Molecular Epidemiology Of Cryptosporidium And Giardia Of Equine In Some Regions Of China

Cryptosporidiosis is an important zoonotic protozoan disease. Cryptosporidiumspp. can infect more than260species of vertebrate animals and can causegastroenteritis and acute and chronic diarrhea, with the prevalence being found in theworld. Cryptosporidium has been found in humans in one hundred and six countries,which is one of the most common six pathogen for human diarrhea and it is one of themost important pathogen in the150water-born diseases. Cryptosporidium can infectdomestic animals and various wildlife, and many animals are the infection source ofzonnotic Cryptosporidium spp. Likewise, Giardia is universal intestinal parasitefound in vertebrate hosts, with a global distribution, which is transmitted by food orwater. Giardia can cause the diarrhea of animals and is one of the common pathogenfor human diarrhea. A few animals such as domestic and wild ruminants, dogs, rabbits,cats, rodents are the reservoir of zonootic Giardia. Therefore, the prevalence andgenotype distribution of Giardia brought to extensive attention.To discuss the prevalence status of Cryptosporidium and Giardia in equines andto assess the public health significance of the two parasites in China, between2008and2011,1063,203, and36fecal samples were collected from donkey, horse, andmule in the partial areas of Henan, Shandong, Neimeng, Gansu, Hebei, and Sichuan.The samples were examined using the Sheather’s sugar flotation technique and theLug’s iodine stain method,921samples were positive for parasites. In which, theinfection rate of Coccidium, Ascaris, nematode, cestode, and other parasiteeggs/oocysts/cysts was7.14%,12.6%,55.99%,3.53%, and4.53%, respectively. Therate of two or more than two parasites mixed infection was2.58%. The overageprevalence of intestinal parasites was70.74%in various equines, ranging31.94%to100%. The mixed infection rate of equines in different sites varied from0to70%.The lowest prevalence was31.94%for the army horses in Neimeng, with the mixedinfection rate of2.08%. The prevalence of nematode and Cryptosporidium spp. inmule was relatively lowest among the horse, donkey, and mule. The infection rate ofnematode and Cryptosporidium spp. was respectively29.21%and86.14%in less than6-month-old equines, which was higher than that in6to12-month-old and adultanimals. The infection rate of parasites in equines under the large scale breedingpattern was higher than that of equines in scattered breeding style in rural area. TheCryptosporidium-positive suspect samples had two hundred and twenty two (17.5%), and the infection intensity was low with the younger equines having the higherprevalence. The oocysts found in equines were close to circular shape, with the size ofabout5μm.A total of114samples were Cryptosporidium positive from the222suspectsamples was successfully amplified and genotyped, which occupied8.76%of the totalnumber of samples investigated. Fifty two PCR products were sequenced.Phylogenetic analysis showed that there were five Cryptosporidium species/genotypes,including Cryptosporidium donkey genotype, C. parvum, horse genotype,C.cuniculus and C. suis, Among which, the Cryptosporidium new genotype was thepredominant parasite and was found in eighty six samples, followed by C. parvum(n=12). All the new genotype samples came from the donkey, thus, the donkeygenotype was proposed in this study. Among the12C. parvum-positive samples, nineand three came from horse and occupied for72.73%and27.27%, respectively. Theresult suggested that horse and donkey were all infected the zonootic C. parvum. Thedonkey genotype was the major Cryptosporidium species in less than half yeardonkey foals, likewise, the two horse genotype samples were all come from donkeyfoals. It was found for the first time that donkey was the new host of C.cuniculus inthe study for the first time.Subtyping based on gp60gene was performed to the Cryptosporidium positivesamples by SSU rRNA. Three subtype famlies were found, including VI, IId, and XII.Two horse samples belonged to VIA11G3, two C. parvum had IIdA19G1, twodonkey genotypes belonged to XIIA16and the other19samples had XIIA16G1,respectively. Among the three subtype families, XII family had21samples andoccupied for84%, whereas the other two subtypes occupied for8%each, which wasidentical to the genotyping results by SSU rRNA gene that donkey genotype was thepredominant Cryptosporidium species. The C. parvum IIdA19G1was the zonooticsubtype. XIIA16and XIIA16G1were first identified subtypes in donkey, and werethe major subtypes found in25samples subtyped by gp60gene. However, thepathogenicity and the possibility of zonootic transmission are still unkown. Thus,further study is needed to better understand these.Giardia was amplified by TPI and GDH genes in partial of samples from donkey.Four and seven samples were Giardia-positive by TPI and GDH genes, respectively.The PCR products were sequenced and the sequences were analyzed using softwaresof ClustalX1.83and Phylip3.69. In the TPI gene, three samples belonged to assemblage B-IV, with only two nucleotide differences, one sample had assemblageA-I. In the GDH gene, seven samples all belonged to assemblage B-IV. Thus, theeight Giardia samples all belonged to the zonootic assemblage A or B, which hadsignificant difference in comparison to previously reported the predominantassemblage E found in horse.The total prevalence of intestinal parasites was70.74%in this study, in which theinfection rate of Strongylid, Coccidium, Ascaris, was55.99%,7.14%,12.6%respectively. The positive and suspected positive rate of Cryptosporidium was17.5%,SSU r RNA gene detected rate was8.76%. Genotyping study suggested that there sixspecies/genotypes isolated from Equus especially form donkey, donkey genotype isthe predominant Cryptosporidium species in equines, followed by zonootic C. parvumand Cryptosporidium horse genotype, the later two were zoonotic species, whileC.cuniculus was found in donkey for the first time. Three subtype famlies were found,including VI, IId, and XII, in which IIdA19G1, VIA11G3, XIIA16, XIIA16G1werefound based on gp60gene analysis, XIIA16、XIIA16G1were new subtypes found indonkey for the first time. There were two genotypes assemblage AI and assemblageBIV were found in Giardia, the later one was the dominate subgenotype. Althoughthe biological characterization such as zonootic transmission possibility was unclear,the finding of C. parvum, Cryptosporidium horse genotype, Giardia assemblage AIand assemblage BIV indicated the equines are the reservoir for humanCryptosporidium and Giardia infections, meanwhile, C. parvum IIdA19G1wassubtype found in human of Henan province. Therefore, although low infection rateand infection intensity of Cryptosporidium and Giardia were observed and noobvious symptom was seen, that equines might play an important role in the humanCryptosporidium and Giardia infections should not be neglected.