| Sarcocystis were common intracellular parasitic protozoa in livestock,which can cause sarcocystosis of hosts and lead to their slow growth even death if seriously infected.China,especially in the Qinghai-Tibet Plateau of this country is the leading area of this world for Yaks'feeding.Currently,two species of Sarcocystis,Sarcocystis poephagicanis(Wei,1983)and S.poephagi(Wei,1983)were found in yak,but its original description was simple and did not provide any molecular data.In the present study,the aims were to investigate the prevalence of Sarccoystis spp.in Yaks from Tibet(Lhasa,Nagqu and Shigatse)based on morphological observation,and molecular analysis based on the four genetic markers(18S rDNA,28S rDNA,mitochondrial COX1 gene and ITS1 region)Muscle samples were collected from 101 yak in this experiment.Microscopic observation revealed that the natural infection rate of Sarcocystis was 63.37%(64/101).Five species of Sarcocystis,Sarcocystis hominis(Railliet and Lucet,1891),S.heydorni(Dubey,2015),S.hirsuta(Moule,1888),S.poephagicanis,and Sarcocystis sp.were diagnosed and identified based on their morphological features and analysis of the four loci.By light microscopy(LM),the wall of S.hominis sarcocysts was thick and surrounded by finger-like protrusion(3.1-11.2?m in length,and mean 7.1?m);Ultrastructurally,sarcocysts of S.hominis had palisade-like protrusions with microtubules within them.The similarities of 18S rDNA,28S rDNA,mitochondrial COX1 gene and ITS1 region among different clones of S.hominis were 98.9%(n=9),99.0%(n=9),98.7%(n=9),91.0%(n=9)identity,respectively.The most similar sequences of the new obtained 18S rDNA sequences of S.hominis in GenBank were those of S.hominis from cattle(on average 98.5%identity);and the new obtained mitochondrial COX1 gene sequences of S.hominis shared the high identity of 98.3%with that of S.hominis isolated from human feces in GenBank.By LM,the thin-wall of S.heydorni sarcocysts was surrounded by short cone protrusions(1.0-1.8?m in length,and mean 1.4?m);Ultrastructurally,sarcocysts of S.heydorni had stubby protrusions,and the gap between the continuous protrusions was larger than the width of the protrusion.The similarities of 18S rDNA,28S rDNA,mitochondrial COX1 gene and ITS1 region among different clones of S.heydorni were 99.2%(n=7),98.4%(n=9),99.2%(n=12),95.8%(n=8),respectively.The most similar sequences of the new obtained 18S rDNA and mitochondrial COX1 gene sequences of S.heydorni in GenBank were those of S.heydorni from cattle,i.e.,99.2%and 99.3%identity,respectively.By LM,sarcocysts of S.hirsuta was thick and surrounded by incline figure-like protrusions(3.0-5.0?m,mean 4.1?m in length,)and there existed a longitudinal continuous black line within the protrusion.Under electron microscopy,sarcocysts of S.hirsuta had the figure-like protrusions with minute invaginations on its surface.The protrusions were enlarged in the middle,and slightly narrow at both ends.The similarities of 18S rDNA,28S rDNA,mitochondrial COX1 gene and ITS1 region among different clones of S.hirsuta were 99.0%(n=9),98.6%(n=8),99,7%(n=12),and 96.2%(n=9),respectively.The most similar sequences of the new obtained 18S rDNA,28S rDNA,mitochondrial COX1 and ITS1 region sequences of Shirsuta in GenBank were those of S.hirsuta from cattle,i.e,99.0%,99.0%,99.4%and 94.8%identity,respectively.Sarcocysts of S.poephagicanis was thin and surrounded by hair-like protrusions with the aid of LM.Ultrastructurally,sarcocysts of this parasite had the hirsute protrusions.The similarities of 18S rDNA,28S rDNA,mitochondrial COX1 gene and ITS1 region among different clones of S.poephagicanis were 99.3%(n=7),98.7%(n=8),98.7%(n=9)and 96.8%(n=8)identity,respectively.The most similar sequences of the new obtained 18S rDNA and ITS1 region sequences of S.poephagicanis in GenBank were those of S.cruzi(Hasselmann,1926)from cattle,i.e.,98.2%and 77.2%identity,respectively.The new obtained 28S rDNA and mitochondrial COX1 gene sequence shared the most similar to those of S.rangi(Gjerde,1984)(on average 91.7%identity)and S.levinei(Dissanaike and Kan,1978)(on average 97.2%identity)in GenBank,respectively.By LM,sarcocysts of Sarcocystis sp.was surrounded by incline finger-like protrusions(1.9-4.7?m in length,and mean 3.4?m).Ultrastructurally,sarcocysts of Sarcocystis sp.was surrounded by wavy multi-layered protrusions with electron dense layer on the surface,and microtubules bundled into bundles deepened into the ground substance layer.The similarities of 18S rDNA,28S rDNA,mitochondrial COX1 gene and ITS1 region among different clones of Sarcocystis sp.were 98.7%(n=7),98.3%(n=9),99.6%(n=12),95.8%(n=9),respectively.The most similar sequences of the new obtained 18S rDNA,28S rDNA and mitochondrial COX1 gene sequences of Sarcocystis sp.in GenBank were those of S.fusiformis(Railliet,1897)(on average 92.4%identity),S.buffalonis(Huong,Dubey,Nikkila and Uggla,1997)(on average 90.5%identity)and S.rommeli(Dubey,2015)(on average 981.3%identity),respectively.Phylogenetic analysis inferred from four loci revealed that S.hominis from yak formed an individual clade with S.hominis from cattle(intermediate hosts)or human(definitive host)within a group comprising Sarcocystis spp.with ruminants as intermediate hosts and felids as definitive hosts.Sarcocystis heydorni from yaks formed a clade with S.heydorni from cattle,which clustered together Sarcocystis spp.using canids as definitive hosts.Sarcocystis hirsuta from yaks and S.hirsuta from cattle formed a clade within a group comprising Sarcocystis spp.with felids definitive hosts.Sarcocystis poephagicanis formed a clade with Sarcocystis spp.with canids definitive hosts.Sarcocystis sp.of yaks,morphologically differentiate from above-mentioned species of Sarcocysits in yaks clustered together with Sarcocystis spp.in ruminants,which employed felids as definitive hosts.In conclusion,based on morphological observation and analysis of four molecular markers,S.hominis,S.heydorni and S.hirsuta should be shared by yaks and cattle.However,Sarcocystis sp.and S.poephagicanis were most likely in yak only. |
PDF Full Text Request