Phylogenetic And Multilocus Genotypes Of Cryptosporidium And Enterocytozoon Bieneusi In Horses

Cryptosporidium and Enterocytozoon bieneusi are two important intestinal protozoa,which can infect a variety of animals,including humans.To date,these two species of parasites have been reported in humans,livestock,companion animals,rodents,birds and even giant pandas in China.However,there are few reports about the infection rate and genotype of Cryptosporidium and E.bieneusi in horses in China.Between August 2015 and May 2017,a total of 441 fecal samples from horses were collected from seven Equestrian Club in Sichuan and Yunnan Province,southwestern China.A total of 6 positive fecal samples Cryptosporidium were detected by nested polymerase chain reaction(PCR)amplification of 18 S rRNA gene,and the overall infection rate of Cryptosporidium was 1.4%;By sequence analysis of 18 S rRNA of Cryptosporidium has identified two species of Cryptosporidium spp: Cryptosporidium hominis and Cryptosporidium andersoni;The subtype IIdA15 of C.hominis was determinded by sequence analysis of gp60(60 kDa glycoprotein gene)gene.The subtypes of C.andersoni were A6,A5,A2,A1 by multilocus sequence typing analysis(MS1,MS2,MS3,MS16),these subtypes that have been considered to be zoonotic and also have been identified in camel in Henan province and Sichuan Province in China.A total of 87 E.bieneusi-positive fecal samples were identified by sequence analysis of ITS gene of E.bieneusi.The overall infection rate was 19.7% and a total of 10 genotypes were found:horse1,horse2,D and SC02 were known genotypes,SCH1-4 and YNH1-2 were novel genotypes.Phylogenetic analysis of E.bieneusi showed that genotypes D,SC02,horse1 and SCH1-3 were clustered into Group 1,which had the possibility of human zoonotic;the genotype SCH4 belonged to the Group2 with the host specific group,and the genotype horse2 and YNH1-2 were clustered into the Group 6.A total of 13,5,9 and 14 samples were success amplified at MSl,MS3,MS4,MS7 loci,respectively,and 4 samples were successfully amplified at four loci which formed 3 MLGs(MLG1-3).In conclusion,C.hominis and C.andersoni were identified in horses in Sichuan and Yunnan provinces.The subtype IIdA15 of C.hominis was expanded the host range and database of the C.hominis.The C.andersoni subtypes A6,A5,A2 and A1 are consistent with previously reported in cattle and the camel in the same area,so there may be the possibility of transmission between horses and other animals in this area.The presence of genotypes D and SC02 of E.bieneusi,which were previously identified in humans and other animals,this result indicate that horses are a potential source of human and animals E.bieneusi infection in China.