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.

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.

Co-infection with Opisthorchis viverrini and Haplorchis taichui detected by human fecal examination in Chomtong district, Chiang Mai Province, Thailand.

Diseases caused by the liver fluke, Opisthorchis viverrini and the minute intestinal fluke, Haplorchis taichui, are clinically important, especially in the Northeast and North regions of Thailand. It is often difficult to distinguish between these trematode species using morphological methods due to the similarity of their eggs and larval stages both in mixed and co-infections. A sensitive, accurate, and specific detection method of these flukes is required for an effective epidemiological control program. This study aimed to determine the prevalence of O. viverrini and H. taichui infections in human feces by using formalin-ether sedimentation and high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR methods. Fecal specimens of people living along the Mae Ping River, Chomtong district were examined seasonally for trematode eggs using a compound microscope. Positive cases were analyzed in HAT-RAPD, DNA profiles were compared with adult stages to determine the actual species infected, and specific DNA markers of each fluke were also screened. Our results showed that out of 316 specimens, 62 were positive for fluke eggs which were pre-identified as O. viverrini and H. taichui. In addition, co-infection among these two fluke species was observed from only two specimens. The prevalence of H. taichui infections peaked in the hot-dry (19.62%), gradually decreased in the rainy (18.18%), and cool-dry seasons (14.54%), respectively. O. viverrini was found only in the hot-dry season (6.54%). For molecular studies, 5 arbitrary primers (Operon Technologies, USA) were individually performed in HAT-RAPD-PCR for the generation of polymorphic DNA profiles. The DNA profiles in all 62 positives cases were the same as those of the adult stage which confirmed our identifications. This study demonstrates the mixed infection of O. viverrini and H. taichui and confirms the extended distribution of O. viverrini in Northern Thailand.

Seasonal variation of metacercariae in cyprinoid fish from Kwae Noi Bamroongdan Dam, Phitsanulok Province, northern Thailand.

Abstract. A seasonal investigation of the occurrence of metacercariae in cyprinoid fish was conducted at the Kwae Noi Bamroongdan Dam, Phitsanulok Province, northern Thailand during December 2008-August 2009. A total 361 cyprinoid fish from 10 species were examined by artificial digestion method evaluating for metacercariae. The metacercariae determined were Haplorchis taichui, Haplorchis pumilio, Haplorchoides sp and Centrocestus caninus. The prevalence of metacercariae ranged from 76.5% to 82.6%, with an intensity of infection of 23.4 to 36.6. H. taichui, Haplorchoides sp and C. caninus had the highest prevalence in the cool season, while H. pumilio was more prevalent during the hot season. The highest infection prevalence (100%) was seen in Rasbora metallicus, Barbodes gonionotus and R. metallicus. No significant differences in the type of metacercariae were found between seasons, indicating year-round infestation of cyprinoid fish.