Jutharat Kulsantiwong

Temperature dependence of Opisthorchis viverrini infection in first intermediate host snail, Bithynia siamensis goniomphalos

Determining of the success of a parasites 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<0.01). A temperature of 34°C gave the highest rate of infection of 44.14%. We also found that the odds of infection in small sized snails was 39.8% higher relative to medium sized snails (P<0.05). Relative to day 1, the decrease in the odds of infection was detected when the day post infection was longer (P<0.01). Proportion of infection in female was not different to male significantly.

Effects of Bayluscide on Bithynia siamensis goniomphalos, the first intermediate host of the human liver fluke, Opisthorchis viverrini, in laboratory and field trials

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 24h 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.

DNA Barcode Identification of Freshwater Snails in the Family Bithyniidae from Thailand

Freshwater snails in the family Bithyniidae are the first intermediate host for Southeast Asian liver fluke (Opisthorchis viverrini), the causative agent of opisthorchiasis. Unfortunately, the subtle morphological characters that differentiate species in this group are not easily discerned by non-specialists. This is a serious matter because the identification of bithyniid species is a fundamental prerequisite for better understanding of the epidemiology of this disease. Because DNA barcoding, the analysis of sequence diversity in the 5’ region of the mitochondrial COI gene, has shown strong performance in other taxonomic groups, we decided to test its capacity to resolve 10 species/ subspecies of bithyniids from Thailand. Our analysis of 217 specimens indicated that COI sequences delivered species-level identification for 9 of 10 currently recognized species. The mean intraspecific divergence of COI was 2.3% (range 0-9.2 %), whereas sequence divergences between congeneric species averaged 8.7% (range 0-22.2 %). Although our results indicate that DNA barcoding can differentiate species of these medically-important snails, we also detected evidence for the presence of one overlooked species and one possible case of synonymy.

RNA-Seq Reveals Infection-Induced Gene Expression Changes in the Snail Intermediate Host of the Carcinogenic Liver Fluke, Opisthorchis viverrini

Background Bithynia siamensis goniomphalos is the snail intermediate host of the liver fluke, Opisthorchis viverrini, the leading cause of cholangiocarcinoma (CCA) in the Greater Mekong sub-region of Thailand. Despite the severe public health impact of Opisthorchis-induced CCA, knowledge of the molecular interactions occurring between the parasite and its snail intermediate host is scant. The examination of differences in gene expression profiling between uninfected and O. viverrini-infected B. siamensis goniomphalos could provide clues on fundamental pathways involved in the regulation of snail-parasite interplay. Methodology/Principal Findings Using high-throughput (Illumina) sequencing and extensive bioinformatic analyses, we characterized the transcriptomes of uninfected and O. viverrini-infected B. siamensis goniomphalos. Comparative analyses of gene expression profiling allowed the identification of 7,655 differentially expressed genes (DEGs), associated to 43 distinct biological pathways, including pathways associated with immune defense mechanisms against parasites. Amongst the DEGs with immune functions, transcripts encoding distinct proteases displayed the highest down-regulation in Bithynia specimens infected by O. viverrini; conversely, transcription of genes encoding heat-shock proteins and actins was significantly up-regulated in parasite-infected snails when compared to the uninfected counterparts. Conclusions/Significance The present study lays the foundation for functional studies of genes and gene products potentially involved in immune-molecular mechanisms implicated in the ability of the parasite to successfully colonize its snail intermediate host. The annotated dataset provided herein represents a ready-to-use molecular resource for the discovery of molecular pathways underlying susceptibility and resistance mechanisms of B. siamensis goniomphalos to O. viverrini and for comparative analyses with pulmonate snail intermediate hosts of other platyhelminths including schistosomes.

Experimental infection of Opisthorchis viverrini cercariae to the cyprinid fish, Barbonymus gonionotus

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<0.001) age (P<0.001, ƛLR(2)=954.72) and size (P<0.001, ƛLR(2)=1204.84) were significantly associated with number of O. viverrini metacercariae recovery with in every additional fish age, the O. viverrini metacercarial recovery rate increased by 5.4% (P<0.001). Most metacercariae were found in the body of the fish (81.4%). We concluded that O. viverrini cercarial infection dosage, and age and size of fish were important determinants for a successful infection to fish.

Species-Specific Primers Designed from RAPD Products for Bithynia funiculata, the First Intermediate Host of Liver Fluke, Opisthorchis viverrini, in North Thailand

Abstract: Bithynia funiculata is the first intermediate host of the human liver fluke Opisthorchis viverrini in northern Thailand but its identification through morphological analysis is often problematic due to the shortage of gastropod taxonomists. As a consequence, we focused on the development of species-specific primers for use as an identification tool. Our work involved recovery of a 502-base pair (bp) amplicon of unknown function through species-specific primers whose effectiveness was tested by analyzing specimens of B. funiculata from 3 locations in northern Thailand. This primer set did not amplify other species in the Bithyniidae or in other gastropod families. By providing a tool to confirm morphological identifications of B. funiculata, and by enabling the identification of juvenile specimens and those with damaged shells, these primers will improve estimates of the prevalence of parasitic infections in this snail.

Infectivity and development of Opisthorchis viverrini metacercariae in immunosuppressed Barbonymus gonionotus fingerlings (Cyprinidae)

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.