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Opisthorchis viverrini and other food-borne trematode infections are major health problems in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. Recently, loop-mediated isothermal amplification (LAMP) has been widely used for detection and identification of trematode for its simple method that is useful in low-resource or field settings. We have reported ITS1-LAMP assay to detect O. viverrini infection from human feces. The sensitivity and specificity of the test was 100% and 61.5%. The sensitivity of the test appeared to be higher than microscopic egg examination; however non-specific amplification from other parasites could not be ruled out. We therefore targeted microsatellites of O. viverrini that is a species specific sequence. By using hydroxyl naphthol blue (HNB)-LAMP, O. viverrini microsatellite 6 (OVMS6) could specifically amplify DNA from O. viverrini genome, but not other parasites such as C. sinensis , Opisthorchis felineus , Centrocestus caninus , Haplorchis taichui , Fasciola gigantica and Haplorchoodes sp. The detection limit of the test is 1 ng genomic DNA, which was 1000 times lower than the ITS1-LAMP, but targeting microstellites showed more specific detection of O. viverrini . In addition, the colorimetric LAMP assay was simple and effective; this makes it potentially applicable for point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People''s Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10−3 ng DNA/μL. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis. [Copyright &y& Elsevier]

Abstract: Opisthorchis viverrini, a carcinogenic liver fluke, requires Bithynia snails as the first intermediate host, which release cercariae after ingesting fluke eggs from contaminated water. Fecal bacterial contamination and O. viverrini-infected Bithynia snails were investigated in samples collected from natural water reservoirs in Ban Phai, Chonnabot and Muang Districts (Ban Lerngpeuy) in Khon Kaen Province, northeast Thailand, where there is a high incidence of cholangiocarcinoma. Water was sampled and examined six times (February, April, June, August, October and December 2006). The most probable number (MPN) index and coliform counts were utilized to evaluate fecal contamination; the cercarial shedding method was conducted for detecting infected snails. The data revealed that all water samples had a high MPN index number, and fecal coliform levels above the WHO standard. This indicated that water in these reservoirs was contaminated with feces or manure constituents. Water sampling from Ban Lerngpeuy showed full-scale bacterial contamination (>1609 MPN index) throughout the year. This finding was correlated with the highest prevalence of O. viverrini-infected snails, which were found nearly all year round in this area. Slightly lower fecal contamination levels were detected in water samples from Chonnabot and Ban Phai, with high MPN index numbers and coliform counts from April to October. This corresponded with the higher recovery of infected snails in June and August, but with relatively lower prevalence than those found in Ban Lerngpeuy. Among the sampling sites, the people in Ban Lerngpeuy live nearer to the reservoir than do those in Ban Phai and Chonnabot. These results indicate that fecal bacterial contamination in natural water reservoirs is an important indicator of seasonal transmission of O. viverrini eggs to snail intermediate hosts. Sanitation improvement is essential and future investigations on the sources of contamination are needed. [Copyright &y& Elsevier]

Abstract: Opisthorchis viverrini requires Bithynia snails as the first intermediate host and cyprinid fish as the second intermediate host. Very low natural infection rates have been reported in Bithynia snails, but very high rates have been found in cyprinid fish in the same endemic region. This study investigated the effect of light intensity, the most important stimulus, on the quantity of O. viverrini cercariae shed from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos snails. Snails were evaluated for cercariae output every hour after exposure to various light intensities for a total period of 7h. The same infected snail was tested under different intensities of light: in the dark, and at 1000, 3000 and 5000lx. The data showed that under exposure to 1000 and 3000lx of light, the average percentage and number of cercariae released were higher than that exposed to 5000lx during the first 2h of the experiment. In contrast, under higher illumination (5000lx) a longer time (6h) was required to stimulate the peak emergence of cercariae. Darkness was not able to induce O. viverrini cercariae emergence. Among the three intensities of light, exposure at 1000lx induced the highest average number of released cercariae per snail and the highest percentage of cercarial emergence within the first 2h (125, 54.86%), followed by exposure at 3000lx (69, 25.58%) and 5000lx (12, 7.78%). The results suggest that the light intensity of 1000lx for 2h would be optimal for O. viverrini cercarial shedding from naturally infected B. (D.) siamensis goniomphalos snails. [Copyright &y& Elsevier]

• Praziquantel treatment for opisthorchiasis alleviates parasite burden • 30.8% of participants showed no relapse of hepatobiliary abnormality over five years • 37.5% of participants showed persistent hepatobiliary abnormality Infection of the liver fluke, Opisthorchis viverrini (OV) is an important public health problem in northeast Thailand and adjacent countries, where people have a habit of eating raw or undercooked fish. A community case-control study was carried out with 8,936 participants from 89 villages, in Khon Kaen province, Thailand. There were 3,359 OV-infected participants all of whom underwent ultrasonography of upper abdomen for the evaluation of hepatobiliary morbidity. The participants with advanced periductal fibrosis (APF) by ultrasound (n = 785) were invited to undergo annual follow-up ultrasonography for five years after praziquantel treatment. The sonographer was blinded with respect to status of OV infection at each visit. The study findings revealed variability in the study population profile of the hepatobiliary morbidities before and after praziquantel treatment over the follow up interval. At the end of the study, 32 (30.8%) out of 104 participants showed no relapse of APF whereas, by contrast, 39 (37.5%) participants showed relapse or persistent APF since the outset of the study (≥ two consecutive visits). The APF in most follow-up visits was significantly associated with male sex, with intrahepatic duct stones, with the width of the gallbladder "pre" minus "post" fatty meal, and with the ratio of left lobe of the liver to aorta. Five cases of suspected cholangiocarcinoma were observed over the five years of follow-up. This long-term ultrasound follow-up study demonstrates a significant incidence of persistent APF in over one-third of opisthorchiasis cases after praziquantel treatment, findings that support the prospect of ongoing cholangiocarcinogenesis long after successful elimination of liver fluke infection among the population. [Display omitted] [ABSTRACT FROM AUTHOR]

Graphical abstract 25-word summary: Reconstructing environmental transmission of Opisthorchis viverrini in six villages between 2008–2016 during the Lawa Project control program using longitudinal infection and treatment data. Highlights • Using longitudinal host infection data for liver fluke modeling. • Decrease in infection prevalence heterogeneous across villages. • Lawa Lake system is hydrologically and developmentally dynamic. • Modeling can be used to suggest effects of environmental and development drivers. Abstract Human infection with the Southeast Asian liver fluke Opisthorchis viverrini and liver fluke-associated cholangiocarcinoma cause significant disease burden in Southeast Asia. While there has been considerable work to understand liver fluke pathology and to reduce infection prevalence, there remains a limited understanding of the environmental determinants of parasite transmission dynamics to inform treatment and control programs. A particular setting where targeted control efforts have taken place is the Lawa Lake complex in northeast Thailand. Here, we describe the recent history of host infections, as well as the hydrologic characteristics of this floodplain ecosystem that influence the extent of snail habitat and fish mobility and the transport of human waste and parasite cercariae. Using mathematical modeling, we outline a framework for reconstructing environmental transmission of O. viverrini over the course of the Lawa Project control program from its inception in 2008 until 2016, using locally acquired but fragmentary longitudinal infection data for both humans and environmental hosts. The role of water flow in facilitating movement between snail, fish, human, and reservoir hosts is a particular focus with respect to its relevant scales and its impact on success of interventions. In this setting, we argue that an understanding of the key environmental drivers of disease transmission processes is central to the effectiveness of any environmental intervention. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Transmissibility is a significant factor in parasite fitness. The rate and magnitude of parasite transmission affect prevalence and infection intensity in individual hosts and are influenced by environmental factors. In this context, the objectives of this study were: (i) to experimentally assess Opisthorchis viverrini miracidia survival and infectivity over time and across temperatures; and (ii) to combine these experimental results with environmental data to build a key component of a transmission model, identifying seasonal windows of transmission risk in hyper-endemic northeastern Thailand. Five replicates of 50 O. viverrini eggs were randomly distributed and maintained under four temperature conditions (25 °C, 30 °C, 35 °C, 40 °C). Microscopic observations were performed on all experimental units over a period of 3 months to record miracidia motility and mortality trends. Six infection trials were also conducted to assess infectivity of miracidia over time and across temperatures, using observations of egg hatching success and infection rates. Upon completion of experiments, data were integrated into a transmission model to create a transmission risk index and to simulate seasonal transmission risk. Miracidia survival rate and motility decreased steadily with 50% mortality observed after 2 weeks. Hatching and infection success also decreased significantly after 3 weeks. Temperatures over 30 °C were associated with increased mortality and decreased infectivity. When incorporating local environmental parameters into our model, we observed low transmission risk during the dry season and increasing transmission risk at the onset of the rainy season, culminating with the highest risk in September. We believe that our results provide the first estimates of O. viverrini miracidia survival and transmission potential under variable temperature conditions and suggest that high temperature treatment (>40 °C) of fecal waste could be an efficient control strategy. [ABSTRACT FROM AUTHOR]

Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA – hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products ( Ov ES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south-eastern Asia. [ABSTRACT FROM AUTHOR]

Chronic infection with the food-borne liver fluke, Opisthorchis viverrini , frequently induces cancer of the bile ducts, cholangiocarcinoma. Opisthorchiasis is endemic in Thailand, Lao PDR, Cambodia and Vietnam, where eating undercooked freshwater fish carrying the juvenile stage of this pathogen leads to human infection. Because inhibition of apoptosis facilitates carcinogenesis, this study investigated modulation by thioredoxin from O. viverrini of apoptosis of bile duct epithelial cells, cholangiocytes. Cells of a cholangiocyte line were incubated with the parasite enzyme after which they were exposed hydrogen peroxide. Oxidative stress-induced apoptosis was monitored using flow cytometry, growth in real time and imaging of living cells using laser confocal microscopy. Immunolocalization revealed liver fluke thioredoxin within cholangiocytes. Cells exposed to thioredoxin downregulated apoptotic genes in the mitogen activated protein kinases pathway and upregulated anti-apoptosis-related genes including apoptosis signaling kinase 1, caspase 9, caspase 8, caspase 3, survivin and others. Western blots of immunoprecipitates of cell lysates revealed binding of thioredoxin to apoptosis signaling kinase 1. Together the findings indicated that thioredoxin from O. viverrini inhibited oxidative stress-induced apoptosis of bile duct epithelial cells, which supports a role for this liver fluke oxidoreductase in opisthorchiasis-induced cholangiocarcinogenesis. [ABSTRACT FROM AUTHOR]

Human liver fluke, Opisthorchis viverrini , a food-borne trematode is a significant public health problem in Southeast Asia, particularly in Thailand. Despite a long history of control programmes in Thailand and a nationwide reduction, O. viverrini infection prevalence remains high in the northeastern provinces. Therefore, a new strategy for controlling the liver fluke infection using the EcoHealth/One Health approach was introduced into the Lawa Lake area in Khon Kaen province where the liver fluke is endemic. A programme has been carried using anthelminthic treatment, novel intensive health education methods both in the communities and in schools, ecosystem monitoring and active community participation. As a result, the infection rate in the more than 10 villages surrounding the lake has declined to approximate one third of the average of 50% as estimated by a baseline survey. Strikingly, the Cyprinoid fish species in the lake, which are the intermediate host, now showed less than 1% prevalence compared to a maximum of 70% at baseline. This liver fluke control programme, named “Lawa model,” is now recognised nationally and internationally, and being expanding to other parts of Thailand and neighbouring Mekong countries. Challenges to O. viverrini disease control, and lessons learned in developing an integrative control programme using a community-based, ecosystem approach, and scaling-up regionally based on Lawa as a model are described. [ABSTRACT FROM AUTHOR]

Abstract: Infection by Opisthorchis viverrini (liver fluke) is a major public health problem in southeastern Asia, resulting in hepatobiliary disease and cholangiocarcinoma. Fluke surface glycoconjugates are prominently presented to the host, thereby constituting a crucial immunological interface that can determine the parasite’s success in establishing infection. Therefore, N- and O-linked glycoprotein glycan profiles of the infective metacercarial stage and of the mature adult were investigated by nanospray ionisation-linear ion trap mass spectrometry (NSI-MS n ). Glycan immunogenicity was investigated by immunoblotting with serum from infected humans. Metacercariae and adult parasites exhibit similar glycan diversity, although the prevalence of individual glycans and glycan classes varies by stage. The N-glycans of the metacercaria are mostly high mannose and monofucosylated, truncated-type oligosaccharides (62.7%), with the remainder processed to complex and hybrid type glycans (37.3%). The N-linked glycan profile of the adult is also dominated by high mannose and monofucosylated, truncated-type oligosaccharides (80.0%), with a smaller contribution from complex and hybrid type glycans (20.0%). At both stages, complex and hybrid type glycans are detected as mono-, bi-, tri-, or tetra-antennary structures. In metacercariae and adults, O-linked glycans are detected as mono- to pentasaccharides. The mucin type core 1 structure, Galβ1–3GalNAc, predominates in both stages but is less prevalent in the adult than in the metacercaria. Immunogenic recognition of liver fluke glycoproteins is reduced after deglycosylation but infected human serum was unable to recognise glycans released from peptides. Therefore, the most potent liver fluke antigenic epitopes are mixed determinants, comprised of glycan and polypeptide elements. [Copyright &y& Elsevier]

Abstract: A cross sectional study on hepatobiliary abnormalities in opisthorchiasis was performed in 8936 males and females aged from 20 to 60years from 90 villages of Khon Kaen province, Northeast Thailand. All were stool-examined for Opisthorchis viverrini infection by standard quantitative formalin/ethyl acetate concentration technique. Of these, 3359 participants with stool egg positive underwent ultrasonography of the upper abdomen. The hepatobiliary abnormalities detected by ultrasound are described here. This study found a significantly higher frequency of advanced periductal fibrosis in persons with chronic opisthorchiasis (23.6%), particularly in males. Risks of the fibrosis included intensity of infection, and age younger than 30years. Height of left lobe of the liver, cross-section of the gallbladder dimensions post fatty meal, sludge, and, interestingly, intrahepatic duct stones were significantly associated with the advanced periductal fibrosis. Eleven suspected cholangiocarcinoma (CCA) cases were observed. This study emphasizes the current status of high O. viverrini infection rate and the existence of hepatobiliary abnormalities including suspected CCA in opisthorchiasis endemic areas of Thailand. [Copyright &y& Elsevier]

Abstract: A cathepsin B-like cysteine protease belonging to family C1 is abundantly expressed in the transcriptome and proteome of the carcinogenic liver fluke of humans, Opisthorchis viverrini. This enzyme is present in excretory/secretory (ES) products released by parasites cultured in vitro. This study evaluated the performance of recombinant O. viverrini cathepsin B1 (rOv-CB-1) as an antigen for immunodiagnosis of opisthorchiasis. The full length Ov-CB-1 cDNA was cloned and recombinant protein was produced in catalytically active form in Pichia pastoris. The recombinant Ov-CB-1 (rOv-CB-1) was affinity purified via nickel-NTA chromatography and tested in enzyme-linked immunosorbent assays (ELISA) with human sera from an opisthorchiasis endemic area. Sera from egg-positive O. viverrini infections produced a strong IgG antibody response to rOv-CB-1 both in ELISA and immunoblot analysis. The sensitivity and specificity of the ELISA test was 67% and 81%, respectively. These findings support the feasibility of using recombinant Ov-CB-1 in ELISA for the serodiagnosis of human opisthorchiasis. [Copyright &y& Elsevier]

Abstract: Cholangiocarcinoma (CCA) is a crucial health problem in northeastern part of Thailand, which is caused by a combination of Opisthorchis viverrini infection and nitrosamine. A better understanding of its molecular mechanism is an important step to discover and develop the new diagnostics and therapies for CCA. To reveal the involvement of potential genes in the development of CCA, the present study investigated the expression kinetics of platelet-derived growth factor alpha (Pdgfa) and its receptor (Pdgfra) during the tumorigenesis of CCA induced by O. viverrini infection with quantitative RT-PCR, and confirmed the expression with immunohistological staining. The results showed that in the hamster model of opisthorchiasis-associated CCA, the expression of Pdgfa was increased after infection plus N-nitrosodimethylamine (NDMA) administration, reached its peak at 2months post infection, and remained at the high level until 6months. Similarly, the expression of Pdgfra was increased time-dependently. The positive immunostaining for PDGFA proteins was observed in the cytoplasm of epithelial tumor cells of hamster CCA. Moreover, the analysis of the expression of these genes in 10 cases of human opisthorchiasis-associated CCA showed that Pdgfa was overexpressed in 80%, and Pdgfra was overexpressed in 40% cases (>3.0 folds, compared with the expressions of adjacent normal tissues). This result suggests that PDGFA is likely involved in the tumorigenesis of opisthorchiasis-associated CCA, and may be a promising candidate biomarker for diagnosis and treatment strategies of CCA. [Copyright &y& Elsevier]

Abstract: Opisthorchis viverrini can develop mitogenic substances into the excretory/secretory product (ESP) that may play an important role in promoting the genesis of cholangiocarcinoma (CCA). In the present study, glutathione S-transferase (GST) is identified as being secreted into Ov-ESP and acting as one of the parasitic mitogens. Its proliferative effect and possible mechanism were explored and its association with the tumor development is proposed. Ov-ESP was concentrated and purified by gel filtration chromatography. SDS-PAGE, 2-DE, and LC-MS/MS identified GST predominantly expressed in the proliferative ESP fraction. The recombinant OvGST (rOvGST) was produced by wheat germ cell-free expression and confirmed by an MTS assay to have a proliferative function on NIH-3T3 murine fibroblasts and MMNK1 non-tumorigenic human bile duct epithelial cells in a dose dependent manner with different optimal doses. The cell surface binding of rOvGST was confirmed in vitro and the activation of both pAKT and pERK was revealed as the mechanism of OvGST-mediated cell proliferation. With support from the observation of secreted OvGST on the biliary cells surrounding the parasites, it is suggested that OvGST can promote cell proliferation that consequently may accelerate the genesis of CCA. [Copyright &y& Elsevier]

Abstract: The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilizing systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 amino acid residues, of molecular mass 11.63kDa. The predicted protein has similarity to previously characterized thioredoxins with 26–51% identity. Recombinant O. viverrini Trx (Ov-Trx-1) was expressed as soluble protein in E. coli. The recombinant protein showed insulin reduction activity and supported the enzymatic function of O. viverrini thioredoxin peroxidase. Expression of Ov-Trx-1 at mRNA and protein levels was observed in all obtainable developmental stages of the liver fluke. Ov-Trx-1 was also detected in excretory–secretory products released by adult O. viverrini. Immunohistochemistry, Ov-Trx-1 was expressed in nearly all parasite tissue excepted ovary and mature sperms. Interestingly, Ov-Trx-1 was observed in the infected biliary epithelium but not in normal bile ducts. These results suggest that Ov-Trx-1 is essential for the parasite throughout the life cycle. In the host–parasite interaction aspect, Ov-Trx-1 may support thioredoxin peroxidase in protecting the parasite against damage induced by reactive oxygen species from inflammation. [Copyright &y& Elsevier]

Abstract: Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20ms of 125V in the presence of 50μg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen. [Copyright &y& Elsevier]

Abstract: Biliary tract infection with the Group I carcinogenic liver fluke Opisthorchis viverrini is associated with severe inflammation leading to cholangiocarcinoma—a major biliary cancer in Southeast Asia. However, mechanism(s) by which the liver fluke induces host mucosal immune/inflammatory responses is unclear. In the present study we address whether a normal immortalized human cholangiocyte cell line (H69 cells) recognizes and responds to O. viverrini excretory/secretory products (OVES). Expression of multiple TLRs, activation of NF-κB, and expression of pro-inflammatory cytokines were monitored in the presence and absence of OVES. Our results showed that OVES induced increased cholangiocyte TLR4 mRNA expression, induced IκB-α degradation in a MyD88-dependent manner, and activated NF-κB nuclear translocation. Moreover, OVES induced expression and secretion of the strong chemoattractant chemokine interleukin 8 (IL-8) and pro-inflammatory cytokine IL-6. These results demonstrate that secreted/excreted products of O. viverrini are recognized by human cholangiocytes and initiate innate mucosal immunity/inflammatory cascades, a primary event in the pathogenesis of opisthorchiasis and cholangiocarcinoma. [ABSTRACT FROM AUTHOR]

Abstract: The human liver fluke Opisthorchis viverrini is endemic in Thailand, Laos and Cambodia where long standing infection is associated with cancer of the bile ducts, cholangiocarcinoma. Here we describe a cathepsin D-like aspartic protease from the gut and other tissues in O. viverrini. Phylogenetic analysis indicated that Ov-APR-1 is cathepsin D-like, conforming with Clan AA, Family A1 of the MEROPS classification. Ov-APR-1 is expressed in the gut of the mature hermaphroditic parasite, in the reproductive tissues including the testis and immature spermatids, and the developing miracidium within the eggshell. The enzyme was also detected in the excretory/secretory products of cultured adult flukes, indicating a role in host-parasite relationships. A recombinant form of the enzyme expressed in Escherichia coli and refolded from denatured inclusion bodies underwent autocatalytic activation and demonstrated hydrolytic activity against the peptide substrate 7-methoxycoumarin-4-acetyl-GKPILFFRLK(DNP)-d-Arg-amide with a k cat/K m =1.7×104 M−1 s−1 and a pH optimum around pH 2.5–3.0. The recombinant enzyme digested hemoglobin and bovine serum albumin. Forty-six serum albumin peptides were detected after digestion with recombinant Ov-APR-1 and sequenced. Like many other aspartic proteases, Ov-APR-1 displayed promiscuous preferences for residues accommodated at the key subsites of the binding pocket although hydrophobic (Leu, Ala, Ile), positively charged (Lys) and bulky aromatic (Phe) residues, in that order, were preferred at P1. Similar residues were accommodated at P1′ although even less selectivity was exerted at this position. [Copyright &y& Elsevier]

Abstract: The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for many years. The mechanisms by which it avoids oxidative damage are unknown. In this study, thioredoxin peroxidase (TPx), a member of the peroxiredoxin superfamily, was cloned from an O. viverrini cDNA library. O. viverrini TPx cDNA encoded a polypeptide of 212 amino acid residues, of molecular mass 23.57kDa. The putative amino acid sequence shared 60–70% identity with TPXs from other helminths and from mammals, and phylogenetic analysis revealed a close relationship between TPxs from O. viverrini and other trematodes. Recombinant O. viverrini TPx was expressed as soluble protein in Escherichia coli. The recombinant protein dimerized, and its antioxidant activity was deduced by observing protection of nicking of supercoiled plasmid DNA by hydroxyl radicals. Antiserum raised against O. viverrini TPx recognized native proteins from egg, metacercaria and adult developmental stages of the liver fluke and excretory–secretory products released by adult O. viverrini. Immunolocalization studies revealed ubiquitous expression of TPx in O. viverrini organs and tissues. TPx was also detected in bile fluid and bile duct epithelial cells surrounding the flukes 2 weeks after infection of hamsters with O. viverrini. In addition, TPx was observed in the secondary (small) bile ducts where flukes cannot reach due to their large size. These results suggested that O. viverrini TPx plays a significant role in protecting the parasite against damage induced by reactive oxygen species from inflammation. [Copyright &y& Elsevier]

Abstract: Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and the Lao People''s Democratic Republic (Lao PDR; Laos). Currently, more than 600 million people are at risk of infection with these fish-borne trematodes and/or their close relatives. Opisthorchiasis has been studied extensively in Thailand, where about 8 million people are infected with the liver fluke. Here we review the pathogenesis, control and re-emergence of O. viverrini infection, in particular in Thailand and, to a lesser extent in Lao PDR given the contiguous geographical range of O. viverrini through these two regions. We also review the association of O. viverrini infection and cholangiocarcinoma, bile duct cancer, and highlight new findings on pathogenesis of liver fluke-induced cholangiocarcinogenesis. Last, we comment on national control strategies in Thailand for the control of O. viverrini infection aimed at reduction in the prevalence of O. viverrini-associated liver cancer in the longer term. [Copyright &y& Elsevier]

Summary: Objectives: To isolate and characterize an asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and evaluate its expression profile, biochemical activity, and potential as an immunodiagnostic antigen. Methods: The full length mRNA encoding an asparaginyl endopeptidase (family C13), Ov-aep-1, was isolated by immunoscreening of a cDNA bacteriophage library of adult O. viverrini using sera from patients infected with O. viverrini. Investigation of Ov-aep-1 transcripts in developmental stages of the parasite, and phylogenetic analysis, immunohistochemical localization, and recombinant protein expression and enzymology were employed to characterize the Ov-AEP-1 protein. Immunoblotting was used to assess the potential of this enzyme for immunodiagnosis of human opisthorchiasis. Results: Ov-AEP-1 is characteristic of the C13 cysteine protease family. Ov-aep-1 transcripts were detected in adult and juvenile worms, eggs, and metacercariae. Phylogenetic analysis indicated that Ov-AEP-1 is closely related to homologous proteins in other trematodes. Recombinant Ov-AEP-1 was expressed in bacteria in inclusion bodies and refolded to a soluble form. Excretory–secretory (ES) products derived from adult O. viverrini and refolded recombinant Ov-AEP-1 both displayed catalytic activity against the diagnostic tripeptide substrate, Ala–Ala–Asn-aminomethylcoumarin. Rabbit antiserum raised to recombinant Ov-AEP-1 identified the native AEP-1 protease in both somatic extract and ES products of adult worms. Anti-Ov-AEP-1 IgG immunolocalized the anatomical site of expression to the gut of the fluke, implying a physiological role in digestion of food or activation of other digestive enzymes. Recombinant Ov-AEP-1 was recognized by serum antibodies from patients with opisthorchiasis but not other helminth infections, with a sensitivity and specificity of 85% and 100%, respectively. The positive and negative predictive values are 100% and 67%, respectively. Conclusions: The liver fluke, O. viverrini, has a gut-localized asparaginyl endopeptidase. Refolded recombinant Ov-AEP-1 is catalytically active and has potential for immunodiagnosis of human opisthorchiasis. [Copyright &y& Elsevier]