昭通地区不同宿主体内斯氏并殖吸虫的鉴定及亲源性分析

Identification of Paragonimus skrjabini in different hosts and analysis of their phylogeny in Zhaotong in Yunnan,China

目的对云南省昭通市流行的并殖吸虫虫种进行鉴定,分析其亲源关系,为防治提供参考。方法根据临床病例感染地线索在昭通市8个县区采集溪蟹1851只,分离并殖吸虫囊蚴;用采自盐津普洱溪蟹的囊蚴感染犬,待发育后从犬肺中获取成虫。对囊蚴和成虫分别进行形态特征分析,提取其基因组DNA。PCR扩增并殖吸虫部分核糖体基因第二间隔区(ITS2)和线粒体细胞色素C氧化酶亚单位I基因(COI)并测序。将核苷酸序列输入Genbank中Blast比对,通过ITS2和COI的同源性判断并殖吸虫种类。结果并殖吸虫囊蚴在溪蟹中的分布密度差异较大呈现为非常不均一的聚集性分布,平均感染度为3.78个/只。14个ITS2基因和14个COI基因DNA序列均分别聚类在UPGMA最优树的一个分支内,且与其他参比序列分开,13个来自昭通不同县区的囊蚴和1条来自昭通盐津的成虫与斯氏并殖吸虫同源性高,综合形态学特征判定昭通地区流行的并殖吸虫虫种为斯氏并殖吸虫。结论昭通地区为斯氏并殖吸虫病自然疫源地,该虫种在形态和基因上接近四川并殖吸虫(Paragonimus szechuanensis),可为当地并殖吸虫病的防治提供参考。

Objective To analyze and study the species of Paragonimus endemic in Zhaotong,Yunnan and to discuss their phylogenetic relationships in order to provide a reference for prevention and treatment.Methods In accordance with clues regarding the site of infection from clinical cases,1,851 crabs were collected from 8 counties in Zhaotong.Adults were obtained from dogs infected with the metacercariae in Puer,Yanjin.After morphological analysis,genomic DNA was extracted from the metacercariae and adults.Internal transcribed spacer 2(ITS2)and mitochondrial cytochrome C oxidase subunit I(COI)genes of the metacercariae and the adults were amplified using PCR.Nucleotide sequences were input into GenBank for Blast alignment,and the homology of ITS2 and COI was used to determine the species of endemic Paragonimus.Results With an average infection rate of 3.78 per crab,the distribution density of metacercariae in crabs differed considerably,and metacercariae were unevenly distributed.Fourteen ITS2 gene and 14 COI gene sequences of DNA were in a branch of the optimal tree according to UPGMA clustering and separate from other reference sequences.The 13 metacercariae from different counties in Zhaotong and 1 adult from Yanjin,Zhaotong are highly similar to Paragonimus skrjabini.According to morphological and molecular features,the species of Paragonimus in the Zhaotong area is P.skrjabini.Conclusion The Zhaotong area is a natural focus of P.skrjabini,which is remarkably similar to that in Sichuan Province according to morphometric and genetic data.This finding can provide a reference for local paragonimiasis control.