Application of environmental DNA analysis for the detection of Opisthorchis viverrini DNA in water samples

Accepted Manuscript Title: Application of environmental DNA analysis for the detection of Opisthorchis viverrini DNA in water samples Authors: Hiroki Hashizume, Megumi Sato, Marcello Otake Sato, Sumire Ikeda, Tippayarat Yoonuan, Surapol Sanguankiat, Tiengkham Pongvongsa, Kazuhiko Moji, Toshifumi Minamoto PII: DOI: Reference:

S0001-706X(16)30556-3 http://dx.doi.org/doi:10.1016/j.actatropica.2017.01.008 ACTROP 4179

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

1-8-2016 12-1-2017 12-1-2017

Please cite this article as: Hashizume, Hiroki, Sato, Megumi, Sato, Marcello Otake, Ikeda, Sumire, Yoonuan, Tippayarat, Sanguankiat, Surapol, Pongvongsa, Tiengkham, Moji, Kazuhiko, Minamoto, Toshifumi, Application of environmental DNA analysis for the detection of Opisthorchis viverrini DNA in water samples.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.01.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Application of Environmental DNA Analysis for the Detection of Opisthorchis viverrini DNA in Water Samples

Hiroki Hashizumea, Megumi Satob, Marcello Otake Satoc, Sumire Ikedab, Tippayarat Yoonuand, Surapol Sanguankiatd, Tiengkham Pongvongsae, Kazuhiko Mojif, Toshifumi Minamotoa, *

a

Graduate School of Human Development and Environment, Kobe University, Japan;

3-11, Tsurukabuto, Nada-ku, Kobe, 657-8501 Japan b

Graduate School of Health Sciences, Faculty of Medicine, Niigata University, Japan;

757, Ichibancho, Asahimachidori, Chuo-ku, Niigata 951-8510, Japan c

Department of Tropical Medicine and Parasitology, Dokkyo Medical University,

Japan; 880 Kitakobayashi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan d

Department of Helminthology, Faculty of Tropical Medicine, Mahidol University,

Thailand; 420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand e

Savannakhet Provincial Health Department, Lao PDR; Phonsavang Neua, KM6,

Kaisone District, Savannakhet Province, Lao PDR f

Graduate School of Tropical Medicine and Global Health, Nagasaki University, Japan;

1-12-4 Sakamoto, Nagasaki 852-8523, Japan

*Corresponding author: Toshifumi Minamoto

1

Graduate School of Human Development and Environment, Kobe University 3-11, Tsurukabuto, Nada-ku, Kobe, 657-8501 Japan Tel/Fax: +81-78-803-7743 E-mail: [email protected]

2

Abstract Opisthorchiasis, which can lead to cholangiocarcinoma in cases of chronic infection, is a major public health problem in Southeast Asian countries. The trematode, Opisthorchis viverrini, is the causative agent of the disease. Accurate and rapid monitoring of O. viverrini is crucial for disease prevention and containment. Therefore, in this study we sought to develop a novel species-specific real-time PCR assay for detecting O. viverrini using environmental DNA (eDNA). The diagnostic sensitivity of the newly developed real-time PCR assay was similar to that of the traditional PCR assay for 50 fecal samples collected in Lao PDR (21 and 19 samples were positive by real-time PCR and traditional PCR, respectively). The efficacy of eDNA analysis and its applicability in the field were tested using a total of 94 environmental water samples collected from 44 sites in Savannakhet, Lao PDR during May and October 2015 and February 2016. O. viverrini eDNA was detected in five samples by real-time PCR, indicating the presence of the fluke in the area and the risk of infection for individuals consuming fish from these water sources. The application of eDNA analysis would facilitate the identification of O. viverrini endemic hotspots and contribute to the ecological control of opisthorchiasis, and this strategy can be applied to other eukaryotic water pathogens.

Keywords: environmental DNA (eDNA), Lao PDR, liver fluke, Opisthorchis viverrini, real-time PCR

3

1. Introduction Opisthorchiasis is a foodborne trematode infection that occurs in Thailand, Lao PDR, Vietnam, and Cambodia (Andrews et al. 2008). It is caused by the Southeast Asian liver fluke, Opisthorchis viverrini. Although no or little subjective symptoms appear during early stages of infection, severe symptoms such as cholangitis, cholecystitis, and cholangiocarcinoma, develop after a few decades during the chronic stage of the disease (Sripa et al. 2007; Kaewpitoon et al. 2008). The anthelmintic, praziquantel, is an effective treatment used for mass drug administration of schoolchildren in endemic areas (Tomokawa et al. 2012). However, O. viverrini has other final hosts, including dogs and cats, and therefore the complete elimination of O. viverrini from the environment has proven difficult (Sato et al., submitted). Campaigns to raise awareness of food culture of eating raw fish and health education have been conducted as preventive measures among local people (Grundy-Warr et al. 2012); however, the challenge of diet culture transformation results in frequent re-infection following de-worming (Saengsawang et al. 2015). The lifecycle of O. viverrini is complex; it has two intermediate hosts and mammals serve as its final host (Figure 1). O. viverrini eggs are passed into the environment via the feces of the final hosts (including human) and are consumed by freshwater snails of the genus Bithynia, the first intermediate host. Cercaria (O. viverrini larvae) are released into the water subsequent to several life stages in snails and then infect cyprinid fish, the second intermediate host, where they encyst and reach the infective stage of metacercaria. Mammals, such as humans, dogs, and cats, become the

4

final hosts by ingesting raw or undercooked infected fish (Kaewkes 2003). Ecological surveys of O. viverrini in the environment are usually conducted by collecting the intermediate hosts, performing shedding tests with snails (Phongsasakulchoti et al. 2005; Kiatsopit et al. 2012), and artificial infection of fish (Waikagul 1998; Rim et al. 2008). However, intensifying rainy seasons and the expansion of irrigation and agricultural practices in recent years have hindered the study of the changing habitat and intermediate hosts of O. viverrini by such time-consuming methods. In addition, the distribution shift of O. viverrini is thought to have been altered due to climate changes (Conlan et al. 2011; Utaaker & Robertson 2015). Thus, rapid and inexpensive survey methods are required to elucidate the current distribution of O. viverrini. Environmental DNA (eDNA) analysis is a technique for examining the biota in environmental water or soil samples by detecting the DNA of micro- and macro-organisms (Ficetola et al. 2008; Minamoto et al. 2012; see Figure 2). This method is used for ecological surveys of different types of organisms and has been reported to be a useful ecological tool for detecting target DNA in the environment, reducing time and costs (Rees et al. 2014b). Although several studies have reported the detection of the eDNA of pathogenic organisms (e.g. Minamoto et al. 2009, 2015; Pitula et al. 2012; Hyman & Collins 2012; Huver et al. 2015; Hall et al. 2015), to date, only a single study has examined the eDNA of human trematode pathogens; the detection of Schistosoma japonicum cercaria from water samples (Worrell et al. 2011). In this study, we developed a real-time PCR detection assay by designing

5

species-specific short-amplicon primers and a TaqMan probe targeting the mitochondrial cytochrome c oxidase subunit I (COI) region of O. viverrini. After confirming the specificity and sensitivity of the assay, we determined the efficacy of eDNA analysis for surveying O. viverrini in Savannakhet, Lao PDR.

2. Materials and Methods 2.1 Primer and probe design PCR primer sets used for eDNA studies are generally designed to yield short amplicons (approximately 100 bp) because eDNA is thought to be easily degraded (Dejean et al. 2011) and short-amplicon PCR assays have high detection sensitivity (Huver et al. 2015). Therefore, we designed primers that amplify short target regions. The mitochondrial COI DNA sequences of trematodes (GenBank accession numbers: O. viverrini [EU022351], Clonorchis sinensis [KJ204589], O. felineus [JX913371], Metagonimus yokogawai [AF096230], Haplorchis taichui [JX174394], H. pumilio [KF044303], Fasciola hepatica [AB300704], F. gigantica [AJ628032], Echinostoma revolutum [KF793291], Schistosoma mekongi [U82263], and Paramphistomum epiclitum [JX678271]) were obtained from NCBI GenBank and a primer set and probe were designed for eDNA analysis and to distinguish between O. viverrini and other trematodes. The forward primer, OV-COI-F (5′-GCTGG ATTTG GGCAC CG-3′), reverse primer, OV-COI-R (5′-AGTAC CCGCA AGCAT ATACA ACC-3′), and TaqMan MGB probe (Thermo Fisher Scientific, Waltham, MA, USA), OV-COI-P (5′-FAMTAGCT CGGTT ACTAT GATTA T -NFQ-MGB-3′), were designed using the Primer

6

Express Software 3.0 (Applied Biosystems, Foster City, CA, USA) with default settings; the predicted designed amplicon size was 103 bp. To determine specificity, the newly designed real-time PCR assay (OV-COI) was conducted to detect O. viverrini and distinguish it from Clonorchis sinensis using DNA obtained from adult worms. Real-time PCR was performed with a StepOnePlus thermocycler (Thermo Fisher Scientific). The reaction was carried out in a 20 μl final volume containing 10 μl of 2×Taqman Gene Expression Master Mix (Thermo Fisher Scientific), 0.5 μl of DNA template, 900 nM each of the OV-COI-F/R primers, and 125 nM of the OV-COI-P probe. The PCR conditions were 2 min at 50 °C and 10 min at 95 °C as initial steps followed by 55 cycles of 15 s at 95 °C and 60 s at 60 °C.

2.2 Diagnostic sensitivity test To confirm the diagnostic sensitivity of OV-COI, we compared the detection rates of an end-point PCR (epPCR) method with primer set OV-6 that produces a 330 bp amplicon (Wongratanacheewin et al. 2001) and real-time PCR with OV-COI using fecal DNA samples as the template. The fecal samples were collected from students at the Savannakhet Teachers Training College, Savannakhet Province, Lao PDR in September 2014 with the approval of the National Institute of Public Health National Ethics Committee (approval No. 037 NIOPH/NECHR, 2014). After providing oral informed consent, the students received instructions for collecting and transporting the fecal samples. Approximately 2 g of feces were collected, preserved in RNAlater Stabilization Solution (Thermo Fisher

7

Scientific) and transported to the laboratory for further analyses. The parasite eggs were disrupted with a μT-12 beads crusher (TAITEC Co., Koshigaya, Japan). DNA was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer’s instructions. Final DNA elution was conducted with 30 μl of elution buffer. Traditional epPCR with primer set OV-6 PCR was conducted using a T100 Thermal Cycler DNA thermocycler (BIORAD, Hercules, CA, USA). The reaction was carried out in a final volume of 25 μl containing PCR reagent (TOYOBO, Osaka, Japan) and 1 µl of DNA preparation as template. The DNA samples were initially denatured at 94 °C for 4 min, followed by 30 amplification cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 30 s, and elongation at 72 °C for 2 min. Amplicons were electrophoresed on a 2.0% agarose gel and positive samples showed amplicons of the proper size. Three replicates were conducted for each sample. The real-time PCR conditions for primer set OV-COI were the same as described above. Three replicates were conducted for each sample including the negative PCR control (PCR blank) and diluted tissue DNA, which served as the positive control. DNA samples with negative results for all three replicates were further analyzed by additional real-time PCR assays for all three replicates. Detected DNA samples were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, USA) and commercially sequenced.

2.3 Detection of O. viverrini DNA from environmental water samples

8

Three water collection field surveys were conducted in Savannakhet Province, Lao PDR throughout the dry and rainy seasons. Water samples were collected from 30 sites on the 8th and 9th of May 2015, immediately prior to the rainy season; 32 sites on the 6th to 8th of October 2015, at the end of the rainy season; and from 32 sites on the 26th to 28th of February 2016, in the middle of the dry season. In total, 94 water samples from 44 sites were collected from the surface water of ponds, rice fields, and rivers. Approximately 600 ml of water were collected at each site into plastic bottles, following a careful prewash with water from the collection site to reduce the risk of contamination (Fukumoto et al. 2015), and stored on ice for a few hours until filtered. The water samples were filtered under vacuum through two glass fiber filters with a mesh size of 0.7 μm (GE Healthcare, Chicago, IL, USA) until clogging occurred (final volume was 100-600 ml per site). Following filtration, 5 ml of 70% ethanol were added, fixed for 1 minute, and then filtrated (Minamoto et al. 2016). For every sampling day, bottled drinking water (600 ml) was filtrated as a negative control (equipment blank). The filters were wrapped in aluminum foil and stored at room temperature until processing at the DNA laboratory. DNA was extracted from the filter samples using the commercial DNA extraction kit, PowerSoil DNA Isolation Kit (MO BIO, Carlsbad, CA, USA), to remove PCR inhibitors. Each filter was placed in a centrifuge tube with a suspended insert (Salivette; Sarstedt, Germany) tube and centrifuged at 5,000 ×g for 2 min; the filtrate was then discarded. Next, 120 μl of Solution C1 were added to the Power Beads Tube solution and this mixture was then added to each of the filters. The filters were then

9

incubated at 56 °C for 30 min and then centrifuged at 5,000 ×g for 3 min. To elute the remaining DNA from the filters, 200 μl of 1×Tris-EDTA buffer (pH 8.0) was added, incubated for 1 min, and then centrifuged at 5,000 ×g for 3 min. The remaining steps of the extraction protocol were performed according to the manual provided with the kit. During DNA extraction, one negative control sample (extraction blank) was included per 20 field samples and treated using the same protocol. Real-time PCR was performed with the OV-COI primers and probe set using a StepOnePlus thermocycler. The reaction conditions were the same as described in section 2.2, except for template DNA volume; each reaction contained 2 (October and February samples) or 5 μl (May samples) of sample DNA as template. Three replicates were conducted for each sample including the negative PCR control (PCR blank) and positive control (diluted tissue DNA). Positive PCR amplicons were directly sequenced as described in section 2.2.

2.4 Discrimination of O. viverrini and O. lobatus To further confirm the results of our survey, we designed another set of species-specific PCR primers that amplifies O. viverrini DNA but not O. lobatus DNA. O. lobatus is a closely related species of O. viverrini; however, there is no evidence that it is distributed in the surveyed area. The forward primer, OV-COI-F2 (5′- GGTG GTTT GGGC TCAT CATA -3′), and the reverse primer, OV-COI-R2 (5′- GAAC CCAA CTAT CCAC CACA TAA -3′), contain two and one O. viverrini specific nucleotide substitutions within five bases from their 3′ end, respectively, which are sufficient for specific eDNA

10

amplification (Fukumoto et al. 2015). The region amplified by this primer set (138 bp excluding the primers) includes two species-specific nucleotides differentiating O. viverrini and O. lobatus. The five eDNA samples positive for O. viverrini based on the real-time PCR test (see Table 2 for the details of the five samples) were subjected to PCR amplification using the OV-COI-F2 and OV-COI-R2 primers and the OV-COI-P probe. The reaction was carried out in a 20 μl final volume containing 10 μl of 2×Taqman Gene Expression Master Mix (Thermo Fisher Scientific), 5 μl of DNA template, 900 nM each of the OV-COI-F2/R2 primers, and 125 nM of the OV-COI-P probe. The PCR conditions were 2 min at 50 °C and 10 min at 95 °C as initial steps followed by 55 cycles of 15 s at 95 °C and 80 s at 60 °C. Positive PCR amplicons were directly sequenced as described in section 2.2.

3. Results 3.1 Specificity and sensitivity of the designed real-time PCR assay The OV-COI real-time PCR assay conducted with tissue-derived DNA resulted in adequate amplification of O. viverrini DNA without any cross-reaction with C. sinensis DNA (not amplified using OV-COI; data not shown). Next, detection sensitivity was compared between epPCR and the newly designed real-time PCR assay using 50 fecal samples in a blind test. O. viverrini DNA was detected by both methods in 14 samples, in seven samples solely by the OV-COI real-time PCR method, and in five samples solely by the epPCR method with OV-6; O. viverrini DNA was undetectable by either method in 24 samples (Table 1). McNemar’s

11

test performed with R 3.1.1 (R Core team 2014) showed no significant difference between the diagnostic sensitivity of both assays (p = 0.77). All DNA samples amplified by real-time PCR were sequenced and confirmed as O. viverrini.

3.2 Detection of O. viverrini DNA from environmental water samples Total DNA was extracted from the filters used for the filtration of all water samples collected from 44 sites (94 samples in total). The results of the real-time PCR assay detected five samples from three sites as positive for O. viverrini DNA (Table 2; Figure 3). Notably, the water samples from site No.1 (rice field) were positive for three successive seasons, throughout the dry and rainy seasons. The negative filtration (equipment blanks), DNA extraction (extraction blanks), and PCR (PCR blanks) controls were all negative. The five positive samples were directly sequenced and confirmed as O. viverrini DNA.

3.3 Confirmatory tests of positive samples Of the five eDNA samples determined as positive for O. viverrini DNA by real-time PCR with primer set Ov-COI-F and Ov-COI-R, three (eDNA samples collected from site 1 in October, site 34 in October, and site 21 in February) were also positive by real-time PCR with primers Ov-COI-F2 and Ov-COI-R2. The amplicon sequences were examined, and two (eDNA from sites 21 and 34) were confirmed as O. viverrini DNA, based on species-specific site matches, while one (eDNA from site 1) showed 99% similarity to both O. viverrini and O. lobatus and possessed one species-specific site

12

corresponding to O. viverrini and one corresponding to O. lobatus.

4. Discussion In this study, we successfully detected O. viverrini DNA directly from environmental water samples and identified a site where O. viverrini is present during several seasons. These results indicate that eDNA analysis coupled with simple water sampling can be used to determine the distribution of O. viverrini. The OV-COI primers were designed to yield a 103 bp amplicon in the COI region that is generally used for DNA barcoding. Previous studies have reported species-specific

primers

that

produce

162

to

380

bp-size

amplicons

(Wongratanacheewin et al. 2001; Intapan et al. 2008; Lovis et al. 2009; Sato et al. 2010; Sanpool et al. 2012) for the detection of O. viverrini DNA in feces, liver tissue, and intermediate hosts. The most suitable amplicon size depends on the purpose and methods; however, primers for short amplicons are more appropriate when the target DNA is subject to degradation, as is the case with eDNA samples. In addition, mitochondrial DNA is thought to be detected more easily in environmental samples because there are more copies of mitochondrial DNA than nuclear DNA in a single cell (Alberts et al. 2002). No significant differences in diagnostic sensitivity were apparent between the well-established epPCR with OV-6 and the newly-designed real-time PCR with OV-COI using fecal samples. Therefore, we conclude that the new real-time PCR assay with OV-COI could be useful for the detection and diagnosis of O. viverrini infection in

13

fecal samples because this system has less risk of contamination and is easy to replicate. Three of the 44 sites tested, including ponds, rice fields, and rivers, were positive for O. viverrini eDNA. However, new findings on opisthorchiid liver flukes suggest that our detection system using OV-COI-F and OV-COI-R would not be able to distinguish between O. viverrini and the recently reported avian liver fluke O. lobatus (Thaenkha et al. 2011; Nawa et al. 2015). The detailed prevalence and distribution of O. lobatus, including in our survey area, have yet to be determined; thus, we developed an additional primer set that distinguishes between the two opisthorchiid species in silico and analyzed five eDNA samples from three O. viverrini positive sites (based on the OV-COI-F and OV-COI-R primers). The amplified DNA from two sites (sites 21 and 34) were confirmed as O. viverrini, while the DNA from the other site (site 1) could not be determined based on the sequence data (see section 3.3 for details). However, fish from a pond connected to the three OV-COI positive rice fields (site 1: Table 2) collected in February 2016 were highly infected with O. viverrini metacercariae, as confirmed by microscopic examination of the fish followed by infection experiments of hamsters and morphological confirmation of adult worms (Tippayarat Yoonuan, unpublished data). Based on these results, we conclude that the eDNA samples from all three sites included O. viverrini DNA. This suggests that the eDNA technique would be a powerful tool for rapid identification of areas with a high infection rate of host fish. However, because of insufficient accumulation of genetic information of O. lobatus, the complete differentiation between these two Opisthorchis species was difficult in the present situation. Also, it should be noted that we did not survey the infectious status of

14

fish in O. viverrini DNA negative sites in this study; negative results do not necessarily signify the absence of O. viverrini. Thus, the eDNA approach for detecting O. viverrini risk areas needs further development before applying to field surveys. Especially, the sensitivity and specificity of the primer-probe sets need to be clarified more precisely in the future studies. The greatest benefit of the eDNA method is the compilation of species distribution data from several sites within a short period because sampling only requires approximately 5 minutes per site. The cost for consumables and reagents needed for DNA concentration, extraction, and real-time PCR is not high (approximately 10 USD per sample). However, the method described in this paper does require sophisticated and expensive equipment as well as well-trained personnel in the endemic area. Therefore, the development of simpler and less expensive methods (e.g. using traditional PCR instead of real-time PCR) is desirable. It should be noted that this is a proof-of-concept study and we were able to demonstrate the applicability of the eDNA approach for detecting O. viverrini risk areas. This eDNA method will provide a useful means for determining the infection risk at each site and is especially applicable for infectious risk screening of rice fields because farmers have been reported to consume intermediate-host fish from their immediate environment, the rice fields (Sato et al. 2015). The one challenge this study faced was the relatively rapid clogging of the glass fiber filters; in some cases only ~50 ml could be filtrated per filter. This might be caused by water containing clay and soil common in tropical areas in Southeast Asia

15

(previous studies have demonstrated that 1~2 liter of temperate zone water could be filtrated per filter). Future efficient protocols should utilize larger-pore-size filters or include a pre-filtering step (Turner et al. 2014; Huver et al. 2015; Robson et al. 2016). The egg and cercaria of O. viverrini are thought to float in water environments. However, we were unable to identify O. viverrini DNA from either cercaria (body 154×75 μm, tail 392×26 μm), eggs (27×15 μm) (Kaewkes 2003), or cell fragments (dead tissue) in the environmental water samples in this study. Tissue > 0.7 μm (the filter pore size that we used) in size could provide a possible source of eDNA. However, it was unlikely that the eDNA detected in this study originated from eggs or cercaria but rather from fragmented cells, because the real-time PCR assay Ct values were high (around 40). If whole eggs or cercaria were filtrated onto the filter, the Ct values should have been lower (20s ~ low 30s) because such tissue types contain many copies of mitochondrial DNA. Theoretically, we can detect intermediate host DNA as well as parasite DNA from a single water sample. A number of eDNA studies targeting mammals, fish, and snails have been reported to date (Ficetola et al. 2008; Minamoto et al. 2012; Foote et al. 2012; Thomsen et al. 2012; Takahara et al. 2013; Goldberg et al. 2013; Rees et al. 2014a). A rapid determination of the distribution of bithynid snails and cyprinid fish would be possible if specific eDNA detection assays were designed for these species. In addition, metabarcording may prove useful for determining the presence of potential host fish in a single eDNA sample (Miya et al. 2015) because a broad range of cyprinid species can serve as potential intermediate hosts. This might provide a solution for

16

elucidating the lifecycle of O. viverrini in various environments yet to be studied. The advantage of the eDNA method is that it allows for the monitoring of ecological changes to O. viverrini and its intermediate hosts as well as environmental changes; thus, we predict that future research of eDNA will provide strategies for the prevention of infections in humans. In addition, eDNA methods are efficient tools for surveying and monitoring eukaryotic water pathogens and their lifecycle (Bass et al. 2015); these methods could be applied to other water parasites for rapid and wide range detection (for example, schistosomes). Collecting water samples from multiple sites and analyzing the distribution of pathogens would be helpful for disease control strategies, for example, constructing pathogen risk maps as part of local health education initiatives.

Acknowledgements This study was supported by RIHN R-04 project (EcoHealth project) from the Research Institute for Humanity and Nature, Japan (KM). We wish to thank the students of the Teachers Training College in Savannakhet for their kind cooperation. In addition, we wish to thank Mr. Nirandorn Homsuwan of the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, and Mrs. Virayporn Phanhanan of the Savannakhet Provincial Health Department, Lao PDR, for their assistance with the field surveys.

17

References Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P.,. 2002. Molecular biology of the cell, 4th edition. Garland Science, New York. Andrews, R.H., Sithithaworn, P., Petney, T.N., 2008. Opisthorchis viverrini: an underestimated parasite in world health. Trends Parasitol. 24, 497–501. Bass, D., Stentiford, G.D., Littlewood, D.T.J., Hartikainen, H., 2015. Diverse applications of environmental DNA methods in parasitology. Trends Parasitol. 31, 499–513. Conlan, J.V., Sripa, B., Attwood, S., Newton, P.N., 2011. A review of parasitic zoonoses in a changing Southeast Asia. Vet. Parasitol. 182, 22–40. Dejean, T., Valentini, A., Duparc, A., Pellier-Cuit, S., Pompanon, F., Taberlet, P., Miaud, C., 2011. Persistence of environmental DNA in freshwater ecosystems. PLoS One. 6, e23398. Ficetola, G.F., Miaud, C., Pompanon, F., Taberlet, P., 2008. Species detection using environmental DNA from water samples. Biol. Lett. 4, 423–425. Foote, A.D., Thomsen, P.F., Sveegaard, S., Wahlberg, M., Kielgast, J., Kyhn, L.A., Salling, A.B., Galatius, A., Orlando, L., Gilbert, M.T.P., 2012. Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals. PLoS One. 7, e41781. Fukumoto, S., Ushimaru, A., Minamoto, T., 2015. A basin-scale application of environmental DNA assessment for rare endemic species and closely related exotic

18

species in rivers: a case study of giant salamanders in Japan. J. Appl. Ecol. 52, 358–365. Goldberg, C.S., Sepulveda, A., Ray, A., Baumgardt, J., Waits, L.P., 2013. Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum). Freshw. Sci. 32, 792–800. Grundy-Warr, C., Andrews, R.H., Sithithaworn, P., Petney, T.N., Sripa, B., Laithavewat, L., Ziegler, A.D., 2012. Raw attitudes, wetland cultures, life-cycles: socio-cultural dynamics relating to Opisthorchis viverrini in the Mekong Basin. Parasitol. Int. 61, 65–70. Hall, E.M., Crespi, E.J., Goldberg, C., Brunner, J.L., 2015. Evaluating environmental DNA-based quantification of ranavirus infection in wood frog populations. Mol. Ecol. Resour. 16, 423–433. Huver, J.R., Koprivnikar, J., Johnson, P.T.J., Whyard, S., 2015. Development and application of an eDNA method to detect and quantify a pathogenic parasite in aquatic ecosystems. Ecol. Appl. 25, 991–1002. Hyman, O.J., Collins, J.P., 2012. Evaluation of a filtration-based method for detecting Batrachochytrium dendrobatidis in natural bodies of water. Dis. Aquat. Organ. 97, 185–195. Intapan, P.M., Thanchomnang, T., Lulitanond, V., Phongsaskulchoti, P., Maleewong, W., 2008. Detection of Opisthorchis viverrini in infected bithynid snails by real-time fluorescence resonance energy transfer PCR-based method and melting curve analysis. Parasitol. Res. 103, 649–655.

19

Kaewkes, S., 2003. Taxonomy and biology of liver flukes. Acta Trop. 88, 177–186. Kaewpitoon, N., Kaewpitoon, S.J., Pengsaa, P., Sripa, B., 2008. Opisthorchis viverrini: the carcinogenic human liver fluke. World J. Gastroenterol. 14, 666–674. Kiatsopit, N., Sithithaworn, P., Saijuntha, W., Boonmars, T., Tesana, S., Sithithaworn, J., Petney, T.N., Andrews, R.H., 2012. Exceptionally high prevalence of infection of Bithynia siamensis goniomphalos with Opisthorchis viverrini cercariae in different wetlands in Thailand and Lao PDR. Am. J. Trop. Med. Hyg. 86, 464–469. Lovis, L., Mak, T.K., Phongluxa, K., Soukhathammavong, P., Sayasone, S., Akkhavong, K., Felger, I., 2009. PCR diagnosis of Opisthorchis viverrini and Haplorchis taichui infections in a Lao community in an area of endemicity and comparison of diagnostic methods for parasitological field surveys. J. Clin. Microbiol. 47, 1517–1523. Minamoto, T., Honjo, M.N., Yamanaka, H., Uchii, K., Yamanaka, H., Suzuki, A.A., Kohmatsu, Y., Iida, T., Kawabata, Z., 2009. Detection of cyprinid herpesvirus 3 DNA in river water during and after an outbreak. Vet. Microbiol. 135, 261–266. Minamoto, T., Naka, T., Moji, K., Maruyama, A., 2016. Techniques for the practical collection of environmental DNA: filter selection, preservation, and extraction. Limnology. 17, 23–32. Minamoto, T., Pu, X., Xie, J., Dong, Y., Wu, D., Kong, H., Yang, X., Takahara, T., Honjo, M.N., Yamanaka, H., Kawabata, Z., 2015. Monitoring of fish pathogenic viruses in natural lakes in Yunnan, China. Limnology. 16, 69–77.

20

Minamoto, T., Yamanaka, H., Takahara, T., Honjo, M.N., Kawabata, Z., 2012. Surveillance of fish species composition using environmental DNA. Limnology. 13, 193–197. Miya, M., Sato, Y., Fukunaga, T., Sado, T., Poulsen, J.Y., Sato, K., Minamoto, T., Yamanaka, S., Yamanaka, H., Araki, H., Kondoh, M., Iwasaki, W., 2015. Mifish, a set of universal PCR primers for metabarcording environmental DNA from fishes: detection of more than 230 subtropical marine species. R. Soc. Open Sci. 2, 150088. Nawa, Y., Doanh, P.N., Thaenkham, U., 2015. Is Opisthorchis viverrini an avian liver fluke? J. Helminthol. 89, 255–256. Phongsasakulchoti, P., Sri-Aroon, P., Kerdpuech, Y., 2005. Emergence of Opisthorchis viverrini cercariae from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos. Southeast Asian J. Trop. Med. Public Health. 36, 189–191. Pitula, J.S., Dyson, W.D., Bakht, H.B., Njoku, I., Chen, F., 2012. Temporal distribution of genetically homogenous ‘free-living’ Hematodinium sp. in a Delmarva coastal ecosystem. Aquat. Biosys. 8, 16. R Core Team, 2014. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project.org/. Rees, H.C., Bishop, K., Middleditch, D.J., Patmore, J.R.M., Maddison, B.C., Gough, K.C., 2014a, The application of eDNA for monitoring of the Great Crested Newt in the UK. Ecol. Evol. 4, 4023–4032. Rees, H.C., Maddison, B.C., Middleditch, D.J., Patmore, J.R.M., Gough, K.C., 2014b.

21

The detection of aquatic animal species using environmental DNA – a review of eDNA as a survey tool in ecology. J. Appl. Ecol. 51, 1450–1459. Rim, H.J., Sohn, W.M., Yong, T.S., Eom, K.S., Chai, J.Y., Min, D.Y., Lee, S.H., Hoang, E.H., Phommasack, B., Insisengmay, S., 2008. Fishborne trematode metacercariae detected in freshwater fish from Vientiane municipality and Savannakhet province, Lao PDR. Korean J. Parasitol. 46, 253–260. Robson, H.L., Noble, T.H., Saunders, R.J., Robson, S.K., Burrows, D.W., Jerry, D.R., 2016. Fine-tuning for the tropics: application of eDNA technology for invasive fish detection in tropical freshwater ecosystems. Mol. Ecol. Resour. 16, 922–932. Saengsawang, P., Promthet, S., Bradshaw, P., 2016. Reinfection by Opisthorchis viverrini after treatment with Praziquantel. Asian Pac. J. Cancer Prev. 17, 857–862. Sanpool, O., Intapan, P.M., Thanchomnang, T., Janwan, P., Lulitanond, V., Doanh, P.N., Hien, H.V., Dung, D.T., Maleewong, W., Nawa, Y., 2012. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis. Parasitol. Res. 111, 89–96. Sato, M., Pongvongsa, T., Sanguankiat, S., Yoonuan, T., Dekumyoy, P., Kalambaheti, T., Keomoungkhoun, M., Phimmayoi, I., Boupha, B., Moji, K., Waikagul, J., 2010. Copro-DNA diagnosis of Opisthorchis viverrini and Haplorchis taichui infection in an endemic area of Lao PDR. Southeast Asian J. Trop. Med. Public Health. 41, 28–35. Sato, M., Pongvongsa, T., Sanguankiat, S., Yoonuan, T., Kobayashi, J., Boupha, B.,

22

Nishimoto, F., Moji, K., Sato, M.O., Waikagul, J., 2015. Patterns of trematode infections of Opisthorchis viverrini (Opisthorchiidae) and Haplorchis taichui (Heterophyidae) in human populations from two villages in Savannakhet Province, Lao PDR. J. Helminthol. 89, 439–445. Sripa, B., Kaewkes, S., Sithithaworn, P., Mairiang, E., Laha, T., Smout, M., Pairojkul, C., Bhudhisawasdi, V., Tesana, S., Thinkamrop, B., Bethony, J.M., Loukas, A., Brindley, P.J., 2007. Liver fluke induces cholangiocarcinoma. PLoS Med. 4, 1148–1155. Takahara, T., Minamoto, T., Doi, H., 2013. Using environmental DNA to estimate the distribution of an invasive fish species in ponds. PLoS One 8, e56584. Thaenkham, U., Nuamtanong, S., Vonghachack, Y., Yoonuan, T., Sanguankiat, S., Dekumyoy, P., Prommasack, B., Kobayashi, J., Waikagul, J., 2011. Discovery of Opisthorchis lobatus (Trematoda: Opisthorchiidae): a new record of small liver flukes in the Greater Mekong Sub-region. J. Parasitol. 97, 1152–1158. Thomsen, P.F., Kielgast, J.O.S., Iversen, L.L., Wiuf, C., Rasmussen, M., Gilbert, M.T.P., Orlando, L., Willerslev, E., 2012. Monitoring endangered freshwater biodiversity using environmental DNA. Mol. Ecol. 21, 2565–2573. Tomokawa, S., Kobayashi, T., Pongvongsa, T., Nisaygnang, B., Kaneda, E., Honda, S., Moji, K., Boupha, B., 2012. Risk factors for Opisthorchis viverrini infection among schoolchildren in Lao PDR. Southeast Asian J. Trop. Med. Public Health. 43, 574–485.

23

Turner, C.R., Barnes, M.A., Xu, C.C.Y., Jones, S.E., Jerde, C.L., Lodge, D.M., 2014. Particle size distribution and optimal capture of aqueous macrobial eDNA. Methods Ecol. Evol. 5, 676–684.

Utaaker, K.S., Robertson, L.J., 2015. Climate change and foodborne transmission of parasites: A consideration of possible interactions and impacts for selected parasites. Food Res. Int. 68, 16–23.

Waikagul, J., 1998. Opisthorchis viverrini metacercaria in Thai freshwater fish. Southeast Asian J. Trop. Med. Public Health. 29, 324–326.

Wongratanacheewin, S., Pumidonming, W., Sermswan, R.W., Maleewong, W., 2001. Development of a PCR-based method for the detection of Opisthorchis viverrini in experimentally infected hamsters. Parasitology. 122, 175–180.

Worrell, C., Xiao, N., Vidal, J.E., Chen, L., Zhong, B., Remais, J., 2011. Field detection of Schistosoma japonicum cercariae in environmental water samples by quantitative PCR. Appl. Environ. Microbiol. 77, 2192–2195.

24

Figure legends

Figure 1.

Life cycle of Opisthorchis viverrini. Parasite eggs are passed via the feces

of infected mammals. The eggs are ingested by the first intermediate host (bithynid snails) and develop into free-swimming cercariae following several growth stages in the snails. The cercariae swim to and colonize the second intermediate hosts (cyprinid fish) and encyst in their skin and fins as metacercariae. Humans and other mammals are infected by eating raw or undercooked fish. Following ingestion, the metacercariae excyst in the duodenum and move into the bile ducts, where they develop into adult worms and lay their eggs (Adapted from http://www.dpd.cdc.gov/DPDx/HTML/Opisthorchiasis.htm).

Figure 2. The four steps of environmental water eDNA analysis performed in this study. Water was sampled from the surface using plastic bottles and vacuum-filtrated with glass fiber filters. eDNA was extracted from the filters and amplified by real-time PCR.

Figure 3. The 44 sampling sites in Savannakhet, Lao PDR. The three closed circles (No.1, No.21, and No.34) represent sites positive for O. viverrini DNA in environmental water.

25

Table 1. Comparison of the detection sensitivities of the two PCR methods for O. viverrini DNA from human fecal samples Sample

epPCR

Real-time PCR

Sample

epPCR

Real-time PCR

No.

(3rep)

(3~6rep)

No.

(3rep)

(3~6rep)

1/6

26

3/3

1/3

1

2

3

27

2/3

2/6

28

4

29

5

30

6

31

2/3

1/3

1/6

32

2/3

1/3

2/3

33

2/3

34

1/3

35

2/3

1/3

7

8

1/3

9

10

1/3

11

1/3

12

1/3

36

1/3

13

37

38

14

2/3

39

15

1/6

40

16

41

17

3/3

18

3/3

42

1/3

43

26

19

3/3

44

20

45

21

46

22

1/3

23

3/3

24

2/3

25

1/3

2/6

2/3

2/6

1/6

47

2/3

3/3

48

1/3

1/3

49

2/3

2/3

50

27

Table 2. Filtration volume and real-time PCR detection results for O. viverrini DNA from three field samplings (May and October 2015 and February 2016) May-15

Oct-15

Feb-16

Site No.

Site

Vol (ml)

Ov DNA

Vol (ml)

Ov DNA

Vol (ml)

Ov DNA

1

Rice Field

140

◯

600

◯

750

◯

2

Pond

150

550

575

3

Pond

125

200

200

4

Pond

80

5

Pond

100

400

250

6

River

100

400

550

7

River

160

200

450

8

Canal

140

9

Rice Field

150

10

Pond

50

11

Pond

150

12

Pond

125

13

Pond

160

600

14-1

Pond

100

150

14-2

Pond

15

Pond

120

200

650

16

Pond

125

375

90

160

200

70

90

28

17

Pond

130

18

Pond

150

19

Pond

100

100

100

20

Pond

140

500

700

21

Pond

100

235

220

22

Pond

100

200

145

23

Pond

150

250

310

24

Pond

80

25

Pond

100

26

Pond

100

230

100

27

Pond

100

200

700

28

Pond

100

475

80

29

Pond

140

400

100

30

Pond

145

100

100

31

Pond

400

150

32

Pond

200

100

33

Canal

400

34

Lotus Pond

500

35

Rice Field

200

36

Rice Field

250

37

Rice Field

500

38

Pond

400

29

◯

650

140

◯

39

Rice Field

450

40

Pond

250

41

Rice Field

200

42

Mekong River

800

1000

43

Mekong River

800

1000

44

Mekong River

600

1000

30

100

31

32

33