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Opisthorchis viverrini is a major public health concern in Southeast Asia. Various reports have suggested that this parasite may represent a species complex, with genetic structure in the region perhaps being dictated by geographical factors and different species of intermediate hosts. We used four microsatellite loci to analyze O. viverrini adult worms originating from six species of cyprinid fish in Thailand and Lao PDR. Two distinct O. viverrini populations were observed. In Ban Phai, Thailand, only one subgroup occurred, hosted by two different fish species. Both subgroups occurred in fish from That Luang, Lao PDR, but were represented to very different degrees among the fish hosts there. Our data suggest that, although geographical separation is more important than fish host specificity in influencing genetic structure, it is possible that two species of Opisthorchis, with little interbreeding, are present near Vientiane in Lao PDR.

Genetic differentiation between two synonymous echinostomes species, Artyfechinostomum malayanum and Artyfechinostomum sufrartyfex was determined by using the first and second internal transcribed spacers (ITS1 and ITS2), the non-coding region of rDNA as genetic makers. Of the 699 bp of combined ITS1 and ITS2 sequences examined, 18 variable nucleotide positions (2.58 %) were observed. Of these, 17 positions could be used as diagnostic position between these two sibling species, whereas the other one variation was intraspecific variation of A. malayanum. A clade of A. malayanum was closely aligned with A. sufrartyfex and clearly distance from the cluster of other echinostomes. Our results may sufficiently suggest that the current synonymy of these species is not valid. [ABSTRACT FROM AUTHOR]

Opisthorchis viverrini is considered as a carcinogenic parasite which is responsible for cholangiocarcinoma in Southeast Asia. Effective treatment and control of the parasite to reduce the risk of cancer requires efficient diagnostic methods. Because of the limitations involved in human studies, the present work is aimed at comparing diagnostic performance of copro-DNA detection by PCR and fecal examination by formalin-ethyl acetate concentration technique (FECT) during the course of O. viverrini infection and postcurative chemotherapy in experimentally infected hamsters. A manual method of DNA preparation from fecal specimens previously established in human studies was used in PCR analysis. Following infection with varying doses of metacercariae (5, 10, 25, and 50 cysts/animal), PCR analyses were positive as early as 3 weeks post-infection while FECT were negative. PCR tests were comparable to FECT regardless of intensity of infection beginning from 4 to 12 weeks post infection. In chemotherapeutic experiments, with reference to the presence of worm in liver, treatment failures were detected by PCR but not FECT in a group of hamsters infected with 10 cysts/animal. PCR and FECT both detected residual infection at 1 and 2 weeks post-treatment in the group of animals infected with five cysts per animal. High concordant results between diagnoses by PCR, FECT, and worm burden indicated that PCR is suitable for an early diagnosis, evaluation of drug efficacy, and also re-infection post-treatment. [ABSTRACT FROM AUTHOR]

Re-infection with liver fluke, Opisthorchis viverrini, increases proinflammatory molecules involved in inflammation-mediated disease and carcinogenesis in an animal model. To clarify whether these genes respond to parasite antigen in peripheral blood leukocytes (PBL) of opisthorchiasis patients, we examined the transcriptional level of oxidant-generating (toll-like receptor 2 (TLR-2), nuclear factor-kappa B (NF-KB), and cyclooxygenase 2 (COX-2)), anti-oxidant-generating (manganese superoxide dismutase 2 (SOD-2) and catalase (CAT)), proinflammatory cytokine (interleukin (IL)-1β), and anti-inflammatory cytokine (IL-10), in PBL exposed to parasite antigen in O. viverrini-infected patients compared with healthy individuals in an in vitro experiment. After O. viverrini antigen-treated PBL, quantitative RT-PCR analysis revealed that increased expression of cytokines and oxidant-generating genes in PBL was similar between O. viverrini-infected and healthy groups. Interestingly, compared with healthy subjects, increase of TLR-2, COX-2, and SOD-2 and decreased CAT mRNA expression levels were observed in O. viverrini-infected group. The results indicate that O. viverrini antigen induces upregulation of TLR-2, COX-2, and SOD-2 and downregulation of CAT genes in opisthorchiasis patients, suggesting that imbalance of oxidant/anti-oxidant transcripts during re-infection may be involved in the inflammatory-driven carcinogenesis. These molecules may be used as the chemopreventive target for intervention of opisthorchiasis patients in an endemic area. [ABSTRACT FROM AUTHOR]

Echinostomatidae are common, widely distributed intestinal parasites causing significant disease in both animals and humans worldwide. In spite of their importance, the taxonomy of these echinostomes is still controversial. The taxonomic status of two species, Echinostoma revolutum and Echinoparyphium recurvatum, which commonly infect poultry and other birds, as well as human, is problematical. Previous phylogenetic analyses of Southeast Asian strains indicate that these species cluster as sister taxa. In the present study, the first internal transcribed spacer (ITS1) sequence was used for genetic characterization and to examine the phylogenetic relationships between an isolate from Thailand with other isolates available from GenBank database. Interspecies differences in ITS1 sequence between E. revolutum and E. recurvatum were detected at 6 (3%) of the 203 alignment positions. Of these, nucleotide deletion at positions 25, 26, and 27, pyrimidine transition at 50, 189, and pyrimidine transversion at 118 were observed. Phylogenetic analysis revealed that E. recurvatum from Thailand clustered as a sister taxa with E. revolutum and not with other members of the genus Echinoparyphium. Interestingly, this result confirms a previous report based on allozyme electrophoresis and 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 Asian countries needs to be confirmed and revised using more comprehensive analyses based on morphology and other molecular techniques. [ABSTRACT FROM AUTHOR]

Recent systematic studies of Opisthorchis viverrini based on multilocus enzyme electrophoresis (MEE) have shown that there are at least five genetic groups and possibly two cryptic species occurring in Thailand and Lao PDR each associated with a specific wetland system. A study based on mitochondrial DNA (mtDNA) gene analyses of an O. viverrini population from Savannakhet (SV, Lao PDR) clustered with several isolates from Thailand and Lao PDR although they originated from different river wetland systems. We used MEE to re-examine whether O. viverrini from SV was similar genetically to isolates from Thailand and Lao PDR. The allelic profiles of O. viverrini from SV and five different wetlands representing defined genetic groups of O. viverrini were recorded at 24 enzyme loci as opposed to only two loci of mtDNA. Contrary to previous studies, O. viverrini from SV was found to have fixed genetic differences at six to eight of the 24 loci examined (24.50-35.42%). Allelic data indicated that O. viverrini from SV differed from isolates in the Nam Ngum River wetland in Lao PDR (29.33% fixed genetic differences) and clustered with O. viverrini from Nakhon Phanom and Sakon Nakhon within the Songkram River wetland in Thailand but had fixed genetic differences from these at 24.5% of loci examined. Our data confirm the association between genetic groups of O. viverrini and specific wetland systems, and raise important questions regarding the significance of the genetic differences and relationships of O. viverrini from these wetlands. [ABSTRACT FROM AUTHOR]

Genetic variation in Opisthorchis viverrini adults originating from different locations in northeast Thailand and Laos, People's Democratic Republic (PDR), was examined using random amplified polymorphic DNA (RAPD) analyses. In an initial analysis, the genomic DNA of one fluke from each of ten localities was amplified using 15 random primers (10-mers); however, genetic variation among O. viverrini specimens was detected reliably for only four primers. A more detailed RAPD analysis using these four primers was conducted on ten individuals from nine localities. Considerable genetic variation was detected among O. viverrini from different geographical areas and among some individuals from the same collecting locality. Comparison of the RAPD profiles revealed that O. viverrini adults from Laos PDR were genetically distinct from those from northeast Thailand. The taxonomic significance of this finding needs to be explored in more detail. The RAPD markers established in the present study provide opportunities to examine the biology and epidemiology of O. viverrini and fish-borne trematodes within the region. Additionally, application of these markers in such studies could have important implications in relation to the prevalence of cholangiocarcinoma in different regions of Asia.