Phylogenetic Analysis Of Three Enteric Protozoa In Dairy Cattle In Xinjiang,china And Population Genetics Study Of Cryptosporidium Spp.

Cryptosporidium spp.,Giardia duodenalis,and Enterocytozoon bieneusi are three of the most important identified gastro-intestinal zoonotic protozoas worldwide that can be transmitted via either direct contact or ingestion of contaminated food or water.Cattle is considered to be an important source of infection for humans of three protozoas,especially in calves fecals with thoese parasitic pathogen which contamination food and water are the main factors leading to human cryptosporidiosis and giardiasis outbreaks.Xinjiang Uygur Autonomous Region in China is an important route of the Silk Road since ancient times,with the unique advantage of the geographical in the process of domestication and breeding of cattle.Cattle is an important livestock and which is the animal used for economic and trade in Xinjiang,however,none genetic characteristics study of three opportunistic protozoa heve been reported in cattle in this area.Recently study showed that C.parvum IId family was probably dispersed from Western Asia to other geographical regions,and the subtype structure of C.andersoni with the unique geographical in China.In the present study,we made the study on epidemiological survey of dairy cattle in Xinjiang,and made the study on molecular epidemiology,species/genotypes identification and phylogenetic analysis of Cryptosporidium spp.,G.duodenalis,and E.bieneusi of dairy calves in Xinjiang,and made the study on population genetic structure analysis of C.andersoni and C.parvum IId,which from dairy cattle in Xinjiang and Xinjiang/Ningxia,respectively.Sheather's sugar flotation technique was used to detect the Cryptosporidium oocysts of dairy cattle in fecal samples from nine cities in Xinjiang.The overall infection rate of Cryptosporidium spp.was 4.9%,among of He cattle all aged adult cattle(> 450 d)with infection rate of Cryptosporidium spp.was 2.3%.The infection rate of Cryptosporidium spp.in Holstein cattle was 5.8%,with the highest infection rate of 12.2%(29/237)in pre-weaned calves(< 60 d).The infection rate of Cryptosporidium spp.was 7.7%,5.4% and 2.6% in post-weaned(61-180 d),young cattle(181-450 d)and adult cattle(> 450 d),respectively.Significant difference was observed among age groups(P<0.01).514 dairy calves fecal samples on 15 different intensive farms were randomly collecte and 82 samples were positive Cryptosporidium with the overall prevalence being 16.0%(82/514)based on SSU r RNA gene.C.andersoni was found as the dominant species.The prevalence of calves in pre-weand and post-weand was 15.6% and 16.2,respectively.But the difference was not statistically significant(P > 0.05).C.parvum was only found in pre-weand calves.Two zoonotic genotypes of them were identified baesd on gp60 gene: IId A14G1 and IId A15G1.And there was a significant correlation between the geographic distribution and genotypes of C.parvum and C.Reitman.64 samples were positive G.duodenalis with the overall prevalence being 13.4.0%(64/514)based on SSU r RNA gene.The post-weand calves were found more prone to infect G.duodenalis than pre-weand calves.Two assemblages were detected in all samples: assemblage A and assemblage E.The assemblage A was considered as the zoonotic genotype.Based on tpi,gdh and bg genes.Eight,eleven and seven new subtypes of assemblage E were found,respectively.Five novel multilocus genotypes E(MLGs E1–E5)were identified.The phylogenetic analysis showed all assemblage E MLGs clustered broadly with previous ones and formed an independent branch.85 samples were positive E.bieneusi with the overall prevalence being 16.5.0%(64/514)based on ITS gene.The calves aged 61-90 days were found to have the highest prevalence with 36.0%.Six E.bieneusi genotypes were found,of which five reported zoonotic genotypes(BEB4 ? D ? Ebp C ? I and J)and one new genotype(NEW1.Phylogenetic analysis declared that three genotypes(D?Ebp C and NEW1)belong to Group1,while I?J and BEB4 genotype belong to cattle-special Group2.Group1 is zoonotic pathogenic group,which suggested these calves have an important public health significance.70 of 1827 dairy cattles fecal samples were identified as C.andersoni by used of microscopy,the overall prevalence was 3.8%.The amplification based on SSU r RNA gene locus and the result of sequence analysis showed all seventy samples was C.andersoni.Among of He cattle with infection rate of C.andersoni was 2.3%.The infection rate of C.andersoni on Holstein cattles was 4.3%,with the highest prevalence of 8.4% being found on post-weaned calves.Significant difference was observed among age groups(P<0.01).Eight MLST subtypes in C.andersoni were formed in 70 isolates,of which the MLST subtype A2,A4,A2,A1 had the most number of isolates.Three new MLST subtypes(A4,A5,A2,A1?A4,A5,A2,A1 and A7,A4,A2,A1)were identified.C.andersoni MLST subtypes of adult Holstein cattles showed that were varied,there are seven MLST subtypes in 12 isolates.Linkage disequilibrium analysis showed C.andersoni from cattles in Xin Jiang were the epidemic populations.To better understand the family population genetic structure characteristics of C.parvum,we collected 41 IId sub-genotype cattle-drived isolates from Xinjiang and Ninxia province and made the multilocus DNA sequence analysis based on 12 genes.Four MLST subtypes were formed.These results,combining with different MLST subtypes compared to the previous MLST subtypes drived from cattle,horse and mice in China along with all MLSTs in the current study was included.Of the 7 MLST subtypes,MLST1-6 belong to a single group,while MLST7 subtype on horse formed another single group.That suggested the host-adaptation existed between different MLST subtypes with IId sub-genotype cattle-drived isolates from China being clonal population structure.Significant geographical segregation were both observed in the MLST subtypes.In conclusion,the study analyzed the species and genotype structure characteristics of Cryptosporidium spp,G.duodenalis and E.bieneusi on cattles from Xin Jiang and will help to understand the public health significance.C.andersoni MLST subtypes from cattles showed that were varied and geographical segregation.The population genetics of C.parvum IId isolates was conducted and our obserbations indicated C.parvum IId had a clonal population structure and which were host-adaptation and geographical segregation.The study will play an important role in promoting the development of Cryptosporidium spread and transmission.