导　　师： 宁长申;张龙现

学科专业： I0602

授予学位： 硕士

作　　者： ;

机构地区： 河南农业大学

摘　　要： 本研究对河南省鹌鹑隐孢子虫病进行了较广泛地流行病学调查,获得239个鹌鹑隐孢子虫分离株;对45个分离株进行nested PCR检测和PCR-RFLP分析;基于核糖体小亚基/(18S rRNA/)基因、肌动蛋白/(Actin/)基因的种类与基因型分析,鉴定河南省鹌鹑隐孢子虫分离株种类、基因型//基因亚型,确定河南省鹌鹑隐孢子虫分离株和其他种类隐孢子虫之间的分子种系进化关系,探讨鹌鹑隐孢子虫病的感染来源和传播机制,为预防隐孢子虫病提供理论基础。 1、为全面、系统地掌握河南省鹌鹑隐孢子虫的流行状况,从2006年9月至2007年8月,采用饱和蔗糖溶液漂浮法和改良抗酸染色法对河南省鹌鹑养殖量较大的5个地市47个鹌鹑养殖场/(户/)近20万只鹌鹑的1818份粪便样品进行检测,结果显示隐孢子虫感染率为13.15/%/(239//1818/),依据形态学检测,初步鉴定为贝氏隐孢子虫/(Cryptosporidium baileyi/)和火鸡隐孢子虫/(C. meleagridis/)。调查结果显示,河南省鹌鹑品种与隐孢子虫感染率无显著差异/(P>0.05/),但具有一定的地区、季节及年龄差异性。本调查为鹌鹑隐孢子虫病的防治及公共卫生安全提供了依据。 2、肠道寄生虫感染一直是影响养禽业发展的重要因素。为查明鹌鹑寄生虫感染情况,对鹌鹑隐孢子虫进行流行病学调查的同时,还检测了相关肠道寄生虫的感染情况。发现鹌鹑共感染3种寄生虫,其中隐孢子虫阳性样品总数为239份,总阳性率为13.15/%/(239//1818/);球虫阳性样品总数1473份,总阳性率为81.02/%/(1475//1818/);线虫阳性样品总数217,总阳性率为11.88/%/(217//1818/)。调查结果表明,河南省鹌鹑肠道寄生虫感染存在一定的年龄、地区和季节差异。本调查为鹌鹑肠道寄生虫的防治提供了较详细的参考。 3、应用nested-PCR扩增45个分离株,用限制性内切酶SspI和VspI对PCR产物进行消化酶切确定种类和基因型。SspI酶切结果显示,45个分离株分为2种,一种酶切片段长度为573bp,267bp;另一种酶切片段长度为450bp,267bp,108bp,11bp,11bp。VspI酶切结果与SspI酶切结果对应,酶切片段长度同样分为2种,一种酶切片度长度为621bp,115bp,104bp;另一种酶切片段为457bp,171bp,115bp,104bp。据酶切片段长度初步鉴定出两种隐孢子虫,即C. baileyi和C. meleagridis,其中42个样品为C. baileyi,2个样品为C. meleagridis,41号样品为两种隐孢子虫混合感染。 4、基于小亚基核糖体/(18S rRNA/)基因,对42株鹌鹑隐孢子虫进行nested-PCR扩增、测序,产物序列长度均为820bp左右。应用Clustal X 1.81、phylip 3.67软件进行种系发育分析,Treeview 1.65绘制进化树,DNAstar 4.0进行同源性分析。结果显示40个隐孢子虫分离株为C. baileyi,其中3个分离株与其他分离株有1个碱基的差异;对40个C. baileyi分离株序列进行同源性分析,其中37个分离株与GenBank上下载的C. baileyi同源性达到100/%,3个分离株与GenBank上下载的C. baileyi同源性达到99.9/%;其他2个分离株为C. meleagridis,其序列与GenBank上下载的C. meleagrids的同源性达到99.5/%。 5、对初步确定的2个C. melesgridis分离株进行基于18S rRNA基因和Actin基因的nested-PCR分析,利用Clustal X 1.81、phylip 3.67等生物学软件进行分析。结果显示在这两个基因位点上,2个鹌鹑隐孢子虫分离株与C. meleagrids的遗传进化关系最近。在18S rRNA基因位点和Actin基因位点上,利用NJ法绘制的进化树节点支持值分别为98、100,利用MP法绘制的进化树节点支持值为85和100。利用DNAstar 4.0进行同源性分析,在这两个基因位点上,同源性分别达到99.3/%、99.9/%。 基于PCR-RFLP分析和基因型//基因亚型的种系发育分析,鉴定出本研究获得的鹌鹑源隐孢子虫分离株分别为C. baileyi和C. meleagridis,推断鹌鹑是人隐孢子虫病的重要感染来源,为今后预防隐孢子虫病提供了理论基础。 The epidemiological investigation of quail cryptosporidiosis was carried out in Henan province. The 239 Cryptosporidium isolates derived from quail.Selecting 45 isolates in the total were amplified by nested-PCRRFLP detection and analysed; genotypic//genotype analysis of the 18S rRNA gene and Actin gene, determing molecular phylogenetic relationship between isolates drived from quail of this province and other species, concluding quail infection source and transmission mechanism in order to provide theoretical basis for prevention of cryptosporidiosis. To study the epidemiology of Cryptosporidium in quail of Henan Province, a total of 1818 fecal samples were collected from 47 quail farms which breed about 200,000 quails in 5 areas, with the Sheather’s sugar flotation and modified acid fast stain techniques. The infection rate of Cryptosporidium in quail is 13.15/%/(239//1818/). By morphology determined, two Cryptosporidium species were found, which are C. baileyi and C. meleagridis. The data shown that the infection rate of Cryptosporidium has no relative with quail’species, however, which is correlation with territory, season and age. It provids the detail information to prevention the cryptosporidiosis of quail and public health security. Intestinal parasitism is one of the important factors restricting the development of poultry industry, so we did not only research the cryptosporidium of quail, but also detect the other intestinal parasites. In result , Cryptosporidium, Coccidium , Nematod were detected from the samples of quail, the infection rate are 13.15/%/(239//1818/), 81.02/%/(1475//1818/), 11.88/%/(217//1818/), respectively. The data shown that the Intestinal parasites has relative with territory, season and quail’age.This investigation has provid the detail information to prevention and cure the intestinal parasitosis of quail. The18S rRNA specific fragments of 45 isolates were amplified by nested PCR. The PCR products were digested by SspI restriction enzyme and VspI restriction enzyme to determine species and genotype. Fragments of 573,267bp and 621,115,104bp were got after digested with SspI and fragments of 621,115,104bp and 457,171,115,104bp were got after digested with VspI. Based on length of restriction fragments, the 45 isolates were initially considered to be C. baileyi and C. meleagridis, in which the sample 41was infected both Cryptosporidium spp. In order to determine species// genotype of cryptosporidium isolates from quail of Henan province, specific fragments of 18S rRNA gene, were amplified by nested PCR and sequenced. Then, Blast or Fasta methods was used to search homological sequences in NCBI, DDBJ and EMBL, after that, homological sequences were alignmented. Phylogenetic tree and homological analysis were made by some biological softwares such as Clustal X 1.81, and DNAstar 4.0. Based on the phylogenetic analysis of 18S rRNA gene the 40 isolates were identified as C.baileyi,in which 3 isolates has one base difference from the other. The 2 isolates were identified as C.meleagridis . To the further reseach the C.meleagridis from quail, 18SrRNA gene and Actin gene specific fragment of the 2 isolates were amplified by nested PCR, and sequenced, then, Blast or Fasta was used to search homological sequences in NCBI, DDBJ and EMBL. After that, homological sequences were alignmented. Phylogenetic tree and homological analysis were made by some biological softwares such as Clustal X 1.81, and DNAstar4.0. Based on the phylogenetic analysis, the two were identified as C.meleagridis. Based on PCR-RFLP, genotypic and subgenotypic analysis,the species//genotype of isolates in this experiment were reached consensus and identified as C.baileyi and C.meleagridis. it can be concluded that quail cryptosporidium is one important source to the human cryptosporidiosis .

关 键 词： 分子生物学特性 隐孢子虫 种类 鹌鹑

分 类 号： [S8]

领　　域： [农业科学]