: In order to observe the antigenic fractions in saline extract of adult Paragonimus westermani, proteins in the crude extract were separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) in reducing conditions. The separated protein fractions were transferred to nitrocellulose paper on which 20 sera from human paragonimiasis were reacted and immunoblotted. Out of 15 stained protein bands in SDS-PAGE, 7 reacted with the sera. Of 14 reacted bands, 30 kilodalton(kDa) band was the most frequently reacted (95%) and was a strong antigen. Protein bands of 23 and 46 kDa were also strong antigens. Bands of over 150 kDa, 120 kDa, 92 kDa, 86 kDa, 74 kDa, 62 kDa, 51 kDa, 32 kDa, 28 kDa, 16.5 kDa and 15.5 kDa were also reactive but their frequencies of the reaction were variable.
parasitology-helminth-trematoda ; Paragonimus westermani ; immunology ; antigen ; electrophoresis

In order to observe the protein compositions of soluble extracts of P. westermani, and their changes during early developmental stages, the crude saline extracts of 4, 6, 8, 10 and 12 week-old worms which were harvested from experimentally infected dogs were analysed by disc-PAGE. The results were as follows: A total of 15 bands were identified from electrophoregrams of respective developmental stages. Of them, 5 bands were recognized throughout the developmental stages. The number and protein amount of identified bands changed according to the worm development from 4 weeks to 12 weeks. However, the banding patterns of 4 and 6 week-old worms and 8 and 10 week-old worms were similar each other. Of 15 identified bands, band 1 was recognized only in 12 week-old worms whereas bands 3, 4, 8, 9, 10, 11 and 15 gradually lowered their amount according to development to disappear in 12 week-old. In addition, band 5 became a major band in 12 week-old while band 6 turned to a minor band at tha same age. The possible relations of changing patterns of protein bands with worm development were discussed.
parasitology-helminth-trematoda ; Paragonimus westermani ; immunology ; protein ; electrophoresis

parasitology-helminth-trematoda ; Paragonimus westermani ; paragonimiasis ; ELISA ; immunology

As epidemiological parameters of human paragonimiasis, the positive rates of intradermal test and the sputum/stool examination have long been employed in population surveys. However, both the specificity of the intradermal test and the sensitivity of sputum/stool examination have been gradually declined as the endemicity was lowered; thus the gap between above two parameters widened. In such context, the development of a new epidemiological parameter or tool which makes it possible to accurately discriminate the active paragonimiasis cases was necessary. In the present study, the detection rate of Paragonimus-specific IgG antibody by micro-ELISA was evaluated as an indicator of epidemiologic status of human paragonimiasis in a population. A total of 4,285 students and inhabitants living in Bukpyeong Myeon and Bukil Myeon, Haenam Gun, Jeonlanam Do was surveyed in October 1983 by intradermal test first. Out of them, 244 case (5.7 percent) were found positively reacted to VBS antigen of P. wetermani. Out of 168 positive reactors, 7 cases (4.2 percent) were egg positive either by two times of sputum examination or by one stool examination. That indicated that only 0.16 percent of total surveyed were confirmed as active paragonimiasis by egg detection. When sera collected from 239 positive reactors of intradermal test were tested by micro-ELISA for their specific IgG antibody, 40 cases (16.7 percent) were found to be positive. All of 7 eggs positive cases were again positive for specific IgG antibody. Among remaining 232 intradermal test positive cases, 33 cases were positive for IgG antibody. In contrast to those, none of 42 positive reactors to intradermal test for Clonorchis and of 128 intradermal test negative cases showed positive for Paragonimus- specific IgG antibody. The rate of specific IgG antibody as detected by micro-ELISA appeared to be increased with the wheal size of the intradermal test. When the wheal size was over 13 mm in diameter, about 50 percent of them were positive for specific IgG antibody. Thirty-one specific antibody positive cases were clinically evaluated by laboratory examinations (repeated sputum examination, peripheral eosinophil count and chest roentgenogram) and by history taking. Out of them 24 cases were associated with one or more positive laboratory findings; thus considered as active paragonimiasis cases. Out of 7 lab. finding-free cases 3 revealed past history of typical paragonimiasis symptoms, suggesting that they were in chronic or in convalescent stages. The remaining 4 cases were considered as either mild or ectopic infection cases; the possibility of cross-reaction with other helminthiases could not be ruled out. From the above results, it was inferred that the detection of Paragonimus-specific IgG antibogy by micro-ELISA was very much helpful in detecting the active cases as well as in proper evaluation of the endemicity of human paragonimiasis in a population. The convenience of mass handling of sera in micro-ELISA was considered another superiority as an epidemiologic tool.
parasitology-helminth-trematoda ; Paragonimus westermani ; paragonimiasis ; ELISA ; immunology ; diagnosis ; IgG

In order to observe the complement fixation test and immuno-diffusion test of paragonimiasis, the sera taken at 10 days intervals up to 150 days from cats infected with Paragonimus westermani were examined by the above two immunological methods. The resultant findings were as follows: The complement fixation test showed positive reaction 20 days after the infection with 20 metacercariae, and 40-50 days after the infection with 10 metacercariae. The highest titer was observed 110 days later following the acceleration at 80 days later. In immuno-diffusion test, one are appeared 30 days after the infection with 20 metacercariae, but 60 days after the infection with 10 metacercariae. However, more than two arcs were observed since 70 days after infection. A relatively wide band appeared by the antigens of Fresh worm material and Somatic material. But relatively clear precipitin lines were observed in the diffusion test with V.B.S. antigen, increasing to 3-4 arcs after 110days. In general, complement fixation test showed earlier and higher sensitive reaction than immuno-diffusion test, and was considered to be more valuable method forr immunological diagnosis.
parasitology-helminth-trematoda ; Paragonimus westermani ; immunology ; cat ; complement fixation test ; immuno-diffusion test

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

To evaluate the immature eggs of Paragonimus westermani as a source of diagnostic antigen, about a million eggs which were excreted by 104 adult worms were collected; their saline extract (soluble egg antigen; PwSEA) was prepared. The specific IgG and IgM antibody levels were observed in experimental dog paragonimiasis by micro-ELISA, using PwSEA as well as whole worm extract of 12 week-old P. westermani (PwWWE). The protein composition of the PwSEA was observed by disc-PAGE. The results could be summarized as follows: Specific IgG antibody to PwSEA began to increase on 8 weeks after the experimental infection; it maintained its high level until the observation period of 13 weeks. The levels of IgM antibody to PwSEA, however, did not show any significant change. Specific IgG antibody to PwWWE began to increase earlier from 2 weeks after the infection and continued to increase until the observation period of 13 weeks. Its level was much higher than that to PwSEA. Specific IgM antibody to PwWWE increased temporarily during 2-8 weeks after the infection. By disc-PAGE, PwSEA showed 2 protein bands of very low motility. The bands of PwSEA corresponded to the first and second bands in the electrophoretic pattern of PwWWE of the 12 week-old worms. The above results indicated that the PwSEA induced antibody production in dog paragonimiasis but its antigenicity was weaker than PwWWE to be used as a diagnostic antigen.
parasitology-helminth-trematoda ; immunology ; Paragonimus westermani ; antigen ; enzyme-linked-immunosorbent assay ; serum ; IgG

Enzyme-linked immunosorbent assay (ELISA) using crude and affinity-purified antigens of adult worms of Paragonimus westermani was performed for infected cat sera with different worm burden, from preinfection to 18th week after infection. Crude antigen was used with supernatant of homogenated worms by freezing-thawing method, and the supernate was centrifuged for l hour at 10,000 rpm at 4C. Affinity-purified antigen (antibody-bound antigen) was prepared from fractions (bound and unbound) of crude antigen by affinity chromatography on CNBr-activated sepharose 4B, and IgG as a ligand was prepared from paragonimiasis cat serum (6 months infected) obtained by ammonium sulfate (40-45 per cent saturated) precipitation method. By SDS-PAGE, crude antigen showed 22 polypeptide fractions while purified antigen showed 4 fractions: 36, 400, 34,700,27,600 and 11,500 in molecular weights. All cats were divided into five groups(G1-G5) by different worm burdens. The mean of recovered worms (+/-SD) and the number of cats in each group are as follows:G1, 2 worms(0) and 4 cats; G2, 4.75(+/-0.66) and eight; G3, 10.75(+/-1.92) and four; G4, 25.20(+/-3.43) and five; G5, 48(+/-12.63) and five cats. The results were summarized as follows: The antibody levels(OD value) increased by worm burden in G1 to G4 generally. However, individual antibody levels were not exactly related with worm burden in all groups, especially there was a wide difference in G4 and G5. These results suggested that the worm burden in G4 (about 20 - 30 worms) is enough to produce antibody maximum in cats of 2~3 kg weight. The antibody levels increased significantly (p<0.05) compared to control sera at the 3rd week in G1 and G2, at the 2nd week in G3, and at the 1st week in G4 and G5. Especially in the 4th week, OD value increased more in G1(p<0.001) and in G2 to G5(p<0.01). In the pattern of antibody levels by ELISA in each group, OD in G1 increased to the 18th week continuously, in G2 OD was maintained same after the 16th week, but in G3 it decresed after the 16th week, and it was maintained same in G4 and G5 after the 14th week. The antibody levels by ELISA with the affinity-purified antigen were higher than those with crude antigen in all groups generally. Especially, the difference of OD values between two antigens was larger from the 4th to the 10th week. In G1 and G2 OD with purified antigen was higher than that with crude one to the 18th week. It was also higher in G3 than that with crude antigen to the 16th week and OD of G4 and G5 were higher before the 14th week than that with crude antigen, however became lower at the 16th week. Consequently, the antibody level in ELISA with affinity-purified antigen was more sensitive at the early weeks after infection and in light infection groups than that with crude antigen.
parasitology-helminth-trematoda ; immunology ; enzyme-linked immunosorbent assay ; Paragonimus westermani ; paragonimiasis ; cat

This study was designed to evaluate the partially purified antigens which were fractionated from crude extract of Paragonimus westermani and to monitor the enzyme-linked immunosorbent assay (ELISA) in experimental cat paragonimiasis during the course of infection as well as before and after chemotherapy. Crude extract of 6-month-old adult P. westermani was fractionated to 5 antigens by successive applications of ammonium sulfate precipitation, ion exchange chromatography and gel filtration. And the cats, 10 in each group, were infected with 60, 30, 15, and 5 metacercariae, then the half of each group was treated with praziquantel 2 times in one day of 100 mg per kilogram of weight on 150 days after the infection. Sera were collected every 10 days. ELISA was performed with the concentration of 2 microgram/ml antigen, 100 times diluted sera and 1,000 times diluted alkaline phosphatase conjugated anti-cat IgG. The results were as follows: Absorbance by ELISA with proteins precipitated by differential concentration of ammonium sulfate was the highest at 51-65 per cent precipitate (PA2), followed by 0-50 per cent precipitate (PA1), 66-80 per cent precipitate (PA3), and 81-90 precipitate (PA4). Unprecipitated protein over 90 per cent ammonium sulfate (PA5) showed the lowest antigenicity. Fractionation of PA1, PA2, and PA3 through the DEAE-cellulose column did not differentiate the antigenic proteins. By passing through the Sephadex G-200 column, PAl and PA2 were fractionated to high molecular weight proteins and those of low molecular weight which showed high absorbance by ELISA (PA1-I, II and PA2- I, II). But PA3 was shown to have a fraction of high molecular weight proteins (PA3-I) which showed high antigenicity. SDS-polyacrylamide gel electrophoresis of PA1-I, PA1-II, PA2-I, PA2-II, PA3-I, and crude extract was performed. Fraction PA1-I was composed of proteins which had the molecular weight of 270 kilodaltons (KD) to 196 KD; of them 220 KD protein was major band. Fraction PA2- I was composed of 255-225 KD, and PA3-I, 255-240 KD, respectively. Fraction PA1-II and fraction PA2-II consisted of 30 KD proteins. Absorbance by ELISA began to increase within 10-20 days after the infection and reached the highest on 140-180 days, then made plateau thereafter. Absorbance by ELISA decreased after praziquantel treatment. In 60 metacercariae infection group, the absorbance had been decreasing, but remained within the positive range during observation period, while those of 30, 15, and 5 metacercariae infection groups turned to negative range. Fraction PA1-II showed the highest antigenicity in ELISA, then fraction PA2-I, fraction PA1-I , fraction PA2-II, fraction PA3-I and crude extract followed. In early phase of infection, the absorbance of fraction PA1-II showed more rapid increase than those of the other fractions and it came to positive range at 20-30 days after infection.
parasitology-helminth-trematoda ; immunology ; enzyme-linked immunosorbent assay ; Paragonimus westermani ; antigen

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