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].

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).

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.