Molecular Identification of Parasitic Protozoa Sarcocystis in Water Samples

SIMPLE SUMMARY: Members of the genus Sarcocystis are protozoan parasites having two-host prey–predator cycle. These parasites are widespread in farm animals. Sarcocystis species are characterized morphologically in intermediate hosts, and these parasites are identified in definitive hosts by molecular methods. Thus far, only few studies have been conducted on Sarcocystis parasites in environmental samples. The aim of the present work was to evaluate several sample preparation and polymerase chain reaction methods for the identification of several Sarcocystis species in water samples. Overall, 114 samples collected from various water sources, ponds, canals, lakes, lagoons, and rivers in Lithuania were tested for the presence of Sarcocystis spp. Based on molecular methods, eight Sarcocystis species, S. bovifelis, S. cruzi, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. bertrami, and S. miescheriana, were identified. The main intermediate hosts of detected Sarcocystis parasites are cattle, sheep, goats, horses, and pigs. Further, more sensitive molecular techniques are needed for the development of the diagnosis of Sarcocystis species in water bodies. ABSTRACT: Sarcocystis parasites are among the most common parasitic protozoa in farm animals. So far, the diversity of these parasites has been mainly studied in animal carcasses by morphological or molecular methods. Research on parasitic protozoa in environmental samples is scarce due to the lack of an appropriate methodology and low concentrations of parasites. For these reasons, there is a paucity of validated methods for Sarcocystis identification from environmental samples. Therefore, the present study aims to investigate various molecular methods for Sarcocystis parasite identification in water samples. In the present study, the sample volume, sporocysts isolation, and various conventional PCR were evaluated, and species-specific primers for the identification of different Sarcocystis species have been developed. Of the methods studied, based on data the most appropriate method for the identification of analyzed Sarcocystis spp. in water bodies is nested PCR, using species-specific primers targeting the cox1 gene. Sarcocystis DNA was detected in 111 out of 114 (97.4%) samples. This paper represents the first identification of S. bovifelis, S. cruzi, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. bertrami, and S. miescheriana by PCR and sequencing in environmental water samples. Our pilot study is useful in developing techniques for the identification of Sarcocystis species from water samples.