Table of Content

30 April 1999, Volume 17 Issue 2

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论著

PURIFICATION AND SPECIFIC DETECTION OF TWO MAJOR SCHISTOSOMA GUT ASSOCIATED CIRCULATING ANTIGENS, CAA AND CCA *

QIANZhongli;WANGZhaojun;LUPing;G.J.VanDam;A.M.Deelder

1999, 17(2):  1-69. 

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　AIM: To investigate the specificity differences of the 2 major gut associated diagnostic circulating molecules in Schistosoma japonicum infection, CAA and CCA, and to obtain sufficient purified material for setting up a standard series in quantitative determinations. METHODS: Isolation and purification of the two worm fractions from a trichloroacetic acid (TCA) soluble preparation of S.japonicum adult worm antigen (AWAj TCA) by Mono Q anion exchange chromatography were performed and the specific reactivity of the eluted fractions by antigen capture ELISA (specific for CAA or CCA) with reference to affinity purified preparations of S.mansoni CAA and CCA was analysed. RESULTS: By using an ionic strength gradient, CCA was eluted in two major peaks, an unbound fraction CCA 1, and a major bound fraction CCA 2. Two additional minor peaks, CCA 3 and CCA 4, were eluted at higher ionic strengths. CAA was only detected in a bound fraction, partly overlapping with CCA 3. In the CCA 1 and CCA 2 fractions reactivity was only found in the antigen capture ELISA using anti CCA McAbs both for capture and detection. The CAA fraction was predominantly found to be positive in the antigen capture ELISA using anti CAA McAbs both for capture and detection. However, when using combinations of anti CCA and anti CAA McAbs for capture and detection by ELISA this fraction showed some reactivity. CONCLUSION: Two CCA fractions contain molecules which bear at least two CCA epitopes; while the CAA fraction contains molecules which contain at least two CAA epitopes, and one CCA epitope.


OBSERVATION ON THE EFFECT OF Sj32DNA VACCINE OF SCHISTOSOMA JAPONICUM *

LIChuanming;SHIYouen

1999, 17(2):  2-73. 

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　AIM: To explore the protective immunity against cercariae challenge in mice immunized with Sj32DNA vaccine of Schistosoma japonicum. METHODS: Primers were designed and the full length and part cDNA encoding 32 kDa(Sj32) amplified from pSj32 by PCR were cloned into pBK CMV. Eukaryotic expression vector pBK Sj32 1 containing full length Sj32 cDNA and pBK Sj32 2 containing fragment of Sj32 cDNA were constructed. After selection and identification, two plasmids were transfected into NIH 3T3 cells. The expression of Sj32 in transfected cells was confirmed by IFA. After identification, the two plasmids were prepared and purified on batches. Each BALB/c mouse was vaccinated at weeks 0,3, and 5, by injection with 100 μg of purified Sj32DNA into quadriceps muscle. Mice immunized with constructs and the control mice were challenged four weeks after final DNA injection, and worms and the number of eggs in the liver tissue were counted at 8 weeks after challenge. RESULTS: Perfusion and egg counts showed significant differences in terms of worm reduction rate 38 6%-30 9% and egg reduction rate 55 7%-55 2% between the vaccinated and control mice. CONCLUSION: Sj32 DNA vaccine could result in protection against a subsequent challenge of S.japonicum in mice.


STUDIES ON THE FEATURES OF PROTECTIVE IMMUNE RESPONSE INDUCED BY RECOMBINANT Sjc26GST OF SCHISTOSOMA JAPONICUM *

ZHOUShenghua;LIUShuxian;SONGGuangcheng;XUYuxin

1999, 17(2):  3-77. 

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　AIM: To compare the differences in the protective immune response induced by reSjc26GST between C57BL/6 and BALB/c mice. METHODS: Mice were immunized subcutaneously with reSjc26GST emulsified with Freund’s adjuvant. The specific antibody isotypes elicited by reSjc26GST, in vitro lymphocyte proliferation and cytokine responses to reSjc26GST were examined. RESULTS: The level of anti reSjc26GST IgG was higher in immunized C57BL/6 mice than in BALB/c mice, and examination of the IgG subclasses demonstrated that reSjc26GST immunized C57BL/6 mice resulted in predominantly IgG1 and IgG2a/IgG2b antibody responses, whereas in BALB/c mice, IgG1 antibody. Cytokine production assays revealed that the quantity of IFN γ and IL 2 and IL 5 released from the reSjc26GST primed splenocytes were significantly higher in C57BL/6 mice than in BALB/c mice. These results showed that the immunization with reSjc26GST in C57BL/6 mice elicited a mixed immune reaction of Th1 and Th2 cell responses as demonstrated by the elevated levels of IgG1, IgG2a/IgG2b and IL 2, IFN γ,IL 5 , whereas in BALB/c mice, reSjc26GST immunization elicited Th2 response as demonstrated by the elevated levels of IgG1, IL 2 and IFN γ. CONCLUSION: Immunization with reSjc26GST could elicit higher humoral and cellular immune responses in C57BL/6 mice than in BALB/c mice.


EVALUATION OF DIAGNOSTIC VALUE OF 18 kDa ANTIGEN IN ALVEOLAR ECHINOCOCCOSIS *

JIANGLi;WENHao;LIXiong;Islayin·Osman

1999, 17(2):  4-80. 

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　AIM: To evaluate the 18 kDa antigen in diagnosis of human alveolar echinococcosis. METHODS: A total of 214 sera from patients with alveolar echinococcosis (AE, 33), cystic echinococcosis (CE, 69), cysticercosis (30) and healthy controls (82) were examined by Western blotting to detect the antibodies against 18 kDa antigen using both antigens from protoscoleces of E.granulosus (Eg) and E.multilocularis (Em). All the sera were tested simultaneously by ELISA to detect the antibodies against hydatid cystic fluid antigens of sheep as a comparison. RESULTS: The positive rate by ELISA was 93 9% in AE, 85 5% in CE, 50% in cysticercosis and 6 1% in healthy controls, respectively, suggesting a critical cross reaction and a certain degree of nonspecific reaction. The serum positive rates by Western blotting with the 18 kDa antigen of Eg and Em were 90 9% and 90 9% in AE, 10 1% and 13% in CE, 13 3% and 16 7% in cysticercosis, respectively. No positive was found in the sera from healthy controls. CONCLUSION: The 18 kDa antigen may be used in the differential diagnosis of AE from CE.


SEQUENCING OF CYTOCHROME C OXIDASE 1 GENE OF ANCYLOSTOMA DUODENALE AND NECATOR AMERICANUS *

LITiehua;ZHANBin;JohnMHawdon;GONGXin;XIAOShuhuaSHANQiang;FENGZheng;PeterJHotez

1999, 17(2):  5-83. 

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　AIM: To identify the genetic diversity between Ancylostoma duodenale and Necator americanus. METHODS : Mitochondrial cytochrome C oxidase subunit 1 (CO1) gene was amplified from genomic DNA of human hookworms collected from infected patients in Hejiang County, Sichuan Province , and the purified PCR products were directly sequenced by using Licor auto sequencer. RESULTS: The PCR products were about 700 bp. Alignment of CO1 gene fragment sequences showed 89 7% similarity between Ancylostoma duodenale and Necator americanus, but still certain nucleotide variations (10 3%) existed. CONCLUSION: CO1 gene sequence can be used as a marker to identify the two species of human hookworms.


STUDIES ON ALLOZYME OF GAMMATRICULA

ZENGXiaopeng;CHENCuie;DINGJianzu;George.M.Davis

1999, 17(2):  6-86. 

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　AIM: To furnish molecular genetic evidences for taxonomy of Gammatricula. METHODS: A total of 24 enzymes of 6 populations of Gammatricula songi and 1 population of Gammatricula chinensis collected from Kaihua County and Chunan County of Zhejiang Province were studied using horizontal starch gel electrophoresis. RESULTS: 29 loci were found. The percentages of polymorphic loci of G.songi populations were 6 9%-13 8%. All loci of G.chinensis were monomorphic. The Neis distance among G.songi populations did not exceed 0 12. The Neis distance between G.songi and G.chinensis was 0 73. CONCLUSION: The allozyme variations of inter G.songi are limited,but the allozyme variation between G.songi and G.chinensis is significant.


ANTI HEMOLYTIC AND ANTI MEMBRANOUS LIPID PEROXIDATION EFFECTS OF DAPHNETIN *

NIYichang;XUYueqin;WANGMingjie;LIUYunguang

1999, 17(2):  7-89. 

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　AIM: To investigate the anti hemolytic and anti membranous lipid peroxidation effects of daphnetin. METHODS: The inhibition rates of daphnetin on AQD induced hemolysis and erythrocytic membranous lipid peroxidation were determined by routine in vitro assay for detecting hemolytic toxicity and by HPLC for detecting membranous lipid peroxidation products. RESULTS: Daphnetin at a range of 10-80 μmol/L inhibited dose dependently AQD induced hemolysis and lipid peroxidation by 33 0%-69 2% and 11 9%-58 2%, respectively. CONCLUSION: Daphnetin has anti hemolytic and anti erythrocytic membranous lipid peroxidation effects.


STUDIES ON RECOMBINANT CHITINASE AND SXP 1 ANTIGENS AS ANTIMICROFILARIAL VACCINES *

WANGShihai;ZHENGHuijun;TAOZenghou;WillyF.Piessens

1999, 17(2):  8-94. 

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　AIM: To determine whether immunization with recombinant filarial chitinase or a fragment containing the epitope recognized by McAbMF1 and SXP 1 could protect jirds against microfilaremia resulting from infection with B.malayi. METHODS: Test jirds were immunized with the following recombinant parasite antigens: filarial chitinase, the c terminal fragments F 7R 2 or F 8R 2 of r chitinase, filarial SXP 1, myosin or maltose binding protein (MBP). Employing immunochemical techniqe (SDS PAGE, Western Blotting) and serology (ELISA) measured antifilarial antibodies level. RESULTS: Immunization of jirds with recombinant chitinase induced partial protection against microfilaremia resulting from subsequent infection with B.malayi, but did not reduce adult worm burdens. Vaccination was much less effective when administered during the prepatent stage of infection and was ineffective when given to microfilaremic jirds.Immunization of jirds with SXP 1, an antigen present in multiple worm stages also reduced microfilaremia and, in some experiments, adult worm burdens. CONCLUSION: The recombinant chitinase, fragments F 7R 2 and F 8R 2 and SXP 1 could provide partial protection against microfilaremia in jirds.


FIRST DISCOVERY OF TAENIA SAGINATA ASIATICA INFECTION IN YUNNAN PROVINCE *

ZHANGLili;TAOHong;ZHANGBingxiang;WANGHuizhen;WANGYunjiLIZengrong;YANGJikun;YANGBingxing;LIYanzhongPANGYankun;ZHANGHaoming;LIYing;WUYing

1999, 17(2):  9-96. 

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　AIM: To investigate the aetiology, species and epidemiological factors of Taenia infection in a pilot area of Lanping County, Yunnan Province.METHODS: Two patients with taeniasis were treated with antiscolex capsule and praziquantel, respectively. Five local weaning pigs free from tapeworm infection were fed with gravid proglottids obtained from the patients. After 2-3 months, the pigs were necropsied to examine cysticerci. In addition, one pig naturally infected with Taenia was treated as well. RESULTS: Based on morphological characteristics of the scolex and gravid proglottid of four tapeworms obtained from the patients, the worms were similar to T.saginata. 23 cysticerci recovered from two experimentally infected pigs were found in the livers, omentum majus and mesenterium, and 3 cysticerci were found in the liver and omentum majus in a naturally infected pig as well. The protoscolex of mature Cysticercus had two rows of rudimentary hooklets, one rostellum and four acetabula resembling to C.cellulosae. According to morphological characteristics and the location of cysticerci, the tapeworm was identified as Taenia saginata asiatia. CONCLUSION: T.saginata asiatica infection was first reported in Yunnan Province.


ASSOCIATION EXPRESSION OF GENES ENCODING GST OF SCHISTOSOMA JAPONICUM AND ENTEROTOXIGENIC ESCHERICHIA COLI *

ZHANGWei;ZHANGJingliu;LIUShuxian

1999, 17(2):  10-100. 

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　AIM: To study the association expression of two genes encoding GST of Schistosoma japonicum and K99 of enterotoxigenic Escherichia coli. METHODS: PCR technique was used to gain the K99 gene. After digestion with BamHⅠ and EcoRⅠ, the gene was cloned into the plasmid vector pUC18 by using recombinant DNA techniques. The other target gene of GST in pSj5 plasmid was obtained by EcoRⅠ digestion, and was then ligated into the recombinant plasmid pUC18 K99. The expressed product was assayed by SDS PAGE, Western blotting, reversal IHA, ELISA and transmission electron microscopy. RESULTS: Co expression product of around 50 kDa was obtained. The protein was recognized by anti GST and anti K99 antibodies (ELISA and IHA), and acted as pilus on the surface of transformed DH5α E.coli bacteria. CONCLUSION: Co expression of the genes encoding GST and K99 was successfully achieved.


STUDIES ON IMMUNOPROTECTION OF MONOCLONAL ANTIBODIES AGAINST CRYPTOSPORIDIUM PARVUM

ZONGHaihong;LIWeiming;ZHANGXue;YUShurong;BAIBanjun

1999, 17(2):  11-105. 

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　AIM: To explore the immune protection of the monoclonal antibody (McAb) againstCryptosporidium parvum.METHODS: On the basis of Madin Darby canine kidney (MDCK) cell culture, the immunoprotection of McAb against C.parvum was screened by means of neutralization test in vitro, and verified by rat model and transmission electron microscopy of infected MDCK cells. RESULTS: The number of C.parvum sporozoites recovered on the surface of rat intestinal epithelial cells and the mean output of oocyst were significantly reduced by McAb Z3D2 (P<0 001, P<0 05). The decreased Cryptosporidium at each development phase within infected MDCK cells and less damage of cell ultrastructure caused by Cryptosporidium were found in Z3D2 treated group. CONCLUSION: McAb Z3D2 possesses highly effective protection against C.parvum.


EFFECT OF IL 6 ON THE MULTIPLICATION OF TOXOPLASMA GONDII

FANGYanqiu;TANYan;ZHANGYongsheng;LIJing

1999, 17(2):  12-108. 

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　AIM: To observe the effect of IL 6 on the multiplication of Toxoplasma gondii.METHODS: Peritoneal macrophages from C57BL/6 mouse were incubated with 3 H uracil labelled T.gondii in vitro . RESULTS: Pretreatment (but not post treatment) of MΦ with IL 6 enhanced T.gondii multiplication in a dose dependent manner. Pretreatment with IFNγ resulted in active killing of parasites whereas the addition of IL 6 resulted in a partial reversal of IFNγ mediated toxoplasmacidal activity. Combining TNFα with IL 6 and IFNγ pretreatment resulted in restoration of toxoplasmacidal activity. Addition of polyclonal anti TNFα antibodies to IL 6 and IFNγ pretreatment resulted in enhancement in the IL 6 mediated impairment of IFNγ function. CONCLUSION: IL 6 could enhance intraperitoneal multiplication of T.gondii and reverse IFNγ mediated toxoplasmacidal activity.


实验研究

ESTABLISHMENT OF A MULTIPLEX PCR SYSTEM TO DETECT PLASMODIUM *

SUNMinglin;XUECaifang

1999, 17(2):  13-112. 

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　AIM: To establish a simple, rapid and practical multiplex PCR system to detect Plasmodium vivax(P.v) and Plasmodium falciparum(P.f). METHODS: A common upper primer S1 and two species specific lower primers of P.v and P.f, S2, S3 were designed according to the sequences of the small subunit ribosomal DNA(SSUrDNA )fragments of the two Plasmodium species. Using these three oligonucleotide primers, the two temperature point multiplex PCR system was established and applied to detect P.v and P.f in the stock blood samples of clinically confirmed patients.RESULTS: DNA fragments of about 705 and 575 base pairs were successfully amplified by multiplex PCR from the genomic DNA of P.v and P.f, but no fragments were obtained from that of P.knowlesi, P.cynomolgi and blood of healthy persons. By means of restriction endonuclease digestion, the amplified fragments were confirmed to be the SSUrDNA fragments of P.v and P.f as expected. This method was successfully used in detecting parasitemia 2-10 parasite/μl whole blood. Of 104 samples tested by this system, 81 were coincident with microscopic examination. The multiplex PCR system also found 17 samlpes of mixed infection, which were not detected microscopically. Another 2 samples detected as P.v by microscopic examination were verified to be P.f infection by the multiplex PCR. CONCLUSION: The ease of operation together with high sensitivity and specificity, particularly the sensitive detection for mixed infection in a single run of amplification suggests that the multiplex PCR system might be a useful tool for malaria diagnosis.


PREPARATION OF DNA PROBE FOR CRYPTOSPORIDIUM PARVUM

GUOBuping;ZHANGJianbin;LIANDerun

1999, 17(2):  14-114. 

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　AIM: To prepare a probe with high specificity and sensitivity for the detection of Cryptosporidium parvum. METHODS: Using PCR method, a fragment from the DNA of C.parvum was amplified . The PCR product, 452 bp DNA, was labeled with hapten digoxigenin.RESULTS: Examination of sensitivity showed that the DNA probe could detect as low as 2 pg DNA from C.parvum. The dot blot hybridizition assay showed that the probe hybridized with the DNA of C.parvum, but not hybridized with DNA of E.histolytica, G.lamblia, E.coli and D.bacilli. CONCLUSION: The probe was highly specific and sensitive for the detection of C.parvum.


PRELIMINARY STUDIES ON MICROWAVE IRRADIATION ELISA FOR DIAGNOSIS OF SCHISTOSOMIASIS JAPONICA

GUQizhang;ZHANGHongying;ZHUShanji;XUWenjuanDirector:JINGuoliang

1999, 17(2):  15-116. 

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　AIM: To explore a fast and highly efficient method for the diagnosis of schistosomiasis japonica. METHODS: Using microwave irradiation ELISA(MWI ELISA)and fast ELISA to detect specific antibodies in sera from 118 cases with schistosomiasis japonica, 61 healthy individuals and 12 paragonimiasis cases. RESULTS: The positive rates of schistosomiasis cases were 88 1% (104/118) by MWI ELISA and 91 5% (108/118) by fast ELISA, respectively (χ 2=0 74, P>0 05). The false positive reaction of healthy individuals was found in 2 cases(3 3%) by MWI ELISA and 1 case (1 6%) by fast ELISA, respectively (χ 2=0 34, P>0 05). No false positive reaction was found in paragonimiasis cases. CONCLUSION: The sensitivity and specificity of the two tests were similar, however, MWI ELISA was faster than fast ELISA.


DETECTON OF CIRCULATING ANTIGEN IN SERA FROM MICE INFECTED WITH TOXOPLASMA TACHYZOITES

JINWuguan;LIYunzhu;YUShancang;YANGHuizhen;QIANZongli

1999, 17(2):  16-118. 

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　AIM: To explore an assay method for the early diagnosis of Toxoplasma infection. METHODS: Serum samples collected from three groups of mice infected experimentally with different doses of Toxoplasma trophozoites were detected for the presence of Toxoplasma circulating antigen by using fast ELISA.RESULTS: Toxoplasma circulating antigen was detected on days 4, 4 and 3after infection in light, moderate and heavy infection groups, respectively. CONCLUSION: The level of circulating antigen was in parallel with the duration of infection. The determination of circulating antigen is useful in the early diagnosis of Toxoplasma infection.