【作者】 戴荣四；

【导师】 薛立群； 刘毅；

【作者基本信息】 湖南农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 能寄生于犬的蠕虫约有120余种,其中人畜共患的约50余种。这些寄生虫不仅常引起犬腹泻、消瘦、贫血,尤其是幼犬,不仅使其生长发育受到影响,导致对其他疾病的抵抗力减弱,从而感染其它疾病,而且严重危害人类的健康。本研究的第一部分首次对湖南省来自9个地区的438只犬蠕虫感染情况进行了调查,结果显示绦虫感染率为42.68%,蛔虫感染率为45.43%,吸虫感染率为21.92%,犬钩虫感染率为7.3%。绦虫、蛔虫复合感染率为23.06%,绦虫、吸虫复合感染率19.18%,蛔虫与吸虫的混合感染率为21.92%。同时发现湖南犬绦虫的分布规律为犬复孔绦虫以岳阳、怀化和张家界感染率最高,达60%～100%;阔节裂头绦虫主要分布在湖南的湘西,且感染强度不大;曼氏迭宫绦虫以山区的张家界及湘西最高,感染率达60%;水泡带绦虫在山区的感染情况比较普遍,以张家界地区最多,感染率达30%;多头带绦虫和带形带绦虫主要分布在山区的怀化和湘西,感染率达20%～25%。豆状带绦虫整体感染率不高,仅3.42%。其它犬蠕虫在湖南的分布规律为钩虫在郴州的感染率最高,达29.03%,蛔虫的感染很普遍,主要是犬弓首蛔虫和狮弓蛔虫,在益阳感染率最高,分别达58%和41%。吸虫的感染主要为华枝睾吸虫,各地区均有分布,以郴州感染率最高,达51.61%。为更好地防治犬蠕虫病提供了重要的基础材料。本研究的第二部分对犬绦虫核糖体DNA（rDNA）内转录间隔区（ITS）序列进行了克隆及序列分析。首次获得了犬曼氏迭宫绦虫、水泡绦虫、多头绦虫的rDNA的ITS序列,其ITS序列总长为1200bp～1300bp（ITS-1为720bp,ITS-2为572bp）,G+C含量为53.99%～59.33%%。序列比对结果显示7个代表性犬绦虫样品（DM4XX,DM6YY,DM15ZJ,MM11JT,TH5XX,TH28CS,TPW2XX）的ITS-1、ITS-2序列存在较大差异,可分为四个种群,且种间相似性最高仅达41.6%和7.4%。即使是不同地方的同种也存在差异,种内相似性最高为98.3%和97.4%。由此可见,种间差异明显,种内也有一定差异。证明ITS序列可作为绦虫的遗传标记,填补了我国部分省份地区犬绦虫ITS序列的遗传变异情况研究的空白。本研究的第三部分对犬绦虫线粒体nad4基因进行了研究。根据绦虫的部分序列设计的一对引物,经过PCR扩增犬绦虫线粒体nad4基因序列,克隆及测序。获得6个代表性样品（MM11JT,TH29CS,TPW32CZ,TPW33CZ,TH45XX,TPW45）的核苷酸序列,序列长度均为523bp,经核苷酸序列互对比较,结果显示它们存在三个种群,且其种间差异为2.1%～30.1%,比种内变异（2.2%）大。种内变异均小于其与其它种种间的差异,能为种间鉴别提供遗传标记。本研究的第四部分对犬绦虫线粒体cox1基因进行了研究。根据绦虫的部分序列设计的一对引物,经过PCR扩增犬绦虫线粒体cox1基因序列,克隆及测序。获得8个代表性样品（TPW2XX,DM4XX,TH5XX,DM6YY,MM11JT,DM15ZJ,TH28XX,DM31CZ）的核苷酸序列,序列长度均为341bp,经核苷酸序列互对比较,结果显示它们存在四个种群,且四个种间cox1序列存在比较大的差异,为12.9%～30.3%,为7～85个碱基差异。种内差异曼氏迭宫绦虫碱基差异为0.0%～1.8%,其它种种内也仅存在1～4个碱基差异。由此说明,cox1基因序列为一段非常保守的基因序列,说明cox1基因非常适合作为绦虫分类的遗传标记。本试验首次对绦虫nad4和cox1基因的遗传变异情况进行研究,从线粒体基因的角度,开展绦虫线粒体遗传变异和DNA多态性方面的研究,找到了研究绦虫分子分类学、种群遗传关系有效的遗传标记,填补了我国寄生绦虫线粒体全基因组相关研究的空白。本研究的第五部分在国内首次建立了犬绦虫特异PCR检测方法,确定了对犬绦虫进行PCR检测时的最佳退火温度为65℃,最佳MgCl₂浓度为3.0mM,此时,检测条带亮且没有非特异性条带出现。并且当样品DNA浓度被稀释640倍时,仍可出现清晰的条带。为该病的诊断和流行病学调查等提供了分子生物学方法,研究结果也为对绦虫进一步的深入研究奠定了基础。更多还原

【Abstract】 The species of helminths were about 120 in dogs,while about 50 in human and animals.These parasites caused dogs diarrhea,weight loss,anemia.Especially,it not only affected the growth and development of puppies,leaded to reduce disease resistance,infected by other diseases,but also badly harmed human health.In partⅠof the study,The prevalence of helminths in adult dogs was firstly investigated in Hunan Province.A total of 438 adult farm dogs slaughtered in local abattoirs from 9 representative administrative regions in Hunan Province were examined for the presence of helminths using a helminthological approach.All collected worms were counted and identified to species according to existing keys and descriptions.It resulted that infection rate was cestode 42.68%,toxocara 45.43%, trematode 21.92%,ancylostoma 7.3%,respectively.Mixed infection rate was cestode and toxocara 23.06%,cestode and trematode 19.18%,trematode and toxocara 21.92 %.At the same time,the distribution law of cestodes of the Hunan dog was discovered. Dipylidium caninum of dog was mainly distributed in Yueyang,Zhangjiajie and Huaihua.The infection rate was 60-100%.Diphyllobothrium latum of dog was mainly distributed in Xiangxi and it’s infection intensity was a little.The highest infection rate of Spirometra mansoni was 60%,mainly distributed in Zhangjiajie and Xiangxi of mountain area.The infection of Taenia hydatigena was common in mountain area,especially in Zhangjiajie,and the infection rate was 30%.Taenia multiceps and Taenia taeniaformis were mainly distributed in Xiangxi and Huaihua of mountain area and the infection rate was 20%～25%.Overall infection rate of Taenia pisiformis was not high,only 3.42%.The distribution law of other dog’ s helminth in Hunan was that the highest infection rate of ancylostoma was in Chen Zhou,amounts to 29.03%;the toxocara was distributed widely,mainly including Toxocara canis and Toxascaris leonine,which the highest infection rates were in Yiyang,and were up 58%and 41%respectively;the trematode was distributed widely,mainly being Clonorchis sinensis,which the highest infection rates were in Chenzhou,51.61%.It provided important data in order to control worm disease of dog.In partⅡof the study,the internal transcribed spacer（ITS） sequences of ribosomal DNA（rDNA） of dog cestodes were cloned and sequenced.The ITS sequences of Spirometra mansoni,Taenia hydatigena and Taenia multiceps were obtained firstly.There were ITS sequences of seven cestodes samples.The total length of those ITS sequence was 1200bp～1300bp（ITS-1 720bp,ITS-2 572bp）.The content of G + C was 59.33%～53.99%.The seven ITS sequences（DM4XX,DM6YY, DM15ZJ,MM11JT,TH5XX,TH28CS,TPW2XX） were compared.It showed that ITS-1 and ITS-2 sequences of those samples were basically inconsistency.They separated into four population.The similarity of four ITS-1 and ITS-2 sequences were only 41.6%and 7.4%.Even if they were same population from different area,there were differences.The most inter-species similarity was 98.3%and 97.4%.It showed that there were apparent differences between population and there were certain inter-species differences.So ITS sequence could be used for genetic marker of cestodes,which have filled in the research gaps of ITS sequence of dog cestodes.In partⅢof the study,mitochondria DNA（mtDNA） nad4 of the dog cestodes have been studied.The mtDNA nad4 were cloned and sequenced.The nad4 sequences of six cestodes samples（MM11JT,TH29CS,TPW32CZ,TPW33CZ, TH45XX,TPW45） were obtained and their sequence length was 523bp.The six nad4 sequences comparison revealed that they separated into three population.The difference among three population were 2.1%～30.1%,more than the inter-species differences（2.2%）.So nad4 could be used as genetic marker for population genetic studies of cestodes.In partⅣof the study,mitochondria DNA（mtDNA） coxl of the dog cestodes have been studied.The mtDNA cox1 were cloned and sequenced.The cox1nad4 sequences of eight cestodes samples（TPW2XX,DM4XX,TH5XX,DM6YY, MM11JT,DM15ZJ,TH28XX,DM31CZ） were obtained and their sequence length was 341bp.The eight cox1 sequences comparison revealed that they separated into four population.The difference among four population were bigger,12.9%～30.3%, 7～85bp.The inter-species differences of Spirometra mansoni were 0.0%～1.8%,and that the inter-species differences of other population were 1～4bp.It showed that cox1 gene sequence were a section of very conservative gene sequence.So it was very suitable that cox1 was used for genetic marker of cestodes classification.In this experiment,nad4 and cox1 genetic variation of cestodes were firstly studied.Genetic variation and DNA polymorphism of mitochondria were carried out from mitochondrial gene.So the effective genetic markers were found which studied molecular taxonomy and Phylogenetic relationship of cestodes.This study have filled in the blank space of research that was related to China’s cestodes mitochondrial genome research.In partⅤof the study,the specific PCR detection method of dog cestodes was firstly established in China.When done specific PCR detection of dog cestodes,the best annealing temperature was 65℃and the best MgCl₂ concentration was 3.0 mM. When the DNA samples were diluted 640 times,strip still clearly appeared.This was the molecular biology methods of cestodes diagnosis and epidemiological investigation.The researching results has founded the basis of cestodes for further research.更多还原