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 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

In an attempt to investigate the sensitivity of immunodiagnosis in cats experimentally infected with Paragonimus westermani, agar-gel precipitin reaction were studied. Metacercariae of P. westermani were administered to cats in various doses(2-100 metacercariae per cat) and antisera were obtained at an interval of a week. Precipitin bands appeared in homologous antigen-antibody in experimental paragonimiasis between 3 and 5 weeks after infection in all the cats. Almost all the cases in which a large number of worms were detected, showed strong reactions as revealed by deeply stained bands. Precipitin reactions did not necessarily parallel with the number of worms detected. This may be attributable to the individual difference of a cat's conditions. Very weak precipitin reactions were noticed between Clonorchis antigen and Paragonimus antisera of cats, but no reactions were noticed between Paragonimus antigen and Clonorchis antisera of cats or rabbits.
parasitology-helminth-trematoda ; paragonimiasis ; Paragonimus westermani ; immunology ; Clonorchis sinensis ; cat ; rabbit

Paragonimiasis is one of the most important endemic diseases in Korea. However, recognition of the limitation of microscopic examination of sputum and feces for Paragonimus eggs has led to the investigation of immunoserological techniques for paragonimiasis. The other hand, bithionol preparation has been used as a drug of choice in paragonimiasis but recently niclofolan preparation has introduced as a new agent. By the time, the authors attempted the evaluation of above both agents against experimental rat paragonimiasis by immunoelectrophoresis and Ouchterlony test. The immunoeletrophoresis and Ouchterlony tests were performed according to the method Tsuji and Grabar et Williams respectively, with antigen extracted from lyophilized worm of P. westermani with 0.1 per cemt saline solution. Meanwhile, rats were infected with per os 20 metacercariae of P. westermani above two kinds of serologic tests and were undertaken at biweekly intervals. Then, bithionol was administered every other day for 5 successively for 2 weeks or 6 weeks after infection. Another groups, niclofolan was administered per os single dose same as above. The sera from the rats infected with P. westermani, after treatment with bithionol or niclofolan preparations began to show the precipitin bands against P. westermani antigen 8 weeks after the infection in immunoelectrophoresis and Ouchterlony test, but from 10 or 12 weeks after infection, the number of bands were decreased or disappeared gradually. In genera1, the sera from the rats treated with bithionol or niclofolan showed the precipitin bands delaying 2 weeks than control rat groups. The sera from the rats administered with hydrocortisone showed precipitin bands neither in immunoelectrophoresis nor in Ouchterlony test.
parasitology-helminth-trematoda ; Paragonimus westermani ; paragonimiasis ; rat ; immunoelectrophoresis ; immunology ; bithionol-chemotherapy ; bithionol

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

In order to obtain more specific antigenic preparation for the diagnosis of human paragonimasis, crude saline extract of whole worm (=PwWWE), secretory-excretory components (PwSEC) and secretion-excretion-free somatic extract (PwSM) of 12 week-old Paragonimus westermani were filtrated through Sephadex G-200 gel column. The adult Paragonimus worms were obtained from experimentally infected dogs. A total of 11 antigenic solutions was lyophilized or diluted to adjust protein content of 1 mg/ml. To evaluate the antigenicity of crude antigens and fractions, micro-ELISA was done with the sera from P. westermani infected cases, C. sinensis infected cases and non-infected control cases to detect Paragonimus specific IgG antibody. The results were as follows: When the PwWWE was filtrated through Sephadex G-200 gel, it was separated into three fractions; PwWWE Fr. 1, PwWWE Fr. 2 and PwWWE Fr. 3. The percentage of protein content was 28.0 percent, 21.6 percent and 50.4 percent respectively. The PwSM was also separated into three fractions; PwSM Fr. 1, PwSM Fr. 2, PwSM Fr. 3 and their percentage of protein content was 41.3 percent, 38.6 percent and 20.1 percent. However, the PwSEC showed different fractionation pattern; i.e. fraction 1 (=PwSEC Fr. 1) and 3 (PwSEC Fr. 3) without fraction 2. The percentage of protein content was 14.0 percent in PwSEC Fr. 1 and 86.0 percent in PwSEC Fr. 3. When the antigenicity of each Paragonimus crude antigen and fractionated antigen was evaluated for specific IgG antibody by micro-ELISA in 10 human paragonimiasis sera, PwSEC Fr. 1 was the most potent antigen showing the mean absorbance 1.98. The PwWWE Fr. 1, PwSEC, PwWWE were next to that; their mean absorbance were 1.72, 1.38 and 0.83, respectively. The antigenicity of fractions 2 and 3 was much weaker in binding specific IgG antibody. When the antigens were reacted in micro-ELISA with 10 human clonorchiasis sera, most antigens showed weak reactivity. Each fraction 1 of crude antigens reacted higher than other fractions or crude antigens; the mean absorbance was 0.17 in fraction 1, but in others the absorbances were about 0.06. With non-infected control sera, the result of micro-ELISA revealed almost same pattern with those of the clonorchiasis sera. From the above results, it became apparent that PwWWE Fr. 1, especially PwSEC Fr. 1 was the most potent antigen reacted with Paragonimus specifc IgG antibody.
parasitology-helminth-trematoda ; Paragonimus westermani ; paragonimiasis ; ELISA ; immunology ; diagnosis ; IgG ; purification

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