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.

Influence of limestone and anthropogenic activities on land snail communities in Satun Province, Thailand.

Southeast Asia is renowned for its rich biodiversity and serves as a global hotspot for organisms. Satun Province, located in southern Thailand, represents a significant biogeographic and biodiversity region situated in the transition zone between the Indochinese and Sundaland hotspots. Thus, Satun Province has high diversity and abundance of organisms, especially land snails. However, Satun Province has been largely understudied for both diversity and biogeography. Our study aimed to investigate land snail communities, emphasizing comparisons between the mainland and island sites, and the influence of limestone and other rock types in Satun Province. Our exploration efforts identified a diverse land snail community comprising 17 families, which updated the total reported species in the province from 22 to 58 species. The results showed that islands had significantly greater land snail species richness and abundance, and larger Shannon index values in comparison to the mainland. Anthropogenic activities on the mainland led to habitat transformation and had detrimental impacts on native land snail diversity while promoting the spread of invasive species. In contrast, the presence of little-disturbed limestone habitats on some of the islands contributed to higher richness of land snails, as limestone areas are known for their high diversity and endemism. Snail communities varied along site characteristics; especially, limestone versus non-limestone areas and human activity levels (which were associated with national park protection), with limestone and protected areas having the highest diversity. Our study illustrates the value of protected areas as potential hotspots of biodiversity, especially for islands that are little disturbed by human activities and that have habitats that support high diversity, such as limestone outcrops. This study provides valuable insights into the biogeography of land snails in Satun Province and Southeast Asia and contributes to our understanding of the factors influencing land snail diversity.

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.

Molecular classification of rumen fluke eggs in fecal specimens from Suphanburi Province, Thailand, based on cytochrome C oxidase subunit 1.

Rumen fluke infections have been known to cause paramphistomiasis in both wild and domestic animals worldwide. Occasionally, coinfections of rumen flukes (Carmyerius, Fischoederius, and Paramphistomum) with liver flukes (Fasciola) have been observed due to the similar life cycles that these two species share. This study involved an alternative approach that was developed to classify and distinguish rumen fluke eggs from other genera by using the polymerase chain reaction (PCR) method based on cytochrome c oxidase subunit 1 (COI). Thirty-eight fecal specimens of Bos taurus from Suphanburi Province, Central Thailand were examined using the formalin-ether sedimentation technique. PCR detection was then performed using COI-specific primers that were developed in this study. The results showed that this primer set can classify and distinguish the egg specimens into a separate clade of the genera comprising Gastrothylax, Carmyerius, Fischoederius, Paramphistomum, Explanatum, and Fasciola. Moreover, epidemiological mapping revealed coinfections of three genera of rumen flukes at some collection sites, leading to the need to further investigate Paramphistomoidea infection along with Fasciolidae infection within the endemic area. This data is important for monitoring the outbreak of these parasites in Suphanburi Province, Thailand. It can be applied for initiating surveillance programs of paramphistomiasis and fascioliasis in veterinary studies.

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.

New Record of Thapariella anastomusa (Trematoda: Thapariellidae) Metacercariae in Northern Thailand.

The family Thapariellidae has been reported in only 3 countries since 1990. The objective of this study was to identify Thapariella anastomusa metacercariae in snails in Thailand based on morphological traits using a light (LM) and a scanning electron microscope (SEM). A total of 94 Filopaludina snails were collected and identified as 50 F. martensi martensi and 44 F. doliaris. Metacercariae of T. anastomusa were recovered from the snails by the crushing method. The overall prevalence was 22.3% (21/94), and the mean intensity was 17.0 per snail. The prevalence in F. martensi martensi was 24.0% (12/50) and F. doliaris 20.5% (9/44) with the mean intensity of 18.8 and 14.8 per snail, respectively. SEM revealed traits such as a concave ventral body and well-developed oral and ventral suckers. This study represents the first report of T. anastomusa in South East Asia. While LM and SEM observations provide novel insights into T. anastomusa metacercarial morphology and life history, the trematode's life cycle remains unclear. To date, there has been no report of T. anastomusa causing infections in humans. However, the snails F. martensi martensi and F. doliaris carrying the infective stages of T. anastomosa are frequently consumed by Thai people. This consumption, particularly uncooked snails, may present a risk of Thapariella infections in humans.

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.

Occurrence and HAT-RAPD analysis of gastrointestinal helminths in domestic chickens (Gallus gallus domesticus) in Phayao province, northern Thailand.

The present study determined the prevalence and distribution of gastrointestinal helminths in domestic chickens (Gallus gallus domesticus) between November 2012 and August 2013. One hundred and twenty domestic chickens were purchased from villages in four districts of Phayao province; Mae Chai, Dok Khamtai, Chun and Chiang Kham. Morphological differences were used to identify the helminth species, and HAT-RAPD technique was used to differentiate among closely related species. The results revealed that the total prevalence of infection was 99.2%. Cestode and nematode infections showed the highest prevalence in rainy season, while trematode infections were low and only found in hot season. The species and their prevalence were: Ascaridia galli (50.8%), Heterakis gallinarum (86.7%), Prosthogonimus macrorchis (1.7%), Echinostoma revolutum (0.8%), Raillietina echinobothrida (48.3%), Raillietina tetragona (57.5%), Raillietina cesticillus (12.5%), Raillietina sp. (35.8%), Cotugnia chiangmaii (14.2%) and Cotugnia sp. (32.5%). The prevalence of helminth infections did not differ significantly between male and female chickens. HAT-RAPD analysis, the specific fragment of 400 and 250 bp indicated that Raillietina sp. and Cotugnia sp. found, respectively, differ from other closely related species. This study has confirmed that HAT-RAPD technique can be used to differentiate among related species combined with morphological observations.

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.

Morphology and Molecular Phylogeny of Raillietina spp. (Cestoda: Cyclophyllidea: Davaineidae) from Domestic Chickens in Thailand.

Raillietina species are prevalent in domestic chickens (Gallus gallus domesticus) in Phayao province, northern Thailand. Their infection may cause disease and death, which affects the public health and economic situation in chicken farms. The identification of Raillietina has been based on morphology and molecular analysis. In this study, morphological observations using light (LM) and scanning electron microscopies (SEM) coupled with molecular analysis of the internal transcribed spacer 2 (ITS2) region and the nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) gene were employed for precise identification and phylogenetic relationship studies of Raillietina spp. Four Raillietina species, including R. echinobothrida, R. tetragona, R. cesticillus, and Raillietina sp., were recovered in domestic chickens from 4 districts in Phayao province, Thailand. LM and SEM observations revealed differences in the morphology of the scolex, position of the genital pore, number of eggs per egg capsule, and rostellar opening surface structures in all 4 species. Phylogenetic relationships were found among the phylogenetic trees obtained by the maximum likelihood and distance-based neighbor-joining methods. ITS2 and ND1 sequence data recorded from Raillietina sp. appeared to be monophyletic. The query sequences of R. echinobothrida, R. tetragona, R. cesticillus, and Raillietina sp. were separated according to the different morphological characters. This study confirmed that morphological studies combined with molecular analyses can differentiate related species within the genus Raillietina in Thailand.

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.

Ethnomedicinal plants used for digestive system disorders by the Karen of northern Thailand.

BACKGROUND: Digestive system disorders have a substantial effect on worldwide morbidity and mortality rates, including in Thailand, where the majority of the rural areas have a lack of proper sanitation and awareness about disease prevention. This has led to the prevalence of different types of digestive diseases. Karen people in Thailand still use medicinal plants as first aid remedies in treating these diseases. Therefore, this study aimed at documenting the plants used to cure and prevent different types of digestive system disorders by Karen people of Chiang Mai Province, northern Thailand. METHODS: Ethnomedicinal data were collected from six key informants and 172 non-specialist informants regarding their traditional knowledge of medicinal plants. Quantitative approaches were used to determine Use Value (UV), Informant Consensus Factor (ICF) and Fidelity Level (FL) values. RESULTS: The study revealed that 36 medicinal plant species belonging to 31 genera and 24 families were used to treat digestive system disorders. The most prevalent plant families were Zingiberaceae (6 species), Euphorbiaceae (4 species) and Fabaceae (4 species). Leaves were the most commonly used plant part accounting for 32.6% of the plants, followed by the bark (18.6%). About 60% of the administrations were given orally by potion (60%) and consumption as food was also indicated (14%). The highest ICF values were recorded for carminative disorders, stomachaches, geographic tongue, constipation, appetite stimulants and food poisoning (1.00 each) indicating the best agreement among the informants knowledge of medicinal plants that were used to treat aliments in these categories. The highest fidelity level values were recorded for Punica granatum (100.00), Psidium guajava (95.45), and Gymnopetalum integrifolium (90.91) showing conformity of knowledge on species with the best healing potential. CONCLUSION: Medicinal plants still play an important role among Karen culture. The present information on these medicinal plants, which have high UV and FL values, may serve as the baseline data to initiate further research for the discovery of new compounds and the biological activities of these potential plant remedies. Further research on these plants may provide some important clues for the development of new drugs for the treatment of digestive system diseases.

Experimental life history and biological characteristics of Fasciola gigantica (Digenea: Fasciolidae).

This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.

Molecular phylogeny of trematodes in Family Heterophyidae based on mitochondrial cytochrome c oxidase subunit I (mCOI).

OBJECTIVE: To analyze a phylogenetic tree for understanding the molecular systematic of trematode in Family Heterophyidae, which are highly distributed in Thailand. METHODS: Based on thirteen sequences of mitochondrial cytochrome c oxidase subunit I (mCOI) gene from six genera of heterophyid trematodes, viz. Haplorchis, Stellantchasmus, Centrocestus, Metagonimus, Pygidopsis, and Haplorchoides were aligned automatically using the Clustal × 2.0 program. A phylogenetic tree was constructed by maximum likeinghood (ML) and neighbor-joining (NJ) methods, with 1 000 bootstrap using the 5.0 program. RESULTS: The phylogenetic relationship from both methods was similar and separated into three groups consisting of Haplorchoides pumilio group, Haplorchoides taichui group and another heterophyid genera. CONCLUSIONS: The sequence data of mtCOI can be used to investigate the phylogenetic relationships of trematodes at the genus level. Each clade of different genera of heterophyid trematodes can be separated into sister groups that correlated with the morphological characteristic, kind of secondary intermediate host and geographic distribution.

Molecular analysis of echinostome metacercariae from their second intermediate host found in a localised geographic region reveals genetic heterogeneity and possible cryptic speciation.

Echinostome metacercariae are the infective stage for humans and animals. The identification of echinostomes has been based until recently on morphology but molecular techniques using sequences of ribosomal RNA and mitochondrial DNA have indicated major clades within the group. In this study we have used the ITS2 region of ribosomal RNA and the ND1 region of mitochondrial DNA to identify metacercariae from snails collected from eight well-separated sites from an area of 4000 km2 in Lamphun Province, Thailand. The derived sequences have been compared to those collected from elsewhere and have been deposited in the nucleotide databases. There were two aims of this study; firstly, to determine the species of echinostome present in an endemic area, and secondly, to assess the intra-specific genetic diversity, as this may be informative with regard to the potential for the development of anthelmintic resistance and with regard to the spread of infection by the definitive hosts. Our results indicate that the most prevalent species are most closely related to E. revolutum, E. trivolvis, E. robustum, E. malayanum and Euparyphium albuferensis. Some sites harbour several species and within a site there could be considerable intra-species genetic diversity. There is no significant geographical structuring within this area. Although the molecular techniques used in this study allowed the assignment of the samples to clades within defined species, however, within these groupings there were significant differences indicating that cryptic speciation may have occurred. The degree of genetic diversity present would suggest the use of targeted regimes designed to minimise the selection of anthelmintic resistance. The apparent lack of geographic structuring is consistent with the transmission of the parasites by the avian hosts.

Prevalence of infection and molecular confirmation by using ITS-2 region of Fasciola gigantica found in domestic cattle from Chiang Mai province, Thailand.

OBJECTIVE: To investigate the infection of Fasciola gigantica (F. gigantica) in domestic cattle from Chiang Mai province and molecular confirmation using ITS-2 region. METHODS: The liver and gall bladder of Bubalus bubalis (B. bubalis) and Bos taurus (B. taurus) from slaughterhouses were examined adult worms and prevalence investigation. The species confirmation with phylogenetic analysis using ITS-2 sequences was performed by maximum likelihood and UPGMA methods. RESULTS: The total prevalences of infection in B. bubalis and Bubalus taurus (B. taurus) were 67.27% and 52.94% respectively. The respective prevalence in both B. bubalis and B. taurus were acquired from Doi-Saket, Muang, and Sanpatong districts, with 81.25%, 62.50% and 60.00% for B. bubalis and 62.50%, 50.00% and 47.06% for Bos taurus respectively. The species confirmation of F. gigantica and some related species by basing on maximum likelihood and UPGMA methods used, 4 groups of trematodes were generated, first F. gigantica group including specimen of Chiang Mai, second 2 samples of F. hepatica, third group of 3 rumen flukes; Orthocoelium streptocoelium, F. elongatus and Paramphistomum epliclitum and fourth group of 3 minute intestinal flukes; Haplorchis taichui, Stellantchasmu falcatus, Haplorchoides sp. and liver fluke; Opisthorchis viverrini respectively. CONCLUSIONS: These results can be confirmed the Giant liver fluke which mainly caused fascioliasis in Chiang Mai was identified as F. gigantica and specimens were the same as those of F. gigantica recorded in other different countries. Nucleotide sequence of ITS-2 region has been proven as effective diagnostic tool for the identification of F. gigantica.

Epidemiology and molecular genotyping of echinostome metacercariae in Filopaludina snails in Lamphun Province, Thailand.

OBJECTIVE: To analyze the prevalence of echinostome metacercariae in Filopaludina dorliaris (F. dorliaris) and Filopaludina martensi martensi (F. martensi martensi) and genotype variation of echinostome metacercariae by using random amplified polymorphic DNA (RAPD) analysis. METHODS: Filopaludina sp. snails were collected from eight localities of Lamphun Province, Northern Thailand and examined for echinostome metacercariae. RAPD-PCR was used to analyze genotype variation of echinostome metacercariae. RESULTS: A total of 3 226 F. dorliaris and F. martensi martensi snails were collected from eight localities. The overall prevalences of echinostome metacercariae in F. dorliaris and F. martensi martensi were 40.89% and 36.27%, while the intensity of infection was 20.37 and 12.04, respectively. The dendrogram constructed base on RAPD profiles, 4 well supported domains were generated; (i) group of metacercariae from Ban Hong, Mae Ta, Meaung, Pa Sang, Toong Hua Chang, and Weang Nong that were clustered in the group of E. revolutum, (ii) Ban Thi, (iii) Lee, and (iv) 3 adults of an out group. CONCLUSIONS: This research demonstrated RAPD profiling has been a useful tool to detect DNA polymorphisms to determine genetic relationship between echinostome metacercariae in Lamphun Province, Northern Thailand.