High Prevalence of Clonorchis sinensis and Other Zoonotic Trematode Metacercariae in Fish from a Local Market in Yen Bai Province, Northern Vietnam.

A small survey was performed to investigate the recent infection status of Clonorchis sinensis and other zoonotic trematode metacercariae in freshwater fish from a local market of Yen Bai city, Yen Bai province, northern Vietnam. A total of 118 fish in 7 species were examined by the artificial digestion method on March 2016. The metacercariae of 4 species of zoonotic trematodes, i.e., C. sinensis, Haplorchis pumilio, Haplorchis taichui, and Centrocestus formosanus, were detected. The metacercariae of C. sinensis were found in 62 (69.7%) out of 89 fish (5 species), and their intensity of infection was very high, 81.2 per fish infected. Prevalences of 3 intestinal flukes, H. pumilio, H. taichui and C. formosanus, were 75.0%, 47.6%, and 31.7% in positive fish species, respectively, with the metacercarial intensities of 15.5, 10.3, and 2.2 per fish infected. From the above results, it has been confirmed that various species of freshwater fish continue to play the role of the infection source of C. sinensis and other zoonotic trematodes in Yen Bai city, Yen Bai province, northern Vietnam. It is of particular note that the prevalence and intensity of C. sinensis metacercariae are much higher than those reported in previous studies in fish in northern Vietnam.

Fishborne zoonotic trematode (FZT) infections, including Clonorchis sinensis, affect the health of more than 30 million people throughout the world and are particularly prevalent in Asian countries, such as Lao PDR, Vietnam, Cambodia, Thailand, Philippines, Taiwan, China, and Korea [1-4]. Human infections with FZT are almost entirely caused by the habitual consumption of raw and/or undercooked fish meat containing infective larvae, the metacercariae [4-6]. Surveys on the infection status of fish with zoonotic trematode metacercariae (ZTM) have been conducted by many workers to obtain useful information on the epidemiology of zoonotic trematodes in particular areas [7-11]. In Vietnam, epidemiological surveys for ZTM were performed on fish intermediate hosts in local areas by many workers [8,12-22]. Especially, Chai et al. [8] surveyed the infection status of ZTM in wild fish from 2 localities of northern Vietnam, Hanoi city and Nam Dinh province, and they also described the morphological characteristics of ZTM and their adults recovered from experimental animals. However, most of the studies were performed on fish from adjacent and/or lower latitude areas of Hanoi [8,12-22].

Yen Bai province is an agricultural-based province located in the northern part of north-central Vietnam. The Red River (midle reach) and the Thac Ba Lake are ecologically important for the source of fish in inhabitants of this province [23]. Furthermore, there are no available information to know the infection status of ZTM in fish intermediate hosts, which is a kind of valuble protein source for the people in this region. Therefore, we performed a small survey to know the infection status of ZTM among wild fish purchased from a local market of Yen Bai city, Yen Bai province, northern Vietnam.

A total of 118 freshwater fish in 7 species were collected from a local market of Yen Bai city (latitude: 21.699533; longitude:104.879628), Yen Bai province, northern Vietnam on March 2016 (Fig. 1) [23]. All collected fish were transferred with ice to the laboratory of Department of Parasitology and Tropical Medicine, Gyeongsang National University College of Medicine, Jinju, Korea. Their length and weight were individually measured and the fish species were identified with the aid of a Korean ichthyologist and Search FishBase site in internet (Table 1) [24]. Individual fish was finely ground in a mortar with pestle, the ground fish meat was mixed with artificial gastric juice, and the mixture was incubated at 36˚C for about 2 hr. The digested material was filtered through a 1×1 mm2 of mesh, and washed with 0.85% saline until the supernatant became clear. The sediment was carefully examined under a stereomicroscope. The metacercariae of each species (only ZTM) were separately collected viewing from the general feature and counted to get hold of the prevalence (%) and intensity of infection (no. of ZTM per fish infected) by fish species [5].

Fig. 1

The surveyed area (red region), Yen Bai province, northern Vietnam (from Wikipedia website [23]).

Table 1

Fish collected from a local market in Yen Bai city, Yen Bai province, Vietnam

Species of fish	No. of fish examined	Length		Weight
		Range	Average	Range	Average
Chanodichthys erythropterus	30	9.6–13.2	11.2	10.9–31.3	19.3
Toxabramis houdemeri	30	7.5–9.4	8.1	5.4–10.2	6.6
Carassius auratus	27	8.1–12.9	9.9	12.3–50.6	23.9
Saurogobio immaculatus	10	6.4–11.2	9.3	3.9–17.2	10.8
Opsariichthys bidens	10	-	-	14.0–28.0	21.8
Squalidus atromaculatus	9	7.3–8.0	7.7	6.2–9.0	7.6
Garra imbarbatus	2	-	7.0	7.4–7.5	7.5

Total 118 fish in 7 species were examined.

Total 4 species of trematode metacercariae, i.e., C. sinensis, Haplorchis pumilio, Haplorchis taichui, and Centrocestus formosanus, were detected. The prevalence of C. sinensis metacercariae were very high to be positive in 62 (69.7%) out of 89 fish in 5 species, i.e., Chanodichthys erythropterus (Fig. 2A), Toxabramis houdemeri (Fig. 2B), Saurogobio immaculatus, Opsariichthys bidens, and Squalidus atromaculatus, with the average intensity of 81.2 per fish infected. The metacercariae of 3 intestinal flukes, i.e., H. pumilio, H. taichui, and C. formosanus, were detected in 75.0%, 47.6%, and 31.7% of positive fish groups, respectively, and their mean intensities were 15.5, 10.3, and 2.2 per fish infected. The infection status of each ZTM by fish species is designated in (Table 2).

Fig. 2

Susceptible fish hosts, Chanodichthys erythropterus (A), Toxabramis houdemeri (B), to C. sinensis metacercariae in the surveyed area, Yen Bai city, Yen Bai province, northern Vietnam.

Table 2

Infection status of zoonotic trematode metacercariae (ZTM) by the fish species from a local market in Yen Bai city, Yen Bai province, Vietnam

ZTM and fish species infected	No. of fish examined	Infection rate (%)	No. of ZTM detected
			Range	Average
Clonorchis sinensis
Chanodichthys erythropterus	30	93.3	2–521	105.5
Toxabramis houdemeri	30	100	8–179	69.1
Saurogobio immaculatus	10	2.0	-	1.0
Opsariichthys bidens	10	10.0	-	1.0
Squalidus atromaculatus	9	11.1	-	1.0
Subtotal	89	69.7	1–521	81.2
Haplorchis pumilio
Chanodichthys erythropterus	30	100	2–152	28.6
Carassius auratus	27	70.4	1–14	4.9
Toxabramis houdemeri	20	75.0	1–19	4.5
Squalidus atromaculatus	9	11.1	-	1.0
Garra imbarbatus	2	50.0	-	3.0
Subtotal	88	75.0	1–152	15.5
Haplorchis taichui
Chanodichthys erythropterus	30	36.7	1–8	3.3
Saurogobio immaculatus	10	70.0	1–44	9.4
Garra imbarbatus	2	100	16–90	53.0
Subtotal	42	47.6	1–90	10.3
Centrocestus formosanus
Chanodichthys erythropterus	30	100	1–4	1.8
Toxabramis houdemeri	20	5.0	-	1.0
Saurogobio immaculatus	10	50.0	1–9	3.4
Subtotal	60	31.7	1–9	2.2

The metacercariae (n=20) of C. sinensis were elliptical and 140–175 (159 in average) by 120–160 (142) μm in size. They had nearly equal sized oral and ventral suckers, brownish pigment granules in the body, and an O-shaped excretory bladder in the posterior portion. The adult worms (40-day-old from an experimental hamster) were flat, elongate leaf-like, tapering anteriorly and somewhat pointed posteriorly, and 5.75–12.0 (av. 8.98) mm long and 1.55–2.15 (1.84) mm wide. Eggs were small, yellowish, mature, operculate, and 25–28 (26) by 13–14 (13) μm in size.

In the present study, 4 species of zoonotic trematode metacercariae, i.e., C. sinensis, H. pumilio, H. taichui, and C. formosanus, were detected in fish from a local market of Yen Bai city, Yen Bai province, northern Vietnam. Among these, C. sinensis metacercaria (CsMc) was the dominant species with regard to the intensity of infection in fish hosts (81.2 CsMc per fish infected). Chai et al. [8] found 6 species of ZTM, i.e., H. taichui, H. pumilio, C. formosanus, Procerovum varium, Stellantchasmus falcatus, and Heterophyopsis continua, in fish from Hanoi and Nam Dinh province, Vietnam. They were all intestinal flukes belonging to the family Heterophyidae, and H. pumilio was the dominant species. Phan et al. [19] also detected 6 species of ZTM, i.e., H. pumilio, H. taichui, H. yokogawai, C. formosanus, P. varium, and C. sinensis, in cultured and wild-caught freshwater fish from the Red River delta, in Nam Dinh province. H. pumilio was also the dominant species, and CsMc were found in only 1 out of 1,543 fish examined. Hung et al. [22] also detected 6 species of ZTM, i.e., H. pumilio, H. taichui, H. yokogawai, C. formosanus, P. varium, and C. sinensis, in fish from Ninh Binh province. Among them, Haplorchis spp. metacercariae were dominant, and CsMc were found in only 1 (12.5%) out of 8 Hemiculter leucisculus examined. In our study, CsMc were highly prevalent in several species of fish from Yen Bai city, Yen Bai province unlike in previous studies performed in adjacent localities of the other northern parts of Vietnam, i.e., Hanoi, Nam Dinh, and Ninh Binh province.

Two species of human-infecting liver flukes, C. sinensis and Opisthorchis viverrini, are known to be distributed in Vietnam. The former is prevalent in northern provinces, whereas the latter is mainly found in central and southern provinces [25]. In previous studies, the metacercariae of liver flukes were very rarely detected in fish from Vietnam. Only 19 O. viverrini metacercariae in total were found in 1.9% of fish from An Giang province, in the Mekong Delta of Vietnam [12]. In addition, Thien et al. [13] could not find CsMc at all in 13 species of cultured fish from the Mekong Delta area. Also, in Nghe An province, no liver fluke metacercariae were detected among farmed fish [15].

With regard to C. sinensis, the metacercariae were found in 1 of 1,185 silver carps, Hypophthalmichthys molitrix, from small-scale farms in northern parts, and in 1 out of 1,543 freshwater fish from Red River delta in Nam Dinh province [18,19]. Hung et al. [22] also detected CsMc in 1 (12.5%) of 8 H. leucisculus examined. In other northern provinces, prevalences of CsMc were very low, 0.1–0.4% [21]. However, in the present study, we detected a very high intensity of CsMc (81.2 per fish infected) from 69.7% freshwater fish in 5 species, i.e., C. erythropterus, T. houdemeri, S. immaculatus, O. bidens, and S. atromaculatus. In addition, it has been first confirmed in this study that 2 fish species, C. erythropterus and T. houdemeri, are highly susceptible for CsMc.

Clonorchiasis is known to be endemic among the residents of northern provinces of Vietnam. Especially, this disease was known to be prevalent in Nam Dinh and Ninh Binh provinces [25]. However, since the FIBOZOPA (Fish-Borne Zoonotic Parasite) project (2004–2012), it became known that various species of minute intestinal trematodes, i.e., H. pumilio, H. taichui, H. yokogawai, C. formosanus, S. falcatus, and P. varium, coexisted in these areas [3,8,18,19,21,22]. Dung et al. [3] reported 64.9% positive rate of minute trematode eggs from 615 residents of Nam Dinh province, and they recovered 6 species of trematode adults, i.e., C. sinensis, H. pumilio, H. taichui, H. yokogawai, S. falcatus, and Fasciolopsis buski, from 33 residents positive for small trematode eggs after treatment with praziquantel and purging with MgSO4. Adult worms of C. sinensis were recovered from 17 (51.5%) residents, and the intensity of infection was 4 worms in average, whereas adults of H. pumilio and H. taichui were recovered from all 33 (100%) and 23 (69.7%) residents, with the infection intensities of 416 and 40 in average, respectively. Likewise, in endemic areas of clonorchiasis in northern Vietnam, the coexistence of minute intestinal trematodes has been confirmed by ZTM studies performed in the same survey areas [8,18,19,21,22].

Several working groups reported the morphological characteristics of ZTM with their infection status in fish hosts from the specific regions in Southeast Asian countries [8-11]. Especially in Vietnam, Chai et al. [8] described the morphological characteristics of 6 species of ZTM, i.e., H. taichui, H. pumilio, C. formosanus, P. varium, S. falcatus, and H. continua, from fish in Hanoi city and Nam Dinh province, and they also described the morphologies of adult flukes, i.e., H. taichui, H. pumilio, C. formosanus, P. varium, and S. falcatus, recovered from experimentally infected animals. In our study, C. sinensis metacercariae were nearly the same size (140–175×120–160 μm) as those of the Korean-origin (150–170×130–150 μm), but they are, more or less, smaller than those of O. viverrini (190–250×150–220 μm) [5,6,11]. Because of the general morphology of the metacercariae, including the elliptical shape, nearly same sized 2 suckers, brownish pigment granules in the body, and O-shaped excretory bladder, of 2 species of liver flukes is very similar except for the cyst size, it is difficult to distinguish one from the other if they are mixed-infected. Therefore, when the metacercariae of liver flukes are detected in fish from somewhere of Vietnam, it would be better to infect them to experimental animals to obtain adult flukes for species identification. Adult worms of C. sinensis in this study were more slender compared to those of the Korean-origin. The ratio of the body length to body width was higher (4.87–5.01 in this study) than that of the Korean-origin (4.36) (Table 3). Other morphological characters of C. sinensis adults were almost the same in both origins. However, the egg size was more or less smaller in this study (25–28×13–14 μm) than those of the Korean-origin (27–33×15–19 μm).

Conclusively, it has been confirmed in this study that several species of freshwater fish continue to play the role of the second intermediate host and infection source of zoonotic trematodes, including C. sinensis and 3 species of intestinal flukes in Yen Bai province, Vietnam. In particular, the prevalence and intensity of CsMc were much higher in this study than those of previous studies. To reveal the detailed epidemiological situation of zoonotic trematode infections in Yen Bai province, Vietnam, more profound studies on intermediate (snail and fish) and definitive hosts (humans and reservoir host animals) should be performed in the near future.