enable, for the first time, the diagnosis and differentiation of infections with S. tenella and S. arieticanis in living sheep during the acute phase of the disease and facilitate comprehensive studies on the epidemiology and importance of infections with pathogenic Sarcocystis species in sheep.