Morhpology And Molecular Characteristics Of 18s RDNA And Coxl Of 10 Species Of Genus Sarcocystis Species From Cattles And Buffaloes

Sarcocysts,some isolated from cattle beef in the Kunming farm market and some collected from cattle and water buffalo sampled previously,were observed and identified using light microscope(LM).In present study,a total 10 species of Sarcocystis was identified in cattle and water buffalo.Among the 10 species of Sarcocystis,the 5 were Sarcocystis species from cattle,i.e.,S.cruzi,S.hominis,S.hirsuta,S.rommeli,and S.heydorni;the remaining 5 were from water buffalos,i.e.,S.hominis-like,S.buffalonis,S.fusiformis,S.sinensis,and S.deho,ngensis n.sp.Under LM,cysts of S.cruzi were microscopic,and had thin wall with hair-like protrusions;cysts of S.hominis were microscopic,upright finger-like protrusions;cysts of S.hirsuta were macroscopic,leaning palisade-like protrusions;cysts of S.rommeli were microscopic,leaning finger-like protrusions;cysts of S.heydorni were microscopic,and had smooth thin wall;cysts of S.hominis-like and S.buffalonis were morphological similar to those of S.hominis and S.hirsuta,respectively;cysts of S.fusiformis were macroscopic,palisade-like protrusions;cysts of S.sinensis were microscopic,leaning finger-like protrusions;cysts of S.dehongensis n.sp.were macroscopic,leaning finger-like protrusions.By transmission electron microscope,ultrastructure of S.rommeli cysts showed that the cysts had finger-like protrusions;these protrusions spaced each other,and contained numerous scattered microtubules,which deepen into the middle of the ground substance layer.2 cats were inoculated with 300 S.rommeli sarcocysts from cattle,respectively.In 15 days post infection,oocysts and sporocysts were observed in the feces of experimental cats.oocysts and sporocyst of Sarcocystis were also found in the small intestinal of the experimental cats on 30-day post infection.However,the oocysts/sporocysts were not observed in the feces and intestinal mucosa of the two domestic cats in the control group.18S rDNA and mitochondrial coxl were amplified using DNAs extracted from individual sarcocysts of the 10 species of Sarcocystis in cattle and water buffalo as PCR templates.Meanwhile,only mitochondrial coxl was amplified using DNA obtained from oocysts/sporocysts of S.rommeli from experimental cats.All PCR products were purified,cloned,sequenced and analyzed.Comparing the sequences of 18S rDNA and coxl for Sarcocystis spp obtained from this experiment with other sequences deposited in GenBank using Basic Local Alignment Search Tool(BLAST).Phylogenetic trees construction based on the sequences of 18S rDNA and mitochondria cox1 for Sarcocystis species could be a useful tool to infer the definitive hosts of Sarcocystis.In the present study,S.rommeli formed a clade with S.bovini,S.sinensis and S.bovifelis,and the later 2 species has been proved to use felids as their definitive hosts.The animal infection here also supported that cat served as definitive host of S.rommeli.Sarcocystis heydorni formed a clade with S.tenella,S.alces,S.gracilis,and these 3 species has been proved to use a canid as definitive hosts.However,human has been regarded as the definitive host of S.heydorni based on insufficient proof,so the definitive host of this species of Sarcocystis need further investigation in the future.Sarcocystis denhongensis formed a clade with S.ovalis that cycled between ruminant intermediate hosts and corvid bird,therefore,this species probably uses a corvid bird as its definitive host.On the basis of the morphological and molecular characteristics of Sarcocystis spp.in cattle and water buffalo,several thoughts could be concluded,that is:(1)S.hominis from cattle and S.hominis-like from water buffalo should represent a same species of Sarcocytis,and water buffalo was probably the new intermediate host for S.hominis;(2)the morphological similar Sarcocystis species in cattle and water buffalo,S.cruzi and S.levinei,S.hirsuta and S.buffalonis should represent separate Sarcocystis species in cattle and water buffalo,respectively;(3)S.bovini should be the synonym of S.rommeli,and S.rommeli distinct from S.sinensis;and(4)Sarcocystis denhongensis is amed as a new species based on the its low identities of the 2 genetic markers with all other sequences of Sarcocystis species,probably it is a rare species for transmitted by birds in buffalo.