| Cryptosporidum spp.,Giardia duodenalis and Enterocytozoon bieneusi are common detected zoonotic intestinal protozoa species.Humans or animals get infected by consuming oocysts,cysts or spores of these three pathogens from infected hosts through contaminated food or water.Infection with Cryptosporidum spp.,G.duodenalis and E.bieneusi cause gastrointestinal symptoms,such as diarrhea,bellyache,nausea or vomiting.To date,zoonotic intestinal protozoa species have been detected in numerous rodent species,indicating their potential risk to be the infection source of intestinal protozoa.Himalayan marmots(Marmota himalayana)and Alashan ground squirrels(Spermophilus alashanicus)are common rodents in Qinghai-Tibetan Plateau area(QTPA),and they often share the same pastures with humans,herbivorous animals,and other wild animals,and can be hosts or reservoirs for a number of zoonotic infectious diseases.Currently,to the best of our knowledge,no infection information,molecular characterization or genetic structure analysis on Cryptosporidum spp.,G.duode nalis or E.bieneusi in the two rodents was available worldwide.Objective:The aim of the present study was to understand the occurrence and genetic characterizations of Cryptosporidum spp.,G.duodenalis and E.bieneusi in wild Himalayan marmots and Alashan ground squirrels in the QTPA,Gansu Province,China.In addition,E.bieneusi isolates derived from these two rodents were characterized by MLST and genetic structure analysis to understand the genetic structure and substructure.Methods:In QTPA of western China’s Gansu Province,five sample collection sites including Luqu of Gannan Tibetan Autonomous Prefecture,Yugur Autonomous County of Sunan,Xiahe of Gannan Tibetan Autonomous Prefecture,Zhangye City,Huining County of Baiyin City were selected randomly.From June to September 2017,498 wild rodents were captured alive by mousetraps,among them,399 from Himalayan marmots,including 99 captured in Luqu,100 captured in Sunan,102 captured in Xiahe,99 captured in Zhangye.While 99 Alashan ground squirrels were all captured in Huining.Intestinal contents of the two animals were indirectly collected after euthanizing with a high dose of CO2 according to biosafety operation specification.Then genomic DNA were extracted.Based on the small subunit(SSU)rRNA gene locus,nested PCR amplification of Cryptosporidium spp.was performed.Then the positive DNA samples were subtyped by further amplification of the 60 kDa glycoprotein(gp60)gene locus.The specificity gene loci β-giardin(bg),glutamate dehydrogenase(gdh)and triosephosphate isomerase(tpi)of G.duodenalis were analyzed by nested PCR amplification.Nested PCR amplification and genotyping of E.bieneusi were performed based on the internal transcribed spacer(ITS)region of rRNA gene.MLST and genetic structure were then analyzed by nested PCR amplification for the satellite loci of MS1,MS3,MS4 and MS7 of the positive DNA samples.Results:The overall detection rate of the Cryptosporidium spp.,G.duodenalis and E.bieneusi in Himalayan marmots and Alashan ground squirrels was 13.25%(66/498).Among them,the overall prevalence of Cryptosporidium spp.was 2.2%(11/498),with 2.5%(10/399)for Himalayan marmots and 1.0%(1/99)for Alashan ground squirrels.Based on sequence analysis of the SSU rRNA products of 11 the Cryptosporidium-positive samples,four Cryptosporidium genotypes were identified,including three novel genotypes marmot genotype Ⅰ,marmot genotype Ⅱ;and marmot genotype Ⅲ in Himalayan marmot samples,and one known horse genotype in Alashan ground squirrel samples.Horse genotype samples further subtyped by gp60 gene,which successfully amplified and identified as a novel subtype VIbA10.On the basis of at least one locus,a total of eight G.duodenalis-positive samples.were detected(1.6%,8/498),detection rate of G.duodenalis of Himalayan marmots and Alashan ground squirrels were 1.5%(6/399)and 2.0%(2/99),respectively.G.duodenalis assemblages A,B and E were identified,with assemblages B showing a predominance.The gdh locus was successfully amplified in five samples,with four samples identified as assemblage B and one sample was identified as assemblage E.The bg locus was successfully amplified in seven samples,with one sample identified as assemblage A,five samples identified as assemblage B and one sample identified as assemblage E.However,amplification of tpi locus failed despite repeated attempts at molecular-experiments using different primersThe overall detection rate of E.bieneusi was 10,0%(50/498),with 11.8%(47/399)for Himalayan marmots and 3.0%(3/99)for Alashan ground squirrels.Based on the sequence analysis of ITS region of rRNA gene,seven genotypes were identified from the 50 E.bieneusi positive samples:one known genotype YAK1,and six novel genotypes ZY37,HN39,HN96,SN45,XH47 and ZY83,with genotype ZY37 showing a predominance.Phylogenetic analysis showed that all the genotypes obtained in this study were fallen into zoonotic Group 1.A total of in three samples of Himalayan marmots from Zhangye had mixed infections of the intestinal protozoa,one had mixed infection of E.bieneusi and Cryptosporidium,two had mixed infection of E.bieneusi and G.duodenalis.Of the 50 E.bieneusi isolates analyzed in this study,25,26,22 and 31 positive samples were amplified successfully at the MS1,MS3,MS4 and MS7 loci,and formed two,two,eight and five types,respectively.A total of 18 E.bieneusi samples were amplified and sequenced successfully at five loci(ITS,MS1,MS3,MS4,MS7),forming nine MLGs.Genetic structure analysis indicated the presence of a clonal population structure of E.bieneusi in the investigated areas.Phylogenetic analysis and network analysis identified two subgroups,subgroup 1 was epidemic population structure,and subgroup 2 was clonal population structure,which suggesting that the epidemic and clonal transmission modes were existed in E.bieneusi in Himalayan marmots.Conclusion:Cryptosporidium spp.,G.duodenalis and E.bieneusi were detected in wild Himalayan marmots and Alashan ground squirrels in QTPA,Gansu Province,China.Cryptosporidium horse genotype and E.bieneusi genotype YAK1 were identified in rodents for the first time,expanded the host range of these genotypes.In addition,three novel Cryptosporidium genotypes and six novel E.bieneusi genotypes were identified in this study.Genetic structure analysis indicated the presence of a clonal population structure of E.bieneusi in the investigated areas.Study results obtained in this study provide data support for the assessment of zoonotic risk,risk factors and disease burden of the two rodent animals and supply reference for local parasite prevention and control. |
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