วารสาร​มหาวิทยาลัย​ราชภัฏ​ยะ​ลา 9 ​Journal of Yala Rajabhat University

Prevalence of taichui Infection in Snails from MaeTaeng Basin, Chiang Mai Province, by Using Morphological and Molecular Techniques

Thapana Chontananarth* and Chalobol Wongsawad* Abstract

The investigation of biological diversity of intestinal trematode in snails which the report concerned is rarely and not extended in current status. Especially in minute intestine trematode, Witenberg, 1930, family of , which was found in the small intestine of mammals including human, and causes serious clinical problem worldwide. So, this study was aimed to investigate H. taichui infection in freshwater snails from Mae Taeng basin, Chiang Mai, Thailand. Total of 1,836 snails were collected during April 2008 to August 2012. Cercarial infection was examined by crushing method. Molecular identification of H. taichui were conducted by a DNA specific primer which amplified the mCOI gene. The PCR product of mCOI were sequenced and confirmed by BLAST program. Six types of cercariae were found viz. megalurous, furcocercous, monostome, pleurolophocercous, parapleurolophocercous, and virgulate cercaria. The parapleurolophocercous cercaria in Melanoides tuberculata, Tarebia granifera, and Thiara scabra were larval stages of H. taichui which yield the specific fragment of 256 bp. Overall of H. taichui infection of snails was 42.71%. The mCOI sequences had 99% similarity with the 29 isolated gene references of the H. taichui in Genbank data base. The molecular method had suitability as an epidemiological tool for suitable control programs against the dissemination of trematodes.

Keywords : Intestinal trematodes Prevalence Molecular identification mCOI gene

* Department of Biology Faculty of Science Chiang Mai University Chiang Mai 50202 Thailand.

ป​ที่ 8 ฉบับ​ที่ 1 มกราคม-มิถุนายน 2556 Vol.8 No.1 January-June 2013 10 ความชุกของพยาธิใบไม้ในลำ�ไส้ Intestinal trematodes prevalence

ค่าความชุกของการติดพยาธิใบไม้ชนิด Haplorchis taichui ในหอยเดียวจากลุ่มแม่น�้ำแม่แตง จังหวัดเชียงใหม่ โดยเทคนิคทางสัญฐานวิทยาและอณูชีววิทยา

ฐาปนา ชลธนานารถ* และ ชโลบล วงศ์สวัสดิ์* บทคัดย่อ

การศึกษาความหลากหลายทางชีวภาพของพยาธิใบไม้ในล�ำไส้ (intestinal trematode) มีรายงาน น้อยมากและยังไม่ครอบคลุม โดยเฉพาะพยาธิใบไม้ในล�ำไส้ขนาดเล็ก Haplorchis taichui Witenberg, 1930 ในวงศ์ Heterophyidae เป็นปรสิตในสัตว์เลี้ยงลูกด้วยนมรวมถึงมนุษย์ การติดเชื้อพยาธิใบไม้ก่อให้ เกิดปัญหาที่ส�ำคัญทางสาธารณสุข และมีรายงานการระบาดในประเทศไทยอย่างต่อเนื่อง ดังนั้นการศึกษา นี้มีวัตถุประสงค์เพื่อศึกษาการติดเชื้อพยาธิใบไม้ H. taichui ในหอยฝาเดียวจากลุ่มแม่น�้ำแม่แตงจังหวัด เชียงใหม่ โดยท�ำการเก็บหอยฝาเดียวทั้งหมด 1,836 ตัว ระหว่างเดือนเมษายน 2008 ถึง เดือนสิงหาคม 2012 เพื่อตรวจสอบการติดตัวอ่อนพยาธิใบไม้โดยวิธี crushing ส�ำหรับการระบุอัตลักษณ์ของพยาธิใบไม้ ชนิด H. taichui จะถูกศึกษาโดยเทคนิคทางอณูชีววิทยาด้วยไพร์เมอร์แบบจ�ำเพาะและถูกเพิ่มปริมาณ ดีเอ็นเอของยีน mCOI (mitochondrial cytochrome c oxidase subunit I) เพื่อหาล�ำดับเบสและตรวจ สอบด้วยโปรแกรม BLAST จากผลการศึกษาพบเซอร์คาเรียทั้งหมด 6 รูปแบบ คือ megalurous cercaria furcocercous cercaria monostome cercaria pleurolophocercous cercaria parapleurolophocercous cercaria และ virgulate cercaria การตรวจสอบการติดพยาธิ H. taichui ระยะเซอร์คาเรียในหอยโดย เทคนิคทางอณูชีววิทยาพบว่าเซอร์คาเรียรูปแบบ parapleurolophocercous cercaria ให้ผลการทดสอบ เป็น positive result โดยสามารถเกิดแถบดีเอ็นเอขนาด 256 bp ที่มีความจ�ำเพาะต่อพยาธิ H. taichui มี ค่าความชุกรวมร้อยละ 42.71 ส่วนล�ำดับเบสของยีน mCOI มีความคล้ายคลึงเท่ากับร้อยละ 99 จาก 29 ไอโซเลทของ H. taichui ในฐานข้อมูลของ Genbank การศึกษาในครั้งนี้ชี้ให้เห็นว่าเทคนิคทางอณูชีววิทยา สามารถน�ำมาประยุกต์เป็นเครื่องมือในการศึกษาด้านระบาดวิทยาเพื่อใช้ในการวางแผนควบคุมการกระ จายตัวของพยาธิใบไม้อย่างมีประสิทธิภาพยิ่งขึ้น

ค�ำส�ำคัญ : พยาธิใบไม้ ค่าความชุก การระบุชนิดเชิงโมเลกุล ยีนไซโคโครมซี ออกซิเดส

* ภาควิชาชีววิทยา คณะวิทยาศาสตร์ มหาวิทยาลัยเชียงใหม่ จังหวัดเชียงใหม่ 50200

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Introduction fish-eating mammals as definitive hosts (7, 8). Mae Taeng basin is located in Chiang The pleurolophocercous and parapleurolopho Mai province, northern Thailand (Figure 1). It cercous cercariae have been reported is serves as an important area for both aquatic heterophyid trematodes including H. taichui, culture and fisheries. Much of this area is in H. pumilio, Stellantchasmus falcatus, and Mae Ngad Somboonchon Dam irrigating Centrocestus caninus, which are abundantly channels which have high biodiversity of recovered from freshwater snails in northern aquatic that are associated in the Thailand (9-11). completion of trematode life cycles (1, 2). The traditional method of H. taichui There have been several reports the heterophyid detection in snails is usually performed by trematode infections in freshwater animals in exposing the snails to light or by dissection, Chiang Mai (3), especially, the minute intestinal in which cercariae may be observed. These trematode, Haplorchis taichui, Witenberg, 1930 methods are difficult to detect specific parasite which have a high prevalence of infection in stages to the species because of similar cyprinoid fishes (4-6). morphological traits in closely related species The life cycle of H. taichui, involves the Heterophyidae. An accurate identification freshwater snails as the first intermediate host, of cercarial stage is very important for cyprinoid fishes as second host, and with understanding the epidemiological situation

Figure 1 A map of Thailand showing the collecting sites in Mae Taeng basin, Chiang Mai province, Thailand. (1) irrigation canal (2-3) rice paddys (4-5) Mae Taeng River (6) Mae Ngad Somboonchon Dam.

ป​ที่ 8 ฉบับ​ที่ 1 มกราคม-มิถุนายน 2556 Vol.8 No.1 January-June 2013 12 ความชุกของพยาธิใบไม้ในลำ�ไส้ Intestinal trematodes prevalence and for developing effective control measures. providing new data on the recent distribution Various PCR methods have been and prevalence of trematode infections and developed for detection of trematode species for developing effective control measures. in intramolluscan stages namely, Amplified Fragment Length Polymorphism (AFLP), High Method Annealing Temperature – Random Amplified Study sites Polymorphic DNA (HAT-RAPD), Polymerase Six sites were selected based on areas Chain Reaction – Restriction Fragment Length with public health problems with trematode Polymorphism (PCR-RFLP), and gene infections in and around the Mae Ngad sequencings (12-14). The development of Somboonchon Dam, Mae Taeng district, aspecific DNA primer for H. taichui detection Chiang Mai province. These areas include an has been rarely successful. Some report have irrigation canal (N 21//12.864/ E 0.50//08.488/), been established specific primers for the rice paddies (N 21//13.134/ E 0.50//07.982/ detection of H. taichui by generating the 256 and N21′′16.486′ E 0.4′′09.359′), Mae Taeng River bp specific fragment as a positive result (6). (N 21//12.992/ E 0.50//07.862/ and N21′′12.992 Data from mitochondrial-based PCR E0.50′′07.862′), and the dam (N21′′19.725′E0. have been extensively used in studies of 50′′03.645′) (Figure 1). identification, evolution, phylogeny, biogeography, population genetics and Snail samples and cercarial infections systematic because the mitochondrial Snail specimens were collected by genomes are readily adundantly copied and hand during April 2008 to June 2010. After not strikingly different in related species (15). collection, the snails were identified by using Lee et al. compared the phylogenetic Brandt (17) and were examined for cercarial relationship of these heterophyid trematodes, infection using a crushing method. Each viz. Metagonimus yokogawai, M. takahashii, groups of cercaria were stained in Delafield’s and M. miyatai, using 28S rDNA and haematoxylin, dehydrated in an alcohol series, cytochrome c oxidase subunit I (mCOI) gene. cleared with xylene, and mounted in permount. The results showed that the phylogenetic tree The cercariae were then identified to family from 28S ribosomal DNA was similar with level and in some cases to genus (18, 19). mCOI sequence data (16). The pleurolophocercous and parapleurolo Our study provides data on the phocercous cercariae were preserved in 70% prevalence of H. taichui infections in alcohol for DNA extraction since these freshwater snails from Mae Taeng basin, cercarial types have been reported as minute Chiang Mai, Thailand by morphological and intestinal heterophyid trematodes (20). PCR-based methods. This is the first step in

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Genomic DNA extraction and fragment, were amplified the barcoding region PCR amplification mCOI gene by a pair of primers according to The genomic DNA was extracted and Yu et al. (21). It included (JB3) 5′′′′ purified individually from both cercarial types TTTTTTGGGCATCC TGACGTTTAT 3′′′, as a found in each snail using a commercial GF-1 forward primer, and (JB 4,5) 5′′′ tissue DNA extraction kit (Vivantis, Malaysia) TAAAGAAAGAACATAATGAAAATG 3′′′′, as a according to the manufacturer’s instructions. reverse primer. The specific DNA fragment of mCOI Molecular identification by were direct sequenced. And confirmed by DNA specific primer BLAST (Basic Local Alignment Search Tool) PCR was performed to detect the program in the NCBI (National Center for H. taichui infections in snails. The protocol Biotechnology Information). followed Wondsawad et al. (6). Sequences of the primers used in this PCR amplification Results were Forward 5′′′-GGCCAACGCAATCGTCATCC Prevalence of infection -3​′′ and Reverse 5′​′-GCGTCGGGTTTCAGACA 1,836 snails of 11species were TGG-3′′. Using these primers, genomic DNA collected, viz. Bithynia siamensis, Clea helena, was amplified in 20 µl final volume, including Eyriesia eyriesi, Filopaludina doliaris, F. 50 ng of DNA template, 50 pM/ µl of each martensi, F. sumatrensis, Lymnaea auricularia, primer (forward and reverse), 1.5 mM of Melanoides tuberculata, Pila polita, Tarebia MgCl2, 200 µ of dNTPs and 0.5 unit of Taq granifera, and Thiara scabra. The results DNA polymerase, and volume made to 20 µ​ l showed that 315 out of 1,836 snails with deionized water. had cercarial infection, with an overall Amplification was perform in a prevalence of 17.16% (Table 1). M. tuberculata, thermocycles with the initial denaturation step L. auricularia, and T. granifera were the most at 95o​ºC for 5 minutes, then 35 cycles including infected and susceptible hosts for cercarial denaturation at 95oºC for 45 seconds, primer infections with a prevalence of 35.71%, annealing at 68oºC for 45 seconds, extension 33.33%, and 30.50%, respectively. C. helena, at 72oºC for 1 minutes, and final extension at F. sumatrensis, F. doliaris, and P. polita were 72oºC for 7 minutes. PCR products were not infected. analyzed through 1.8% agarose gels Six groups of cercariae were detected: electrophoresis separation at 50 volts for 40 megalurous, furcocercous, monostome, minutes in TBE buffer, stained with ethidium pleurolophocercous, parapleurolophocercous, bromide, and visualized with a Kodak digital and virgulate cercaria. They were subdivided camera (Gel Logic 100). into 7 types based on morphological The genomic DNA of positive results, characteristics and internal organ arrangement. which gave an amplification of the 256 bp They are described here (Figure 2).

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Table 1 Prevalence of cercarial infections in snails from the Mae-Taeng basin, Chiang Mai, Thailand

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Figure 2 Seven types of cercaria were found infect to snails. A – distome cercaria: Philophthalmidae cercaria, B – furcocercouse cercaria, Strigeidae cercaria, C – furcocercous cercaria: Transversotrema sp., D – monostome cercaria: Notocotylidae cercaria, E - redia of Notocotylidae, F – pleurolophocercous cercaria, Heterophyidae cercaria, G – parapleurolophocercous cercaria: Haplorchis sp. cercaria (body), H - anterior part of Haplorchis sp., I –virgulate cercaira: Lecithodendriidae cercaria (ag - adhesive gland, df - dorsoventral finfold, ep - eye spots, fu - furca, lf - lateral finfold, os - oral sucker, pg - penetrated gland, ph - pharynx, st - stylet, ts – tail stem, tt – testis, vo - virgulate organ, vs – ventral sucker ) (scale bar 0.1 mm).

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Megalurous cercaria: Philophthalmidae An oral sucker is absent while a ventral sucker This parasite only infected T. granifera. is present medially. Testes are symmetrical The megalurous cercaria is ovoid with and within the intestinal ring, while the genital yellowish-brown granules. The body surface pore is located medially. The tail is longer is covered with longitudinal and fine traverse than the body and with arm like processes wrinkles. Minute spines cover the surface of at the anterior end of the tail-stem (Figure the posterior half of the body. The esophagus 2C). bifurcated midway between the pharynx and the ventral sucker, and the intestinal ends Monostome cercaria: Notocotylidae blindly near the posterior end of body. The Notocotylidae cercariae were found ventral sucker is slightly larger than the oral only in B. siamensis and T. granifera. The sucker, and is located medially on the the cercaria body is ovoid and the anterior quarter body. The tail is as long as the body, elastic, of the body is highly pigmented, especially and slender. Long tail tips numerous adhesive around the eyespots. There are three black gland cells and lacks an excretory canal in pigmented eyespots arranged in the form of the tail base (Figure 2A). a triangle in the anterior dorsal region of the mature cercaria (Figure 2D). Furcocercous cercaria: Strigeidae This parasite was found in B. siamensis, Pleurolophocercous cercaria: Heterophyidae F. martensi, and M. tuberculata. The cercaria This type of cercaria was found in body is ovoid and the body surface is covered B. siamensis, L. auricularia, M. tuberculata, with minute spines. The oral sucker is T. granifera, and T. scabra. The cercaria is subterminal. The pharynx is very small and ovoid and entirely covered with minute spines. circular. The large intestine is composed of A pair of conspicuous eyespots rectangular two ceca. The tail is forked and longer than are found at the end of the anterior third of the body. The tail stem is longer than the the body. The pharynx lies just behind the furca, and each furca has a dorsal and ventral oral sucker. Intestinal ceca bifurcate and finfold (Figure 2B). extend posteriorly to the excretory vesicle level. Seven pairs of penetration glands are Furcocercous cercaria: Transversotrema sp. arranged in 2 bundles anterolaterally to the Transversotrema cercaria was found genital premordium, which is a relatively large only in T. scabra. The cercaria body is short, triangular mass of cells located just anterior flat, discord, and with yellowish-brown to the excretory vesicle. The tail is almost the pigmentation. The body surface is covered as long as body, slender, and usually attached with scale-like spines. The pharynx is very to the dorso-ventral finfolds. A brownish large and the esophagus is short and narrow. pigment is dispersed throughout the body There are one pair of eyespots which are large except in the oral sucker (Figure 2F). spherical, located posterior to the pharynx.

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Parapleurolophocercous cercaria : Haplorchis sp. The oral sucker extends to a pharynx; This type of cercaria was found in B. oesophagus bifurcating preacetabularly into siamensis, E. eyriesi, M. tuberculata, T. an intestinal caeca which encircles the granifera, and T. scabra. The cercaria is ovoid acetabulum, terminating postacetabularly. The and is entirely covered with minute spines. acetabulum is smaller than the oral sucker. The small pharynx lies just behind the oral The virgulate organ is butterfly–shaped, sucker. Two square eyespots are located on situated below the stylet. There are three pairs each side above the pharynx. The anterior of penetration glands, the two anterior pairs part of the body is covered with fine spines. have fine granules and the posterior pair with Seven pairs of penetration glands are located coarser granules medially. The tail is shorter between the pharynx and the posterior part than the body (Figure 2I). of the body, surrounding mainly the genital premordium and arranged into two columns H. taichui infection in snails with the ventral sucker. The excretory vesicles H. taichui specific primers were are elongate. The tail is slender about 3 times performed to amplify a 256 bp fragment in the body length with two lateral finfolds the PCR technique for the identification of extending along the anterior third of the tail H.taichui cercariae infecting snails. Amplification A dorsoventral finfolds extends along the of H. taichui cercariae could detect only the posterior two- thirds and around tip of the 256 bp specific fragment. Genomic DNA of tail (Figure 2 G). adults from this parasite were used as a positive control, which also gave 256 bp Virgulate cercaria: Lecithodendriidae specific fragment. This carcaria was found in B. siamensis, As shown in Table 2, 199 of 1,836 F. martensi, M. tuberculata, T. granifera, and snails were infected by parapleurolophocercous T. scabra. The cercaria is small shaped with cercaria including B. siamensis, E. eyriesi, a prominent stylet and develops in sporocysts. M. tuberculata, T. granifera, and T. scabra.

Table 2 Prevalence of H. taichui infections, using specific PCR.

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Figure 3 Gel electrophoresis profiles of parapleurolophocercous cercariae isolated from thiarid snails. Lane M: molecular size maker, Lane N: negative control, Lane Ht: adult of H. taichui, DNA samples of parapleurolophocercous cercariae form snails (Lane Mt: Melanoides tuberculata, Lane Ta: Tarebia granifera, Land Ts : Thiara scabra, Lane Bs: Bithynia siamensis; Lane Ee: Eyriesia eyriesi).

Figure 4 Sequence of mCOI nucleotides of Haplorchis taichui. After testing with H. taichui specific primers, Discussion only three species were infected this trematode, Eleven snail species from the Mae viz. M. tuberculata, T. granifera, and T. scabra Taeng basin are intermediate hosts of (Figure 3). The overall prevalence of H. taichui digenetic trematodes. We found, 7cercarial infections based on PCR detection was types in 4 snail families, viz. Thiaridae, 42.71%. T. granifera was the most susceptible Viviparidae, Bithyniidae, and Lymnaeidae) from snail host for H. taichui infections (Table 2). 7 snail species (Table 1). The prevalence of The length of partial mCOI nucleotide cercarial infections found in our study sequence data was 340-bp. The mCOI (17.16%) was higher than Mard-arhin et al. sequence showed 99% similarity with the found in five provinces in northern Thailand reference sequence of the 29 isolated (6.20 %) (22). Dechruruksa et al. reported that H. taichui in the Genbank data base (Figure 4). the prevalence of cercarial infections in thiarid

ป​ที่​8​ฉบับ​ที่​1​มกราคม-มิถุนายน​2556​ Vol.8​No.1​January-June​2013 วารสาร​มหาวิทยาลัย​ราชภัฏ​ยะ​ลา 19 ​Journal of Yala Rajabhat University snails from the Khea River, Phitsanulok indicated that the mCOI sequence products province, Thailand was only 0.9% (11). The good to species level and applicable for Transversotrema sp. cercaria infected only molecular identification. According to T. scabra which agrees with Wongsawad and Thaekham et al. investigated the PCR-RFLP Kumchoo (23). patterns of cytochrome c oxidase subunit I In our study, parapleurolophocercous (mCOI) gene amplification to distinguish O. and pleurolophocercous cercariae were viverrini from H. taichui (12). dominant. They have been reported previously Our results established that three as heterophyid trematodes, viz. C. caninus, species of snails (M. tuberculata, T. granifera, H. taichui, H. pumilio, and S. falcatus (11, 13, and T. scabra) serve as the first intermediate 24). Our findings agree with Radomyos et al. hosts of H. taichui. Dechruksa et al. reported who reported that heterophyid trematodes are that thiarid snails were infected with intestinal widely distributed and with high prevalence and blood fluke larvae (11). The snails, M. in northern Thailand (25). Kumchoo et al. tuberculata and T. granifera are medically reported a high prevalence of H. taichui important since they can serve as the first infections (91.4%) in cyprinoid fish from Mae intermediate host of minute intestinal flukes Taeng district, Chiang Mai province (5). (13, 24). Nithikathkul and Wongsawad (2008) indicated The PCR-based method is a more a high prevalence of H. taichui and effective approach for the identification of Haplorchoides sp. infections (83.90%) in H. taichui cercariae including other medically Chiang Mai province (26). important species, which can be used for For the PCR-based assay, parapleurolo epidemiological studies, and developing a phocercous cercariae were confirmed to be control program. H. taichui. Overall H. taichui infections based on specific PCR amplification was 42.71% of Acknowledgements all parapleurolophocercous cercariae We greatly appreciate the Applied populations. The prevalence of the Parasitology Research Laboratory, Institute morphological method was higher than the for Science and Technology Research and molecular method because the PCR-based Economic Plants Genome Research and method is more exact specific than the Service Center, Chiang Mai University, identification by using only morphology. Thailand for providing research facilities. Pleurolophocercous and parapleurolo phocercous cercariae can develop to be Special thanks are given to Mr. Sukson several heterophyid species. Our study Chuboon for his helpful and suggestions. We provides significant evidence for the would like to thank Dr. J.F. Maxwell for editing identification and epidemiological situation of our manuscript. Finally, we acknowledge the larval trematodes in their intermediate hosts. graduate school, Chiang Mai University for The high similarity of mCOI with 29 isolates supporting the publication fee of this paper. of H. taichui from Vietnam and Thailand ป​ที่ 8 ฉบับ​ที่ 1 มกราคม-มิถุนายน 2556 Vol.8 No.1 January-June 2013 20 ความชุกของพยาธิใบไม้ในลำ�ไส้ Intestinal trematodes prevalence

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