This study aimed to determine and identify Sarcocystis spp. infection in sheep of Mashhad city, Iran. From October 2018 to May 2019, the entire esophagus and diaphragm from 100 slaughtered sheep were collected from the Mashhad abattoir. Initially, samples were inspected by the naked eye for the presence of macrocysts. Also, all samples were examined for Sarcocystis spp. by tissue impression smear, histopathology, and PCR tests. Additionally, eight samples were inspected by transmission electron microscopy (TEM) and gene sequencing to confirm species identification. The infection rate of sarcocystosis by impression smear, histopathology, and PCR methods were 69%, 96%, and 100%, respectively. Histopathological examination revealed the existence of S. gigantea macrocyst with PAS-positive secondary cyst wall in 26% of sheep. Also based on cyst wall morphology, two types of microcysts including S. tenella with striated thick cyst wall and S. arieticanis with smooth thin cyst wall were identified in 47% and 11% of sheep, respectively. By TEM, the cyst wall of S. gigantea had cauliflower-like, S. tenella had finger-like and S. arieticanis had hair-like villar protrusions. Comparative analyses of the sequencing of the 18S rRNA gene revealed S. gigantea, S. tenella, and S. arieticanis in PCR samples. The results showed that the infection rate of Sarcocystis spp. was very high by the PCR method. Also, the existence of S. gigantea, S. tenella, and S. arietcanis species was confirmed by histopathology, TEM, and DNA sequencing methods in sheep of this area.

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