【作者】 卢庆斌；

【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】 本试验对河南部分地区水牛、肉牛和奶牛隐孢子虫病进行了广泛的流行病学调查,对部分牛源隐孢子虫分离株进行卵囊形态鉴定,巢式PCR检测和PCR-RFLP分析,基于核糖体小亚基(18S rRNA)基因的种类与基因型分析,鉴定出河南部分地区牛源隐孢子虫分离株种类、基因型,确定本地区牛源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,为预防隐孢子虫病提供理论依据。为掌握河南信阳地区水牛隐孢子虫的流行动态,我们在鸡公山地区采集散养水牛粪便样品257份,经饱和蔗糖溶液和改良抗酸染色法检测,结果显示隐孢子虫总感染率为16.73%(43/257)。依据形态学数值初步鉴定为出2种隐孢子虫即微小隐孢子虫(C.parvum)和安氏隐孢子虫(C.andersoni)。其中微小隐孢子虫为人兽共患隐孢子虫种类,其感染率为0.78%(2/257),2个C.parvum分离株均在1岁多的水牛粪便样品发现。由于本次调查的水牛常在河边放养,这对当地的水源安全构成了威胁。利用饱和蔗糖溶液漂浮法和改良抗酸染色法对4个肉牛场208份河南南乐县肉牛粪便进行隐孢子虫病流行病学调查,结果发现隐孢子虫总感染率为4.32%(9/208)。根据显微镜观察,初步鉴定感染肉牛的隐孢子虫只有1种:安氏隐孢子虫。为了详细了解肉牛源隐孢子虫的遗传特征多样化情况,利用巢式PCR扩增9个肉牛源隐孢子虫分离株18S rRNA基因部分序列,进行PCR-RFLP分析和基于18S rRNA基因种系发育分析,结果表明本次分离于肉牛的9个隐孢子虫分离株均为安氏隐孢子虫(C.andersoni)。在本课题组对奶牛隐孢子虫研究的基础上,针对奶牛养殖集中的郑州、开封、济源和鹤壁4个地区9个奶牛场采集12月龄以内的奶牛粪便样品582份,经饱和蔗糖溶液漂浮法和改良抗酸染色法检测,结果显示隐孢子虫总感染率为26.12%(152/582)。其中,断奶前犊牛(5日龄至2月龄)隐孢子虫感染率为30.91%(51/165),断奶后(3月龄至12月龄)感染率为24.22%(101/417)。依据形态数值初步鉴定为2种隐孢子虫即微小隐孢子虫和安氏隐孢子虫。其中微小隐孢子虫为人兽共患隐孢子虫种类,在断奶前奶牛中的感染率为50.98%(26/51),在断奶后奶牛中为9.90%(10/101)。本次调查发现微小隐孢子虫具有较高的感染率,确定了奶牛隐孢子虫的重要公共卫生意义;同时还发现断奶前犊牛不同饲养方式对2种隐孢子虫感染率有显著影响。本试验利用巢式PCR扩增19个隐孢子虫分离株18S rRNA基因特异片段,PCR产物进行测序,产物序列长度均为837bp;用限制性内切酶Ssp I和Vsp I对PCR产物进行消化酶切确定种类和基因型。Ssp I酶切结果:2个隐孢子虫分离株酶切后产生110bp、265bp、420-430bp三个片段,17个隐孢子虫分离株酶切产生380-400bp、440-460bp ;VspI酶切结果:2个隐孢子虫分离株酶切后产生100-110bp、600-630bp两个片段,2个隐孢子虫分离株酶切后产生110-180bp、440-470bp两个片段,15个隐孢子虫分离株酶切后产生110-120bp、720-740bp两个片段;根据酶切片段初步确定这2个隐孢子虫分离株为牛隐孢子虫(C.bovis),2个隐孢子虫分离株为隐孢子虫鹿基因型(Cryptosporidium cervine genotype),15个隐孢子虫分离株为安氏隐孢子虫(C.andersoni)。基于种系发育关系分析确定河南部分地区奶牛源隐孢子分离株的种类或基因型,利用巢式PCR对19个隐孢子虫分离株进行18S rRNA基因特定片段扩增,并对扩增片段进行测序,测序后的序列用Blast或Fasta在NCBI、EMBL和DDBJ三大核酸序列数据库搜索同源序列,然后利用Clustal X1.81、Phylip3.65和DNAstar4.0等生物学软件对序列进行比对、构建分子进化树以及同源性分析。根据在18S rRNA基因位点种系进化关系分析,表明2个奶牛源隐孢子虫分离株为牛隐孢子虫(C.bovis),2个隐孢子虫分离株为隐孢子虫鹿基因型(Cryptosporidium cervine genotype),15个隐孢子虫分离株为安氏隐孢子虫(C.andersoni)。本次从奶牛体内分离出2个隐孢子虫鹿基因型分离株尚属首次,而隐孢子虫鹿基因型能够感染包括人在内的多种哺乳类动物,这为进一步研究隐孢子虫鹿基因型的生物学特性等提供了重要的参考资料。更多还原

【Abstract】 The epidemiological survey of water buffaloes ,beef catlle and dairy cattle Cryptosporidiosis were carried out in Henan Province. Some Cryptosporidium isolates derived from water buffaloes ,beef catlle and dairy cattle were identified by microscope, nested PCR-RFLP detection and nested PCR-RFLP analysis ;genotypic analysis of the Small-Subunit rRNA (SSU rRNA) gene were carried out to indentify species/ genotype, determine molecular phylogenetic relationship between zoonotic cryptosporidium species/ genotypes, and to deduce animals infection source and transmission route.To investigate the prevalence of Cryptosporidium in water buffaloes, 257 fecal samples ,which were collected from water buffaloes in Jigong Mountain of Henan Province ,were examined by saturated sucrose solution flotation technique and the modified acid-fast staining method. The results showed that the total infection rate was 16.73%. Cryptosporidium parvum and C.andersoni were discovered by microsc- ope. C. parvum is the only zoonotic species/genotype,and the infection of C. parvum is 0.78%.Two C.bovis isolates were founded in water buffaloes more than 12 moths. The results of the present study confirm that these water buffaloes should be consider- ed as a potential zoonotic reservoir for drinking water source.In order to understand the infected status of Cryptosporidium and its epidemiological characteristics in beef catlle of Henan Provice, samples of fresh stool obtained from beef catlle were detected for oocysts of Cryptosporidium by the saturated sucrose solution and the modified acid-fast staining method. The results revealed that the total infection rate of Cryptosporidium was 4.32%(9/208). C.andersoni were only founded in beef catlle by microscope. To elucidate the genetic diversity, the partial sequences of the cryptosporidium 18S ribosomal RNA gene were targeted by nested PCR. Nine isolates from beef catlle were identificated species/ genotype/ subgenotype by nested PCR-RFLP analysis and genotypic analysis of the Small-Subunit rRNA (SSU rRNA) gene .The results show that nine isolates from beef catlle were C.andersoni .To further understand the prevalence of cryptosporidiosis among dairy frams in Henan Province,a total of 582 fecal samples were collected from less than 12-month-old calves in nine dairy farms from Zhengzhou, Kaifeng, Jiyuan and Hebi districts, the results showed the total infection rate was 26.12(152/582) by the Sheather’s sucrose flotation technique and the acid-fast staining method. Thereinto, the infection rate of pre-weaned(5 days to 2 months) calves was 30.91%(51/165), and 24.22%(101/417) in post-weaned (3–12 months)calves respectively. Two species of Cryptosporidium were discovered, namely, Cryptosporidium parvum and C.anderson.. C.parvum, the only zoonotic species/genotype, constituted 50.98% of the parasite infections in pre-weaned calves ,but only 9.90%of the Cryptosporidium infections in post-weaned calves. The result of present study showed that C.parvum was of high infection rate in Henan, to verify Cryptosporidium in dairy is of important role in public health, and suggested that the type of confinement for pre-weaned dairy calves had significant influence to infection rate of two species of Cryptosporidium.The Small-Subunit rRNA(SSU rRNA) gene specific fragments of isolates from were amplifie dairy catlle by nested PCR. The PCR products were sequenced and digested by Ssp I restriction enzyme and Vsp I restriction enzyme respevtively to determine species and genotype. The amplified fragments length of two isolates were 837bp. Two of neneteen isolates’fragments of 110bp ,265bp and 420-430bp were got after digested with Ssp I ,the others fragments of 380-400 bp and 440-460 bp were got after digested with Ssp I . Two of neneteen isolates’fragments of 100-110bp and 600-630 bp were got after digested with Vsp I , two isolates’fragments of 110-180bp ,440-470 bp were got fter digested with Vsp I and the others fragments of 110-120bp、720-740bp were got after digested with Vsp I .Based on length of restriction fragments, two isolates were considered to be C.bovis , two isolates were considered to be named Cryptosporidium cervine genotype and the others were considered C.andersoni.In order to determine species/ genotype of cryptosporidium isolates from dairy catlle of Henan Province, specific fragments of 18S rRNA gene was amplified by nested PCR, and sequenced. Then, Both Blast and 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, two isolates were idenfied as C.bovis , two isolates were idenfied as Cryptosporidium cervine genotype and the others were idenfied as C.andersoni.This is the first identification of a genotype of Cryptosporidium cervine genotype oocyst in the feces of dairy cattle . Cryptosporidium cervine genotype can infecton mammalian including human .The results of this experiment lend an important data for further studying the biological characteristics of Cryptosporidium cervine genotype.更多还原