Thin layer immunoassay was carried out to demonstrate antibodies against Clonorchis sinensis in sera from clonorchiasis patients. Saline extract of adult worm was used as antigen. TIA technique was performed as described earlier by Elwing et al. (1976), but agarose was used instead of agar. The antibody titres of sera in 60 clonorchiasis cases were higher than that of 10 healthy and 10 amoebiasis cases, but not different comparing with that of 10 paragonimiasis cases. Antibody titres in clonorchiasis gave no differences according to the age, sex, EPG in feces, eosinophilia degree of blood, level of alkaline phosphatase and transaminase (SGOT, SGPT) in sera. It is suggested that, after evaluation, the TIA might supplement or be used as an alternative to other immunodiagnostic tests already in use for the diagnosis of clonorchiasis.
parasitology-helminth-trematoda ; Clonorchis sinensis ; clonorchiasis ; immunology

Thin layer immunoassay was carried out to demonstrate antibodies against Clonorchis sinensis in sera from clonorchiasis patients. Saline extract of adult worm was used as antigen. TIA technique was performed as described earlier by Elwing et al. (1976), but agarose was used instead of agar. The antibody titres of sera in 60 clonorchiasis cases were higher than that of 10 healthy and 10 amoebiasis cases, but not different comparing with that of 10 paragonimiasis cases. Antibody titres in clonorchiasis gave no differences according to the age, sex, EPG in feces, eosinophilia degree of blood, level of alkaline phosphatase and transaminase (SGOT, SGPT) in sera. It is suggested that, after evaluation, the TIA might supplement or be used as an alternative to other immunodiagnostic tests already in use for the diagnosis of clonorchiasis.
parasitology-helminth-trematoda ; Clonorchis sinensis ; clonorchiasis ; immunology

This study was undertaken to evaluate the role of peritoneal exudate cells in the transfer of immunity against the liver fluke, Clonorchis sinensis in the inbred BALB/c mice. Ten donor mice were divided into 2 groups. One group consisted of 5 mice was infected orally with 20 metacercariae of C. sinensis, and the other group was injected intraperitoneally with 20 excysted larvae. Thirty days after immunization, the peritoneal exudate cells were obtained from the donor mice. Twenty recipient mice were divided into 4 equal groups for the purpose of primary immunization. The mice of Group I were injected intraperitoneally with 2 x 10(6) peritoneal exudate cells of the donor mice infected orally, those of Group III were injected intraperitoneally with 2 x 10(6) peritoneal exudate cells of the donor mice injected intraperitoneally. Those of Group II were injected orally with 20 metacercariae of C. sinensis. The group IV mice served as controls. Four days after the primary immunization all recipient mice were challenged orally with 20 metacercariae of C. sinensis, and then killed 30 days after the challenging infection. When the peritoneal exudate cells were injected into the recipient mice, pronounced reduction in eggs per gram of the feces was found in the mice of Group I and Group II, but no reduction in those of Group III. In the worm burdens of C. sinensis, the number of flukes found in the mice of Group II was only significantly less than those in the control group(IV). In addition the number of plaque forming cells per spleen in the mice of Group II was found larger than those in Group I. It is likely that donor peritoneal exudate cells transferred to the recipients might result in the production of relative immunity.
parasitology-helminth-trematoda ; Clonorchis sinensis ; immunology ; mouse

The present study attempted to demonstrate an acquired immunity against Clonorchis sinensis in albino rat. Three separate experiments were carried out to determine acquired resistance developed by stimulating procedure followed by challenging infection with metacercariae. Acquired resistance was evaluated by the rate of recovery or the average number of flukes recovered from the liver of challenged albino rats, compared with the controls. In drder to demonstrate the rate of recovery of the fluke, three experimental groups of rats were challenged with 50 metacercariae per rat- 7,15 and 30 days after single injection of worm extract. The recovery rate was ranged from 33.2% to 38.0% in experimental group and their control group harbored from 37.8% to 42.6%. No significant difference was found on statistical analysis. In experimental groups received two immunizing injections with worm extract followed by challenging infection of metacercariae. Statistically significant difference was recovered between experimentals and controls. It was noted that reduction of the recovery rate was prominent in Group 5 and 6, which were challenged 15 and 30 days after two stimulating injections. From the third experiment which was consisted of single immunizing infection with 20 metacercariae followed by challenging infection with 30 metacercariae, no significant reduction was found between experimental rats and their controls. The number of recovered worms ranged from 16.2 to 18.5 worms in experimental group, while that of control group ranged from 18.9 to 19.8 worms. The evidence of delayed hypersensitivity reaction was observed in the groups with acquired immunity developed rats, by histopathological study of host hepatic tissue.
parasitology-helminth-trematoda ; Clonorchis sinensis ; immunology ; rat ; histology

The sensitivity and specificity of crude and affinity-purified antigens of Clonorchis sinensis obtained from the infected rabbits were studied. Stage-specific antigenic proteins from the eggs, metacercariae and adult worms were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA) The results were as follows: The antibody-binding antigen (ABA) purified from whole worm crude antigen (WWA) by CNBr-activated Sepharose 4B affinity chromatography made 4 specific bands against rabbit anti-sera on Ouchterlony gel diffusion plate, while WWA made 7 bands. Major WWA protein bands by SDS-PAGE were found at 16,300-18,500 and 28,000-29,000 daltons, while major ABA protein bands were at 18,000-21,000 and 29,000-31,000 daltons. The reactivity of ABA with rabbit anti-sera in ELISA was remarkably less sensitive than that of WWA. Molecular weights of egg antigen (EGA), metacercarial antigen (MEA) and adult worm antigen (WWA) of C. sinensis ranged from 15,000-200,000 daltons, 15,000-100,000 daltons and 11,000-80,000 daltons, respectively. Major WWA proteins consisted mainly of polypeptide bands of low molecular weight, less than 31,000 daltons, while those of EGA and MEA consisted of higher molecular weights than 30,000 daltons. The ELISA reactivities of WWA to rabbit anti-sera were remarkably greater than those of MEA. EGA showed negative reaction throughout the experiments. WWA showed higher optical density (O.D.) than 1.0, when reacted with rabbit anti-sera obtained at 4-6 weeks after the infection. In the rabbit anti-sera later than 12 weeks after the infection, the O.D. reacting with WWA showed a plateau without variation. MEA showed relatively low O.D. values (<0.6), when reacted with anti-sera from lightly infected groups throughout the experiments, although there were some weak positive cases (O.D.>0.6) in heavily infected groups. MEA reacted with rabbit anti-sera showed negative results on Ouchterlony gel diffusion plates. Summarizing the above results, it is suggested that the whole worm antigen prepared from the adult worms of C. sinensis is most highly antigenic. However, this antigen might reveal cross reactions with other trematodes such as Paragonimus westermani, therefore, purification of antigenic proteins from the crude antigen is essential to increase the sensitivity and specificity for the immunodiagnosis of clonorchiasis.
parasitology-helminth-trematoda ; Clonorchis sinensis ; clonorchiasis ; rabbit ; immunology ; diagnosis ; antigen

In an attempt to investigate the specific antigenic substance of Fasciola hepatica, Ouchterlony tests and immunoelectrophoretic analyses were carried out. Crude Fasciola antigen was prepared and fractionated by Sephadex G-200 column to Antigen I, II and III according to protein content. Crude antigens of Paragonimus westermani, Clonorchis sinensis and Paramphistomum sp. were also prepared for control and absorption study. Antiserum was prepared by injecting 0.5 ml of crude Fasciola antigen with same amount of complete Freund's adjuvant in rabbits, 10 times at an interval of l week. The results obtained in this study were as follows: Crude Fasciola antigen reacted with antiserum with 9 precipitin bands by Ouchterlony test and with 11 bands by immunoelectrophoresis. Cross reaction was observed between Paragonimus, Clonorchis and Paramphistomum antigens and anti-Fasciola rabbit serum respectively. By Ouchterlony test, 3-4 cross reacting bands were found. Anti-Fasciola sera which were absorbed with respective Paragonimus, Clonorchis and Paramphistomum antigens, reacted with Fasciola crude antigen. Ouchterlony test gave 5-6 precipitin bands. Further reaction between Fasciola antigen and antiserum absorbed with the above 3 antigens concomitantly gave 5 precipitin bands by Ouchterlony test and 7 bands by immunoelectrophoretic analyses. Fractionated Fasciola antigens (Antigens I, II and III) reacted with anti-Fasciola rabbit serum in immunoelectrophoresis. Antigen I, II and III gave 2, 3 and 5 precipitin bands respectively. Anti-Fasciola rabbit serum which was absorbed with 3 trematodes antigens gave, by immunoelectrophoresis, 1 band with Antigen I, 2 bands with Antigen II and III of Fasciola hepatica. From the above results, it is concluded that Fasciola hepatica possessed the specific antigenic substance not cross-reacted with other trematodes.
parasitology-helminth-trematoda ; Fasciola hepatica ; Paragonimus westermani ; Clonorchis sinensis ; Paramphistomum sp. ; antigen ; immunology ; electrophoresis

In order to understand the effect of prednisolone injection on the histopathological changes of the mouse liver and the chronological changes of the worm structure of Clonorchis sinensis, when this fluke was inoculated to the mouse intraperitoneally. The recovery rate, survival rate, location and size of the inoculated worms as well as the histopathological changes of the liver were investigated for the comparison among the groups of mice, which were classified by number of worms and the duration of observation period. The result obtained were summarized as follows: The recovery rate and survival rate of the worms decreased especially 28 days after the inoculation. Most of worms (45.5 percent) were collected from the peritoneal cavity, and some of worms were found tightly adherent to the capsules of the liver, spleen, intestine and diaphragm. The mean worm size after inoculation was constantly smaller than that before inoculation. Remarkable atrophy in the reproductive organs of the worm, such as spermatheca, testes, vitelline gland and ovary was frequently observed at the 10th day of inoculation. Histopathologically the liver failed to show any parasitic worm inside the intrahepatic biliary system. However, multiple well formed egg-containing granulomas were present along the liver capsule. These necrotic granulomas were occasionally found under the fibrotic liver capsule. Focal necrosis and focal phlebitis together with vascular dilatation were prominent features of the liver. The worms recovered in the capsule of the liver were degenerated and necrotized. Usually, there were remarkable capsulitis and granuloma formation around the eggs.
parasitology-helminth-trematoda ; Clonorchis sinensis ; histology ; pathology ; mouse-liver ; immunology ; immunesuppression ; necrosis ; phlebitis ; dilatation ; capsulitis ; granuloma

Agar-gel diffusion test (AGD), counterimmunoelectrophoresis (CIEP) and enzyme-linked immunosorbent assay(ELISA) were examined with the sera of skin test positives for paragonimiasis. The crude antigen(Paragonimus whole worm extracts: protein concentration, 7.56mg/ml) and human sera were used in AGD and CIEP. And in ELISA test, diluted antigen with 1:40,000 of crude antigen and diluted sera with 1:100, 1:200 were used in the test. The positive identical ratio between AGD and CIEP reactions is 98 percent and negative identical ratio is 100 percent. One or three precipitin bands are observed in AGD. One to seven precipitin bands are also revealed in CIEP. Especially, deeply stained bands are observed in CIEP than those of AGD. The positive identical ratios between AGD and ELISA tests are 96 percent in 1:100 diluted sera, and 94 percent in 1:200 diluted sera. But the negative identical ratios between AGD and ELISA tests are 97 percent and 99 percent respectively in 1:100 and 1:200 diluted sera. The positive identical ratios between CIEP and ELISA tests are 98 percent and 96 percent respectively in 1:100 and 1:200 diluted sera, but also 97 percent and 99 percent in 1:100 and 1:200. Control sera, such as clonorchiasis, amoebiasis and toxoplasmosis, revealed all negatives with Paragonimus antigen in AGD, CIEP and ELISA tests. By above results, ELISA was most sensitive, next CIEP and AGD. But AGD test appears to be more useful when used to crude antigen without cross reaction with other parasitic infections. CIEP test is basically equal in terms of precipitin reaction, but CIEP is able to be detected more sensitively and rapidly though less simple in handiwork than AGD. Consequently, three methods for immunological tests of paragonimiasis have good correlations with one another. Also, each of these has both merits and demerits in immunological test for paragonimiasis. But the ELISA test was proved to be the most sensitive and convenient tool for mass screening test, especially in case of using purified antigen.
parasitology-helminth-trematoda ; Clonorchis sinensis ; clonorchiasis ; ELISA ; immunology ; diagnosis ; paragonimiasis ; Paragonimus westermani ; agar-gel diffusion ; counterimmunoelectrophoresis

A comparison was made of a new serological method, thin layer immunoassay (TIA), and an established method, enzyme-linked immunosorbent assay (ELISA), in the detection and quantification of antibodies in clonorchiasis. Saline extract of lyophilized Clonorchis sinensis adult worm was used as antigen, and TIA by the method of Elwing et al. (1976) and ELISA by Voller et al. (1974) were performed. Using sera from known clonorchiasis cases, 100 percent of the sera tested were positive by TIA and 88.3 percent by ELISA. TIA produced false positive results in 14 out of 36 cases, which were 10 amoebiasis cases, 16 paragonimiasis cases and 10 healthy controls. ELISA, however, produced a small number of false positives, 7 out of 55 cases. There was correlation between immunoglobulin G level in sera and ELISA value (correlation coefficient, 0.69), whereas no correlation between immunoglobulin G level and TIA result. The performance of TIA and ELISA was not correlated in the results using homologous antigen.
parasitology-helminth-trematoda ; Clonorchis sinensis ; clonorchiasis ; ELISA ; immunology ; diagnosis ; thin-layer immunoassay

Serum IgE level and Clonorchis specific IgE in individuals with Clonorchis sinensis were determined by radioimmunosorbent(RIST) and radioallergosorbent technique(RAST) respectively. Highly significant elevations of serum IgE (P<0.001) and specific IgE antibodies (P<0.01) were observed in area from individuals with clonorchiasis. The mean values of serum IgE in individuals with clonorchiasis and healthy individuals were 2,372 IU/ml and 364 IU/ml respectively and specific IgE antibodies of both groups were 52.0 and 4.4%. A close correlation(r=0.9451) between serum IgE level and specific IgE antibodies were observed and correlation (r=0.6056) between serum IgE and EPG and between specific IgE and EPG(r=0.5693) were also observed.
parasitology-helminth-trematoda ; Clonorchis sinensis ; clonorchiasis ; immunology ; radioimmunosorbent test ; radioallergosorbent test ; IgA ; IgE ; serum