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The two parasitic trematodes, Clonorchis sinensis and Opisthorchis viverrini, have a major impact on the health of tens of millions of humans throughout Asia. The greatest impact is through the malignant cancer ( = cholangiocarcinoma) that these parasites induce in chronically infected people. Therefore, both C. sinensis and O. viverrini have been classified by the World Health Organization (WHO) as Group 1 carcinogens. Despite their impact, little is known about these parasites and their interplay with the host at the molecular level. Recent advances in genomics and bioinformatics provide unique opportunities to gain improved insights into the biology of parasites as well as their relationships with their hosts at the molecular level. The present study elucidates the transcriptomes of C. sinensis and O. viverrini using a platform based on next-generation (high throughput) sequencing and advanced in silico analyses. From 500,000 sequences, >50,000 sequences were assembled for each species and categorized as biologically relevant based on homology searches, gene ontology and/or pathway mapping. The results of the present study could assist in defining molecules that are essential for the development, reproduction and survival of liver flukes and/or that are linked to the development of cholangiocarcinoma. This study also lays a foundation for future genomic and proteomic research of C. sinensis and O. viverrini and the cancers that they are known to induce, as well as novel intervention strategies.

Background: The liver fluke Opisthorchis viverrini infects several million people in Southeast Asia. Adult flukes live in the bile ducts of humans, where they cause hepatobiliary pathology, including cholangiocarcinoma. Here, we investigated the potential of extracellular vesicles (EVs) secreted by the fluke and defined recombinant proteins derived from EVs to generate protective immunity in a hamster vaccination-challenge model. Methodology/Principal findings: EVs isolated from the excretory-secretory products of O. viverrini and two recombinant EV surface proteins encoding the large extracellular loops (LEL) of Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 (rOv-TSP-3) were adjuvanted and used to vaccinate hamsters intraperitoneally followed by challenge infection with O. viverrini metacercariae. The number of adult flukes recovered from hamsters immunized with EVs, rOv-TSP-2, rOv-TSP-3 and rOv-TSP-2+rOv-TSP-3 were significantly reduced compared to control animals vaccinated with adjuvant alone. The number of eggs per gram feces was also significantly reduced in hamsters vaccinated with rOv-TSP-2 compared to controls, but no significant differences were found in the other groups. The average length of worms recovered from hamsters vaccinated with EVs, rOv-TSP-2 and rOv-TSP-3 was significantly shorter than that of worms recovered from the control group. Anti-EV IgG levels in serum and bile were significantly higher in hamsters vaccinated with EVs compared to control hamsters both pre- and post-challenge. In addition, levels of anti-rOv-TSP antibodies in the serum and bile were significantly higher than control hamsters both pre- and post-challenge. Finally, antibodies against rOv-TSP-2 and rOv-TSP-3 blocked uptake of EVs by human primary cholangiocyte in vitro, providing a plausible mechanism by which these vaccines exert partial efficacy and reduce the intensity of O. viverrini infection. Conclusion/Significance: Liver fluke EVs and recombinant tetraspanins derived from the EV surface when administered to hamsters induce antibody responses that block EV uptake by target bile duct cells and exert partial efficacy and against O. viverrini challenge. [ABSTRACT FROM AUTHOR]

Opisthorchis viverrini is distinct among helminth infections as it drives a chronic inflammatory response in the intrahepatic bile duct that progresses from advanced periductal fibrosis (APF) to cholangiocarcinoma (CCA). Extensive research shows that oxidative stress (OS) plays a critical role in the transition from chronic O. viverrini infection to CCA. OS also results in the excision of a modified DNA lesion (8-oxodG) into urine, the levels of which can be detected by immunoassay. Herein, we measured concentrations of urine 8-oxodG by immunoassay from the following four groups in the Khon Kaen Cancer Cohort study: (1) O. viverrini negative individuals, (2) O. viverrini positive individuals with no APF as determined by abdominal ultrasound, (3) O. viverrini positive individuals with APF as determined by abdominal ultrasound, and (4) O. viverrini induced cases of CCA. A logistic regression model was used to evaluate the utility of creatinine-adjusted urinary 8-oxodG among these groups, along with demographic, behavioral, and immunological risk factors. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive accuracy of urinary 8-oxodG for APF and CCA. Elevated concentrations of 8-oxodG in urine positively associated with APF and CCA in a strongly dose-dependent manner. Urinary 8-oxodG concentrations also accurately predicted whether an individual presented with APF or CCA compared to O. viverrini infected individuals without these pathologies. In conclusion, urinary 8-oxodG is a robust ‘candidate’ biomarker of the progression of APF and CCA from chronic opisthorchiasis, which is indicative of the critical role that OS plays in both of these advanced hepatobiliary pathologies. The findings also confirm our previous observations that severe liver pathology occurs early and asymptomatically in residents of O. viverrini endemic regions, where individuals are infected for years (often decades) with this food-borne pathogen. These findings also contribute to an expanding literature on 8-oxodG in an easily accessible bodily fluid (e.g., urine) as a biomarker in the multistage process of inflammation, fibrogenesis, and infection-induced cancer. [ABSTRACT FROM AUTHOR]

Background: The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens. Methodology/Principal Findings: The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Conclusions/Significance: This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed. Author Summary: Liver fluke infection is a fish borne disease that afflicts millions of residents in Thailand and Laos. Infection results from eating undercooked freshwater fish contaminated with larvae of the worm Opisthorchis viverrini. Infection can lead to cancer of the bile ducts (cholangiocarcinoma). Indeed, O. viverrini is designated as a Group 1 carcinogen by the World Health Organization, i.e. a definitive cause for cancer. Proteins produced at the surface and/or released from this parasite play pivotal roles in maintaining the infection and disease. These proteins are valid targets for development of vaccines and new drugs. Tetraspanins are prominent in the tegument (the surface covering) of parasites closely related to O. viverrini where they are exposed to immune responses. Similar molecules on the surface of O. viverrini may be vital for the parasite's survival and may make effective vaccines. Here the gene coding for O. viverrini tetraspanin-1 (Ov-TSP-1) was investigated. We used electron microscopy to show that Ov-TSP-1 is expressed in the tegument. We then silenced expression of the gene encoding Ov-TSP-1 and showed that this resulted in malformation of the tegument, highlighting the importance of this molecule for parasite development and its potential as a vaccine target. [ABSTRACT FROM AUTHOR]

Genome sequences are available for the human blood flukes, Schistosoma japonicum, S. mansoni and S. haematobium. Functional genomic approaches could aid in identifying the role and importance of these newly described schistosome genes. Transgenesis is established for functional genomics in model species, which can lead to gain- or loss-of-functions, facilitate vector-based RNA interference, and represents an effective forward genetics tool for insertional mutagenesis screens. Progress toward routine transgenesis in schistosomes might be expedited if germ cells could be reliably localized in cultured schistosomes. Vasa, a member of the ATP-dependent DEAD-box RNA helicase family, is a prototypic marker of primordial germ cells and the germ line in the Metazoa. Using bioinformatics, 33 putative DEAD-box RNA helicases exhibiting conserved motifs that characterize helicases of this family were identified in the S. mansoni genome. Moreover, three of the helicases exhibited vasa-like sequences; phylogenetic analysis confirmed the three vasa-like genes—termed Smvlg1, Smvlg2, and Smvlg3—were members of the Vasa/PL10 DEAD-box subfamily. Transcripts encoding Smvlg1, Smvlg2, and Smvlg3 were cloned from cDNAs from mixed sex adult worms, and quantitative real time PCR revealed their presence in developmental stages of S. mansoni with elevated expression in sporocysts, adult females, eggs, and miracidia, with strikingly high expression in the undeveloped egg. Whole mount in situ hybridization (WISH) analysis revealed that Smvlg1, Smvlg2 and Smvlg3 were transcribed in the posterior ovary where the oocytes mature. Germ cell specific expression of schistosome vasa-like genes should provide an informative landmark for germ line transgenesis of schistosomes, etiologic agents of major neglected tropical diseases. Author Summary: Schistosomes, the blood flukes, are responsible for the major neglected tropical diseases termed schistosomiasis, which afflicts >200 million people in impoverished regions of the developing world. The genome sequence of these parasites has been decoded recently. The DEAD-box family is the largest of RNA helicase families. These enzymes play roles in RNA metabolic processes—transcription, pre-mRNA splicing, ribosome biogenesis, transport, initiation of translation, organelle gene expression, and RNA decay. Database searches indicated that S. mansoni has at least 33 DEAD-box helicases. A DEAD-box helicase known as Vasa is a determinant in germ line segregation and maintenance. Three schistosome DEAD-box RNA helicases exhibited Vasa-like sequences, and phylogenetic analysis confirmed they were members of the Vasa/PL10 DEAD-box subfamily. Quantitative real time PCR revealed that all three, termed Smvlg1, Smvlg2, and Smvlg3, were expressed in developmental stages of S. mansoni, with elevated expression in adult females, eggs, miracidia and sporocysts. Moreover, whole mount in situ hybridization analysis confirmed that these vasa-like genes were expressed in the schistosome ovary. These findings provide insights into the mechanisms underlying development of germ cell lines and gonads in schistosomes and have implications for understanding the role of RNA helicases, germ and stem cell biology. [ABSTRACT FROM AUTHOR]

Background: Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA interference techniques that, if available, promise to guide development of new interventions for schistosomiasis haematobia. Methods/Findings: Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs, messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S. haematobium eggs in 50 ng/µl Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square wave electroporation of the schistosome eggs and adult worms in 150 ng/µl of mRNA. RNA interference knockdown (silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs, schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser scanning confocal microscopy to view the adult stages of S. haematobium. Conclusions: These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen. Author Summary: More people are infected with Schistosoma haematobium than other major human schistosomes yet it has been less studied because of difficulty in maintaining the life cycle in the laboratory. S. haematobium might be considered the 'neglected schistosome' since minimal information on the genome and proteome of S. haematobium is available, in marked contrast to the other major schistosomes. In this report we describe tools and protocols to investigate the genome and genetics of this neglected schistosome. We cultured developmental stages of S. haematobium, and investigated the utility of introducing gene probes into the parasites to silence two model genes. One of these, firefly luciferase, was a reporter gene whereas the second was a schistosome gene encoding a surface protein, termed Sh-tsp-2. We observed that both genes could be silenced – a phenomenon known as experimental RNA interference (RNAi). These findings indicated that the genome of S. haematobium will be amenable to genetic manipulation investigations designed to determine the function and importance of genes of this schistosome and to investigate for novel anti-parasite treatments. [ABSTRACT FROM AUTHOR]