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Occurrence and Molecular Identification of Liver and Minute Asian Biomedicine Vol. 7 No. 1 February 2013; 97-104 DOI: 10.5372/1905-7415.0701.155 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 Wongsawada, b, Pheravut Wongsawada, Somboon Anuntalabhochaia, Jong-Yil Chaic, Kom Sukontasond aDepartment of Biology, Faculty of Science, bApplied Technology in Biodiversity Research Unit, Institute of Science and Technology, Chiang Mai University, Chiang Mai 50202, Thailand, cDepartment of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul 110799, Korea, dDepartment 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 2010. 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 agriculture. Trematode life cycle is completed when agro-economics area in northern Thailand located in metacercariae (infective stage) infect fish, which are Fang and Mae Ai districts, Chiang Mai province. The eaten by a definitive host such as piscivorous birds topography consists of mountains over 1000 m. and fish-eating mammals (including humans) [1]. elevation and important watershed resources for There are many aquatic animals distributed in this area, especially snails, crabs, and fishes. They serve Correspondence to: Dr. Chalobol Wongsawad, Department of as the first and second intermediate hosts of several Biology, Faculty of Science, Chiang Mai University, Chiang Mai trematode species. Chiang Mai province is known as 50202, Thailand. E-mail: [email protected] an endemic area of minute intestinal fluke infection. 98 C. Wongsawad, et al. Some monogenetic trematodes, viz. Dactylogyrus sp., Agricultural Basin, and identification of metacercariae Gyrodactylus sp., and Paradiplozoon sp.; plus using the HAT-RAPD PCR method for comparing three species with metacercariae, viz. Centrocestus DNA profiles of these parasites [10]. caninus, Haplochis taichui, and Haplorchoides sp. and one species of nematode: Rhabdochona sp. were Materials and methods recovered from the Mae Ngat Somboonchon reservoir Fish specimens were seasonally collected with gill [2]. Recently, Centrocestus caninus, Haplorchis nets from eight sampling sites (Figure 1, site 1-4 Mae taichui, and Stellantchasmus falcatus were found Jai Stream, Fang district; site 6 Mae Kok stream; site in cyprinoid fish from Mae Sa Stream, Mae Rim 5, 7, and 8 Mae Fang stream, Mae-Ai district) in the district, Chiang Mai province [3]. The prevalence of Fang-Mae Ai Agricultural Basin between October 2009 H. taichui and Haplorchoides sp. metacercariae and December 2010. This experiment followed the infecting cyprinoid fish at Mae Ngat and Mae Kuang guidelines of animal experiment by Chiang Mai Udomtara was 62.16% and 48.65%, respectively [4]. University. Fish specimens were identified by species Parasitic transmission has never been studied in using the handbook of Fishes of the Cambodian the Fang-Mae Ai Agricultural Basin. Molecular Mekong [11]. Each fish was digested with 1% of approaches, such as PCR have been developed for acid pepsin solution (99 ml of 0.85% sodium chloride detection of different parasite species. Specific DNA solution, 1 g of pepsin, and 1 ml of concentrated probes have been investigated for the detection of O. hydrochloric acid) and incubated at 37οC for one hour viverrini [5, 6] and Paragonimus heterotremus [7] and thirty minutes. The digested material was filtered in Thailand. A mitochondrial based multiplex PCR for through graded sieves to remove large particles and the detection and discrimination of Clonorchis rinsed twice with normal saline solution (0.85% NaCl). sinensis and O. viverrini has been documented [8]. The metacercariae were collected and counted under Specific primers derived from HAT-RAPD marker a stereo microscope and identified based on have been developed for detection and identification morphological characters. The prevalence and mean of H. taichui [9]. Our study was performed to density of metacercarial infections were also determine the occurrence of trematode infection, calculated. The seasonal variation on the prevalence especially liver fluke and minute intestinal flukes of metacercarial was analyzed by using ANOVA. metacercariae in fresh water fish from Fang-Mae Ai Figure 1. Map of the eight sampling sites in the Fang-Mae Ai Agricultural Basin Vol. 7 No. 1 Molecular identification of intestinal metacercariae 99 February 2013 Genomic DNA of all parasites was extracted the presence of metacercariae. Eleven species of and purified from adult flukes and metacercariae, cyprinoid fish, viz. Barbonymus gonionotus, using the GF-1 Tissue DNA extraction kit (Vivantis, Esomus metallicus, Hampala macrolepidota, Malaysia), according to the manufacturer’s Henicorhynchus siamensis, Labiobarbus instructions. All extracted genomic DNA was detected siamensis, Mystacoleucus marginatus, Osteochilus by HAT-RAPD PCR using arbitrary primers to hasselti, Puntius brevis, Raiamas guttatus, Systomus compare DNA profiles between adult flukes and orphoides, and Systomus stoliczkanus and one metacercariae. The six arbitrary primers used were; species of Anabantid fish, Anabas testudineus, OPP 11, OPN 09, OPA 01, OPA 02, OPN 01, and were infected with metacercariae. Five species OPN 02. The reaction of HAT-RAPD PCR was of metacercariae were found; one species of liver carried out in a final volume of 20 L, with common fluke, Opisthorchis viverrini, and four species of PCR composition. The reactions were performed in minute intestinal flukes, viz. Haplorchis taichui, a MyCyclerTM Thermocycler (Bio-RAD) and PCR Haplorchoides sp., Centrocestus caninus and conditions performed as follows; 1 cycle of 94οC for Stellantchasmus falcatus. The highest total 2 minutes, 35 cycle of 94οC for 30 seconds, 48οC for prevalence of metacercarial infection was observed 45 seconds, 72οC for 1 minutes and 1 cycle of final in the cool-dry season with 78.30%, followed by the extension at 72οC for 7 minutes. HAT-RAPD PCR rainy and hot-dry seasons with 72.84%, and 69.01%, products were separated on 1.4% TBE agarose gel respectively. electrophoresis, stained with ethidium bromide, and The highest prevalence of metacercarial infection photographed with a Kodak Digital Camera Gel Logic based on each trematode species found were: 100. The amplified HAT-RAPD markers were Haplorchoides sp. (37.43%), H. taichui (35.66%), scored as present (1) or absent (0) for each sample. C. caninus (3.80%), and S. falcatus (1.40%). Ambiguous bands that could not be easily distinguished Contrastingly, O. viverrini was for the first time were not scored [12]. Phylogenetic relationships recorded from this area in the hot-dry season with a among each species were analyzed using the UPGMA prevalence of 0.44% (Figure 2). For statistical method in the Clustal W2. analysis, there were no significant differences (p >0.05) among the prevalence of metacercarial Results infections found in each season. Two thousand two hundred eighty two fish belonging to 27 species were investigated for Figure 2. Prevalence of metacercarial infections found in each season 100 C. Wongsawad, et al. Haplorchoides sp. metacercaria were also found A molecular study using HAT-RAPD PCR was with H. taichui in the same fish species
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