Metacercaria Infection Status of Fishborne Zoonotic Trematodes,Except for Clonorchis sinensis in Fish from the Heilongjiang Province,China

Abstract

To investigate the metacercarial infections of fishborne zoonotic trematodes (FZT), a total of 6815 freshwater fish (in representing 13 species of 5 families) were collected from Songhua river (n = 2636), Nenjiang river (n = 1935), Mudanjiang river (n = 301), and other lakes or ponds (n = 1943) in 36 representative regions in Heilongjiang Province, China, from August 2012 to December 2015. Metacercariae of four FZT species, that is, Clonorchis sinensis, Metorchis orientalis, Isthmiophora hortensis, and Metagonimus yokogawai, metacercariae were detected in the examination by the artificial digestion method. As the partial data for C. sinensis were previously reported, the remaining three FZT species are to be treated in this study. The overall prevalence of M. orientalis, I. hortensis, and M. yokogawai, metacercariae was 10.54%, 0.28%, and 1.35%, respectively. Metacercariae of M. orientalis were detected in seven fish species, that is, Pseudorasbora parva, Hemiculter leucisculus, Saurogobio dabryi, Rhynchocypris lagowskii, Carassius auratus, Rhodeus ocellatus and Perccottus glehnii. Their prevalences were the highest in false dace, P. parva (26.81%), and in fish from Songhua river (17.94%). Metacercariae of I. hortensis were detected in only one fish species, Misgurnus anguillicaudatus, from Nenjiang river only. Metacercariae of M. yokogawai were detected in three fish species, that is, P. parva, H. leucisculus and S. dabryi. Their prevalences were the highest in sharpbelly, H. leucisculus (6.05%), and in fish from Mudanjiang river (5.65%). This study first demonstrated the existence of M. orientalis, I. hortensis, and M. yokogawai in freshwater fish from Heilongjiang Province, posing a major public health concern. Eight fish species, namely M. anguillicaudatus, P. parva, H. leucisculus, S. dabryi, R. lagowskii, C. auratus, R. ocellatus, and P. glehnii, cannot be eaten raw. Moreover, the findings of this study not only extended the second intermediate host range of FZT, but also improve the information of the distribution of FZT in China.

Introduction

Fishborne zoonotic trematodes (FZT), the well-known causative agents of fluke disease in humans, consist of four families, such as Opisthorchiidae, Heterophyidae, Nanophyetidae, and Echinostomatidae (Hung et al., 2013). Isthmiophora hortensis, Metagonimus yokogawai, and Metorchis orientalis are the most important FZT species, and frequently reported worldwide. I. hortensis and M. yokogawai commonly inhabit in the small intestine of mammals (Shimazu and Kino, 2015), and their metacercarial stages cannot be easily differentiated with other species in the same genera or families, respectively. M. orientalis is usually parasitic in the bile duct and gallbladder of humans and animals (Na et al., 2016).

Recently, FZT infection is of increasing concern because FZT not only widely distribute all over the world, but also threaten human health seriously. According to statistics, >18 million people are infected and for about half a billion people it is an especially worrisome problem worldwide (Hung et al., 2013). The problems of FZT infection are even worse in Asian countries.

In view of this background, attention has recently been focused on freshwater fish because of their potentially important role in the transmission of FZT to definitive hosts, including humans. Therefore, large number of investigations have been conducted on the prevalence and genotypes of FZT in freshwater fish worldwide (Chai et al., 2009; Li et al., 2013a, b; Pitaksakulrat et al., 2013; Cho et al., 2014; Eom et al., 2015). In China, information about prevalence of fishborne trematodes in freshwater fish is limited, however, these cases were only reported in markets in Hubei (Shen et al., 2015) and Guangxi (Zhang et al., 2006; Sohn et al., 2009), and in rivers in Guangdong (Li et al., 2013a), Lancang river basin (Jiang et al., 2013), and Heilongjiang (Huang et al., 2015), and most of these were published in Chinese journals. More importantly, only few investigations regarding Clonorchis sinensis metacercariae in freshwater fish have been reported in the Heilongjiang province (Liu et al., 2014; Sun et al., 2014; Zhang et al., 2014). However, few data concerning M. orientalis, I. hortensis, and M. yokogawai infection in freshwater fish were available.

In the present study, a total 6815 fish meat samples from 13 freshwater fish species from Songhua river, Nenjiang river, Mudanjiang river, and other lakes or ponds from representative cities in Heilongjiang Province were examined for the prevalence of M. orientalis, I. hortensis, and M. yokogawai for the first time. Moreover, identified metacercariae were also experimentally infected on dogs and ducks to obtain adult worms. The results would provide basedata for further prevention and control measures to avoid FZT infection in freshwater fish, other animals, and humans in these endemic areas.

Materials and Methods

Samples collection

A total of 6815 freshwater fish (1626 Pseudorasbora parva, 529 Hemiculter leucisculus, 1129 Saurogobio dabryi, 752 Rhynchocypris lagowskii, 832 Carassius auratus, 393 Rhodeus ocellatus, 156 Xenocypris davidi, 85 Cyprinus carpio, and 153 Hemibarbus maculatus; 430 Perccottus glehnii taxonomically belonging to the family Eleotridae; 476 Misgurnus anguillicaudatus; 79 Silurus asotus; and 175 Tachysurus fulvidraco) were randomly collected from 36 regions according to representative rivers and administrative regions between August 2012 and December 2015 (Table 1). All the fish are commonly in the market and are widely distributed in China. The investigated specimens included partial of samples from a previous study (Zhang et al., 2014). All the fish were collected or purchased primarily from markets, fish farms, or fish grounds. Moreover, wild fish were captured by fishermen from Mudanjiang river, Nenjiang river, Songhuajiang river, and some other lakes or ponds. All samples were then stored on ice and transported within 24 h to the Parasitology Laboratory, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University for examination. All information on the examined fish, that is, species, length, weight, fish farms or wilderness, geographical region, and sampling times, were recorded.

Table 1.

Metagonimus yokogawai, Isthmiophora hortensis, And Metorchis orientalis Metacercariae In Different Freshwater Fish Species In Different River Basins In Heilongjiang Province, China

		M. orientalis		I. hortensis		AM. yokogawai
Characteristics	Fish tested (N)	Number positive (n)	% (95% CI)	Number positive (n)	% (95% CI)	Number positive (n)	% (95% CI)
Species
Pseudorasbora parva	1626	436	26.81 (24.66–28.97)	0	0 (—)	9	0.56 (0.19–0.91)
Hemiculter leucisculus	529	47	8.89 (6.46–11.31)	0	0 (—)	32	6.05 (4.02–8.08)
Saurogobio dabryi	1129	85	7.53 (5.99–9.07)	0	0 (—)	51	4.52 (3.31–5.73)
Rhynchocypris lagowskii	752	62	8.25 (6.28–10.21)	0	0 (—)	0	0 (—)
Carassius auratus	832	45	5.41 (3.87–6.95)	0	0 (—)	0	0 (—)
Rhodeus ocellatus	393	4	1.02 (0.03–2.01)	0	0 (—)	0	0 (—)
Perccottus glehnii	430	39	9.07 (6.36–11.78)	0	0 (—)	0	0 (—)
Misgurnus anguillicaudatus	476	0	0 (—)	19	4.0 (2.23–5.75)	0	0 (—)
Xenocypris davidi	156	0	0 (—)	0	0 (—)	0	0 (—)
Cyprinus carpio	85	0	0 (—)	0	0 (—)	0	0 (—)
Silurus asotus	79	0	0 (—)	0	0 (—)	0	0 (—)
Tachysurus fulvidraco	175	0	0 (—)	0	0 (—)	0	0 (—)
Hemibarbus maculatus	153	0	0 (—)	0	0 (—)	0	0 (—)
Collection sites
Zhaoyuan	673	157		0	0 (—)	0	0 (—)
Zhaodong	1262	297		0	0 (—)	0	0 (—)
Harbin	179	9		0	0 (—)	0	0 (—)
Bayan	48	0	0 (—)	0	0 (—)	0	0 (—)
Mulan	115	0	0 (—)	0	0 (—)	0	0 (—)
Yilan	67	0	0 (—)	0	0 (—)	0	0 (—)
Jiamusi	50	0	0 (—)	0	0 (—)	0	0 (—)
Suibin	43	0	0 (—)	0	0 (—)	0	0 (—)
Fujin	208	10		0	0 (—)	50
Tongjiang	22	0	0 (—)	0	0 (—)	0	0 (—)
Nenjiang	91	2		0	0 (—)	17
Nehe	435	5		0	0 (—)	0	0 (—)
Fuyu	145	0	0 (—)	0	0 (—)	0	0 (—)
Gannan	208	48		0	0 (—)	0	0 (—)
Qiqihaer	1056	84		19		0	0 (—)
Mudanjiang	203	0	0 (—)	0	0 (—)	18
Ningan	55	0	0 (—)	0	0 (—)	0	0 (—)
Hailin	43	0	0 (—)	0	0 (—)	0	0 (—)
Yichun	96	0	0 (—)	0	0 (—)	0	0 (—)
Lindian	97	8		0	0 (—)	0	0 (—)
Dumeng	153	8		0	0 (—)	0	0 (—)
Daqing	160	4		0	0 (—)	0	0 (—)
Anda	46	0	0 (—)	0	0 (—)	0	0 (—)
Zhaozhou	115	24		0	0 (—)	0	0 (—)
Shangzhi	232	2		0	0 (—)	2
Wuchang	150	7		0	0 (—)	0	0 (—)
Shuangyashan	73	0	0 (—)	0	0 (—)	0	0 (—)
Suihua	110	0	0 (—)	0	0 (—)	0	0 (—)
Muling	107	0	0 (—)	0	0 (—)	0	0 (—)
Jixian	31	0	0 (—)	0	0 (—)	0	0 (—)
Huma	242	32		0	0 (—)	0	0 (—)
Heihe	94	0	0 (—)	0	0 (—)	0	0 (—)
Hegang	40	0	0 (—)	0	0 (—)	0	0 (—)
Jixi	59	0	0 (—)	0	0 (—)	0	0 (—)
Fuyuan	63	21		0	0 (—)	5
Qitaihe	75	0	0 (—)	0	0 (—)	0	0 (—)
Rivers
Songhua river	2636	473	17.94 (16.48–19.41)	0	0 (—)	50	1.90 (1.38–2.42)
Nenjiang river	1935	139	7.18 (6.03–8.33)	19	0.98 (0.54–1.42)	0	0 (—)
Mudanjiang river	301	0	0 (—)	0	0 (—)	17	5.65 (3.04–8.26)
Other lakes or ponds	1943	106	5.46 (4.45–6.47)	0	0 (—)	25	1.29 (0.79–1.79)
Types
Farm-raised fish	1943	106	5.46 (4.45–6.47)	0	0 (—)	25	1.29 (0.79–1.79)
Wild fish	4872	612	12.56 (11.63–13.49)	19	0.39 (0.22–0.57)	67	1.38 (1.05–1.70)
Total	6815	718	10.54 (9.81–11.26)	19	0.28 (0.15–0.40)	92	1.35 (1.08–1.62)

Examination of metacercariae

The collected fish were preserved at 4°C and were processed within 3 days. Fish fillet were used to examine the encysted metacercariae of FZT using digestion technique, and all the test operations were carried out according to previously reported procedures (Zhang et al., 2014). The sediment was carefully examined under a stereomicroscope, and the metacercariae were identified based on morphological characteristics. Moreover, the infection intensity of metacercariae in each fish was also recorded.

Molecular identification

Genomic DNA of all metacercariae samples were extracted using the Stool DNA Kit (OMEGABIOTEK, Inc.) according to the manufacturer's instructions, stored at −20°C until used. The partial sequence of the second internal transcribed spacer (ITS-2) of trematode metacercariae was amplified using polymerase chain reaction (PCR) with previously reported primers BD1 and BD2 (Zhang et al., 2014) to determine the FZT species. The PCR reaction and their cycling conditions were performed as a previous study (Zhang et al., 2014). Positive and negative controls were included in each test. Amplification products were tested by using 1% agarose gel containing ethidium bromide. The positive products were sent to Life Technology Company (Beijing, China) for sequencing.

Statistical analysis

The variation in FZT prevalence of fish in different species, and different river basins was analyzed by qui-square test using SAS 9.3 (SAS Institute, Cary, NC) (Hallewell et al., 2016).

Results

In the present study, in a total of 6815 freshwater fish, 829 (12.16%) were tested FZT positive by morphological method combined with PCR (Table 1). Metacercariae of four FZT species (C. sinensis, M. orientalis, I. hortensis, and M. yokogawai) were detected in the investigation by the artificial digestion method. As the data for C. sinensis were previously reported in Zhang et al. (2014), the remaining species are to be examined in this study. The overall prevalences of three FZT metacercariae, M. orientalis, I. hortensis, and M. yokogawai, were 10.54% (718/6815), 0.28% (19/6815), and 1.35% (92/6815) (Table 1). The present study also showed that the three pathogens have various prevalences (Table 1) and infection intensity (Supplementary Tables S1–S3; Supplementary Data are available online at www.liebertpub.com/fpd) in different fish species. Metacercariae of M. orientalis were detected in seven fish species, with the highest prevalence in P. parva (26.81%) (Table 1). Metacercariae of I. hortensis were detected in only one fish species, M. anguillicaudatus, from only Nenjiang River. Metacercariae of M. yokogawai were detected in three fish species, that is, P. parva, H. leucisculus, and S. dabryi (Table 1). Their prevalences were highest in sharpbelly, H. leucisculus (6.05%), and in fish from Mudanjiang river (5.65%) (Table 1). However, coinfection with M. orientalis and M. yokogawai was only detected in the investigated fish (0.15%) (Table 2). Interestingly, wild fish has a significant higher M. orientalis prevalence than farm-raised fish in the investigation (Table 1). The representative sequences of rDNA-ITS-2 gene were deposited in GenBank with accession numbers of KX832894 and KX857496 for M. orientalis, KX832895 and KX857497 for M. yokogawai, and KX832896 for I. hortensis. All the sequences showed >99% similarity with the corresponding reference sequences.

Table 2.

Prevalence Of Metorchis orientalis, Metagonimus yokogawai, And/Or Isthmiophora hortensis In Heilongjiang Province, China

Agent(s)	Number positive	% positive	Total positive for any listed agent	% coinfected out of fishes with any listed agent
M. orientalis	718	10.53	718	NA
M. yokogawai	92	1.35	92	NA
I. hortensis	19	0.28	19	NA
M. orientalis and M. yokogawai	10	0.15	800	1.25
M. orientalis and I. hortensis	0	0	741	0
M. yokogawai and I. hortensis	0	0	111	0
M. orientalis, M. yokogawai, and I. hortensis	0	0	833	0

NA, not applicable.

Discussion

FZT are the most common causative agents of fluke disease in humans (Hung et al., 2013). Fifty-nine species of FZT have been found all over the world (Hung et al., 2013), and only nine species, such as, C. sinensis, M. orientalis, M. yokogawai, I. hortensis, Procerovum varium, Haplorchis pumilio, Haplorchis taichui, Stellantchasmus falcatus, and Centrocestus formosanus, were found in mainland China (Sohn et al., 2009; Li et al., 2013a). However, only three species namely M. orientalis, M. yokogawai, and I. hortensis have been detected in this study, which suggests that M. orientalis, I. hortensis, M. yokogawai, together with C. sinensis previously found in Heilongjiang (Zhang et al., 2014), were endemic in freshwater fish in Heilongjiang province.

In this investigation, the overall prevalence of M. orientalis in the examined fish was 10.53% (718/6815), which was lower than that in P. parve (40%) and S. dabryi (36%) in Baicheng, China (Ye et al., 2008), but higher than that in P. parva (7.13%) and Pseudogobio rivularis (3.38%) in Anhui Province (Zhan et al., 2015), in C. auratus (10%) and in Ctenopharyngodon idellus (9.5%) in Baicheng, China (Ye et al., 2008), and in P. parva (6.7%) in Huaihe basin (Zhu et al., 2006). The different prevalences may be due to different geo-ecological conditions, age distribution of samples, management measure, sample collection time, animal husbandry practices, sample collection quantity, as well as the susceptibility of different fish species. Moreover, perhaps because wild fish have more chance (there are many stray animals, such as ducks, stray dogs and cats, wandering along the river banks, shedding feces with FZT eggs into rivers) to contact with M. orientalis compared with farm-raised fish, wild fish (12.56%, confidence interval [95% CI] 11.63–13.49) investigated in this study were more than two times (odds ratio [OR] = 2.49, 95% CI 2.01–3.08, p < 0.0001) at risk of acquiring the M. orientalis infection compared with farm-raised fish (5.46%, 95% CI 4.45–6.47), which suggests that it is essential to implement integrated strategies with efficient management measures to prevent and control stray animals in these regions of China. M. orientalis was found in all four rivers (Songhua river, Nenjiang river, Mudanjiang river, and other lakes or ponds), and M. yokogawai was detected in three rivers (Songhua river, Mudanjiang river, and other lakes or ponds); however, I. hortensis was only found in Nengjiang river. This may be related to the different pet management measures, sample collection time, as well as sample collection quantity.

Prevalences of M. yokogawai infection in fish have been recorded in different countries (Chai et al., 1991, 2015; Shimazu and Kino, 2015). The reported prevalence for M. yokogawai infection in fish was 56.7% (182/321) in Taiwan (Li et al., 2013b) and 51.48% in Japan (Kino et al., 2006). In the present study, in 6815 fish, only 92 (1.35%) fish were detected positive for M. yokogawai. Moreover, 0.28% of the investigated fish were positive for I. hortensis, which is much lower than that in mudfish (29.52%) in Harbin, China (Fan et al., 1992). Many factors may affect the detection rate, such as selection biases, diagnostic methods, sample size, and sampling time. Therefore, it is difficult to explain the reasons of actual discrepancy in different investigations.

M. orientalis, I. hortensis, and M. yokogawai are important FZT species worldwide. Of these, I. hortensis and M. yokogawai are parasitic in the small intestine of mammals (Shimazu and Kino, 2015), otherwise, M. orientalis commonly inhabits in the bile duct and gallbladder of hosts (Na et al., 2016). More importantly, all of them can cause serious economic losses and public health problems. Since these pathogens were first recorded, many animals were also identified as the hosts of these pathogens, such as humans, quails, guinea pigs, ducks, chickens, Grus japonensis, and dogs (Jiang et al., 1988; Wu et al., 1990; Yao et al., 1991; Wu and Li, 1995; Lin et al., 2001; Fan and Zhang, 2003; Chen et al., 2008; Liu and Shi, 2011). These findings suggest that these pathogens' infection is likely to be widespread and we should pay more attention to cross-transmission of these pathogens among fish, livestock, and humans.

Coinfections with different FZT pathogens are common in fish in Guangxi Zhuang Autonomous Region, China (Sohn et al., 2009), Korea (Sohn, 2009), and Vietnam (Chai et al., 2009). Thus, 10 fish coinfected with two agents (M. orientalis and M. yokogawai) were found in this study as we expected. To our knowledge, in China, 28 fish species have been recorded as the second intermediate hosts of M. yokogawai, including H. leucisculus and C. auratus (Sohn et al., 2009); four fish species (C. idellus, crucian, P. parva, and P. rivularis) have been identified as infected with M. orientalis (Ye et al., 2008); only mudfish have been investigated as the second intermediate hosts of I. hortensis (Fan et al., 1992). In the present study, M. orientalis metacercariae were detected in seven fish species (P. parva, H. leucisculus, S. dabryi, R. lagowskii, C. auratus, R. ocellatus, and P. glehnii). M. yokogawai metacercariae were only identified in P. parva, H. leucisculus, and S. dabryi. I. hortensis was only found in M. anguillicaudatus. More importantly, because of the habit of eating grilled/raw fish by the residents of Heilongjiang, it is increasing the risk of infection from FZT to humans. Therefore, the results of the present study not only extend a new second intermediate host of FZT in China, but also suggest that all eight fish species (M. anguillicaudatus; P. parva, H. leucisculus, S. dabryi, R. lagowskii, C. auratus, R. ocellatus, and P. glehnii) cannot be eaten raw. Furthermore, the different prevalence of the trematodes found in the study may be due to the differences in the sample sizes for each fish species. Further study should be done on these pathogens in a larger sample size.

FZT are important zoonotic agents. These trematodes have similar and complex life cycle. Many animals are hosts for these pathogens. However, only 13 fish species have been investigated for prevalences of FZT in the present study; prevalences of FZT in other fish and animals should be conducted in further studies.

In summary, this is the first report of M. orientalis, I. hortensis, and M. yokogawai in freshwater fish from Heilongjiang Province, China, which raises a major public health concern. The present study not only extends second intermediate host range of FZT, but also improves the information of the distribution of FZT in China. More importantly, habits of eating raw fish should be abolished in Heilongjiang Province.

Footnotes

Acknowledgments

This work was supported, in part, by the Fund for Imported Talents in Heilongjiang Bayi Agricultural University (XDB2013-42). The authors thank Prof. Xing-Quan Zhu at the State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, and Chinese Academy of Agricultural Sciences, for improving the English language of the article.

Disclosure Statement

No competing financial interests exist. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies.

References

Chai

, Jung

, Kim

, Lim

, Shin

, Lee

, Kim

, Han

, Yeom

, Park

, Hwang

. Heterophyid trematodes recovered from people residing along the Boseong River, South Korea. Acta Trop, 2015; 148:142–146.

Chai

, Shin

, Lee

, Rim

. Foodborne intestinal flukes in Southeast Asia. Korean J Parasitol, 2009; 47:S69–S102.

Chai

, Sohn

, Kim

, Hong

, Lee

. Three morphological types of the genus Metagonimus encysted in the dace, Tribolodon tazamowskii, caught from the Sumjin river. Korean J Parasitol, 1991; 29:217–225.

Chen

, Zhang

, Jiang

. Susceptibility study of experimental animals to infection with Metorchi orientalis . J Appl Prev Med, 2008; 2:8. (In Chinese.)

Cho

, Lee

, Kim

, Seok

, Lee

, Jeong

, Na

, Sohn

. Prevalence of zoonotic trematode metacercariae in freshwater fish from Gangwon-do, Korea. Korean J Parasitol, 2014; 52:399–412.

Eom

, Park

, Lee

, Sohn

, Yong

, Chai

, Min

, Rim

, Insisiengmay

, Phommasack

. Infection Status of zoonotic trematode metacercariae in fishes from Vientiane Municipality and Champasak Province in Lao PDR. Korean J Parasitol, 2015; 53:447.

Fan

, Li

, Wang

, Wen

. Investigation of Isthmiophora hortensis in commercially mudfish in Harbin and experimental infection in rat. Chin J Parasitol Parasit Dis, 1992; 2:148–149. (In Chinese.)

Fan

, Zhang

. A case of Isthmiophora hortensis infection. Chin J Parasit Dis Control, 2003; 16:182. (In Chinese.)

Hallewell

, Reuter

, Stanford

, Topp

, Alexander

. Monitoring seven potentially pathogenic Escherichia coli serogroups in a closed herd of beef cattle from weaning to finishing phases. Foodborne Pathog Dis, 2016; 13:661–667.

10.

Huang

, Zeng

, Yang

, Zhuang

, Xiang

, Du

, Zhang

. Investigation on metacercaria infection rates and infection sites of 6 kinds of freshwater fish in Qiqihar. Chin Trop Med, 2015; 15:812–815. (In Chinese.)

11.

Hung

, Madsen

, Fried

. Global status of fish-borne zoonotic trematodiasis in humans. Acta Parasitol, 2013; 58:231–258.

12.

Jiang

, Du

, Wang

, Wu

. Foodborne trematode metacercariaein in intermediate hosts in the lower Lancang River basin. J Pathog Biology, 2013; 2:167–169. (In Chinese.)

13.

Jiang

, Xu

, Chang

, Yang

, Yu

, Luo

, Zhou

, Xu

, Tang

, Zhang

, Miao

. Three cases of human Metagonimus yokogawai in Anhui province. Chin J Zoonoses, 1988; 2:40. (In Chinese.)

14.

Kino

, Suzuki

, Oishi

, Suzuki

, Yamagiwa

, Ishiguro

. Geographical distribution of Metagonimus yokogawai and M. miyatai in Shizuoka Prefecture, Japan, and their site preferences in the sweetfish, Plecoglossus altivelis, and hamsters. Parasitol Int, 2006; 55:201–206.

15.

, Clausen

, Murrell

, Liu

, Dalsgaard

. Risks for fishborne zoonotic trematodes in tilapia production systems in Guangdong province, China. Vet Parasitol, 2013a;198:223–229.

16.

, Huang

, Chen

, Huang

, Chen

, Ooi

. Metagonimus yokogawai: Metacercariae survey in fishes and its development to adult worms in various rodents. Parasitol Res, 2013b;112:1647–1653.

17.

Lin

, Li

, Chen

, Xu

, Lin

, Chen

, Xiu

, Li

, Pan

, Peng

. Report on human body experimental infection with Metorchis orientalis . Strail J Prev Med, 2001; 7:9–11. (In Chinese.)

18.

Liu

, Sun

, Zhang

, Li

. Prevalence of metacercariae of Clonorchis sinensis in wild freshwater fishes from Nenjiang River around Qiqihaer city. Chin J Parasitol Parasit Dis, 2014; 32:292–294. (In Chinese.)

19.

Liu

, Shi

. Two cases of Isthmiophora hortensis infection in Guangxi. Chin J Parasitol Parasit Dis, 2011; 29:77.(In Chinese.)

20.

, Gao

, Liu

, Fu

, Su

, Yue

, Gao

, Zhang

, Wang

. The complete mitochondrial genome of Metorchis orientalis (Trematoda: Opisthorchiidae): Comparison with other closely related species and phylogenetic implications. Infect Genet Evol, 2016; 39:45–50.

21.

Pitaksakulrat

, Sithithaworn

, Laoprom

, Laha

, Petney

, Andrews

. A cross-sectional study on the potential transmission of the carcinogenic liver fluke Opisthorchis viverrini and other fishborne zoonotic trematodes by aquaculture fish. Foodborne Pathog Dis, 2013; 10:35–41.

22.

Shen

, Zhang

, Sun

, Yang

, Chen

, Li

, Guo

, Li

, Yang

, Li

. Investigation on infection of flukes metacercaria in the commercial fresh-water fish and shrimp in shiyan. J Hubei Univ Med, 2015; 34:548–551. (In Chinese.)

23.

Shimazu

, Kino

. Metagonimus yokogawai (Trematoda: Heterophyidae): From discovery to designation of a neotype. Korean J Parasitol, 2015; 53:627–639.

24.

Sohn

, Eom

, Min

, Rim

, Hoang

, Yang

, Li

. Fishborne trematode in freshwater fish from Guangxi Zhuang Autonomous Region, China. Korean J Parasitol, 2009; 47:249–257.

25.

Sohn

. Fish-borne zoonotic trematode metacercariae in the Republic of Korea. Korean J Parasitol, 2009; 47:S103.

26.

Sun

, Zhang

, Liu

. Investigation of Clonorchis sinensis metacercaria infection in freshwater fish in integrated markets of Qiqihar city. J Qiqihar Univ Med, 2014; 6:860–861. (In Chinese.)

27.

, Lv

, Kang

. Investigation of Opisthorchiidae trematodes inducks of Anhui Fengyang. Chin J Vet Med, 1990; 16:30–31. (In Chinese.)

28.

, Li

. Distribution and epidemic situation of known human parasites in Hubei Province. Hubei J Prev Med, 1995; 6:19–22. (In Chinese.)

29.

Yao

, Chen

, Zhang

, Xu

, Ye

, Lei

. A report on the first case of human infection Metagonimus yokogawai in Zhejiang Province. Chin J Parasitol Parasit Dis, 1991; 4:144–145. (In Chinese.)

30.

, Li

, Wang

, Li u

, Wu

. Investigation on commercial freshwater fish infection with metacercaria of Metorchis orientalis . Practical Prev Med, 2008; 5:1326–1327. (In Chinese.)

31.

Zhan

, Li

, Zhao

, Wang

, Gu

. Metorchis orientalis founded in basin of Wuhu City, Anhui Province. Chinese J Schistosomiasis Control, 2015; 27:299–301. (In Chinese.)

32.

Zhang

, Li

, Tan

, Lan

, Yang

, Lin

, Xu

, Min

, Sun

, Feng

. A survey on the Heterophid trematodes metacercariae carried by fresh water fish in Guangxi. Chin J Zoonoses, 2006; 22:111–113. (In Chinese.)

33.

Zhang

, Chang

, Zhang

, Na

, Wang

, Xu

, Gao

, Liu

, Wang

, Zhu

. Prevalence of Clonorchis sinensis infection in freshwater fishes in northeastern China. Vet Parasitol, 2014; 204:209–213.

34.

Zhu

, Sun

, Li

, Qin

. Survey on natural nidus of Metorchis orientalis in Huaihe river basin. Chin J Parasitol Parasit Dis, 2006; 1:74–75. (In Chinese.)

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