Parasitol Res (2011) 108:751–755 DOI 10.1007/s00436-010-2180-8 SHORT COMMUNICATION Genetic characterization of Echinostoma revolutum and Echinoparyphium recurvatum (Trematoda: Echinostomatidae) in Thailand and phylogenetic relationships with other isolates inferred by ITS1 sequence Weerachai Saijuntha & Chairat Tantrawatpan & Paiboon Sithithaworn & Ross H. Andrews & Trevor N. Petney Received: 2 November 2010 /Accepted: 17 November 2010 /Published online: 1 December 2010 # Springer-Verlag 2010 Abstract Echinostomatidae are common, widely distribut- an isolate from Thailand with other isolates available from ed intestinal parasites causing significant disease in both GenBank database. Interspecies differences in ITS1 se- animals and humans worldwide. In spite of their impor- quence between E. revolutum and E. recurvatum were tance, the taxonomy of these echinostomes is still contro- detected at 6 (3%) of the 203 alignment positions. Of these, versial. The taxonomic status of two species, Echinostoma nucleotide deletion at positions 25, 26, and 27, pyrimidine revolutum and Echinoparyphium recurvatum, which com- transition at 50, 189, and pyrimidine transversion at 118 monly infect poultry and other birds, as well as human, is were observed. Phylogenetic analysis revealed that E. problematical. Previous phylogenetic analyses of Southeast recurvatum from Thailand clustered as a sister taxa with Asian strains indicate that these species cluster as sister E. revolutum and not with other members of the genus taxa. In the present study, the first internal transcribed Echinoparyphium. Interestingly, this result confirms a spacer (ITS1) sequence was used for genetic characteriza- previous report based on allozyme electrophoresis and tion and to examine the phylogenetic relationships between mitochondrial DNA that E. revolutum and E. recurvatum in Southeast Asia are sister species. Hence, the taxonomic status of E. recurvatum in Thailand, as well as in Southeast W. Saijuntha Walai Rukhavej Botanical Research Institute (WRBRI), Asian countries needs to be confirmed and revised using Mahasarakham University, more comprehensive analyses based on morphology and Maha Sarakham 44150, Thailand other molecular techniques. C. Tantrawatpan (*) Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Introduction Rangsit Campus, Pathumthani 12121, Thailand Echinostomiasis is a significant disease in domestic e-mail: [email protected] animals, wildlife, and humans (Graczyk and Fried 1998). : P. Sithithaworn R. H. Andrews Human echinostomiasis, which is caused by at least 19 Department of Parasitology, Liver Fluke and Cholangiocarcinoma species of echinostomes from eight genera, has been Research Centre (LFCRC), Faculty of Medicine, reported in China, India, Indonesia, Japan, Korea, Malay- Khon Kaen University, Khon Kaen 40002, Thailand sia, the Philippines, Russia, Taiwan, including Thailand (Graczyk and Fried 1998; Miliotis and Bier 2003; Fried et T. N. Petney al. 2004). Morbidity and mortality due to echinostomiasis Department of Ecology and Parasitology, are related to parasite load, with anemia, headache, Karlsruhe Institute of Technology, Kornblumen Strasse 13, dizziness, stomach and abdominal pain, and loose stools Karlsruhe, Germany being most often reported for light-to-moderate infections. 752 Parasitol Res (2011) 108:751–755 Heavy infections are associated with eosinophilia, abdom- inal pain, profuse watery diarrhea, anemia, edema and anorexia, and can potentially be lethal (Huffman and Fried 1990; Graczyk and Fried 1998; Chai et al. 2005). The life cycle of echinostomes involves eggs that are passed with host faces into the water where they reach the fully developed miracidial stage, whereupon the miracidia hatch, seek out and penetrate into their first intermediate snail hosts. They then transform from sporocysts to radiae, represents an identical nucleotide and then cercariae which emerge to seek a second dot intermediate host, e.g., other gastropods, bivalves, fish, or tadpoles. Definitive host infection by humans occurs sp. A through eating raw or uncooked snails or tadpoles contain- 111111111 ing echinostome metacercariae (Huffman and Fried 1990). ...T.....T ...T.....T Echinostomiasis is difficult to control, because a wide range of aquatic animals, namely snails, bivalves, crustaceans, fish, and amphibians serve as the second intermediate hosts Echinoparyphium (Huffman and Fried 1990; Graczyk and Fried 1998). A , and – high rate of echinostome infection in domestic and wild – poultry and water birds would be expected, as aquatic A A animals are common components of their diets. The taxonomic characters used to distinguish between E. recurvatum species and genera of the echinostomes include the structure , of the circumoral disc, the number and arrangement of rows of T ...T.....T uninterrupted crown spines, the number of collar spines, as well T ...T.....T as testicular characters (Miliotis and Bier 2003). However, a E. revolutum large number of morphologically very similar adult stages from many species exist, due to a long history of inadequate description, poor specific diagnoses, and extensive synonymy (Kostadinova et al. 2003). The taxonomy of this group is therefore confused and controversial. In addition, E. revolu- tum shares some major, distinguishing morphological charac- ters with E. recurvatum, e.g., they have a well-developed circumoral dish and collar spine. The gross morphology is different in testes, which are multilobed in E. revolutum and ––– ––– ––– –– oval in E. recurvatum. However, considerable variation in ––– – testicular characters in E. revolutum endemicinThailandhas 1222223455555788001223358 been reported with the oval shape as in E. recurvatum being Alignment position: observed (Petleart et al. 2005). The major difference between 87567890302457417578782359 these two species is the number of collar spines, 37 and 43–50 collar spines in E. revolutum and E. recurvatum, respectively a (Fried and Graczyk 2004). However, collar spines are very small and light and electron microscopy are needed for determining number and arrangement. Moreover, spine loss or retraction of spines may occur by improper handling, leading ThailandUK HM747938 C A AY168931Thailand A HM747939 T G A T A T T C C C T G C T G G T A T T C T A G T G A G T T C T C G . A . G A . USA GQ463130 USA AF067850 Australia ERU58102 to mistaken identification (Fried et al. 2009). isolate from Thailand As a consequence, a variety of genetic markers have been developed and used effectively to differentiate among sp.sp. USA USA GQ463136 GQ463135 T G T T C T C T C C T T T C T T A T A T C C C C . A . A G G A . represents nucleotide deletion echinostome species and explore their population genetic b b and phylogenetic relationships between/among different “–” E. recurvatum genera, species, and geographic areas, as well as temporally Variable positions in the nucleotide sequence alignments of the partial ITS1 sequences of and in relation to intermediate hosts (Kostadinova et al. sample examined in this study accession number Echinoparyphium E. revolutum E. recurvatum Echinoparyphium Table 1 a b to that in Parasite sample Isolate AC no. E. recurvatum E. revolutum E. revolutum E. revolutum 2003, Detwiler et al. 2010, Saijuntha et al. 2010a, b). The symbol Parasitol Res (2011) 108:751–755 753 Genetic variation in echinostomes in Southeast Asia has slaughterhouse in Khon Kaen Province, Thailand. The been studied using allozyme markers and the cytochrome c species of adult worms were identified using a light oxidase subunit 1 (CO1) sequence (Saijuntha et al. 2010a, microscope according to the body size, size of the b). Interestingly, this study revealed that E. revolutum was circumoral disc, testes morphology, and the number of more closely aligned with E. recurvatum than the other collar spines (Miliotis and Bier 2003). The adult worms species of genus Echinostoma (e.g., E. malayanum), were then washed extensively in normal saline and frozen contradicting traditional morphological taxonomy (Saijun- at −80°C for subsequent electrophoretic analyses. tha unpublished). Thus, this finding needs to be confirmed by a more comprehensive genetic investigation using other ITS1 sequence analysis molecular genetic markers. The ITS region has been shown to be valuable for characterizing and discriminating After being crushed using a tissue grinder and lysed with between species having similar characteristics, including lysis buffer, proteinase K (200 μg/ml), genomic DNA the phylogenetic relationships of many parasitic trematodes, (gDNA) from adult worms was extracted using a DNA e.g., opisthorchid liver flukes (Park 2007; Kang et al. extraction kit (QIAGEN, Hilden, Germany). PCR was 2008), Fasciola spp. (Ichikawa and Itagaki 2010) and 37- used for amplification using primers: forward BD1 (5′- collar-spine group of echinostomes (Morgan and Blair GTCGTAACAAGGTTTCCGTA-3′) and reverse BD2 1995). In this study, genetic characterization of E. revolu- (5′-TATGCTTAAATTCAGCGGGT-3′)(Morganand tum and E. recurvatum from Thailand and the taxonomic Blair 1995) in a PTC-200 thermal cycler (MJ Research, and phylogenetic relationships with other isolates are Watertown, Massachusetts, USA), and reaction volumes investigated based on ITS1 sequence analysis. of 25 μl containing 25 mM MgCl2, 10× buffer, 2.5 mM dNTPs,and0.7uofTaq DNA polymerase (iNtRON Biotechnology, Korea). The PCR conditions used were: Materials and methods 96°C for 1 min,