Molecular Epidemiology Of Enterocytozoon Bieneusi And Cryptosporidium In The Bai Ethnic People And Wild Rodents From Dali Area

ObjectiveThe sensitivity,specificity and accuracy of nested PCR reaction were used to investigate the infection of Enterocytozoon bieneusi and Cryptosporidium.To study whether there are zoonotic genotypes of Enterocytozoon bieneusi in Bai ethnic people and wild rodents,and the infected species of Cryptosporidium,which can provide reference to further research of Microsporidiosis and Cryptosporidiosis.MethodsFrom April 2016 to July 2017,a total of 599 fecal samples from the Bai ethnic people and 287 fecal samples from wild rodents were collected in the surrounding areas and villages of Dali City.DNA samples were extracted from fecal samples by OMG Kit.The internal transcribed spacer(ITS)of ribosomal DNA of Enterocytozoon bieneusi and the polymorphic region of the small subunit of ribosomal RNA(SSU rRNA)gene of Cryptosporidium were amplified by nested PCR,respectively.The successfully amplified samples were sequenced to determine the positive rate.Homologous sequence search in the NCBI database,the genotypes and epidemiological distribution of Enterocytozoon bieneusi and Cryptosporidium were compared and analyzed by Cluster X 1.83,SPSS19.0 and MEGA6.Results1.Among 599 stool samples collected from Bai ethnic group in Dali area,and 12 samples were tested as E.bieneusi positive by nested PCR after sequencing.The overall prevalence of E.bieneusi infection was 2.00%(12/599,95%CI 0.88~3.13).The prevalence was 2.91%(8/275,95%CI 0.92~4.90)in male and the prevalence was 1.24%(4/324,95%CI 0.03~2.44)in female.There was no significant difference in infection rate between male and female groups(P>0.05).As for age group,no E.bieneusi positive sample was detected in under 18 years old group.Two samples was tested as E.bieneusi positive in 18~40 years old group,and the infection rate was 1.36%(2/147,95%CI 0.00~3.23).Seven samples was tested as E.bieneusi positive in 41~65 years old group,with an infection rate of 2.30%(7/304,95%CI 0.62~3.99).Among the group of over 65 years old,the infection rate was 3.49%(3/86,95%CI 0.00~7.37).There was no significant difference in infection rate among different ages(P = 0.44).The infection rate of E.bieneusi in rural residents(2.97%,11/370,95%CI 1.19~4.52)was significantly higher than that in the urban population(0.44%,1/229,95%CI 0.00~1.38)and the difference was statistically significant(P<0.05).Out of 12 positive samples,4 known genotypes were identified: EbpC(n=5),SC02(n=1),Henan-?(n=2)and D(n=1).Three new genotypes named as Yunnan1,Yunnan2 and Yunnan3 were identified.2.Twelve samples were tested as E.bieneusi positive from 287 fecal samples of wild rodents,with an infection rate of 4.18%(12/287,95%CI 1.87~6.50).The infection rate of Rattus norvegicus was 4.44%(12/270),the infection rate of Rattus flavipectus was 0(0/17).The infection rate was 12.50%(2/16,95%CI 0.00~28.70)in juvenile group,1.37%(1/73,95%CI 0.00~4.04)in junior group and 4.55%(9/198,95%CI 1.64~7.45)in adult group.The difference was not statistically significant(P>0.05).Female infection rate was 4.57%(8/175,95%CI 1.48~7.67)and male infection rate was 3.57%(4/112,95%CI 0.13 ~7.00).The difference was not statistically significant.Besides the same genotypes D and EbpC found in the fecal samples of people,there is a new genotype named as YNM1.3.In the study of the infection with Cryptosporidium in Bai ethnic people,only one positive sample was detected,the infection rate was 0.17%(1/599),it represented Cryptosporidium muris.4.Thirteen samples were tested as Cryptosporidium positive in the feces of wild rodents.The infection rate was 4.53%(13/287,95%CI 2.12~6.94).The infection rate in juvenile group(31.25%,5/16,95%CI 8.54~53.96)was significantly higher than that in the junior group(6.85%,5/73,95%CI 1.06~12.64)and the adult group(1.52%,3/198,95%CI 0.00 ~ 3.22),respectively.Age is an important risk factor for Cryptosporidium infection in wild rodents,and the difference is statistically significant(P<0.001).The infection rate of female was 6.29%(11/175,95%CI 2.69~9.88)and that of the male rats was 1.79%(2/112,95%CI 0.00~4.24).The difference was not statistically significant(P>0.05).In the wild rodents,only one juvenile wild rodent was co-infected with E.bieneusi and Cryptosporidium.ConclusionThis study was the first time to analyze the genotypes and species of E.bieneusi and Cryptosporidium in the Bai ethnic people and rodents in Dali area.The infection rates of E.bieneusi and Cryptosporidium were 2.00% and 0.17% in the Bai ethnic people,4.18% and 4.53% in wild rodents,respectively.The E.bieneusi genotypes were successfully identified: EbpC,SC02,Henan-?,D,Yunnan1,Yunnan2,Yunnan3 and YNM1;The same genotypes were also found in wild rodents as those in human: D and EbpC.The Yunnan1,Yunnan2,Yunnan3 and YNM1 were identified as new genotypes in this study.Cryptosporidium which was found in this study in human and wild rodents was identified as C.muris.