Ultrastructural and Molecular Identification of the sarcocysts of Sarcocystis tenella and Sarcocystis arieticanis Infecting Domestic Sheep (Ovis aries) from Egypt.

Background: In spite of the global economic significance of sheep production, little is known about the prevalence of various Sarcocystis spp. infecting the domestic sheep (Ovis aries) in Egypt.
Materials and Methods: Muscle samples were collected from 175 sheep (> 2 years) slaughtered at El-Mahalla El-Kubra abattoir, Gharbia governorate, Egypt. Samples were initially examined by naked eye for the existence of macrosarcocysts. The microscopic sarcocysts were detected and identified using the light microscopy and the Transmission electron microscopy (TEM). Different microscopic species of ovine Sarcocystis were molecularly confirmed by PCR, sequence analyses and phylogeny.
Results: Preliminary light microscopic inspection of the muscle specimens revealed the existence of only the microscopic sarcocysts of Sarcocystis tenella and Sarcocystis arieticanis in 152 (86.8%) out of the175 examined animals. Sarcoysts of S.tenella had striated thick cyst wall that amounted from 3.5-5.5 μm in thickness whereas, S.arieticanis sarcocysts had a thin cyst wall that ranged from 1-3 μm in thickness. S.tenella sarcocysts were detected in 115 sheep (65.7%), and were more prevalent than those of S.arieticanis, observed only in 68 sheep (38.8%). No macroscopic sarcocysts were observed in any of the examined carcasses. Transmission electron microscopy (TEM) of the cyst wall of S.tenella revealed the existence of the short stubby villar protrusions (VP) with the characteristic disk-like structures at the tips of the (VP). While, TEM of S.arieticanis showed that the cyst wall had elongated tubular protrusions that measured approximately 5-7 μm in length. Each (VP) consisted of a dome-shaped base (0.3-0.9 μm in diameter), a relatively thick middle portion (0.1-0.3 μm) in width, and a thin hair-like distal portion that measured about (0.03 x 1-4.5 μm).
Conclusion: Comparative analyses of the sequences of the four genetic markers (18S rRNA, 28S rRNA, mitochondrial cox1 and ITS-1) for S.tenella and S.arieticanis isolates detected herein, revealed genetic variations of 95% and 95- 96% among the different isolates on the level of the 18S rRNA and 28S rRNA, respectively. Whereas, the cox1 and ITS-1 shared sequence identities of 76-78% and 70-73%, respectively. S.tenella was strongly related to S.capracanis infecting goats (Capra hircus). Sequence identity of 98% on the level of 18S rRNA, 28S rRNA genes was observed between the currently identified isolates of S.tenella and the formerly GenBank deposited isolates of S.capracanis. While, cox1 sequences shared identities of 92-93%. Furthermore, S.arieticanis isolates identified here were closely related to the formerly published sequences of S.hircicanis. The 18S rRNA and 28S rRNA sequences of S.arieticanis shared 98% and 94-95% identities with those of S.hircicanis, respectively. However, 87-88% homologies were observed between the cox1 sequences of S.arieticanis and S.hircicanis. Consequently, cox1 and ITS-1 gene sequences act as better genetic markers than 18S rRNA and 28S rRNA sequences for the characterization of ovine Sarcocystis spp. Maximum parsimony analyses based on the sequences of three genetic markers, (18S rRNA, 28S rRNA and mitochondrial cox1), yielded the same placement of the currently identified isolates of the two taxa (S.tenella and S.arieticanis) within a clade of Sarcocystis species with carnivorous animals as known, or assumed, final hosts.