Morphological And Molecular Characteristics Of Sarcocystis In Some Wild Small Mammals From Yunnan And Domestic Dogs From Guangxi

From March 2017 to December 2018,1153 samples of wild small mammals were collected from 35 sampling sites in Kunming,Dali,Diqing,Dehong and Chuxiong prefectures,all located in Yunnan Province,southwestern China.These speciments included 1037 rodents(23 speceis),96 insectivores(4 speceis)and 20 shrews(1 speceis).During October 2017,skeletal muscle samples from 37 dogs were purchased from a peddlers' market in YulinCity,Guangxi,located in southwestern China.Sarcocysts were observed in 193 of 1153(16.74%)specimens of small wild mammals.Eight species of Sarcocystis were identified from the skeletal muscles of these specimens based on the morphological and molecular characteristics.(1)Sarcocystis losea n.sp.Intermediate hosts included Rattus losea,R.flavipectus and R.norvegicus.By light microscopy(LM),sarcocysts of S.losea were bounded by numerous palisade-like protrusions.Under tranmission electron micrograph(TEM),S.losea sarcocsyts had palisade-like villar protrusions,similar to type "10".The sequences of 18S rDNA for this parasite shared the most similar with S.zuoi(98.24%identity),that was found from rats with snakes as definitive hosts;the sequences of COX1 gene for this parasite had the most identity of 94.97%with Sarcocystis sp.,which was only diagnosed from definitive host,Pantherophis alleghaniensis.(2)S.cymruensis.Intermediate host included R.rattus,R.norvegicus,R.yunnanensis,R.flavipectus and R.nitidus.Under light microscopy(LM),the cyst wall of sarcocysts of S.cymruensis was smooth.By tranmission electron micrograph(TEM)of S.cymruensis sarcocsyts are bounded by numerous massif-like villar protrusion,similar to type "T".The 18S rDNA sequences for S.cymruensis shared the most similar with S.Ratti(99.96%identity).And the COX1 gene sequences had the most identy with S.cymruensis(95.26%identity).(3)S.confucianus n.sp..Intermediate hosts included Niviventer con.fucianus and R flavipectus.By light microscopy(LM),sarcocysts of S.confucianus are bounded by numerous finger-like protrusion.Under tranmission electron micrograph(TEM),S.confucianus sarcocsyts are bounded by numerous irregular villar protrusion,sometimes with "T" shape or "mushroom" shape,but there are without microtubules in it,which was similar to type "27".The most similar 18S rDNA sequences in GenBank was that of S.zuoi from rats with snakes as definitive hosts(97.14%identity);the sequences of COX1 gene for this parasite had the most identy of 95.15%with Sarcocystis sp.,which was only diagnosed from definitive host,P.alleghaniensis.(4)S.zuoi.Intermediate hosts included R.norvegicus,R.yunnanensis,R.nitidus and R.rattus.Under light microscopy(LM),sarcocysts of S.zuoi had long finger-like villar protrusion.(5)S.chevieri n.sp..Intermediate hosts was Apodemus chevieri.By light microscopy(LM),the sarcocysts cyst wall of S.chevieri was smooth.Under tranmission electron micrograph(TEM),sarcocsyts of S.chevieri were bounded by numerous irregular villar protrusions,similar to type "18".The sequences of 18S rDNA for this parasite shared the most similar with Sarcocystis sp.(98%identity),that was found from the blood of Procyon lotor in Japan;the sequences of COX1 gene for this parasite had the most identy of 99.57%with Sarcocystis sp.,which was only diagnosed from definitive host,P.alleghaniensis.(6)S.eothenomysi.Intermediate hosts was Eothenomys miletus.Under light microscopy(LM),the sarcocysts cyst wall of S.eothenomysi was smooth.The COX1 gene sequences for this parasite had the most identy of 99.8%,which was only diagnosed from definitive host,P.alleghaniensis.(7)S.clethrionomyelaphis.Intermediate hosts was E.miletus.By light microscopy(LM)of S.clethrionomyelaphis are bounded by numerous with long finger-like villar protrusion.The COX1 gene sequences for this parasite had the most identy of 94.47%which was only diagnosed from definitive host,P.alleghaniensis(8)S.attenuata n.sp..Intermediate hosts was C.attenuata.By light microscopy(LM),sarcocysts of S.attenuata are bounded by numerous with petaloid or tongue-like villar protrusion.Under tranmission electron micrograph(TEM),S.attenuata sarcocsyts had numerous irregular villar protrusions,which was similar to type "9".The sequences of 18S rDNA for this parasite shared the most similar with S.zuoi(94.47%identity),that was found from rats with snakes as definitive hosts;the sequences of COX1 gene for this parasite had the most identy of 95.18%with Sarcocystis sp.,which was only diagnosed from definitive host,Pantherophis alleghaniensis.The prevalence of S.caninum in a peddlers' market in Yulin,Guangxi is 5.4%.Under light microscopy(LM),the sarcocsyts of S.caninum had numerous short wedge-like villar protrusions.By tranmission electron micrograph(TEM),S.caninum sarcocsyts were bounded by numerous pleomorphic,anastomosed villar protrusions,similar to typeu "9".The most similar sequences in GenBak of 18S rDNA,28S rDNA,mitochondrial COX1 gene,ITS-1 gene and RpoB gene for this parasite was S.arctica,which was from foxes or wolves.The sequences of the five loci shared high similarities of 99.98%?99.98%?99.8%?99.91%,and 99.68%,respectively,with those from S.arctica found in the gray wolf,Arctic fox,and red fox from the USA and/or Europe.Phylogenetic analysis inferred from sequences of the two loci(18S rDNA and COX1 gene)reveald that the four new species of Sarcocystis,S.losea n.sp.in S.losea,R.flavipectus and R.norvegicus,S.chevieri n.sp.in A.chevieri,S.confucianus n.sp.in N.confucianus and R.flavipectus,and S.attenuata n.sp.in C.attenuate closely related with Sarcocystis spp.that cycled between rodents and snakes.Therefore,we surmise that these speceis probabaly use snakes as their definitive hosts.Based on the phylogenetic analysis,S.caninum formed an individual clade with S.arctica from foxes within a group containing Sarcocysits spp.with raptorial birds as definitive hosts.Therefore,a bird probabaly acts as their definitive host.