Investigation Of Molecular Epidemiology And Genotypic Identification Of Four Enteric Protozoa In Tan Sheep In The Ningxia Hui Autonomous Region

Enterocytozoon bieneusi(E.bieneusi),Giardia duodenalis(G.duodenalis),Blastocystis and Cryptosporidum spp.are zoonotic parasites which are considered to be opportunistic pathogens of humans and animals.A number of studies have reported infections of these four parasites in various hosts.These parasites colonized in intestines and caused gastrointestinal diseases.They can cause host self-limited diarrhea,weight loss and even death in severe cases.Intestinal protozoa bring great influence and harm to public health and animal husbandry,however,no information is available on the occurrence and genotype diversities of them in the Tan sheep,which is a unique indigenous sheep species in the Ningxia Hui Autonomous Region,China.The objectives of the present study were to examine the prevalence and identify the genotypes of these four parasites in Tan sheep in China.These findings provide baseline data and have implications for the epidemiology and control of infection with the four parasites in Tan sheep.A total of 1014 fecal specimens of Tan sheep from six farms in the Ningxia Hui Autonomous Region were examined by nested PCR amplification of the internal transcribed spacer(ITS)for E.bieneusi,and the multilocus genotyping was performed for ITS positive samples based on the satellite loci of MS1,MS3,MS4 and MS7.Nested PCR amplification of G.duodenalis was performed on three gene loci: ?-giardin(bg),glutamic dehydrogenase(gdh)and triglyceride isomerase(tpi),multi-loci typing was carried out based on the above three loci.Blastocystis was amplified by PCR at conserved SSU rRNA locus;Cryptosporidium was amplified by nested PCR at SSU rRNA locus.All results were statistically analyzed for positive results in different regions,ages,and genders to assess the statistical differences caused by different factors.The overall prevalence of E.bieneusi was 12.2%(124/1014).Sequence analysis identified 10 genotypes of E.bieneusi,including three known genotypes,BEB6,COS-I,and CHG13,and seven novel genotypes designated as NX1 to NX7,which all belonged to group 2 by phylogenetic analysis.The overall prevalence of G.duodenalis in Tan sheep was 14.5%(147/1014),two assemblages(assemblage A and E)were detected,including one novel assemblage A at bg locus and one novel assemblage A at tpi locus,and 10 and 11 novel subtypes of assemblage E were detected at the bg and gdh loci,respectively.One assemblage A MLGs were formed based on sequence variation among the three loci.A total of 259 samples identified as Blastocystis were positive,with overall prevalence rate of 25.5%(259/1014).Six different genotypes were identified,including ST1,ST4,ST5,ST10,ST12,and ST14;among which ST10 had the largest number and may have certain species-genus specificity.The overall prevalence rate of Cryptosporidium was 1.2%(12/1014),two Cryptosporidium species(C.ubiquitum and C.xiaoi)were identified through sequence analysis.This study is the first to report the prevalence of E.bieneusi,G.duodenalis,Blastocystis and Cryptosporidium in Tan sheep,which expanded the distribution of genotypes of these four parasites.These results have provided data support and reference for public health prevention and control of these parasite diseases in Tan sheep.