【作者】 宋军科；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2017， 博士

【摘要】 寄生于人和动物肠道的原虫由于种类众多,分布广泛,危害严重,而日益成为全球寄生虫学以及公共卫生领域研究的热点。其中的一些人兽共患种类可通过水、食物等多种方式在人和动物之间传播,造成人和动物严重的胃肠道相关疾病,给动物养殖和人类健康带来巨大危害。隐孢子虫(Cryptosporidium)、蓝氏贾第虫(G.lamblia)和芽囊原虫(Blastocystis)是重要的人兽共患肠道原虫病病原。目前,关于这3种肠道原虫的形态学、传播方式及其生物学特性等方面的研究已有较多报道,但其在山羊中的流行情况、基因分型及种群结构研究却鲜有报道。因此,本文对陕西省部分地区山羊隐孢子虫、贾第虫和芽囊原虫进行了感染情况调查和种群结构研究。(1)山羊隐孢子虫、蓝氏贾第虫和芽囊原虫感染情况。通过分子学方法和形态学方法,对采集自陕西省榆林市榆阳区、延安市宝塔区、渭南市富平县、宝鸡市千阳县、商洛市洛南县及杨陵区6个县(区)的874份山羊粪便样品进行了隐孢子虫、贾第虫和芽囊原虫形态学研究和感染情况调查。显微镜观察结果表明,分离到的隐孢子虫卵囊大小约为5μm,呈卵圆形,内含一个明显的卵囊残体和4个祼露的子孢子。粪便中的贾第虫多以近椭圆的包囊形式存在,包囊大小约为15μm,内可见2个核。基于SSU rRNA、gp60、tpi、bg、gdh等特异性基因位点对山羊粪便样品进行分子学检测,结果显示,芽囊原虫感染率为58.0%(458/789),显著高于隐孢子虫的感染率11.6%(101/874)和蓝氏贾第虫的感染率9.8%(86/874),成为优势感染虫种。其中有105份样品为混合感染,占阳性样品总数的16.4%(105/639)。统计学表明,3种肠道原虫的感染率也因山羊品种、年龄和地理位置不同而存在差异。断奶前山羊(0~2月龄)的隐孢子虫和贾第虫感染率最高,分别为27.6%(53/192)和14.6%(28/192);而芽囊原虫在7~11月龄的山羊中感染率最高,达66.5%(127/191)。(2)山羊隐孢子虫基因分型。基于SSU rRNA基因部分片段序列,对采集的874份山羊源隐孢子虫阳性样品进行了种类鉴定,鉴定出了肖氏隐孢子虫(Cryptosporidium xiaoi)、微小隐孢子虫(Cryptosporidium parvrm)、贝氏隐孢子虫(Cryptosporidium baileyi)和泛在隐孢子虫(Cryptosporidium ubiquitum)5个种。统计结果显示,在检出的101份隐孢子虫阳性样品中,C.xiaoi 88份,C.parvum 7份,C.baileyi 5份,C.ubiquitum 1份,分别占总阳性样品的87.1%、6.9%、5.0%和1.0%。提示陕西省山羊隐孢子虫感染的优势种为C.xiaoi。鉴定的C.parvum和C.ubiquitum为人兽共患虫种,存在感染人的风险。进一步应用隐孢子虫gp60基因对获得的7个C.parvum分离株进行亚型分析,结果有4个分离株属于亚型ⅡdA19G1,3个属于ⅡdA20G1亚型。(3)山羊蓝氏贾第虫集聚体分析和多位点分型。根据蓝氏贾第虫的tpi、bg和gdh基因设计并合成引物,对采集的874份山羊粪便进行套式PCR扩增和序列分析。结果表明,基于tpi特异性引物鉴定出65份蓝氏贾第虫集聚体E阳性样品和6份蓝氏贾第虫集聚体A阳性样品,形成了7个集聚体E亚型和3个集聚体A亚型;基于bg特异性引物鉴定出50份蓝氏贾第虫集聚体E阳性样品,4份蓝氏贾第虫集聚体A阳性样品和3份集聚体B阳性样品,形成了5个集聚体E亚型、3个集聚体A亚型和3个集聚体B亚型;基于gdh特异性引物鉴定出12份蓝氏贾第虫集聚体E阳性样品和2份蓝氏贾第虫集聚体A阳性样品,形成了4个集聚体E亚型和1个集聚体A亚型。进一步通过tpi、bg和gdh基因的多位点序列分型,结果表明共有14份蓝氏贾第虫分离株在3个位点均扩增成功,分别形成2、4、5个集聚体亚型,构成7个不同的MLGs,而且均集中在榆林白绒山羊和富平关中奶山羊中,其中0~2月龄中的山羊有4个不同的MLGs,3~6月龄有1个MLGs,7~11月龄有4个MLGs。(4)山羊芽囊原虫基因分型。基于SSU rRNA部分基因片段,对陕西省不同地区、不同用途的山羊品种来源的芽囊原虫的458个阳性样品进行了基因亚型分析。测序和序列分析表明,山羊源芽囊原虫存在6个已知亚型(ST1,3,4,5,10,14)和1个新发现的亚型ST novel(Sd26),其中ST10亚型为山羊源的优势亚型,占到总亚型数的63.8%(292/458)。其中,ST1、ST3、ST4和ST5这4个基因亚型属于已报道的人兽共患亚型。综上所述,通过对陕西省不同地区、不同品种山羊重要人兽共患肠道原虫感染情况的调查和种群结构的研究,阐明了陕西省山羊隐孢子虫、蓝氏贾第虫和芽囊原虫的种类、基因型、亚型分布和种群遗传结构,为相关研究提供了科学依据。更多还原

【Abstract】 Protozoan parasites inhabiting intestinal tract of humans and animals are increasingly becoming one of research focuses in the field of global parasitology and public health because of their species varieties,wide distribution and serious damage.Among them,zoonotic parasites could be transmitted between humans and animals through various routes including water-and food-borne,causing severe gastrointestinal diseases,leading to significant damages to human health and animal production.Cryptosporidium,G.lamblia and Blastocystis have been recognized as important zoonotic intestinal protozoa.At present,numerous studies were focused on morphology,transmission ways and biological characteristics of these three parasites,but there are few reports about the epidemiology,genotyping and population structure in goats.Here,the prevalence,species and genotypes/assemblages/subtypes of Cryptosporidium,G.lamblia and Blastocystis were studied in goats from partial areas in Shaanxi province to evaluate the population structures of these zoonotic intestinal protozoa in goats from Shaanxi province.(1)Prevalence of Cryptosporidium,G.lamblia and Blastocystis in goatsTo explore the infection status of Cryptosporidium,G.lamblia and Blastocystis in goats in Shaanxi province,874 goat fecal samples were collected from Yuyang district of Yulin city,Baota district of Yan’an city,Fuping county of Weinan city,Qianyang county of Baoji city,Luonan county of Shangluo city and Yangling district in Shaanxi province to examine prevalence by using molecular methods and morphological characters.Microscopic observation showed that the size of the isolated Cryptosporidium oocysts was about 5 μm,and oocysts were oval and contained an obvious oocyst residue and four naked sporozoites,while most G.lamblia existed in the form of nearly oval cysts with two nuclei,with the size of 15 μm.Using molecular methods based on species-specific loci of 18 S rRNA,gp60,tpi,bg,gdh,the prevalence of Blastocystis was 58.0%(458/789),which was significantly higher than that of Cryptosporidium(11.8%)and G.lamblia(9.8%)(86/874),and it was identified as the dominant species.Mix infections were detected in 105 samples,accounting for 16.4%(105/639)of the total number of positive samples.Statistical analysis showed that the infection rates of three intestinal protozoa are significantly affected by goat breeds,age groups and geographical origins.The highest prevalences of Cryptosporidium(27.6%,53/192)and G.lamblia(14.6%,28/192)were found in pre-weaned goats(0-2 months),while the highest infection rate(66.5%,127/191)of the Blastocystis was detected in goats aged of 7 to 11 months old.(2)Species/genotypes of Cryptosporidium in goatsCryptosporidium species of 874 fecal samples of goats were identified based on the sequences of SSU rRNA gene fragment.Four species were found,including C.xiaoi,C.parvum,C.baileyi and C.ubiquitum.Of 101 Cryptosporidium positive samples,the numbers of C.xiaoi,C.parvum,C.baileyi and C.ubiquitum were 88,7(,5,and 1,respectively,with C.xiaoi as the predominant species.Two zoonotic species(C.parvum and C.ubiquitum)would pose potential to transmit Cryptosporidium to humans.Subtyping of C.parvum using the gp60 gene indicated four subtype IIdA19G1 and three subtype IIdA20G1.(3)Assemblages and multilocus genotypes(MLGs)of G.duodenalis in goatsAssemblages and MLGs of G.lamblia in 874 goat feces in Shaanxi province were identified based on loci of tpi,bg and gdh.Based on tpi-specific primers,a total of 65 assemblage E and six assemblage A positive samples were identified,belonging to seven assemblage E subtypes and three assemblage A subtypes.The bg-specific primers successfully amplified 49 assemblage E,four assemblage A and three assemblage B samples,forming five assemblage E subtypes,three assemblage A subtypes and three assemblage B subtypes.16 G.lamblia-positive samples were amplified at the locus gdh,and sequence analysis indicated 12 of assemblage E and two of assemblage A samples,forming four assemblage E subtypes and one assemblage A subtype.Multilocus sequence typing based on loci tpi,bg and gdh genes showed that 14 G.lamblia-positive isolates of assemblage E were successfully amplified at all three gene loci,respectively forming two,four,five assemblage subtypes and seven different MLGs.These MLGs were all observed in white cashmere goats of Yulin city and Guanzhong dairy goats of Fuping county.Among them,four,one,and four different MLGs were respectively detected in goats aged 2 months,3-6 months and 7-11 months.(4)Subtypes of Blastocystis in goatsSubtypes of 458 Blastocystis positive samples from different origins and production categories were identified based on the partial 18 S rRNA gene fragment.Six known(ST1,3,4,5,10,14)and one novel subtypes(Sd26)were identified,with the ST10 as the predominant subtype in goats from this area,accounting for 63.8%(292/458)of total subtypes.Among them,four subtypes,namely ST1,ST3,ST4 and ST5,were zoonotic subtypes.Collectively,investigation on infection status of intestinal protozoan parasites in different goat breeds from different areas of Shaanxi province clearly illustrated distribution of species,genotypes,subtypes and population structure of Cryptosporidium,G.lamblia and Blastocystis in goats in Shaanxi province,which provided valuable basic data for correlation research.更多还原