Identification in immuno-electron microscopy of solitary multi-polar peripheral neurons of adult Opisthorchis viverrini by antibody against rat diacylglycerol kinase / Identificación por microscopía imuno-electrónica de las neuronas periféricas multipolares solitarias del Opisthorchis viverrini adulto mediante anticuerpo contra la diacilglicerol kinasa de ratas

By utilizing the antibody for rat DGKz a substantial number of immunopositive cells were found in the OV (Opisthorchis viverrini). The immunopositive cells appeared solitarily and they were distributed rather symmetrically to the longitudinal axis of the OV. Some of them were located in close proximity to internal organs such as uterus, ovary, testes, vitelline glands and guts. The immunostained cells extended tapering processes horizontally or obliquely to the OV longitudinal axis. In immuno-electron microscopy, the immunopositive cells were characterized by intensely immunostained mitochondria and weakly immunostained cytoplasm and immunonegative chromatin-poor nucleus. Vacuoles of various sizes without the immunoreactivity were also contained in the cells. Thin cellular processes without the immunoreactivity were found to enclose thinly the entire surfaces of the immunostained cells and processes, and they were in continuity with the interstitial partition-like processes which contained nuclei and aggregation of microfibrils at some distance from the cytoplasmic envelopes. The present finding suggests the possibility that the immunostained cells were peripheral neurons enveloped by peripheral glia and that the glia are of mesenchymal origin because of their cytoplasmic continuity to the interstitial partition-like processes. The motor or sensory nature of the neurons remains to be elucidated.

Mediante el uso del anticuerpos DGK para rata se determinó un número considerable de células inmunopositivas en el Opisthorchis viverrini (OV). Las células inmunopositivas aparecían solitarias y se distribuían simétricamente al eje longitudinal de la OV. Algunas estaban ubicadas en las proximidades de los órganos internos como el útero, ovarios, testículos, glándulas vitelinas e intestino. Las células inmunoteñidas extendían sus procesos horizontalmente u oblicuamente al eje longitudinal de la OV. Por microscopía inmunoelectrónica, las células inmunopositivas se caracterizaron por presentar mitocondrias intensamente teñidas, citoplasma con tinción débil e inmunonegatividad en núcleos pobres en cromatina. También se observó en las células, vacuolas de diversos tamaños sin inmunorreactividad. Se encontraron procesos celulares sin inmunorreactividad para cerrar finamente todas las superficies de las células y procesos, y se continuaron con los procesos de partición intersticiales que contenían núcleos y agregación de microfibrillas a cierta distancia de las envolturas citoplásmicas. El presente hallazgo sugiere la posibilidad de que las células inmunoteñidas son neuronas periféricas envueltas por glia periférica y que la glía presenta origen mesenquimal debido a su continuidad citoplasmática con los procesos de partición intersticiales. La naturaleza motora o sensorial de las neuronas aún no se ha dilucidado.

Studies on concomitant antigens of Bithynia funiculata for detection of antibody to Opisthorchis viverrini: effect of different centrifugal speeds on antigen preparation.

Antigens derived from somatic extracts of Bithynia funiculata, an intermediate snail host of O. viverrini, have been demonstrated to be highly heterogeneous in molecular weight (MW). These antigens have been suggested to be of potential use for serodiagnosis. In this study, B. funiculata somatic antigens were extracted using five different centrifugal speeds, namely 10,000 (C1); 20,000 (C2); 30,000 (C3); 40,000 (C4) and 50,000 (C5) rpm, with the aim of removing some non-specific antigens and determining the optimal centrifugal speed to obtain the highest efficiency of the test for which they will be used. The enzyme-linked immunosorbent assay technique was used to compare the reactivity of the five different centrifugal speed-prepared antigens. The sensitivity and specificity of all five antigens were compared by testing against sera from 81 opisthorchiasis patients, 30 parasite-free healthy individuals and 50 individuals infected with other helminthic infections, using mean + 4SD of all healthy individuals as the cut-off value. The sensitivity of these antigens was 69.1, 84.0, 80.2, 84.0 and 70.4, respectively; while the specificity was 66.2, 76.2, 82.5, 86.2 and 71.2, respectively. Immunoreactive components of each antigen were analyzed by SDS-PAGE and Western blot technique. The assay showed that three pairs of antigens with MW of 29 and 30, 47 and 50, and 86 and 90 kDa of all five antigens, which have previously been shown to be highly immunogenic, still reacted with a pooled serum from patients with opisthorchiasis. However, the C4 antigens gave more distinct components. Our results showed that 40,000 rpm is the optimal speed for antigen preparation for use in the serological diagnosis of opisthorchiasis, as demonstrated by the most satisfactory results of both sensitivity and specificity in the indirect ELISA and Western blot technique.

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda)

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.

Parasites elicited cross-reacting antibodies to Opisthorchis viverrini.

Two batches of crude antigens extracted from adult Opisthorchis viverrini worms were compared. One was derived from adult worms harvested from the livers of laboratory infected hamsters and another was obtained from worms sedimented from the faeces of opisthorchiasis patients following treatment with Praziquantel. SDS-PAGE and Coomassie brilliant blue staining revealed that the two preparations had similar protein components of which the predominant ones were the 17-18 kDa doublet. The antigens were used in an indirect ELISA for the detection of antibodies against O. viverrini in the sera of four groups of patients, ie. patients with opisthorchiasis (group 1), patients with mixed infections of O. viverrini and other parasites (group 2), patients with other parasitic infections (group 3), and normal-heathy, parasite-free individuals (group 4). The sensitivity of the test was high (91-92%), regardless of the batch of the antigen used. However, its specificity was relatively low (70-80%). Cross-reaction was observed with patients infected with Paragonimus heterotremus, Schistosoma spp.; Taenia spp.; Trichinella spiralis; Strongyloides stercoralis; hookworms; Plasmodium spp.; hookworms and Plasmodium spp.; S. stercoralis, Blastocystis hominis and yeast; and hookworms, Ascaris lumbricoides, Trichuris trichiura and P. falciparum. Western blot analysis revealed that sera of patients infected with these heterologous organisms contained antibodies reactive to O. viverrini antigenic components ranging from Mr 15.5 to 144.

Serological differentiation of human small fluke infections using Opisthorchis viverrini and Haplorchis taichui antigens.

Sera from 642 inhabitants of Vientiane Province (Laos) were examined by enzyme-linked immunosorbent assay (ELISA) using cytoplasmic and membranous antigens prepared from adult worms. Worms of Opisthorchis viverrini originated from liver of dissected cats, Haplorchis taichui were obtained from a stool specimen of a Laotian patient after praziquantel treatment. The sera were divided into five groups according to the intensity of infection expressed as egg count per gram of patients stool (EPG). Correlation between intensity of infection and the level of antibodies in serum was recorded. Reactions obtained using the cytoplasmic antigens were more sensitive and more specific compared to those with membranous antigens. Cross-reactions between antigens of both helminth species were found. Highly positive sera were examined using electroimmunotransfer blots (EITB) with cytoplasmic antigens of both species, which enabled the species differentiation. Antigens of both species yielded several shared fractions; however, differences between them were found: homologous sera reacted specifically with O. viverrini antigen in the area of 70 kDa and with H. taichui antigen in the area of 10 kDa. Thirty-one of 122 tested sera had specific antibodies against O. viverrini, 77 sera against H. taichui and 14 sera against both species. The results confirmed our assumption about predominant occurrence of heterophyid flukes in the human population living in studied area, compared with the occurrence of opisthorchid flukes. Hence, serology seems to be helpful tool for correct diagnosis of small fluke infections.

Diagnosis of opisthorchiasis by enzyme-linked immunosorbent assay using partially purified antigens.

Opisthorchis viverrini antigens were partially purified from adult worms collected from liver and extrahepatic biliary system of infected hamsters. Tegument fraction was obtained by chemical extraction, whereas other fractions were purified by Sephadex G-200 gel filtration chromatography. Five fractions of O. viverrini antigens were obtained, namely tegument extract, somatic extract, fraction 1 (P1), fraction 2 (P2) and fraction 3 (P3), respectively. The enzyme-linked immunosorbent assay technique was used to compare the reactivity of the five partially purified antigens. The sensitivity and specificity of all five antigens were compared by testing against the sera of 78 O. viverrini-infected individuals from O. viverrini endemic areas and 70 individuals from non-endemic areas infected with hookworm, Trichuris and Ascaris including 49 individuals with negative stool examination. The assays performed with tegument extract, somatic extract and P1 fraction were found to have 100% sensitivity, whereas the sensitivities of those with P2 and P3 were 96.1% and 83.3%, respectively. The tegument extract had the highest specificity as demonstrated by the lowest cross-reactivity with other parasites. Our results indicated that surface tegument is the most suitable antigen for use in immunological diagnosis of opisthorchiasis.

Detection of antibodies against Opisthorchis viverrini in patients before and after treatment with praziquantel.

Levels of antibody in sera of 78 patients with opisthorchiasis, 30 patients with other liver diseases, 10 patients with schistosomiasis and 30 healthy individuals were compared using three serodiagnostic tests, namely indirect haemagglutination (IHA), enzyme-linked immunosorbent assay (ELISA) and lectin immuno test (LIT). The geometric mean reciprocal titer in sera of opisthorchiasis patients was significantly higher than patients with other diseases, patients with schistosomiasis and healthy individuals (p less than 0.00001). After treatment with praziquantel, the antibody titers were decreased and became lowest 120 days after treatment. A statistically significant decrease from the pre-treatment sample was observed only at 120 days after infection and not earlier and only with ELISA (p = 0.03) and not with IHA and LIT (p greater than 0.05). Even with ELISA, significant decrease in antibody titer was apparent only when the pre-treatment sera had high enough antibody titer. ELISA was therefore better than the other two tests for the assessment of cure provided that the titer of pre-treatment sera was high.

Effect of praziquantel treatment on antibody levels and lymphoproliferative responses in patients with opisthorchiasis.

The purpose of this study was to explore alternative method(s) to monitor the efficacy of anthelmintic treatment of patients with opisthorchiasis. Therefore, in our initial attempt, we studied the changes in antibody levels and lymphoproliferative responses in O. viverrini infected patients before and 2 months after successful praziquantel treatment. The results showed that although a substantial reduction of the antibody levels occurred after such a treatment, it did not occur in all patients. In those showing reduction, the final level were still above 2 standard deviations of the normal mean value. The reduction was more profound for IgG antibody. With regard to the IgA antibody isotype, the reduction was not as marked. In contrast, IgE antibody levels in most patients not only failed to decline, but instead, showed a tendency to be elevated after praziquantel treatment. Unlike the antibody levels, there was no alteration in the lymphoproliferative response to PHA stimulation and therefore this parameter is not useful for our intended objective. It was suggested that studies of a more specific O. viverrini component may be more reliable than the current method of parasitological examination of eggs in the feces of suspected individuals.

Analysis of Opisthorchis viverrini antigens: physicochemical characterization and antigen localization.

Antigens of Opisthorchis viverrini were identified and characterized by enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis following radioimmuno-precipitation, and indirect immunofluorescence. The immunoreactive protein with a relative molecular weight (Mr) of 89 kD was the predominating component of the parasite metabolic products. An immunofluorescence study showed it to be associated with parasite eggs, linings of the reproductive system and secretions therein. Protein of the surface tegument had Mr of greater than 116, 108, 64, 38, 34, 20 and 16-17 kD. The 16-17 kD surface molecule was the predominant protein, representing approximately 50% of the total protein in crude aqueous extracts of adult worms. However, it was poorly immunogenic when compared with the 89 kD molecule. Together with data presented previously, it appears that in addition to the 89 kD protein, several tegumental molecules are also specific for O. viverrini and have immuno-diagnostic potential.

Immunization of hamsters against Opisthorchis viverrini infection.

Attempts were made to induce acquired immunity against Opisthorchis viverrini infection in hamsters by immunizing them with aqueous somatic extract and metabolic products of adult worms, crude adult worm homogenates and metacercarial somatic extracts via either the intraperitoneal or combined intraperitoneal and oral routes. These procedures failed to stimulate significant protective response in animals that had never been exposed to O. viverrini. However, the protective response reached a significant level (30% worm reduction) in hamsters that had been infected with a small member of flukes prior to immunization with aqueous somatic extract of adult worms. Although these findings indicate that it may be possible to reduce reinfection in people in the endemic area by immunization, it appears that a better method currently available for the control of O. viverrini infection is health education aimed at changing food habits and improving sanitation and personal hygiene.

Enzyme-linked immunosorbent assay for detection of Opisthorchis viverrini infection.

Antibodies to O. viverrini in the sera of people from endemic and non-endemic areas were investigated using indirect ELISA technique. For the patients from the endemic area, 92.8% who passed eggs in the stool were found to be positive for O. viverrini antibody. In addition, 46.5% of the people who did not pass eggs in the stool were also found to have low titer of O. viverrini antibody. On the other hand only 2.4% of the people from the non-endemic area with other intestinal parasite infections were found to have O. viverrini antibody in their sera. It was concluded that positive reaction of O. viverrini antibody is not cause by cross-reaction with other parasites but low liter of antibody is probably due to low-level or past infection. There is a positive correlation between the titer of O. viverrini antibody and intensity of infection as indicated by number of eggs excreted per milligram of feces. Patients with a few O. viverrini eggs in feces, but biopsy-proved-cholangiocarcinoma had very high titer of antibody.

Humoral immune responses in hamsters infected with Opisthorchis viverrini.

The kinetics and nature of humoral immune responses to somatic and excretory-secretory (ES) antigens were investigated in hamsters experimentally infected with different numbers of Opisthorchis viverrini. ES antigens were obtained from the in vitro culture of adult flukes and somatic antigens were aqueous extracts of adult flukes. Antibodies in the serum and bile of infected animals were determined by the microhaemagglutination technique, using glutaraldehyde fixed sheep red blood cells sensitized with these parasite antigens. Antibody responses to both somatic and ES antigens were detected in the serum from the second week of infection onward. The peak response was noted at the end of the second month and declined slowly thereafter. Antibody levels in animals with heavy infections (100 metacercariae) appeared earlier but declined more rapidly than in animals with light infections (25 metacercariae). The serum antibodies were highly sensitive to mercaptoethanol throughout the course of infection (23 weeks). Antibodies also appeared in the bile obtained at the time of sacrifice but their titres were rather low compared with those in the serum. Like serum antibodies, biliary antibodies were reactive with both somatic and ES antigens. Biliary antibodies were of the secondary IgA type. These findings are discussed in relation to pathogenesis of the disease process and to the possible usefulness in immunodiagnosis.

Anti-P1, allohemagglutinins associated with Clonorchis sinensis and opisthorchis viverrini infections in patients from Southeast Asia.

An automated assay of anti-P1 allohemagglutinins has been carried out on sera of 61 individuals from Southeast Asia : 28 with clonorchiasis, 18 with opisthorchiasis and 15 control subjects. Anti-P1 activity was detected in 61% of the opisthorchiasis sera, 57% of the clonorchiasis sera and in 26.6% of the control subjects. Their concentration, in the sera, was low in control subjects and exceptionally high in clonorchiasis and opisthorchiasis (up to 13 and 22 times the maximum concentration of the control subjects, respectively). In all cases the anti-P1 antibodies were of IgM class. The results suggested that Clonorchis and Opisthorchis were responsible for immunization of the patients, with P1 alloantigen.