Introduction

Chronic opisthorchiasis caused by Opisthorchis viverrini (Leiper 1915) infection is generally accepted as the cause of bile duct cancer, cholangiocarcinoma (CCA) (IARC 2002; Bouvard et al. 2009). Prevalence of the O. viverrini infection has been reported in various parts of the Lower Mekong subregion countries, especially in Thailand and Lao PDR (WHO 1995; Sithithaworn et al. 2012; Aung et al. 2017). The northeast region of Thailand and the central and south regions of Lao PDR were found to have the highest prevalence than elsewhere in the countries (Jongsuksuntigul and Imsomboon 2003; Sithithaworn et al. 2012; Suwannatrai et al. 2018). In the transmission cycle, the parasite requires freshwater snails in Genus Bithynia (MolluscaBase 2021) and freshwater fish in Family Cyprinidae as first and second intermediate hosts, respectively. Humans get infected with the parasite by consuming raw or undercooked freshwater fish contaminated with infective metacercariae. Despite the freshwater fish’s prominent role in transferring infective metacercariae to humans, Bithynia snails still play a crucial role in disease propagation as amplifier hosts, capable of releasing cercariae into the environment (Wykoff et al. 1965; WHO 1995; Sithithaworn and Haswell-Elkins 2003; Prasopdee et al. 2019). Three taxa of the Bithynia have been reported as natural hosts for O. viverrini and are in different geographical habitats. Bithynia funiculata (Walker 1927) is found in northern Thailand and Lao PDR, and B. siamensis siamensis (Lea 1856) in Central, South, and Northern Thailand; Southern Vietnam; Cambodia; and Myanmar. Bithynia siamensis goniomphalos (Morelet 1866) in Northeastern Thailand; Lao PDR; Southern Vietnam; and Cambodia (Brandt 1974; Harinasuta and Harinasuta 1984; Sithithaworn et al. 2008; Kulsantiwong et al. 2013; Miyamoto et al. 2014; Dao et al. 2017; Prasopdee et al. 2019). Among the habitats of the Bithynia species, the B. siamensis goniomphalos is in the regions where opisthorchiasis was highly reported. In a natural field survey, the prevalence of O. viverrini infection in B. siamensis goniomphalos was ranged from 0.45 to 8.37% (Sri-Aroon et al. 2005; Kiatsopit et al. 2012; Kulsantiwong et al. 2015; Prasopdee et al. 2020), whereas 0.3% and 1.6% were reported in B. funiculata and B. siamensis siamensis, respectively (Upatham and Sukhapanth 1980; Ngern-klun et al. 2006). Recently, in laboratory conditions, Prasopdee et al. (2015a) revealed susceptibility of B. siamensis goniomphalos to O. viverrini infection was 32.96%. In addition, it was shown that the infection rate decreased by the time post-infection increased. Although B. siamensis goniomphalos is the primary species responsible for O. viverrini transmission in endemic areas, B. siamensis siamensis and B. funiculata remain potential hosts for transmission. This study aims to determine the susceptibility of B. siamensis siamensis and B. funiculata to O. viverrini to assess the risk of O. viverrini transmission in non-endemic areas. This study would greatly benefit parasitologists, malacologists, and epidemiologists to comprehend the biology of snail-borne opisthorchiasis.

Materials and methods

Preparation of Bithynia snails

Bithynia siamensis siamensis and B. funiculata were collected from Klong Luang, Pathum Thani, Thailand (14° 37′ 32.7″ N, 100° 20′ 28.0284″ E) and San Kamphaeng, Chiangmai, Thailand (18° 30′ 39.744″ N, 99° 14′ 34.836″ E), respectively. The snails were sorted and identified based on shell morphology following available protocols (Brandt 1974; Upatham et al. 1983; Chitramvong 1992). Briefly, B. siamensis siamensis is a more slender shape, narrower umbilicus, and weaker carina when compared with B. funiculata. The shell of B. funiculata is a subovate conic (almost narrowly subovate conic), dull, green to reddish-brown (olive-brown). It is slightly larger than B. siamensis siamensis. The umbilicus is funnel-shaped with a strong carina (Fig. 1).

Fig. 1
figure 1

Shell morphology of Bithynia species. A B. siamensis siamensis; B B. funiculata

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The snail samples were examined for trematode infection based on the cercarial shedding method (Prasopdee et al. 2020). Briefly, the snails were placed individually into plastic containers filled with 6 ml de-chlorinated tap water. Release of cercariae was induced by exposing the snails to 8-W electric light for at least 3 h, followed by covering the containers with black plastic sheets overnight. Uninfected snails of each species were used for further experiments.

Preparation of O. viverrini eggs

Two golden Syrian hamsters (Mesocricetus auratus) were experimentally infected with each 50 O. viverrini metacercariae. At 6 weeks post-infection, the infected hamsters were euthanized, and after that, O. viverrini adults were retrieved from biliary tracts and gallbladders. The recovered O. viverrini adults were washed several times with 0.85% sodium chloride solution and then subjected to obtain mature eggs from the distal part of the uterus under a stereoscope (Khampoosa et al. 2012). To undergo full maturation, the eggs were washed with distilled water and kept at room temperature for 2 weeks before experimental infection (Chanawong and Waikagul 1991).

Experimental infection and testing of susceptibility

For experimental infection, full-grown, medium-sized (0.61–1.00 cm) snails were used. In each species, 80 snails were placed individually in transparent plastic containers filled with 6 ml of de-chlorinated tap water and 50 embryonated O. viverrini eggs. The snails were infected in the plastic containers covered with porous lids and activated to ingest the parasite eggs by exposure to 8-W electric light for 24 h (Prasopdee et al. 2015a). Subsequently, the snails were washed and reared in the 15 × 20 cm glass containers according to their species, with a maximum of 50 snails per container density. Examination of egg hatching in snail feces was determined for snail infection (by observing the opening of the egg’s operculum) (Khampoosa et al. 2012). The infected snails were raised at room temperature (approximately 28 °C) with a 12:12 dark and light cycle and fed synthetic snail food (Sumethanurungkul 1970). All exposed snails were checked weekly for shedding of cercariae for a total of 56 days. At 1, 7, 14, 28, and 56 days post-infection (dpi), 16 snails per time point were randomly selected and examined pre-patent infection using specific primer PCR if the snails were negative for cercarial shedding. The specific primer PCR was carried out as described by previous study (Prasopdee et al. 2015a). Briefly, soft body of the snail was retrieved and homogenized in CTAB buffer (2% w/v CTAB, 1.4 M NaCl, 0.2% v/v beta-mercaptoethanol, 20 mM EDTA, 100 mM Tris–HCl, pH 8.0, 0.2 mg/ml proteinase K) (Winnepenninckx et al. 1993). The snail homogenates were incubated at 55 °C for 6 h. Snail homogenate proteins were first precipitated with phenol/chloroform, centrifuged at 12,000 × g for 10 min at 4 °C followed by doubly precipitated with phenol/chloroform/isoamyl alcohol, centrifuged at 12,000 × g for 10 min at 4 °C. DNA was precipitated with isopropanol then washed twice with 70% ethanol followed by absolute ethanol. The DNA pellet was air-dried, re-dissolved with TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), diluted to 10 ng/µl, and used as a template for PCR. The specific primers, OV-6F (5′-CTG AAT CTC TCG TTT GTT CA-3′) and OV-6R (5′-GTT CCA GGT GAG TCT CTC TA-3′) (Wongratanacheewin et al. 2001), were used to amplify the pOV-A6 specific region of 330 bp. The PCR reaction was carried out using a DNA Thermal cycler, performed in a final volume of 10 μl with 0.04 μl TaKaRa Ex Taq 250 U, 1 μl dNTP mixture, 1 μl 10 × Ex Taq buffer, 3 μl DNA sample, 5 pmol of each primer, and adjusted to 10 μl with distilled water. The PCR was carried out with cycling conditions of initial denaturation at 94 °C for 5 min followed by 35 cycles of 94 °C denaturations for 1 min, 55 °C annealings for 1 min, and 72 °C extensions for 1 min, followed by a final extension at 72 °C for 7 min. PCR products were analyzed by 1.5% TBE agarose gel electrophoresis. Numbers of susceptible Bithynia snails to O. viverrini infection were obtained from the presence of either released cercariae or 330 bp specific band of PCR product as described in the previous study (Prasopdee et al. 2015a).

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics for Windows, version 22.0. (IBM Corp, Armonk, NY, USA). The number of susceptible snails to O. viverrini infection was reported as % susceptibility. The association between dpi (predictor) and the susceptibility to O. viverrini infection (binary outcomes) of (1) B. siamensis siamensis, (2) B. funiculata, and (3) Bithynia spp. (B. siamensis and B. funiculata) were gauged by crude odds ratios obtained from binary logistic regression analysis. The binary logistic regression analysis was also used to find the association between the susceptibility to O. viverrini infection (binary outcomes) and species of the Bithynia snails (predictor).

Results

Determination of infectious status

There was no release of O. viverrini cercariae detected throughout the time-course of infection. However, the Bithynia snails were susceptible to the O. viverrini infection determined by specific primer PCR-based detection (Fig. 2).

Fig. 2
figure 2

Agarose gel electrophoresis showing 330 bp of PCR product generated with specific primers (pOV-A6) for O. viverrini. Lane M: 100 bp DNA ladder; N: Bithynia snail negative for trematode infection (based on cercarial shedding); lane P: O. viverrini adult; lane 1–4: experimental infection with O. viverrini in Bithynia snails

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Susceptibility of Bithynia snails to infection with O. viverrini

The highest percentage of susceptibility to O. viverrini infection of B. siamensis siamensis and B. funiculata was shown at 1 dpi with 94% and 82%, respectively. The susceptibility seemed to decrease as the dpi was increased (Fig. 3). The odds ratio (OR) for the association between susceptibility to O. viverrini infection of Bithynia snails and dpi is presented (Tables 1 and 2). In addition, there was evidence that B. funiculata was associated with a 64.5% decrease in odds of susceptibility to O. viverrini infection relative to B. siamensis siamensis (Twald = 4.737, df = 1, P = 0.03).

Fig. 3
figure 3

Percent of snails of the B. siamensis siamensis and B. funiculata species infected by O. viverrini versus day post-infection (dpi)

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Table 1 Association between infection and day post-infection of B. siamensis siamensis
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Table 2 Association between infection and day post-infection of B. funiculata
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B. siamensis siamensis vs O. viverrini

There was evidence of an association between dpi and susceptibility of B. siamensis siamensis to O. viverrini infection that at least two groups differed (Twald = 11.309, df = 4, P = 0.023). Relative to day 1 (as baseline), there was a decrease in odds of susceptibility for 14 dpi (P = 0.003), showing an association with a 97% decrease in the odds of susceptibility. At 7 dpi, the susceptibility to infection was similar to 1 dpi (P = 0.2). Interestingly, there was no infection detected at 28 and 56 dpi.

B. funiculata vs O. viverrini

There was evidence of an association between dpi and susceptibility to infection of B. funiculata that at least two groups differed (Twald = 16.065, df = 4, P = 0.03). Relative today 1 (as baseline), there was a decrease in odds of susceptibility for 7, 28, and 56 dpi (P < 0.05). There was evidence that 7 and 56 dpi were associated with a 92.3% decrease in the odds of susceptibility (P = 0.003). At 28 dpi, a 98.5% decrease in the odds of susceptibility was shown associated (P = 0.001). However, at 14 dpi, statistical evidence of susceptibility due to susceptible snails was not detected.

B. siamensis siamensis and B. funiculata (Bithynia spp.) vs O. viverrini

There was evidence of an association between dpi and susceptibility to infection of Bithynia spp. that at least two groups differed (Twald = 44.089, df = 4, P < 0.01). Relative to day 1, there was a decrease in odds of susceptibility for every extra dpi (Table 3).

Table 3 Association between infection and day post-infection of Bithynia spp.
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Discussion

Infectivity of O. viverrini in Bithynia snails was demonstrated to be temperature-dependent (Prasopdee et al. 2015a). In the present study, the chosen temperature of 28 ± 2 °C (room temperature) was applied to test the susceptibility to O. viverrini infection in B. siamensis siamensis and B. funiculata as it was demonstrated as an optimum range of temperature (22 to 34 °C) for O. viverrini infection in the sister taxa B. siamensis goniomphalos (Prasopdee et al. 2015a). In addition, as reported by the Thai Meteorological Department, this temperature is comparable with an average weather temperature of Thailand in the recent year 2019: 28 ± 1 °C. This was recorded as the highest average weather temperature of Thailand in the past 69 years (1951–2019), not only indicating global warming at effect, but also increasing the risk of snail-borne opisthorchiasis in humans and animals in the coming decades. Although the Bithynia snails have separate male and female individuals, the sex was not included in the current experimental infection setting since the susceptibility patterns to O. viverrini infection between male and female B. siamensis goniomphalos was not significantly different (Prasopdee et al. 2015a).

In a natural field survey for O. viverrini infection, among three taxa of Bithynia in Thailand, B. siamensis goniomphalos was reported to have the highest prevalence in comparison to other species (Kulsantiwong et al. 2015). Nevertheless, in experimental infection, B. funiculata and B. siamensis siamensis were more susceptible to O. viverrini than B. siamensis goniomphalos (Chanawong and Waikagul 1991). However, unlike the present study, the infection of O. viverrini in Bithynia snails of the previous studies was evaluated for only patent infection using a microscopic method cercarial shedding. In this study, observing of elicited O. viverrini cercariae and detecting specific bands of PCR product (330 bp) using specific primers to O. viverrini, both pre-patent and patent infections were determined throughout the time course of 1, 7, 14, 28, and 56 dpi. Interestingly, the susceptibility patterns of B. siamensis siamensis to O. viverrini infection demonstrated that the snail was highly susceptible to the parasite in the early period after exposure (94% and 75% for 1 and 7 dpi, respectively), before significantly becoming more resistant and finally, completely immune to the parasite during the later periods of infection. This trend is similar to the susceptibility patterns of B. funiculata to O. viverrini infection, where the susceptibility was found significantly higher at 1 dpi (82%) than other periods of infection (Fig. 1; Tables 1 and 2). This is under a previous study in B. siamensis goniomphalos, demonstrating that the odds of susceptibility were decreased when the dpi was longer (Prasopdee et al. 2015a). In this phenomenon, recent studies revealed several proteins and transcripts of Bithynia snails upon infection with O. viverrini involved in immune response (Prasopdee et al. 2014, 2015b; Suwannatrai et al. 2016). Moreover, as the snail hemocyte was reported involved in defense mechanism by killing invaded parasites (Adema et al. 1994; Humbert and Coustau 2001), several hemocyte proteins of O. viverrini-infected Bithynia snail were found differentially expressed at 1 and 2 dpi compared to uninfected Bithynia (Suwannatrai et al. 2016). This was possibly involved in the defense response of the Bithynia that could eliminate initial parasite invasion, which led to diminished and complete elimination of the parasite, respectively. Hence, the susceptibility to O. viverrini infection of the Bithynia was decreased over time. In addition, relative to day 1, the result presented herein demonstrated the odds of susceptibility of Bithynia spp. at 56 dpi was decreased approximately 98% (Table 3). This indicates less chance for further development and production of O. viverrini cercariae.

In natural field survey, the prevalence of O. viverrini infection (determined by detection of free cercariae) in B. funiculata was shown extremely low at 0.3% compared to 1.6% in B. siamensis siamensis (Upatham and Sukhapanth, 1980; Ngern-klun et al. 2006), likewise demonstrated in this study where the odds of susceptibility of B. siamensis siamensis were found 64.5% higher relative to B. funiculata. However, the Bithynia spp. act as the amplifier host for the transmission of O. viverrini to humans. In other words, the O. viverrini eggs are ingested by the snails, and miracidia hatch from the enclosed O. viverrini eggs in the snail digestive tract. The miracidia after that penetrate through the snail tissue and transform into sporocysts. The sporocysts undergo asexual reproduction through the redial stage to produce numerous free-swimming cercariae, which ultimately infect and develop to infective metacercariae in intermediate fish hosts (Wykoff et al. 1965). Despite the low infection of O. viverrini in its Bithynia hosts, it is essential and sufficient for transmission. Thus, in addition to endemic areas, awareness of opisthorchiasis should be increased in other regions, especially in Central, South, and Northern Thailand; Southern Vietnam; Cambodia; and Myanmar, where the B. siamensis siamensis was found (Brandt 1974; Harinasuta and Harinasuta 1984; Sithithaworn et al. 2008; Kulsantiwong et al. 2013; Miyamoto et al. 2014).

Conclusion

We can conclude that susceptibility patterns of B. siamensis siamensis and B. funiculata to O. viverrini infection were similar where the susceptibility was high in early infection and decreased over time. Nevertheless, the B. siamensis siamensis was more susceptible to O. viverrini infection than B. funiculata. This provides a basis on the O. viverrini-intermediate snail host relationship that could help epidemiologists and parasitologists to indicate risk and raise awareness of opisthorchiasis in other regions besides the endemic areas.