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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Blast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0.5~2 h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,泡囊狸殖吸虫不具有虫种独立性。
OBJECTIVE: To study the independence of Paragonimus ovipositor through morphological observation, life cycle test and molecular biology. Methods The metacercariae and metacercariae of Enterobius sibirica were isolated from crabs collected from the ternary area for morphological observation. In order to infect the crabs in the infected areas of the infected blisters, the crabs were infected with crabs and crabs were examined later. Toxoplasma gondii metacercaria approximate. Genomic DNA was extracted from metacercaris of T. smegmatis and metacercariae of Paragonimus japonicum, and ITS2 gene was amplified by PCR using specific primers. The PCR products were purified and sequenced. The sequence of the gene was analyzed by GenBank Blast program Homology, and application of ME program MEGA3.0 software to build a phylogenetic tree. Results The morphological differences of metacercariae between Paecilomyces brucei and Paenibacillus serrata were significant, but it was observed that 57.14% of the metacercariae of Paecilomyces butei Approximate characteristics of the metazoan metacercariae. Cyclocarya gonorrhoeae eggs were put into the natural environment to carry on the life history cycle test, from the collected crabs only isolated from the genus Sri Lankan with genus Rectus serrata did not find the genus Bubra genus metacercaria. The results of gene homology showed that the ITS2 gene homology of B. gonorrhoeae and T. gondii was 100%, base difference was 0, and there was no genetic difference. Conclusions Although the morphological characteristics of metacercariae of the genus Bupleurum and that of the genus Schistisioi have significant differences, after a certain period of time, 57.14% Cysticercosis similar to the characteristics. From the cycle of living history experiments using the cycle of buccal gonococcal eggs collected crab body did not find Cysticercus metazoan metacercaria. ITS2 gene sequence homology comparative analysis found that the genera of the genus Schizochyma oviparous and the Bovine Ovipariasis gene highly homologous, and the phylogenetic tree also showed no significant genetic differentiation between the two. Therefore, the genus Clostridium spp. And the same species of the genus Bubra are not identical, and the genus Bubra is not insectivorous.