Analysis Of Epidemiological Characteristics And Zoonotic Risk For Parasitic Intestinal Protozoan From Ruminants From Partial Areas Of Western China

Cryptosporidium spp.,Giardia duodenalis,Enterocytozoon bieneusi and Blastocystis sp.are common zoonotic intestinal protozoan parasites worldwide.These pathogens have broad host ranges,including humans,a great number of livestock and wild animals,significantly threatening the health of humans and livestock.The western region of China is an important breeding region for ruminants,e.g.,cattle,sheep and camels.However,there are only a few regional studies on infections of intestinal parasites for ruminants in western China,especially for data about the infection,species and population structure of zoonotic intestinal protozoan parasites,partly hindering the prevention and control of parasitic diseases in this area.Based on this,the present study selected main ruminants(including sheep,goats,beef cattle,Camelus bactrianus,alpacas,red deer,Moschus chrysogaster and blue sheep)in Inner Mongolia,Shaanxi,Ningxia and Gansu as the research objects to investigate the infection,species/genotype/subtype distribution and the zoonotic risk of Cryptosporidium spp.,G.duodenae,E.bieneusi and Blastocystis sp.by using microscopy,polymerase chain reaction(PCR)and multilocus sequence genotyping(MLST).The following results were obtained:1.The infection of intestinal parasites from main ruminants in partial regions of western China was clarified.A total of 2209 fecal samples were collected from 31 breeding areas and a nature reserve in Inner Mongolia,Shaanxi,Ningxia and Gansu,including 797 of sheep,813 of goats,108 of beef cattle,407 of bactrian camels,10 of alpacas,38 of red deer,20 of M.chrysogaster and 16 of blue sheep.1872 samples were found to be infected with intestinal parasites,with the total infection rate of 84.7%(1872/2209).Eight species of intestinal parasites were detected,including coccidia,Strongyloides spp.,whipworm,Moniezia spp.,pinworm,Nematodirus spp.,Cryptosporidium spp.and Giardia spp.,with coccidia to be dominant(54.7%,1209/2209).PCR amplification showed that the infection rate of zoonotic E.bieneusi,Blastocystis sp.,G.duodenalis,and Cryptosporidium spp.,were 15.2%(336/2209),8.1%(178/2209),7.6%(168/2209),and 1.6%(36/2209),respectively.These results showed that the infection of intestinal parasite infection was widespread in investigated ruminants in western China and the zoonotic intestinal protozoan parasites were found in these animals.2.Species/subtypes and zoonotic risk of Cryptosporidium in main ruminants from partial regions of western China were determined.Through analyzing sequences of the SSU r RNA gene for 36 Cryptosporidium-positive samples,five Cryptosporidium species were identified,namely C.xiaoi(n = 16),C.ubiquitum(n = 11),C.andersoni(n = 5),C.bovis(n = 2),and C.parvum(n = 2).Subtyping zoonotic C.xiaoi,C.ubiquitum and C.parvum,based on the 60 k Da glycoprotein(gp60),found C.ubiquitumⅫa(n = 5),and C.xiaoi XⅫⅠc(n = 1)XⅫf(n = 3)XⅫg(n = 5),with C.xiaoi XⅫg firstly identified in sheep.These results showed that Cryptosporidium infection was widespread in investigated ruminants in western China,and there were zoonotic Cryptosporidium species with genetic diversity,suggesting that ruminants may have important public health significance in the local transmission of cryptosporidiosis.3.The assemblage distribution and zoonotic risk of G.duodenalis in main ruminants from partial regions of western China were characterized.A total of 168 G.duodenalis SSU r RNA-positive samples were subjected to PCR amplification and sequence analysis based on β-giardidin(bg),glutamate dehydrogenase(gdh)and triphosphate isomerase(tpi)genes.Three assemblages were identified,including zoonotic assemblage A(n = 11),animal-adapted assemblage E(n = 118),and a mixed assemblage B/E(n = 1).99,28 and 52 sequences were obtained at loci bg,gdh and tpi,respectively.Five multi-locus genotypes(MLGs)were identified,namely MLG-E1-MLG-E5.These findings showed genetic diversity within G.duodenalis assemblages for investigated ruminants in western China and existence of zoonotic G.duodenalis assemblages.4.Subtypes and zoonotic risk of Blastocystis subtypes in main ruminants from partial regions of western China were determined.Through analyzing sequences of 178 Blastocystis SSU r RNA-positive samples,eight known genotypes(namely ST5,ST10,ST14,ST21,ST24,ST25,ST26,and ST30)and a new genotype(namely PT-ST)were identified.There are eight subtypes(namely ST10,ST14,ST21,ST24,ST25,ST26,ST30 and PT-ST)in Camelus bactrianus,and the dominant subtype was PT-ST.Three(namely ST5,ST10,and ST14),and five(namely ST5,ST10,ST14,ST21,and ST26)in sheep and goats were identified,respectively.Among these,ST10 was the dominant subtype(47.7%,85/178).These results indicated genetic diversity of Blastocystis subtypes from investigated ruminants in western China.Notably,the zoonotic subtype ST5 was found in this study,and the subtypes ST21 and ST26 were firstly identified in goats in China.5.Genotypes,population structure and zoonotic risk of E.bieneusi in main ruminants from partial regions of western China were determined.A total of 13 E.bieneusi ITS genotypes were identified,including 10 known genotypes(namely BEB6,COS-I,J,CHC8,I,CHG1,BEB4,CHG3,CHS7,and NCF2)and three novel genotypes(namely CNR1-CNR3).Among these,the genotype BEB6 was to be dominant.Phylogenetic analysis showed that most of ITS genotypes were clustered into the Group 2,but a(NCF2)genotype was clustered into the zoonotic Group 1,indicating zoonotic risk of investigated ruminants to transmit E.bieneusi.Population genetic analysis showed that E.bieneusi isolates from different animals formed different subpopulation structures,and the results of LD analysis supported the genetic recombination of E.bieneusi among different animal populations,possibly caused by the high infection rates and genetic diversities of E.bieneusi.In conclusion,the present study found that main ruminants investigated in western China were widely infected with Cryptosporidium spp.,G.duodenalis,Blastocystis sp.and E.bieneusi,with genetic diversity.In addition,zoonotic C.ubiquitum,C.parvum,G.duodenalis assemblage A,Blastocystis ST5 subtype,and E.bieneusi NCF2 genotype were found in this study,and ruminants have important public health significance in the transmission of zoonotic intestinal protozoan parasites in these regions.These findings provide data for the prevention and control and the assessment of zoonotic risks for intestinal parasitic diseases in ruminants in western China as well as other regions in China.