Original article. Chalobol Wongsawad a, b, Pheravut Wongsawad a, Somboon Anuntalabhochai a, Jong-Yil Chai c, Kom Sukontason d a

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1 Asian Biomedicine Vol. 7 No. 1 February 2013; DOI: / Original article Occurrence and molecular identification of liver and minute intestinal flukes metacercariae in freshwater fish from Fang-Mae Ai Agricultural Basin, Chiang Mai province, Thailand Chalobol Wongsawad a, b, Pheravut Wongsawad a, Somboon Anuntalabhochai a, Jong-Yil Chai c, Kom Sukontason d a Department of Biology, Faculty of Science, b Applied Technology in Biodiversity Research Unit, Institute of Science and Technology, Chiang Mai University, Chiang Mai 50202, Thailand, c Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul , Korea, d Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50202, Thailand Background: Fang-Mae Ai Agricultural Basin is located in Fang and Mae Ai districts, Chiang Mai province. There are many aquatic species distributed in this area, especially snails, crabs, and fish, which can serve as the first and second intermediate hosts of several trematodes. The roles of these intermediate hosts as related to parasitic infections in the area are not known. Objective: We determined the occurrence of liver flukes and minute intestinal fluke metacercariae in freshwater fish from Fang-Mae Ai Agricultural Basin. We also identified of metacercariae by using HAT-RAPD PCR method comparing DNA profiles of parasites. Materials and methods: Liver flukes and minute intestinal flukes were studied from the Fang-Mae Ai Agricultural Basin between October 2009 and September Fish specimens were seasonally collected and each fish was digested and filtered. The metacercariae were collected and counted under a stereo microscope and identified based on morphological characters. The genomic DNA of all parasites was extracted and purified from adult flukes and metacercariae. All extracted genomic DNA was detected by HAT-RAPD PCR using arbitrary primers to comparing DNA profiles between adult flukes and metacercariae. Results: Five species of metacercariae were found. There were one species of liver fluke, Opisthorchis viverrini, and four species of minute intestinal flukes, viz. Haplorchis taichui, Haplorchoides sp., Centrocestus caninus, and Stellantchasmus falcatus. The prevalence of metacercarial infection was observed in the cool-dry season at 78.30%, followed by rainy and hot-dry seasons at 72.84% and 69.01%, respectively. The prevalence of trematodes were Haplorchoides sp. (37.43%), H. taichui (35.66%), C. caninus (3.80%), S. falcatus (1.40%), and O. viverrini (0.44%). Conclusion: Minute intestinal flukes accounted for high infection rates while, liver fluke, O. viverrini was of low infection rate. DNA profiles among metacercariae in Fang-Mae Ai Agricultural Basin correctly identified adult stages. Keyword: Liver flukes, metacercaria, minute intestinal flukes, molecular identification, occurrence Fang-Mae Ai Agricultural Basin is an important agro-economics area in northern Thailand located in Fang and Mae Ai districts, Chiang Mai province. The topography consists of mountains over 1000 m. elevation and important watershed resources for Correspondence to: Dr. Chalobol Wongsawad, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50202, Thailand. wchalobol@gmail.com agriculture. Trematode life cycle is completed when metacercariae (infective stage) infect fish, which are eaten by a definitive host such as piscivorous birds and fish-eating mammals (including humans) [1]. There are many aquatic animals distributed in this area, especially snails, crabs, and fishes. They serve as the first and second intermediate hosts of several trematode species. Chiang Mai province is known as an endemic area of minute intestinal fluke infection.

2 98 C. Wongsawad, et al. Some monogenetic trematodes, viz. Dactylogyrus sp., Gyrodactylus sp., and Paradiplozoon sp.; plus three species with metacercariae, viz. Centrocestus caninus, Haplochis taichui, and Haplorchoides sp. and one species of nematode: Rhabdochona sp. were recovered from the Mae Ngat Somboonchon reservoir [2]. Recently, Centrocestus caninus, Haplorchis taichui, and Stellantchasmus falcatus were found in cyprinoid fish from Mae Sa Stream, Mae Rim district, Chiang Mai province [3]. The prevalence of H. taichui and Haplorchoides sp. metacercariae infecting cyprinoid fish at Mae Ngat and Mae Kuang Udomtara was 62.16% and 48.65%, respectively [4]. Parasitic transmission has never been studied in the Fang-Mae Ai Agricultural Basin. Molecular approaches, such as PCR have been developed for detection of different parasite species. Specific DNA probes have been investigated for the detection of O. viverrini [5, 6] and Paragonimus heterotremus [7] in Thailand. A mitochondrial based multiplex PCR for the detection and discrimination of Clonorchis sinensis and O. viverrini has been documented [8]. Specific primers derived from HAT-RAPD marker have been developed for detection and identification of H. taichui [9]. Our study was performed to determine the occurrence of trematode infection, especially liver fluke and minute intestinal flukes metacercariae in fresh water fish from Fang-Mae Ai Agricultural Basin, and identification of metacercariae using the HAT-RAPD PCR method for comparing DNA profiles of these parasites [10]. Materials and methods Fish specimens were seasonally collected with gill nets from eight sampling sites (Figure 1, site 1-4 Mae Jai Stream, Fang district; site 6 Mae Kok stream; site 5, 7, and 8 Mae Fang stream, Mae-Ai district) in the Fang-Mae Ai Agricultural Basin between October 2009 and December This experiment followed the guidelines of animal experiment by Chiang Mai University. Fish specimens were identified by species using the handbook of Fishes of the Cambodian Mekong [11]. Each fish was digested with 1% of acid pepsin solution (99 ml of 0.85% sodium chloride solution, 1 g of pepsin, and 1 ml of concentrated hydrochloric acid) and incubated at 37 ο C for one hour and thirty minutes. The digested material was filtered through graded sieves to remove large particles and rinsed twice with normal saline solution (0.85% NaCl). The metacercariae were collected and counted under a stereo microscope and identified based on morphological characters. The prevalence and mean density of metacercarial infections were also calculated. The seasonal variation on the prevalence of metacercarial was analyzed by using ANOVA. Figure 1. Map of the eight sampling sites in the Fang-Mae Ai Agricultural Basin

3 Vol. 7 No. 1 February 2013 Molecular identification of intestinal metacercariae 99 Genomic DNA of all parasites was extracted and purified from adult flukes and metacercariae, using the GF-1 Tissue DNA extraction kit (Vivantis, Malaysia), according to the manufacturer s instructions. All extracted genomic DNA was detected by HAT-RAPD PCR using arbitrary primers to compare DNA profiles between adult flukes and metacercariae. The six arbitrary primers used were; OPP 11, OPN 09, OPA 01, OPA 02, OPN 01, and OPN 02. The reaction of HAT-RAPD PCR was carried out in a final volume of 20 L, with common PCR composition. The reactions were performed in a MyCycler TM Thermocycler (Bio-RAD) and PCR conditions performed as follows; 1 cycle of 94 ο C for 2 minutes, 35 cycle of 94 ο C for 30 seconds, 48 ο C for 45 seconds, 72 ο C for 1 minutes and 1 cycle of final extension at 72 ο C for 7 minutes. HAT-RAPD PCR products were separated on 1.4% TBE agarose gel electrophoresis, stained with ethidium bromide, and photographed with a Kodak Digital Camera Gel Logic 100. The amplified HAT-RAPD markers were scored as present (1) or absent (0) for each sample. Ambiguous bands that could not be easily distinguished were not scored [12]. Phylogenetic relationships among each species were analyzed using the UPGMA method in the Clustal W2. Results Two thousand two hundred eighty two fish belonging to 27 species were investigated for the presence of metacercariae. Eleven species of cyprinoid fish, viz. Barbonymus gonionotus, Esomus metallicus, Hampala macrolepidota, Henicorhynchus siamensis, Labiobarbus siamensis, Mystacoleucus marginatus, Osteochilus hasselti, Puntius brevis, Raiamas guttatus, Systomus orphoides, and Systomus stoliczkanus and one species of Anabantid fish, Anabas testudineus, were infected with metacercariae. Five species of metacercariae were found; one species of liver fluke, Opisthorchis viverrini, and four species of minute intestinal flukes, viz. Haplorchis taichui, Haplorchoides sp., Centrocestus caninus and Stellantchasmus falcatus. The highest total prevalence of metacercarial infection was observed in the cool-dry season with 78.30%, followed by the rainy and hot-dry seasons with 72.84%, and 69.01%, respectively. The highest prevalence of metacercarial infection based on each trematode species found were: Haplorchoides sp. (37.43%), H. taichui (35.66%), C. caninus (3.80%), and S. falcatus (1.40%). Contrastingly, O. viverrini was for the first time recorded from this area in the hot-dry season with a prevalence of 0.44% (Figure 2). For statistical analysis, there were no significant differences (p >0.05) among the prevalence of metacercarial infections found in each season. Figure 2. Prevalence of metacercarial infections found in each season

4 100 C. Wongsawad, et al. Haplorchoides sp. metacercaria were also found with H. taichui in the same fish species and were found at all sampling site in all three seasons. Seven fish species: Systomus orphoides, Henicorhynchus siamensis, Mystacoleucus ectypus, Labiobarbus siamensis, Osteochilus hasselti, Barbodes gonionotus, and Puntius brevis were recognized as hosts of Haplorchoides sp. H. taichui was also found at all sampling sites in all three seasons and infected six fish species: S. orphoides, H. siamensis, M. ectypus, O. hasselti, B. gonionotus, and Raiamus guttatus. C. caninus and S. falcatus were rare at each sampling site in all three seasons. C. caninus and S. falcatus were found only Rasbora metallicus and Anabas testudineus, respectively. O. viverrini metacercariae were also found for the first time in S. stoliczkanus at only one sampling site (site 3) during the hot-dry season. The prevalence and mean density of metacercariae found in each fish species are shown in Table 1. A molecular study using HAT-RAPD PCR was performed to generate DNA profiles and analyze phylogenetic relationships among metacercariae collected. This was done by comparing DNA profiles of metacercariae found in adult stages. The pattern of DNA profiles obtained from the primer OPP-11, OPN-09, OPA-01, OPA-02, OPN-01, and OPN-02 showed total polymorphism (Table 2) while the highest and lowest fragment number generated were observed from OPA-02 and OPN-02 with 13 and seven fragments respectively. DNA profiles of all metacercarial found in this study were the same as those from adult stages. For O. viverrini metacercaria found in S. stoliczkanus, the DNA profile showed the same pattern as those of adult flukes from northeast Thailand (Figures 3 and 4). UPGMA phylogram analysis, based on HAT-RAPD profiles showed that all metacercariae found in this study were of the same branch as those of their adult clusters (Figure 4). Table 1. Prevalence and mean density of metacercariae found in each fish species Fish species Species of metacercaria No. infected/ % Mean examined Prevalence density Anabas testudineus Stellantchasmus falcatus 11/ Barbonymus gonionotus Haplorchis taichui 73/ Haplorchoides sp. 135/ Esomus metallicus Centrocestus caninus 29/ Hampala macrolepidota Haplorchis taichui 54/ Haplorchoides sp. 75/ Henicorhynchus siamensis Haplorchis taichui 103/ Haplorchoides sp. 136/ Labiobarbus siamensis Haplorchis taichui 86/ Haplorchoides sp. 126/ Mystacoleucus marginatus Haplorchis taichui 233/ Haplorchoides sp. 299/ Osteochilus hasselti Haplorchis taichui 96/ Haplorchoides sp. 88/ Puntius brevis Centrocestus caninus 13/ Raiamas guttatus Haplorchoides sp. 8/ Systomus orphoides Haplorchis taichui 74/ Systomus stoliczkanus Haplorchoides sp. 18/ Opisthorchis viverrini 3/

5 Vol. 7 No. 1 February 2013 Molecular identification of intestinal metacercariae 101 Table 2. Characteristics of random oligonucleotide primers used in the HAT-RAPD analyses. Primers Sequence No. of No. of % Polymorphism fragments polymorphic OPP-11 AACGCGTCGG OPN-09 TGCCGGCTTG OPA-01 CAGGCCCTTC OPA-02 TGCCGAGCTG OPN-01 CTCACGTTGG OPN-02 ACCAGGGGCA Figure 3. DNA profiles generated by using six arbitrary primers: OPP-11 (a) OPN-09 (b) OPA-01 (c) OPA-02 (d) OPN-01 (e) OPN-02 (f). Lane M, 100 bp ladder; Lane 1, O. viverrini (adult); Lane 2, H. taichui (adult); Lane 3, C. caninus (adult); Lane 4, S. falcatus (adult); Lane 5, Haplorchoides sp. (adult); Lane 6, F. gigantica (adult);lane 7, P. epiclitum (adult); Lane 8, O. viverrini (metacercaria); Lane 9, H. taichui (metacercaria); Lane 10, C. caninus (metacercaria); Lane 11, S. falcatus (metacercaria); Lane 12, Haplorchoides sp. (metacercaria).

6 102 C. Wongsawad, et al. Figure 4. Cladogram constructed with 55 polymorphic fragments generated with the HAT-RAPD technique using UPGMA method from Clustal W2 Discussion The occurrence of trematode infections in the Fang-Mae Ai Agricultural Basin was already determined and the most common was the group of minute intestinal flukes with H. taichui, Haplorchoides sp., C. caninus, and S. falcatus. The liver fluke, O. viverrini was rarely found in this area. The highest prevalence (100%) was found in three species of trematodes, viz. Haplorchoides sp., C. caninus, and S. falcatus. Haplorchoides sp. metacercaria were always mixed infections along with H. taichui populations and it is a dominant species in cyprinoid fish [13]. These two flukes were distributed at all sampling site in all three seasons, whereas C. caninus and S. falcatus were rarely distributed but found in all seasons and in all sampling sites. C. caninus was found only from Rasbora metallicus, but previously reported in P. brevis [14]. In our study, S. falcatus was found in A. testudineus, agreeing with study from San Sai district, Chiang Mai province whereas other reports recovered this parasite from the half-beaked fish, Dermogenus pusillus [15, 16]. For the mean density of trematode infection, H. taichui was the highest in B. gonionotus with mean density of 24.0 individuals / fish while 21.0 individuals / fish in He. siamensis. Mean density of trematode infection in Fang and Mae Ai districts were lower than in Chom Thong and Mae Taeng districts [13]. The cause of lower infection in Fang and Mae-Ai district than Chom Thong district may be the location of this area. Fang and Mae-Ai districts are located in the basin of the Kok river, which is a mainstream river in this area. The Kok river originates in a mountainous area and is running through small communities and farmland, while the Ping river is mainstream of Chom Thong district, which longer and goes through numerous communities and farmland. The other reason for lower infection may be the intermediate host diversity of these parasites. The number of snail intermediate host in the Kok river quite lower than the Ping river. Metacercarial infection of the minute intestinal flukes: H. taichui, Haplorchoides sp., C. caninus and S. falcatus were similar to previous reports, which claimed that these trematodes were distributed in the northern region [3-4, 13]. Importantly, we found O. viverrini for the first time from S. stoliczkanus at only one sampling site. This finding provides evidence that O. viverrini infection extended to northern Thailand. The epidemiology and ecological distribution of these trematodes depend on several snail species and cyprinoid fish that are the first and second intermediate host respectively. They are commonly found in all rivers in this basin. Raining and flooding are the main factors that affect the dispersion of intermediate hosts. This combination of first and second intermediate host interaction enhances the possibility of parasitic infections. Our findings are supported by reports of host-parasite relationships that indicate that the number of parasite infection depends

7 Vol. 7 No. 1 February 2013 Molecular identification of intestinal metacercariae 103 on the host abundance [3]. Liver fluke infection in humans is relatively low, but intestinal fluke is very high in the northern region [17]. The risk of liver fluke infection in humans due to eating raw fish is much lower than that of intestinal flukes. The extended distribution of liver flukes in the northern region may be due to migration of people from the northeast region. The detection by molecular methods using HAT-RAPD PCR was performed to compare DNA profiles and analyze phylogenetic relationships among adult flukes and metacercariae in the Fang-Mae Ai Agricultural Basin with correctly identified adult stages. The pattern of DNA profiles of metacercariae were compared with those of adult stages, which were correctly identified as to species. Our method produced a high magnification of HAT-RAPD PCR for the identification of trematode larvae especially for O. viverrini metacercaria found in S. stoliczkanus, and with the same species as adult stage found in northeast Thailand. This confirms the extended distribution of O. viverrini infections in the northern region. The minute intestinal flukes reported in this study account for high infection rates since they are found in several fish species at all sampling site and in all seasons. In addition to the distribution of trematodes found in this study, H. taichui and Haplorchoides sp. were the most abundant and widely distributed species in the Fang-Mae Ai Agricultural Basin. To support our results, other specific molecular markers such as nucleotide sequences of conserved gene regions should be required for the determination of genetic differences at specific level. A combination of morphology and molecular analyses is the most effective means for the identification of trematodes. Acknowledgements We thank to the staff of the Parasitology Research Laboratory, Department of Biology, Faculty of Science, Chiang Mai University for use of their facilities, and the Institute for Science and Technology Research for financial support. Specially, thank to Dr. J.F. Maxwell from Herbarium and Flora Database, Faculty of Science, Chiang Mai University and Assistant Professor Dr. Choosak Nithikathkul, Faculty of Medicine, Mahasarakham University for manuscript corrections. The authors have no conflict of interest to report. References 1. Gjurcevic E, Petrinec Z, Kozaric Z, Kuzir S, Gjurcevic KV, Vucemilo M, et al. Metacercariae of Centrocestus formosanus in goldfish (Carassius auratus L.) imported into Croatia. J Helminthol. 2007; 44: Boonchot K, Wongsawad C. A survey of Helminths in Cyprinoid Fish from The Mae Ngad Somboonchon Reservoir, Chiang Mai Province, Thailand. Southeast Asian J Trop Med Public Health. 2005; 36: Wongsawad C, Rojtinnakorn J, Wongsawad P, Rojanapaibul A. Helminths of vertebrates from Maesa stream. Southeast Asian J Trop Med Public Health. 2004; 35 (Suppl 1): Nithikathkul C, Wongsawad C. The occurrence of heterophyid metacercariae in freshwater fish from reservoirs. Asian Biomed. 2008; 2: Sermswan R, Mongkolsuk S, Pamyim S, Siresinha S. Isolation and Characterization of Opithorchis viverrini specific DNA Probe. J Mol Cell Probes. 1991; 5: Maleewong W, Intapun PM, Wongkam C, Wongsaroj T, Komsuwan T, Phumidonming, W, Pongsakulchoti P. Detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes by PCR-based method. J Parasitol. 2003; 126: Intapun PM, Wongkham C, Imtawit KJ, Phumidonming W, Prasongdee TK, Miwa M, Maleewong W. Detection of Paragonimus heterotremus egg in experimentally infested cats by a polymerase chain reaction-based method. J Parasitol. 2005; 91: Le TH, De NV, Blair D, Sithithaworn P, McManus DP. Clonorchis sinensis and Opisthorchis viverrini. Development of a mitochondrial-based PCR for their identification and discrimination. J Exp Parasitol. 2006; 112: Wongsawad C, Wongsawad P, Chai JY, Anantalabhochai S. Haplorchis taichui Witenberg, 1930: Development of HAT-RAPD marker for the detection of minute intestinal fluke infections. J Exp Parasitol. 2009; 123: Chundet R, Cutler RW, Tasanon M, Anuntalabhochai S. Hybrid Detection in Lychee (Litchee chinensis Sonn.) Cultivars Using HAT-RAPD Markers. Sci Asia. 2007; 33: Rainboth WJ. Fishes of the Cambodian Mekong. Rome: FAO; Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA Polymerphisms ampliwed by arbitrary primers are useful as genetic markers. Nucl Acids Res. 1990; 18:531-5.

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