一种新型无钩豆状囊尾蚴的鉴定和18S DNA序列分析

Identification and 18S DNA Sequence Analysis of a New Type No-hook Cysticercus pisiformis

旨在鉴别无钩豆状囊尾蚴与有钩豆状囊尾蚴的区别。用扫描电镜对自然感染的豆状囊尾蚴头节进行超微结构观察,对其18S DNA进行PCR扩增和序列分析,并与其他动物的囊尾蚴作了进化树比较。结果显示:超微结构观察无钩和有钩豆状囊尾蚴的主要区别位于顶突,而吸盘和原始体节的结构是相同的。从囊泡中取出的豆状囊尾蚴多处于相对静止状态。无钩豆状囊尾蚴的顶突如同数层圆饼叠放在一起,表面比较平坦,中央有一个较大的结节,结节的直径约为20μm,周边有数层齿轮状花纹,形成重合式齿轮状构造。有钩豆状囊尾蚴顶突的前端比较钝,通过皮肌柱与形似鸡爪样的小钩相连。小钩表面光滑,富有光泽,具有坚硬的外观,长120~150μm。通过用18S-P1和18S-P2引物对无钩和有钩豆状囊尾蚴的目的基因、感受态细胞的培养液和菌体质粒的PCR检测,均获得相同的目的基因条带。测序结果表明18S-P1引物扩增的无钩豆状囊尾蚴与有钩豆状囊尾蚴750 bp序列的差别比18S-P2引物扩增的666 bp序列的区别明显。与其他动物囊尾蚴相关序列的进化比较显示,无钩豆状囊尾蚴与牛囊尾蚴有亲缘性,而有钩豆状囊尾蚴则与猪囊尾蚴有亲缘关系。总之,无钩和有钩豆状囊尾蚴虽然同属豆状囊尾蚴,但无钩豆状囊尾蚴是一种新发现的豆状囊尾蚴。

This study was designed to identify the difference between no-hook Cysticercus pisiformis(C. pisiformis) and hook C. pisiformis. Scolex’s ultrastructures of C. pisiformis were observed by scanning electron microscope;PCR amplification and sequences analysis of 18 S DNA of C. pisiformis were conducted and evolutionary tree comparison was performed among C. pisiformis and other animals’ cysticercus in this study. The results showed that different ultrastructures of no-hook and hook C. pisiformis were mainly observed on rostellum, but the ultrastructures of suck and somite were alike. Most of the C. pisiformis taken out from the vesicles were in relative rest state. The rostellum of no-hook C. pisiformis looked like several oblate pastries stacked up together. Its surface was flatter and had a bigger nodule in the center, in which the diameter was about 20 μm. There were several layers of gear like patterns around the rostellum,forming an overlapping gear like structure. The front end of rostellum of hook C. pisiformis was blunt, and was connected to the chicken claw like hamula through the tegument muscular columns. The hamula were about 120-150 μm long, and its surface was smooth, rich luster and possessed hard appearance. Using 18 S-P1 and 18 S-P2 primers, the same target gene bands were obtained by PCR in the target genes of no-hook and hook C. pisiformis, culture medium of competent cells and bacterial plasmid. The sequencing results showed that the difference of the 750 bp sequences amplified by 18 S-P1 primers was more significant than that of the 666 bp sequences amplified by 18 S-P2 primers. Compared with evolution of Cysticercus of other animals, the no-hook C. pisiformis was related to C. saginate, while the hook C. pisiformis was related to C. solium. In conclusion, although both no-hook and hook C. pisiformis belong to the same genus, no-hook C. pisiformis is a new C. pisifomis being discovered.