Molecular Epidemiology Study And Zoonotic Risk Assessment Of Three Enteric Protozoa In Yak In Tibet, China

Cryptosporidium spp.,Giardia duodenalis,and Enterocytozoon bieneusi are common gastro-intestinal zoonotic protozoas that are highly prevalent worldwide,with varied routes of transmission.A large number of studies have shown that livestock is the main source of human three protozoas infection,among which Cattle is considered to effect mostly.With abundant livestock resources but less intensive farming,the breeding industry is still dominated by grazing in Tibet.Yak is an indispensable livestock species serving for the herdsman life and economic development.Despite being reared outdoors,Yak is in close contact with local inhabitants in Tibet.However,the data of epidemiological and phylogenetic relationships about Cryptosporidium spp.,Giardia duodenalis,and Enterocytozoon bieneusi from Yak in Tibet remain are rather scarce.The objectives of the present study were to analyze the potential zoonotic risk and phylogenetic relationships of the three intestinal protozoa based on genotypic identification and molecular epidemiological data,we collected samples of yak from three regions of Nyingchi,Shannan and Shigatse in Tibet.Part 1: Investigation on the Prevalence of Intestinal Parasites in Yak in TibetUsing Lugo's iodine staining and Sheaher's sugar floatation technique,we investigate the prevalence of intestinal parasites of 577 fecal samples from Nyingchi,Shannan and Shigatse in microscope.10 species of intestinal parasites were detected and the overall infection rate was 82.7%.46.4% of samples were mixed infected by 2 to 5 kinds of parasites.The infection rates of Cocidium spp.,Giardia duodenalis,Cryptosporidium spp.,Strongylid spp.,whipworm,Trematoda spp.,tapeworm,Amoebida spp.,Ascaris and Balantidium coli were 45.4%,0.4%,1.6%,36.6%,3.8%,1.6%,3.1%,53.4%,3.6% and 1.0%,respectively.The infection rates of Mainling,Gongbujiangda,Bayi District,Gyaca and Xietongmen were 95.2%,65.6%,85.1%,82.1% and 93.1%,respectively.Statistical difference is extremely significant(P<0.01).Part 2: Study on Molecular Epidemiology of Cryptosporidium in Yak in Tibet8 samples were positive Cryptosporidium with the overall prevalence being 1.4% based on SSU r RNA gene.A total of two Cryptosporidium species: C.andersoni and C.bovis were found and C.andersoni as the dominant species.The infection rates of Cryptosporidium were 0 to 6.9% in different regions.Yaks in Xietongmen had a highest infection rate,6.9%,whereas Cryptosporidium was not identified in Mainling and Gyaca.The infection rates of Gongbujiangda and Bayi District were all0.8%.Statistical difference is extremely significant(P<0.01).Part 3: Study on Molecular Epidemiology of Giardia duodenalis in Yak in Tibet10 samples were positive Giardia duodenalis with the overall prevalence being 1.7% based on SSU r RNA and tpi genes.Sequence analysis of the isolates revealed assemblage E was detected in all10 positive samples.The infection rates of Giardia duodenalis were 0 to 3.6% in different regions.Yaks in Gyaca had a highest infection rate,3.6%,whereas Giardia duodenalis was not identified in Mainling.The infection rates of Xietongmen,Bayi District and Gongbujiangda were 1.4%,1.5% and2.4%.Statistical difference is not significant(P>0.05).Part 4: Molecular Characterization of Enterocytozoon bieneusi in Yak in Tibet29 samples were positive Enterocytozoon bieneusi with the overall prevalence being 5.0% in 577 samples based on ITS gene.The infection rates of Enterocytozoon bieneusi were 1.6 to 12.9% in different regions.Yaks in Mainling had a highest infection rate,12.9%,whereas Yaks in Gongbujiangda had a lowest infection rate,1.6%.The infection rates of Bayi District,Gyaca and Xietongmen were 4.9%,5.4% and 5.6%.Statistical difference is significant(0.01<P<0.05).Sequence analysis of the isolates revealed 6 genotypes were detected in all 29 positive samples,inclouding:D(n=5),I(n=6),J(n=2),CHC8(n=1),BEB4(n=14),BEB6(n=1),and the dominant genotype was BEB4.Genotype D was clustered into Group 1 with high zoonotic potential in the phylogenetic analysis.Genotype I,J,BEB4,CHC8 and BEB6 were clustered into Group 2.In conclusion,the intestinal parasites of fecal samples were detected in microscope firstly,then the species,assemblages and genotypes of Cryptosporidium spp.,Giardia duodenalis and Enterocytozoon bieneusi were identified by molecular characterization.Genotypic identification and molecular epidemiological data were used to analysis the potential zoonotic risk and phylogenetic relationships of the three intestinal protozoa.The present study will play an important role in promoting the development of Cryptosporidium,Giardia duodenalis,and Enterocytozoon bieneusi spread and transmission.