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Digenetic trematodes are important parasites of humans and animals. They have complex life cycles and typically infect a gastropod as the first intermediate host. Bithynia siamensis goniomphalos , the first intermediate host of the liver fluke, Opisthorchis viverrini , harbours a wide variety of other trematode species. Morphological details of cercariae of 20 trematode taxa from B . s . goniomphalos , collected mainly in Thailand from 2009 to 2014, were provided in an earlier paper. Correct identification to the species or genus level based on morphology of these cercariae is generally not possible. Therefore, we used molecular data to improve identification and to investigate the diversity of the species of trematodes infecting B . s . goniomphalos . We were successful in extracting, amplifying and sequencing portions of the 28S ribosomal RNA (rRNA) gene for 19 of these 20 types of cercaria, and the internal transcribed spacer 2 region for 18 types. BLAST searches in GenBank and phylogenetic trees inferred from the 28S rRNA sequences identified members of at least nine superfamilies and 12 families. Only a few cercariae could be assigned confidently to genus or species on the basis of the sequence data. Matching sequence data from named adult trematodes will be required for definitive identification. There is clearly a great diversity of trematode species utilizing B . s . goniomphalos in Thailand.

In total, 160 ticks infesting cattle in the northeast region of Thailand were collected and used for molecular investigation. Three tick species-Rhipicephalus microplus Canestrini, Rhipicephalus haemaphysaloides Supino and Haemaphysalis bispinosa Neumann-were identified based on morphology and DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) and 16S ribosomal RNA (16S rRNA). In total, 26 and seven unique haplotypes of the CO1 and 16S rRNA genes, respectively, were recovered. Phylogenetic analysis using the CO1 sequence revealed that the R. microplus from northeastern Thailand were grouped into the previously described clades A and C, whereas the 16S rRNA phylogenetic tree assigned all isolates of R. microplus from Northeast Thailand into the previously described clade B. Clade C of the CO1 phylogenetic tree is a new genetic assemblage recently discovered from India and Malaysia, which has now been detected in our study. The haplotype network also demonstrated that R. microplus is divided into two haplogroups corresponding to the assemblage of the CO1 phylogenetic tree. Our findings strongly support the previous genetic assemblage classification and evidence that R. microplus from Northeast Thailand is a species complex comprising at least two genetic assemblages, i.e., clades A and C. However, further investigation is needed and should involve more comprehensive genetic and morphological analyses and cover a larger part of their distributional range throughout Southeast Asia., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Several trematodes including Opisthorchis viverrini utilize Bithynia siamensis goniomphalos as a snail intermediate host in their life cycles. In order to capture a comprehensive range of host–parasite interactions and their transmission dynamic patterns, B. s. goniomphalos were sampled monthly over 4 consecutive years in an irrigated paddy-field habitat in northeast Thailand. Using a standard cercarial shedding method, a high diversity of trematodes (17 types) was recovered. Virgulate xiphidiocercariae were the most prevalent (7.84%) followed by O. viverrini (0.71%). In addition to seasonal and environmental factors, the quantity of irrigation water for rice cultivation correlated with transmission dynamics of trematodes in B. s. goniomphalos. The peak prevalence of all trematode infections combined in the snails shifted from the cool-dry season in 2010–2012 to the hot-dry season in 2013 associated with an increasing quantity of water irrigation. A low frequency of mixed trematode infections was found, indicating that the emergence of virgulate cercariae, but not of O. viverrini, was negatively impacted by the presence of other trematodes in the same snail. Taken together, the observed results suggest that interactions between host and parasite, and hence transmission dynamics, depend on specific characteristics of the parasite and environmental factors including irrigated water for rice cultivation.

The group 1 carcinogen, the liver fluke Opisthorchis viverrini is the causative agent of opisthorchiasis and subsequent bile duct cancer (cholangiocarcinoma; CCA), which is an important public health problem in Southeast Asia. Bithynia snails are known to be the sole intermediate host of O. viverrini, and distributed throughout endemic areas of opisthorchiasis. Since 2001, the genetic variation investigation of O. viverrini has progressively been investigated. Comprehensive genetic variation studies of O. viverrini and Bithynia snails were undertaken and consecutively published in 2007 by Saijuntha and colleagues. These studies provided genetic evidence that O. viverrini and Bithynia snails are both species complex with evidence of co-evolution. Later, several studies have provided data in support of this finding, and have continuously to date reinforced that both O. viverrini and Bithynia are species complexes. Moreover, studies have shown that genetic variation of O. viverrini is related to geographical, temporal, fish host species including geographical genetic variation of its snail host, Bithynia siamensis sensu lato. This is significant and important in our understanding of the evolution and phylogenetic relationships between species within the O. viverrini and Bithynia species complexes. A comprehensive knowledge of the systematics and population genetics of O. viverrini and Bithynia snails provides a sound basis to instigate and develop effective prevention and control programs targeting opisthorchiasis and CCA in the endemic areas of Southeast Asia. Thus, this review examines the historical series of investigations of the systematics and population genetics of O. viverrini including Bithynia spp. in Southeast Asia since molecular genetic investigations commenced some 20 years ago., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Haemonchus contortus is one of the most economically important parasitic nematodes affecting small ruminant livestock worldwide. This study was conducted to elucidate the genetic diversity and population structure of this nematode in Thailand based on mitochondrial DNA markers, the nicotinamide dehydrogenase subunit 4 (nad4) and the cytochrome c oxidase subunit 1 (cox1) genes. One hundred and thirty-six adult worms were obtained from 86 abomasa of slaughtered goats from 13 different localities in 5 regions of Thailand. Identification to the genus Haemonchus was done using morphology. DNA sequences of the nuclear ribosomal second internal transcribed spacer (ITS2) identified each specimen to species: three fixed nucleotide (SNP) differences distinguished H. contortus from H. placei. Genetic analysis defined 118 and 122 unique haplotypes in partial sequences of nad4 (alignment length 723 bp) and cox1 (645 bp) genes, respectively. Nucleotide diversities were 0.031 and 0.043 for nad4 and cox1 genes, respectively. Low genetic differentiation was observed among H. contortus samples from various provinces in Thailand. This is the first study on the genetic diversity and population structure of H. contortus of goats in Thailand. This study has provided insights into the transmission dynamics of this parasitic nematode, information which is essential for farm management and parasite control., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The freshwater snails, Bithynia are the first intermediate hosts of the liver fluke, Opisthorchis viverrini, the causative agent of cholangiocarcinoma (CCA) in Southeast Asia. In Thailand, there are three traditionally recognized taxa of Bithynia: Bithynia funiculata; B. siamensis siamensis; B. s. goniomphalos. This study examines the geographical distribution and genetic structure of Bithynia species from five previously reported water catchments and six new catchments in Thailand. Of these, three new catchments Kok, Wang, and Nan are from the north and the remaining three new catchments are Phetchaburi, Prachuap Khiri Khan Coast, Mae Klong from the west of Thailand. We sampled 291 Bithynia snails from 52 localities in 11 catchment systems in the northern, western and central regions of Thailand. Mitochondrial cytochrome c oxidase subunit 1 (COI) and 16S ribosomal DNA (16S rDNA) sequences were used to examine genetic diversity of Bithynia snails which revealed 200 and 27 haplotypes of COI and 16S rDNA, respectively. However, as 16S rDNA is a conserved gene, it is not suitable to distinguish Bithynia at the species and sub-species levels in our study. The phylogenetic tree and haplotype network analyses included sequences of COI from GenBank. B. funiculata was found only in the north of Thailand and the genetic structure did not differ among populations. Genetic differentiation (Φ ST ) analyses showed that B. s. goniomphalos contained three distinct lineages. Lineage I contained B. s. goniomphalos from the vast majority of catchment systems in Thailand and Lao PDR. Lineage II contained all B. s. goniomphalos from the Prachin Buri and Bang Pakong catchment systems in eastern and central Thailand, including samples from all catchment systems in Cambodia. While lineage III contained B. s. goniomphalos from the Songkram and Nam Kam catchment systems in Thailand and the Nam Ngum and Huai Som Pak catchment systems in Lao PDR. Furthermore, results showed that all samples of B. s. siamensis were classified into one lineage and placed phylogenetically between B. s. goniomphalos lineages I and II. Thus, the taxonomic status of B. s. goniomphalos and B. s. siamensis requires reassessment, and they should be reclassified as belonging to the species complex "Bithynia siamensis sensu lato"., (Copyright © 2021. Published by Elsevier B.V.)

Infection by the small liver fluke, Opisthorchis viverrini, causes serious public health problems, including cholangiocarcinoma, in Thailand and southeastern Asian countries. Previous studies have reported that O. viverrini represents a species complex with varying levels of genetic differentiation in Thailand and Lao PDR. In this study, we re-examined population genetic structure and genetic diversity of O. viverrini using extensive samples of the parasite collected over 15 years from 12 geographical localities in Thailand and eight localities in Lao PDR. Parasite life-cycle stages of 721 individuals of O. viverrini (91 cercariae, 230 metacercariae and 400 adult worms) were genotyped using 12 microsatellite loci. Metacercariae exhibited genetic diversity comparable with that of experimentally raised adults: metacercariae can therefore be used to represent O. viverrini populations without the need for laboratory definitive hosts. Data obtained from larval as well as adult worms identified two distinct genetic clusters of O. viverrini. Sequences of a portion of the mitochondrial cox1 gene strongly supported the existence of these two clusters. One, the widespread cluster, was found at all sampled sites. The second cluster occurred only in Phang Khon District, Sakon Nakhon Province (SPk), within the Songkram River wetland in Thailand. A striking feature of our data relates to the temporal dynamics of the SPk cluster, which was largely replaced by representatives of the widespread cluster over time. If the SPk cluster is excluded, no marked genetic differences were seen among O. viverrini populations from Thailand and Lao PDR. The underlying causes of the observed population structure and population dynamics of O. viverrini are not known., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Opisthorchis viverrini is a fish-borne zoonotic trematode that causes significant public health problems in Southeast Asia. Its life cycle requires Bithynia snails as the first intermediate hosts, fish, and human and/or carnivore hosts. This study assessed impacts of land use practice for rice cultivation and seasonality on the transmission dynamics of O. viverrini in Bithynia siamensis goniomphalos in rice paddy field habitats. The transmission of O. viverrini cercariae in B. s. goniomphalos was monitored at monthly intervals over a 4-year period from January 2010 to December 2013. From a total of 59,727 snails examined by standard cercarial shedding, the prevalence of O. viverrini was 0.7% (range, 0.0-4.1%). The prevalence of O. viverrini infection in B. s. goniomphalos varied with the amount of rainfall, with peaks of infection occurring in the cool-dry season, that is, after each rainy season. A shift of peak prevalence from cool-dry to hot-dry season observed in 2013 was associated with the increase in preceding water irrigation to support the production of second annual rice crop. Significant positive correlations were found between the prevalence and intensity of cercarial infection and the size of snails. Our results revealed substantial variation between years so that to have a clear understanding of the population dynamics of this complex system, studies should be conducted over an extended period (> 1 year). Results from this study highlight that water irrigation schemes in rice paddy cultivation and seasonality have a significant effect on the prevalence of O. viverrini in B. s. goniomphalos .

Background: Patients with cholangiocarcinoma (CCA) usually have no specific symptoms until an advance stage of the disease and curative treatment is not possible. Patients with early stage, operable disease can be found using ultrasonography (US). A US-screening program was implemented in Thailand where CCA incidence is the highest worldwide. Here we evaluate the effectiveness of the program by comparing the proportion of individuals with early stage CCA in the screening group with that of the walk-in group presenting at hospitals with clinical symptoms., Methods: All patients had a pathological diagnosis of CCA. The difference in the proportions and the 95% confidence interval (CI) were obtained using binomial regression., Results: Of the 762 histologically proven CCA cases, 161 were from the screening group and 601 from the walk-in group. The proportion of early stage CCA (stages 0 to II) diagnosed was 84.5% in the screening and 21.6% in the walk-in groups. After adjustment age, gender, and liver fluke infection, there was a significantly higher proportion (P < 0.001) and higher chance (P < 0.001) of having early stage CCA in the screening group than in the walk-in group., Conclusions: US-screening is an effective tool for detecting early stage, operable CCA in high incidence areas., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Infection with the liver fluke Opisthorchis viverrini is the major causative factor inducing cholangiocarcinoma in the Mekong region of Southeast Asia. Northeast Thailand has the highest incidence of this cancer worldwide leading to about 20,000 deaths every year. Infection with the liver fluke comes from eating raw or undercooked fish, a tradition in this area that can potentially be countered by education programs at school level. Here we develop a school-based health education model, based on protection motivation theory (PMT), including module design, learning materials, student activities, and capacity building amongst teachers. This education program was applied and tested in primary school to pupils (9-13 years) in Khon Kaen province, northeast Thailand. Using a randomized control trial, four schools served as intervention groups ( n = 118 pupils) and another four acted as controls ( n = 113 pupils). Based on PMT constructs, we found that the pupils in the intervention group had significantly greater knowledge and perceived the severity, vulnerability, response efficacy, and self-efficacy parameters concerning the dangers of eating raw fish and of developing cholangiocarcinoma than those in the control schools ( p < 0.05). All of the PMT constructs measured were significantly intercorrelated with each other ( p < 0.001). At the same time, some background knowledge, from community-based education programs, was present in the control schools. The result from this initial study suggests that PMT can be used to predict protective attitude as well as behavior changes in evaluating the consequence of school health intervention programs.

The infection dynamics of Opisthorchis viverrini metacercariae was analyzed in cyprinid fish from endemic areas in Mukdahan Province, Thailand, and Khammouane Province, Lao PDR. The fish were collected during the cool-dry (November-February), hot-dry (March-May), and rainy (June-October) seasons in 2017. They were examined by the digestion method, and the infection status was statistically analyzed by study area, season, and fish size. The prevalence (no. of fish positive/no. of fish examined × 100) and metacercarial intensities (no. of metacercariae detected/no. of fish positive) of O. viverrini in both study areas depended on season, being high in the cool-dry season and varying in the hot-dry and rainy seasons. In Mukdahan Province, the average prevalence was 18.3% (range 11.0-46.7%, n = 420) and the intensity was 4.07 ± 5.86 cysts/fish (mean ± SD), whereas in Khammouane Province, the prevalence was 51.9% (range 9.1-70.6%, n = 673) and the intensity was 6.67 ± 12.88 cysts/fish. Among the cyprinid fish species examined, the infection was associated with fish body size and predominantly found in Hampala dispar (86.5%), Cyclocheilichthys armatus (73.2%), and Puntius brevis (42.7%). The distribution of O. viverrini metacercariae in fish was skewed, with most of the fish having a low worm burden with an average of four to six cysts/fish. The findings that seasonality, sampling locality, fish size, and species of fish play roles in the risk of O. viverrini infection imply that these host and environmental factors are important for the transmission dynamics and control of O. viverrini .

The liver fluke Opisthorchis viverrini sensu lato causes serious public-health problems in Northeast Thailand and Southeast Asian countries. A hypothesis has been proposed that O. viverrini represents a species complex with varying levels of genetic differentiation in Thailand and Lao PDR. This study aimed to clarify whether O. viverrini populations can be genetically divided into separate taxa. We collected O. viverrini s.l. from eight different locations in Lao PDR and Thailand. The results of nad1, cox1, CF-int6, Pm-int9, ITS2 and 28S rDNA sequence analysis revealed that sub-structuring occurred between the eight populations. We found that O. viverrini s.l. from Sakon Nakhon (SK), Thailand, shows significant genetic differentiation (P < .05) from all other isolates from different localities in Thailand and Lao PDR. This was supported by haplotype and phylogenetic tree analyses in which the SK isolate was separated from all other isolates. This suggests that O. viverrini s.l. from SK is a cryptic species. The data, however, also confirm the association between genetic groups of O. viverrini s.l. and specific wetland systems, and raise important questions regarding the epidemiological significance of these genetic differences., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The northeast of Thailand, which is the poorest region of the country, has the highest incidence of cholangiocarcinoma (CCA) worldwide. This is associated with infection with the liver fluke Opisthorchis viverrini. Although an estimated 20,000 people die every year of this disease, the socioeconomic impact of this mortality on the victims' family and the community in which he or she lived remains unknown. Here, we provide background information on the socioeconomic groups most effected by CCA and provide a qualitative estimate of the likely financial burden on the family and community. Most victims of CCA are small-scale farmers. Mortality occurs most commonly in males between the ages of 40 and 65, having either children or grandchildren to support. Costs can be divided between premortality with the family paying for transport and accommodation to the hospital, as well as costs not covered by the Thai Universal Health Coverage scheme. The main costs, however, are likely to be postmortem with loss of income and potentially the loss of a major contributor to farm work. What is urgently required is a quantitative estimate of the costs of CCA and long-term studies of the families and communities affected to determine where and how the burden of CCA falls., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Opisthorchiasis is a neglected tropical disease, caused by infection with the fish-borne trematode Opisthorchis viverrini sensu lato that afflicts more than 10million people in Southeast Asia, including Thailand, Lao PDR, Vietnam and Cambodia. The disease is characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. This chronic inflammation eventually leads to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in approximately 1% of O. viverrini-infected individuals. In Thailand alone, CCA kills up to 20,000 people every year and is therefore of substantial public health importance. Its socioeconomic impacts on impoverished families and communities are considerable. To reduce O. viverrini-associated morbidity and CCA, the primary intervention measures focus on opisthorchiasis control and elimination. Accurate diagnoses of O. viverrini infection, in both mammalian, snail and fish intermediate hosts, are important for achieving these goals. Despite extensive efforts over several decades to find sensitive and specific diagnostics for opisthorchiasis, a simple and robust diagnostic method is still required. Here we review earlier and current developments in the search for new diagnostics for opisthorchiasis, with practical applications in the research laboratory, the clinic and the field. Of the methods currently available, the urine antigen assay shows considerable potential for the diagnosis and screening of opisthorchiasis. Nevertheless, these new assays require validation, determination of their cost-effectiveness when applied for mass screening in an endemic setting in support of policy decisions for national public health programs aimed at the control and elimination of opisthorchiasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The "37 collar-spined" or "revolutum" group of echinostomes is recognized as a species complex. The identification of members of this complex by morphological taxonomic characters is difficult and confusing, and hence, molecular analyses are a useful alternative method for molecular systematic studies. The current study examined the genetic diversity of those 37 collar-spined echinostomes which are recognized morphologically as Echinostoma revolutum in Thailand and Lao PDR using the cytochrome c oxidase subunit 1 (CO1) and the NADH dehydrogenase subunit 1 (ND1) sequences. On the basis of molecular investigations, at least two species of 37 collar-spined echinostomes exist in Southeast Asia, namely E. revolutum and Echinostoma miyagawai. The specimens examined in this study, coming from ducks in Thailand and Lao PDR, were compared to isolates from America, Europe and Australia for which DNA sequences are available in public databases. Haplotype analysis detected 6 and 26 haplotypes when comparing the CO1 sequences of E. revolutum and E. miyagawai, respectively, from different geographical isolates from Thailand and Lao PDR. The phylogenetic trees, ND1 haplotype network and genetic differentiation (ɸST) analyses showed that E. revolutum were genetically different on a continental scale, i.e. Eurasian and American lineages., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Seasonal changes play roles in the transmission success of fish-borne zoonotic trematodes (FZT). This study examined the seasonal transmission patterns of Opisthorchis viverrini sensu lato (s.l.) and a virgulate cercaria (family Lecithodendriidae) in the snail intermediate host, Bithynia siamensis goniomphalos in northeast Thailand. Snail samples were collected monthly during the rainy, cool, and hot seasons during 2012-2013 to determine the prevalence and intensity of larval trematode infections. The prevalence of O. viverrini s.l. varied significantly with season, being 0.31%, 1.05%, and 0.37% in the rainy, cool, and hot seasons, respectively (P < 0.05). Similarly, the prevalence of virgulate cercariae was 3.11%, 6.80%, and 1.64% in the rainy, cool, and hot seasons, respectively (P < 0.05). The intensity of larval trematode infections also varied between seasons and peaked in the hot season (P < 0.05) in both species. The snails infected with O. viverrini s.l. were significantly smaller (P < 0.05) and those infected with virgulate cercariae were significantly larger (P < 0.05) than uninfected snails. Seasonal variation and the different sizes of B. s. goniomphalos parasitized by O. viverrini s.l. and virgulate trematodes indicate complex host-parasite interactions with important implications for the epidemiology of O. viverrini s.l., (© The American Society of Tropical Medicine and Hygiene.)

Background: Cholangiocarcinoma (CCA) is an extremely aggressive cancer that is usually fatal. Although globally morbidity and mortality are increasing, knowledge of the disease remains limited. The Mekong region of Southeast Asia, and particularly the northeast of Thailand, has by far the highest incidence of CCA worldwide with 135.4 per 100,000 among males and 43.0 per 100,000 among females being reported in Khon Kaen Province. Most patients are first seen during late stage disease with 5-year survival being less than 10%. Starting in 1984, control and prevention strategies have been focused on health education. Although early detection can substantially increase 5-year survival, there are currently no strategies to increase early diagnosis., Methods/design: The Cholangiocarcinoma Screening and Care Program (CASCAP) is a prospective cohort study comprising two cohorts- the screening and the patient cohorts. For the screening cohort, ultrasound examination will be carried out regularly at least annually to determine whether there is current bile duct and/or liver pathology so that the optimal screening program for early diagnosis can be established. This cohort is expected to include at least 150,000 individuals coming from high-risk areas for CCA. For the patient cohort, it is estimated that about 25,000 CCA patients will be included during the 5-year recruitment period. All CCA patients will be treated according to routine clinical care and followed so that effective surgical treatment can be formulated. This cohort is indeed a conventional cancer registry. Thus, CASCAP is an ongoing project in which the number of participants changes dynamically., Discussions: This is the first project on CCA that involves screening the at risk population at the community level. At the time of preparing this report, a total of 85,927 individuals have been enrolled in the screening cohort, 55.0% of whom have already undergone ultrasound screening, and 2661 CCA cases have been enrolled in the patient cohort. Among the participants of the screening, whose mean age was 53.8±9.8 years, 55.6% were female, 77.5% attained primary school as the highest level of education, 79.9% were farmers, 29.9%, reported having relatives with CCA, 89.1% had eaten uncooked fish, and 42.2% of those who had been tested for liver fluke were found to be infected.

Human–wildlife interactions have reached unprecedented levels, and humans are influencing the earth’s ecosystems more rapidly and extensively than ever before. This situation is cause for serious concern, especially since disease interactions between wildlife and humans have been recognized as major conservation threats. In this study, long-tailed macaques, Macaca fascicularis, from 2 forest parks located in north-eastern Thailand were investigated to determine the influence of habitat modification by humans on helminth parasite associations in non-human primates. Macaque populations with contact to anthropogenically modified environments were compared with sylvatic groups in nearby natural environments. In order to test for human–non-human primate transmission of parasites, the local human populations were also examined. Humans were infected with a number of potentially pathogenic parasites, including Opisthorchis viverrini and Strongyloides stercoralis. However, eggs of these helminths were not detected in macaque feces. Thus, no direct parasite transfer from humans to non-human primates could be confirmed. However, macaque groups with more frequent contact with human modified habitats, and a higher portion of human-provided food in their diet, had significantly higher prevalences and intensities of Strongyloides fuelleborni and of an intestinal fluke (probably Haplorchis sp.) than sylvatic groups. Positive correlations were found between the time foraging on the ground and infection with S. fuelleborni, and the amount of human-provided food and intestinal fluke infection. Human alteration of habitat and associated modifications in nonhuman primate behaviour are likely to play a role in determining the occurrence, prevalence and intensity of zoonotic helminth infection of wild non-human primates.

Bithynia snails are important hosts of the human liver fluke, Opisthorchis viverrini. Allozyme markers were used to examine genetic variation and the systematics of Bithynia snails from different geographical localities in Thailand and Lao PDR. Our results show that Bithynia siamensis goniomphalos contains two major evolutionary clades with at least nine morphologically similar but genetically distinct "cryptic" species (taxa). Clade A consists of snails from the Chi and the Mun River wetlands, Thailand and clade B consists of snails from the Nam Ngum River wetland, Lao PDR, as well as snails from some localities in the Songkram River wetland, Thailand. There is an association between O. viverrini cryptic taxa with B. s. goniomphalos cryptic taxa within the defined wetlands. Bithynia species are the critical amplifying component of O. viverrini and they control transmission from humans to fish second intermediate hosts. These findings are particularly important for understanding the transmission dynamics of O. viverrini. They are, in addition, a link in the life cycle of O. viverrini which can be used for developing and implementing programs to eliminate and control opisthorchiasis and its associated cholangiocarcinoma., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Throughout Southeast Asia and China, eating raw and or partially cooked cyprinid fish causes liver (hepatobiliary) disease and cancer (cholangiocarcinoma) due to fishborne zoonotic trematodes (FZT), in particular Clonorchis sinensis and Opisthorchis viverrini. The primary source of transmission is by native fish, but aquaculture fish are also reported to have high infective potential. Here, a cross-sectional survey of FZT in fish farms was conducted in an endemic area in Khon Kaen Province, Thailand. By using conventional and polymerase chain reaction (PCR) methods, we detected O. viverrini and FZT metacercariae (Centrocestus formosanus and Haplorchis taichui) in two popular fish species, Barbonymus gonionotus (silver barb) and Cirrhinus mrigala (mrigal), from aquaculture farms. Both species were infected in five of six farms examined by PCR but not by conventional methods, yet the prevalence of FZT metacercariae in aquaculture fish was high (46.9%). In addition to O. viverrini (17.1%), the native fish Cyclocheilichthys armatus and Hampala dispar had a prevalence of FZT of 81.4%, which included 5.7% for C. formosanus and 17.1% for H. taichui by conventional method. To our knowledge, this is the first discovery of O. viverrini in aquaculture fish in Thailand. More comprehensive studies are required to determine if human-induced disease transmission coupled with natural transmission cycle occurs throughout the aquaculture industry in the region. This has significant impact on food quality and safety, and provides the basis for the development of an effective strategy for the prevention and control of foodborne diseases.

Together with host and environmental factors, the systematics and population genetic variation of Opisthorchis viverrini may contribute to recorded local and regional differences in epidemiology and host morbidity in opisthorchiasis and cholangiocarcinoma (CCA). In this review, we address recent findings that O. viverrini comprises a species complex with varying degrees of population genetic variation which are associated with specific river wetland systems within Thailand as well as the Lao PDR. Having an accurate understanding of systematics is a prerequisite for a meaningful assessment of the population structure of each species within the O. viverrini complex in nature, as well as a better understanding of the magnitude of genetic variation that occurs within different species of hosts in its life cycle. Whether specific genotypes are related to habitat type(s) and/or specific intermediate host species are discussed based on current available data. Most importantly, we focus on whether there is a correlation between incidence of CCA and genotype(s) of O. viverrini. This will provide a solid basis for further comprehensive investigations of the role of genetic variation within each species of O. viverrini sensu lato in human epidemiology and genotype related morbidity as well as co-evolution of parasites with primary and secondary intermediate species of host., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The carcinogenic liver fluke, Opisthorchis viverrini, requires Bithynia snail intermediate hosts in its life cycle. However, the prevalence of O. viverrini in snail intermediate hosts is typically low (< 1%). Here, we examined B. siamensis goniomphalos from 48 localities in Thailand and The Lao People's Democratic Republic (Lao PDR) and reported high-prevalence levels of O. viverrini. The highest-prevalence levels per locality were 6.93% (mean = 3.04%) in Thailand and 8.37% (mean = 2.01%) in Lao PDR; 4 of 13 localities examined showed prevalence higher than any prevalence previously recorded. The number of cercariae infecting snails and their prevalence were positively correlated with the size of the snails. High prevalence occurred in the Songkram River wetland (Thailand) and the Nam Ngum River wetland (Lao PDR). Our results show that transmission of O. viverrini from humans as well as animal reservoir hosts to snail intermediate hosts is ongoing and potentially increasing in endemic areas across Thailand and Lao PDR.

Khon Kaen Province in northeast Thailand is known as a hot spot for opisthorchiasis in Southeast Asia. Preliminary allozyme and mitochondrial DNA haplotype data from within one endemic district in this Province (Ban Phai), indicated substantial genetic variability within Opisthorchis viverrini. Here, we used microsatellite DNA analyses to examine the genetic diversity and population structure of O. viverrini from four geographically close localities in Khon Kaen Province. Genotyping based on 12 microsatellite loci yielded a mean number of alleles per locus that ranged from 2.83 to 3.7 with an expected heterozygosity in Hardy-Weinberg equilibrium of 0.44-0.56. Assessment of population structure by pairwise F(ST) analysis showed inter-population differentiation (P<0.05) which indicates population substructuring between these localities. Unique alleles were found in three of four localities with the highest number observed per locality being three. Our results highlight the existence of genetic diversity and population substructuring in O. viverrini over a small spatial scale which is similar to that found at a larger scale. This provides the basis for the investigation of the role of parasite genetic diversity and differentiation in transmission dynamics and control of O. viverrini.

We echo the viewpoints presented in the recent editorials and forums in ecohealth in 2009 and 2010 that understanding the links between water and health using biogeography can provide insights into the patterns of and the processes that give rise to the distribution of disease prevalence. In particular, we underscore the need to integrate disease ecology and biogeography using landscape ecological approaches. We use opisthorchiasis, a major public health problem in Southeast Asia, to illustrate our opinions.

Snails are the critical amplifying hosts of the liver fluke Opisthorchis viverrini, the causative agent of hepatobiliary disease and cholangiocarcinoma in the Mekong area of Southeast Asia. Bithynia funiculata, B. siamensis goniomphalos and B. s. siamensis are the first intermediate hosts of O. viverrini in Thailand. Morphological similarity between Bithynia species and subspecies creates problems for their taxonomic identification and an understanding of Bithynia systematics. In this study, multilocus enzyme electrophoresis (MEE) was applied to define genetic markers that could prove useful for investigating the systematics and population genetics of this genus in Thailand. Of the 34 enzymes examined, 20 encoding a presumptive 24 loci showed sufficient staining intensity and resolution for genetic interpretation. Of these, three loci were monomorphic and eight loci were diagnostic among the three Bithynia taxa. The remaining 13 loci were diagnostic between combinations of the three taxa. Fixed genetic differences were detected at 67-73% of loci among these taxa which in turn differed from a closely related species, Hydrobioides nassa, at 88% of loci. Seventy three percent fixed genetic differences were detected between B. funiculata and the two sub-species B. s. siamensis and B. s goniomphalos. Our data reveals similarly large genetic divergence, 67% fixed genetic differences, between B. s. siamensis and B. s. goniomphalos, which may well represent different species rather than subspecies as currently defined. The genetic markers detected will form the basis for subsequent comprehensive studies on the systematics and population genetics of Bithynia snails as well as for their role in the transmission of O. viverrini and opisthorchiasis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A total of 314 individual Echinostoma revolutum were collected at different locations and times from domestic ducks from Khon Kaen Province, Thailand and Vientiane Province, the Lao People's Democratic Republic (PDR). Genetic variation of these parasites was analyzed using multilocus enzyme electrophoresis at three polymorphic loci namely, glucose-6-phosphate dehydrogenase (G6pd), malic enzyme (Me) and peptidase valine-leucine (PepA). High levels of genetic variability were found within and between populations. Significant heterozygote deficiencies compared with the predictions under Hardy-Weinberg equilibrium were detected in populations from Thailand and the Lao PDR for all loci except G6pd-1. Significant genetic differentiation was observed between spatially separated populations from Thailand and the Lao PDR. This as also true for some samples collected at different times in Thailand. The variability found may be consistent with a Wahlund effect, genetic drift and/or other factors such as the population structure of snail hosts. Our data provide further insight into the process of genetic divergence within and among geographically and temporally isolated populations of E. revolutum, and potentially other medically important echinostomes in Southeast Asia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Previous molecular genetic analyses indicate that Opisthorchis viverrini is a complex of at least 2 cryptic species in Thailand and the Lao People's Democratic Republic (PDR). Opisthorchis viverrini sensu lato (s.l.) populations can be divided into 6 genetic groups, which correlate with 5 different river wetlands systems. The present study examines the infectivity, growth, fecundity, and body size of O. viverrini s.l. isolates from these systems. Worm recovery was lowest in the Songkhram River, which differed significantly from all other wetland systems. In addition, the Chi River and Nam Ngum River populations also differed significantly. A similar pattern was found for fecundity. These results support the genetic/molecular data indicating a subdivision of O. viverrini populations on the basis of watershed and that propose the existence of cryptic species in Thailand and the Lao PDR.

The population genetics of 317 individual Opisthorchis viverrini from Khon Kaen Province Thailand, from 4 different years and 4 cyprinid fish species was examined using multilocus enzyme electrophoresis of enolase (Enol), phosphoglucomutase (Pgm) and triose phosphate isomerase (Tpi). Allele and genotype frequencies for Enol and Pgm were consistent irrespective of year or host species. No heterozygote deficiency was detected for Enol. Significant heterozygote deficiencies were detected in 3 of 4 years for Pgm. For Tpi, allele frequencies of the most common allele and genotype frequency varied between years and among individuals from different host species. Heterozygote deficiencies for Tpi were detected in 2 years. No significant heterozygous deficiencies were detected among O. virerrini from different fish species in 2005, except at Pgm and Tpi from Puntioplites protozsron. There was no statistical significance in pairwise FST values between O. viverrini from Cyclocheilichthys armatus in different years or different host species in 2005. Significant departures from Hardy-Weinberg expectations and a high rate of gene flow in a population of O. viverrini are discussed in terms of self- and cross-fertilisation, natural selection, non-random mating, the Wahlund effect, presence of null alleles, intensity of infection, biology and ecology of their intermediate cyprinid hosts.

Genetic variation among 231 individuals of Opisthorchis viverrini from the Ban Phai District, Khon Kaen Province (Thailand) was examined at three polymorphic enzymes: enolase (ENOL), phosphoglucomutase (PGM), and triose phosphate isomerase (TPI). Four alleles were detected for TPI and PGM, whereas only two alleles were detected for ENOL. The inferred genotype frequencies for both TPI and ENOL were not significantly different from Hardy-Weinberg equilibrium. In contrast, the inferred genotype frequencies for PGM showed a significant departure from Hardy-Weinberg equilibrium, with a lack of heterozygous individuals. This heterozygote deficiency suggests non-random mating and/or potentially high self fertilization.

The present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0.3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0.5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13-31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.

The food-borne trematodes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis, have long been recognized as the cause of major human health problems, with an estimated 40 million infected persons. Of the three species of liver fluke, only O. viverrini is classified as a type 1 carcinogen because of its role as an initiator of chronic inflammation and the subsequent development of cholangiocarcinoma. At present, there are no techniques for the early diagnosis of cholangiocarcinoma and it is fatal for most patients. There is considerable variation in parasite prevalence and disease presentation in different geographical areas, the latter of which may be associated with genetic differences among parasites. In the present study, multilocus enzyme electrophoresis was used to provide a comprehensive genetic characterization of O. viverrini from different geographical localities in Thailand and the Peoples' Democratic Republic of Laos. Parasites from different localities were compared genetically at 32 enzyme loci. The results of the genetic analyses are sufficient to reject the null hypothesis that O. viverrini represents a single species. Therefore, O. viverrini consists of at least two genetically distinct, yet morphologically similar (i.e. cryptic) species. Moreover, there was also separation of the different populations of snails (i.e. the first intermediate hosts) into two distinct genetic groups that corresponded with the delineation of O. viverrini into two species. This suggests that there may be a history of co-evolution in this host-parasite lineage. Additionally, five distinct genetic groups of parasites were detected, each of which occurred within a different and independent river wetland system. Our findings have major implications for the implementation of effective control and surveillance programs targeted to these medically important food-borne parasites.

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.

The liver fluke, Opisthorchis viverrini, is one of the major food borne trematodes in Southeast Asia, where infection causes hepatobiliary disease and subsequent development of cholangiocarcinoma. In Thailand, O. viverrini is most prevalent in the northeast where there is marked regional variation in the rate of infection in humans at provincial, district and village levels. To date, the roles of genetic variation of O. viverrini on this observed variability in infection, transmission and associated disease are not known. We have applied multilocus enzyme electrophoresis (MEE), specifically allozyme electrophoresis, to isolates of O. viverrini from Thailand and Laos to establish genetic markers to examine its systematics and population structure. Forty-six enzymes commonly found useful for genetic characterisation in parasitic helminths were screened, and of these, 33 enzymes gave sufficient staining and resolution to act as potential genetic markers. Sixteen enzymes were monomorphic and 17 enzymes were polymorphic in the pools of worms examined. Whether they are indicative of different enzyme loci, heterozygosity or unique genotypes within the pools of worms examined remains to be determined. Preliminary investigations examining five individual worms at enzyme loci where pools of worms showed multiple bands have confirmed the diagnostic value of the enzyme loci established as well as providing evidence of potential population sub structuring and heterozygosity. For the first time, we have established at least 17 enzymes that provide the basis to undertake comprehensive genetic analyses of the systematics and population structure of O. viverrini, a medically important food borne trematode in Southeast Asia.

We conducted an allozyme electrophoretic study to explore potential enzyme markers to distinguish Opisthorchis viverrini in Thailand and Lao PDR. Twenty-eight enzymes encoding presumptive 32 loci were established. The enzymes glucose-6-phosphate dehydrogenase and pyruvate kinase were diagnostic between two geographically separate isolates from Thailand. Twelve enzymes, ie, aconitate hydratase, aldolase, creatine kinase, enolase, esterases, fumarate hydratase, aspartate aminotransferase, glucose-phosphate isomerase, alanine aminotransferase, isocitrate dehydrogenase, malic enzyme, and pyruvate kinase, also provided diagnostic markers for these two isolates from Thailand and one isolate from Lao PDR.

The changes taking place within the societies, cultures and the environments in which we live are massive and complex. By referring to simple epidemiological models it is possible to build an objective framework with which to look at these changes in terms of their likely impact on the epidemiology of parasitic diseases within human communities. These parameters are listed for hosts and both micro- and macroparasites, as are epidemiologically significant cultural, social and environmental variables. Changes in these variables may be either detrimental or beneficial to human health and may, in addition, interact in complex ways. Examples of the complexity of changes which can influence epidemiology are provided for a cultural template of the population living in the north-east of Thailand.