Effects of physicochemical factors on development and survival of Opisthorchis viverrini uterine eggs

Objective: To investigate the maturity development of miracidia in uterine eggs from four portions of the Opisthorchis viverrini uterus and environmental factors possibly affected in maturation and infectivity of distal part uterine eggs. Methods: Uteri of adult worms were divided into 4 equal parts. Development of eggs was determined under light microscope. Only embryonated eggs were used to evaluate the effects of physicochemical factors: temperature, salinity, acidity, ultraviolet A, B, C. Infection success was evaluated by feeding treated eggs to intermediate host snails and determining by using a PCR approach. Results: Eggs obtained from the uterus closest to the ovary (regions 1 and 2) failed to develop in vitro. Eggs from region 4 of the uterus (close to the genital pore) were used to study effects of physicochemical factors. The highest survival and infection success was in groups of eggs kept at 30 ℃ (95.20％). The calculated period of loss infection success (LI50 and LI95) on miracidia in distal uterine eggs by exposure to UV-A, UV-B and UV-C were 73 and 1523 d; 8 and 20 d; 1 and 2 d, respectively. Lethal concentrations (LC50 and LC95) of salinity, HCl and NaOH on miracidia in distal uterine eggs were 45.43 and 120.09 ppt, 0.01 and 0.25 M; 0.01 and 0.11 M, respectively, after 24 h exposure. Conclusions: Opisthorchis viverrini eggs display a high tolerance to environmental conditions, especially after snail host eating for infection.

Field investigation of Bithynia funiculata, intermediate host of Opisthorchis viverrini in northern Thailand.

A survey of freshwater snail, Bithynia funiculata, was conducted in four locations, Doi Saket, Mueang, Saraphi and Mae Rim Districts of Chiang Mai Province, northern Thailand, between June and October, during the rainy season of 2004. A total of 2,240 snails was collected and classified into 7 families and 15 genera; of which 352 B. funiculata were obtained. B. funiculata was found most abundant in July and September. The infection rate of trematode cercariae in B. funiculata was 9.6% (19/352), while that of pleurolophocercous was 0.3% (1/352). Virgulate cercaria was the most common type, followed by lophocercous, monostome and pleurolophocercous. B. funiculata prefers habitats with clear water, which was less than 30 cm depth, temperatures between 24.48 and 31.78 degrees C, dissolved oxygen 2.03-7.66 mg/l, saturated dissolved oxygen 26.70-95.00%, conductivity 0.000-0.2642 mS/cm, turbidity 16.00-288.00 NTU and pH 6.58-7.56. Geographic Information System (GIS) analysis using loose soil mixed with clay revealed that the breeding grounds of this snail species was in the paddy fields and village environs of the Ping, Kuang and Fang river basins.

Molecular cloning and characterization of a glutathione S-transferase encoding gene from Opisthorchis viverrini.

An adult stage Opisthorchis viverrini cDNA library was constructed and screened for abundant transcripts. One of the isolated cDNAs was found by sequence comparison to encode a glutathione S-transferase (GST) and was further analyzed for RNA expression, encoded protein function, tissue distribution and cross-reactivity of the encoded protein with other trematode protein counterparts. The cDNA has a size of 893 bp and encodes a GST of 213 amino acids length (OV28GST). The most closely-related GST of OV28GST among those published for trematodes is a 28 kDa GST of Clonorchis sinensis as shown by multiple sequence alignment and phylogenetic analysis. Northern analysis of total RNA with a gene-specific probe revealed a 900 nucleotide OV28GST transcriptional product in the adult parasite. Through RNA in situ hybridization OV28GST RNA was detected in the parenchymal cells of adult parasites. This result was confirmed by immunolocalization of OV28GST with an antiserum generated in a mouse against bacterially-produced recombinant OV28GST. Both, purified recombinant and purified native OV28GST were resolved as 28 kDa proteins by SDS-PAGE. Using the anti-recOV28GST antiserum, no or only weak cross-reactivity was observed in an immunoblot of crude worm extracts against the GSTs of Schistosoma mansoni, S. japonicum, S. mekongi, Eurytrema spp. and Fasciola gigantica. The enzyme activity of the purified recombinant OV28GST was verified by a standard 1-chloro-2, 4-dinitrobenzene (CDNB) based activity assay. The present results of our molecular analysis of OV28GST should be helpful in the ongoing development of diagnostic applications for opisthorchiasis viverrini.

Diversity of mollusks in the Lam Ta Khong reservoir, Nakhon Ratchasima, Thailand.

Sampling surveys to study the diversity of mollusks in the Lam Ta Khong reservoir, Nakhon Ratchasima Province, northeast Thailand, were carried out in the hot, rainy and cold seasons. The study area was divided into: Area I--the upper part where the Lam Ta Khong river drains; Area II--the mid-section of the reservoir; Area III--behind the dam. Mollusks were collected from four locations on each bank (to the right and left) of each area. Each location was sampled to include 6 cross-sectional stations; in total, 144 stations were sampled. In the deep water, an Ekman dredge was used to collect samples; the scoop or manual method was used at the water's edge. Ten species of snails and four species of clams were found. The dominant species of snails were: Clea helena, Bithynia siamensis goniomphalos and Melanoides tuberculata; clams were dominated by Corbicula sp. The intermediate host of the human blood fluke was not found. The population of most mollusk species increased during the cold season while that of clams and that of some species of snails increased during the rainy season. Clams and operculate snails predominated in Areas II and III. Pulmonate snails were mostly found close to the bank and on aquatic plants especially in Areas I and II. Operculate snails and clams mainly inhabited water 1 to 10 m deep. Two species of edible mollusks were found: Filopaludina martensi martensi and large numbers of Corbicula. Neither shedding light nor digestion with pepsin A revealed any human parasites in the mollusks sampled.