The Investigation Of Intestinal Parasites Based On Amplicon Sequencing And Molecular Epidemiological Research Of Two Intestinal Parasites In Bos Frontalis

Objective:The intestinal zoonotic parasites is a potential pathogen causing mutual infection between human and Bos frontalis.This study focused on Bos frontalis in Nujiang Prefecture,Yunnan Province,the intestinal parasitic pathogens composition and community diversity of it were investigated by amplicon sequencing.The infection of Cryptosporidium spp.and Entamoeba spp.in Bos frontalis was investigated by nested PCR and conventional PCR,and the species were identified to determine whether there was a risk of zoonotic infection.The results provide a theoretical basis for the prevention and control of local parasites,especially zoonotic parasites,and for their study.Methods:In the first part,100 fecal samples of Bos frontalis were collected in the summer from five groups(Dulongjiang Township,Jiumendang village,Yazuoluo village,Guquan village and Cikai Township),and the composition of parasitic pathogens and community diversity of fecal samples from Bos frontalis at various taxonomic levels were comprehensively investigated and analysed by extracting total genomic DNA and amplifying standard eukaryotic 18S r RNA gene V9,based on amplicon high-throughput sequencing.In the second part,625 fecal samples were collected in four seasons from Jiumendang village,Yazuoluo village,Guquan village and Cikai Township of Nujiang River Basin and Dulongjiang Township of N’Mai River Basin in Nujiang Prefecture,Yunnan Province.Fecal samples DNA was extracted and amplified by nested PCR based on the SSU r RNA gene of Cryptosporidium spp.,lastly,the positive samples were sequenced and analysed.In the third part,625 fecal samples DNA was extracted and amplified by conventional PCR based on the 18S r RNA gene of Entamoeba spp.,lastly,the positive samples were sequenced and analysed.Results:The results of the first part showed that the community species richness and diversity of the five groups of fecal samples were high.At the phylum level,Nematoda and Apicomplexa were detected.At the class level,Spirotrichea,Chromadorea,Conoidasida,and Heterolobosea were detected.At the order level,Sporadotrichida,Colpodida,Rhabditida,and Eucoccidiorida were detected.At the family level,Colpodidae,Rhabditidae,Oxytrichidae,Sarcocystidae,Entamoebidae,and Acanthamoebidae were detected.At the genus level,Cercomonas,Colpoda,Blastocystis and Tetramitus,Diploscapter,Gonostomum,Oxytricha,and Naegleria.At the family and genus level,Entamoebidae,Acanthamoebidae,Blastocystis and Naegleria all have the risk of zoonotic infection.The results of the second part showed that 39 samples were positive for Cryptosporidium spp.,with an overall infection rate of 6.24%(39/625).Among the infection rate of five sampling locations,the highest of 10.04%(23/229)was detected in Cikai Township,and the lowest of 1.27%(1/79)in Yazuoluo village,with a highly significant difference between the five sampling locations(P<0.01,χ~2=16.849).The infection rates were 6.28%(35/557)in Nujiang River basin and 5.88%(4/68)in N’Mai River basin,and there was no significant difference between the two basins(P>0.05,χ~2=0.017).Among the four seasons,the highest infection rate was 15.60%(34/218)in spring,the lowest was 0 in autumn,with a highly significant difference between four seasons(P<0.01,χ~2=50.634).In all positive samples,Cryptosporidium andersoni(10/39),Cryptosporidium sp.(28/39),and Cryptosporidium ryanae(1/39)were identified.C.andersoni has a risk of zoonotic infection.The results of the third part showed that 61 samples were positive for Entamoeba spp.,with an overall infection rate of 9.76%(61/625).Among the infection rate of five sampling locations,the highest of 13.54%(31/229)was detected in Cikai Township,and the lowest of 4.12%(4/97)in Jiumendang village,with a significant difference between the five sampling locations(P<0.05,χ~2=10.187).The infection rates in the Nujiang River basin and N’Mai River basin were 10.41%(58/557)and 4.41%(3/68),respectively,with non-significant differences(P>0.05,χ~2=2.478).Among the four seasons,the highest infection rate was 23.66%(22/93)in summer,the lowest was3.33%(4/120)in autumn,with a highly significant difference between four seasons(P<0.01,χ~2=32.955).In all positive samples,Entamoeba bovis(51/61)and Entamoeba sp.MG107/BEL(10/61)were identified,which were not zoonotic parasites.Conclusion:The parasite composition at different taxonomic levels in the fecal samples of Bos frontalis showed that Entamoebidae,Acanthamoebidae,Blastocystis and Naegleria might be potential pathogens that could cause infection in humans.This study was the first to investigate and identify the infection situation and species of Cryptosporidium spp.and Entamoeba spp.in Bos frontalis in this area,filling the gap in related studies of them.Location and season were the risk factors of infection,with the highest infection rates of both Cryptosporidium spp.and Entamoeba spp.were found in Cikai Township,and the highest infection of that in spring and summer,respectively.C.andersoni identified in this study is a zoonotic parasite and a potential pathogen of mutual infection between Bos frontalis and human,threatening public health security potentially.