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Opisthorchiasis is endemic in parts of Southeast Asia, including the northern and northeastern regions of Thailand. In these regions, the transmission by fish intermediate hosts has received little attention. We investigated the intensity of Opisthorchis viverrini metacercariae (OV MC) in wild cyprinid fishes from five districts within the Nakhon Phanom Province, Northeast Thailand. Fishes were procured from local markets in five districts throughout three different seasons (hot, rainy and cold) between February 2018 and January 2019. The samples were identified, counted and weighed before metacercariae detection was performed via the artificial digestion method. A total of 2,149 freshwater fishes, representing 20 species were collected. The fish most commonly contaminated with OV MC were Anematichthys repasson and Hampala dispar. The intensity of OV MC in Nakhon Phanom was 0.23 OV MC/fish and varied among districts, ranging from 0.07 to 0.52. A low intensity of OV MC/fish (defined as=1 cyst) was found in all three different seasons in the Nakhon Phanom Province; hot season (0.55), cold season (0.22) and the rainy season (0.13). The intensity of OV MC/fish was moderate (defined as1 cyst) in the Renu Nakhon district (2.5) in the hot season. By fish species, H. dispar yielded the highest, with a moderate intensity of 2.1. In natural freshwater cyprinid fish in Nakhon Phanom, OV MC infection is endemic with intensity rates varying according to district, season and fish species.

The snail Bithynia siamensis goniomphalos acts as the first intermediate host of the human liver fluke, Opisthorchis viverrini, a major cause of cholangiocarcinoma in Northeast Thailand. In this study, we investigated host-parasite interactions in wet- and dry-season rice paddy plantation. The snails that aestivated through 4-month-dried period after rice harvest had average mortality rate of 36.45% and monthly increasing from 17.76% to 54.57%. Surprisingly, at depths greater than 5 cm experienced a higher mortality rate than upper level. Average mortality rate at the depth more than 5 cm, was 42.97%. In the initial month of aestivation, mortality rate of 0–5 and 5–10 cm depth was 9.13% and 26.39% then increase to 57.58% and 51.97%, respectively in the last 4th month. The average prevalence O. viverrini infection in snails during the study period was 0.44%. The highest prevalence of O. viverrini infection was found in the cool dry and wet season. The odds of O. viverrini infection in female snails and large snails were higher relative to male snails and medium-sized snails. The physicochemical factors involved in increased prevalence of O. viverrini infection in snail hosts were mean daily air temperature and average monthly rainfall. Short aestivation period made lower mortality in irrigation area than the previous report of non-irrigation area, that is, the aestivation is one of snail population control.

Cholangiocarcinoma (CCA) is a serious health challenge with low survival prognosis. The liver fluke, Opisthorchis viverrini, plays a role in the aetiology of CCA, through hepatobiliary abnormalities: liver mass (LM), bile duct dilation, and periductal fibrosis (PDF). A population-based CCA screening program, the Cholangiocarcinoma Screening and Care Program, operates in Northeast Thailand. Hepatobiliary abnormalities were identified through ultrasonography. A multivariate zero-inflated, Poisson regression model measured associations between hepatobiliary abnormalities and covariates including age, sex, distance to water resource, and history of O. viverrini infection. Geographic distribution was described using Bayesian spatial analysis methods. Hepatobiliary abnormality prevalence was 38.7%; highest in males aged > 60 years (39.8%). PDF was most prevalent (20.1% of males). The Standardized Morbidity Ratio (SMR) for hepatobiliary abnormalities was highest in the lower and upper parts of the Northeast region. Hepatobiliary abnormalities specifically associated with CCA were also more common in males and those aged over 60 years and distributed along the Chi, Mun, and Songkram Rivers. Our findings demonstrated a high risk of hepatobiliary disorders in Northeast Thailand, likely associated with infection caused by O. viverrini. Screening for CCA and improvement of healthcare facilities to provide better treatment for CCA patients should be prioritized in these high-risk areas.

At a time when Bhutan is on the verge of malaria elimination, the aim of this study was to identify malaria clusters at high geographical resolution and to determine its association with local environmental characteristics. Malaria cases from 2006–2014 were obtained from the Vector-borne Disease Control Program under the Ministry of Health, Bhutan. A Zero-Inflated Poisson multivariable regression model with a conditional autoregressive (CAR) prior structure was developed. Bayesian Markov chain Monte Carlo (MCMC) simulation with Gibbs sampling was used to estimate posterior parameters. A total of 2,062 Plasmodium falciparum and 2,284 Plasmodium vivax cases were reported during the study period. Both species of malaria showed seasonal peaks with decreasing trend. Gender and age were not associated with the transmission of either species of malaria. P. falciparum increased by 0.7% (95% CrI: 0.3%, 0.9%) for a one mm increase in rainfall, while climatic variables (temperature and rainfall) were not associated with P. vivax. Insecticide treated bed net use and residual indoor insecticide coverage were unaccounted for in this study. Hot spots and clusters of both species were isolated in the central southern part of Bhutan bordering India. There was significant residual spatial clustering after accounting for climate and demographic variables.

Infections due to soil-transmitted helminths (STHs), i.e. Ascaris lumbricoides, Trichuris trichiura, hookworms, and Strongyloides stercoralis, are widely distributed in tropical and subtropical areas in which approximately 1.5 billion people are infected. A clear understanding of the epidemiology and distribution of diseases is an important aid for control and prevention. The aim of our study was to identify the effects of environmental and climatic factors on distribution patterns of STHs and to develop a risk map for STH infections under current environmental and climate regimes in Thailand. Geographical information systems (GIS), remote sensing, and Maximum Entropy (MaxEnt) algorithm software were used to determine the significant factors and to create predictive risk maps for STH infections in Thailand. The disease data from Thailand covered the years from 1969 to 2014, while environmental and climatic data were compiled from the Worldclim database, MODIS satellite imagery, Soilgrids and ISCGM. The models predicted that STHs occur mainly in southern Thailand. Mean annual precipitation was the factor most affecting the current distribution of A. lumbricoides, T. trichiura, and S. stercoralis. Land cover class was the main predictor for distribution of S. stercoralis and important for hookworms. Altitude was the dominant factor affecting the distribution of hookworms, and mean temperature of the wettest quarter was significantly associated with A. lumbricoides distribution. A predicted distribution map of STHs to identify environmental risk factors in Thailand is presented. This work provides a model for use in STH monitoring and health planning not only in Thailand but also in other countries with similar disease conditions.

The Greater Mekong Subregion (GMS) is a trans-national region of the Mekong River basin in Southeast Asia comprising Cambodia, the People's Republic of China (specifically Yunnan Province and Guangxi Zhuang Autonomous Region), Lao People's Democratic Republic (Lao PDR), Myanmar, Thailand, and Vietnam home to more than 340 million people or almost 4.5% of the global population. Human liver fluke infections caused by Opisthorchis viverrini and Clonorchis sinensis have been major public health problems in this region for decades. Opisthorchiasis caused by O. viverrini is prevalent in Thailand, Lao PDR, Cambodia and central-southern Vietnam with more than 12 million people infected. Clonorchiasis caused by C. sinensis is endemic in northern Vietnam and Guangxi with estimated 3.5 -5 million infected. The infections can cause several liver and biliary diseases such as cholangitis, periductal fibrosis, gallstones, and cholangiocarcinoma (CCA), a fatal bile duct cancer. Key determinants of the geographical distribution differences of the two liver fluke species are snail species and geographic barriers. Main risk behaviour of the infections is the culture of eating raw fish "the raw attitude" of people in the GMS, especially the Tai/Dai/Thai/Laos ethnic groups, the major population in the GMS. Over the past 20 years, there is a big change in prevalence of the infections. Opisthorchiasis has long been endemic, particularly in northern and northeastern Thailand and Lao PDR with over 8-10 million cases estimated. However, after several rounds of national campaign against opisthorchiasis using integrated control approach in Thailand over the past three decades, the prevalence of O. viverrini infection has reduced from over 15% in 1996 to 2.2% in 2019. High prevalence of O. viverrini infection continues in Lao PDR and central Vietnam. Emerging high prevalence, up to a maximum of 47.5%, has been noted in Cambodia during the past 10 years possibly due to more studies being conducted rather than increasing prevalence. O. viverrini infection has now also been reported in Lower Myanmar in recent years. Clonorchiasis has been known in northern Vietnam and southern China for a long time. Several surveys have reported infections in Guangxi in the last 10 years, and until now liver fluke infected cases have not been reported in Yunnan. Overall, nowadays, there is a shift in high risk areas for GMS liver fluke infection from northeastern Thailand to Lao PDR, Cambodia, Vietnam, Myanmar, and Guangxi P.R. China. Urgent systematic disease mapping and integrated liver fluke control using One Health approaches should be implemented nationwide in GMS countries.

Parasitic infections caused by Opisthorchis viverrini and Strongyloides stercoralis remain a major public health threat in the Greater Mekong Sub-region. An understanding of climate and other environmental influences on the geographical distribution and emergence of parasitic diseases is a crucial step to guide targeted control and prevention programs. A parasitological survey was conducted from 2008 to 2013 and included 12,554 individuals (age between 20 and 60 years) from 142 villages in five districts in Khon Kaen Province, Thailand. Geographical information systems, remote sensing technologies and a Bayesian geostatistical framework were used to develop models for O. viverrini and S. stercoralis mono- and co-infections in areas where both parasites are known to co-occur. The results indicate that male sex, increased age, altitude, precipitation, and land surface temperature have influenced the infection rate and geographical distribution of mono- and co-infections of O. viverrini and S. stercoralis in this area. Males were 6.69 times (95% CrI: 5.26–8.58) more likely to have O. viverrini - S. stercoralis co-infection. We observed that O. viverrini and S. stercoralis mono-infections display distinct spatial pattern, while co-infection is predicted in the center and southeast of the study area. The observed spatial clustering of O. viverrini and S. stercoralis provides valuable information for the spatial targeting of prevention interventions in this area.

Helminthiases are common neglected tropical diseases in Thailand, thus regular surveillance is necessary for their control. During fiscal year 2019, the Thailand Ministry of Public Health carried out a cross-sectional nationwide survey in people of all age groups from the 12 Regional Health Offices in 76 provinces of Thailand. Multi-stage cluster random sampling design was employed to assess the prevalence of helminth infections and certain behavioural risk factors. A total of 16,187 stool samples and demographic data were obtained from the participants. Stool examination was done and parasite eggs/lavae were identified microscopically by experienced technicians. Positive stool samples for Opisthorchis viverrini, hookworms, or Ascaris lumbricoides were further quantified and expressed in eggs per gram feces (EPG). The results revealed an overall prevalence of helminthic infections of 9.79% with over 14 species identified. The highest prevalence was hookworms (4.47%) followed by O. viverrini (2.2%) with mean infection intensities of 222.7 EPG and 120.9, respectively. The majority of the infections were low intensity (97.4% for hookworms and 99.1% for O. viverrini). Similarly for A. lumbricoides, 93.9% of the positive cases were low infections. Two major helminthiases caused by hookworms and O. viverrini were highlighted in this report. While the liver fluke was highly endemic in Northeast Thailand, the hookworms were prevalent in the southmost region of the country. Association with demographic characteristics and risk behaviors of the two parasites were analyzed and presented in this study. Overall, this countrywide survey provides basic information of the current status of helminth infections in Thailand. Moreover, the data clearly indicates a dramatic reduction of O. viverrini prevalence likely due to extensive control activities under the national campaign against the liver fluke over the past five years.

Background Opisthorchis viverrini infection is highly prevalent in northeast Thailand. This liver fluke is classified as a carcinogen due to its causal links with cholangiocarcinoma (CCA) development. Although treatment with praziquantel (PZQ) effectively cures O. viverrini infection, the prevalence remains high due to the traditional consumption of raw fish. Therefore, re-infection is common in the endemic community, leading to severe hepato-biliary morbidities including the fatal CCA. In this study, we evaluate the association between the frequency of previous PZQ treatment and current O. viverrini infections among Thai adults living in the endemic area of northeast Thailand. Methods This study includes all participants who were screened for O. viverrini infection in the Cholangiocarcinoma Screening and Care Program (CASCAP), northeast Thailand. History of PZQ treatment was recorded using a health questionnaire. O. viverrini infections were diagnosed using urine antigen detection. Associations between PZQ and O. viverrini were determined by adjusted odds ratio (aOR) and 95% confidence interval (CI) using multiple logistic regression. Results Among participants, 27.7% had previously been treated once with PZQ, 8.2% twice, 2.8% three times, and 3.5% more than three times. Current O. viverrini prevalence was 17% (n = 524). Compared with participants who never used PZQ, the aOR for infection among those who used the drug once was 1.09 (95% CI: 0.88–1.37), twice was 1.19 (95% CI: 0.85–1.68), three times was 1.28 (95% CI: 0.74–2.21), and more than three times was 1.86 (95% CI: 1.18–2.93; P = 0.007). Conclusions The population with a frequent history of PZQ use and still continued raw fish consumption showed high levels of repeated reinfection with O. viverrini. They were infected, treated and re-infected repeatedly. These findings suggest that certain participants continue raw fish consumption even after previous infection. This is a particular problem in highly endemic areas for O. viverrini and increases the risk of CCA.

The Bithynia snails of B. funiculata, B. siamensis siamensis and B. siamensis goniomphalos, are first intermediate hosts of Opisthorchis viverrini. The success of parasitic infection in snails is related to the host species and efficiency of their internal defense system. Parasitic infections in snails exhibited significant variations in the number of hemocytes in hemolymph. This study investigated hemocyte counts in Bithynia snails for various durations of O. viverrini infection. The three species/subspecies snails were infected with O. viverrini and hemocytes were counted at post-exposed periods of 2, 4, 6, 12, 24, 48, 96 h, and 1, 4 and 8 wk to evaluate the hemocyte response to the infection. Three major hemocyte morphotypes, namely, agranulocytes, semigranulocytes and granulocytes, were observed. After infection, the differential hemocyte morphotype counts in all species/subspecies significantly increased in granulocytes at 2 h (p < 0.05) and decreased in semigranulocytes and agranulocytes at 2 to 4 h (p < 0.05). The total hemocyte counts were significant increased at 2 h after exposure in B. s. siamensis and B. funiculata (p < 0.05). Additionally, the number of hemocytes was reduced after exposure at 4, 6 and 12 h in B. s. goniomphalos and at 12, 24 and 48 h in B. funiculata (p < 0.05). From 96 h to 8 wk, the number of hemocytes was reestablished in the hemolymph, indicating that defensive cells in the host species have different mechanisms for their susceptibility or resistance to parasitic infections. Further studies on molecular functions will be done.

Table S1. Crude and adjusted odds ratio between O. viverrini infection and periductal fibrosis and 95% confidence interval adjusted for all other factors using multiple logistic regression. (DOCX 16 kb)

Multilingual abstracts in the five official working languages of the United Nations. (PDF 263 kb)

This article reviews the past and present scientific reports regarding Bithynia spp. focusing on the biology, ecology and life cycle of Bithynia snails and their responses to Opisthorchis viverrini infection. Moreover, new data regarding comparative molecular genomics and proteomic approaches have recently revealed novel molecular components involved in the immune defence responses from Bithynia spp., providing additional perspectives for future studies. Studies on the specific interaction between Bithynia snails and their trematodes will contribute to further understanding the snail-parasite relationship with regards to epidemiology and control of Opisthorchiasis and broaden the scope on comparative immunology of gastropod snails.

The snail Bithynia siamensis goniomphalos acts as first intermediate host of the liver fluke, Opisthorchis viverrini, which causes opisthorchiasis in humans. In this study, we used a geographic information system (GIS), remote sensing (RS) and software using the maximum entropy (MaxEnt) algorithm to predict the distribution of B. s. goniomphalos in Thailand on the basis of environmental and climatic factors. The MaxEnt model for B. s. goniomphalos was excellent, with average test AUC values of 0.89. The predicted distribution of B. s. goniomphalos was affected by altitude, land cover, normalized difference vegetation index (NDVI), precipitation in the driest month (BIO 14), land surface temperature (LST) and soil pH. The areas suitable for B. s. goniomphalos were mostly in Northeast Thailand and some northern parts of the country. The presence of B. s. goniomphalos decreases with increasing altitude and increasing NDVI value. Bithynia s. goniomphalos is most likely to occur in paddy fields and cropland. Opisthorchiasis prevalence was directly proportional to the likelihood of snail occurrence as predicted by the model. This is the first time this ecological niche model has been used to predict Bithynia snail distribution and hence to provide a basis for future work of opisthorchiasis prevention in opisthorchiasis-endemic countries.

Opisthorchiasis in the Lower Mekong Subregion is a parasitic disease caused by the liver fluke Opisthorchis viverrini. This parasite has a well-documented distribution in Thailand, Lao PDR, Cambodia, Myanmar and Southern Vietnam. In this chapter, we describe the current knowledge of the epidemiology of O. viverrini infection, highlighting advances in control efforts made in the last four decades in Thailand and identifying ongoing gaps in our epidemiological knowledge which need to be filled to support efforts to permanently overcome the heavy morbidity and mortality burden caused by these parasites within their endemic regions.

Opisthorchis viverrini is a zoonotic liver fluke that causes opisthorchiasis among humans in endemic areas, such as northeastern Thailand. The objective of this study was to determine the prevalence and risk factors for O. viverrini infection among cats and dogs in six districts surrounding Ubolratana Dam. Fecal samples of 1,018 dogs and 249 cats were collected between 2008 and 2013 to examine for O. viverrini infection using a modified formalin-ether concentration technique. The prevalence of O. viverrini infection among cats (77 of 249, 30.92%) was higher than dogs (2 of 1,018, 0.20%). Age and the eating habits of cats were associated with O. viverrini infection. Cats aged3 years were more likely than those aged1 year [Odds ratio (OR)=2.96; 95% confidence interval (95% CI): 1.01- 8.35; p = 0.044] of being infected. Cats that consumed raw fish were significantly more likely to be infected than those that consumed other foods (OR=1.82, 95% CI: 1.05-3.16; p = 0.032). Cats had a higher prevalence of O. viverrini infection than dogs and may play an important role in the transmission and maintenance of this disease in the study area.

Opisthorchiasis in the Lower Mekong Subregion is a parasitic disease caused by the liver fluke Opisthorchis viverrini. This parasite has a well-documented distribution in Thailand, Lao PDR, Cambodia, Myanmar and Southern Vietnam. In this chapter, we describe the current knowledge of the epidemiology of O. viverrini infection, highlighting advances in control efforts made in the last four decades in Thailand and identifying ongoing gaps in our epidemiological knowledge which need to be filled to support efforts to permanently overcome the heavy morbidity and mortality burden caused by these parasites within their endemic regions.

Opisthorchis viverrini infection and cholangiocarcinoma are serious problems in South East Asia. This study aimed to find the prevalence of opisthorchiasis in various hosts in Udon Thani Province. Total fecal samples were collected from 14,766 participants. The epidemiological data collected and analysed included prevalence and intensity of infection. Odds ratios (OR) were calculated to determine the associations between cross sectional data and to predict possible risk factors. The prevalence of O. viverrini infection in Udon Thani Province averaged 15.3% (eggs per gram (epg.) = 48.9 and range; 12-1,320), with differences between villages (range; 3.8%-79.8%). An age-dependence for infection was observed to increase from ages 25 to 50 years and then decrease for older participants. A univariate analysis identified risk parameters including age (p = 0.040; OR = 3.9 (95% CI = 1.2-7.5)), education (p

Opisthorchis viverrini, a carcinogenic fish borne fluke, requires freshwater snails and fish as intermediate hosts. Opisthorchiasis is endemic in parts of Southeast Asia, including Central and South Vietnam. In this region the transmission by intermediate hosts has received little attention. Therefore, freshwater snails and wild fish from Bau My Tho, an opisthorchiasis endemic area in Binh Dinh Province were collected for examination of O. viverrini cercariae and metacercariae, respectively. A total of 12,000 snails belonging to six families, of which 1616 Bithynia snails representing Bithynia siamensis goniomphalos and Bithynia funiculata; as well as 754 fish representing 12 species were examined. Shedding of O. viverrini cercariae was observed only in B. s. goniomphalos and B. funiculata, at infection rates of 0.86% and 0.14%, respectively. O. viverrini infection in Bithynia spp. was significantly associated with the habitat but not with the species and the shell size of Bithynia spp. O. viverrini metacercariae were found in 10 fish species representing both Cyprinidae and non-Cyprinidae families. The prevalence of O. viverrini infection in fish was significantly associated with species. Carassius auratus, a fish species commonly eaten raw, Rasbora aurotaenia and Puntius brevis had the highest prevalence of 74.0%, 55.8% and 31.6%, respectively. Sharing of the same snail and fish intermediate host species was found for O. viverrini and a O. viverrini duck-genotype that are sympatric in the study region. This study is the first to report on the intermediate host species of O. viverrini in Central Vietnam and indicates a high risk of acquiring opistorchiasis when eating raw fish dishes.

Opisthorchis viverrini is an important public health problem, a major cause of cholangiocarcinoma in the Greater Mekong subregion including Lao PDR, Cambodia, Vietnam and Thailand. Humans acquire the infection by consumption of raw, fermented or partially cooked freshwater cyprinid fish containing infective metacercariae. This study examined the effect of cercarial infection dosage (25-200 cercariae), age (1-60 day) and size (1-24mm) of Barbonymus gonionotus fish on infection success of O. viverrini cercariae. Additionally, the site of cyst formation of O. viverrini cercariae and subsequent development to the metacercariae was examined. The result showed that cercarial infection dose (F4,95=8.52, P

Snails and fishes from ponds in an area endemic for human liver fluke (Opisthorchis viverrini) in Khon Kaen Province, northeast Thailand, were surveyed for the presence of zoonotic helminth parasites during May-November 2007. The surveys were performed to obtain information on the potential for transmission of these parasites to fish cultivated for human consumption. Sixteen species/subspecies of snails from 7 families and 14 species of fish from 6 families were collected for parasitological examination. Four species of snails were found to be infected with a range of trematode species. Bithynia siamensis goniomphalos, the first intermediate host of O. viverrini, was the most commonly infected with trematodes, with a prevalence of infection of 1.69-25.00% in 10 ponds. O. viverrini was found in 1 of 1065 (0.09%) B. siamensis goniomphalos individuals examined in one pond. Metacercariae of Echinostoma revolutum, another agent of human infection, was found in the snail Idiopoma umbilicata in one pond. The fish Luciosoma bleekeri was infected with the trematode Centrocestus caninus in one pond. An environmental management trial was conducted in selected ponds in the endemic area to measure the effects of exclusion of snails and thus trematode parasites. After modifications to the ponds and boundaries, including the exclusion of snails, the ponds were stocked with Barbonymus gonionotus fingerlings. Batches of the fingerlings from the same nursery farm were determined to be free of parasites prior to their introduction to the ponds. The experimental ponds were examined monthly for the presence of snails. Fish were also sampled monthly for parasites. After 8 months, fish grown in environmentally managed ponds were larger than those of the control pond. However, fish in all ponds were found to be infected with the trematode Haplorchis taichui. The source of this zoonotic species is uncertain and will require further investigation and potential environmental management, both at farms and at hatcheries. (C) 2014 Elsevier B.V. All rights reserved.

ObjectivesTo assess associations between periductal fibrosis (PDF) and bile duct dilatation (BDD) in ultrasonography (US) screening of population at risk of cholangiocarcinoma (CCA) due to residence in an endemic area for Opisthorchis viverrini. CCA survival rates are low, and early identification of risk factors is essential. BDD is one symptom that can identify patients at risk of CCA. Detection of PDF by US can also identify at-risk patients, at an earlier stage of CCA development. Identification of association between PDF and BDD will inform screening practices for CCA risk, by increasing the viability of PDF screening for CCA risk.SettingNine tertiary care hospitals in Northeast Thailand.DesignCross-sectional study.ParticipantsStudy subjects in the Cholangiocarcinoma Screening and Care Program (CASCAP) in Northeast Thailand. CASCAP inclusion criteria are all residents of Northeast Thailand aged ≥40 years. Participants are recruited through CCA screening centres and through primary healthcare units. So far, 394 026 have been enrolled.MethodsPDF and BDD were identified through US. PDF was categorised into three groups, PDF1, 2 and 3, depending on their high echo locality in the peripheral, segmental and main bile duct, respectively. Associations between PDF and BDD were determined by adjusted OR and 95% CI using multiple logistic regression.ResultsBDD was found in 6.6% of PDF3, 1.7% of PDF2 and 1.4% of PDF1 cases. Among PDF cases, especially in PDF3, BDD was found in men more than in women (8.9% and 4.6%, respectively). Compared with non-PDF, the association between PDF3 and BDD was highly significant (adjusted OR=5.74, 95% CI 4.57 to 7.21, pConclusionsOur findings reveal that there is a relationship between PDF and BDD, which is associated with CCA. Therefore, PDF can also be an indicator for suspected CCA diagnosis through US.

Bithynia siamensis goniomphalos is a freshwater snail that serves as the first intermediate host of the human liver fluke Opisthorchis viverrini. This parasite is a major public health problem in different countries throughout the Greater Mekong sub-region (Thailand, southern Vietnam, Lao PDR and Cambodia). Chronic O. viverrini infection also results in a gradual increase of fibrotic tissues in the biliary tract that are associated with hepatobiliary diseases and contribute to cholangiocarcinoma (a fatal type of bile duct cancer). Infectivity of the parasite in the snail host is strongly correlated with destruction of helminths by the snail’s innate immune system, composed of cellular (hemocyte) and humoral (plasma) defense factors. To better understand this important host-parasite interface we applied sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS) to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini and compare them to non-infected snails (control group). A total of 362 and 242 proteins were identified in the hemocytes and plasma, respectively. Of these, 145 and 117 proteins exhibited significant differences in expression upon fluke infection in hemocytes and plasma, respectively. Among the proteins with significantly different expression patterns, we found proteins related to immune response (up-regulated in both hemocyte and plasma of infected snails) and proteins belonging to the structural and motor group (mostly down-regulated in hemocytes but up-regulated in plasma of infected snails). The proteins identified and quantified in this work will provide important information for the understanding of the factors involved in snail defense against O. viverrini and might facilitate the development of new strategies to control O. viverrini infection in endemic areas., Author Summary The control of snails involved in transmission of trematodes plays a key role in the fight against these parasitic diseases. While some snails have been studied thoroughly, only few genomic and proteomic studies have focused on Bithynia siamensis goniomphalos, the snail intermediate host for Opisthorchis viverrini, and none of them separately analyzed the cellular and humoral arms of the snail’s immune system. Here, we applied a label-free proteomic approach to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini. We identified several immune response proteins that could play critical roles at the snail-trematode interface, generating important new information on the molecular interactions between flukes and their intermediate snail hosts. This work will contribute towards generating a body of knowledge that will ultimately result in the control of O. viverrini transmission in endemic areas.

Global climate change is now regarded as imposing a significant threat of enhancing transmission of parasitic diseases. Maximum entropy species distribution modeling (MaxEnt) was used to explore how projected climate change could affect the potential distribution of the carcinogenic liver fluke, Opisthorchis viverrini, in Thailand. A range of climate variables was used: the Hadley Global Environment Model 2-Earth System (HadGEM2-ES) climate change model and also the IPCC scenarios A2a for 2050 and 2070. Occurrence data from surveys conducted in 2009 and 2014 were obtained from the Department of Disease Control, Ministry of Public Health, Thailand. The MaxEnt model performed better than random for O. viverrini with training AUC values greater than 0.8 under current and future climatic conditions. The current distribution of O. viverrini is significantly affected by precipitation and minimum temperature. According to current conditions, parts of Thailand climatically suitable for O. viverrini are mostly in the northeast and north, but the parasite is largely absent from southern Thailand. Under future climate change scenarios, the distribution of O. viverrini in 2050 should be significantly affected by precipitation, maximum temperature, and mean temperature of the wettest quarter, whereas in 2070, significant factors are likely to be precipitation during the coldest quarter, maximum, and minimum temperatures. Maps of predicted future distribution revealed a drastic decrease in presence of O. viverrini in the northeast region. The information gained from this study should be a useful reference for implementing long-term prevention and control strategies for O. viverrini in Thailand.

Objective To explore larvicidal effects of anthelmintic drugs on Opisthorchis viverrini (O. viverrini) for alternative approach to interrupting its cycle for developing a field-based control program. Methods The larvicidal activities of albendazole (Al), artesunate (Ar), praziquantel (Pzq) and miltefosine (Mf) on O. viverrini cercariae and mature metacercariae were investigated. Lethal concentrations (LC50 and LC95) of these drugs were determined. Mature metacercariae previously exposed to various concentrations of the drugs were administered to hamsters. Worms were harvested 30 d post infection and worm recovery rates calculated. Al, Ar, Pzq and Mf produced morphological degeneration and induced shedding tails of cercariae after 24 h exposure. Results The LC50 and LC95 of Al, Ar, Pzq and Mf on cercariae were 0.720 and 1.139, 0.350 and 0.861, 0.017 and 0.693, and 0.530 and 1.134 ppm, respectively. LC50 and LC95 of Ar on mature metacercariae were 303.643 and 446.237 ppm and of Mf were 289.711 and 631.781 ppm, respectively but no lethal effect in Pzq- and Al-treated groups (up to 1 ppt). No worms were found in hamsters administered Pzq-treated metacercariae. The adult worms from Al-treated metacercariae were significantly bigger in size compared to the control group (P Conclusions The larvicidal effects of these drugs were high efficacy to O. viverrini cercariae but lesser efficacy to metacercariae. It should be further studied with the eventual aim of developing a field-based control program.

Opisthorchis viverrini (Ov) is a fish-borne parasite endemic in parts of Lao PDR, Cambodia, southern Vietnam and Northeast Thailand (Isaan) where an estimated 10 million people are infected. Human Ov infection, associated with hepatobiliary complications, including cholangiocarcinoma (CCA), occurs when infected fish are consumed raw or undercooked, a longstanding cultural tradition in the region. This mixed- methods descriptive study was carried out in Isaan villages around Lawa Lake, Khon Kaen Province, known for their Ov endemicity. Focus group discussions (FGDs) and in depth interviews (IDIs) were used to explore socio-cultural determinants underlying raw fish consumption practices, and global positioning system (GPS) devices to map local fish distribution networks. Qualitative data affirmed major socio-cultural and dietary lifestyle transitions occurring consequent on recent decades of modernization policies and practices, but also the persistence of Isaan traditional raw-fish eating practices and incorrect beliefs about infection risk avoidance. Fish traders/middlemen purchase most of the catch at the lakeshore and play the dominant role in district market fish distribution networks, at least for the larger and less likely infected, fish species. The lower economic value of the small potentially-infected cyprinid fish means local fishermen typically distribute them free, or sell cheaply, to family and friends, effectively concentrating infection risk in already highly Ov infected villages. Our study confirmed the persistence of traditional Isaan raw-fish meal practices, despite major ongoing socio-cultural lifestyle transitions and decades of Ov infection health education programs. We contend that diffuse socio-cultural drivers underpin this practice, including its role as a valued cultural identity marker. A "fish economics" factor was also evident in the concentration of more likely infected fish back into local villages due to their low economic value at district market level. The complexity of factors supporting "risky" fish-eating traditions in Isaan underscores the importance of integrated liver fluke infection control strategies to draw on transdisciplinary knowledge beyond biomedicine and also embrace participatory protocols for engaging communities in developing, implementing and evaluating interventions.

Effects of surface salt on distribution of snails in family Viviparidae in Khon Kaen province, northeast Thailand were analyzed the association model by using geographic information system. Mollusks were collected in twenty localities base on various levels of surface salt, 16 – 20 stations/reservoir. Three species of viviparid snails were found in 9 out of 20 reservoirs, i.e. Filopaludina (Siamopaludina) martensi martensi, F. (Filopaludina) sumatrensis speciosa and Idiopoma umbilicata. Nine families (11 species) of sympatric mollusks shared the same habitat with viviparid snails. Water quality in the viviparid habitat revealed that, pH: 6.79 - 8.01, temperature: 23.06- 27.33 oC, turbidity: 3.26 – 163.00 NTU, conductivity: 0.42 - 10.25 mS/cm, salinity: 0.20 - 5.69 ppk and dissolved oxygen (DO): 0.43 – 6.01 mg/L. Dominant planktons in food content from digestive tracts of F. (S.) martensi martensi consisted of diatoms and they were the same type to Bithynia siamensis goniomphalos, first intermediate host of human liver fluke, Opisthorchis viverrini. That is viviparid snails may play important role in food competition with B. siamensis goniomphalos. The number of B. siamensis goniomphalos was negative correlation to the number of F. (S.) martensi martensi (r = - 0.182, p 0 - 50% of surface areas revealed that the index of 0.079, 0.027, 0.001 and 0, respectively. The geographic information system analysis model was assessed and approved by mollusk survey. ผลของเกลอผวดนตอการกระจายและความหนาแนนของหอยในวงViviparidaeในจงหวดขอนแกน ประเทศไทย โดยวเคราะหขอมลดวยระบบสารสนเทศภมศาสตร ศกษาโดยเกบตวอยางหอยในแหลงนำทมสภาพเกลอผวดนทระดบแตกตางกน ทงหมด 20 แหลงนำ ทำการสมเกบตวอยางในแตละแหลงนำ 16-20 สถาน พบหอยในวงศ Viviparidae 3 ชนดคอFilopaludina (Siamopaludina) martensi martensi, F. (Filopaludina)sumatrensis speciosa และ Idiopoma umbilicataในแหลงนำท9 แหลงจากจำนวนททำการเกบตวอยาง20 แหลงพบหอยรวมนเวศ 9 วงศ (จำนวน 11 ชนด)ลกษณะทางภายภาพนำทพบหอยวงศ Viviparidae ม pH 6.79 -8.01 อณหภม 23.06- 27.33 องศา,คาความขน 3.26 - 163.00 NTU,คาการนำไฟฟา 0.42 - 10.25 mS/cm,คาความเคม 0.20 - 5.69 คาการละลายของออกซเจน (DO) 0.43 - 6.01 mg/L. ในระบบทางเดนอาหารของหอย F. (S.) martensi martensi ทเปนประชากรสวนใหญในการศกษาครงน พบชนดของแพลงกตอนเหมอนกนกบBithynia siamensis goniomphalos ซงเปนโฮสตกลางของพยาธใบไมตบของคนชนดOpisthorchis viverrini จงนาจะเปนสตวทแกงแยงอาหารกน ซงพบวาจำนวนหอย B. siamensis goniomphalos มความสมพนธเชงลบกบหอย martensi martensi (r = - 0.182, p 0 - 50% ของพนทผวดนโดยม คาดชนเทากบ .079,0.027, 0.001และ 0 ตมลำดบ คาดชนไดทำการประเมนความถกตองโดยการสำรวจในพนท ทไดทำการวเคราะหแลวไดคาทถกตอง

Opisthorchis viverrini is restricted to and requires for its aquatic life cycle only Bithynia snail as first intermediate host but many species of cyprinid fish as second intermediate hosts. A survey in Thailand of trematode infection in freshwater snails of the family Bithyniidae carried out during October 2008 - July 2009 found a total of 5,492 snails, classified into ten species distributed in various geographic areas. Bithyniafuniculata and Gabbia pygmaea were localized to the north, B. s. goniomphalos, Wattebledia siamensis and W. crosseana to northeast and B. s. siamensis, Hydrobioides nassa and G. wykoffi to central region. W. baschi and G. erawanensis was found only in the south and Erawan waterfall, Kanchanaburi Province, respectively. Trematode infection rate was 3.15%. Cercariae were identified as belonging to six types, namely, amartae , monostome, mutabile, O. viverrini, virgulate, and unknown. The prevalence of cercarial infection in B. s. goniomphalos of amartae, mutabile, O. viverrini, virgulate, and unknown type cercaria was 0.55%, 0.74%, 1.07%, 2.87%, and 0.37%, respectively, and in B. s. siamensis monostome (1.10%) and virgulate (0.55%). Only virgulate cercariae were shed from W. crosseana (3.85%) and W. siamensis (5.19%). Cercariae of the unknown type were found in G. wykoffi (1.69%). No infection of O. viverrini cercariae was detected in the other species.

Opisthorchiasis caused by Opisthorchis viverrini infection, is a serious public health problem in Lao PDR, Cambodia, Vietnam and Thailand. Cyprinid fish are required for the transmission of O. viverrini metacercariae to fish eating-mammal hosts including humans. The transmission of infective metacerariae is an important event, potentially resulting in the development of cholangiocarcinoma in cases of chronic opisthorchiasis. For a better understanding of the development of O. viverrini metacercariae in the second intermediate host, Barbonymus gonionotus (Cyprinidae), the immune response of infected B. gonionotus through fibrous tissue formation and superoxide dismutase (SOD) activity were investigated. The immunosuppressant (prednisolone) was administered to B. gonionotus infected with O. viverrini cercariae to study infectivity and development of the metacercariae. Additionally, the effects of prednisolone were studied by the investigation of fibrous tissue formation around the metacercariae, as well as SOD activity. In our study, prednisolone administered prior to infection in B. gonionotus had higher metacercarial recovery when compared to the non-administered control group. On the other hand, the administration of prednisolone after the O. viverrini infection allowed metacercariae to develop to the infective stage. Energy consumption from encystation to the infective stage required 443.9-463.7degree-days. The results show that immunosuppression decreases fibrous tissue and SOD activity in infected fish, and also affects the ability of O. viverrini metacercariae to develop to the infective stage. The immune response to metacercarial development of O. viverrini in cyprinid fish should be further studied to gain a greater understanding of this process, and hence, a better understanding of the mechanism(s) involved in metacercarial wall formation.

Opisthorchiasis in northeastern Thailand is an important etiology of cholangiocarcinoma. To form the infectious stage, free swimming cercariae penetrate cyprinid fish, shed their tails, and then secret a cystic substance to cover their larval stage to form metacercariae in the fish body. We determined the location of the cystogenous glands in Opisthorchis viverrini cercariae. The cercariae and metacercariae were obtained from the naturally infected snail host, Bithynia siamensis goniomphalos and from cyprinid fish, respectively. The cyst walls of the metacercariae were separated and used to immunize inbred male BALB/c mice to obtain cyst wall antibodies. The general characteristics of the O. viverrini cercariae and metacercariae were studied by hematoxylin and eosin (HE) staining of sections. The location and ultrastructure of the cystogenous glands of cercariae were studied by immunoperoxidase, immunofluorescence and transmission electron microscopy. The structures and organelles of cercariae and metacercariae could be identified, but the cystogenous glands could not be detected in HE sections. The immunoperoxidase and immunofluorescence sections revealed positive reactions for cystogenous glands predominanted in the lateral part of the cercariae and were clearly seen in the cyst wall of the metacercariae. The ultrastructure of the cystogenous glands contained semitranslucent electron dense oval shaped granules. If interference occurs during the formation of the cysts by fish immune response, the metacercariae may not develop to maturity. It may be easily digested or degraded by human stomach acid and pepsin. This may be an efficient method for control of O. viverrini infection which requires further detailed study.

Determining of the success of a parasite's infectiveness in its snail host clearly depends on environmental conditions. Temperature, one of the most influential factors impinging on metabolism of cold-blooded animals, is believed to be an important factor in parasitic infection in snails. In order to elucidate the influence of temperature, sex and size of snails on infectivity of Opisthorchis viverrini to its first intermediate host, Bithynia siamensis goniomphalos, 960 snails were divided into 2 groups by sex. Each group was subdivided by their size into small and medium sub-groups. Each snail was fed with embryonated uterine-eggs of O. viverrini at different temperatures (16-37°C, 3°C intervals). Dissections were carried out 1, 7, 14, 28 and 56 days thereafter and detection of O. viverrini infection was undertaken by PCR using specific primers. Infection was strongly temperature-dependent, as temperature increases of 1°C resulted in increased odds of infection 5.4% (P

Eggs of most species digenean flukes hatch in the external environment to liberate larvae that seek and penetrate a snail intermediate host. Those of the human liver flukes, Opisthorchis viverrini, hatch within the gastrointestinal canal of their snail hosts. While adult parasites are primarily responsible for the pathology in cases of human opisthorchiasis, their eggs also contribute by inducing granulomata and in serving as nidi for gallstone formation. In view of the peculiar biology of O. viverrini eggs and their contribution to pathology, we investigated embryogenesis in this species by light and transmission electron microscopy. Egg development was traced from earliest stages of coalescence in the ootype until full embryonation in the distal region of the uterus. Fully mature eggs were generally impermeable to resin and could not be examined by conventional electron microscopy methods. However, the use of high-pressure freezing and freeze-substitution fixation of previously fixed eggs enabled the internal structure of mature eggs, particularly the subshell envelopes, to be elucidated. Fertilization occurs in the ootype, and the large zygote is seen therein with a single spermatozoon wrapped around its plasma membrane. As the zygote begins to divide, the spent vitellocytes are pushed to the periphery of the eggs, where they progressively degrade. The early eggshell is formed in the ootype by coalescing eggshell precursor material released by approximately six vitelline cells. The early eggs have a thinner eggshell and are larger than, but lack the characteristic shape of, mature eggs. Characteristic shell ornamentation, the "muskmelon" appearance of eggs, appears after eggshell polymerization in the ootype. Pores are not present in the shell of O. viverrini eggs. The inner and outer envelopes are poorly formed in this species, with the outer envelope evident beneath the eggshell at the opercular pole of the mature egg. The miracidium has a conical anterior end that lacks the distinctive lamellar appearance of the terebratorium of other digeneans, such as the schistosomes. The miracidium is richly glandular, containing an apical gland in the anterior end, large cephalic gland, and posterior secretory glands. Each gland contains a secretory product with different structure. The paucity of vitelline cells associating with eggs, the reduced size of eggs, and reduced complexity of the extraembryonic envelopes are interpreted as adaptations to the peculiar hatching biology of the miracidia.

The molluscicidal effects of Bayluscide (niclosamide) were investigated on Bithynia siamensis goniomphalos, the first intermediate host of human liver fluke, Opisthorchis viverrini . Lethal concentrations of 50% (LC 50 ) and 95% (LC 95 ) against young and adult males were 0.38 and 0.80, 0.42 and 0.86 ppm, respectively. The LC 50 and LC 95 against young and adult females were 0.42 and 0.86, 0.46 and 0.97 ppm, respectively. No significant differences in mortality rate between sexes or snail size (p > 0.05) was detected. Bayluscide-related tissue damage in B. siamensis goniomphalos included detachment of cilia of the epithelial layer of the digestive tract and decreased number of calcium cells. In tests of lethal concentrations of Bayluscide on non-target animals, no lethal effect was observed on Filopaludina martensi martensi (Viviparous snail) but high mortality rates were recorded in Puntius gonionotus fingerling, Ricefish ( Oryzias mekongensis ) and shrimp ( Macrobrachium lanchesteri ), but lower in guppy fish ( Poecilia reticulata ) after 24 h exposure. For field trials, sufficient Bayluscide was sprayed in 3 roadside ditches to result in final concentrations of 5, 10 or 20 ppm, with mortality rates on B. siamensis goniomphalos of 10.94, 20.00 and 31.25%, respectively. Non-target snails died in small numbers but no effect was observed in other aquatic vertebrate animals. Field trials of Bayluscide on B. siamensis goniomphalos revealed low mortality rates, suggesting the need for application methods of higher efficacy or that Bayluscide is not suitable for application to operculate snails or snails which are able to escape by burying in mud.

Opisthorchis viverrini infection is associated with human cholangiocarcinoma and northeast Thailand has the highest incidence of this disease in the world. Bithynia siamensis goniomphalos is the major freshwater snail intermediate host of O. viverrini in this area and an analysis based on geographical information systems was used to determine the effect of variation in soil surface salt on the density and distribution of this snail. A malacological survey was carried out in 56 water bodies in the Khorat basin, northeast Thailand at locations with various soil surface salt levels. Mollusk samples were collected from 10 ecologically representative water body sites with 10-20 sampling stations in each. The shoreline of clear, shallow water bodies was found to be the preferred B. s. goniomphalos habitat. The snails were exclusively found in water with salinity levels ranging between 0.05 and 22.11 parts per thousand (ppt), which supports the notion that B. s. goniomphalos prefers water with some saline content over pure, freshwater. The highest snail population densities were in rice fields, ponds, road-side ditches and canals within a water salinity range of 2.5-5.0 ppt. However, the presence of B. s. goniomphalos was negatively correlated with water salinity (P ≤0.05), both with regard to density and distribution. The areas with the highest density of B. s. goniomphalos were those with less than 1% soil surface salt (potential index = 0.314), while the lowest densities were found in areas exceeding 50% soil surface salt (potential index = 0.015).