Table of Content

30 December 2002, Volume 20 Issue 6

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论著

Molecular Identification of Anopheles maculatus Complex from China

MAYa-jun;QUFeng-yi;DONGXue-shu;ZHOUHong-ning

2002, 20(6):  1-324. 

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　Objective To establish the molecular identification of five members in Anopheles maculatus complex from China. Methods Different rDNA-ITS2 regions of An. maculatus complex were sequenced and analyzed. The species specific primers were designed, and PCR assay was used for the identification. Results The length and GC contents of ITS2 were 328 bp, 58.54% in An. pseudowillmori, 330 bp, 57.85% in An. maculatus, 337 bp, 59.05% in An. willmori, 334 bp, 58.68% in An. dravidicus, and 338 bp, 57.69% in An. sawadwongporni, respectively. The intra-species ITS2 sequences were conservative. The ranges of divergence level among five members were from 9.7% to 18.9% . Five distinct specific fragments were amplified by PCR assay using five species specific primers and 5. 8S primer. The length was 119, 186, 231, 327 and 406 bp respectively. Conclusion The diagnostic PCR assay based on ITS2 divergence to distinguish five members of An. maculatus complex was simple and reliable.


Analysis and Cloning of Proteinase Cathepsin L1 Gene Coding Sequence of Schistosoma japonicum

LEIZhi-gang;MENGJin-xiu;HEAi;LIZhuo-ya;YIBing;ZHANXi-mei

2002, 20(6):  2-327. 

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　Objective To analyze the full coding sequence of proteinase cathepsin L1 (SjCL1) of Schistosoma japoni-cum, and clone it into the eukaryotic expression vector pcDNA3. Methods Total RNA was isolated from adult worms of S. japanicum, the sequence of SjCL1 gene 5'-end was attained by performing averse nested PCR, and the sequence of SjCL1 gene 5'-end was determined by sequencing after being cloned into T vector. The coding region gene of SjCL1 was amplified by PCR, and the fragment from PCR was cloned into eukaryotic expression vector pcDNA3 via Bam HI and Xho I sites. The resulting construct was determined by PCR, restriction analysis and sequencing. Results A 320 bp sequence of SjCL1 gene 5'-end of Schistosoma japonicum was obtained by using averse nested PCR. After combined with the reported segment of SjCL1 gene, an integral coding sequence was obtained. The coding region of SjCL1 gene was specifically amplified by PCR, with a size of about 1 kb. The expression plasmid pcDNA-SjCL1 contained the amplified fragment, which is validated by PCR, restriction analysis and sequencing. Conclusion The eukaryotic expression plasmid containing the coding sequence of SjCL1 gene was constructed.


Further Studies on the Life Cycle of Thelazia callipaeda in China

WANGZeng-xian;SHENJi-long;WANGKe-can;WANGHong-yan;YANGZhao-xin;DUJi-shuang;JIANGBao-ling

2002, 20(6):  3-331. 

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　Objective To further investigate the life cycle and intermediate host of Thelazia callipaeda in China. Methods Dogs and rabbits were experimentally infected with larvae of T. callipaeda from naturally infected houseflies Musca spp. and fruit flies Amiota okadai. Houseflies and A. okadai bred in laboratory were fed with newborn larvae of T. callipaeda to define the intermediate host of the eye worm. Results Two rabbits and one dog were infected with 34 larvae of T. callipaeda taken from 493 naturally infected A. okadai. As a consequence, 11 adult worms were harvested from the experimentally infected animals 18 to 44 days after infection. The development process of T. callipaeda larvae in A. okadai included three successive stages. The infective larvae migrated through the hemocoel to the head and proboscis of A. okadai. Complete larval development in A. okadai required 14 - 17 days under appropriate temperature. Infective larvae entered the conjunctiva sac of the definitive hosts (dogs, cats, and man) when infected A. okadai sucked their eye secretions. The larvae grew into adults, with two ecdyses in the process of development. The adult females began to produce newborn larvae in 35 days after infection. The longest life-span of T. callipaeda was more than 30 months. Conclusion A. okadai is the intermediate host of T. callipaeda in China.


Passage Cultivation and Immunological Identification of Schistosoma japonicum Cercaria Cells

ZHANGZhong-yong;ZENGXian-fang;LIJing-ru;YIXin-yuan;ZENGQing-ren;ZHANGJing;YANGan-wei;ZHANGJie

2002, 20(6):  4-334. 

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　Objective To study the methods of in vitro proliferation and passage cultivation of the cells of Schistosoma japanicum cercariae. Methods Between 5 000 and 10 000 cercariae of Schistosoma japonicum were collected under aseptic condition and placed into RPMI 1640 medium containing 10% fetal bovine serum. The cercariae were disrupted swiftly using a tissue tearor, and the disrupted material was incubated for 30 min in 250 U crab collagenase at 26℃. After centrifugation, the enzyme solution was removed, and modified RPMI 1640 medium was added containing penicillin (100 U/ ml), streptomycin (0.1 mg/ ml), and amphotericin B (0.25 μg/ ml), and certain amount of cell growth enhancing material. When adhesion cells proliferated and grew fully on the bottom, subculture was in progress according to 1:2 split ratio. Cells cultivated by 5 passages were used to detect the specific antibody in sera of patients with chronic schistosomiasis through ELISA. Results On the 3rd day of primary cultivation, bright cells, both individual and clustered, were seen around the disrupted cercariae. A monolayer of cells formed on the 10th day. Adhesion cells grew fully on the bottom and subculture was in progress on the 14th day. Cells were found to grow evenly in passage cultivation, within every 7-14 days another passage could be made. Using cells of the fifth passage cultivation as antigen, the antibody positive rate was 90.3% in patients with chronic schistosomiasis and the false positive rate was 6.7% in healthy controls. Conclusion The in vitro passage cultivation of S. japonicum cercaria cells has been successful to the 5th generation and the cultured material could be used in the immunological research.


Effect of Inhibitors of Cell Signal Transduction on Egg Granuloma Formation in Mice Infected with Schistosoma japonicum

XIAChao-ming;GONGWei;LUOWei;ZHOUWei-fang;LIYun-he;XIONGSi-dong;ZHAXi-liang

2002, 20(6):  5-338. 

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　Objective To observe the effect of signaling inhibitors of tyrosine-protein kinase (TPK), protein kinase C (PKC) and phosphatidylinositol-3-kinase (PI3-K) (tyrphostin-25, D-sphingosine and wortmannin, respectively) on the egg granuloma formation of Schistosama japonicum , and probe the mechanism of the effect. Methods Three signaling inhibitors were injected by tail vein of mice from the thirty-fifth day after infection for five successive days. The liver egg granuloma measurement was performed by histological examination and the kits of ELISA and NO assay were used for the quantitative determination of IFN-γ, IL-4 and NO respectively in murine serum at 6 and 8 weeks after infection. Results The egg granuloma formation of liver tissue was significantly reduced by the specific inhibitors of TPK and PKC in vivo. The ratio of egg granuloma inhibition was up to 56.2% - 63.4% by the effects of PKC inhibitor D-sphingosine. The PKC inhibitor mainly inhibited the expression of IL-4 and the detection of NO level further demonstrated the inhibition. Conclusion The egg granuloma formation could be significantly inhibited by PKC inhibitor in the early stage of Schistosama japonicum infection in mice. These findings suggest that PKC inhibitor might inhibit the Th2 bias and mediate a deviation from Th2 to Th1.


Cloning, Sequencing and Expression of Trichinella spiralis p49 Gene

WENYan;LAOWei-de;GAOHong;ZHANGChuan-sheng;LIUSi-guo;GANShao-bo

2002, 20(6):  6-341. 

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　Objective To conduct cloning, sequencing and expression of Trichinella spiralis ES antigen p49 gene. Methods RT-PCR was used to amplify the specific gene fragment from the total RNA of Trichinella spirais larvae. The PCR product was ligated to the T-vector and the recombinant plasmid was verified by sequencing. T-p49 and pGE-4T-3 were treated by both BamHI and XhoI. The ligation reaction was catalyzed by T4 DNA ligase. Results The p49 gene was cloned by using RT-PCR. Sequence analysis showed that the p49 gene obtained was consistent to the p49 sequence reported in the database. The expressed protein was shown as a new band at SDS-PAGE. BLAST analysis demonstrated that this p49 gene was 99% identical to the p49 gene reported and to the 43 kDa secreted glycoprotein gene in the database. Conclusion p49 gene from Trichinella spiralis larvae was cloned, sequenced and expressed.


Studies on Cytotoxicity of Nitric Oxide to Schistosomula of Schistosoma japonicum

LONGXiao-chun;LIYong-long;FANGZheng-ming

2002, 20(6):  7-344. 

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　Objective To study the in vitro larvicidal activity of nitric oxide (NO) to the juvenile Schistosoma japoni-cum. Methods Macrophages were induced by LPS or LPS + IFN-γ to produce NO, schistosomula obtained mechanically from cercariae were added to the medium with activated macrophages, the larvicidal activity was observed within 48 h . In order to further confirm the effect of NO, an inhibitor of iNOS,L-NNA (Nω-nitro-L-arginine), was used to inhibit the production of NO, larvicidal activity was measured by the same methods and the difference of dead larvae ratio was compared between the inhibited and uninhibited groups. Results LPS and LPS + IFN-γ can induce macrophages effectively, with the NO production of (109.96±3.70)μmol/L and (113.50±7.38) μmol/L respectively, accordingly the larvicidal effect reached to 91.07% ±2.92% and 96.86%±2.36% respectively. This activity can be inhibited by L-NNA. NO production and dead larvae ratio were reduced significantly in the inhibited group than in the uninhibited one. Conclusion NO produced by activated macrophages can kill schistosomula of Schistosoma japonicum.


Curative Effect of Praziquantel on Juvenile Paragonimus westermani and the Dynamic Changes of T Lymphocytes in Infected Mice after Treatment

YANTao;LIGuoliang

2002, 20(6):  8-347. 

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　Objective To study the curative effect of praziquantel against juveniles of Paragonimus westermani and the dynamic changes of T lymphocytes and their subpopulations in infected mice after treatment. Methods Mice infected with metacercariae of P. westermani were treated with praziquantel 10 days after infection for examining the curative effect. Results Mice infected orally each with 30 metacereariae of F. westermani were treated with praziquantel at doses of 400,500 and 600 mg/(kg·d) respectively for 3 d, the juvenile reduction rates were 71.01 % ± 10.38% ,82.29% ± 7.92% and 95.83% ± 3.24% respectively; when the mice were infected with 80 metacercariae and treated with a dose of praziquantel 600 mg/(kg·d) for 3 d, the worm reduction rate was 84.49% ±10.91%. T lymphocytes and their subpopulations began to increase at 1 wk and returned to normal at 8 wk after praziquantel treatment. Conclusion The efficacy of praziquantel against juvenile P. westermani was poorer than that against adult worms ; the efficacy was relevant to the drug dosage and the number of metacercariae infected. The T lymphocytes and their subpopulations may be regarded as an index for curative effect.


Studies on Th2 Type Cytokines in Patients with Neurocysticercosis

XUHong-xiu;JIAFeng-ju;LIUYu-bing;XUJing;WEIDong-dong

2002, 20(6):  9-350. 

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　Objective To explore the level of Th2 type cytokines including IL-4, IL-10 and IL-13, and the immunoreg-ulation of cytokines in patients with neurocysticercosis. Methods Lymphocytes in patients with neurocysticercosis were separated from the blood sample with density gradient centrifugation and the total RNA was extracted by guanidine isothiocynate method. cDNA was synthesized by reversed transcription reaction. The target gene was then amplified by PCR. The PCR products were analyzed by electrophoresis. Results Results of RT-PCR showed that cytokines mRNA in lymphocytes of peripheral blcod were detected in 27 patients with neurocysticercosis but not in the other 3 cases. Among the positive cases, mRNA of IL-4, IL-10 and IL-13 was detected in 16, 17 and 14 cases respectively and mRNA of IL-4, IL-10 and/or IL-13 was detected in all the 27 cases. In the detection of lymphocytes in peripheral blcod of 10 healthy subjects, expression of IL-4 and IL-10 was found in only one case at low level. Conclusion The study revealed that Th2 associated cytokines were expressed at high level and the humoral immunocompetence was relatively strong in patients with neurocysticercosis.


Restudy on a Model for Estimating the Sporozoite Infection Rate in Mosquitoes Using Pooled Sampling

SUNQing-wen;ZHUHuai-min;LULiu;GUZheng-cheng;CHENGXiang;FANGYing

2002, 20(6):  10-353. 

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　Objective To analyze the pooled sampling method for estimating the vector prevalence theoretically and provide the pooling schemes. Methods The criterion of mean square error (MSE) was used to determine the pool size and the number of pools. Results In comparison with the traditional method based upon testing samples one by one, the pooling method suggested in the present paper greatly reduced the laborious work load. Conclusion Based on the criterion of MSE and the predicted vector infection rate, the method was proved feasible in determining pool size and number of pools. The estimator x/mn is more manageable and precise than the traditional unbiased estimator.


Reduction of Total Antioxidant Capacity in Artemether-treated Female Schistosoma japonicum

ZHAIZi-li;MEIJing-yan;JIAOPei-ying;XIAOShu-hua

2002, 20(6):  11-357. 

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　Objective To study the effect of artemether (Art) on total antioxidant capacity (T-AOC) in adult Schisto-soma japonicum. Methods In vitro, the T-AOC was determined in five-week old worms incubated without or with Art and/ or hemin for 24 h, and the worms were continuously incubated for 96 h, then worm survival was assessed. In vivo, T-AOC was determined in worms freshly recovered from mice 6 - 24 h after treatment with Art 300 mg/kg. Results Throughout 96 h incubation no worms were killed by 50 μmol/L Art or 50 μmol/L hemin alone, but approximately 80% of them were killed by Art plus hemin. Addition of reduced glutathione and vitamin E could significantly block the tidal action of the combined treatment. No effect on T-AOC was seen in the worms exposed to Art or hemin alone for 24 h, but the combined treatment led to a pronounced T-AOC reduction in female worms in vitro. Such a drug effect on female worms was demonstrated in vivo. After female worms were exposed to Art for 6 - 24 h in vivo, their T-AOC was significantly reduced by 40% - 64%. However, no drug effect on male worms' T-AOC was observed in vivo and in vitro exposed to Art plus hemin. Conclusion Art-induced T-AOC reduction in female worms may sensitize them to lethal damages of endogenous and exogenous oxygen radicals.


Recognition of Specific Antigens by Specific IgG and IgE During Anaphylactic Shock Induced by Echinococcus granulosus in Sheep

ZHENGHong;XUZhi-xin;YANGGe-xiong;WENHao

2002, 20(6):  12-360. 

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　Objective To determine the specific recognition of Echinococcus granulosus (E.g.) cyst fluid crude antigen (EgCF) and antigen B (EgB) by serum specific IgG and IgE using Western blotting during anaphylactic shock induced by E. g. in sheep, and to investigate the importance of defined characteristics and molecular weight of the specific antigens. Methods EgCF was obtained through local slaughterhouses in Urumqi from the cysts of infected sheep liver. Western blotting was used to analyze total specific IgG and IgE antibodies in serum from sheep infected with E.g. using either crude antigen of E. g. and EgB, and to understand specific recognition of hydatid cyst antigens by serum total IgG and specific IgE. Results SDS-PAGE and Western blotting showed that there were differences between EgB and EgCF in electrophoresis pattern. EgB was recognized by IgG from sera of infected sheep in a series of bands with molecular weight ranging from 31, 43 to 66.2 kDa. No binding of IgG against EgCF was observed in any serum from the infected sheep during anaphylactic shock. In contrast, specific IgE antibodies in E. g. infected sheep obviously recognized the single 43 kDa subunit of EgCF, but no binding of specific IgE against EgB was observed in sera of the infected sheep. Conclusion EgCF is consisted of antigenic components in which there is a specific antigen against IgE with a molecular weight of over 43 kDa. This component may lead to an anaphylactic shock induced by E. g. . EgB is a specific antigen against the total IgG but not to the specific IgE.


实验报道

Analysis of Trace Elements in Lung,Liver and Spleen of Rats Infected with Pneumocystis carinii

DUANYi-nong;LIRong;ZHOUQuan;PENGGuang-ren

2002, 20(6):  13-363. 

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　Objective To study the effect of Pneumocystis carinii (Pc) infection on the level of six trace elements (Ca2+ , Mg2+ , Fe2 + , Cu2 + , Zn2+ , Mn2+ ) in the lung, liver and spleen of the rats. Methods 30 rats were randomly divided into two groups:20 rats in the experimental group and 10 in the control group. Each rat in the experimental group was injected subcutaneously with dexamethasone (1 mg per rat) twice a week. All rats in the experiment group (Pc infected and PC negative) and the control group were killed to obtain lungs, livers and spleens after 10 weeks and the atomic absorption method was used for element analysis. Results Compared with the Pc negative group and the control group, the level of Zn2+ in the lung in Pc infected group was significantly reduced (P<0.05). The amount of Ca2+ and Mg2+ in the infected rats were higher than that of the control (P<0.05). No difference was determined in the content of Fe2+ , Cu2+ and Mn2+ among the Pc infected group, the Pc negative group and the control group. The level of Zn2+ in liver in the Pc infected group was significantly reduced (P<0.05). The amount of Mg2+ in the Pc infected rats was higher than that of the control (P<0.05), but no difference was found in the content of Ca2+ , Fe2+ , Cu2+ and Mn2 + among the groups. The level of Zn2+ and Cu2+ in spleen in Pc infected group was significantly reduced (P < 0. 05 ) , and no difference was found in the content of Ca2+ , Mg2+ , Fe2+ and Mn among the three groups. Conclusion Pneumocystis carinii infection might play a role in the changes of trace elements in the lung, liver and spleen of rats.