Southeast Asian J Trop Med Public Health

TREMATODE INFECTION OF FRESHWATER SNAIL, FAMILY BITHYNIIDAE IN THAILAND

Jutharat Kulsantiwong1,2, Sattrachai Prasopdee1,3, Supawadee Piratae1, Panita Khampoosa1, Chalida Thammasiri1, Apiporn Suwannatrai1, Thidarut Boonmars1, Vithoon Viyanant4, Jiraporn Ruangsitichai5, Pairat Tarbsripair6 and Smarn Tesana1

1Food-Borne Parasite Research Group, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen; 2Department of Biology, Faculty of Science, Udon Thani Rajabhat University, Udon Thani; 3Chulabhorn International College of Medicine, Thammasat University, Klong Luang, Pathum Thani; 4Center of Excellence for Research in Biomedical Sciences, Thammasat University, Klong Luang, Pathum Thani; 5Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok; 6Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand

Abstract. Opisthorchis viverrini is restricted to and requires for its aquatic life cycle only Bithynia snail as first intermediate host but many species of cyprinid fish as second intermediate hosts. A survey in Thailand of trematode infection in freshwater snails of the family Bithyniidae carried out during October 2008 - July 2009 found a total of 5,492 snails, classified into ten species distributed in various geographic areas. Bithynia funiculata and Gabbia pygmaea were localized to the north, B. s. goniomphalos, Wattebledia siamensis and W. crosseana to northeast and B. s. siamensis, Hydrobioides nassa and G. wykoffi to central region. W. baschi and G. erawanensis was found only in the south and Erawan waterfall, Kanchana- buri Province, respectively. Trematode infection rate was 3.15%. Cercariae were identified as belonging to six types, namely, amartae , monostome, mutabile, O. viverrini, virgulate, and unknown. The prevalence of cercarial infection in B. s. goniomphalos of amartae, mutabile, O. viverrini, virgulate, and unknown type cercaria was 0.55%, 0.74%, 1.07%, 2.87%, and 0.37%, respectively, and in B. s. sia- mensis monostome (1.10%) and virgulate (0.55%). Only virgulate cercariae were shed from W. crosseana (3.85%) and W. siamensis (5.19%). Cercariae of the unknown type were found in G. wykoffi (1.69%). No infection of O. viverrini cercariae was detected in the other species. Keywords: Opisthorchis viverrini, cercarial type, family Bithyniidae, snail, Thailand

INTRODUCTION Correspondence: Dr Smarn Tesana, Food- Borne Parasite Research Group, Department of Freshwater snails of the family Bithy- Parasitology, Faculty of Medicine, Khon Kaen niidae serve as intermediate hosts for the University, Khon Kaen 40002, Thailand. multiplication of intramolluscan stages Tel: +66 (0) 43 363 434; Fax: +66 (0) 43 202 475 of many types of trematodes. Snails in E-mail: [email protected], tessmarn@yahoo. this family were first described for native com snails in Europe and Asia in the early

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1870s (Brandt, 1974; Mills et al, 1994). A Thailand and Vietnam) (IARC, 1994; Keiser number of species have been reported as and Utzinger, 2005; IARC, 2011). The intermediate hosts for trematode parasites aquatic life cycle of O. viverrini requires (Wykoff et al, 1965; Chitramvong and Bithynia snail as the first intermediate Upatham, 1989; Ellen et al, 2008). host and many species of cyprinid fish as In Thailand, three genera of Bithy- second intermediate hosts (Wykoff et al, niidae were classified by Brandt (1974), 1965; Harinasuta et al, 1984; WHO, 1995; viz. Bithynia (2 subgenera of Digoniostoma Waikagul, 1998). In the endemic regions and Gabbia), Hydrobioides and Wattebledia. of northeast Thailand, the prevalence of O. Chitramvong and Upatham (1989) clas- viverrini infection in humans and cyprinid sified Gabbia as a valid genus. According ranges from 2.1% to 97% (Brockelman et al, to Chitramvong (1991, 1992), 10 species/ 1986; Haswell-Elkins et al, 1992; Sriamp- subspecies, namely, Bithynia funiculata, orn et al, 2004), but is lower in snail hosts B. siamensis goniomphalos, B. s. siamensis, (0.083%-1.6%) (Wykoffet al, 1965; Upatham Gabbia wykoffi, G. pygmaea, G. erawanen- and Sukhapanth, 1980; Upatham et al, sis, Wattebledia crosseana, W. siamensis, W. 1983). Moreover, epidemiological surveys baschi and Hydrobioides nassa make up during 1981-1991 indicated that there is an the family Bithyniidae as identified by increase in incidence of human O. viverrini morphological characteristics of shells, infection in the north and central regions internal organs and radula cusp patterns. of the country, whilst in the northeastern Their ecological habitats have been re- region the incidence is decreasing due ported in various types of water bodies in part to the implementation of a drug and a number of species share the same control strategy (Jongsuksuntigul and ecological niches. Within Bithyniidae, Imsomboon, 1998). only the genus Bithynia has been identified Experimental infection studies show- as being of medical importance (Wykoff ed that B. funiculata and B. s. siamensis have et al, 1965; Harinasuta et al, 1984; WHO, higher susceptibility to O. viverrini than 1995; Waikagul, 1998; Sri-aroon et al, 2005). B. s. goniomphalos, and within the same In Thailand, two species of snails, B. taxon immature laboratory bred snails funiculata and B. siamensis (2 subspecies are more susceptible to O. viverrini infec- of B. s. goniomphalos and B. s. siamensis, tion than mature field snails (Chanawong respectively) have been reported to serve and Waikagul, 1991). Trematode infection as the first intermediate hosts of human in B. s. goniomphalos showed that it could liver fluke, Opisthorchis viverrini (Wykoff serve as an intermediate host for 7 types of et al, 1965; Brockelman et al, 1986; Sri-aroon cercariae (amartae, amphistome, furcocer- et al, 2005). Opisthorchiasis is believed to cariae, monostome, O. viverrini, virgulate predispose the onset of cholangiocarci- and xiphidiocercaria) when shedding is noma , a cancer of the bile duct (Thamavit induced by illumination (Adam et al, 1993; et al, 1978; Haswell-Elkins et al, 1992; IARC, Nithiuthai et al, 2002; Sri-aroon et al, 2005). 1994; Sithithaworn et al, 1994; Vatanasapt Four types of cercariae (lophocercous, et al, 2000; Watanapa and Watanapa, 2002; monostome, pleurolophocercous and Honjo et al, 2005; IARC, 2011). O. viverrini is virgulate) were found in B. funiculata in a medically important parasite of humans the north but no O. viverrini infection was in the Greater Mekong subregion (Cam- detected (Ngern-klun et al, 2006). bodia, Lao People’s Democratic Republic, There is no report of human liver

Vol 46 No. 3 May 2015 397 Southeast Asian J Trop Med Public Health fluke, O. viverrini, or other cercarial in- time shedding. Cercariae were preserved fections in the other species of family in 10% formalin and then observed under Bithyniidae apart from the genus Bithynia a stereo microscope (40x magnification). even though they are in the same family Snail specimens were observed for emerg- and share the same ecological niche. The ing cercariae at least 2 times within one present study examined the prevalence of month. Morphology study of cercariae trematode infections particularly O. viver- was undertaken using both unstained rini, in snail species of the family Bithy- and stained [with 0.1% (w/v) Fast Red B niidae in various localities in Thailand. or 0.1% (w/v) Malachite Green dye] under a light microscope and photographed. MATERIALS AND METHODS Cercaria types were identified based on available morphological description Snail collection (Wykoffet al, 1965; Schell, 1970; Frandsen The study localities for snail collec- and Christensen, 1984). For confirmation tion were in the north, south, west, north- of O. viverrini cercariae, species specific east and central areas of Thailand. The PCR was used (Wongratanacheewin et al, locations were selected based on informa- 2001). tion from previous reports (Brandt, 1974; Chitramvong, 1989, 1992; Nithiuthai et al, RESULTS 2002; Ngern-klun et al, 2006). The snail A total of 5,492 snails were collected survey was conducted during October from 17 locations of Thailand: Nong 2008 to July 2009. The locations of snail Khai, Nong Bua Lam Phu, Khon Kaen, collection were recorded using the Global Maha Sarakham, Yasothon, and Roi Et in Positioning System (Garmin model Nuvi the northeast; Chiang Mai, Lamphun in 203, Taiwan) and plotted on a map (Fig the north; Phitsanulok, Phichit, Nakhon 1). All snails were collected by wire-mesh Sawan, Sing Buri, Saraburi, Suphan Buri, scoop or by hand, and each specimen Bangkok in central; Kanchanaburi in the was cleaned, air-dried, labeled and kept west; and Surat Thani in the south (Fig 1). in porous plastic bags. The snail samples Snails were classified into ten species/sub- were brought back to the laboratory to be species in family Bithyniidae (Fig 2). The examined for trematode infection. Snails distribution of the snails were as follows: were identified based on shell morphol- B. funiculata (n = 120) and G. pygmaea (120) ogy for species following available keys from the north; B. s. goniomphalos (2,159), and descriptions (Brandt, 1974; Upatham W. siamensis (154) and W. crosseana (104) et al, 1983; Chitramvong, 1992). from the northeast; B. s. siamensis (1,813), Examination for trematode infection H. nassa (160) and G. wykoffi (592) from Trematode infection of snails were central; W. baschi (150) from the south conducted using a cercarial shedding and G. erawanensis (120) from Erawan method (day time and night time shed- waterfall, Kanchanaburi Province in the ding) as follows. Snails were placed in west (Table 1). plastic containers with 50 ml of dechlori- No night time cercarial shedding was nated tap-water, exposed to electric light observed. B. s. goniomphalos, B. s. siamensis, for 2 hours for day time shedding, or G. wykoffi, W. crosseana and W. siamensis were covered with a black plastic sheet shed cercariae in day time, with 6 types of overnight at room temperature for night cercariae, namely, amartae, monostome,

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no fin fold. Mutabile cercaria has oral and ventral suckers of equal sizes and no tail. O. viverrini cer- caria has a pair of eye spots and fin fold. Virgulate cercaria has virgule organs, ventral suckers smaller than oral suckers, and tail without a dorsoventral fin fold. The unknown type cercaria has a pair of eye spots located laterally to the oral sucker and the tail with a fin-fold. Cercarial emergence was ob- served in 173 (3.15%) snails, the most frequent being in B. s. goniom- phalos (5.60%), followed by W. sia- mensis (5.19%), W. crosseana (3.85%), G. wykoffi (1.69%) and B. s. siamensis (1.65%). The cercarial types found in B. s. goniomphalos were amartae (0.55%), mutabile (0.74%), O. viver- rini (1.07%), virgulate (2.87%), and unknown type cercaria (0.37%). Only monostome (1.10%) and vir- Fig 1–Map of Thailand showing collection localities in gulate (0.55%) cercariae were found five regions of Thailand. 1, Nong Khai; 2, Nong in B. s. siamensis, virgulate cercariae Bua Lam Phu; 3, Khon Kaen; 4, Maha Sarakham; (3.85% and 5.19%) in W. crosseana 5, Yasothon; 6, Roi Et; 7, Chiang Mai; 8, Lamphun; 9, Phitsanulok; 10, Phichit; 11, Nakhon Sawan; 12, and W. siamensis, respectively, and Sing Buri; 13, Saraburi; 14, Suphan Buri; 15, Bang- unknown type cercariae (1.69%) in kok; 16, Kanchanaburi; 17, Surat Thani. G. wykoffi(Table 2). mutabile, O. viverrini, virgulate, and un- DISCUSSION known type cercaria, being identified (Fig The distribution of snail species is 3). Identification of O. viverrini cercariae mostly limited to certain geographical ar- was verified by PCR amplification of a 330 eas and along river wetlands. In Thailand, bp fragment (data not shown) (Wongrata- B. funiculata distribution is restricted to nacheewin et al, 2001). the north of the country but B. s. siamensis The morphological characteristics of is found not only in the north but also the cercaria types are as follows. Amartae distributed throughout the central region cercaria has oral and ventral suckers of (Brandt, 1974), similar to the findings of equal sizes or ventral sucker that is larger the present study. B. s. goniomphalos is lim- than the oral sucker, a tail without a dor- ited to the northeast and no other Bithynia soventral fin fold, and no virgule organ. species are found as determined by DNA Monostome cercaria presents distinct analysis (Kulsanthiwong et al, unpub- three-eye spots, and with the tail having lished data). In this study H. nassa was

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were found only in the northeast, but in previous reports they have been de- tected in the north, central and northeast(Brandt, 1974; Chitramvong, 1991, 1992). As for W. baschi and G. erawanensis, their distribu- tion was limited to the south and the Erawan waterfall, Kanchanaburi Province as has been previously report- ed (Brandt, 1974; Chitram- Fig 2–Shell morphology of ten snail species of the family Bithyni- vong and Upatham, 1989). idae. (A) B. funiculata; (B) B. s. goniomphalos; (C) B. s. siamensis; Field investigation in (D) H. nassa; (E) W. baschi; (F) W. crosseana; (G) W. siamensis; this study indicated that G. wykoffi G. pygmaea G. erawanensis (H) ; (I) ; (J) . Scale bars Bithyniidae snails were = 1 mm. infected with a number of trematodes, namely, amar- tae, monostome, mutabile, O. viverrini, virgulate, and unknown type cercaria. The species of snails infected were B. s. goniomphalos, B. s. siamensis, G. wykoffi, W. crosseana, and W. siamensis. To date, reports of natural infection have only been available for B. s. goniom- phalos. In the present study Fig 3–Morphology of cercariae found in snails collected in Thai- six cercarial types (amar- land. (A) mutabile; (B) monostome; (C) O. viverrini; (D) tae, monostome, mutabile, amartae; (E) unknown; (F) virgulate. ES, eye-spot; ff, fin O. viverrini, virgulate, and fold; OS, oral sucker; VS, ventral sucker. Scale bar = 1 µm. unknown) were found in B. s. goniomphalos. In previous discovered downstream of Chao Phraya reports, 9 cercarial types (amartae, am- River and its tributaries, in Sing Buri phistome, furcocercariae, lophocercous, Province and in the northern provinces monostome, O. viverrini, pleurolopho- as previously reported (Brandt 1974; Chit- cercous, virgulate, and xiphidiocercaria) ramvong, 1991, 1992). G. wykoffi was pres- were found in B. s. goniomphalos (Adam ent in central, northern and northeastern et al, 1993; Nithiuthai et al, 2002; Sri-aroon Thailand, which is different from previous et al, 2005). Mutabile and unknown cercar- reports (Brandt, 1974; Chitramvong, 1991, iae were found in our study but no furco- 1992), but only G. pygmaea was found in cercariae, lophocercous, pleurolophocer- the north. W. siamensis and W. crosseana cous, or xiphidiocercaria were detected.

400 Vol 46 No. 3 May 2015 Trematode Infection in Snails of the Family Bithyniidae 120 120 120 592 160 104 154 150 Total 1,813 5,492 2,159 0 0 0 0 0 0 0 0 0 17 (1) 150 South 150 0 0 16 120 (12) West 0 0 0 15 (9) 0 0 0 0 150 0 0 0 0 0 14 (12) 482 135 250 482 285 520 0 0 0 0 0 0 0 52 50 13 (10) 302 404 0 0 0 0 0 12 50 50 100 0 0 0 0 0 0 0 0 Central 11 (2) (4) 20 65 85 0 0 0 0 0 0 38 38 10 (1) 0 0 0 34 (7) 0 0 0 0 120 0 0 0 0 0 Region [number of sites(s)] (3) 178 154 178 Table 1 Table 0 0 North 7 8 9 52 120 120 (12) 0 0 0 0 0 0 0 0 317 0 6 20 609 20 0 (1) 0 0 0 0 0 0 0 0 0 5 87 87 0 0 0 0 0 0 (4) (1) 121 121 0 0 0 0 0 0 3 4 34 Northeast 134 120 (30) 2,045 1,757 0 0 0 0 0 0 0 2 88 20 20 48

a 0 0 0 0 0 0 0 0 0 Snails collected from five regions of Thailand during October 2008 - July 2009. July - 2008 October during Thailand of regions five from collected Snails 1 (3) 126 126

Species B. funiculata B. s. siamensis B. s. goniomphalos crosseana Wattebledia siamensis W. baschi W. Gabbia wykoffi G. pygmaea G. erawanensis nassa Hydrobioides Total 1, Nong Khai; 2, Nong Bua Lam Phu; 3, Khon Kaen; 4, Maha Sarakham; 5, Yasothon; 6, Roi Et; 7, Chiang Mai; 8, Lamphun; 9, Phitsanulok; Et; 7, Chiang Mai; 6, Roi Yasothon; 5, Phu; 3, Khon Kaen; 4, Maha Sarakham; Lam 1, Nong Khai; 2, Bua a Nakhon Sawan; 12, Sing Buri; 13, Saraburi; 14, Suphan 15, Bangkok; 16, Kanchanaburi; 17, Surat Thani. 10, Phichit; 11,

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Table 2 Cercarial infection rates in freshwater snails collected during October 2008 - July 2009.

Species No. of snails No. of Type of cercaria No. of snails with infected snails (%) cercarial type (%)

B. s. gomiomphalos 2,159 121 (5.60) Virgulate 62 (2.87) O. viverrini 23 (1.07) Mutabile 16 (0.74) Amartae 12 (0.55) Unknown 8 (0.37) B. s. siamensis 1,813 30 (1.65) Monostome 20 (1.10) Virgulate 10 (0.55) B. funiculata 120 0 - 0 W. crosseana 104 4 (3.85) Virgulate 4 (3.85) W. siamensis 154 8 (5.19) Virgulate 8 (5.19) W. baschi 150 0 - 0 G. wykoffi 592 10 (1.69) Unknown 10 (1.69) G. pygmaea 120 0 - 0 G. erawanensis 120 0 - 0 H. nassa 160 0 - 0 Total 5,492 173 (3.15) - 173 (3.15)

O. viverrini cercariae have been found G. wykoffi, W. crosseana and W. siamensis previously in the genus Bithynia in Thai- in Thailand. land (Brockelman et al, 1989; Adam et al, The infection rates of trematodes 1993; Nithiuthai et al, 2002; Sri-aroon et al, varied and were dependent on snail spe- 2005; Ngern-klun et al, 2006; Kiatsopit et al, cies, locality, season and cercarial type. 2012) and not in other genera, which was Trematode infections in B. s. gonimophalos, confirmed by our study. Our results also B. s. siamensis, G. wykoffi W. crosseana, and showed that no trematode infection was W. siamensis examined by cercarial shed- detected in B. funiculata from the north. ding gave infection prevalence of 5.60%, Nithiuthai et al (2002) and Ngern-klun 1.65%, 1.69%, 3.85%, and 5.19%, respec- et al (2006) reported that B. funiculata in tively. In previous reports, infection of B. the north of Thailand is infected with the s. siamensis has a similar rate (0.083% to trematode cercariae, with virgulate being 1.6%) (Upatham and Sukhapanth, 1980). the most common type. However, in our study only 3.15% of the The distribution of B. s. siamensis snails collected were infected with trema- snails is only in the central and north of todes as examined by cercarial shedding, Thailand and is not detected in the north- with the highest infection rate in B. s. go- east using morphological or PCR-based niomphalos (5.60%), which is higher than analysis (Kulsanthiwong et al, unpub- that previously reported (Nithiuthai et al, lished data). However, previous studies 2002), but the prevalence of each type of have detected these cercariae only in cercarial infection is not different. Kiasopit Bithynia snails while our results showed et al (2012) have recently reported that the for the first time trematode infection in total prevalence of O. viverrini infection in

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B. s. goniomphalos is 3.04% in northeastern between different geographical locali- Thailand, higher than other previous ties, between reservoir and human hosts, reports (Wykoff et al, 1965; Upatham density of snail population, water quality, et al, 1983; Brockelman et al, 1986). In the temperature and rainfall (Upatham et al, present study, O. viverrini was found only 1983; Nithiuthai et al, 2002; Ngern-klun in B. s. goniomphalos with a prevalence et al, 2006). Furthermore, Sri-aroon et al of 1.07%, similar to previous reports in (2007) reported that changes in irrigation Yang Talat District, Kalasin Province practice and particularly the development (1.3%) but higher than in Kamalasai Dis- of large-scale irrigation systems are as- trict, Kalasin Province (0.61%) and Khon sociated with increasing risk of infection, Kaen Province (0.03%-0.36%) (Lohachit, possibly due to the ideal habitat they form 2001; Sri-aroon et al, 2005). Differences for snails. in the overall prevalence of trematode In summary, ten species/subspecies of infections may involve many factors, in- bithynid snails were found in this study cluding different collection time, locality, and the distribution varied depending season, type and number of parasite and on the species and ecology of the water intermediate host, availability of infected bodies. Three snail species/subspecies in definitive or reservoir hosts. Brockelman this family served as intermediate hosts et al (1986) reported that under conditions of human liver fluke. This study revealed of adequate rainfall, two generations of that currently B. siamensis and especially B. s. goniomphalos normally are produced B. s. goniomphalos from northeastern Thai- per year, occurring in the first summer land served as intermediate hosts of O. rains in April or May and after the fall viverrini with varying levels of prevalence monsoon floods. A number of snails are of infection. Knowledge of the infection seen in November and have migrated to rates and distributions of the host snail the edge of a new environment within a populations is essential and must be few months. Therefore, the percentage of taken into consideration when develop- infected snails may be remarkably low ing future control strategies. The results between November and March, which of this study can be used as a data base to overlaps the sample collection period in examine the diversity of snail hosts and our study. to introduce control strategies regionally. Generally, the populations of snails fluctuate according to rainfall, with snail ACKNOWLEDGEMENTS populations declining as a result of heavy rains in the rainy season, which cause The authors thank the Office of the flushing of snail habitats. The snails that Higher Education Commission for a CHE survive from flooding take a few months PhD Scholarship to Miss Jutharat Kulsan- to resettle and subsequently migrate to tiwong; the Department of Parasitology, the edge of reservoirs and begin to repro- Faculty of Medicine, Khon Kaen Univer- duce, reaching a carrying capacity of the sity; Thammasat University; and Udon new environment within a few months Thani Rajabhat University. Additionally, (Upatham et al, 1983; Nithiuthai et al, this work was supported by the Higher 2002). Many factors are involved in the Education Research Promotion and Na- prevalence of trematode infection in snail tional Research University Project, Office hosts, with prevalence usually varying of Higher Education Commission, Thai-

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