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

doi:10.12140/j.issn.1000-7423.2024.04.008

Abstract

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

CLC Number:

R532.23

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.

Figures/Tables 4

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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）