我国多个地区麦穗鱼华支睾吸虫囊蚴感染情况及其鉴定分析

doi:10.12140/j.issn.1000-7423.2024.04.008

中国寄生虫学与寄生虫病杂志 ›› 2024, Vol. 42 ›› Issue (4): 475-480.doi: 10.12140/j.issn.1000-7423.2024.04.008

我国多个地区麦穗鱼华支睾吸虫囊蚴感染情况及其鉴定分析

王居锋(), 薛诗杰, 刘江永, 赖德华^*(), 伦照荣

中山大学生命科学学院，水产动物疫病防控与健康养殖全国重点实验室，广东省水生经济动物良种繁育重点实验室，广东广州 510275

收稿日期:2024-03-29 修回日期:2024-05-17 出版日期:2024-08-30 发布日期:2024-08-20
通讯作者: *赖德华（1981—），男，博士，副教授，从事寄生虫学研究。E-mail：laidehua@mail.sysu.edu.cn
作者简介:王居锋（1994—），男，博士研究生，从事寄生虫学研究。E-mail：wangjf9@mail2.sysu.edu.cn
基金资助:
广州市基础与基础应用研究项目(202201011831);国家自然科学基金(32170470)

Infection and identification of Clonorchis sinensis metacercariae in Pseudorasbora parva in multiple regions of China

WANG Jufeng(), XUE Shijie, LIU Jiangyong, LAI Dehua^*(), LUN Zhaorong

State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, Guangdong, China

Received:2024-03-29 Revised:2024-05-17 Online:2024-08-30 Published:2024-08-20
Contact: *E-mail: laidehua@mail.sysu.edu.cn
Supported by:
Science and Technology Projects in Guangzhou(202201011831);National Natural Science Foundation of China(32170470)

摘要/Abstract

摘要：

目的了解我国多个地区麦穗鱼体内华支睾吸虫囊蚴的感染情况及其形态学和分子生物学鉴定。方法 2022年8月至2024年1月，在华东、华南、华中、西北和西南地区水域的12个采样点采集麦穗鱼。消化鱼肉后，分离吸虫囊蚴进行形态学鉴定。每个采样地点选取部分阳性麦穗鱼中的囊蚴，提取单个囊蚴DNA，PCR扩增16S rDNA并测序，采用BLAST进行序列比对，用MEGA7和Fasttree2以最大似然法构建系统进化树。取镜检吸虫囊蚴阳性的麦穗鱼饲喂12只SD大鼠（约200个囊蚴/鼠）和10只昆明小鼠（约100个囊蚴/鼠）。感染后25、90、300 d，分别收集大鼠和小鼠粪样中的虫卵和肝胆管中的成虫，并进行鉴定形态。采用Microsoft Excel 2021软件对数据进行统计学分析，组间比较采用Fisher精确检验。结果共采集麦穗鱼665尾，检出吸虫囊蚴感染277尾，感染率为41.6%。麦穗鱼囊蚴感染率较高的地区分别为广西横州（6/6）、河南濮阳（100%，22/22）、山东烟台（98.4%，122/124）、安徽阜阳（75.0%，24/32）、陕西汉中（49.2%，95/193）、福建宁德（5/19）。分离的吸虫囊蚴经形态学鉴定为华支睾吸虫囊蚴。PCR扩增和测序结果显示，扩增出的约400 bp大小条带为华支睾吸虫16S rDNA。序列比对结果显示，与华支睾吸虫（GenBank登录号：MT607652.1）16S rDNA序列一致性为100%。系统进化树结果显示，获得的囊蚴与华支睾吸虫聚为一支。感染后25、90 d，小鼠粪样中未检出虫卵，胆管和胆囊中未检出成虫；大鼠粪样中检出虫卵。感染后300 d，2只大鼠粪样检出虫卵，3只大鼠肝胆管中分离到成虫。虫卵和成虫均符合华支睾吸虫形态学特征。结论广西横州、河南濮阳、山东烟台、安徽阜阳、陕西汉中和福建宁德地区的麦穗鱼存在不同程度的华支睾吸虫感染。16S rDNA基因结合形态学特征可以鉴定麦穗鱼中的华支睾吸虫囊蚴。

关键词: 华支睾吸虫, 麦穗鱼, 吸虫, 囊蚴, 16S rDNA

Abstract:

Objective To understand the infection, morphological and molecular identification of Clonorchis sinensis metacercariae in Pseudorasbora parva in multiple regions of China. Methods From August 2022 to January 2024, P. parva samples were collected from 12 sampling sites of water bodies in East, South, Central, Northwest, and Southwest China. After digesting the fish, metacercariae were isolated for morphological identification. From each sampling site, individual metacercariae in infected fishes were isolated individually to extract DNA， of which 16S rDNA was amplified with PCR and sequenced. Sequence alignment was conducted using BLAST, and a phylogenetic tree was constructed using MEGA7 and Fasttree2 with the maximum likelihood method. SD rats (n = 12) and Kunming mice (n = 10) were fed with metacercariae-positive P. parva (approximately 200 metacercariae per rat and 100 metacercariae per mouse). Eggs and adult worms from fecal samples and the bile ducts were collected, respectively, for morphological and identification at 25, 90 and 300 days post-infection. Data was analyzed by Microsoft Excel 2021 software. Fisher’s exact test was used for comparison between groups. Results Out of the 665 P. parva samples examined, 277 were positive for metacercariae, resulting in an average infection rate of 41.6%. Higher infection rates of P. parva were found in Hengzhou of Guangxi (6/6), Puyang of Henan (100%, 22/22), Yantai of Shandong (98.4%, 122/124), Fuyang of Anhui (75.0%, 24/32), Hanzhong of Shaanxi (49.2%, 95/193), and Ningde of Fujian (5/19). The isolated trematode metacercariae samples were morphologically identified as of C. sinensis. PCR amplification and sequencing results showed that the amplified about 400 bp fragment matched to the sequence of 16S rDNA of C. sinensis, revealing 100% identity (GenBank accession no.: MT607652.1). Phylogenetic analysis indicated that the isolated metacercariae clustered with C. sinensis in the same branch. At 25 and 90 days post-infection, no eggs or adults were found in mouse feces or bile ducts, while eggs were found in rat feces. At 300 days post-infection, eggs were found in the feces of two rats, and adults were isolated from the bile ducts of three rats. The eggs and adults exhibited typical morphological characteristics of C. sinensis. Conclusion The present study domonstrated that C. sinensis infection at varying degrees was found in P. parva from Hengzhou, Guangxi, Puyang, Henan, Yantai, Shandong, Fuyang, Anhui, Hanzhong, Shaanxi, and Ningde, Fujian. The 16S rDNA gene combined with the morphological characteristics can effectively identify C. sinensis metacercariae in P. parva.

Key words: Clonorchis sinensis, Pseudorasbora parva, Trematode, Metacercariae, 16S rDNA

中图分类号:

R532.23

王居锋, 薛诗杰, 刘江永, 赖德华, 伦照荣. 我国多个地区麦穗鱼华支睾吸虫囊蚴感染情况及其鉴定分析[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(4): 475-480.

WANG Jufeng, XUE Shijie, LIU Jiangyong, LAI Dehua, LUN Zhaorong. Infection and identification of Clonorchis sinensis metacercariae in Pseudorasbora parva in multiple regions of China[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(4): 475-480.

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采样点 Sampling site	采样时间 Sampling time	检查数量/尾 No. examined	阳性数/尾（阳性率/%） No. positive (Positive rate/%)
华东地区 East China
安徽阜阳 Fuyang, Anhui	2023.04.28	12	12（12/12）
安徽阜阳 Fuyang, Anhui	2024.01.20	20	12（60）
福建宁德 Ningde, Fujian	2023.05.13	19	5（5/19）
浙江衢州 Quzhou, Zhejiang	2023.05.13	40	0（0）
山东烟台 Yantai, Shandong	2023.04.26	51	49（96.1）
山东烟台 Yantai, Shandong	2023.09.24	23	23（100）
山东烟台 Yantai, Shandong	2023.11.27	50	50（100）
山东青岛 Qingdao, Shandong	2022.11.07	23	1（4.3）
上海 Shanghai	2023.11.20	40	0（0）
华南地区 South China
广东汕头 Shantou, Guangdong	2022.11.04	50	0（0）
广东汕头 Shantou, Guangdong	2023.05.29	32	0（0）
广西横州 Hengzhou, Guangxi	2022.08.01	6	6（6/6）
华中地区 Central China
河南濮阳 Puyang, Henan	2022.10.15	22	22（100）
湖北武汉 Wuhan, Hubei	2023.01.19	30	0（0）
湖北武汉 Wuhan, Hubei	2023.04.25	36	0（0）
西北地区 Northwest China
陕西汉中 Hanzhong, Shaanxi	2022.10.25	60	60（100）
陕西汉中 Hanzhong, Shaanxi	2023.04.28	50	33（66）
陕西汉中 Hanzhong, Shaanxi	2023.06.02	32	1（3.1）
陕西汉中 Hanzhong, Shaanxi	2023.09.18	35	1（2.9）
陕西汉中 Hanzhong, Shaanxi	2023.11.20	16	0（0）
西南地区 Southwest China
四川成都 Chengdu, Sichuan	2023.05.13	30	2（6.7）

我国多个地区麦穗鱼华支睾吸虫囊蚴感染情况及其鉴定分析

Infection and identification of Clonorchis sinensis metacercariae in Pseudorasbora parva in multiple regions of China

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