Table of Content

28 February 2000, Volume 18 Issue 1

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论著

SEQUENCE ANALYZING AND GENOTYPING OF THE GENE ENCODING GLUTAMATE RICH PROTEIN OF GEOGRAPHICALLY DIFFERENT PLASMODIUM FALCIPARUM ISOLATES OBTAINED FROM DIFFERENT MALARIA ENDEMIC AREAS

ZHUXinping;ZHANGXinmei;ZHOULei;GAOXin

2000, 18(1):  1-4. 

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　Objective] To sequence a gene encoding GLURP and identify the genotypes of geographically different Plasmodium falciparum isolates from Yunnan and Hainan Provinces, China. [Methods] The gene of R2 repeat region of GLURP was amplified by the nested polymerase chain reaction and cloned into T vector. The nucleotide sequence of the GLURP gene was determined using automatic sequencer (dideoxy chain termination method), and analyzed by DNA Star software. [Results] At least 7 different GLURP genotypes ranging from 600 bp to 1 500 bp were found in different P falciparum isolates from Yunnan and Hainan Provinces. R2 region of GLURP gene consisted of several repeat units, each was composed of 19～20 residues which were shown to be highly conserved. The GLURP gene was also size polymorphic due to differences in the number of repeat units, whereas the repeat sequence was conserved. Sequence analysis showed that DNA sequences and deduced amino acid sequences were highly homologous among the geographically dispersed isolates or various isolates from the same geographical region. No obvious differences were found in the GLURP gene sequences among geographically different isolates. [Conclusion] TheGLURP gene of geographically different P falciparum isolates is highly conserved and size polymorphic, being useful in searching for malaria vaccine candidate antigen and developing a genotyping method for malaria research.


EFFECT OF PYRONARIDINE, MEFLOQUINE AND QUININE ON ARTESUNATE-SENSITIVE AND ARTESUNATE-RESISTANT PLASMODIUM FALCIPARUM

YANGHenglin;GAOBaihe;

2000, 18(1):  2-7. 

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　Objective] To compare the sensitivity of artesunate sensitive and artesunate resistant P falciparum to pyronaridine,mefloquine and quinine and to understand the effect of artesunte combined with the above mentioned 3 drugs respectively on artesunate resistant P falciparum . [Methods] Rieckmanns in vitro miorotechnique was used. [Results] The ID 50 values of pyronaride,mefloquine, quinine and artesunate were 59 0, 69 7, 283 8 and 9 6 nmol/L to artesunate sensitive P falciparum ;the ID 50 of the 4 drugs mentioned above were 170 6, 63 2, 272 4 and 85 1 nmol/L to the artesunate resistant P falciparum , respectively. In artesunate pyronaridine combinaton, the ID 50 values were 1/3 7 (22 8/85 1) and 1/4 7 (36 6/170 6) of the 2 drugs singly used.In artesunate mefloquine combinaton,the ID 95 is 1/125 (3 2/400) and 1/16 (80/128) of the 2 drugs singly used, respectively. [Conclusion] The artesunate resistant P falciparum isolate has no cross resistance to mefloquine and quinine.When artesunate was used in combination with the 2 drugs mentioned above respectively,the efficacy proved to be enhanced.


STUDIES ON THE SENSITIVITY OF PLASMODIUM FALCIPARUM TO PYRONARIDINE /SULFADOXINE/PYRIMETHAMINE IN VITRO

HANXianglu;LIUDequan;FENGXiaoping

2000, 18(1):  3-10. 

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　Objective] To develop an in vitro method for the assessment of drug response in P falciparum to pyronaridine/sulfadoxine/pyrimethamine(PND/S/P).[Methods]The PND/S/P microtest plate was designed,Rieckmann in vitro microtest(WHO standard kit) was used to test the sensitivity of P falciparum in continuous culture(FCC1/HN strain), and the data obtained were analyzed using a computer programme. Fractional inhibitory concentration (FIC) was calculated to test the possible synergy between PND and S/P. [Results]:The effect of the PND/S/P plates was fairly stable and the ED 50 values of pyronaridine,sulfadoxine and pyrimethamine were 0 11,215 12 and 2 9 pmol,respectively. The FIC obtained confirmed the synergism between PND and S/P. [Conclusion]The in vitro method can be used to assess the sensitivity of P. falciparum to pyronaridine /sulfadoxine/pyrimethamine.


RELATIONSHIP BETWEEN HEMOLYMPH PHENOL OXIDASE AND MELANIZATION OF OOCYSTS OF PLASMODIUM YOELII IN ANOPHELES STEPHENSI

SHIChaomei;HUANGFusheng;KUANGMingshu;DUANJianhua

2000, 18(1):  4-13. 

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　Objective] To explore the relationship between the hemolymph phenol oxidase and melanization of oocysts. [Methods] Anopheles stephensi Plasmodium yoelii system was used to determine the activity of monophenol oxidase (MPO) and o diphenol oxidase (o DPO) in the hemolymph collected from 4 groups of mosquitoes by polyacrylamide gel electrophoresis (PAGE) followed by density scanning. The 4 groups of mosquitoes were: non blood fed (N), normal blood fed (B), infected blood fed (I) and nitroquine administrated (D), respectively. [Results] No significant difference was found in the activities of MPO and o DPO between groups N and B.The activities of MPO and o DPO were not obviously modified in group I, but were significantly increased on day 10 and decreased on day 15 after blood feeding in the group D as compared with those in the groups N and B. [Conclusion]The alteration in the mosquito hemolymph PO activity coincided at each time point with the melanization of Plasmodium yoelii oocysts.


STUDIES ON HUMAN CYTOKINE RESPONSES BEFORE AND AFTER PRAZIQUANTEL CHEMOTHERAPY IN AN ENDEMIC AREA OF SCHISTOSOMIASIS JAPONICA

SHENLei;WUHaiwei;ZHANGZhaosong;RosemaryWeir;SHAOLijun;XIEZhangwu;HULinsheng;CHENShuzheng;SUChuan;TaobiZhang;MartinGTaylor;WUGuanling

2000, 18(1):  5-17. 

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　Objective] To observe the cellular immune responses in a population of an endemic area of schistosomiasis japonica and the influence of praziquantel treatment.[Methods] Blood was taken from 129 residents (64 cases were egg positive,65 cases were egg negitive) of an endemic area of Poyang Lake before and 45 days after praziquantel treatment. Cytokines induced by the schistosome soluble egg antigen (SEA) and soluble worm antigen preparations (SWAP) in the peripheral blood cells including IL 5, IL 10 and IFN γ were measured. [Results] Among 129 cases, the cytokine levels were found much higher in egg negative individuals than in egg positive individuals.The cytokine levels induced by both antigens were increased significantly after praziqantel treatment especially IL 5 and IFN γ.[Conclusion] The cellular immune responses in the population in schistosomiasis japonica endemic area exhibited a general trend of down regulation and were elevated significantly after praziquantel treatment.


STUDIES ON RISK FACTORS FOR LIVER FIBROSIS OF SCHISTOSOMIASIS JAPONICA

LIUYing;YUANHongchang;LINDandan;LIUYuemin;HUFei;ZHAOGeming;JIANGQingwu;ZHANGShaoji

2000, 18(1):  6-20. 

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　Objective] To explore the risk factors for schistosomal liver fibrosis.[Methods] 192 hepatitis negative patients with schistosomiasis were selected and divided into 3 groups according to the result of B ultrasound examination,that is,grade 2 and 3 fibrosis group(81 patients), grade 1 fibrosis group(61 patients) and control group (non fibrosis, 50 patiets).The univariate and multivariate ordinal regression model was made to analyse the possibly harmful factors influencing the liver damages of these patients. [Results] Four factors were found to be positively associated with schistosomal liver fibrosis.They were : number of treatments of schistosomiasis (OR=1 75),interval of schistosomal infection(OR=1 40),history of drinking wine(OR=1 95) and familial history of advanced schistosomiasis (OR=2 11). [Conclusion] Patients with repeated schistosome infection,long duration of schistosomal infection, long history of drinking and familial history of advanced schistosomiasis had higher risk for liver fibrosis than schistosomiasis patients without these factors.


ALLOZYME-BASED GENETIC VARIATION WITHIN AN UNSTABLE “POPULATION” OF CHINESE ONCOMELANIA HUPENSIS (GASTROPODA: RISSOACEA: POMATIOPSIDAE)

ZHANGYi;GeorgeMDavis;LIUHexiang;FENGTing

2000, 18(1):  7-23. 

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　Objective] To answer the following questions:① For Oncomelania snails collected two years apart from the same locality, has there been genetic divergence? ②How much experimental error has there been in studying subsets of these populations? ③As this is an unstable population, what has the net effect been on Hardy Weinberg equilibrium (HWe)? [Methods]Allozymes were studied using horizontal starch gel electrophoresis. Data collected from numbers of experiments were compiled. Data from each collection were divided into two equal subsets based on chronology of the experiments. Thirty four loci were studied using 72 to 180 snails per subset. [Results] The mean number of alleles per locus ranged from 1 5 ～1 9. With each consecutive subset,the % polymorphic loci dropped from 38 2 to 17 6.The mean heterozygosity was very low: 0 033 to 0 049 and not significantly different from Hardy Weinberg expectations. Ten loci and 11 alleles exclusive to the first group were eliminated from the overall study reducing the number of polymorphic loci from 19 to 10. There were significant departures from HWe at five loci having a substantial number of individuals for each allele. Neis and Wrights D were 0 003±0 001 and 0 054±0 006 respectively. [Conclusion] ① There were significant errors seen primarily in the results scored in the earliest experiments.② These earlier errors involving scoring difficult to resolve loci, and interpretation of rare alleles that were not found in later experiment had no significant effect on overall genetic distance.③ The use of Wright's D for closely related populations is explained. Results with Nei's D indicated no significant difference among the four subunits; Wright's D yielded significant difference between the collections made two years apart, attributed to the annual flooding of the Yangtze River mixing snails from different localities. ④ Major polymorphic loci were not in Hwe as predicted using the unstable population model. ⑤ One must study 25 or more individuals to find relatively rare alleles and study population genetics.


THE DIAGNOSTIC VALUE OF COMBINED DETECTION OF CIRCULATING ANTIGENS AND ANTIBODIES IN URINE OF PATIENTS WITH SCHISTOSOMIASIS JAPONICA

ZHANGEnying;LOUWenxian;XUEChunliang;WANGZhaojun

2000, 18(1):  8-25. 

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　Objective] To assess the value of detecting circulating antigens and antibodies in urine as a noninvasive method for the diagnosis of schistosomiasis. [Methods] A sandwich ELISA and ELISA using McAb were applied to detect circulating schistosomal antigens and specific antibodies in the urine of patients with acute and chronic schistosomiasis. [Results] When the urine samples from 10 cases of acute schistosomiasis and 61 cases of chronic schistosomiasis were examined, the positive rates of circulating antigens and specific antibodies were 60%,40%, and 80%, 60 1%, respectively; when both detection was combined, the positive rates were 100% and 71 7% respectively, whereas a false positive rate of 3% of CAg or CAb was detected in the urine of 100 normal controls. [Conclusion] The detection of circulating antigen in urine is a practicable and noninvasive method for the diagnosis of schistosomiasis.


CONSTRUCTION AND ANALYSIS OF cDNA LIBRARY OF NECATOR AMERICANUS THIRD STAGE LARVAE

ZHANBin;JohnHawdon;SHANQiang;RENHainan;QIANGHuiqing;XIAOShuhua;LITiehua;FENGZheng;PeterHotez

2000, 18(1):  9-29. 

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　Objective] To obtain the genetic information on Necator americanus and to search for the purpose genes.[Methods] mRNA was isolated from the third stage larvae of Necator americanus maintained in hamsters. Double strand cDNA was synthesized and ligated to λZAPII vector to construct the cDNA library. Expressed sequence tages (ESTs) were obtained by single pass sequencing of randomly isolated cDNA clones from the established library. [Results] A cDNA library of N americanus was successfully constructed with high recombinant efficiency. The titer of unamplified library was 1×10 7. The insert size was about 750～3 000 bp. Of 11 ESTs obtained from the library, 7 have a significant homology with certain functional genes.[Conclunsion] A high quality and high representative cDNA library of N.americanus was constructed at the first time and some functional genes were identified from the library by ESTs.


CLONING AND SEQUENCE ANALYSIS OF A GENE ENCODING AMASTIN FROM LEISHMANIA MAJOR

CHENJun;SIChongwen;WANGQinhuan;LIUYan;ZHONGYanwei;YANGJizhen;HONGWeiguo

2000, 18(1):  10-32. 

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　Objective]To clone a gene encoding surface protein from Leishmania major .[Methods] Using T cruzi amastin DNA sequence as a reference,computer search was done on GenBank and dbEST databases by using BLAST path. A Leishmania major DNA library has been constructed and screened by in situ colony hybridization.[Results] A 309nt DNA fragment from Leishmania major was found in dbEST. Leishmania major DNA library was screened using specific primers synthesized according to 309 nt DNA sequence, and a full length coding sequence for Leishmania major amastin was cloned. The coding sequence consisted of 552 nt, and translated into 183 amino acid residues. The homology is 23 5% at amino acid sequence level between Leishmania major and T cruzi amastins. [Conclusion] A full length amastin coding gene for Leishmania major has been cloned.


THE PROTECTIVE IMMUNITY IN MICE IMMUNIZED WITH FhGST OR AsGST AGAINST SCHISTOSOMA JAPONICUM CERCARIAE

SUNWenyu;LIUShuxian

2000, 18(1):  11-36. 

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　Objective] To study the protective immunity against Schistosoma japonicum (Sj) cercariae challenge in mice immunized with FhGST or AsGST. [Methods] Three groups of Kunming mice were immunized three times with rSjGST, FhGST and AsGST, respectively. Then 30 cercariae of Schistosoma japonicum were given per mouse by abdominal skin inoculation. Six weeks later, all mice were killed to collect adult worms, liver, spleen and large intestine for worm count and egg count. [Results] The worm burden was reduced by 27 8%～36 4% in the three immune groups compared with the two control groups ( P <0 05). rSj26GST, FhGST and AsGST significantly decreased the number of eggs deposited in the tissues. [Conclusion]: FhGST and AsGST could induce protection against S japonicum cercariae infection.


CLONING AND EXPRESSION OF CYSTICERCUS CELLULOSAE ANTIGEN cC1 IN E.coli

CHENRuiwen;LINYi;SUNShuhan

2000, 18(1):  12-39. 

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　Objective]To clone and express Cysticercus Cellulosae antigen cC1 in E.coli . [Methods] cC1cDNA fragment was cloned to BamHI and PstI sites of pGEM 3Z vector.After alteration of the restriction sites,the fragment was cloned to EcoRI and XhoI sites of pGEX 5T with a synthetic linker to construct recombinant expression vector pGEX 5T cC1. [Results] The clone produced the largest yield of cC1 protein expression when incubated in 2YT culture medium for 3 h or induced by IPTG for 6 h.Detected by scanning optical densitometry, cC1 constituted 57% of the total bacterial proteins. Western blotting analysis revealed that the GST cC1 fusion protein exhibited a specific reactive band.[Conclusion] High level expression of Cysticercus cellulosae antigen cC1 was obtained in E.coli .


IMMUNE RESPONSE IN PNEUMOCYSTIS CARINII PNEUMONIA IN RATS

LIUBin;HELixian;QUJieming;HUBijie;WANGBaoqing;LIXiying

2000, 18(1):  13-42. 

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　Objective] To study the inflammatory immune response to Pneumocystis canii pneumonia(PCP) in rats induced by glucocorticoid(GC).[Methods] The model of PCP was set up by injecting GC subcutanously to SD rats. Lymphocyte proportion in peripheral blood, CD4 +/CD8 + T cell ratio of PBL and lymphocyte proportion in the BALF were measured. The levels of sIL 2R and TNF α in the BALF were detected.[Results]① After the rats were immunospressed, the lymphocyte proportion in the peripheral blood and CD4 +/CD8 + ratio of PBL, and the lymphocyte proportion in the BALF were decreased, and the levels of sIL 2R, TNF α in BALF were reduced. ② The lowest levels of TNF α in BALF and CD4 +/CD8 + T cells of PBL were observed in PCP group; ③ The lymphocyte proportion in the BALF was significantly higher in PCP group than in PC negative group.[Conclusion]The reduction in the level of TNF α and CD4 + /CD8 + T cell ratio in rats treated with GC might result in PCP infection under immunosuppressive condition.


DETECTON OF CIRCULATING ANTIGEN IN URINE FROM MICE INFECTED WITH TOXOPLASMA TACHYZOITES BY SPA-ELISA

JINWuguan;LIYunzhu;YUShancang;YANGHuizheng

2000, 18(1):  14-45. 

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　Objective] To explore a simple and convenient immunoassay for early diagnosis of Toxoplasma infection.[Methods] Urine samples collected from three groups of mice infected with different doses of tachyzoites were detected for Toxoplasma circulating antigen (TCA) by dot ELISA using HRP SPA as a second antibody ( SPA ELISA).[Results] Toxoplasma circulating antigens were detected in all three groups of infected mice in contrast with the normal control group. Toxoplasma circulating antigen was detected on d 6 and d 3 after infection in mice of light and moderate infection groups, respectively.[Conclusion]SPA ELISA is a simple and convenient method for early immunodiagnosis of recent Toxoplasma infection.


实验报道

QUALITATIVE AND QUANTITATIVE COMPARISON OF THREE AGGLUTINATION TESTS FOR DETECTING TOXOPLASMA GONDII ANTIBODIES

ZHANGShuyi;WEIMeixiong

2000, 18(1):  15-48. 

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　Objective] To evaluate the diagnostic value of three agglutination tests used in three countries for detection of antibodies to Toxoplasma gondii .[Methods] A total of 288 human serum samples were assayed using modified agglutination test (MAT 1,using selfmade antigen),latex agglutination test (LAT,using Japanese Kit) and modified agglutination test (MAT 2,using French antigen).[Results] The positive rates of MAT 1 (≥1∶20), LAT (≥1∶32) and MAT 2 (≥1∶20) were 9 7% (28/288), 8 9% (10/112) and 8 1% (17/210), respectively. No significant statistical difference was found among these positive rates (χ 2 =0 392, P >0 05). High agreements were found between MAT 1 and LAT (93 7%), LAT and MAT 2 (94 5%), and MAT 2 and MAT 1 (97 3%). Significant correlation were demonstrated in MAT 1 and LAT (r = 0 613), LAT and MAT 2 (r = 0 551), and MAT 2 and MAT 1 (r = 0 841), p <0 001.[Conclusion] The detection efficiency of the three agglutination tests is in good agreement and could alternatively be used for the diagnosis of toxoplasmosis.


防治经验

PRACTICABILITY OF IFAT USING PLASMODIUM CYNOMOLGI AND PLASMODIUM FALCIPARUM ANTIGENS IN DIFFERENT MALARIOUS AREAS

LUOManzhen;ZHENGXiang;SHANGLeyuan;CHENJifeng;ZHUWeidong;TANGLinhua

2000, 18(1):  16-51. 

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　Objective] To compare the practicability of IFAT in different malarious areas using Plasmodium cynomolgi ( P c ) and Plasmodium falciparum ( P f )antigens. [Methods]This survey was carried out in Yaliang Township of Sanya City, Hainan Province, wherea mixed malaria is endemic, and in Tongbo County, Henan Province where only vivax malaria is endemic, and in Weihui City, Henan Province where vivax malaria has been under effective control since 1994～1998.[Results] In Yaliang Township, 310 blood samples were examined, the antibody positive rates with P c and P f were 37 4% and 31 3% ,respectively,the rate of coincidence being 83 9%. In Tongbo County, 300 blood samples were examined The antibody positive rates with P c and P f were 23 0% and 9 7%, respectively ( P <0 01). Another 245 blood samples from children were examined in Weihui City and the antibody positive rates were below 1% with two antigens, while the positive antibody rate was 3 3% with P f antigen.[Conclusion]Both P f and P c antigens could be used in malaria antibody surveillance in mixted endemic areas, while in vivax malaria endemic areas, P c antigen was recommended.