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

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.

Apoptosis-related gene expression in hamster opisthorchiasis post praziquantel treatment.

The aim of this study was to investigate the apoptosis-related gene expression in hamster opisthorchiasis after praziquantel treatment. Hamsters were infected with Opisthorchis viverrini metacercariae then treated with praziquantel. The expression of apoptosis-related genes [i.e., apoptosis gene Bcl-2-associated protein X (BAX), caspase 9, p53, and protein kinase B (PKB)] was detected by real-time reverse transcription polymerase chain reaction. Histopathological analyses of liver tissues were studies by staining the sections with hematoxylin and eosin using light microscopy. Apoptotic assay was used to localize the apoptotic cell death. The results show that BAX, Akt/PKB, p53, and caspase 9 expression level were significantly increased on day 30 post infection and at 6 h post treatment and gradually decreased nearly to the uninfected control and 24 h post treatment, perhaps due to a decrease in inflammatory cells. Apoptotic staining was positive reaction at inflammatory cells and nuclei of epithelial bile ducts. Although using praziquantel has an advantage in killing parasites, our results show the effect of praziquantel treatment from host immune response that induces increased apoptosis-related genes in the short term due to an increase in inflammatory cells surrounding the bile ducts.

The ELISA-based detection of anti-Opisthorchis viverrini IgG and IgG4 in samples of human urine and serum from an endemic area of north-eastern Thailand.

The levels of correlation between the number of Opisthorchis viverrini eggs excreted in the faeces and levels of anti-Opisthorchis IgG and IgG(4) in the serum and urine (as indicated by absorbances in ELISA) have recently been evaluated in north-eastern Thailand. The 225 subjects investigated in detail, all of whom came from an endemic village in Chaiyaphum province, were selected on the basis of the numbers of O. viverrini eggs that they were excreting. ELISA based on a crude antigen extract of the trematode were then used to determine the levels of specific IgG and IgG(4) in serum and urine samples. Compared with the egg-negative, the villagers who were found to be egg-positive for O. viverrini had significantly higher levels of specific IgG in their urine and serum and significantly higher levels of specific IgG(4) in their serum. The serum levels of specific IgG and IgG(4) and the urine levels of specific IgG all correlated with the numbers of O. viverrini eggs/g faeces [with correlation coefficients (r) of 0.251, 0.121 and 0.142, respectively]. Although the serum levels of IgG were positively correlated with the urine levels of IgG (r=0.098), there was no significant relationship between the serum and urine levels of specific IgG(4) (r=0.051). When the 225 subjects investigated in the ELISA were divided according to whether they had no detectable Opisthorchis eggs in their faeces (N=57), or 1-100 (N=154), 101-1000 (N=5), 1001-1500 (N=5) or >1501 (N=4) eggs/g faeces, the serum and urine levels of specific IgG and the serum (but not urine) levels of specific IgG4 were also found to correlate significantly with the infection-intensity categories (with r-values of 0.550, 0.146 and 0.578, respectively). When the results of the faecal examinations were treated as the 'gold standard', the ELISA for the detection of (Opisthorchis-specific) serum IgG, serum IgG(4), urine IgG and urine IgG(4) had sensitivities of 99.2%, 23.1%, 43.0% and 45.9% and specificities of 93.0%, 29.6%, 45.9% and 67.2%, respectively. Although the study was limited by the small number of subjects with intense infections, it appears worth investigating urine samples for subclasses of specific IgG other than IgG(4).

Apoptosis-related gene expressions in hamsters re-infected with Opisthorchis viverrini and re-treated with praziquantel.

Our objective was to reveal whether host immune response in hamster opisthorchiasis post-praziquantel treatment could induce apoptotic cell death in inflammatory cells. We, therefore, investigated apoptosis-related gene expression in hamsters re-infected with Opisthorchis viverrini (OV) and re-treated with praziquantel. Hamsters were re-infected with OV metacercariae then re-treated with praziquantel. The expression of apoptosis-related genes (i.e. apoptosis gene Bcl-2 associated protein X [BAX], caspase 9, p53 and protein kinase B [PKB]) was detected by real-time reverse transcription-polymerase chain reaction. Histopathological analyses of liver tissues were performed by staining the sections with haematoxylin and eosin using light microscopy. The results show that BAX, Akt/PKB, p53 and caspase 9 expression levels were significantly increased on day 30 post-infection and at 6 h post-treatment and gradually decreased to a level near the uninfected control and at 24 h post-treatment, perhaps because of a decrease in inflammatory cells. Apoptotic cell death was observed at the nuclei of epithelial cells of the bile ducts and of T cells. Our results suggest that repeated infection with OV and re-treatment with praziquantel induces a host immune response that increases inflammatory cells, which in turn leads to increase, apoptosis-related gene expression in the short term post-treatment.

Nucleotide sequence of mitochondrial CO I and ribosomal ITS II genes of Opisthorchis viverrini in northeast Thailand.

The mitochondrial cytochrome c oxidase subunit I (CO I) gene and the second internal transcribed spacer region (ITS II) gene of Opisthorchis viverrini were compared among O. viverrini from various areas in northeast Thailand. The nucleotide sequences of partial CO I gene (417 bp) of O. viverrini differed among O. viverrini originated from Ubon Ratana, Leongpleuy, Ban Phai, Maha Sarakham, and Chatturat. These intraspecific variations were classified into 5 patterns but no area-specific pattem was observed. Amino acid sequence deduced from the nucleotide sequences of these genes was identical. Nucleotide sequences of a region of the O. viverrini ITS II gene (296 bp) from different areas were identical. However, they were different from those of Clonorchis sinensis, Haplorchis taichui, H. pumilio, Fasciola gigantica, Echinostoma malayanum and Centrocestus sp..

Seasonal variation of Opisthorchis viverrini infection in cyprinoid fish in north-east Thailand: implications for parasite control and food safety.

Reported is the seasonal pattern of Opisthorchis viverrini metacercariae in cyprinoid fish in north-east Thailand. Samples of fish were collected in 1991-92 at monthly intervals from two areas-Khon Kaen Province, where the opisthorchiasis transmission rate was high, and Mahasarakham Province, where the rate was low. Metacercarial loads in both study areas had similar seasonal patterns. High burdens occurred in the late rainy season and winter (July to January) with low burdens during the summer (March to June). The average burden for Puntius leiacanthus in Khon Kaen was 1.68 metacercariae per fish (127.43 per kg), higher than for all species of cyprinoid fish from the low transmission area. The intensities of infection among P. leiacanthus and Cyclocheilichthys armatus collected in Mahasarakham were comparable, but lower than the intensity of Hampala dispar (0.75 metacercariae per fish) concurrently sampled from the same area (P < 0.05). There was no significant difference in metacercarial load per kg between fish species from Mahasarakham. The results indicate that seasonal variation in metacercariae was a common phenomenon in areas with both high and low endemicity of infection. Also, the metacerarial load in fish was positively associated with infection levels among humans.

PIP: Food-borne trematode infections such as Opisthorchis viverrini are a major cause of morbidity in Asia. Samples of cyprinoid fish collected at monthly intervals during 1991-92 in two provinces (Khon Kaen and Mahasarakham) in North East Thailand revealed substantial seasonal variation of O viverrini metacercariae. The former province is a low transmission area (29.7% average infection rate), while the latter is a high transmission area. Metacercarial intensity was highest from January-March 1991 and October 1991-April 1992, with maximum intensities during February and December 1991. Puntius leiacanthus fish from Khon Kaen bore the highest density of metacercariae per individual fish (1.68). Differences in the intensity of infection for the same species of fish captured in two different geographic areas presumably reflect variations in environmental conditions such as local levels of human and snail infections. Fecal contamination of the study reservoirs is greatest during the early parts of the rainy season as a result of run-off drainage and coincides with the rapid increase in snail populations. The optimal time for large-scale community-based parasite control programs is when the probability of rapid reinfection is lowest and the prevalence and intensity of infection in people are highest (e.g., April-June or the summer months). Most effective is a program that integrates parasite control, environmental management, health education, and food safety.

Metacercariae isolated from cyprinoid fishes in Khon Kaen District by digestion technic.

Several kinds of fresh-water fishes collected from natural water resources in Khon Kaen District were digested with 0.25% pepsin A to obtain the infective stage or metacercariae of fish-borne trematodes. Most of them harbored at least 3 species of trematodes. Two families of medical trematodes, i.e., Family heterophyidae and Family Opisthorchiidae, were isolated. The heterophyid flukes consisted of Haplorchis taichui, H. pumilio and Centrocestus formosanus. Opisthorchis viverrini was the only parasite of the Family Opisthorchiidae found in these fish. Among these four species, H. taichui was predominant and found in all kinds of fish examined, especially Hampala dispar.

Centrocestus formosanus: surface morphology of metacercaria, adult and egg

The surface morphology of metacercariae isolated from Puntius spp., adult worms from infected hamsters and eggs of Centrocestus formosanus (Digenea: Heterophyidae) were studied using scanning electron microscopy. It was found that the surfaces of the metacercariae and adult worms were closely similar in appearance. The oral sucker was surrounded by a circumoral expansion with two rows of 32 spines (16 spines each). The ventral sucker, with six large nonciliated papillae on the lip, and the genital opening were located mid-ventrally. The excretory pore was terminal. The body surface was covered with pectinate scale-like spines of varying sizes. The scales in the middle area were larger than those in the anterior and posterior parts of the body. Sensory papillae, mostly uniciliated, were present in greater abundance at the anterior region of both stages. However, morphological variations were observed among the adults depending on the age of the worms. At 4 weeks, the oral sucker and circumoral expansion were cobblestone-like in structure and the grooves of spines split compared with their smooth surfaces at 10 days. In addition, the ventral sucker became depressed and its large papillae on the lip disappeared at 10 weeks post infection. Multiciliated papillae were also present in 10-week old worms. Each egg of this parasite possessed a prominent operculum and a latticed design on the egg shell.