Characteristics of granuloma formation and liver fibrosis in murine schistosomiasis mekongi: a morphological comparison between Schistosoma mekongi and S. japonicum infection.

A histopathological study was performed to clarify the characteristics of granuloma formation and liver fibrosis in Schistosoma mekongi infection in comparison with S. japonicum infection. Mice were exposed to S. mekongi (Laotian strain) and S. japonicum (Japanese strain) cercariae, and were dissected at 6, 8, 12, 16, and 20 weeks post-exposure. In the liver, granulomas in S. mekongi infection were cellular, initially organized with foam cells, and continuously appeared in the intralobular area, while granulomas in S. japonicum infection were fibrous and did not continuously appear in the intralobular area. Portal fibrosis was not seen in S. mekongi infection, but was commonly seen in S. japonicum infection in the later weeks. Granulomas in the small intestine were seen mainly in the submucosa with foam cells in S. mekongi infection and without foam cells in S. japonicum infection. The lung granulomas contained mainly histiocytes in both S. mekongi and S. japonicum infection. The absence of portal fibrosis in S. mekongi infection allows schistosome eggs to infiltrate into the intralobular area continuously, which can be what lies behind the ultrasonographic differences; the echogenic network pattern as was seen in S. japonicum infection, has not been noted in S. mekongi infection.

Affinity purified oval antigen for diagnosis of Opisthorchiasis viverrini.

Monoclonal antibodies (MAb) were raised against an oval antigen of the liver fluke Opisthorchis viverrini which is the causative agent of a parasitosis, i.e. opisthorchiasis in Thailand. The antibodies were used in an affinity column to purify the O. viverrini oval antigen from a crude extract of adult parasites by chromatography. The oval antigen was then used in a membrane (dot) ELISA for detecting antibodies in serum samples of parasitologically confirmed Opisthorchis viverrini infected individuals (adult parasites were found in stools after praziquantel treatment and salt purgation), as well as of individuals infected with other parasites and parasite-free controls. The MAb-based dot-ELISA using the affinity purified O. viverrini oval antigen revealed 100% sensitivity, specificity and accuracy for detecting O. viverrini infection. The test is simple, rapid and highly reproducible. Several samples can be tested at the same time without the requirement for special equipment or much increase in testing time; thus it is suitable for mass screening for O. viverrini exposure, especially in new endemic areas. Furthermore using serum specimens could increase patient and community compliance compared to the conventional parasitological survey which uses stool samples for the detection of O. viverrini ova, without treatment and subsequent salt purgation, this conventional method shows a low sensitivity and is also unpleasant to both the sample donors and the laboratory technicians which has historically shown a further negative impact on the final outcome.

Serum antibody responses in opisthorchiasis.

We evaluated an enzyme-linked immunosorbent assay using crude parasite homogenates as a diagnostic test for Opisthorchis viverrini infection in humans. Serum antibody (Ab) responses to O. viverrini adult worm homogenate (AWH) and metacercaria homogenate (MH) were studied in 83 infected residents of an opisthorchiasis-endemic area in Thailand. Elevated levels of Ab persisted for over 1 year following curative treatment with praziquantel, and cross-reactivity to O. viverrini AWH and MH antigens was observed in sera from individuals with other parasitic infections. Serum Ab to crude AWH and MH are therefore unsuitable for immunodiagnosis since they may be non-specific and would not differentiate between ongoing and past infection.

Possible protective immunity in human opisthorchiasis.

Chronic infections with the liver flukes Opisthorchis viverrini and Clonorchis sinensis affect over 30 million people in southeastern Asia. With ongoing exposure, reinfection readily occurs following curative treatment and cumulative infections result in significant morbidity and a predisposition to cholangiocarcinoma. Though protective immunity has never been described in human opisthorchiasis, heterogeneity in worm burden occurs and a small number of exposed residents of endemic areas remain apparently uninfected. To explore the nature of this heterogeneity, we compared levels of serum antibody (Ab) to O. viverrini measured by an enzyme-linked immunosorbent assay in 83 stool egg-positive and 49 stool egg-negative residents of an O. viverrini-endemic area in Thailand. Compared to the egg-positive residents, the egg-negative group had significantly higher levels of immunoglobulin (Ig)G, IgA and IgM to adult worm homogenate (AWH) and total Ab to metacercaria homogenate (MH). Furthermore, immunoblot analyses revealed that a significantly higher proportion of sera from the egg-negative residents had IgA reactivity against a 38-kDa AWH antigen and IgM reactivity against carbohydrate epitopes of a 42-kDa AWH glycoprotein antigen. These findings support a hypothesis that the egg-negative group includes individuals who may be immunologically resistant to this usually chronic infection.

Separation and characterization of adult worm proteins and glycoproteins from the liver fluke Opisthorchis viverrini.

Detailed studies of liver fluke proteins and antigens are necessary to facilitate further investigation of the human immune responses to these parasites. Accordingly, Opisthorchis viverrini antigens were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. We initially encountered excessive background smearing, vertical streaking, and indistinct bands that were similar to problems previously described by investigators studying this and other trematodes including Schistosoma mansoni. These problems were especially evident with silver staining of proteins and occurred despite the extensive use of protease inhibitors. They were minimized by using mini (vs. large) SDS-PAGE and Coomassie blue protein staining. With the latter 2 techniques, adult worm somatic proteins and excretory-secretory products were separated and characterized. Immunoblots using rabbit anti-adult worm sera demonstrated that some of these proteins were antigens common to both the adult and metacercarial stages. Several of these antigens also corresponded (according to molecular weight) to glycoproteins, detected by concanavalin A blotting. These findings form a base for subsequent studies of the human immune response to liver fluke infection.

Specific monoclonal antibodies to Opisthorchis viverrini.

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.