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Prevalence of Clonorchis sinensis infection in freshwater fishes in northeastern China
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Trematodes
Prevalence of Clonorchis sinensis infection in freshwater fishes in northeastern China Y. Zhang a , Q.C. Chang a , Y. Zhang a , L. Na a , W.T. Wang a , W.W. Xu a , D.Z. Gao a , Z.X. Liu a , C.R. Wang a,∗ , X.Q. Zhu a,b,∗∗ a College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China b State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China
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Article history: Received 16 December 2013 Received in revised form 16 March 2014 Accepted 8 May 2014
Keywords: Clonorchis sinensis Metacercaria Prevalence Freshwater fishes Northeastern China
a b s t r a c t The prevalence of Clonorchis sinensis infection in freshwater fishes was surveyed in Heilongjiang Province, northeastern China, between August 2011 and September 2013. Thirteen species of freshwater fish (n = 3221) and one species of shrimp (n = 93) were collected from Songhua river, Nenjiang river and other lakes or ponds in 37 sites of 15 representative cities in Heilongjiang Province. They were individually examined by digestion technique, and the C. sinensis metacercariae were identified morphologically followed by confirmation using sequences of the second internal transcribed spacer of ribosomal DNA. Ten of the 13 examined species of freshwater fishes were infected with C. sinensis metacercariae, while all shrimps were negative. The overall prevalence of C. sinensis infection in 3221 examined freshwater fishes was 19.96%, with 42.57% (272/639) in Pseudorasbora parva, 22.55% (83/368) in Hemicculter leuciclus, 20.44% (121/592) in Carassius auratus, 17.71% (68/384) in Saurogobio dabryi, 10.85% (23/212) in Rhodeus ocellatus, 10.54% (48/455) in Phoxinus lagowskii, 8.20% (21/256) in Perccottus glehnii, 6.25% (5/80) in Misgurnus anguillicaudatus, 4.55% (1/22) in Xenocypris davidi, and 1.49% (1/67) in Cyprinus carpio. The average infection intensity in P. parva was 103.3 encysted metacercariae per gram of fish meat in Zhaoyuan city. The average prevalence of C. sinensis infection in Songhua river, Nenjiang river and lakes or ponds were 31.96% (503/1574), 11.30% (102/903) and 7.93% (59/744), respectively. The prevalence of C. sinensis infection in Zhaoyuan city (43.68%) was the highest among all sampling locations. These results revealed a high-prevalence of C. sinensis infection in freshwater fishes in Heilongjiang Province, northeastern China, posing significant public health concern. © 2014 Published by Elsevier B.V.
1. Introduction ∗ Corresponding author. Tel.: +86 459 6819099; fax: +86 459 6819090. ∗∗ Co-Corresponding author at: College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China. Tel.: +86 931 8342837; fax: +86 931 8340977. E-mail addresses: [email protected] (C.R. Wang), [email protected] (X.Q. Zhu).
Fishborne zoonotic trematodes (FZT) are well-known causes of fluke diseases in humans (Chai et al., 2005; Phan et al., 2010). These trematodes include the liver flukes Opisthorchis viverrini and Clonorchis sinensis, and intestinal flukes of the families Heterophyidae, Leicithodendriidae and Echinostomatidae (Yu and Mott, 1994). Among these
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Please cite this article in press as: Zhang, Y., et al., Prevalence of Clonorchis sinensis infection in freshwater fishes in northeastern China. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.007
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trematodes, C. sinensis is one of the most harmful to humans, which can cause cholangiocarcinoma. Clonorchiasis is a fishborne trematode infection which is considered one of the major parasitic zoonoses in some parts of Asia, including China, Korea, Japan and Vietnam, with approximately 35 million people being infected globally, of whom approximately 15 million are in China (Lun et al., 2005, Lin et al., 2011). Clonorchiasis can cause a series of hepatic diseases such as periductal inflammation, fibrosis, pyogenic cholangitis, cholecystitis, cholelithiasis and liver cirrhosis, and C. sinensis is currently classified as carcinogenic to humans for cholangiocarcinoma (Chai et al., 2005; Shin et al., 2010; Sun et al., 2011). C. sinensis has been known to be actively transmitted along the rivers, especially among the residents who have the habit of eating raw fish (Kim et al., 2008). Humans, cats, dogs, pigs and some wild animals are definitive hosts for C. sinensis. Some aquatic snails, freshwater fishes and shrimps act as the first and the second intermediate hosts, respectively (Keiser and Utzinger, 2005). The prevalence of C. sinensis metacercariae in freshwater fishes has been reported in many countries, such as South Korea, Vietnam (Cho et al., 2006; Kim et al., 2008; Phan et al., 2010; Van et al., 2012). In China, approximately 140 species of freshwater fishes and four species of shrimp have been recognized as complementary intermediate hosts for C. sinensis (Zhou et al., 2008). However, only limited data is available regarding the prevalence of C. sinensis metacercariae in freshwater fishes in China (Chen et al., 2010; Sohn et al., 2009), and no such survey has been performed in Heilongjiang Province where the residents have the habit of eating raw fishes, resulting in a high C. sinensis prevalence of 4.72%, which ranked the third high in China (Fang et al., 2008). The objective of the present study was to investigate the prevalence of C. sinensis infection in freshwater fishes in different river basins in Heilongjiang Province, northeastern China, which would provide “base-line” data for assess the risk for human infection and for execute control programs. 2. Materials and methods
purchased primarily from fishing grounds, fish farms, fish ponds, and markets, whereas wild fishes were captured by fishermen from Songhua river, Nenjiang river, some lakes and ponds. Songhua river runs through Zhaoyuan, Zhaodong, Harbin, Mulan and Fujin city. Nenjiang river passes through Nehe, Fuyu, Gannan, Qiqihaer city. Daqing, Dumeng, Lindian, Anda, Mudanjiang, and Suihua city have some lakes or ponds. The shrimps examined were sampled from Songhua river and Nenjiang river in four cities mentioned above. All samples were preserved on ice and transported within 24 h to our parasitology laboratory in Daqing (College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University) for examination.
2.2. Examination of encysted metacercariae Collected fish were kept in a refrigerator at 4 ◦ C for no longer than 3 days before being processed. After the length and weight of each fish was recorded, encysted metacercariae of C. sinensis were individually examined by digestion technique (Sohn et al., 2009). In brief, each fish meat (head, scales, bones, viscera excluded) was ground finely with pestle in a mortar, then mixed with artificial gastric juice (8 g of pepsin 1:3000 (Shanghai, China) and 10 ml of concentrated HCl in 1 l of normal saline), and the mixture was incubated at 37 ◦ C for 2–3 h. The fluid containing digested fish meat was filtered with a 40-well/inch copper sieve to remove the large fragments. The filtered fluid was then placed into a 500 ml glass beaker, washed with 0.9% saline until the supernatant became clear. The sediment was carefully examined under a stereomicroscope to count the encysted metacercariae. The metacercariae were identified based on morphological characteristics, such as the size of cysts, folding body displaying vigorous movement within the cyst, and prominent and clearly visible oral and ventral suckers (Scholz et al., 1991; Kaewkes, 2003; Sohn et al., 2009). Finally, the infection intensity of metacrcariae in each fish was recorded. The identity of collected metacercariae was confirmed by molecular identification.
2.1. The study sites for fish collection Heilongjiang Province (longitude, 121◦ 11 to 135◦ 05 , latitude, 43◦ 25 to 53◦ 33 ) is located in the most northeastern end of China, which shares borders with Russia in the North and East, with the Inner Mongolian Autonomous Region in the West, and with Jilin Province in the South. It covers an area of 454,000 km2 , with 13 administrative regions, where have numerous rivers and lakes, including the Songhua river, Nenjiang river and a number of lakes and ponds. From August 2011 to September 2013, a total of 3221 freshwater fishes representing 13 fish species were collected from 37 sites of 15 representative cities. In addition, 93 shrimps representing Caridina nilotica gracilipes were collected from Zhaoyuan (28), Zhaodong (40), Qiqihaer (20) and Mulan city (5). Cluster random sampling was performed according to representative rivers and administrative areas. Aquacutured fishes were collected or
2.3. Molecular identification The partial sequence of the second internal transcribed spacer (ITS-2) of C. sinensis metacercariae was amplified using BD1 and BD2 primers (Luton et al., 1992). One microlitre of DNA template was used in a PCR reaction of 25 l containing 5 l of 5× colorless go Taq flexi buffer (pH 8.5), 2 l of MgCl2 (25 mM), 2 l of dNTP Mixture (2.5 mM), 0.5 l of each primer (10 pmol/l) and 0.13 l of go Taq DNA polymerase (5 U/l) in a thermocycler under the following conditions: 95 ◦ C for 2 min (initial denaturation), followed by 35 cycles of 95 ◦ C for 1 min (denaturation), 50 ◦ C for 1 min (annealing); 72 ◦ C for 1.2 min (extension) for 35 cycles, and then followed by a final extension at 72 ◦ C for 5 min. Each amplicon (25 l) was examined by agarose gel (1%) electrophoresis and ethidium bromide staining. The positive products were sent to Life Technology Company
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(Beijing, China) for sequencing using the same primers used in primary amplification. 2.4. Statistical analysis Statistical analyses of data were performed by SPSS version 16 (Chicago, IL). Chi-squares (2 ) tests were used to compare prevalence of C. sinensis infection between species of fish and different river basins. 3. Results 3.1. Molecular identification Three partial ITS-2 sequences were all 300 bp in length, which have been deposited in the GenBank under the accession numbers KF740423–KF740425. These ITS-2 sequences have 99.68–100% similarity to that of C. sinensis from Russian (GenBank accession No. JQ048577.1). 3.2. Prevalence of C. sinensis infection in freshwater fish and shrimp among different river basins The prevalences of C. sinensis infection in 13 species of freshwater fish are shown in Table 1. The overall prevalence of C. sinensis infection in freshwater fish was 19.96%, but no positive shrimp was found in this study. The prevalence of C. sinensis infection in freshwater fishes in Songhua river (31.96%) was higher than that in Nenjiang river (11.30%) and in the lakes or ponds (7.93%). The prevalent difference between Songhua river and Nenjiang river was statistically significant (P < 0.05), but the prevalent difference between Nenjiang river and lakes or ponds was not statistically significant (P > 0.05). The prevalence of C. sinensis infection in freshwater fish in 15 different cities was 43.68% in Zhaoyuan, 42.82% in Zhaodong, 39.81% in Gannan, 39.67% in Harbin, 24.86% in Fujin, 18.27% in Anda, 13.07% in Dumeng, 9.10% in Lindian, 7.83% in Mulan, 7.83% in Qiqihaer, 5% in Daqing, 3.69% in Mudanjiang, 2.76% in Fuyu, 2.07% in Nehe, and 0% in Suihua. 3.3. Prevalence of C. sinensis infection among various freshwater fish species In the present investigation, 10 species of fishes were positive of C. sinensis metacercariae (Table 1). Among the C. sinensis positive fishes, P. parva was collected in multiple locations and it had the highest prevalence (42.57%) which was significantly higher than that in other fish species. The prevalent differences between P. parva and C. auratus, P. glehnii and C. carpio were statistically significant (P < 0.05). The average infection intensity of C. sinensis metacercariae was also the highest in P. parva which was 103.3 metacercariae per gram of fish meat in Zhaoyuan city. 4. Discussion A number of surveys of freshwater fish infection with C. sinensis have been conducted in many countries and some provinces of China, and the prevalence in fishes was 100% in Korea (Cho et al., 2006), 0.1% in the Red River Delta of
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Vietnam (Phan et al., 2010), 37.09% in the Pearl river delta of Guangdong Province (Chen et al., 2010), and 16.97% in Guangxi Province of China (Sohn et al., 2009). In the present study, the overall C. sinensis prevalence in freshwater fishes was 19.96%, which was higher than that in Vietnam and Guangxi Province, but lower than that in the Pearl river delta of Guangdong Province and Korea. We then analyzed and compared the prevalences of C. sinensis infection in fishes from different geographical locations of China. The highest C. sinensis metacercariae prevalence was in freshwater fish from Zhaoyuan city, which was consistent with the high C. sinensis prevalence of 67.46% in humans (Ge and Wang, 2009). One possible reason is that residents along Songhua river and Nenjiang river have the habit of eating raw fish, especially in Zhaoyuan city. Another possible explanation is that residents along those rivers usually keep dogs and cats as pets. Dogs and cats are reservoir hosts of C. sinensis (Lin et al., 2011), increasing the infection risk of C. sinensis to humans. Our previous survey of helminthes prevalence in adult dogs in Heilongjiang Province showed a high C. sinensis prevalence (26.4%) (Wang et al., 2006). In October 2010, 681 adult worms of C. sinensis were found in the liver and bile duct of a dog in Zhaoyuan city by our laboratory (unpublished data). It is noteworthy that owing to the poor management in rural areas, there are many stray dogs and cats wandering along the river banks, shedding feces with C. sinensis eggs into rivers. This might be the reason why the C. sinensis infection in fishes in lakes or ponds were quite low possibly because the fishes from lakes or ponds were mainly cultured. Clonorchiasis is the most important FZT in China and Korea. In China, 140 species of freshwater fishes have been reported as the second intermediate hosts for C. sinensis (Zhou et al., 2008), and C. sinensis metacercariae have been detected in 42 species of freshwater fish in Korea (Sohn, 2009). In the present study, 10 freshwater fish species were found metacercariae-positive as the complementary hosts of C. sinensis in China. The negative fishes (S. asotus, P. fulvidrac, H. maculates) and shrimp (C. nilotica gracilipes) have been also reported previously as the second intermediate hosts for C. sinensis, but no C. sinensis metacercariae were detected in this investigation. This is likely due to the small numbers of sampled fishes and shrimps. Among the 10 fish species infected with C. sinensis metacercaria, P. parva showed the highest prevalence and infection intensity, similar to that of previous studies in Korea and Pearl river delta of Guangdong Province, China (Kim et al., 2008; Chen et al., 2010). P. parva has been used as an index species for distribution of C. sinensis at a certain unknown areas because of its high susceptibility to C. sinensis metacercaria (Hong and Hong, 2005; Kang et al., 1985). In addition, the size, growth rate and water layer in which fish live may also account for prevalent differences in different fish species. P. parva lives in shallow water, its skin is very thin, C. sinensis cercariae showed intensive positive phototaxis. The features should be the reason why P. parva is the most susceptible species. But P. parva is small in size, not suitable for making “sashimi” (raw fish), so it is not the main source of human infection with C. sinensis. By contrast, C. carpio is larger, and is one of the
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Table 1 Prevalence of Clonorchis sinensis infection in 13 species of freshwater fishes in different river basins in Heilongjiang Province, northeastern China. Species of fish
Total no.
Positive no.
Prevalence (%)
Pseudorasbora parva Hemicculter leuciclus Saurogobio dabryi Phoxinus lagowskii Carassius auratus Rhodeus ocellatus Perccottus glehnii Misgurnus anguillicaudatus Xenocypris davidi Cyprinus carpio Silurus asotus Pelteobagrus fulvidraco Hemibarbus maculatus Total 95% Confidence interval of positive rate
639 368 384 455 592 212 256 80 22 67 19 74 53 3221
272 83 68 48 121 23 21 5 1 1 0 0 0 643
42.57 22.55 17.71 10.54 20.44 10.85 8.20 6.25 4.55 1.49 0 0 0 19.96 9.99–19.77
most favorite fish species being eating raw in Heilongjiang Province. C. sinensis infection has been identified as a risk factor for perihilar cholangiocarcinoma, and the disease is the second most common primary malignant tumor of the liver after hepatocellular carcinoma (Suarez-Munoz et al., 2013). Previous studies have revealed that clonorchiasis patients have a high incidence of cholangiocarcinoma (Choi et al., 1989; Qian et al., 2012). In China, clonorchiasis has been reported in 24 provinces, especially in Guangdong (16.42%), Guangxi (9.75%) and Heilongjiang Provinces (4.72%) (Fang et al., 2008). A total of 15 million people were estimated to be infected with C. sinensis in China (Lun et al., 2005), of which nearly 5000 cholangiocarcinoma patients were attributed to C. sinensis infection (Qian et al., 2013). The present investigation revealed a high C. sinensis prevalence in freshwater fishes in Heilongjiang Province, posing a significant risk for human infection with C. sinensis for local residents. Therefore, inhabitants residing in endemic areas, like Heilongjiang Province, should be aware of such infection risk, and consumption of raw freshwater fish naturally produced in these areas should be avoided. Integrated programs should be executed to prevent and control C. sinensis infection in fresh water fishes in these endemic regions. Conflict of interest statement No conflict of interest. Acknowledgments This work was supported, in part, by the Fund for Imported Talents in Heilongjiang Bayi Agricultural University (XDB2013-42), and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006). References Chai, J.Y., Darwin, Murrell, K., Lymbery, A.J., 2005. Fishborne parasitic zoonoses: status and issues. Int. J. Parasitol. 35, 1233–1254.
No. of fish samples (% of fish sample per district) Songhua river
Nenjiang river
Lakes or ponds
354 (60.73) 214 (32.71) 195 (32.31) 58 (32.75) 377 (26.79) 102 (16.67) 116 (10.34) 40 (12.50) – 62 (1.61) 17 39 – 1574 (31.96) 14.12–32.56
124 (41.13) 99 (13.13) 103 (1.94) 186 (2.15) 78 (14.10) 87 (2.30) 98 (9.18) 40 (0) 22 (4.55) 0 0 13 (0) 53 (0) 903 (11.30) 2.72–18.84
161 (3.72) 55 (0) 86 (3.49) 211 (11.75) 137 (6.57) 23 (17.39) 42 (0) 0 0 5 (0) 2 (0) 22 (0) 0 744 (7.93) 1.94–9.78
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Trematodes
Prevalence of Clonorchis sinensis infection in freshwater fishes in northeastern China Y. Zhang a , Q.C. Chang a , Y. Zhang a , L. Na a , W.T. Wang a , W.W. Xu a , D.Z. Gao a , Z.X. Liu a , C.R. Wang a,∗ , X.Q. Zhu a,b,∗∗ a College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China b State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China
a r t i c l e
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Article history: Received 16 December 2013 Received in revised form 16 March 2014 Accepted 8 May 2014
Keywords: Clonorchis sinensis Metacercaria Prevalence Freshwater fishes Northeastern China
a b s t r a c t The prevalence of Clonorchis sinensis infection in freshwater fishes was surveyed in Heilongjiang Province, northeastern China, between August 2011 and September 2013. Thirteen species of freshwater fish (n = 3221) and one species of shrimp (n = 93) were collected from Songhua river, Nenjiang river and other lakes or ponds in 37 sites of 15 representative cities in Heilongjiang Province. They were individually examined by digestion technique, and the C. sinensis metacercariae were identified morphologically followed by confirmation using sequences of the second internal transcribed spacer of ribosomal DNA. Ten of the 13 examined species of freshwater fishes were infected with C. sinensis metacercariae, while all shrimps were negative. The overall prevalence of C. sinensis infection in 3221 examined freshwater fishes was 19.96%, with 42.57% (272/639) in Pseudorasbora parva, 22.55% (83/368) in Hemicculter leuciclus, 20.44% (121/592) in Carassius auratus, 17.71% (68/384) in Saurogobio dabryi, 10.85% (23/212) in Rhodeus ocellatus, 10.54% (48/455) in Phoxinus lagowskii, 8.20% (21/256) in Perccottus glehnii, 6.25% (5/80) in Misgurnus anguillicaudatus, 4.55% (1/22) in Xenocypris davidi, and 1.49% (1/67) in Cyprinus carpio. The average infection intensity in P. parva was 103.3 encysted metacercariae per gram of fish meat in Zhaoyuan city. The average prevalence of C. sinensis infection in Songhua river, Nenjiang river and lakes or ponds were 31.96% (503/1574), 11.30% (102/903) and 7.93% (59/744), respectively. The prevalence of C. sinensis infection in Zhaoyuan city (43.68%) was the highest among all sampling locations. These results revealed a high-prevalence of C. sinensis infection in freshwater fishes in Heilongjiang Province, northeastern China, posing significant public health concern. © 2014 Published by Elsevier B.V.
1. Introduction ∗ Corresponding author. Tel.: +86 459 6819099; fax: +86 459 6819090. ∗∗ Co-Corresponding author at: College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China. Tel.: +86 931 8342837; fax: +86 931 8340977. E-mail addresses: [email protected] (C.R. Wang), [email protected] (X.Q. Zhu).
Fishborne zoonotic trematodes (FZT) are well-known causes of fluke diseases in humans (Chai et al., 2005; Phan et al., 2010). These trematodes include the liver flukes Opisthorchis viverrini and Clonorchis sinensis, and intestinal flukes of the families Heterophyidae, Leicithodendriidae and Echinostomatidae (Yu and Mott, 1994). Among these
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Please cite this article in press as: Zhang, Y., et al., Prevalence of Clonorchis sinensis infection in freshwater fishes in northeastern China. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.007
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trematodes, C. sinensis is one of the most harmful to humans, which can cause cholangiocarcinoma. Clonorchiasis is a fishborne trematode infection which is considered one of the major parasitic zoonoses in some parts of Asia, including China, Korea, Japan and Vietnam, with approximately 35 million people being infected globally, of whom approximately 15 million are in China (Lun et al., 2005, Lin et al., 2011). Clonorchiasis can cause a series of hepatic diseases such as periductal inflammation, fibrosis, pyogenic cholangitis, cholecystitis, cholelithiasis and liver cirrhosis, and C. sinensis is currently classified as carcinogenic to humans for cholangiocarcinoma (Chai et al., 2005; Shin et al., 2010; Sun et al., 2011). C. sinensis has been known to be actively transmitted along the rivers, especially among the residents who have the habit of eating raw fish (Kim et al., 2008). Humans, cats, dogs, pigs and some wild animals are definitive hosts for C. sinensis. Some aquatic snails, freshwater fishes and shrimps act as the first and the second intermediate hosts, respectively (Keiser and Utzinger, 2005). The prevalence of C. sinensis metacercariae in freshwater fishes has been reported in many countries, such as South Korea, Vietnam (Cho et al., 2006; Kim et al., 2008; Phan et al., 2010; Van et al., 2012). In China, approximately 140 species of freshwater fishes and four species of shrimp have been recognized as complementary intermediate hosts for C. sinensis (Zhou et al., 2008). However, only limited data is available regarding the prevalence of C. sinensis metacercariae in freshwater fishes in China (Chen et al., 2010; Sohn et al., 2009), and no such survey has been performed in Heilongjiang Province where the residents have the habit of eating raw fishes, resulting in a high C. sinensis prevalence of 4.72%, which ranked the third high in China (Fang et al., 2008). The objective of the present study was to investigate the prevalence of C. sinensis infection in freshwater fishes in different river basins in Heilongjiang Province, northeastern China, which would provide “base-line” data for assess the risk for human infection and for execute control programs. 2. Materials and methods
purchased primarily from fishing grounds, fish farms, fish ponds, and markets, whereas wild fishes were captured by fishermen from Songhua river, Nenjiang river, some lakes and ponds. Songhua river runs through Zhaoyuan, Zhaodong, Harbin, Mulan and Fujin city. Nenjiang river passes through Nehe, Fuyu, Gannan, Qiqihaer city. Daqing, Dumeng, Lindian, Anda, Mudanjiang, and Suihua city have some lakes or ponds. The shrimps examined were sampled from Songhua river and Nenjiang river in four cities mentioned above. All samples were preserved on ice and transported within 24 h to our parasitology laboratory in Daqing (College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University) for examination.
2.2. Examination of encysted metacercariae Collected fish were kept in a refrigerator at 4 ◦ C for no longer than 3 days before being processed. After the length and weight of each fish was recorded, encysted metacercariae of C. sinensis were individually examined by digestion technique (Sohn et al., 2009). In brief, each fish meat (head, scales, bones, viscera excluded) was ground finely with pestle in a mortar, then mixed with artificial gastric juice (8 g of pepsin 1:3000 (Shanghai, China) and 10 ml of concentrated HCl in 1 l of normal saline), and the mixture was incubated at 37 ◦ C for 2–3 h. The fluid containing digested fish meat was filtered with a 40-well/inch copper sieve to remove the large fragments. The filtered fluid was then placed into a 500 ml glass beaker, washed with 0.9% saline until the supernatant became clear. The sediment was carefully examined under a stereomicroscope to count the encysted metacercariae. The metacercariae were identified based on morphological characteristics, such as the size of cysts, folding body displaying vigorous movement within the cyst, and prominent and clearly visible oral and ventral suckers (Scholz et al., 1991; Kaewkes, 2003; Sohn et al., 2009). Finally, the infection intensity of metacrcariae in each fish was recorded. The identity of collected metacercariae was confirmed by molecular identification.
2.1. The study sites for fish collection Heilongjiang Province (longitude, 121◦ 11 to 135◦ 05 , latitude, 43◦ 25 to 53◦ 33 ) is located in the most northeastern end of China, which shares borders with Russia in the North and East, with the Inner Mongolian Autonomous Region in the West, and with Jilin Province in the South. It covers an area of 454,000 km2 , with 13 administrative regions, where have numerous rivers and lakes, including the Songhua river, Nenjiang river and a number of lakes and ponds. From August 2011 to September 2013, a total of 3221 freshwater fishes representing 13 fish species were collected from 37 sites of 15 representative cities. In addition, 93 shrimps representing Caridina nilotica gracilipes were collected from Zhaoyuan (28), Zhaodong (40), Qiqihaer (20) and Mulan city (5). Cluster random sampling was performed according to representative rivers and administrative areas. Aquacutured fishes were collected or
2.3. Molecular identification The partial sequence of the second internal transcribed spacer (ITS-2) of C. sinensis metacercariae was amplified using BD1 and BD2 primers (Luton et al., 1992). One microlitre of DNA template was used in a PCR reaction of 25 l containing 5 l of 5× colorless go Taq flexi buffer (pH 8.5), 2 l of MgCl2 (25 mM), 2 l of dNTP Mixture (2.5 mM), 0.5 l of each primer (10 pmol/l) and 0.13 l of go Taq DNA polymerase (5 U/l) in a thermocycler under the following conditions: 95 ◦ C for 2 min (initial denaturation), followed by 35 cycles of 95 ◦ C for 1 min (denaturation), 50 ◦ C for 1 min (annealing); 72 ◦ C for 1.2 min (extension) for 35 cycles, and then followed by a final extension at 72 ◦ C for 5 min. Each amplicon (25 l) was examined by agarose gel (1%) electrophoresis and ethidium bromide staining. The positive products were sent to Life Technology Company
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(Beijing, China) for sequencing using the same primers used in primary amplification. 2.4. Statistical analysis Statistical analyses of data were performed by SPSS version 16 (Chicago, IL). Chi-squares (2 ) tests were used to compare prevalence of C. sinensis infection between species of fish and different river basins. 3. Results 3.1. Molecular identification Three partial ITS-2 sequences were all 300 bp in length, which have been deposited in the GenBank under the accession numbers KF740423–KF740425. These ITS-2 sequences have 99.68–100% similarity to that of C. sinensis from Russian (GenBank accession No. JQ048577.1). 3.2. Prevalence of C. sinensis infection in freshwater fish and shrimp among different river basins The prevalences of C. sinensis infection in 13 species of freshwater fish are shown in Table 1. The overall prevalence of C. sinensis infection in freshwater fish was 19.96%, but no positive shrimp was found in this study. The prevalence of C. sinensis infection in freshwater fishes in Songhua river (31.96%) was higher than that in Nenjiang river (11.30%) and in the lakes or ponds (7.93%). The prevalent difference between Songhua river and Nenjiang river was statistically significant (P < 0.05), but the prevalent difference between Nenjiang river and lakes or ponds was not statistically significant (P > 0.05). The prevalence of C. sinensis infection in freshwater fish in 15 different cities was 43.68% in Zhaoyuan, 42.82% in Zhaodong, 39.81% in Gannan, 39.67% in Harbin, 24.86% in Fujin, 18.27% in Anda, 13.07% in Dumeng, 9.10% in Lindian, 7.83% in Mulan, 7.83% in Qiqihaer, 5% in Daqing, 3.69% in Mudanjiang, 2.76% in Fuyu, 2.07% in Nehe, and 0% in Suihua. 3.3. Prevalence of C. sinensis infection among various freshwater fish species In the present investigation, 10 species of fishes were positive of C. sinensis metacercariae (Table 1). Among the C. sinensis positive fishes, P. parva was collected in multiple locations and it had the highest prevalence (42.57%) which was significantly higher than that in other fish species. The prevalent differences between P. parva and C. auratus, P. glehnii and C. carpio were statistically significant (P < 0.05). The average infection intensity of C. sinensis metacercariae was also the highest in P. parva which was 103.3 metacercariae per gram of fish meat in Zhaoyuan city. 4. Discussion A number of surveys of freshwater fish infection with C. sinensis have been conducted in many countries and some provinces of China, and the prevalence in fishes was 100% in Korea (Cho et al., 2006), 0.1% in the Red River Delta of
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Vietnam (Phan et al., 2010), 37.09% in the Pearl river delta of Guangdong Province (Chen et al., 2010), and 16.97% in Guangxi Province of China (Sohn et al., 2009). In the present study, the overall C. sinensis prevalence in freshwater fishes was 19.96%, which was higher than that in Vietnam and Guangxi Province, but lower than that in the Pearl river delta of Guangdong Province and Korea. We then analyzed and compared the prevalences of C. sinensis infection in fishes from different geographical locations of China. The highest C. sinensis metacercariae prevalence was in freshwater fish from Zhaoyuan city, which was consistent with the high C. sinensis prevalence of 67.46% in humans (Ge and Wang, 2009). One possible reason is that residents along Songhua river and Nenjiang river have the habit of eating raw fish, especially in Zhaoyuan city. Another possible explanation is that residents along those rivers usually keep dogs and cats as pets. Dogs and cats are reservoir hosts of C. sinensis (Lin et al., 2011), increasing the infection risk of C. sinensis to humans. Our previous survey of helminthes prevalence in adult dogs in Heilongjiang Province showed a high C. sinensis prevalence (26.4%) (Wang et al., 2006). In October 2010, 681 adult worms of C. sinensis were found in the liver and bile duct of a dog in Zhaoyuan city by our laboratory (unpublished data). It is noteworthy that owing to the poor management in rural areas, there are many stray dogs and cats wandering along the river banks, shedding feces with C. sinensis eggs into rivers. This might be the reason why the C. sinensis infection in fishes in lakes or ponds were quite low possibly because the fishes from lakes or ponds were mainly cultured. Clonorchiasis is the most important FZT in China and Korea. In China, 140 species of freshwater fishes have been reported as the second intermediate hosts for C. sinensis (Zhou et al., 2008), and C. sinensis metacercariae have been detected in 42 species of freshwater fish in Korea (Sohn, 2009). In the present study, 10 freshwater fish species were found metacercariae-positive as the complementary hosts of C. sinensis in China. The negative fishes (S. asotus, P. fulvidrac, H. maculates) and shrimp (C. nilotica gracilipes) have been also reported previously as the second intermediate hosts for C. sinensis, but no C. sinensis metacercariae were detected in this investigation. This is likely due to the small numbers of sampled fishes and shrimps. Among the 10 fish species infected with C. sinensis metacercaria, P. parva showed the highest prevalence and infection intensity, similar to that of previous studies in Korea and Pearl river delta of Guangdong Province, China (Kim et al., 2008; Chen et al., 2010). P. parva has been used as an index species for distribution of C. sinensis at a certain unknown areas because of its high susceptibility to C. sinensis metacercaria (Hong and Hong, 2005; Kang et al., 1985). In addition, the size, growth rate and water layer in which fish live may also account for prevalent differences in different fish species. P. parva lives in shallow water, its skin is very thin, C. sinensis cercariae showed intensive positive phototaxis. The features should be the reason why P. parva is the most susceptible species. But P. parva is small in size, not suitable for making “sashimi” (raw fish), so it is not the main source of human infection with C. sinensis. By contrast, C. carpio is larger, and is one of the
Please cite this article in press as: Zhang, Y., et al., Prevalence of Clonorchis sinensis infection in freshwater fishes in northeastern China. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.007
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Table 1 Prevalence of Clonorchis sinensis infection in 13 species of freshwater fishes in different river basins in Heilongjiang Province, northeastern China. Species of fish
Total no.
Positive no.
Prevalence (%)
Pseudorasbora parva Hemicculter leuciclus Saurogobio dabryi Phoxinus lagowskii Carassius auratus Rhodeus ocellatus Perccottus glehnii Misgurnus anguillicaudatus Xenocypris davidi Cyprinus carpio Silurus asotus Pelteobagrus fulvidraco Hemibarbus maculatus Total 95% Confidence interval of positive rate
639 368 384 455 592 212 256 80 22 67 19 74 53 3221
272 83 68 48 121 23 21 5 1 1 0 0 0 643
42.57 22.55 17.71 10.54 20.44 10.85 8.20 6.25 4.55 1.49 0 0 0 19.96 9.99–19.77
most favorite fish species being eating raw in Heilongjiang Province. C. sinensis infection has been identified as a risk factor for perihilar cholangiocarcinoma, and the disease is the second most common primary malignant tumor of the liver after hepatocellular carcinoma (Suarez-Munoz et al., 2013). Previous studies have revealed that clonorchiasis patients have a high incidence of cholangiocarcinoma (Choi et al., 1989; Qian et al., 2012). In China, clonorchiasis has been reported in 24 provinces, especially in Guangdong (16.42%), Guangxi (9.75%) and Heilongjiang Provinces (4.72%) (Fang et al., 2008). A total of 15 million people were estimated to be infected with C. sinensis in China (Lun et al., 2005), of which nearly 5000 cholangiocarcinoma patients were attributed to C. sinensis infection (Qian et al., 2013). The present investigation revealed a high C. sinensis prevalence in freshwater fishes in Heilongjiang Province, posing a significant risk for human infection with C. sinensis for local residents. Therefore, inhabitants residing in endemic areas, like Heilongjiang Province, should be aware of such infection risk, and consumption of raw freshwater fish naturally produced in these areas should be avoided. Integrated programs should be executed to prevent and control C. sinensis infection in fresh water fishes in these endemic regions. Conflict of interest statement No conflict of interest. Acknowledgments This work was supported, in part, by the Fund for Imported Talents in Heilongjiang Bayi Agricultural University (XDB2013-42), and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006). References Chai, J.Y., Darwin, Murrell, K., Lymbery, A.J., 2005. Fishborne parasitic zoonoses: status and issues. Int. J. Parasitol. 35, 1233–1254.
No. of fish samples (% of fish sample per district) Songhua river
Nenjiang river
Lakes or ponds
354 (60.73) 214 (32.71) 195 (32.31) 58 (32.75) 377 (26.79) 102 (16.67) 116 (10.34) 40 (12.50) – 62 (1.61) 17 39 – 1574 (31.96) 14.12–32.56
124 (41.13) 99 (13.13) 103 (1.94) 186 (2.15) 78 (14.10) 87 (2.30) 98 (9.18) 40 (0) 22 (4.55) 0 0 13 (0) 53 (0) 903 (11.30) 2.72–18.84
161 (3.72) 55 (0) 86 (3.49) 211 (11.75) 137 (6.57) 23 (17.39) 42 (0) 0 0 5 (0) 2 (0) 22 (0) 0 744 (7.93) 1.94–9.78
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Please cite this article in press as: Zhang, Y., et al., Prevalence of Clonorchis sinensis infection in freshwater fishes in northeastern China. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.007