Morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae from Chinese pipistrelle bats and viviparid snails in Thailand.

This study aimed to describe the morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae isolated in Thailand. Adult flukes were isolated from the Chinese pipistrelle bat (Hypsugo sp.), and cercariae were detected in the viviparid snail (Filopaludina martensi martensi) from Chiang Mai province. The morphological characteristics were observed and described using conventional methods, and the molecular characteristics with internal transcribed spacer 2 (ITS2) and 28S rDNA gene sequences. The adult flukes were fusiform, 0.84-0.98 mm in length, and 0.37-0.49 mm in width, and were distinguishable from other species by the presence of longitudinal uterine coils. The cercariae were nonvirgulate xiphidiocercariae, with the oral sucker bigger than the acetabulum, the tail without fin fold, a body size of 117.5-138.3 × 48.3-52.2 µm, and a tail size of 100.7-103.7 × 15.0-18.9 µm. Molecular studies revealed that the adults and cercariae shared 99.3% (ITS2) and 99.6% (28S rDNA) homology with each other. They were phylogenetically close to P. longiforme with an identity of 94.5% for ITS2 and 98.7% for 28S rDNA. This study provides new information on the natural definitive host and first intermediate host of P. longiforme in Thailand. The discovery of its cercarial stage in Filopaludina snails highlights the importance of monitoring the associated second intermediate host and prevention and control of this potentially zoonotic trematode.

Morphology and Molecular Identification of Echinostoma revolutum and Echinostoma macrorchis in Freshwater Snails and Experimental Hamsters in Upper Northern Thailand.

Echinostome metacercariae were investigated in freshwater snails from 26 districts in 7 provinces of upper northern Thailand. The species identification was carried out based on the morphologies of the metacercariae and adult flukes harvested from experimental hamsters, and on nucleotide sequences of internal transcribed spacer 2 (ITS2) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) genes. Twenty-four out of 26 districts were found to be infected with echinostome metacercariae in freshwater snails with the prevalence of 40.4%. The metacercariae were found in all 6 species of snails, including Filopaludina martensi martensi (21.9%), Filopaludina doliaris (50.8%), F. sumatrensis polygramma (61.3%), Bithynia siamensis siamensis (14.5%), Bithynia pulchella (38.0%), and Anenthome helena (4.9%). The echinostome metacercariae found in these snails were identified as Echinostoma revolutum (37-collar-spined) and Echinostoma macrorchis (45-collar-spined) morphologically and molecularly. The 2-week-old adult flukes of E. revolutum revealed unique features of the cirrus sac extending to middle of the ventral sucker and smooth testes. E. macrorchis adults revealed the cirrus sac close to the right lateral margin of the ventral sucker and 2 large and elliptical testes with slight indentations and pointed posterior end of the posterior testis. The ITS2 and nad1 sequences confirmed the species identification of E. revolutum, and the sequences of E. macrorchis have been deposited for the first time in Gen-Bank. The presence of the life cycle of E. macrorchis is a new record in Thailand and the snail F. doliaris as their second intermediate host seems to be new among the literature.

Morphological and Molecular Characteristics of Anisakis typica Larvae in Two Species of Threadfin Bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand.

The third stage larvae (L3) of Anisakis typica were detected in 2 species of threadfin bream, Nemipterus hexodon and N. japonicus, from the Gulf of Thailand, and were morphologically and molecularly characterized. Total 100 threadfin breams, 50 Nemipterus hexodon and 50 N. japonicus, were examined with naked eyes after the opening of abdominal cavity with scissors. Almost all infected larvae remained alive and active even the fish were transported for 1-2 days. Anisakid larvae were exclusively distributed in the body cavity and rarely in the liver. The prevalence of A. typica L3 were 68.0% and 60.0% in N. hexodon and N. japonicus and their infection intensities were 3.5 and 4.2 per fish infected each. Morphological and morphometric analysis were performed by viewing specimens under both a light microscope and a scanning electron microscope. Interestingly, the protruded mucron of Anisakis typica under SEM showed a distinct cylindrical shape that differed from the cone shape of A. simplex. The protruded mucron could be used to identify A. typica L3 larvae in the future. A comparison of the ITS1-5.8S-ITS2 rDNA nucleotide sequences of these species revealed high blast scores with A. typica. Conclusively, it was confirmed that A. typica L3 are prevalent in threadfin breams from the Gulf of Thailand, and their morphological and molecular characters are something different from those of other anisakid larvae, including A. simplex and A. pegreffii.

Morphological and Molecular Identification of Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae) in Thailand.

We tried a series of morphological and molecular approaches to identify a new species of Stellantchasmus (Digenea: Heterophyidae) originating from the wrestling half-beaked fish, Dermogenys pusillus of Thailand. Adult worm samples of the new species were recovered from hamsters experimentally infected with the metacercariae from D. pusillus in Thailand. Two isolates (Thai and Korean) of Stellantchasmus falcatus were used as comparative control groups. Worm samples of 3 Stellantchasmus groups were morphologically observed and molecularly analyzed with the mitochondrial cytochrome c oxidase 1 gene. The morphological characteristics of S. dermogenysi n. sp. are similar to S. falcatus originating from brackish water fish, but minor difference was noted including the absence of the prepharynx, position of the ovary near the ceca end, smaller body size, and shorter esophageal length. A phylogenetic tree derived from neighborjoining and maximum-likelihood methods suggests that S. dermogenysi n. sp. is separated from S. falcatus supported by high bootstrap values. The relative divergences persist between these host-specific trematodes, which we suggest should be recognized as 2 distinct species. Comparisons of S. dermogenysi n. sp. with S. falcatus isolated from mullets in Thailand and Korea indicate a genetic divergence of mitochondrial DNA of 19.4% and 21.7%, respectively. By the present study, a new species, Stellantchasmus dermogenysi n. sp. (Digenea: Heterophyidae), is proposed in Thailand based on molecular evidences, in addition to minor morphological differences between S. falcatus and the new species.

Prevalence of Centrocestus formosanus Metacercariae in Ornamental Fish from Chiang Mai, Thailand, with Molecular Approach Using ITS2.

The prevalence of Centrocestus formosanus metacercariae was investigated in ornamental fish purchased from a pet shop in Chiang Mai, Thailand, including Carassius auratus (goldfish), Cyprinus carpio (Koi), Poecilia latipinna (Sailfin Molly), Danio rerio (Zebrafish), and Puntigrus tetrazona (Tiger barb). The parasite species was identified by the morphology of worms as well as by a molecular approach using ITS2. The results showed that 50 (33.3%) of 150 fish examined were infected with the metacercariae. The highest prevalence was found in C. auratus (83.3%), and the highest intensity was noted in C. carpio (70.8 metacercariae/fish). The most important morphological character was the presence of 32-34 circumoral spines on the oral sucker. The phylogenetic studies using the rRNA ITS2 region revealed that all the specimens of C. formosanus in this study were grouped together with C. formosanus in GenBank database. This is the first report on ornamental fish, C. carpio, P. latipinna, D. rerio, and P. tetrazona, taking the role of second intermediate hosts of C. formosanus in Thailand. Prevention and control of metacercarial infection in ornamental fish is urgently needed.

Molecular Phylogenetics of Centrocestus formosanus (Digenea: Heterophyidae) Originated from Freshwater Fish from Chiang Mai Province, Thailand.

This study aimed to investigate the morphology and reconstruct the phylogenetic relationships of Centrocestus formosanus originating from 5 species of freshwater fish, i.e., Esomus metallicus, Puntius brevis, Anabas testudineus, Parambassis siamensis, and Carassius auratus, in Chiang Mai province, Thailand. Sequence-related amplified polymorphism (SRAP) and phylogeny based on internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were performed. The results showed similar morphologies of adult C. formosanus from day 5 after infection in chicks. C. formosanus originated from 4 species of freshwater fish had the same number of circumoral spines on the oral sucker, except for those from C. auratus which revealed 34 circumoral spines. The phylogenetic tree obtained from SRAP profile and the combination of ITS2 and CO1 sequence showed similar results that were correlated with the number of circumoral spines in adult worms. Genetic variability of C. formosanus also occurred in different species of freshwater fish hosts. However, more details of adult worm morphologies and more sensitive genetic markers are needed to confirm the species validity of C. formosanus with 34 circumoral spines originating from C. auratus in the future.

Phylogenetic analysis reveals cryptic species diversity within minute intestinal fluke, Stellantchasmus falcatus Onji and Nishio, 1916 (Trematoda, Heterophyidae).

OBJECTIVE: To examine the taxonomic boundaries in the Stellantchasmus populations in different hosts and reveal the cryptic speciation of the Stellantchasmus in Thailand based on both morphological and molecular approaches. METHODS: Stellantchasmus falcatus (S. falcatus) s.l. was collected from different hosts throughout Thailand. The worms were examined and measured. The nuclear ITS2 gene and the mitochondrial COI gene were used to investigate the S. falcatus s.l. from Dermogenus pusillus and Liza subviridis. RESULTS: Stellantchasmus was one of food-borne trematode that widely distribute throughout Thailand. The comparison of S. falcatus s.l. from Dermogenus pusillus and Liza subviridis indicated a genetic divergence of nuDNA with 3.6% and mtDNA with 19.3%, respectively. Morphological characteristics were fairly different in the ratio of body size, length of prepharynx, and ratio of organ size. CONCLUSIONS: Our results provide initial evidence that S. falcatus s.l. from different hosts tend to be a different species based on both molecular and morphological characters. Cryptic species complexes are generally found among parasites that tend to have large populations and/or rapid evolution. The degree of genetic diversity existing would suggest the practice of targeted regimes to design and minimize these lections of anthelmintic resistance in the future.

DISCRIMINATION 28S RIBOSOMAL GENE OF TREMATODE CERCARIAE IN SNAILS FROM CHIANG MAI PROVINCE, THAILAND.

Trematode cercariae are commonly found in many freshwater gastropods. These cercariae can serve to identify the occurrence of such trematodes as Centrocestus formosanus, Haplorchis taichui, Haplorchoides sp, and Stellantchasmus falcatus, which are important parasites in Chiang Mai Province, Thailand. As the species of these cercariae cannot be identified accurately based on morphology, this study employed sequencing of a fragment of 28S ribosomal DNA and phylogenetic analysis to identify the trematode cercariae found in freshwater gastropods in Chiang Mai Province. Eight types of trematode cercariae were identified, namely, distome cercaria (grouped with Philophthalmus spp clade), echinostome cercaria (grouped with Echinostoma spp clade), furcocercous cercaria (grouped with Posthodiplostomum sp/Alaria taxideae/Hysteromorpha triloba clade), monostome cercaria (grouped with Catatropis indicus clade), parapleurolophocercous cercaria (grouped with Haplorchoides sp clade), pleurolophocercous cercaria (grouped with Centrocestusformosanus clade), transversotrema cercaria (grouped with Transversotrema spp clade), and xiphidiocercaria (grouped with Prosthodendrium spp clade). These results provide important information that can be used for identifying these parasites in epidemiological surveys.

Morphological and molecular profiling of Spirogyra from northeastern and northern Thailand using inter simple sequence repeat (ISSR) markers.

Green algae, Spirogyra (Chlorophyta), are found in a wide range of habitats including small stagnant water bodies, rivers, and streams. Species identification of Spirogyra based on morphological characteristics has proven to be a difficult process. An accurate identification method is required to evaluate genetic variations. This study is aimed at investigating the molecular profiling of 19 samples of Spirogyra from northern and northeastern Thailand. The morphological characteristics of each sample were recorded, viz. cell dimensions (width and length), along with the number and arrangement of chloroplast spirals/pyrenoids. With regard to a correlation of the biological and ecological parameters, conductivity was clearly significantly related to the number of pyrenoids. While DO is negatively related to the number of chloroplast spirals. Molecular studies with 10 ISSR primers were amplified to examine the DNA fingerprints. Morphological characters were determined to be significantly different by revealing 5 traits (P < 0.05) for all specimens. In addition, the DNA markers of all specimens were investigated using 10 ISSR primers. The results show that the PCR technique amplified 108 fragments. An analysis of the DNA fragments grouped all samples by ISRR-PCR, which were then separated into two groups according to their distribution.

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand.

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.

Molecular identification and phylogenetic relationship of green algae, Spirogyra ellipsospora (Chlorophyta) using ISSR and rbcL markers.

Spirogyra is found in a wide range of habitats, including small stagnant water bodies, rivers, and streams. Spirogyra ellipsospora is common in northern Thailand. Species identification of the Spirogyra species based only on morphological characteristics can be difficult. A reliable and accurate method is required to evaluate genetic variations. This study aims to apply molecular approaches for the identification of S. ellipsospora using microsatellites and rbcL markers. Based on DNA sequencing, the rbcL gene was sequenced and the data was analyzed using the BLAST (Basic Local Alignment Search Tool) program in the NCBI (National Center for Biotechnology Information) database. The sequence of S. ellipsospora from this study revealed definitive identity matches in the range of 99% for the consensus sequences of S. ellipsospora. The 10 primers of ISSR could be amplified by 92 amplification fragments. The DNA fragments and the rbcL sequence data grouped the Spirogyra specimens into two distinct clusters.

Molecular phylogenetics and character evolution of morphologically diverse groups, Dendrobium section Dendrobium and allies.

It is always difficult to construct coherent classification systems for plant lineages having diverse morphological characters. The genus Dendrobium, one of the largest genera in the Orchidaceae, includes ∼1100 species, and enormous morphological diversification has hindered the establishment of consistent classification systems covering all major groups of this genus. Given the particular importance of species in Dendrobium section Dendrobium and allied groups as floriculture and crude drug genetic resources, there is an urgent need to establish a stable classification system. To clarify phylogenetic relationships in Dendrobium section Dendrobium and allied groups, we analysed the macromolecular characters of the group. Phylogenetic analyses of 210 taxa of Dendrobium were conducted on DNA sequences of internal transcribed spacer (ITS) regions of 18S-26S nuclear ribosomal DNA and the maturase-coding gene (matK) located in an intron of the plastid gene trnK using maximum parsimony and Bayesian methods. The parsimony and Bayesian analyses revealed 13 distinct clades in the group comprising section Dendrobium and its allied groups. Results also showed paraphyly or polyphyly of sections Amblyanthus, Aporum, Breviflores, Calcarifera, Crumenata, Dendrobium, Densiflora, Distichophyllae, Dolichocentrum, Holochrysa, Oxyglossum and Pedilonum. On the other hand, the monophyly of section Stachyobium was well supported. It was found that many of the morphological characters that have been believed to reflect phylogenetic relationships are, in fact, the result of convergence. As such, many of the sections that have been recognized up to this point were found to not be monophyletic, so recircumscription of sections is required.

Opisthorchis viverrini and Haplorchis taichui: development of a multiplex PCR assay for their detection and differentiation using specific primers derived from HAT-RAPD.

Specific primers for the detection of Opisthorchis viverrini and Haplorchis taichui were investigated by using the HAT-RAPD PCR method. Fourteen arbitrary primers (Operon Technologies) were performed for the generation of polymorphic DNA profiles. The results showed that a 319 bp fragment generated from the OPA-04 primer was expected to be O. viverrini-specific while a 256 bp fragment generated from the OPP-11 primer was considered to be H. taichui-specific. Based on each sequence data, two pairs of specific primers were designed and sequences of each primer were as follows; H. taichui; Hapt_F5'-GGCCAACGCAATCGTCATCC-3'and Hapt_R1 5'-CTCTCGACCTCCTCTAGAAT-3' which yielded a 170 bp PCR product. For O. viverrini, OpV-1F: 5'-AATCGGGCTGCATATTGACCGAT-3' and OpV-1R: 5'-CGGTGTTGCTTATTTTGCAGACAA-3' which generated a 319 bp PCR product. These specific primers were tested for efficacy and specific detection for all parasites DNA samples. The results showed that 170 and 319 bp specific PCR products were generated as equivalent to positive result in H. taichui and O. viverrini, respectively by having no cross-reaction with any parasites tested. PCR conditions are recommended at 68°C annealing temperature and with 0.5 mM magnesium chloride (Mg Cl(2)). Additionally, specific primers developed in this study were effective to determine the presence of both parasites in fish and snail intermediate hosts, which the DNA of O. viverrini was artificially spiked since it is rarely found in northern Thailand. The H. taichui and O. viverrini-specific primers successfully developed in this study can be use for epidemiological monitoring, preventing management and control programs.

Identification of dendrobium species used for herbal medicines based on ribosomal DNA internal transcribed spacer sequence.

Stems of genus Dendrobium (Orchidaceae) have been traditionally used as an herbal medicine (Dendrobii Herba) in Eastern Asia. Although demand for Dendrobium is increasing rapidly, wild resources are decreasing due to over-collection. This study aimed to identify plant sources of Dendrobii Herba on the market based on sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. We constructed an ITS1-5.8S-ITS2 sequence database of 196 Dendrobium species, and the database was employed to identify 21 herbal samples. We found that 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) were possibly used as plant sources of Dendrobii Herba, and unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum were also used as sources. Furthermore, it is clear that D. catenatum is one of the most important sources of Dendrobii Herba (5 out of 21 samples).

Infection dynamics and molecular identification of metacercariae in cyprinoids from Chiang Mai and Sakon Nakhon Provinces.

The infection dynamics of metacercariae were assessed in cyprinoid fish (cyprinoids) from Chiang Mai and Sakon Nakhon Provinces, Thailand, during October 2008 to September 2009. The samples were collected during 3 seasons from rivers and local markets. Metacercarial infection was determined by acid-pepsin digestion and confirmed using HAT-RAPD PCR method. Thirteen and 16 species of cyprinoids were collected from Chiang Mai and Sakon Nakhon with overall prevalences of metacercarial infection of 84.5 and 47.6%, respectively. Haplorchis taichui, Haplorchoides sp, and Centrocestus caninus were found in Chiang Mai and 4 species of metacercariae: H. taichui, Haplorchoides sp, O. viverrini and an unknown trematode species in Sakon Nakhon. H. taichui and Haplorchoides sp metacercariae in 3 species of cyprinoids (Henicorhynchus siamensis, Cyclocheilichthys armatus, Amblyrhynchichthy truncatus) had the highest prevalence (100%) in Chiang Mai, while the highest prevalence (100%) of metacercaria in Sakon Nakhon was Haplorchoides sp in 1 species of cyprinoids (Cyclocheilichthys armatus). The overall prevalence from Chiang Mai Province was highest in the rainy season (95.6%), lower in the hot-dry season (88.1%) and lowest in the cool season (72.5%). In Sakon Nakhon Province the highest prevalence was in the hot-dry season (52.7%), and lower in the rainy and cool season, 44.4% and 43.5%, respectively. The HAT-RAPD profiles confirmed the identity of metacercariae and adult stage of H. taichui, Haplorchoides sp, C. caninus and O. viverrini.

Molecular markers for identification of Stellantchasmus falcatus and a phylogenic study using the HAT-RAPD method.

Stellantchasmus falcatus is a minute intestinal fluke in the family Heterophyidae. Metacercariae, the infective stage, were reported in a marine fish, mullet Liza subviridis, and a fresh water fish, Dermogenus pusillus, in Thailand. Adults were found in chicks, rats, cats, and humans. Morphological studies were done for comparing Stellantchasmus sp. worms found in 2 different fish hosts; their shapes and organ arrangements were very similar except for the prepharynx length. Therefore, the present study aimed to compare their DNA fingerprints using the HAT-RAPD method for both types of Stellantchasmus and several other related species. Ten arbitrarily selected primers (OPA-04, OPA-09, OPN-02, OPN-03, OPN-09, OPN-12, OPP-11, OPR-15, OPX-13, and OPAD-01) were used. It was found that OPA-09, OPN-03, and OPAD-01 were able to generate S. falcatus specific fragments in mullets which consisted of 200, 760, and 280 bp, respectively. In addition, the results of morphologic, DNA fingerprinting, and phylogenetic analyses strongly suggest that the fresh water and marine specimens of Stellantchamus may be different species.

Development of PCR-based diagnosis of minute intestinal fluke, Haplorchis taichui.

Haplorchis taichui specific primers were designed using a high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR method and 18 arbitrary primers (Operon Technologies) to generate polymorphic DNA profiles for 13 different parasites. The H. taichui specific fragment was screened. A 256 bp HAT-RAPD marker generated from OPP-11 primer specific for H. taichui was cloned and sequenced. From the sequence data, specific primers were designed that generated a 256 bp amplicon. The minimum DNA template needed for PCR detection was 10 fg. The successful development of the H. taichui specific DNA-based detection will be beneficial in management and epidemiological control programs.

Copro-diagnosis of Haplorchis taichui infection using sedimentation and PCR-based methods.

Fifty-one human fecal specimens were collected from villagers inhabiting along Mae Kuang River, Ban Sob Tha, Pa Sang District, Lamphun Province, Thailand. By the formalin-ether sedimentation technique (FEST) under a light microscope, eggs of 3 helminth species, Haplorchis taichui, Ascaris lumbricoides and unidentified hookworm species were detected with prevalences of 22, 14 and 4%, respectively. PCR amplification with H. taichui specific primers showed that H. taichui specific amplicon 260 bp was generated in all FEST-positive specimens, and also in some FEST negative specimens. This H. taichui specific PCR method can be used to detect this parasite in all developmental stages and in both definitive and intermediate hosts, which should be useful in prevention and control programs.

Haplorchis taichui, Witenberg, 1930: Development of a HAT-RAPD marker for the detection of minute intestinal fluke infection.

Specific primers to determine the presence of an intestinal fluke, Haplorchis taichui, were investigated using the high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR, and 18 arbitrary primers (Operon Technologies), to generate different polymorphic DNA profiles. Thirteen kinds of parasites were used to compare fingerprints. A 256bp HAT-RAPD marker, generated from the OPP-11 primer, was found to be H. taichui-specific, and this marker was cloned, transformed, and sequenced. From the sequence data, a pair of primers were designed with Genetyx-MAC ver.11 and indicated as: Hap-t F 5'-GGC CAA CGC AAT CGT CAT CC-3' and Hap-t R 5'-GCG TCG GGT TTC AGA CAT GG-3'. These specific primers were tested for efficacy and specificity by amplifying them with all 13 parasites DNAs in PCR reaction. A 256bp amplicon was generated, which was shown to have a positive result, only for H. taichui DNA. It revealed no cross-reaction with any of the other tested parasite species. The minimum DNA template, needed for detection by PCR, was 0.1picogram (pg). The successful development of H. taichui-specific primers is expected to be beneficial for epidemiological studies and for prevention and control of these parasitic infections.

High annealing temperature-random amplified polymorphic DNA (HAT-RAPD) analysis of three paramphistome flukes from Thailand.

The rumen flukes of 37 cows (Bos indicus) from Chiang Mai and Lamphun provinces were investigated, and the overall prevalence of infection was 78.38% (29/37). Three species were found: Paramphistomum epiclitum, Orthocoelium streptocoelium, and Fischoederius elongatus with prevalences of infection of 75.68%, 48.65%, and 40.54%, respectively. Genomic DNA was amplified by polymerase chain reaction based on the high annealing temperature-random amplification of polymorphic DNA (HAT-RAPD) technique. Five random 10-mer oligonucleotide primers (OPA2, OPA4, OPB18, OPC9, and OPH11) produced distinct banding patterns in three species. No genetic variations in these three species were identified using 10 arbitary primers.