摘要
目的通过形态观察、生活史循环试验和分子生物学鉴定研究泡囊狸殖吸虫的独立性问题。方法从三元区采集溪蟹分离得到泡囊狸殖吸虫囊蚴和斯氏狸殖吸虫囊蚴，进行形态观察。以泡囊狸殖吸虫虫卵投放到调查过无泡囊狸殖吸虫的疫区中感染螺蛳，继而感染溪蟹，稍后检查蟹体内所获囊蚴是否保持泡囊狸殖吸虫形态还是同斯氏狸殖吸虫囊蚴近似。提取斯氏狸殖吸虫囊蚴和泡囊狸殖吸虫囊蚴的基因组DNA，用特异引物，PCR扩增其ITS2基因并将PCR扩增产物纯化后测序，然后通过GenBank的B1ast程序分析基因序列的同源性，并应用MEGA3．0软件中的ME程序构建种系发生树。结果泡囊狸殖吸虫囊蚴与斯氏狸殖吸虫囊蚴形态差异明显，但观察到泡囊狸殖吸虫囊蚴在从蟹体分离后0．5～2h内有57．14％变成同斯氏狸殖吸虫囊蚴近似的特征。将泡囊狸殖吸虫虫卵投放到自然环境进行生活史循环试验，从收集的溪蟹中只分离到斯氏狸殖吸虫囊蚴而没有发现泡囊狸殖吸虫囊蚴。基因同源性比较结果显示：泡囊狸殖吸虫与斯氏狸殖吸虫的ITS2基因同源性为100％，碱基差异为0，没有基因差异。结论虽然泡囊狸殖吸虫囊蚴和斯氏狸殖吸虫囊蚴的形态特征有较明显的差别，但放置一段时间后，57．14％的泡囊狸殖吸虫囊蚴变成与斯氏狸殖吸虫囊蚴形态相似的特征。从用泡囊狸殖吸虫虫卵进行生活史循环试验中收集的溪蟹体内没有发现泡囊狸殖吸虫囊蚴。ITS2基因序列的同源性比较分析发现斯氏狸殖吸虫和泡囊狸殖吸虫的基因高度同源，而且种系发生树也显示二者之间无明显的遗传分化。因此，斯氏狸殖吸虫和泡囊狸殖吸虫为同一物种，泡震狸殖吸虫不具有虫种独立性。
The problem whether Pagumogonimus veocularis exhibit independence was investigated on the basis of morphological observation, life cycle circulation testing and molecular biological identification. In the present study, the metacarcariae of P. veocularis and P. skrjabini collected in San-yuan district of Fujian province were observed morphologically. Eggs of P. veocularis were put in the epidemic area where no P. veocularis infection was found in order to infect snails and later to infect crabs. Then, the metacercariae obtained from crabs were undergone examination to test whether they would keep the special shape of P. veocularis or their analogs in morphological characters. The genomic DNAs of P. skrjabini and P. veocularis metacercariae were extracted , the ITS2 gene in the DNA fragment was amplified by PCR with special primers and the amplified products were sequenced after purification. Meanwhile, the gene sequence homology was analyzed with Blast program of GenBank, and the phylogenetic tree was constructed from the results using ME program of MEGA 3.0 software. It was found that although there were obvious differences in porphological characters between metacercariae of P. skrjabini and P. veocularis, but 57.14% of P. vsocularis metace.rcariae changed into amalogous metacercarise of P. skrjabini separated from crabs 0. 5-2 hours later. When eggs of P. veocularis were put into the natural emvironments of infected area to perform the life cycle circulation experiment, only metacercariae of P. skrjabini were isolated from the collected crabs, but no metacercariae of P. veocularis was found. It was also demonstrated from the gene identity studies that the ITS2 gene homolog between P. veocularis and P. skrjabini was found to be 100% with a base variation number of 0, showing no any genetic difference in these two parasites. In addition, there was also no obvious hereditary differentiation in these parasites from the analysis of the phylogenetic tree constructed, in which the identities of ITS2 gene among P. veocularis and P. westermani, P. cenocopiosus, P. macrorchis were 90%, 93% and 92% respectively. As a whole, it is evident that P. vercularis and P. skrjabini appear to be the identical species, and P. veocularis exhibits no species independence.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2006年第8期750-754,共5页
Chinese Journal of Zoonoses
基金
国家自然科学基金资助项目(No.30160080)
关键词
泡囊狸殖吸虫
斯氏狸殖吸虫
虫种
独立性
Pagurnogonirnus skrjabini
P. veocularis
species independence
