【作者】 菅复春；

【导师】 张龙现；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2012， 博士

【摘要】 隐孢子虫是一种重要的人兽共患原虫病病原，可以感染260多种脊椎动物，主要引起肠胃炎和急慢性腹泻为主的隐孢子虫病，已在106个国家的人群中发现隐孢子虫病的流行。隐孢子已成为人类6种常见腹泻病的病原之一，同时它也是150种水传疾病病原中最重要的一种。很多动物是人兽共患隐孢子虫的宿主和人感染隐孢子虫的来源。贾第虫是全球分布的脊椎动物普遍存在的肠道寄生虫，该虫主要是通过食物或者饮水传播，可引起动物腹泻，也是人类常见的腹泻病原之一。多种动物如家养和野生反刍动物及犬、猫、兔及其他啮齿动物等是人兽共患贾第虫的贮存宿主，因此，动物的隐孢子虫和贾第虫流行情况和基因型分布受到人们的重视。为研究我国马属动物中隐孢子虫和贾第虫临床感染情况及其公共卫生意义，从2008-2011年间，从河南，山东，内蒙古，甘肃，河北，四川等省区的部分地区分别采集驴、马、骡粪便样品1063、203、36份，经饱和蔗糖溶液漂浮法和卢戈氏碘液染色法检查，共发现寄生虫阳性粪样921份，其中球虫、蛔虫、圆线虫、绦虫、其他寄生虫卵/卵囊/包囊感染率分别为7.14%、12.6%、55.99%、3.53%、4.53%，两种及其以上寄生虫混合感染率2.58%。不同来源的马属肠道寄生虫的阳性率从31.94%-100%之间，总阳性率为70.74%，各地马属动物肠道寄生虫混合感染率从0-70%不等。内蒙古军马场的军马感染率最低为31.94%，混合感染率为2.08%。骡的线虫和隐孢子虫的感染率较马、驴的低。6月龄以下的马属动物隐孢子虫和线虫的感染率分别为29.21%和86.14%，较6-12月龄和成年动物高；集中养殖方式马属动物各种寄生虫的感染率均高于农村散养动物。本次调查共检出隐孢子虫疑似样品222（17.5%）份，感染强度较低，以幼龄马属动物感染率较高。马属动物的隐孢子虫近圆形，大小在5μm左右。用基于SSU rRNA基因的PCR-RFLP分析鉴定以及DNA序列分析鉴定技术对上述222份隐孢子虫阳性或疑似样品进行基因分型，结果发现，222份阳性和疑似样品有114份扩增出目的条带，占调查总数的8.76%。其中51个样品进行序列测定。经RFLP分析和种系发育分析，共鉴定出6个隐孢子虫种类/基因型：Cryptosporidium donkey genotype，C. parvum，horse genotype， C. cuniculus基因型， Cryptosporidium pig1，ferret genotype。其中新基因型有86个样品，donkey genotype为优势种类，其次为C. parvum共12个样品。2个样品属于horse genotype,2个为C. cuniculus，2个为Cryptosporidium pig1。在12个C. parvum样品中，驴的样品占到9份为72.73%，马的样品为27.27%，表明马和驴的隐孢子虫均有人兽共患隐孢子虫种类。donkey genotype是6月龄以下驴驹感染的主要隐孢子虫种类，两个horsegenotype样品均来自驴驹。本试验发现驴是自然感染隐孢子虫C.cuniculus和ferret genotype的新宿主。经SSU rRNA基因扩增鉴定的阳性样品进一步用gp60基因进行亚型分型，发现获得的扩增产物分属三个不同的亚家族：IId、VI和一个新的亚型家族，按照gp60亚型的命名原则将新亚型命名为XII；三种不同亚家族中，共发现四个亚型：horse genotype家族VI中两个样品均是VIA11G3亚型，IId亚家族中两个样品均属IIdA19G1，在新家族中，有两个样品属于XIIA16亚型，其余19个都为XIIA16G1亚型。所属XII家族样品数量为21个，占84%，其余两个亚型所占比例各为8%，和基于SSU rRNA的分类结果一样，donkey genotype为优势种类。其中horse genotype、IIdA19G1是人兽共患隐孢子。基因亚型XIIA16、XIIA16G1是新发现的驴隐孢子虫基因亚型，而且在成功扩增的25个gp60样品中，XIIA16G1是优势亚型，该亚型对动物的致病力、是否对人具有感染性尚不明确，需要进一步的试验探索。对部分驴粪样品进行贾第虫TPI基因和GDH基因扩增并测序，共有4份TPI基因阳性，7份GDH基因阳性，经搜索同源序列、ClustalX1.83比对、phylip3.69进化分析，发现扩增出的4个TPI序列中的3个属于聚集体BIV，与聚集体BIV仅有2个碱基的变异；另一个TPI阳性样品属于聚集体AI。扩增的7个GDH序列均属聚集体BIV，两个基因位点分析的共8个贾第虫样品均属于人兽共患的聚集体A、B，这与文献报道的马的贾第虫主要种类为聚集体E有明显差别。以上研究探明了我国部分地区马属动物肠道寄生虫感染总阳性率70.74%，其中圆线虫，球虫，蛔虫分别为55.99%，7.14%、12.6%。隐孢子虫阳性和疑似阳性率为17.5%，经SSU rRNA基因鉴定的阳性率为8.76%。马属动物尤其是驴隐孢子虫基因分型显示有6个隐孢子虫种类/基因型，其中donkey genotype为新基因型，同时也是我国驴感染的优势虫种，C. parvum，horse genotype为人兽共患种类/基因型，而C.cuniculus和ferret genotype的检出表明驴为这两个种类的新宿主。gp60亚型分析共发现3个不同亚型家族，4个亚型：IIdA19G1、VIA11G3、XIIA16、XIIA16G1，后两者是首次发现的驴隐孢子虫亚型。马属动物贾第虫基因分型结果发现两个基因型：聚集体AI和BIV，后者是优势亚型。尽管donkey genotype生物学特性如是否具有人兽共患性尚不清楚，但C. parvum和horse genotype的及贾第虫聚集体AI和BIV的发现表明我国马属动物是人隐孢子虫和贾第虫感染的贮存宿主，其中C. parvum IIdA19G1也是河南人隐孢子虫流行的亚型。因此，虽然马属动物隐孢子虫和贾第虫的感染率不高，感染强度不大，在动物本身并不表现出临床症状，但作为贮存者和携带者在人隐孢子虫和贾第虫感染中所扮演的角色却不容忽视。更多还原

【Abstract】 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.更多还原