Sirikachorn Tangkawattana

A Portrait of the Transcriptome of the Neglected Trematode, Fasciola gigantica—Biological and Biotechnological Implications

Fasciola gigantica (Digenea) is an important foodborne trematode that causes liver fluke disease (fascioliasis) in mammals, including ungulates and humans, mainly in tropical climatic zones of the world. Despite its socioeconomic impact, almost nothing is known about the molecular biology of this parasite, its interplay with its hosts, and the pathogenesis of fascioliasis. Modern genomic technologies now provide unique opportunities to rapidly tackle these exciting areas. The present study reports the first transcriptome representing the adult stage of F. gigantica (of bovid origin), defined using a massively parallel sequencing-coupled bioinformatic approach. From >20 million raw sequence reads, >30,000 contiguous sequences were assembled, of which most were novel. Relative levels of transcription were determined for individual molecules, which were also characterized (at the inferred amino acid level) based on homology, gene ontology, and/or pathway mapping. Comparisons of the transcriptome of F. gigantica with those of other trematodes, including F. hepatica, revealed similarities in transcription for molecules inferred to have key roles in parasite-host interactions. Overall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of F. gigantica, as well as a basis for applied outcomes such as the development of novel methods of intervention against this neglected parasite.

High prevalence of Opisthorchis viverrini infection in reservoir hosts in four districts of Khon Kaen Province, an opisthorchiasis endemic area of Thailand.

Khon Kaen, a northeastern province of Thailand, has been considered as one of the human opisthorchiasis endemic areas with continuing high prevalence. Unsuccessful eradication of the disease is probably from the culture of eating raw and undercooked fish of local residence and the parasitic persistency in animal reservoir hosts, such as cats and dogs. In cooperation with the other human opisthorchiasis control programs in an endemic area of 29 villages in Ban Haet, Ban Phai, Chonnabot and Muncha Khiri Districts, Khon Kaen, this study investigated the prevalence of Opisthorchis viverrini infection using a formalin-ether sedimentation method as the gold standard, and hematology and blood chemistry of the reservoir hosts in this endemic area. The results showed that cats had much higher prevalence (76 of 214, 35.51%) than dogs (3 of 821, 0.37%). Hematology between the infected and uninfected cats was not different. Complete blood count and biochemistry reflected some altered hepatic functions. However, only severely infected cats showed apparent clinical signs, including lethargy, diarrhea, ocular and nasal discharges. Moreover, the ultrasonogram of infected cats with very high egg per gram (>1500 EPG) showed apparent thickening of the gall bladder wall with hyperechoicity of hepatic parenchyma. This study suggests that cat is the most important animal reservoir of human opisthorchiasis, especially in this endemic area. It is also interesting that villages with infection are mostly located in the vicinity of Chi River and two large water reservoirs (Lawa and Nong Kongkaew Lakes), but people without infection were away from Chi River, on the south of Kudkhow Lake. Further investigation on this particular geofactor is essential for effective opisthorchiasis control programs.

Comparative histopathology of Opisthorchis felineus and Opisthorchis viverrini in a hamster model: An implication of high pathogenicity of the European liver fluke

European liver fluke (Opisthorchis felineus) and Asian liver fluke (Opisthorchis viverrini) are similar in morphology but comparative pathology of the infections has not been described. We therefore did comparative histopathology of both parasites in an experimental animal model. The study was conducted in 3 groups of 105 Syrian golden hamsters; the first and second groups fed with 50 metacercariae of O. felineus (OF) or O. viverrini (OV) and the last group was uninfected controls. Five hamsters in each group were euthanized on weeks 1, 2, 4, 8, 12 and 24 post-infection. The liver tissue was fixed and processed for routine histopathology and immunohistochemistry for proliferation markers (BrdU or PCNA). Overall, the liver histopathology of O. felineus and O. viverrini infection was generally similar. However, various histopathogical features including intense inflammation, fibrosis, biliary and goblet cell hyperplasia and dysplasia occurred earlier in the OF group. In addition, the existence of precancerous lesions such as cholangiofibrosis in a long-term infection was observed only in this group. O. felineus is larger in size than O. viverrini which, together with its excreted and secreted antigens, likely is crucial in the induction of liver fluke induced disease. The differences in nature and timing of the histopathological profile indicate that opisthorchiasis caused by the European liver fluke O. felineus is more pathogenic than its Asian relative O. viverrini.

Decreased risk of cholangiocarcinogenesis following repeated cycles of Opisthorchis viverrini infection-praziquantel treatment: Magnetic Resonance Imaging (MRI) and histopathological study in a hamster model

It has been suggested that repeated infection of Opisthorchis viverrini followed by repeated treatment with praziquantel (PZQ) increases risk of development of cholangiocarcinoma (CCA). Evidence for the prediction has accumulated based on findings of indirect approaches involving molecular changes and epidemiological trends. By contrast, here we directly monitored the impact of repeated liver fluke infection and treatment with PZQ on cholangiocarcinogenesis in a rodent model of human opisthorchiasis, using magnetic resonance imaging (MRI) and histopathology. Twenty five Syrian golden hamsters were assigned to five treatment groups: 1) infection with O. viverrini (OV group), 2) treatment with the carcinogen N-nitrosodimethylamine (NDMA) at 12.5ppm (DMN), 3) O. viverrini infection in tandem with NDMA (OD), 4) O. viverrini infection, NDMA, and treatment with PZQ (ODP), and 5) uninfected, untreated control. The repeated infections were established by intragastric inoculation of 50 metacercariae of O. viverrini to the OV, OD and ODP hamsters at weeks 0, 5 and 10. PZQ at 300mg/kg body weight was given to each hamster of the ODP group on weeks 4, 9 and 13 (four weeks after each infection). Imaging by MRI was undertaken on weeks 5, 10 and 14 (i.e. one week after each PZQ treatment). MRI revealed that the ODP hamsters did not develop CCA, whereas necropsy at week 40 revealed CCA in hamsters of the OD and DMN groups. Findings for histopathology and for proliferating cell nuclear antigen index conformed to the MRI findings. In overview, and notwithstanding that the immune response of individual hosts may play roles in cholangiocarcinogenesis, three cycles of the infection with O. viverrini followed treatment of the infection with PZQ did not increase the risk of bile duct cancer in this hamster model of liver fluke infection-induced CCA.

Chicken IgY-based coproantigen capture ELISA for diagnosis of human opisthorchiasis.

Diagnosis of Opisthorchis viverrini infection by conventional stool examination is increasingly difficult due to the low intensity of the infection after several rounds of control programmes in endemic regions as well as coinfections with intestinal flukes. Therefore sensitive and specific diagnostic test is needed. In this study, a coproantigen sandwich ELISA using recombinant O. viverrini cathepsin F (rOv-CF) was developed. This sandwich ELISA employing chicken IgY raised against rOv-CF in combination with rabbit IgG antibody to the somatic O. viverrini antigens showed a lower detection limit (LLD) of 70ng native O. viverrini somatic antigens by spiking the parasite antigens into control feces. When applied to the diagnosis, the IgY-based sandwich ELISA exhibited sensitivity and specificity of 93.3% and 76.7%, respectively, in an investigation of 90 human cases positive or negative for opisthorchiasis. The positive predictive value (PPV) and negative predictive value (NPV) for this coproantigen detection were 66.7% and 95.2%, respectively. This IgY-based sandwich ELISA using parasite cathepsin F detection shows a promising immunodiagnostic alternative for human opisthorchiasis in endemic regions.

Social network analysis of food sharing among households in opisthorchiasis endemic villages of Lawa Lake, Thailand

Consumption of raw fish is a well-documented risk factor for Opisthorchis viverrini infection. Sharing of food, especially raw fish recipes may influence the spread of disease through a community. Using social network analysis of an ego network, we investigated food sharing among households in an Opisthorchis-endemic area. Network centrality properties were used to explain the differences in O. viverrini transmission and control between villages with a low and high prevalence of infection. Information on demography and O. viverrini infection in 2008 from villagers in the Lawa Lake area was extracted from the Tropical Disease Research Center database. The two villages that had the lowest and the highest O. viverrini infection at the household level were recruited. Ten percent of households of each village were randomly sampled. Participatory epidemiology and face-to-face structured interviews guided by a social network questionnaire were used to collect data on livelihood, agricultural patterns, food sources, raw fish eating habits, and other food sharing during daily life and social gatherings. The number of contacts including in-degree and out-degree varied from 0 to 7 in the low-infection village and 0 to 4 in the high-infection village. The mean number of contacts for the food-sharing network among the low- and high-infection villages was 1.64 and 0.73 contacts per household, respectively. Between these villages, the mean number of out-degree (p=0.0125), but not in-degree (p=0.065), was significantly different. Food-sharing differed in numbers of sharing-in and sharing-out between the two villages. Network analysis of food sharing may be of value in designing strategies for opisthorchiasis control at the community level.

Risk factors for Clonorchis sinensis infection transmission in humans in northern Vietnam: A descriptive and social network analysis study

Clonorchis sinensis is major fish-borne trematode, endemic in North Vietnam. Risk factors described so far include individual eating behaviors and environmental factors. Here, additional to conventional risk factors, we report on socially influenced liver fluke transmission in endemic communities. A cross-sectional study on risk factors and fish sharing networks was conducted in 4 villages of Gia Thinh Commune, Ninh Binh Province. A total of 510 residents in 272 households were recruited for risk factor analysis while 220 households, 28 fishermen and 10 fish-sellers were enrolled for social network study. Fecal examination for C. sinensis eggs was performed. Average C. sinensis infection rate at Gia Thinh commune was 16.5% (range 2% to 34.4%). Higher infection rates were significantly associated with males, lower educational levels, eating raw fish, and location of the villages. Social network analysis (SNA) showed a strong positive correlation between ego network size (number of households in fish sharing network) and quantity of raw fish consumed (r=0.603, P<0.05). The infection rate in people who ate raw-fish caught from a nearby river was significantly higher than those who consumed fish taken from farmed ponds (P<0.05). The amount of raw-fish meal consumed per resident/year was significantly higher in villages that had a strong network of sharing raw-fish food (P<0.001). This study reports for the first time on fish-food sharing among neighbors, proximity to water bodies, frequency of eating raw fish from natural water bodies and low education were key risk factors in C. sinensis infection transmission in northern Vietnam.

Differential Protein Expression in the Hemolymph of Bithynia siamensis goniomphalos Infected with Opisthorchis viverrini

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.

Efficacious and safe dose of praziquantel for the successful treatment of feline reservoir hosts with opisthorchiasis

Opisthorchiasis caused by Opisthorchis viverrini is a major food-borne zoonosis in Greater Mekong sub-region. Even though campaigns discouraging the consumption of raw fish have been launched to public, the disease still remains highly endemic. The unsuccessful eradication of the disease is probably because of the persistence of the parasite in animal reservoir hosts, particularly felids. Praziquantel (PZQ) is the drug of choice for morbidity control of opisthorchiasis in humans and animals. However, there is no specific study on its dosage regimen for feline opisthorchiasis. Thus, the effective treatment dose of PZQ, as well as its adverse effects, was evaluated in O. viverrini infected cats. Twenty-eight infected male and female cats from the endemic area of Khon Kaen and Maha Sarakham Provinces, Thailand were enrolled in this study. Physical, hematological, blood chemical and urine examinations were analyzed, as indicators of health status, on the day before and 30days after treatment. Intensity of the infections was determined by the formalin-ethyl acetate sedimentation technique. Cats were equally allotted into the low infection group of 14 cats with egg count per gram of feces (EPG) <300 and the high infection group of 14 cats with EPG higher than 300. Cats in each group were equally divided into two subgroups of 7 cats; thus, there were two low infection subgroups (L1 and L2 subgroups) and two high infection subgroups (H1 and H2 subgroups). A single dose of 25mg/kg PZQ was orally administered to each cat in the L1 and H1 subgroups and a single oral dose of 40mg/kg PZQ was administered to the L2 and H2 subgroups. Complete clearance of O. viverrini eggs was found in all cats in the L1, L2 and H2 subgroups; thus, the cure rate (CR) and egg reduction rate (ERR) were 100%. However, partial clearance was observed in two cats with high EPG (1502 and 1518) in the H1 subgroup, which received 25mg/kg PZQ. Regards, CR and ERR for these two animals was 71.4 and 99.5%. No significant difference among the 4 subgroups was seen. Almost all hematological, blood chemical and urinalysis data were within normal ranges, except for the eosinophilia and an increase of alanine aminotransferase (ALT). Hookworm infection seen in all cats would cause eosinophilia. As for drug safety, there was no side effect observed in any cats. In conclusion, this study suggested that 40mg/kg PZQ is a highly effective and safe dosage for the treatment of feline reservoir hosts of human opisthorchiasis.

Cell-based polymerase chain reaction for canine transmissible venereal tumor(CTVT) diagnosis

Canine transmissible venereal tumor (CTVT) is the only naturally contagious tumor that is transmitted during coitus or social behaviors. Based on the tumor’s location, the diagnosis of genital TVT (GTVT) is comparably easier than those in the extragenital area (ETVT) that are more easily incorrectly diagnosed. Fortunately, CTVT cells contain a specific long interspersed nuclear elements (LINE), inserted upstream of the myc gene, allowing a diagnostic polymerase chain reaction (PCR) based detection assay. The objectives of this study were aimed to improve the diagnostic accuracy by applying the diagnostic LINE1-c-myc PCR assay and fine needle aspiration (FNA) collection in direct comparison with standard cytological and histopathological analyses. Seventy-four dogs, comprised of 41 and 31 dogs with tumor masses at their external genitalia and extragenital areas (e.g. skin and nasal cavity), respectively, were included in this study. The signalment of these 65 dogs and clinical history of 20 client-owned dogs were collected. Samples were taken by biopsy for both histopathological examination and FNA for cytological examination and diagnostic PCR. The PCR products from 10 apparently CTVT samples were purified and sequenced. Sixty-one CTVT cases were diagnosed by cytological and histological analyses, but 65 were positive by the PCR assay. Overall, the PCR assay improved the accuracy of diagnostic CTVT results, especially for the more difficult ETVT tumors. Moreover, this PCR-based approach can facilitate the decision as to discontinue chemotherapy by discrimination between residual tumor cell masses and fibrotic tissue.