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INFECTION STATUS AND MOLECULAR IDENTIFICATION OF DIGENEAN CERCARIAE IN SNAILS IN KIM SON DISTRICT, NINH BINH PROVINCE AND BA VI DISTRICT, HA NOI

In this study, the molecular method was used to identify digenean cercariae from freshwater snails in Kim Son District (Ninh Binh Province) and Ba Vi District (Ha Noi). A total of 9 snail species were collected and examined for cercarial infection. Three snail species (Radix swinhoei, Angulyagra polyzonata, and Pomacea canaliculata) were not infected, while the other 6 species (Austropeplea viridis, Gyraulus convexiusculus, Parafossarulus striatulus, Bithynia fuchsiana, Melanoides tuberculata, and Tarebia granifera) were infected with digenean cercariae at low infection rates, ranging from 2.3% to 6.3%. Seven cercarial groups were identified: Echinostome, Monostome, Parapleurophocercaria, Xiphidiocercaria, Fucocercaria, Gymnocephalous, and Megalurous. Snails M. tuberculata and P. striatulus were infected with 4 cercarial groups, A. viridis and G. convexiusculus snails were infected with 2 groups, 2 snail species B. fuchsiana and T. granifera were infected with one group of cercaria. The analyses of ITS2 sequences of the cercarial groups identified the larvae of 9 trematode species, namely Echinostoma revolutum, Echinochasmus japonicus, Notocotylus intestinalis, Philophthalmus gralli, Haplorchis pumilio, Procerovum cheni, Fasciola gigantica, Australapatemon burti, and Cyathocotyle prussica. Among them, the last three species, P. cheni, A. burti and C. prussica, were found for the first time in Vietnam. In addition, the ITS-2 sequence of Gymnocephalous cercariae which was previously identified as Sphaeridiotrema monorchis, from P. striatulus snail was 97% similar to that of Sphaeridiotrema pseudoglobulus. Likewise, ITS-2 sequence of Echinostome cercaria from B. fuchsiana snail was 93% similar to that of E. japonicus and that of Xiphidiocercaria cercaria from M. tuberculata snail was 93% similar to Lecithodendrium spathulatum.


INTRODUCTION
Parasitic trematodes have complex life cycles, requiring at least two hosts. Of which, the obligatory first intermediate hosts are mollusks (usually freshwater snails), where the development of larval stages (sporocyst, redia, and cercaria) takes place. Therefore, the identification of trematode larvae in snails helps to understand life cycles of trematodes and assess the trematode infection situation of humans and animals, providing a scientific basis for controlling the intermediate hosts of trematode transmission.
Cercarial larvae of trematode are classified into 38 groups (Schell, 1970). The determination of cercarial groups is based on morphological characteristics, although it is not easy to distinguish cercariae among various species. To overcome this limitation, molecular techniques have been used for the accurate identification of trematode cercariae (Chuboon and Wongsawad, 2009;Chontananarth and Wongsawad, 2010;Anucherngchai et al., 2016).
In Vietnam, surveys on trematode larvae in snails have been carried out in some locations. However, the previous reports mainly published the prevalence of trematode cercariae or morphological identification of cercarial groups (Le et al., 1990(Le et al., , 2000The, 1993;Chau et al., 1996;Kim and Vinh, 1997;Dung et al., 2010;Hung et al., 2015;Clausen et al., 2015;Dung et al., 2019). In this study, we used a molecular technique to identify trematode larvae in freshwater snails in Kim Son District, Ninh Binh Province and Ba Vi District, Hanoi, where livestock and poultry are commonly raised.

Study sites
Kim Son District, Ninh Binh Province and Ba Vi District, Ha Noi.

Methods
Freshwater snails were collected in Kim Son District, Ninh Binh Province and Ba Vi District, Hanoi, and were classified according to Thanh et al. (1980). The snails were examined for cercarial infection using the shedding and crushing methods. Each snail was kept separately in a shedding tube of 2 × 2 cm (diameter × depth). In the next morning, the shedding tubes were observed under a stereoscopic microscope to detect cercariae escaped from the snails. The cercariae were transferred to a glass slide and covered with a cover glass for morphological observation under a microscope. The cercariae were classified into a group level according to the keys described by Schell, 1970, Frandsen andChristensen, 1984. Some cercariae were used for molecular analyses.
Then, the snails were examined to observe young larvae remaining in the snails using the crushing method. For small and soft snail species, the snails were pressed between 2 glass plates. For larger species of snails, a shear was used to remove the hard shell of the back of the screw and immerse it in a small water drop on the slide glass. The slide was checked under a microscope.
Cercarial samples were molecularly analyzed to identify species using ITS2 sequences, according to the following procedure: Genomic DNA from cercariae was extracted using the QIAamp DNA Mini Kit; the ITS2 sequence was amplified by PCR technique using a primer pair: 3S (forward primer, 5'-CGC TGG ATC ACT CGG CTC GT-3') and A28 (reverse primer, 5'-CCT GGT TAG TTT CTC TTC CGC-3') (Bowles et al. 1993); the PCR products were purified using Qiaquick PCR purification Kit (Qiagen Inc., Tokyo, Japan). The forward and reverse strands were sequenced directly using the Genetic Analyzer 3130 using Big-Dye terminator cycle-sequencing kit v3.1 (Applied Biosystems). The obtained sequences were compared with DNA sequences available from GenBank with the BLAST search program.

Identification of cercariae
All cercarial groups were molecularly analyzed using ITS2 sequences as a marker. The results of the BLAST search identified the larvae of 9 trematode species with the similarities over 99% to the sequences deposited in GenBank.

DISCUSSION
According to previous reports, the prevalence of trematode larvae in freshwater snail species was relatively high. The (1993) reported the prevalence of C. sinenis cercariae in M. tuberculata in Nam Dinh Province up to 90%. Le et al. (1995) found trematode larvae in 30.6% of L. swinhoei and 26.3% of L. viridis snails in Ha Tay Province. The survey conducted by Kim and Vinh (1997) showed the prevalence of Fasciola cercariae in 62.1% of Lymnaea snails in Bac Ninh Province. In contrast, recent surveys have revealed low prevalences of trematode larvae in freshwater snails. Dung et al. (2010) reported that 13.3% of M. tuberculata snails in Nghia Hung District, Nam Dinh Province was infected with trematode cercariae. A survey in Thai Binh, Nam Dinh, Ninh Binh, and Thanh Hoa Provinces revealed that the prevalence of trematode larvae in 13 snail species collected was generally low (Clausen et al. 2015). Hung et al. (2015) collected 7 snail species in Gia Vien District, Ninh Binh Province, and found a very low prevalence of cercariae in two snail species, M. tuberculata (7/858 = 0.8%) and B. fuchsiana (2/1,894 = 0.1%). Phuong et al. (2019) reported that the prevalence of cercarial infection in M. tuberculata snail was 14.3% in Ha Trung District, Thanh Hoa Province. Dung et al. (2019) surveyed in some suburban districts of Ha Noi, and found that among 9 snail species collected, 5 snails (M. tuberculata, B. fuchsiana, A. viridis, G. convexiusculus, Stenothyra messageri) were infected with cercarial larvae at low infection rates ranging from 0.3 to 2.6%. The results of the present study also indicated that the infection rates of trematode larvae in freshwater snails were relatively low (2.3% -6.3%). These reflect a significant reduction of trematode infection in humans and animals in comparison to those in the past.
Previously published studies focused only on infection status of snails or identification of the cercarial groups. Le et al. (1995) described 3 cercarial groups (Echinostome, Fucorcercaria, and Xiphidiocercaria) from L. swinhoei and L. viridis snails in Ha Tay Province. Dung et al. (2010)  In the present study, using ITS2 sequence analysis, we accurately identified the larvae of nine trematode species (E. revolutum, E. japonicus, N. intestinalis, P. gralli, H. pumilio, , F. gigantica, A. burti, C. prussica, and P. cheni). Among them, three species, A. burti, C. prussica, and P. cheni, were first detected in Vietnam. It should be noted that M. tuberculata snail has been reported as the first intermediate host of small liver fluke Clonorchis sinensis (rev. by Doanh and Nawa, 2016), of which cercariae belong to the Parapleurophocercaria group. In this study, Parapleurophocercaria group cercariae from M. tuberculata snail were identified as the larvae of small intestinal flukes, H. pumilio and P. cheni. Since cercariae of C. sinensis and small intestinal flukes, H. pumilio and P. cheni, all belong to the Parapleurophocercaria group, it is possible that mis-identification might occur in the previous publications, in which the classification of cercariae from freshwater snails was solely based on the morphological characteristics (Doanh and Nawa, 2016).
In this study, ITS-2 sequence of Gymnocephalous group cercariae from P. striatulus snail was 97% similar to that of Sphaeridiotrema pseudoglobulus. This cercarial group found in Nam Dinh Province was classified as S. monorchis through the experimental infection in ducks (Besprozvannykh et al. 2013). The present results showed that the ITS2 sequence of S. pseudoglobulus from P. striatula snail was 97% similar to that of S. monorchis.
The Echinostome groups from B. fuchsiana and P. striatulus snails were morphologically similar to each other, but the cercariae from P. striatulus was identified as E. japonicus with the similarity over 99%. On the contrary, the cercariae from B. fuchsiana snails showed a lower level of ITS-2 sequence similarity (93%) to E. japonicus. Thus, the Echinostoma groups from B. fuchsiana snails is possibly cercariae of another species, E. beleocephalus, which was reported in Vietnam (Le, 1995).
The Xiphidiocercaria group from M. tuberculata snail has the highest similarity (93%) with Lecithodendrium spathulatum. The trematodes of the genus Lecithodendrium are typical trematodes of bats. In Vietnam, three species of the genus Lecithodendrium (L. daovantieni, L. rohdei, and Lecithodendrium sp.; Le, 1995) were reported in bats. However, molecular data of these species are not available in GenBank database for comparison.

CONCLUSION
Nine snail species were collected in Ba Vi District, Ha Noi and 7 species were collected in Kim Son District, Ninh Binh Province. While three snail species, R. swinhoei, A. polyzonata, and P. canaliculata, were negative for cercariae, the other 6 snail species were infected with cercariae with low infection rates, ranging from 2.3% to 6.3%. By morphology, 7 cercarial groups, Echinostome, Monostome, Parapleurophocercaria, Xiphidiocercaria, Fucocercaria, Gymnocephalous, and Megalurous, were identified. The snail hosts M. tuberculata and P. striatulus were infected with 4 cercarial groups, snails A. viridis and G. convexiusculus were infected with 2 groups, snails B. fuchsiana and T. granifera were infected with one group.