Supawadee Piratae

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.

Molecular characterization of a tetraspanin from the human liver fluke, Opisthorchis viverrini.

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.

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.

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.

Suppression of mRNAs encoding CD63 family tetraspanins from the carcinogenic liver fluke Opisthorchis viverrini results in distinct tegument phenotypes

The liver fluke Opisthorchis viverrini infects 10 million people in Southeast Asia and causes cholangiocarcinoma (CCA). Fluke secreted and tegumental proteins contribute to the generation of a tumorigenic environment and are targets for drug and vaccine-based control measures. Herein, we identified two tetraspanins belonging to the CD63 family (Ov-TSP-2 and Ov-TSP-3) that are abundantly expressed in the tegument proteome of O. viverrini. Ov-tsp-2 and tsp-3 transcripts were detected in all developmental stages of O. viverrini. Protein fragments corresponding to the large extracellular loop (LEL) of each TSP were produced in recombinant form and antibodies were raised in rabbits. Ov-TSP-2 and TSP-3 were detected in whole worm extracts and excretory/secretory products of O. viverrini and reacted with sera from infected hamsters and humans. Antibodies confirmed localization of Ov-TSP-2 and TSP-3 to the adult fluke tegument. Using RNA interference, Ov-tsp-2 and tsp-3 mRNA expression was significantly suppressed for up to 21 days in vitro. Ultrastructural observation of tsp-2 and tsp-3 dsRNA-treated flukes resulted in phenotypes with increased tegument thickness, increased vacuolation (tsp-2) and reduced electron density (tsp-3). These studies confirm the importance of CD63 family tegument tetraspanins in parasitic flukes and support efforts to target these proteins for vaccine development.

Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos

Abstract The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24–28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.

Effects of Surface Salt on Distribution and Density of Snails in Family Viviparidae in Khon Kaen Province, Thailand, Analyses by Using Geographic Information System

Effects of surface salt on distribution of snails in family Viviparidae in Khon Kaen province, northeast Thailand were analyzed the association model by using geographic information system. Mollusks were collected in twenty localities base on various levels of surface salt, 16 – 20 stations/reservoir. Three species of viviparid snails were found in 9 out of 20 reservoirs, i.e. Filopaludina (Siamopaludina) martensi martensi, F. (Filopaludina) sumatrensis speciosa and Idiopoma umbilicata. Nine families (11 species) of sympatric mollusks shared the same habitat with viviparid snails. Water quality in the viviparid habitat revealed that, pH: 6.79 - 8.01, temperature: 23.06- 27.33 oC, turbidity: 3.26 – 163.00 NTU, conductivity: 0.42 - 10.25 mS/cm, salinity: 0.20 - 5.69 ppk and dissolved oxygen (DO): 0.43 – 6.01 mg/L. Dominant planktons in food content from digestive tracts of F. (S.) martensi martensi consisted of diatoms and they were the same type to Bithynia siamensis goniomphalos, first intermediate host of human liver fluke, Opisthorchis viverrini. That is viviparid snails may play important role in food competition with B. siamensis goniomphalos. The number of B. siamensis goniomphalos was negative correlation to the number of F. (S.) martensi martensi (r = - 0.182, p 0 - 50% of surface areas revealed that the index of 0.079, 0.027, 0.001 and 0, respectively. The geographic information system analysis model was assessed and approved by mollusk survey. ผลของเกลอผวดนตอการกระจายและความหนาแนนของหอยในวงViviparidaeในจงหวดขอนแกน ประเทศไทย โดยวเคราะหขอมลดวยระบบสารสนเทศภมศาสตร ศกษาโดยเกบตวอยางหอยในแหลงนำทมสภาพเกลอผวดนทระดบแตกตางกน ทงหมด 20 แหลงนำ ทำการสมเกบตวอยางในแตละแหลงนำ 16-20 สถาน พบหอยในวงศ Viviparidae 3 ชนดคอFilopaludina (Siamopaludina) martensi martensi, F. (Filopaludina)sumatrensis speciosa และ Idiopoma umbilicataในแหลงนำท9 แหลงจากจำนวนททำการเกบตวอยาง20 แหลงพบหอยรวมนเวศ 9 วงศ (จำนวน 11 ชนด)ลกษณะทางภายภาพนำทพบหอยวงศ Viviparidae ม pH 6.79 -8.01 อณหภม 23.06- 27.33 องศา,คาความขน 3.26 - 163.00 NTU,คาการนำไฟฟา 0.42 - 10.25 mS/cm,คาความเคม 0.20 - 5.69 คาการละลายของออกซเจน (DO) 0.43 - 6.01 mg/L. ในระบบทางเดนอาหารของหอย F. (S.) martensi martensi ทเปนประชากรสวนใหญในการศกษาครงน พบชนดของแพลงกตอนเหมอนกนกบBithynia siamensis goniomphalos ซงเปนโฮสตกลางของพยาธใบไมตบของคนชนดOpisthorchis viverrini จงนาจะเปนสตวทแกงแยงอาหารกน ซงพบวาจำนวนหอย B. siamensis goniomphalos มความสมพนธเชงลบกบหอย martensi martensi (r = - 0.182, p 0 - 50% ของพนทผวดนโดยม คาดชนเทากบ .079,0.027, 0.001และ 0 ตมลำดบ คาดชนไดทำการประเมนความถกตองโดยการสำรวจในพนท ทไดทำการวเคราะหแลวไดคาทถกตอง