【作者】 王永立；

【导师】 张龙现； 宁长申；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 隐孢子虫病是一种重要的人兽共患原虫病。本试验从2006年7月到2007年7月对分布在河南省4个地区的5个羊场开展为期一年的流行病学调查,在显微镜检测的基础上对发现的绵羊肠道寄生虫虫卵或卵囊进行形态鉴定;利用巢式PCR和PCR-RFLP技术对隐孢子虫卵囊进行基于核糖体小亚基(18S rRNA)基因的种类与基因型分析,鉴定河南省绵羊隐孢子虫流行种类和基因型,确定河南绵羊隐孢子虫分离株和其它种类隐孢子虫之间的分子种系进化关系;并对某隐孢子虫阳性绵羊场进行跟踪调查,以此来分析绵羊隐孢子虫种类和基因型随不同年龄段、不同季节的动态变化规律,为防控人兽共患隐孢子虫病提供理论依据。为了解河南省绵羊肠道寄生虫感染情况,应用寄生虫常规检测方法对在5个规模化绵羊场共采集的1265份粪样进行了寄生虫检查。共检出虫卵和卵囊5类,其中原虫2类(球虫、隐孢子虫);线虫2类(类圆线虫、鞭虫);绦虫1类。本次调查中共发现1178份粪便样品为寄生虫阳性,寄生虫总感染率为93.12%(1178/1265),5个绵羊场的隐孢子虫平均感染率为3.10%,球虫平均感染率为88.94%,鞭虫平均感染率为10.83%,类圆线虫平均感染率为59.11%,绦虫平均感染率为43.96%。本次调查发现绵羊寄生虫混合感染现象比较严重,其中球虫、绦虫和类圆线虫混合感染的比例达到60.10%;绵羊寄生虫感染率呈现随年龄增长而降低的现象。为掌握河南省绵羊隐孢子虫流行病学资料,应用饱和蔗糖溶液漂浮法和改良抗酸染色法对分布在河南省4个地区的5个羊场共1701份绵羊粪样进行检查,并对绵羊隐孢子虫感染的风险因素进行评估。结果表明:查出阳性样品80份,隐孢子虫总阳性率为4.70%(80/1701), 3周龄羔羊和产后3周母羊的感染率最高分别达41.18%和6.29%。根据隐孢子虫卵囊形态数值学分析结果,将发现于绵羊的隐孢子虫卵囊初步鉴定为微小隐孢子虫(C. parvum)和安氏隐孢子虫(C. andersoni)。同时我们发现绵羊隐孢子虫感染存在年龄差异,但未见明显的季节性变化。本试验利用巢式PCR技术扩增80个绵羊隐孢子虫分离株18S rRNA基因,选择限制性内切酶SspⅠ和VspⅠ分别对PCR产物进行消化酶切,进行RFLP分析。39个PCR产物经测序后,用Clustal X1.81、Phylip3.65和DNAStar4.0等生物学软件对扩增的序列进行种系发育分析及同源性分析,以此来确定河南省绵羊隐孢子虫流行种类及基因型。PCR-RFLP和分子种系发育分析结果表明:绵羊隐孢子虫种类和基因型主要有cervine genotype(35/39)、C. bovis (2/39)、C. andersoni(2/39),其中cervine genotype存在着较大的人兽共患风险。为了分析绵羊隐孢子虫种类和基因型随时间的变化规律,本试验在进行绵羊隐孢子虫流行病学调查的基础上,对阳性羊场的怀孕母羊及其羔羊进行跟踪调查,以此来研究分析绵羊隐孢子虫种类和基因型随年龄和季节的动态变化规律,确定新生羔羊隐孢子虫的来源,探讨羊场隐孢子虫持续存在的机制,最终为研究绵羊隐孢子虫人畜互传机制和制定防控措施提供理论依据。结果显示:Cryptosporidium cervine genotype可以感染每个年龄段的绵羊,绵羊隐孢子虫感染种类和基因型随年龄变化的规律也不如奶牛明显;在绵羊感染的所有隐孢子虫种类和基因型中,cervine genotype一年四季中均为优势基因型,隐孢子虫种类和基因型随季节变化不明显。本试验利用PCR-RFLP技术和分子种系发育分析,首次鉴定国内绵羊隐孢子虫主要有cervine genotype、C. bovis、C. andersoni,优势虫株为cervine genotype。更多还原

【Abstract】 Cryptosporidiosis is an important zoonotic parasitosis. In order to investigate the prevalence of intestinal parasites in sheep in Hennan province, we spent one year to detect sheep faecal samples from 5 farms in 4 counties from July 2006 to July 2007. The samples were identificated by microscope, nested PCR detection and nested PCR-RFLP analysis; genotypic analysis of the Small-Subunit rRNA (SSU rRNA) gene were carried out to indentify species/ genotype, infered molecular phylogenetic relationship between sheep cryptosporidium and other Cryptosporidium.spp. In order to analyze dynamic variation rule of sheep Cryptosporidium.spp with host age and seasons, a trace investigate was carried out in one sheep farm where Cryptosporidium.spp were found in sheep, so that established necessary control rationale of Cryptosporidiosis.For understanding the prevalence of intestinal parasites in sheep in Hennan province, 1265 sheep faecal samples from 5 farms were detected with the convention methods. The results showed that 5 types parasites were detected in stool specimens, 2 types were protozoon (Coccidian, Cryptosporidium), 2 types were nematode (Trichuris ovis, Strongyloides stercoralis), and 1 type was cestode. As a result,the average infection rate was 93.12%(1178/1265), Thereinto, the infection rate of Cryptosporidium sp. was 3.10%, Eimeria sp. was 88.94%, Trichuris sp was 10.83%, Strongyloides sp was 59.11%, cestode was 43.96%, respectively. The results showed that sheep parasites mixed infections are more serious; In addition, the mixed infection of Eimeria sp, tapeworm and Strongyloides stercoralis took up 60.10% of the total infections rate. The results showed that sheep infection rate was decreased with the age increased.An epidemiologic survey was carried out to investigate the prevalence of and to identify factors associated with the risk of Cryptosporidium infection in sheep in Henan province. 1701 sheep faecal samples from 5 farms in 4 regions were detected by the Sheather’s sugar flotation and modified acid-fast staining method. The results showed that oocysts of Cryptosporidium were found in 80 samples, the total infection rate was 4.70%(80/1701), and the percentage of animals shedding oocysts peaked in 3-week-old lambs and in ewes on three week postpartum was 41.18% and 6.29%, respectively. According to the morphometrical values, the Cryptosporidium oocysts were identified as C. parvum and C. andersoni. At the same time, the results showed that there were difference in the infection rates of Cryptosporidium among different ages of sheep, but with no significant difference among seasons.The Small-Subunit rRNA (SSU rRNA) gene fragments of 80 sheep postitive samples were amplified by nested PCR. The PCR products were digested by SspⅠrestriction enzyme and VspⅠrestriction enzyme respevtively to determine species and genotype. After 39 samples sequencing, homological sequences were alignmented. Phylogenetic tree and homological analysis were made by some biological softwares such as ClustalX 1.81, and DNAStar 4.0. Based on the phylogenetic analysis of 18S rRNA gene and PCR-RFLP analysis, 39 isolates were idenfied as cervine genotype(35/39)、C. bovis (2/39)、C. andersoni (2/39), and cervine genotype may be an important zoonotic Cryptosporidium.spp.To analyze variation rule of sheep Cryptosporidium.spp with age and season, on the basis of epidemiological survey. We investigated pregnant ewes and lamb in positive sheep farm to study and analyze sheep Cryptosporidium species and genotypes variation rule with different age groups, different seasons, to infer animals infection source and transmission mechanism in sheep farm.The result showed: Cryptosporidium cervine genotype could infect differenct age groups sheep and was dominance species in all sheep Cryptosporidium.spp in Henan, variation rule with age and seasons was not significant.The present research PCR- RFLP and molecular phylogenetic relationship analysed were employed to identity sheep Cryptosporidium.spp in Henan, China. The result showed that sheep Cryptosporidium.spp included cervine genotype、C. bovis、C. andersoni, and cervine genotype was dominance species in all Cryptosporidium. spp.更多还原