Very low prevalence of Opisthorchis viverrini s.l. cercariae in Bithynia siamensis siamensis snails from the canal network system in the Bangkok Metropolitan Region, Thailand

The liver fluke Opisthorchis viverrini s.l. is associated with a long-term public health problem in Thailand. However, O. viverrini s.l. infection in Bithynia snails in the canal network system (CNS) in the Bangkok Metropolitan Region (BMR) has never been assessed. This study aimed to investigate the occurrence of B. siamensis siamensis and the prevalence of O. viverrini s.l. infection in this snail in the CNS in BMR along with morphological examination and molecular analyses on O. viverrini s.l. cercariae. The snails were randomly sampled from the CNS in all BMR areas from January 2018 to July 2019. Snail specimens were identified and examined for digenean infection by shedding and dissection. The cercariae were identified using morphology and molecular methods, including PCR with a species-specific primer and a Bayesian phylogenetic analysis of ITS2 sequences. Bithynia siamensis siamensis was found in almost all sampling localities, with different quantities and detected frequencies. From a total of 7473 B. s. siamensis specimens, O. viverrini s.l. infections were detected in the Northern Bangkok, Muang Nakhon Pathom, Krathum Baen, and Lam Luk Ka areas with an overall prevalence of 0.05% (4/7473) and prevalence of 0.22% (1/455), 0.21% (1/469), 0.40% (1/253), and 0.16% (1/614) in individual localities with positive snails, respectively. This study is the first investigation of digenean infection in the canal network system-type habitat in Thailand and revealed extremely low O. viverrini s.l. prevalence.


Introduction
The fish-borne zoonotic digenean, Opisthorchis viverrini sensu lato (s.l.) has caused a significant, sustained public health problem in the Lower Mekong Basin [24]. In Thailand, opisthorchiasis is most prevalent in the northeastern region, decreasing in the northern, central, and southern regions, respectively [55]. Opisthorchis viverrini s.l. utilizes three snail taxa, i.e., Bithynia funiculata, B. siamensis goniomphalos, and B. s. siamensis, as the first intermediate host, and central Thailand has broad distribution of B. s. siamensis [23]. The cercarial stage and Bithynia snails play a pivotal role in spreading O. viverrini s.l. in ecosystems and limiting the opisthorchiasis epidemic boundaries [18,26]. Moreover, prevalence data of O. viverrini s.l. infection in snails reflect the transmission of O. viverrini s.l. from definitive hosts to the environment [7,32], and are useful for forecasting the epidemiological situation of infections in fishes and humans for the development of preventive applications for the future [1,55].
Although Bangkok Metropolitan Region (BMR) is a part of central Thailand, it has two unique characteristics that might be of epidemiological significance. First, BMR is the most densely populated area in Thailand and has the most migrant workers from other regions and neighboring countries [5]. Second, the canal network system (CNS) in BMR, which is the most comprehensive and dense CNS in Thailand [33,50], is possibly an ideal habitat for transmissions and dispersions of digeneans throughout their life cycles since: (1) it might have high contamination of helminths [61]; (2) it is a never-dry, aquatic habitat [44,50] in which abundance and diversity of snail and fish hosts could be high all year round [34]; (3) it has excellent distribution routes for aquatic animals [15]; and (4) it is a public water source where people can catch fish hosts. These might reflect the epidemiological importance of the CNS. Nevertheless, BMR is a poorly and only very partially studied area concerning digenean infection in freshwater snails [1,59]. Furthermore, O. viverrini s.l. infection in any Bithynia taxa, including B. s. siamensis, and their occurrences in any of Thailand's CNSs have not been investigated to date.
To assess the epidemiological situation of O. viverrini s.l. infection in B. s. siamensis in the CNS of BMR, the occurrence of B. s. siamensis and the prevalence of O. viverrini s.l. infection in this snail were investigated. In addition, morphological and molecular analyses were performed to identify and describe O. viverrini s.l. cercariae obtained from this uninvestigated O. viverrini special habitat type in the area unconnected to the Mekong River and its tributaries.

Ethics statement
The ethics of using animals to nurture the collected snail specimens and the investigation of O. viverrini s.l. infection in this study were approved by the ethics committee at Kasetsart University (Approval No. ACKU61-SCI-034).

Snail specimen collection
Thirty-five localities of the CNS in BMR's six provinces were chosen as snail-sampling localities; there was one sampling locality per district/area boundary ( Fig. 1 and Table 1). The geographical coordinates in WGS 1984 datum for each sampling locality were marked by the global positioning system. The random samplings of B. s. siamensis from canals were conducted using one collector and 20 min at each locality by hand-picking and scooping based on the counts per unit of sampling time method [38] every three months from January 2018 through July 2019. The collected snails were identified using shell morphological criteria according to the taxonomic keys [8].

Examinations of O. viverrini s.l. infection
The B. s. siamensis specimens were examined for O. viverrini s.l. infections by cercarial shedding and dissection. Each snail individual was placed in a small transparent plastic cup containing de-chlorinated water, and then exposed to light with an intensity of approximately 3000 lx from daylight-LED tube lights (850 lm) from 6:00 AM to 2:00 PM at room temperature (25 ± 2°C). Subsequently, each cup was examined under a dissecting microscope. The living cercariae were fixed with 10% neutral buffered formalin (NBF), and some were stained with 0.5% neutral red dye (NR). They were then investigated and photographed with a brightfield Olympus BX51 fitted to an Olympus DP70 digital camera (Olympus Corporation, Japan). Afterward, snails were dissected, then investigated in the same manner as the shedding procedure described above.
Morphological identification and descriptive study of O. viverrini s.l. cercariae The photomicrographs of mature-cercarial specimens were utilized to identify the O. viverrini s.l species according to the morphological descriptions [23,45,63]. Measurements of 17 morphological characteristics (Table 4 and Fig. 4C) of the 20 photographed O. viverrini s.l. cercariae (from all four infected snails) fixed with 10% NBF, were performed in ImageJ version 1.50e. Metric data in micrometers (lm) of all measured characteristics in the text and Table 4 are represented as the arithmetic mean ± standard deviation followed by range (minimum-maximum) values in parentheses. The multi-planar photomicrographs of O. viverrini s.l. cercariae were used in drawing and labeling to display their characteristic details (Fig. 4).

DNA preparation
Approximately 1200 individuals of the morphologically authenticated O. viverrini s.l. cercaria were collected separately from each infected-B. s. siamensis individual (the total number of infected snails was 4), washed with phosphate buffered saline and ultrapure water, and preserved in absolute ethanol within each 1.5 mL microcentrifuge tube. For use as a comparator in molecular detection, the O. viverrini s.l. adults preserved in 70% ethanol were obtained from the Faculty of Medicine at Khon Kaen University, Thailand. The genomic DNA of the cercarial and adult specimens was extracted separately using a GF-1 Tissue DNA Extraction Kit (Vivantis, Malaysia), according to the manufacturer's protocol.
Molecular detection using species-specific primers PCR amplification of a 330 bp specific region using a pOV-A6 probe was performed four times for the 4 DNA samples of O. viverrini s.l. cercariae and the DNA sample of O. viverrini s.l. adults with the OV-6F and OV-6R primers from Wongratanacheewin et al. [62] in a total reaction volume of 50 lL containing 1Â PCR buffer (ViBuffer A, Vivantis, Malaysia), 2 mM MgCl 2 , 0.7 lM of each primer, 1 mM dNTP mixture, 1 lL Taq DNA polymerase (5 u/lL), and 10 lL DNA template in a thermal cycler (Mastercycler Pro, Eppendorf, Germany), following the operating conditions of Prasopdee et al. [42]. Gel electrophoresis was operated to investigate a 330 bp band of PCR products in 2% agarose gel. PCR products of O. viverrini s.l. adults and ultrapure water were used as positive and negative controls (Fig. 2).

ITS2 amplification
One from a total of 4 DNA samples of cercariae, which were all verified as the O. viverrini s.l. species with molecular detection, was used as a representative to analyze further by phylogenetic analysis. The ITS2 region was amplified by PCR with the ITS3 and ITS4 primers from Barber et al. [3] in a total reaction volume of 50 lL containing 1Â PCR buffer (ViBuffer A, Vivantis, Malaysia), 2 mM MgCl 2 , 0.4 lM of each primer, 0.16 mM dNTP mixture, 0.4 lL Taq DNA polymerase (5 u/lL), and 2 lL DNA template in a thermal cycler (Mastercycler Pro, Eppendorf, Germany) with the following operating conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min each, annealing at 56°C for 1 min, and extension at 72°C for 30 s, then followed by a final extension at 72°C for 10 min. PCR products were inspected using gel electrophoresis in the same manner as molecular detection. The amplified DNA sample was sent to Macrogen (Korea) to purify, and the sequencing was then performed using ITS3 and ITS4 primers.

Molecular identification and phylogenetic analysis
The forward and reverse DNA chromatograms and sequences were processed in BioEdit version 7.2.5 to obtain a consensus sequence OVCLLK18 (Table 1), then submitted to the GenBank database. A dataset of 28 comparative ITS2 sequences was retrieved from GenBank ( Table 1). All sequences were aligned by ClustalW and improved by being trimmed in BioEdit version 7.2.5. The GTR + G model was the best-fit model of nucleotide substitution for the sequence dataset by evaluating with MrModeltest version 2.4 via PAUP* version 4.0a using the Akaike Information Criterion. For phylogenetic analysis using Bayesian Inference (BI), Haplorchis taichui in the Heterophyidae family (accession number KX815126.1 in Table 1) was determined as an outgroup. BI analysis was executed in MrBayes version 3.2.7a using four chains of a Markov chain Monte Carlo algorithm for 15 million generations, with a sample frequency of 100. The first 25% of the sample trees were discarded, and the remaining sample trees were used to build a 50% majority-rule consensus tree and calculate Bayesian posterior probabilities (BPPs).

Occurrences of B. s. siamensis snails
A total of 7473 B. s. siamensis were detected from almost all the sampling localities, except South Bangkok, Central Bangkok, Pak Kret, Phra Samut Chedi, and Muang Samut Prakan ( Fig. 1 and Table 2). Each locality had different detection frequencies and various quantities of B. s. siamensis ( Table 2). The B. s. siamensis were found in all localities of only Nakhon Pathom Province, along with the highest frequency of detecting B. s. siamensis.
The mature free-swimming O. viverrini s.l. cercariae were the bent-billiard tobacco pipe form, concaving on the ventral side and bent outward on the dorsal side at the time of cercarial resting and momentarily hanging in the water (Fig. 3B). Its main structures consisted of a body and tail (Figs. 3 and 4). The body had uneasily visible sensory hairs on the surface, was ovate, and had prominent brownish granular pigments that spread bilaterally and symmetrically over the body. The obvious ovate muscular oral sucker located near the midway anterior end of the body possessed three rows of molar toothlike structures that were quite noticeable when the oral sucker contracted. The ventral sucker located in front of the excretory bladder was small and nearly unnoticeable. Each of 10 penetration glands was a slightly evident translucent sac-like structure and possessed one duct. These ducts located at the median line of the body came out of the glands, passed through the central gap between one pair of the striking dark-brown pigmented eye spots, passed over the less apparent spherical pharynx, continued to the oral sucker, and finally opened toward the orifice of the oral sucker. There were numerous cystogenous glands with a small, relatively flat circular droplet-like shape that spread in dorso-ventral and anterior-posterior directions on both sides of the body, which were easily visible for the stained specimens.
The excretory bladder had a thick wall, especially in the posterior position, and was large and clearly discernible. The shape thereof was different according to the specimen's state: globular in free-swimming specimens, especially in the bentbilliard tobacco-pipe shaped cercariae; an inverted triangle with rounded corners in the alive stained and fixed unstained specimens; and trapezoidal with rounded corners in fixed unstained specimens. At the anterior position close to the excretory bladder wall, a genital primordium was a nearly flattened translucent triangular lump with a base, which has a base and height equal to 4/5 and 1/5 of the bladder length. The genital primordium was effortlessly seen in unfixed stained and fixed unstained specimens if it was neither concealed nor pushed down to the excretory bladder's dorsal location by the glandular penetration sacs when the anterior part of the cercarial body shrinks.
The tail's origin was connected to the posterior end of the excretory bladder within the body. The tail was a long translucent cylindrical structure with a circumference that was gradually reduced from the widest anterior tail portion to the smallest portion at the posterior tip. The tail was significantly longer than the body, averaging 2.5 times the length ( Table 4). The tiny transparent pill-like nuclei appeared within the tail, mostly in the tail's central axis and spreading sparsely from the anterior to the posterior of the tail. These nuclei were easily noticeable in the stained specimens. The translucent, transversely striated, dilated tegument enveloped the anterior tail portion with an average of 1/3 the tail length. The dorso-ventral finfold was like a thin translucent membrane that materializes bilaterally along the tail edges. The finfold location slightly overlapped on the posterior end of the anterior tail portion's tegument until it slightly passes the tail posterior tip. When comparing the NR color intensity on the alive O. viverrini s.l. cercariae structures, most structures within the body were successfully dyed but varied in color intensity, which helped to distinguish these structures, especially the cystogenous glands. Contrarily, the tail and the structures within and on it, particularly the dorso-ventral finfold, were unsuccessfully dyed, except the somewhat successfully dyed tail nuclei. Data from 17 quantitative morphological characteristics of the O. viverrini s.l. cercariae were enumerated and compared to previous studies in Table 4.

Molecular analyses
The morphologically verified O. viverrini s.l. cercariae from all four infected snails were O. viverrini s.l. species based on the presence of a DNA band from PCR products of all four cercarial sources in a position parallel to the 330 bp specific pOV-A6 DNA band from the PCR products of O. viverrini adults (Fig. 4, which displays the example of the one-time gel electrophoresis result from a total of four detection tests). Additionally, phylogenetic analysis of the digeneans according to 394 bp (after the multiple alignment and improvement-by-trimming steps) of the ITS2 region of a representative sample from a total of four samples, was conducted with the BI method for species corroboration among the current and contemporaneous O. viverrini s.l. and other Opisthorchis species. The other   Table 1 and Fig. 5) with the maximum support value (BPP = 1), which phylogenetically substantiated that OVCLLK18 was of the O. viverrini s.l. species.

Discussion
Bithynia s. siamensis snails were not found in five BMR localities, although the BMR CNS provides plenty of habitats and spreading routes for aquatic animals. The urban environment of southern and central Bangkok might not suit the lifestyle of B. s. siamensis [56]. Based on our observations, the strong waves created by frequent high-speed motorboats in Pak Kret may significantly disrupt the growth, reproduction, and lifecycle of B. s. siamensis [19]. In Phra Samut Chedi and Mueang Samut Prakan, B. s. siamensis might be unable to endure the high-salinity water caused by seawater intrusions from the Gulf of Thailand [51]. These reveal a considerably low risk of O. viverrini s.l. infections to fish hosts and, subsequently, humans in these four localities. Interestingly, the four O. viverrini s.l.-detected localities had a high frequency of snail encounters, which might indicate an encountering risk between snail host and O. viverrini s.l. eggs [48].
Interestingly, the prevalences of O. viverrini s.l. infections in Bithynia snails from the CNS in BMR of this study were lower than in previous studies. The 1.6% O. viverrini s.l. prevalence in B. s. siamensis from standing-water bodies in Bangna, Bangkok was reported four decades ago [59]. However, most Bangna areas have changed from agricultural land to urbanized areas [10] that may not suit B. s. siamensis. Ranges of the mean, total, and overall prevalences of O. viverrini s.l. infections in Bithynia spp. from various standing-water habitats in northeastern Thailand, Cambodia, Lao PDR, and southern Vietnam were 1.39-3.04%, 0.0-6.8%, and 0.11-1.73%, respectively [7,9,17,27,28,30,32,35,59]. With respect to the populations living in these opisthorchiasis endemic areas, their lifestyles may be associated with higher-prevalence causes than in the present study. Unhygienic defecation and consumption of undercooked fishes have been reported in some populations, along with high O. viverrini s.l. prevalences in these previously studied areas [16,32,40,60]. Interestingly, there seem to be consistencies among the relatively high O. viverrini s.l. infections in Bithynia snails, cyprinoid fishes, and humans in northern and northeastern Thailand [27,36,41,55], Cambodia [7,12,32], Lao PDR [11,27,60], and Vietnam [16,17]. Moreover, high to very high O. viverrini s.l. prevalences (30.92-53.1%) were reported in cats in these areas [2,60]. These factors possibly lead to continuous transmissions and accumulations of O. viverrini s.l. in ecosystems, which might be reflected by the high infection in Bithynia snails. Similarly, in the BMR, unsanitary excretion by cats and dogs was scarcely reported [21,52], along with lower infection rates (1.6%) [21] and very low prevalence in humans that is steadily decreasing [55].
Another cause of very low O. viverrini s.l. infection in this study might relate to the unique characteristic of inland aquatic ecosystems possessing the CNS in BMR. The highest O. viverrini s.l. prevalences in B. s. goniomphalos collected from standing-water habitats have been reported in the cooldry season in northeastern Thailand [9,28,35] that may be due to the decreased water content, leading to high densities of snails [54] and O. viverrini s.l. eggs concurrently [35]. In contrast, a similar situation might hardly occur in the CNS in BMR since it is supported continuously by enormous water masses from the upper and middle Chao Phraya River systems [33,50]. Although the highest prevalence in snails was reported in the wet season in northeastern Thailand [13], the greater amount of water entering the CNS [33,50] from extreme rainfalls in BMR [31] may lead to dilutions of O. viverrini s.l. eggs and cercariae, resulting in a reduced chance of O. viverrini s.l. eggs being ingested, and cercariae fish-finding success. Furthermore, water currents in the CNS might push the O. viverrini s.l. eggs and cercariae away from B. s. siamensis [37,58] and fish hosts [6,64], especially in the wet season when water flow velocity increases [22,43]. Moreover, the spontaneous water currents might complicate host finding [20] and reduce the life expectancy of O. viverrini s.l. cercariae [6], and might kill them at high velocity with high shear turbulences [64]. These factors may disrupt lifecycles and accumulations of O. viverrini s.l. in ecosystems that might eventually result in decreases of encountering-frequency and infection of O. viverrini s.l. in B. s. siamensis in the BMR CNS [6,22,31,33,43,58,64].
In Thailand, BMR has the highest proportion (35.79%) of migrant workers [5]. High O. viverrini s.l. prevalences have been reported in workers who migrated from northeastern Thailand [53], Lao PDR, and Cambodia [25]. With respect to migrant workers, there might possibly be migrants infecting O. viverrini s.l. in BMR. However, the O. viverrini s.l. infection rate in B. s. siamensis in the CNS in BMR may remain very low due to dilution [37,58] and withdrawal effects [22,43] from canal waters on O. viverrini s.l. eggs. Similarly, the nature of works in the major economic sectors, like the construction      [39,45,49]. In contrast, based on our observation, the tiny numbers of emerged Cystophorous cercariae from the snail (probably due to the parasite's overlong size:~2500 lm) may reduce infection to predatory hosts, unlike O. viverrini s.l., parapleurolophocercous, furcocercous, and monostome cercarial types whose intermediate and definitive hosts exist in different ecosystems [23,39,45].
The identification of samples in this study based on morphological descriptions [23,63] and the detection using species-specific primers [62], which were utilized in previous studies [7, 13, 28-30, 35, 42, 57], and the phylogenetic analysis using the available DNA sequence (i.e., ITS2 region) [7,30,57]   The O. viverrini s.l. cercariae from this study appeared indistinguishable based on qualitative morphological characteristics of O. viverrini s.l. cercariae from B. s. goniomphalos [63]. However, the quantitative morphological characteristics of O. viverrini s.l. cercariae from this study have most structures that were slightly longer and broader than in most previous studies [36,47,63], and more than those described by Boonmekam et al. [7] approximately 2-4 times. The latter difference might be because O. viverrini s.l. cercariae are an immature stage [14]. The former variations possibly reflect parasite adaptations in different habitat types and relationships to genetic variation [4,46]. Data on the additionally measured quantitative morphological characteristics and the qualitative morphological characteristics in this study may facilitate species identification of O. viverrini s.l. cercariae.
In conclusion, this study is the first to report on O. viverrini s.l. infections in any snail host in an uninvestigated habitat for any digenean infections in Thailand, like the network system of flowing-water bodies (i.e., CNS). It is also the first to provide a comprehensive survey in BMR, and revealed very low O. viverrini s.l. prevalence. The study also provides molecularly passed-verified, qualitative and quantitative morphological descriptions and metric measurement guidelines to identify the O. viverrini s.l. cercariae. Environmental data, which might correlate with O. viverrini s.l. infections, should additionally be assessed comprehensively to apply in developing opisthorchiasis-preventive applications in the future.