Membrane proteins and their antigenicity of Toxoplasma gondii

Surface membrane proteins of virulent RH strain and tissue cyst-forming Fukaya strain of Toxoplasma gondii were analyzed by SDS-polyacrylamide gel electrophoresis after LPO-catalyzed surface iodination and lectin blotting, then identified the zoite-specific antigens. Prior to the analyses, purification of RH tachyzoites from mouse peritoneal exudate and of Fukaya bradyzoites from mouse brain tissues were performed by centrifugation on the discontinuous Percoll density-gradient. Tachyzoites were obtained at the interface of 50 per cent and 60 per cent Percoll solution and brain cysts were harvested at the interfaces of 40-50 per cent and 50-60 per cent, then bradyzoites were obtained by treating the cysts with hypertonic solution. The LPO-catalyzed iodination detected 15 KDa and 14 KDa proteins of bradyzoites and 30 KDa protein of tachyzoites as major bands with several other minor bands. But Con A blotting revealed some bands of 200 K-50 KDa glycoproteins of bradyzoites and 52 KDa band as major and minor bands of 33 K-20 KDa of tachyzoites. Phytohemagglutinin did not detect any band in the two forms. EITB with anti-Fukaya antibody and anti-RH antibody revealed cross-reactivities between the two forms. Despite the cross-reactivity, anti-Fukaya antibody reacted with 15 KDa band of bradyzoites specifically and, anti-RH antibody with 52 KDa, 30 KDa, and 25 KDa bands of tachyzoites, respectively. It was identified that 15 KDa protein in bradyzoite, which was not a glycoprotein, was a major membrane protein with sufficient antigenicity, and in the case of tachyzoite, 52 KDa surface glycoprotein (gp52) with specific antigenicity might be added to the major surface protein, p30.

Toxoplasmacidal effect of HL-60 cells differentiated by dimethylsulfoxide

In vitro culture of Toxoplasma gondii in HL-60 cells cnd cell-mediated immunity against Toxoplasma in dimethylsulfoxide(DMSO)-induced HL-60 cells, i.e., differentiation into granulocytes, were pursued. HL-60 cells were treated with various concentrations of DMSO, and 1.3%(v/v) for 3 day incubation was chosen as the optimal condition for differentiation into granulocytes. The degree of differentiation was assayed in physiological and functional aspects in addition to morphological point. When treated with 1.3% DMSO for 3 days, HL-60 cells did not synthesize DNA materials beyond background level, and showed active chemotactic response to chemotactic peptide, formyl-methionyl-leucyl-phenylalanine(FMLP). Morphologically promyelocytes of high nuclear/cytoplasmic(N/C) ratio changed to granulocytes of relatively low N/C ratio. The relationships between HL-60 cells or DMSO-induced HL-60 cells and Toxoplasma were examined after stain with Giemsa and fluorescent dye (acridine orange). HS-60 cells did not show any sign of toxoplasmacidal activity but showed intracellular proliferation of Toxoplasma to form rosette for 72 hr co-culture. In contrast, DMSO-induced HL-60 cells phagocytosed Toxoplasma within 1 hr, and performed a process of intracellular digestion of Toxoplasma thereafter. With the above results, it is suggested that phagosome-lysosome fusion is one of the critical events for the parasitism by Toxoplasma or for susceptibility of host cells. The in vitro culture system of this study has offered a defined condition to study the protozoan parasite-host cell interactions.

Toxoplasma antibodies by indirect latex agglutination tests in zoo animals

Total of 216 animals conserved in Seoul Grand Park were examined on the antibody titers of Toxoplasma by the indirect latex agglutination test, 20 out of 131 cases (15.3%) in mammals, 2 out of 75 cases (2.7%) in birds, and none in reptiles, according to species, 15 out of 68 species (22.1%) in mammals, 2 out of 36 species (5.6%) in birds showed positive antibody titers when the titers of 1:32 or higher were regarded as positive. In mammals, it appeared as positive in 2 out of 6 cases (1 out of 3 species ) in marsupials, 1 out of 15 cases (1 out of 11 species) in primates, 1 out of 1 case in bats, 6 out of 13 cases (5 out of 10 species) in carnivores, 1 in 12 cases (1 species out of 3) in odd-toed ungulates, 9 out of 80 cases (6 species out of 38) in even-toed ungulates, and none in rodents and in whales. In birds, 1 out of 21 cases (1 out of 7 species) in gallinaceous birds and 1 out of 6 (5 species ) in parrots appeared to have the positive antibody titers of Toxoplasma. And, none of reptiles showed positive. Frequencies of positive antibody titers were high in 1: 64, 9 cases in mammals followed by 1: 32, 6 cases, 1: 128, 3 cases, and 1: 256, 2 cases, respectively. Two positive cases in birds appeared to be 1: 64.

Egg detection rates of Enterobius vermicularis in children

The cello-tape anal swab specimens from 936 primary school children in Seoul, 308 primary school children in a rural area and 92 children in Nanjido, were examined for Enterobius vermicularis eggs. Out of 936 specimens from a primary school in Seoul, E. vermicularis eggs were detected in 114 specimens, the egg detection rate being 12.2 percent. In 308 specimens from a primary school in a rural area, 77 (25.0 percent) were positive for E. vermicularis eggs. Among 92 specimens from Nanjido children in Seoul, egg positive rate of E. vermicularis was 28.3 percent. The egg detection rate of primary school children in Seoul was significantly lower than that of primary school children in a rural area and of Nanjido children. The egg detection rate in boys was lower than that of girls. As the age of children increased, the egg detection rate decreased.

Purification of antigenic proteins of Paragonimus westermani and their applicability to experimental cat paragonimiasis

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.

Ultrastructure of the integument of adult Paragonimus westermani

The present study was performed to observe the ultrastructure of the integument of adult Paragonimus westermani. Dogs experimentally infected with 60 metacercariae of P. westermani were autopsied 4 months after the infection. Adult P. westermani were extracted from the dogs and the fine structure was studied by means of scanning and transmission electron microscope. The findings are as follows: Scanning electron microscopic findings showed that the spines and the papillae are distributed at whole body surface but the well developed spines or papaillae are shown around the oral suckrer and ventral sucker. At the end of the body, excretory pore was found, the shape was irregular. Transmission electron microscopic findings showed that plasma membrane, tegument, basal lamina, connective tissue, circular muscle layer, longitudinal muscle layer, nerve axon and tegumental cell were observed. In higher magnification, plasma membrane and bar-shaped granules were found at the outer surface of the tegument.

Prevalence of intestinal helminthic infections and skin tests for Paragonimus and Clonorchis in tuberculosis patients

This study was conducted to evaluate prevalence of intestinal helminthic infections and skin tests and Paragonimus and Clonorchis in tuberculosis(TB) patients in Korea. Stool examinations by formalin-ether technique and intradermal tests were carried out in 2 areas; Masan TB hospital and Kong-Ju TB hospital. The positive rate of intestinal helminths of any kind was 51.7 percent among 329 patients in Masan hospital, but 18.1 percent among 215 patients in Kong-Ju hospital. The infection rates of Trichocephalus trichiurus and Clonorchis sinensis were 20.7 percent and 17.6 percent respectively in Masan hospital patients, but the infection rates of T. trichiurus and C. sinensis were 6.5 percent and 6.0 percent respectively in Kong-Ju hospital patients. In skin tests, positive reactions to Paragonimus and Clonorchis antigens were 22.0 percent and 37.6 percent respectively in Masan hospital patients. On the contrary, Kong-Ju hospital patients showed the positive rates of 15.2 percent for Paragonimus and 27.2 percent for Clonorchis respectively.

Toxoplasma antibodies by indirect latex agglutination tests in National Seoul Mental Hospital patients

A total of 573 patients hospitalized in National Seoul Mental Hospital and 76 of healthy persons as control were examined by indirect latex agglutination test in order to evaluate Toxoplasma antibody titers in mental patients. Throughout this survey, 1:32 or more titers of diluted sera were regarded as positive. The 573 samples of test sera showed negative in 386 cases (67.4 percent), 1:2 in 93 cases (16.2 percent), 1:4 in 57 cases (9.9 percent), 1:8 in 14 cases (2.4 percent), 1:16 in 12 cases (2.1 percent), 1:32 in 5 cases (0.9 percent), 1:64 in 1 case (0.2 percent), 1:128 in 3 cases (0.5 percent) and 1:256 in 2 cases (0.3 percent) respectively. Among total 573 mental patients, 11 cases (19 percent) showed positive, and they were 9 cases (1.8 percent) of schizophrenia and 2 cases (7.4 percent) of manic depression. One case (1.3 percent) out of 76 control sera showed positive result.