Apiporn Suwannatrai

Light and electron microscopy observations of embryogenesis and egg development in the human liver fluke, Opisthorchis viverrini (Platyhelminthes, Digenea).

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.

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.

Modeling impacts of climate change on the potential distribution of the carcinogenic liver fluke, Opisthorchis viverrini, in Thailand

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.

Seasonal and Spatial Environmental Influence on Opisthorchis viverrini Intermediate Hosts, Abundance, and Distribution: Insights on Transmission Dynamics and Sustainable Control

Background Opisthorchis viverrini (Ov) is a complex-life-cycle trematode affecting 10 million people in SEA (Southeast Asia). Human infection occurs when infected cyprinid fish are consumed raw or undercooked. Ov requires three hosts and presents two free-living parasitic stages. As a consequence Ov transmission and infection in intermediate and human hosts are strongly mediated by environmental factors and understanding how environmental variability influences intermediate host abundance is critical. The objectives of this study were 1) to document water parameters, intermediate hosts abundance and infection spatio-temporal variation, 2) to assess their causal relationships and identify windows of transmission risk. Methodology/Principal Findings Fish and snails were collected monthly for one year at 12 sites in Lawa Lake, an Ov-endemic region of Khon Kaen Province in Northeast Thailand. Physicochemical water parameters [pH, temperature (Tp), dissolved oxygen (DO), Salinity, electrical conductivity (EC), total dissolved solid (TDS), nitrite nitrogen (NO2-N), lead (Pb), total coliform bacteria (TCB) and fecal coliform bacteria (FCB)] were measured. Multivariate analyses, linear models and kriging were used to characterize water parameter variation and its influence on host abundance and infection prevalence. We found that sampling sites could be grouped in three clusters and discriminated along a nitrogen-salinity gradient where higher levels in the lake’s southern region predicted higher Bithynia relative abundance (P<0.05) and lower snail and fish species diversity (P<0.05). Highest Bithynia abundance occurred during rainy season (P<0.001), independently of site influence. Cyprinids were the most abundant fish family and higher cyprinid relative abundance was found in areas with higher Bithynia relative abundance (P<0.05). Ov infection in snails was anecdotal while Ov infection in fish was higher in the southern region (P<0.001) at sites showing high FCB. Conclusions/Significance Our results indicate that water contamination and waterways configuration can influence freshwater communities’ assemblages possibly creating ideal conditions for sustained transmission. Sustainable control may require a better appreciation of the system’s ecology with wise governance and development planning particularly in the current context of SEA agricultural intensification and landscape modification.

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.

Opisthorchis viverrini infection in the snail and fish intermediate hosts in Central Vietnam

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.

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.

A Large Scale Study of the Epidemiology and Risk Factors for the Carcinogenic Liver Fluke Opisthorchis viverrini in Udon Thani Province, Thailand

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<0.0001; OR = 7.3 (95% CI = 1.8-21.6)) and eating habits (p = 0.032; OR = 1.6 (95% CI = 0.5-3.7)). Interestingly, most participants were not aware of treatments such as praziquantel (p< 0.0001; OR = 3.5 (95% CI = 1.4-11.6)), had no history of parasitic treatment (p = 0.486; OR = 1.5 (95% CI = 0.5-3.5)) and had eaten raw fish (p = 0.04; OR = 7.4 (95% CI = 1.5-18.6)). Liver fluke infection in dogs (18.1%, epg. = 44.7, range; 32-96) was significantly higher than in cats (11.0%, epg. = 117.8, range; 44-372) (p<0.05). A positive association between O. viverrini infection in dogs and their owners was found. In addition, cyprinid fish dominantly infected by metacercaria including Henicorhynchus siamensis (27.7%), Cyclocheilichthys repasson (21.9%), Hampala dispar (14.1%), and Barbonymus gonionotus (6.9%). This study provides basic information required for the development of future effective and sustainable strategies to reduces infection rates, mainly by providing health education and encouraging behavioural changes.