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Molecular Identification of Larval Trematode in Intermediate Hosts from Chiang Mai, Thailand

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Molecular Identification of Larval Trematode in Intermediate Hosts from Chiang Mai, Thailand SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH MOLECULAR IDENTIFICATION OF LARVAL TREMATODE IN INTERMEDIATE HOSTS FROM CHIANG MAI,THAILAND Suksan Chuboon and Chalobol Wongsawad Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand Abstract. Snail and fish intermediate hosts were collected from rice fields in 3 dis- tricts; Mueang, Mae Taeng and Mae Rim of Chiang Mai Province during April-July 2008. For identification of larval trematode infection, standard (cracked for snail and enzymatically digested for fish) and molecular methods were performed. The re- sults showed that three types of cercariae were found, pleurolophocercus, cotylocercous, and echinostome among 4 species of snail with a prevalence of 29, 23 and 3% respectively. Melanoides tuberculata snail was the most susceptible host for cercariae infection. Four species of metacercariae, Haplorchis taichui, Stellantchasmus falcatus, Haplorchoides sp and Centrocestus caninus, were found with a prevalence of 67, 25, 60 and 20%, respectively. The Siamese mud carp (Henicorhynchus siamensis) was the most susceptible fish host for H. taichui, and half- beaked fish (Dermogenys pusillus) for S. falcatus metacercariae infection, whereas Haplorchoides sp and C. caninus were concomitantly found in Puntius brevis. HAT-RAPD profile confirmed that pleurolophocercus cercariae found in Melanoides tuberculata from Mae Taeng Dis- trict belonged to H. taichui and in Tarebia granifera from Mueang District were S. falcatus. INTRODUCTION among several metacercarial species in the same fish and snail hosts including their In Thailand, heterophyid flukes, morphology, which is particularly similar in Stellantchasmus falcatus, Centrocestus caninus the egg forms and larval stages, it is diffi- and Haplorchis taichui, were reported as en- cult to distinguish such parasites from one demic species in the northern region another by standard methods. Consequently, (Sripalwit et al, 2003). Humans become in- specific and accurate detection is needed for fected with these parasites by eating better definition and epidemiological con- undercooked freshwater fish containing trol program. metacercariae (Kumchoo et al, 2003). Fish- In recent years, molecular approaches eating animals, including dog, cat and ro- using PCR method have been developed for dent, can also become infected and can serve specific detection of different parasites spe- as reservoirs of infection (Le et al, 2006). Be- cies, viz. PCR and filter-hybridization have cause of the sympatric relationship found been introduced to detect bird schistosome cercariae in lakes (Hertel et al, 2002), snail Correspondence: Chalobol Wongsawad, Depart- ment of Biology, Faculty of Science, Chiang Mai host, and in fecal, water and plankton University, Chiang Mai 50200, Thailand. samples (Hamburger et al, 1998; Pontes et al, Tel: +66 (0) 53 943 346 ext 1105 2002). In Thailand, specific DNA probes have E-mail: [email protected] been developed for the detection of 1216 Vol 40 No. 6 November 2009 MOLECULAR INDENTIFICATION OF LARVAL TREMATODE IN INTERMEDIATE HOSTS O.viverrini, (Sermsawan et al, 1991) and was performed using commercially avail- Paragonimus heterotremus (Intapun et al, able arbitary 10-mer primers (Operon tech- 2005). Additionally, a PCR-RFLP methods nology, USA). HAT-RAPD PCR reaction has been developed to detect cercariae of was carried out in a final volume of 20 µl. the large liver fluke, Fasciola hepatica infect- The reaction was performed in MyCyclerTM ing Lymnaea columella snail (Magalhães Thermocycler (Bio RAD) as follows: 1 cycle et al, 2004. Due to their rapidity, accuracy and of 95ºC for 5 minutes, 30 cycles of 95ºC for specificity, PCR-based methods have also 45 seconds, 48ºC for 45 seconds, 72ºC for 2 been introduced for use as coprodiagnosis minutes; and 1 cycle of final extension at in comparison with standard methods in 72ºC for 7 minutes. HAT-RAPD PCR prod- the detection of several parasites, such as ucts were separated by 1.4% agarose gel- Echinococcus multilocularis in the definitive electrophoresis, stained with ethidium bro- host (Dinkel et al,1998) and O. viverrini mide and photographed (Kodak digital (Wongratanacheewin et al, 2002). camera Gel Logic 100). This study is aimed to identify trema- tode species at larval stages in intermediate RESULTS hosts (cercariae in snail and metacercariae Three types of cercariae were found, in fish) using a combination of standard and pleurolophocercus, cotylocercous and molecular (HAT-RAPD) methods, which echinostome, among 3 species of snail should be useful to demonstrate the epide- (Melanoides tuberculata, Tarebia granifera, miological situation and in application in Bithynia funiculata) with a prevalence of 29, control programs. 23 and 3%, respectively (Table 1) M. tuberculata snail was the highest susceptible MATERIALS AND METHODS host for cercariae infection. Interestingly, it was also found that pleurolophocercus Snails and fish were collected from showed a high infection capability as it paddy fields in 3 districts: Mueang, Mae was found in both Melanoides and Tarebia Taeng and Mae Rim of Chiang Mai Province snails. during April-July 2008. For the investigation of larval trematode infection, standard Metacercariae of four parasite species, (cracked for snails and enzymatically di- Haplorchis taichui, Stellantchasmus falcatus, gested for fish) and molecular methods were Haplorchoides sp and Centrocestus caninus, performed. were found among 3 fish species (Heni- corhynchus siamensis, Dermogenys pusillus and HAT-RAPD PCR Puntius brevis) with a prevalence of 67, 25, 60 Parasite genomic DNA was extracted and 20%, respectively (Table 2). The Siamese and purified from adult worms using mud carp, Henicorhynchus siamensis, was the Dneasy Tissue Kit (QIAGEN) according to most susceptible fish host for H. taichui, and instructions of manufacturer. Extracted ge- half- beaked fish, Dermogenys pusillus, for nomic DNA was diluted to a working con- S. falcatus metacercariae infection, whereas centration of 50 ng/µl and stored at -20ºC Haplorchoides sp and C. caninus were con- until used. comitantly found with high specificity in A high annealing temperature-random Puntius brevis. Haplorchoides sp was also amplified polymorphic DNA(HAT-RAPD) found in H. siamensis indicating a higher in- technique (Anuntalabhochai et al, 2000) fection capability than others. Vol 40 No. 6 November 2009 1217 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH Table 1 Prevalence of cercariae infection among snail intermediate host collected from paddy field in 3 districts of Chiang Mai Province. Cercariae No. infected/examined % Prevalence Pleurolophocercous 25/90 29 Cotylocercous 21/90 2 Echinostome 3/90 3 Table 2 Prevalence of metacercariae infection among fish intermediate host collected from paddy field in 3 districts of Chiang Mai Province. Metacercariae No. infected/examined % Prevalence Haplorchis taichui 27/40 67 Stellantchasmus falcatus 10/40 25 Haplorchoides sp 24/40 60 Centrocestus caninus 8/40 20 HAT-RAPD profile confirmed that M 1 2 3 4 5 6 7 8 9 10 11 12 pleurolophocercus cercariae found in Melanoides tuberculata from Mae Taeng Dis- trict (Fig 1, lane 11) belonged to H. taichui (Fig 1, lane 3), whereas pleurolophocercus in Tarebia granifera from Mueng District (Fig 1, lane 12) was identified as S. falcatus (Fig 1, lane 4). From HAT-RAPD profile, it also was found that pleurolophocercus cercariae could infect different snail hosts and impor- tantly, they could come from more than Fig 1–HAT-RAPD profile generated by OPN-09 primer. Lane 1, Haplorchoides sp; lane 2, Ganeo one species. This study revealed that S. tigrinus; lane 3, Haplorchis taichui; lane 4, falcatus and H. taichui were developed from Stellantchasmus falcatus; lane 5, Opisthorchis pleurolophocercus cercariae (Fig 1). viverrini; lane 6, Centrocestus caninus; lane 7, Diplodiscus sp; lane 8, Orthocoelium DISCUSSION streptocoelium; lane 9, Paramphistomum epiclitum; lane 10, Fishoederius elongatus; This study showed that M. tuberculata lane 11, pleurolophocercus cercariae (in snail was the most susceptible host for cer- Tarebia snail); lane 12, pleurolophocercus cer- cariae infection and pleurolophocercus cariae (in Melanoides snail). DNA was ex- showed high infection capability because it tracted from adult worm in lanes 1-10, and was found in both Melanoides and Tarebia from cercariae in lanes 11-12. 1218 Vol 40 No. 6 November 2009 MOLECULAR INDENTIFICATION OF LARVAL TREMATODE IN INTERMEDIATE HOSTS snails. For metacercariae infection, Siamese Chiang Mai University for providing instru- mud carp (H. siamensis) was the most sus- mental facilities. ceptible fish host for H. taichui, half- beaked fish (D. pusillus) for S. falcatus metacercariae REFERENCES infection, and Haplorchoides sp and C. caninus were concomitantly found in P. brevis. It can Anuntalabhochai S, Chiangda J, Chandet R, be assumed that pleurolophocercus is serv- Apawat P. Genetic diversity within lychee (Litchi chinensis Soonn.) based on RAPD ing as a dominant cercarial type found in the analysis [Abstract]. Cairhs, Australia: Inter- study area, while most of the metacercariae national Symposium on Tropical and Sub- belong to family Heterophyidae, similar to tropical Fruit. 26th Nov-1st Dec 2000: 45. results of Sripalwit et al (2003); Kumchoo et al Boonchot K, Wongsawad C. A survey of (2005) and Boonchot et al (2005). helminthes in cyprinoid fish from the Mae HAT-RAPD profile confirmed that Ngad Somboonchon reservoir, Chaing Mai pleurolophocercus cercariae
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