Table of Content

28 February 1991, Volume 9 Issue 1

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MOLECULAR RECOGNITION BETWEEN GLYCOPHORIN A AND PLASMODIVM FALCIPARUM MEROZOITES

1991, 9(1):  1-3. 

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By using purified human erythrocyte membrane glycophorin A (GPA) and glycopeptide of GPA, antibodies against GPA and against GPA- glycopeptide, and SPA-colloidal gold, Plasmodium falciparum FCC-l/HN merozoites were immunolabeled. The labeled samples were observed under transmission electron microscope (TEM). The TEM pictures showed that colloidal gold pellets were distributed over all of the merozoite surface. This is the first report on the direct experimental evidence of molecular recognition and combination between GPA (or glycopeptide of GPA) and Plasmodium falciparum merozoites. The results strongly support the hypothesis that GPA is involved as a recognized receptor on erythrocy-tes for Plasmodium falciparum and the glycopeptide domain of GPA is the receptor site for Plasmodium falciparum.

STUDIES ON RESA IN PLASMODIUM FALCIPARUM CULTURE MEDIUM

1991, 9(1):  4-7. 

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P. falciparum grown in the complete RPMI 1640 medium (containing about 10% normal rabbit serum) for 24 h was collected at about 5% parasitemia. The medium was centrifuged at 500g for 20 min and the supernatant was then centrifuged once more at 40 OOOg for 30 min. Ten serum samples from falciparum malaria patients with anti-RESA antibodies collected from Yunnan Province were mixed with different dilutions of the culture supernatant, incubated at 3℃ for 2h and then at 4℃ overnight, and used for conducting RESA-IFA test. The titers of RESA-IFA test of all samples were reduced as compared with controls. When heated supernatant was used in the test, the inhibitory activity remained unchanged. The results indicate that RES A in the super natants was soluble and heat-stable.To establish the origin of the reacting antigens in the inhibition experiments, the soluble fraction of sonicated merozoite-enriched collection and sonicates of ghost made from normal O phenotype erythrocytes were used in the inhibition RESA -IFA test with sera containing anti-RESA antibodies. The results showed that merozoite- enriched preparations were fully inhibitory at the concentration of 3.1μg/ml, while no inhibition was observed at 0.2 μg/ml. In contrast, similar extracts made from normal erythrocyte ghosts were not inhibitory until concentrations about 30 times higher than that of merozoite antigen.

RESTRICTION FRAGMENT LENGTH DIFFERENCES OF GENOMIC REPETITIVE DNA FROM FIVE SIBLING SPECIES OF ANOPHELES HYRCANUS GROUP

1991, 9(1):  8-11. 

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Genomic DNA were prepared from 5 sibling species of Anopheles hyrcanus group in-eluding An. sinensis (ASS), An. anthropophagus (ALA), An. liangshanensis (ALS),An: crawfordi (ACW) and An. xiaokuanus (AXK). High molecular weight DNA from each species were cut with three restriction endonuleases (Bgl Ⅱ, Hae Ⅲ and PstⅠ) and the DNA fragments analysed by agarose gel electrophoresis and ethidium bromide st?.inin.g. Three enzymes (Bgl Ⅱ, Hae Ⅲ and PstⅠ) produced unique fragments in all sibling species tested. A diagnostic restriction fragment length of DNA from 5 sibling species of An. hyrcanus group may be derived from a single restriction enzyme pattern (i. e. Bgl Ⅱ). The results demonstrated that the restriction fragment length differences of repetitive DNA could used as a tool to distinguish sibling species of An. hyrcanus group.

STUDIES ON THE STRAIN DIFFERENCES OF SCHISTOSOMA JAPONICUM IN THE MAINLAND OF CHINA.——Ⅲ. MORPHOMETRIC DATA

1991, 9(1):  12-16. 

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The present investigation was undertaken to determine whether differences exist in the size of cercariae, adult worms and eggs among different isolates of S. japonicum in the mainland of China. Measurements on the corresponding stages of Japanese and Philippine strains of S. japonicum were also carried out for comparison.The size of cercariae collected from pooled naturally infected snails from different isolates was shown in Table 1. The body index of Yunnan isolate, which is defined as cercaria length ×width (mm) × 1000, was found to be smaller than those of the other 4 isolates, this difference being significant at p0.01.Measurements of adult worms and eggs from different isolates were taken from various hosts infected with the same number of cercariee and for the same duration of infection. Results indicated that the mean length of mature worms and the size of tnature eggs varied not only in different host species but also among host individuals of the same species infected with the same isolate of parasite. The mean length of mature worms of S. japonicum from various hosts infected with Yunnan isolate was considerably smaller than those of the other 4 isolates (Table 2), this difference being significant at p0.05 by analysis of variance.Regarding the size (length ×width) of eggs in mice and hamsters, large size eggs were found from both Yunnan and Guangxi isolates, while in rabbits and rhesus monkeys, large size eggs were found from Sichuan parasites. Judging from the index defined as the ratio of egg width/length × 100, the eggs of the Sichuan isolate were broad and short in shape, giving higher index. Those of Guangxi and Hubei isolates were oblong, giving the lowest index (Table 3). The other two isolates from Yunnan and Anhui laid eggs between these two extremes.

DISTRIBUTION OF ZYMODEMES OF ENTAMOEBA HISTOLYTICA FROM DIFFERENT AREAS OF CHINA

1991, 9(1):  17-20. 

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Using agarose gel electrophoresis, the electrophoretic patterns of four enzymes: ma-late NADP oxidoreducte.se (malic er.zyme) (ME), glucosephosphate isomerase (GPI), phosphogiucomutase (PGM) ar..d hexakinase (HK), were compared and analysed for five strains of Entamoeba histolytica, which had been obtained from patients with acute amebic dysentdry or asymptomatic cyst carrier in Beijing, Tianjin and Fujian Province. The results showed that the electrophoretic patterns of all the five strains of E. histolytica belong to pathogenic zymodeme. zymodeme XIV, which is suggested to be the main pathogenic zymodeme in our country.

APPLICATION OF MONOCLONAL ANTIBODIES AGAINST LEISHMANIA DONOVANI——Ⅱ. DETECTION OF CIRCULATING ANTIGEN IN SERA OF VISCERAL LEISHMANIASIS BEFORE AND AFTER TREATMENT

1991, 9(1):  21-23. 

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Monoclonal antibodies L12F7 against the target antigen of L. doreovani promastigotes were used for the detection of circulating antigen(CAg) from sera of visceral leishmaniasis pati-ents. The results showed that of 118 serum samples from visceral leishmaniasis tested. 105 were positive(88.9%). 55 normal serum samples were negative. No cross reaction with sera from patients with vivax malaria, schistosomiasis. leprosy and brucellosis, was found.The authors suggested that this assay may be used as a sensitive and new serodiagno-stic test for detecting existing infection of visceral leishmaniasis for epidemiological survey and for assessment of cure after effective treatment.

VARIATIONS IN REPRODUCTIVE CAPACITY, GONOTROPHIC CYCLE AND LONGEVITY OF ANOPHELES SINENSIS INFECTED BY BRUGIA MALAY I LARVAE

1991, 9(1):  24-27. 

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Under the experimental conditions of 26±10℃, relative humidity 70-80%, 100±20 Lux and photoperiod 16 hours/day and using a membrane feeding method, the development of filerial larvae and the variations in reproductive capacity, gonotrophic cycle and longevity of An. sinensis infected with microfilariae of Bmgia malayi were observed. The infection rate and infection intensity of filarial larvae in An. sinensis increased with the microfilarial density in the blood meal from 50 to 150 mf/20μl. The mature rate and 1EI decreased when the density rose to 200 mg/20μl. The concentration ratio of mf in blood by An. sinensis was 1.2-1.4.In the 3rd gonotrophic cycle, the feeding ratio of infected mosquitoes became lower when the mf density rose to 150mf/20Al, but the infection of filarial larvae did not affect the number of oviposition, the regularity of egg-production activity and the hatching rate of eggs, while the quantity of egg-production increased when the mf density was 150mf/ 20μl. The egg-production rate, and gonotrophic cycle were not basically influenced by fil-.arial larvae infection. The multifeeding ratio was 16.7-48.4% in An. sinensis.The longevity of An. sinensis was extended by infection with mf density of 100mf/ 20Mμl and shortened in infection with mf density of 200 mf/20μl.The authors conclude that "Brwgia malayi-Anophelesj sinensis" is a highly adapted "pathogen-vector "system.

IN VITRO CULTIVATION OF THE EXOERYTHROCYTIC STAGE OF PLASMODIUM BERGHEI

1991, 9(1):  28-30. 

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This paper reports on an in vitro culture system for the exoerythrocytic (EE) stage of Plasmodium berghei (P.b.) using embryonic lung cells. The system was first developed by our laboratory in China. The embryonic lung cells were isolated by trypsin digestion of a human embryonic lung obtained from a therapeutic abortion case and was designated as cell line Elu 8801. Anopheles stephensi mosquitoes were infected by biting P. b. ANKA strain infected Kunming mice and after 18-21 days were dissected under aseptic conditions for preparation of a sporozoite suspension. This suspension was used to inoculate the monolayer cultures of Elu 8801. Regular examination found that following a cultivation for 48 hours, up to 100 multinuclear EE schizonts of P.b. could be observed on 1×1cm cover slide. Seventy-two hours later mature merozoites were seen among part of the schizonts. An intrape-ritoneal inoculation of the supernatant culture medium to mice could induce malaria infection which could be transferred to other mice by blood inoculation. When the mice infected with the second generation were allowed to feed A. stephensi, sporozoites developed in the mosquitoes. The results demonstrate that the human embryonic lung cell line Elu 8801 established in our laboratory is susceptible to P.b. ANKA sporozoites and can support the developmental maturation of EE stages, producing potentially infectious merozoites.

COMMON ANTIGENIC COMPONENTS IN SOLUBLE ANTIGENS SHARED BY SCHISTOSOMA JAPONICUM EGGS AND PARAGONIMUS WESTERMANI ADULT WORMS

1991, 9(1):  31-34. 

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Using enzyme-linked immunoblot technique (ELIB). the antigenic protein components of water-soluble and urea-scluble antigens of Schistosoma japonicum eggs (JSEA, JEA-U) aad Paragonimus westermani adult worms (PAA, PAA-U) were analysed. The results showed that in both JSEA and JEA-U, 8 and 3 bands of polypeptides could be recogni-zed by sera from patients with schistosomiasis japonica (Sj). In both PAA and PAA-U, there were 9 and 2 bands of polypeptides respectively, which could be recognized by sera from patients with pagumogonimiasis skrjabini (Ps). The components of 36/37kDa in PAA and 20/21 kDa in JSEA were identified to be shared antigenic fractions existing between the two species of trematodes, which gave positive reaction against patient's sera from both S.j. and P.s., while PAA-U caused the reaction only with sera from patients with acute schistosomiasis. PAA-U might be useful to avoid cross reaction in the majority of schistosomiasis patients in the fields.

HISTOPATHOLOGY OF EXPERIMENTAL HEPATIC AMEBIASIS

1991, 9(1):  35-38. 

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The method of intrahepatic inoculation was successfully used to establish an experimental model of hepatic amebiasis in golden hamsters for studying its pathological morphology. Three types of macroscopic liver lesions, i. e. isolated abscess, multinodular abscess and ruptured lesion were described. On light microscopy the alteration and the proliferation type lesions were found as distinct pathological characteristics of the early and advanced hepatic amebiasis respectively. The developmant pattern of hepatic amebiasis and the pathogenicity of Entamoeba histolytica were discussed.

INDIRECT IMMUNOPEROXIDASE STAINING TECHNIQUE WITH FROZEN SECTIONS OF SCHISTOSOME FOR SERODIAGNOSIS OF LATENT SCHISTOSOMIASIS

1991, 9(1):  39-42. 

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Indirect immunoperoxidase staining technique using frozen sections of adult worm as antigen (ⅡP-AWA) was carried out to detect antibodies against schistosome antigens (AWAb) for the diagnosis of existing infection of schistosomiasis in COPT positive cases. Sera from 229 COPT positive and 135 COPT negative cases in Shanghai County, where schis-tosomiasis had been eradicated for more than 5 years, were tested. Sera from 122 patients with positive stool hatching from an endemic area were served as positive controls. The positive rates of the three groups were 96.9%, 5.2%, and 100% respectively. The staining pattern of the worm sections was mainly diffused at serum dilutions 1:4 to 1:16.149 sero-positive cases were treated with pyquiton (60mg/kg-2d) and re-examined 1, 1.5. and 2.5 years post-treatment. The negative conversion rate of HP-AWA was consi-derably higher than that of COPT (80% vs. 61.1%) at the first year, but no significant difference was observed after 2.5 years (85.5% vs. 83.6%). With the decreasing antibody titer, the staining pattern of worm sections changed from diffused to focal pattern, mostly in the gut.The results suggest that the presence of detectable AWAb in untreated patients of patients treated 2 years ago with pyquiton possibly indicate latent schistosomiasis. ⅡP-AWA is of practical value in screening populations for latent schistosomiasis in areas where the disease had been under control.

FUSION PROTEIN Dot-ELISA FOR THE DETECTION OF PLASMODIVM FALCIPARVM ANTIBODIES

1991, 9(1):  43-45. 

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The feasibility of using fusion protein Dot-ELISA to detect the antibodies to Plas-modium falciparum was presented. Fusion protein expressed in the P. falciparum genomic DNA expression library with λgtll. was used as the antigen to coat the solid support nitrocellulose strips. Horseradish peroxidase conjugated with goat antihuman IgG was used as the second antibody. The enzyme activity was read by naked eyes according to the dot colour. The results by Dot-ELISA correlated well with those by IFA. The study indicated that this method was specific, sensitive, simple, and replicable. It would be valuable for further studying the usefulness of fusion protein in the immuncdiagnosis of malaria.

COMPARATIVE STUDIES ON THE REPETITIVE DNA SEQUENCES OF DIPLOID AND TRIPLOID FORMS OF PARAGONIMUS WESTERMANI BY RESTRICTION ENDONUCLEASE AND SOUTHERN BLOTTING

1991, 9(1):  46-49. 

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Restriction enzyme digestion of total genomic DNA of two chromosome forms of Paragonimus westermani showed the presence of homologous highly repeated DNA in both diploid and triploid forms. Southern blotting analysis provided further evidence that the distribution, of restriction enzyme sites (with 3 enzymes) on repetitive sequence of DNA of both forms were similar. However, with PstⅠ, Ddel, HaeⅢ and HpaⅡ, their polymorphism revealed differences which were also found in each form tested separately with the hybridization technique.The present study, at the molecular level, supports the previously reported biological and biochemical results that they might be considered as different isolates or forms.It is suggested that PstⅠ, DdeⅠ, HaeⅢ and HpaⅡdigestion pattern might be useful to distinguish the diploid from the triploid forms of Paragonimus westermani.

OBSERVATION ON INTERLEUKIN-2 IN MICE INFECTED WITH PLASMODIUM YOELII

1991, 9(1):  50-54. 

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C57BL/6J, NIH and ICR mice were inoculated with nonlethal P. yoelii (BY 265 strain) and NIH mice which had recovered from primary infection were reinocizlated with the same parasite. The parasitemia of the mice was observed and IL-2 production by spleen lymphocytes of the mice upon Con A or P. yoelii antigen stimulation was detected at different intervals throughout primary infection and reinfection. The IL-2 level was measured by the microassay bzsed on incorporation of H-TdR into CTLL2. The main results were as follows: (1) There were marked differences in both the Con A-induced IL-2 production and the parasitemia between C57BL/6J, NIH and ICR mice. The Con A-induced IL-2 levels (X± SE of SI) in C57BL/6J, NIH and ICR mice before primary infection were 223.7±19.32, 122.12±28.91 and 7226±30.60 respectively. The maximum parasitemia (X± SE of %) in C57BL/6J, NIH and ICR mice after primary infection were 2.7%±0.29%, 14.50% ±2.75% and 31.30%±1.80% separately. It suggests that the innate Con A-induced IL-2 levels in the three strains of mice might be one of the factors influencing their susceptibility to P. yoelii primary infection. (2) There was significant depression in the capacity of releasing IL-2 upon Con A stimulation 3, 10 and 15 days after NIH mice had been inoculated with P. yoelii; after that, it was followed by the gradual resumption of the Con A-induced IL-2 production. P. yoelii antigen-induced IL-2 was only detected by d34 after inoculation. In contrast, when NIH mice which had been recovered from primary infection were reinoculated with the same parasite, the animals were partly re3istant to reinfection and there was no decrease in Con A-induced IL-2 production after reinocu-lation and the enhancement of IL-2 production upon specific antigen stimulation 3 and 19 days after reinoculation. The findings suggest that the parasitespecific splenic lymphocytes capable of releasing IL-2 upon antigen stimulation appear in the late st?.ge of primary infection and the lymphocytes possessing memory function may elevate their capacity of secreting IL-2 during the course of reinfection. This phenomenon is considered to be related to regulation of protective immunity against malaria.

ULTRASTRUCTURAL OBSERVATION ON THE SPERMIOGENESIS OF PAGUMOGONIMUS SKRJABINI

1991, 9(1):  55-57. 

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The spermiogenesis of Pagumogonimus skrjabini was observed on the ultrastructural' level by trasmission electron microscopy. The development and the morphological characteristics of spermatids and sperm were studied. The Golgi comples was located near the anterior region of the spermatid nucleus and developed into an acrosomal cap of the sperm. Accordingly, an acrosomal structure of P. skrjabini was recognized in this study.The mature sperm of P.s. was composed of head and tail. The head part was filled with dense nucleus and the nucleoplasma extended into the anterior end of the median part of the sperm tail. A conjunction part was seen between the head and the tail. The tail was composed of a median part and a terminal part. At the beginning of median part of the tail, two axial filaments were found on each side. Each axial filament was composed of two central filaments and nine pairs of outer filaments. The two central filaments were surrounded by fibrous sheath, both of which were connected with spoke-like structures. Mitochondria were arranged in a line in the ventral portion of the central filaments. At. the terminal part of the tail, two axial filaments lay closely together.

SCANNING ELECTRON MICROSCOPICAL OBSERVATIONS ON PAGUMOGONIMUS SKRJABINI METACERCARIA AND JUVENILES

1991, 9(1):  58-60. 

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The present paper reports on the results of scanning electron microscopical observations on seven excysted metacercariae and twelve juveniles of Pagumogonimus skrjabini. The former were from the naturally excysted metacercariae in fresh water in Ningquang District of Shaanxi Province and the latter were collected from the dogs and rats experimentally infected with metacercariae from the District mentioned above.Both excysted metacercariae and juveniles have tegumental ridges or folds on the surface of the body. The anterior two-thirds of the body surface are covered with many dome-shaped sensory papillae. There are rings of this kind of papillae on the rim of oral and ventral suckers. The papillae being obviously in decreasing number in the 30-40-day-old juveniles and adult worms.Except for the suckers and excretory pores, the whole body surface of the metacer-cariae and the juveniles are covered with posteriorly pointing tegumental spines which are relatively denser in the forebody than in the hindbody. Spines around the oral sucker r.re bayonet-shaped, and those on the rest part of the body surface are basically chisel-shaped. The authors considered it important that spines of P. skrjabini can be arranged singly or in groups.

STUDIES ON DYNAMIC OF ANTIBODIES DETECTED IN SERA FROM JIRDS INFECTED WITH BRVGIA MALAYI BEFORE AND AFTER TREATMENT

1991, 9(1):  61-64. 

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Indirect immunofluoresent antibody test (IFAT) and immunoenzymatic staining technique (IEST), using frozen section of Brugia malayi and Setaria cervi adult worms, were applied to detect levels of IgG and IgM antibodies in jirds infected with Brugia malayi before and after treatment. Both methods could show the dynamic of antibodies during the course of infection. The peak of IgG and IgM antibodies were at the 12-14th week and 2-6th week after infection respectively. A high correlation was observed between the levels of IgG antibody and the period of infection, whereas the antibody titer had no relation with the density of infection. During 6 months after treatment, the levels of antibodies detected by IFAT and IEST using two antigens decreased to low litre. It is considered that IFAT and IEST using heterologous and homologcus antigens could be equally used for the serodiagnosis and the evaluation of cure in filariasis.

DIAGNOSIS OF NEUROCYSTICERCOSIS BY IMMUNOENZYMATIC STAINING TECHNIQUE (IEST) WITH FROZEN SECTIONS OF CYSTICERCUS CELLULOSAE

1991, 9(1):  65-67. 

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This article deals with the sensitivity and specificity of IEST in neurocysticercosis with frozen sections of Cysticerus cellulosae as antigens. 55 out of 56 ccses (98.2%) of neurocysticercosis showed positive reaction. All serum specimens from healthy human controls (50 cases), clonorchiasis (23 cases) and paragonimiasis (26 cases) were negative, but 18% (4/22) of serum specimens from echinococcosis revealed positive reaotions. The results indicate that IEST is specific, sensitive, economical and easy to perform for the diagnosis of neurocysticercosis.

IgE AND IgE-MEDIATED IMMUNOREACTION IN THE PATHOGENESIS OF HUMAN CYSTICERCOSIS

1991, 9(1):  68-70. 

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Total IgE, specific IgE and histamine in sera from 35 documented cases of cysticercosis and 30 transfusion donors were measured and mast cell degranulation test was perforired. Total IgE levels (730.6IU/ml), the positive rate of specific IgE (85.7%), histamine levels (5.3μg/ml) and the positive rate of mast cell degranulation (42.9%) of the patients were found to be significantly higher than those of the normal group (P0.01).The results suggested that the antigen of Cysticercus cellulosae could induce IgE production and mast cell degranulation, resulting in the release of histamine. It was, therefore, deemed that the IgE and IgE-mediated immunoreaction might be involved in the pathogenesis of human cysticercosis.