Introduction.

Cholangiocarcinoma (CCA) is a lethal cancer arising in the bile ducts within and just outside the liver. It occurs worldwide and falls into two etiologically defined groups, one related to chronic liver fluke infection and the other not. Liver fluke-related CCA is found in continental Southeast Asia (caused by Opisthorchis viverrini with infection leading to opisthorchiasis), East Asia (Clonorchis sinensis), and Eastern Europe and Russia (Opisthorchis felineus). Both O. viverrini and C. sinensis are classified as group one carcinogens, while recent data from O. felineus suggest the same. In Southeast Asia, an estimated 67.3 million people are at risk of O. viverrini infection and subsequently developing CCA. When the three liver fluke species are considered, an estimated 700 million people are at risk of infection and developing CCA globally. The northeast of Thailand (Isan) is the world's hot spot of liver fluke infection and CCA. Early detection, diagnosis, and surgical intervention/curative treatment of CCA are critical to increase life expectancy and quality of life of people in the region and globally. Despite concentrated recent efforts focusing on a multidisciplinary approach to understand the ecology, epidemiology, biology, public health, and social significance of infection by cancer causing liver flukes, it remains an underestimated and under-resourced public health problem. In addition, it is still believed to be a regional problem without global significance-this is not the case. This book focuses on O. viverrini as the main causative agent of CCA in Southeast Asia, but many aspects detailed in the following chapters also relate to the two other liver fluke species. Our aim is to produce a holistic framework including the basic biology of O. viverrini and its relation to the epidemiology of the disease through diagnosis to treatment, including palliative methods, pathology, and control.

Opisthorchis viverrini Life Cycle, Distribution, Systematics, and Population Genetics.

Opisthorchis viverrini plays a key role as the carcinogenic liver fluke causing bile duct cancer in Southeast Asia. A comprehensive understanding of its life cycle, distribution, systematics, and population genetics is critically important as they underpin the effective development and establishment of future prevention and control programs that center on opisthorchiasis and cholangiocarcinoma. This chapter provides detailed information concerning the basic biology and updated information of O. viverrini related to its host life cycle, transmission route via raw, partially cooked or fermented freshwater cyprinid fish, endemic areas, and the discovery of new foci. Previous sequential studies over the last two decades on the phylogenetic and systematic relationships, genetic variation, and population genetics of O. viverrini as well as its snail intermediate host Bithynia spp. are presented and discussed, which have led to the currently known complex species level systematics and population genetics framework of this host-parasite system. Additionally, further directions for comprehensive research are suggested to provide a more complete understanding of liver fluke, O. viverrini-related cholangiocarcinoma.

Synopsis.

Cholangiocarcinoma (CCA) is the second most common primary liver cancer worldwide. Despite the severity of the disease and its impact on individuals, families, and communities, there remains an overall lack of awareness and interest in this disease. The information contained in the chapters of this book shows that this is indeed a significant public health and socioeconomic problem with varying levels of country-specific awareness. In Southeast Asia liver fluke, O. viverrini related CCA is endemic with the highest incidence worldwide in northeast Thailand, yet it is treatable and preventable. The chapters highlight significant advances in our knowledge of the biology and epidemiology of the O. viverrini species complex, intermediate hosts, systematics, population genetics, and the complexity of the three-host life cycle. A comprehensive conceptual framework has been developed to assist in understanding the complexity of molecular mechanisms of CCA carcinogenesis and cancer development which can result in improvement of targeted CCA therapy. There have been many advances in understanding the pathology of CCA in the biliary tract, including advances in prognosis and molecular pathogenesis. The development of different modalities and their advantages for diagnosis have increased diagnostic accuracy, providing reliable information allowing appropriate treatment and management programs to be selected for each patient. Particularly exciting is the recent development of a urine antigen assay which has revolutionized the diagnostic approach of opisthorchiasis due to its simplicity, the non-invasive nature of sample collection, and its ease of use in field settings. Significant in-roads and advances have been made in the surgical and systemic treatment of CCA patients. Additionally, a sophisticated data collection and analysis system, the Isan Cohort, has been developed and established for the treatment and control of CCA. Importantly, a greater understanding has been made of the social, community, religious, and anthropological issues initiating and sustaining the eating behavior of raw, partially cooked, and/or fermented fresh water fish. Specially designed education programs/curricula, based on currently available multidisciplinary hard data targeting school children, have been introduced since the inception of the Cholangiocarcinoma Screening and Care Program (CASCAP) and the subsequent strategic Fluke Free Thailand Model. The education program is being expanded to other provinces in Thailand and in the near future to other Southeast Asian countries, initially to Lao PDR, where the Fluke Free Lao PDR program has already been implemented. Despite advances that have been made in many disciplines focused on O. viverrini related CCA, raising awareness of CCA at all levels, particularly across endemic regions, is still needed, as is raising the awareness of CCA globally. As parasites and parasite related diseases have no borders, it is critical that an effective common strategic plan is instigated and established between all countries where liver fluke, O. viverrini related CCA is a significant public health problem, thereby increasing the quality of life and life expectancy of millions of people who suffer from this insidious disease.

RAW ATTITUDES: Socio-Cultures, Altered Landscapes, and Changing Perceptions of an Underestimated Disease.

Raw attitudes relate to the food cultures, eating habits, and behaviours of people in relation to the consumption of raw, partially cooked, and fermented freshwater fish dishes, which puts people at risk of Ophistochis viverrini and other parasitic infections. The chapter reviews raw attitudes within the countries and across the borders of the greater Mekong region, particularly northeast Thailand, Lao PDR, Vietnam, and Cambodia. Rather than treat each nation-state as an isolated epidemiological box, the chapter explores transborder complexity in relation to multiple anthropogenic transformations to the landscapes of the region and developmental impacts upon ecosystems and life cycles. Economic projects such as multiple hydropower dams, irrigation schemes, water-diversions, roads, and aquaculture ponds have significant impacts on FTZ life-cycle dynamics. In addition, many ecological changes are transboundary ones, and there are added complications relating human mobility, altered agrarian landscapes, and significant numbers of migrant workers. The chapter also examines public health programmes and educational interventions which are altering perceptions of O. viverrini and cholangiocarcinoma over time. A key argument is the need for transdisciplinary scientific and social science strategies alongside multi-pronged health interventions, such as 'the CASCAP model'. There exist many unknowns and gaps relating to widespread anthropogenic modifications upon life cycles, upon lifestyles, livelihoods, and human behaviours, which require research projects that span socio-economic, ecological, geographical, and public health dynamics of disease.

Epidemiology and Control of Opisthorchis viverrini Infection: Implications for Cholangiocarcinoma Prevention.

It is known that Opisthorchis viverrini (OV) is the most significant risk factor for the development of cholangiocarcinoma (CCA); hence, it is also known as carcinogenic parasite. Effective control and elimination of OV infection should significantly reduce O. viverrini-related CCA. This chapter includes details of the three recently developed innovative tools, namely the Isan cohort database software, an OV-RDT for screening of O. viverrini, and an ultrasound telecommunication system. Past and current control programs, i.e., education, medication, and sanitation were discussed and stressed the need for a comprehensive control program which encompasses primary, secondary, and tertiary patient care programs for confirmation and management of suspected CCA cases. The approach of mathematical modeling for control of OV and CCA was also briefly described. Additionally, we highlighted the current progress toward control of OV and CCA in Thailand and potential for expansion into nearby countries in Southeast Asia.

Population dynamics and diversity of trematode infections in Bithynia siamensis goniomphalos in an irrigated area in northeast Thailand.

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 host­parasite 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 2010­2012 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.

Genetic diversity and phylogenetic analyses of ixodid ticks infesting cattle in northeast Thailand: the discovery of Rhipicephalus microplus clade C and the rarely detected R. haemaphysaloides.

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.

Intron sequence variation of the echinostomes (Trematoda; Echinostomatidae): implications for genetic investigations of the 37 collar-spined, Echinostoma miyagawai Ischii, 1932 and E. revolutum (Fröelich, 1802).

Echinostomes are a diverse group of digenetic trematodes that are difficult to classify by predominantly traditional techniques and contain many cryptic species. Application of contemporary genetic/molecular markers can provide an alternative choice for comprehensive classification or systematic analysis. In this study, we successfully characterized the intron 5 of domain 1 of the taurocyamine kinase gene (TkD1Int5) of Artyfechinostomum malayanum and the other two species of the 37 collar-spined group, Echinostoma revolutum and Echinostoma miyagawai, whereas TkD1Int5 of Hypoderaeum conoideum cannot be amplified. High levels of nucleotide polymorphism were detected in TkD1Int5 within E. revolutum and E. miyagawai, but not in A. malayanum. Thus, TkD1Int5 can be potentially used as genetic marker for genetic investigation of E. miyagawai and E. revolutum. We therefore used TkD1Int5 to explore genetic variation within and genetic differentiation between 58 samples of E. miyagawai and five samples of E. revolutum. Heterozygosity was observed in 17 and two samples with 16 and three insertion/deletion (indel) patterns in E. miyagawai and E. revolutum, respectively. Heterozygous samples were then cloned and nucleotide sequence was performed revealing the combined haplotypes in a particular sample. Based on nucleotide variable sites (excluding indels), the 72 E. miyagawai and seven E. revolutum haplotypes were subsequently classified. The haplotype network revealed clear genetic differentiation between E. miyagawai and E. revolutum haplogroups, but no genetic structure correlated with geographical localities was detected. High polymorphism and heterogeneity of the TkD1Int5 sequence found in our study suggest that it can be used in subsequent studies as an alternate independent potential genetic marker to investigate the population genetics, genetic structure, and possible hybridization of the other echinostomes, especially the 37 collar-spined group distributed worldwide.

A comparison of the proportion of early stage cholangiocarcinoma found in an ultrasound-screening program compared to walk-in patients.

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.

Liver Flukes: Clonorchis and Opisthorchis.

Clonorchis sinensis, Opisthorchis viverrini, and O. felineus are liver flukes of human and animal pathogens occurring across much of Europe and Asia. Nevertheless, they are often underestimated compared to other, better known neglected diseases in spite of the fact that many millions of people are infected and hundreds of millions are at risk. This is possibly because of the chronic nature of the infection and disease and that it takes several decades prior to a life-threatening pathology to develop. Several studies in the past decade have provided more information on the molecular biology of the liver flukes which clearly lead to better understanding of parasite biology, systematics, and population genetics. Clonorchiasis and opisthorchiasis are characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. These chronic inflammations eventually lead to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in some infected individuals. In Thailand alone, opisthorchiasis-associated 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 hepatobiliary morbidity and CCA, the primary intervention measures focus on control and elimination of the liver fluke. Accurate diagnosis of liver fluke infections in both human and other mammalian, snail and fish intermediate hosts, are important for achieving these goals. While the short-term goal of liver fluke control can be achieved by praziquantel chemotherapy, a comprehensive health education package targeting school children is believed to be more beneficial for a long-term goal/solution. It is recommended that a transdisciplinary research or multisectoral control approach including one health and/or eco health intervention strategy should be applied to combat the liver flukes, and hence contribute to reduction of cholangiocarcinoma in endemic areas.

Preliminary genetic evidence of two different populations of Opisthorchis viverrini in Lao PDR.

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.

Cercarial emergence patterns for Opisthorchis viverrini sensu lato infecting Bithynia siamensis goniomphalos from Sakon Nakhon Province, Thailand.

UNLABELLED: Opisthorchis viverrini sensu lato is a food-borne trematode which is classified as a class 1 carcinogen, with infection potentially leading to cholangiocarcinoma. Snails of the genus Bithynia act as the first intermediate hosts and an amplifying point in the parasite life cycle. In order to investigate seasonal effect on transmission dynamics of O. viverrini in Bithynia siamensis goniomphalos, cercarial emergence and output profiles were monitored at different season. A total of 4533 snails originating from Sakon Nakhon Province, Thailand, collected during the three main seasons, were analyzed for O. viverrini s.l. INFECTION: Emergence of O. viverrini s.l. cercariae from snails was monitored daily from 06:00 to 18:00 h for seven consecutive days. The prevalence of infection in the snails was highest in the hot-dry season and declined in the rainy and cool-dry seasons. Peak cercarial emergence occurred between 08:00 and 10:00 h during the rainy and cool-dry seasons and between 10:00 and 12:00 h during the hot-dry season. The cercarial output was highest in the hot-dry season, similar to a previous study from Lao People's Democratic Republic (PDR). Average cercarial output/snail in Thailand was higher than in Lao PDR. The number of cercariae emerging from the snails was strongly related to snail size, but the relationship between prevalence of infection and snail size differed between seasons. Observed discrepancies in the emergence patterns and per capita cercarial release may reflect differences in environmental, snail, and/or parasite factors particularly biological characteristics between the cryptic species of O. viverrini s.l. and B. s. goniomphalos from Thailand and Lao PDR.

EVALUATION OF A COMMERCIAL STOOL CONCENTRATOR KIT COMPARED TO DIRECT SMEAR AND FORMALIN-ETHYL ACETATE CONCENTRATION METHODS FOR DIAGNOSIS OF PARASITIC INFECTION WITH SPECIAL REFERENCE TO OPISTHORCHIS VIVERRINI SENSU LATO IN THAILAND.

Opisthorchiasis and cholangiocarcinoma (CCA) are major public health problems in Thailand and countries in the lower Mekong Subregion. Elimination of opisthorchiasis will be an important step toward the prevention, control and reduction of CCA. In order to achieve this goal, a sensitive and robust diagnostic method is required to identify people with current Opisthorchis viverrini sensu lato infection as the parasite is a group 1 carcinogen believed to be an etiology of CCA. To date, sensitive parasitological methods, such as formalin-ethyl acetate concentration technique (FECT) is preferred, but it is not practical in a remote primary care setting. In this study, we evaluated the diagnostic accuracy of a commercial stool concentrator kit with that of a direct simple smear method and a modified FECT. In diagnosing parasite infection and opisthorchiasis, the commercial kit had greater sensitivity (43.8-58.5%) than direct smear method (12.5-31.7%), but was less sensitive than FECT (73.2-75%). In a separate sample population, similar results were obtained when comparing the diagnostic accuracy of the commercial kit and FECT. However, the commercial kit was more effective in a field setting than FECT, and had better accuracy than direct smear method, which suggests that the kit could have potential utility in epidemiological studies and control programs of opisthorchiasis, as well as other parasitic infections. The design of the self-contained one-tube kit plus its long storage time after sample preparation provides a considerable advantage over other methods, such as direct or Kato thick smear method, under similar field conditions.

Cohort profile: cholangiocarcinoma screening and care program (CASCAP).

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.

Susceptibility, metacercarial burden, and mortality of juvenile silver barb, common carp, mrigal, and tilapia following exposure to Haplorchis taichui.

Fish-borne zoonotic trematodes (FZT) including heterophyids and opisthorchiids are prevalent in native and aquaculture fish in Southeast Asia. FZT are reported highly prevalent in juvenile aquaculture fish that belonged to the family Heterophyidae, particularly Haplorchis taichui. Aquaculture fish are reported to have varying levels of natural infection with H. taichui, but data for their susceptibility as well as resistance to infection are not available. The aim of this study was to determine the susceptibility of five aquaculture fish to H. taichui. Experimental infections were performed by exposing fish individually to 0 (control), 50, 100, or 200 H. taichui cercariae for 12 h. Metacercarial burden was measured at 45 days postinfection. Three out of five fish species, silver barb (Barbonymus gonionotus), common carp (Cyprinus carpio), and mrigal (Cirrhinus mrigala), were highly susceptible to H. taichui infection with percentage ranging from 93.33 to 100%. The Nile and red tilapia were not susceptible to infection. Among the susceptible fish species, silver barb had higher intensity of H. taichui metacercariae than common carp and mrigal (P < 0.001). Metacercarial burden significantly correlated with the dose of cercarial infection (P < 0.001). Our findings that common aquaculture fish species have varying degrees of susceptibility to H. taichui infection provide important information to reduce parasite transmission in aquaculture fish.

Analysis of the population genetics of Opisthorchis viverrini sensu lato in the Nam Ngum River wetland, Lao PDR, by multilocus enzyme electrophoresis.

A previous population genetics study of Opisthorchis viverrini from a locality in an endemic area in Thailand found little genetic variation over time and second intermediate fish host species. Since a similar comparative analysis is not available for Lao PDR, we conducted a study of O. viverrini from different endemic foci in Vientiane Province, Lao PDR, based on spatial, temporal and fish host species. A total of 620 adult O. viverrini originating from the Nam Ngum River wetland were analysed at five previously defined polymorphic enzyme loci. Of these worms, 252 were from six different localities (spatial samples), 162 worms from different years (temporal samples) and 206 worms from four different cyprinid fish species. Significant heterozygote deficiency was found in most O. viverrini populations with levels of genetic differentiation ranging between F ST 0.0000 and 0.0197 suggesting that gene flow occurred at a variable rate. The role of temporal factors and fish host species had little influence on the level of genetic differentiation. As for O. viverrini from Thailand, these findings indicate that self-fertilization and/or a clonal distribution of O. viverrini occurs in Lao PDR. Unlike the results for O. viverrini from Thailand, spatial population substructuring may be the underlying population processes for O. viverrini in Lao PDR. These findings indicate that geographical variation may contribute to the transmission dynamics of the parasite with implications for parasite control. However, other host factors, such as snail intermediate hosts and mammal reservoir hosts, as well as human beings, may also play significant roles.

Genetic characterization and phylogenetic analysis of echinostomes.

We conducted this study to identify species and determine the phylogenetic relationships using ribosomal DNA (rDNA) sequences [partial sequences of 28S rDNA and second internal transcribed spacer (IT52)] of echinostomes collected from free-grazing ducks in Phitsanulok Province, Thailand. Four adult echinostomes were morphologically identified as Echinostoma revolutum, 4 as Hypoderaeulm conoideurn and 2 unidentified. Sequences of other species/isolates of echinostomes retrieved from the GenBank database were employed to compare and construct the phylogenetic tree. Three major lineages were found, namely, genus Echinostoma, genus Echinoparyphiulm and genus Hypoderaeulm. One of the unidentified echinostome specimen was 99% identical to and clustered with genus Echinoparyphiurm, whereas the other was located in the "revolutum" roup, but was closely related to the geographical isolates from America rather than from Thailand. This study indicates that 28S rDNA and 1T52 regions are suitable molecular markers for genetic characterization and phylogenetic analysis of echinostomes.

Genetic differentiation of Artyfechinostomum malayanum and A. sufrartyfex (Trematoda: Echinostomatidae) based on internal transcribed spacer sequences.

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.

A cross-sectional study on the potential transmission of the carcinogenic liver fluke Opisthorchis viverrini and other fishborne zoonotic trematodes by aquaculture fish.

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.

Intron Regions as Genetic Markers for Population Genetic Investigations of Opisthorchis viverrini sensu lato and Clonorchis sinensis.

Opisthorchiasis and clonorchiasis are prevalent in Southeast and Far-East Asia, which are caused by the group 1 carcinogenic liver flukes Opisthorchis viverrini sensu lato and Clonorchis sinensis infection. There have been comprehensive investigations of systematics and genetic variation of these liver flukes. Previous studies have shown that O. viverrini is a species complex, called "O. viverrini sensu lato". More comprehensive investigations of molecular systematics and population genetics of each of the species that make up the species complex are required. Thus, other polymorphic genetic markers need to be developed. Therefore, this study aimed to characterize the intron regions of taurocyamine kinase gene (TK) to examine the genetic variation and population genetics of O. viverrini and C. sinensis collected from different geographical isolates and from a range of animal hosts. We screened seven intron regions embedded in TK. Of these, we selected an intron 5 of domain 1 (TkD1Int5) region to investigate the genetic variation and population genetics of theses liver flukes. The high nucleotide and haplotype diversity of TkD1Int5 was detected in O. viverrine. Heterozygosity with several insertion/deletion (indel) regions were detected in TkD1Int5 of the O. viverrine samples, whereas only an indel nucleotide was detected in one C. sinensis sample. Several O. viverrine samples contained three different haplotypes within a particular heterozygous sample. There were no genetic differences between C. sinensis isolated from various animal host. Heterozygous patterns specifically detected in humans was observed in C. sinensis. Thus, TkD1Int5 is a high polymorphic genetic marker, which could be an alternative marker for further population genetic investigations of these carcinogenic liver flukes and other related species from a wide geographical distribution and variety of animal hosts.