Morphological Characteristics And Molecular Phylogeny Based On 18S RRNA And Mitochondria Coxl Of Sarcocystis Spp. In Mouse-deer, Sheep And Goats

Sarcocysts is classed to protozoa that parasitized in almost all kinds of cells of warm-blooded animals. Ruminants are its important intermediate host in the nature, but none of Sarcocystis speceis has been reported from Family Traguliade, meanwhile the molecular characteristics of species of Sarcocystis from goats and sheep are poorly understood until now. So, the present study we described the morphology and molecular characteristics of Sarcocysts detected in mouse deer, sheep and goats in Yunnan Province, China.Here, two new species, Sarcocystis tuagulusi and S. menglaensis, were described in 5 of 12, and 1 of 12 Williamson’s mouse deer from southwest China, respectively. Using Light micrograph (LM), the sarcocysts of S. tuagulusi were bounded by finger-like villar protrusions. Transmission electron microscopy (TEM) revealed the protrusions with elongated lancet-like shapes in longitudinal section. In the core of the villar protrusions, a bundle of microtubules was present, that were generally loosely organized within the distal parts of the villar protrusions, but tightly packed within the middle and proximal parts of the protrusions. In cross section, the cyst walls formed numerous mushroom-like protrusions, which was similar to type 24. By LM, the cysts of S. menglaensis were bounded by palisade-like villar protrusions. TEM showed the protrusions with finger-shaped outline arose from the cyst wall. In the protrusions, there were numerous scattered microtubules; small invaginations appeared on their primary cyst surface. This TEM type was similar to type 10f.Two species of Sarcocystis have been found in the sheep destined for human consumption in Kunming, i.e. S. tenella and S. arieticanis. LM of cyst of S. tenella revealed that the sarcocysts were bounded by palisade-like villar protrusions. TEM of S. tenella showed the protrusions were finger-like or palisade-like, without microtubules in the villi, but plaques appeared on the tip of the protrusions, similar to type 24. LM of cysts of S. arieticanis was found to be bounded by hair-like villar protrusions. TEM of this species showed that the protrusions were hair-like or bone-like, but there werer no microtubules in the villi, similar to type7.Two species of Sarcocystis have been found in the goats destined for human consumption in Kunming:S. capracanis and S. hircicanis. LM of cysts of S. capracanis was found to be bounded by palisade-like villar protrusions. TEM showed the protrusions were finger-like or palisade-like, and there were no microtubules in the villi, similar to type 14. LM of cyst of S. hircicanis was bounded by hair-like villar protrusions. TEM showed the protrusions were hair-like or bone-like, and there were no microtubules in the villi, type 7.18S ribosomal small subunit gene (18S rRNA) and mitochondrial cytochrome c oxidase subunit I gene (cox1) have been used in many investigations to reveal the phylogenetic relationships among species of Sarcocystis in recente years. Particularly the latter seems to be better able to separate species of Sarcocystis from different host but possessed the similar morphology. In this point of view, the aim of this present study were to sequence and analyze the 18S rRNA and coxl of those 6 Sarcocystis species originated from mouse deer, sheep and goats (2 isolates of each specie).The intraspecific identity of 18S rRNA nucleotide sequences of S. tuagulusi was 99.8%. The homology of S. tuagulusi was most close to S. fusiformis from water buffalo in GenBank (93.1%identity). The intraspecific identity of 18S rRNA nucleotide sequences of S. menglaensis was 99.5%, and the identity with S. tuagulusi was 95.8-96%. The homology of S. menglaensis was most close to S. fusiformis from water buffalo in GenBank (94.8-95.2%identity).The intraspecific identity of coxl nucleotide sequences of S. tuagulusi was 99.5%. The homology of S. tuagulusi was most close to S. fusiformis from water buffalo in GenBank (84.2-84.5%identity). The intraspecific identity of coxl nucleotide sequences of S. menglaensis was 99.7%, the identity with S. tuagulusi was 83.8-84.0%. The homology of S. menglaensis was most close to S. gigantea from sheep in GenBank (83.9%identity).The intraspecific identity of 18S rRNA nucleotide sequences of S. tenella was 99.7%, the identity with S. tenella in GenBank was 99.2-99.4%. The intraspecific identity of 18S rRNA nucleotide sequences of S. arieticanis was 99.4%. the identity with S. arieticanis in GenBank was 98.2-98.4%.The intraspecific identity of coxl nucleotide sequences of S. tenella was 98.1%, the identity with S. tenella in GenBank was 96.9-97.5% The intraspecific identity of coxl nucleotide sequences of S. arieticanis was 99.5%, The homology of S. hircicanis was most close to S. grueneri from Rangifer tarandus tarandus in GenBank (84.2-84.3% identity).The intraspecific identity of 18S rRNA nucleotide sequences of S. capracanis was 99.3%, the identity with S. capracanis in GenBank was 99.1-99.2%. The intraspecific identity of 18S rRNA nucleotide sequences of S. hircicanis was 99.6%. The homology of S. hircicanis was most close to S. arieticanis from Ovis aries in GenBank (97.2-97.4%identity).The intraspecific identity of coxl nucleotide sequences of S. capracanis was 99.4%. The homology of S. capracanis was most close to S. tenella from Ovis aries in GenBank (92.3-93.2%identity). The intraspecific identity of coxl nucleotide sequences of S. hircicanis was 99.5%, The homology of S. hircicanis was most close to S. grueneri from Rangifer tarandus tarandus in GenBank (90.0-90.6%identity).Phylogenetic analysis based on both the 18S rRNA and the coxl partial sequences revealed that the 2 species of Sarcocystis in Williamson’s mouse deer were placed into the same group as other species that cycle between ruminant intermediate hosts and felid definitive hosts. Therefore, we surmised that the final hosts of the 2 species of Sarcocystis from Williamson’s mouse deer were probably felids. The sequences of S. tenella were placed within a clade formed by S. capracanis; the sequences of S. arieticanis formed the cluster with S. hircicanis. Both taxa were within a group comprising species with canines as the known definitive hosts.In the present study,2 Sarcocystis species has been identified for the first time from tragulid species. In the meantime,18S rRNA and coxl sequences of two Sarcocystis species from tragulid species and coxl sequenecs of S. arieticanis, S. capracanis, and S. hircicanis have been provided for the first time.