Table of Content

30 April 2003, Volume 21 Issue 2

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论著

Study on DNA Sequences of Paragonimus skrjabini Populations from Five Provinces in China

GUIAi-li;CHANGZheng-shan;CHENMing-gang;DavidBlair;ZHANGYong-nian;CHENShao-hong;FENGZheng

2003, 21(2):  3-75. 

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　Objective To study differences among Paragonimus skrjabini populations from five provinces (Guangdong, Fujian, Yunnan, Hubei, Sichuan) and Paragonimus szechuanensis and to analyze the taxonomic status of P. heterotremus and P. veocularis in the Genus Paragonimus Braun, 1899. Methods DNA sequences were obtained from the ITS2 and CO1 genes and phylogenetic trees were built. Results Difference of the DNA sequences among P. skrjabini populations from five provinces were minor. P. veocularis and P. skrjabini were similar in DNA sequences. There were also some resemblances between P. miyazakii from Japan and the Fujian isolates of P. skrjabini. In addition, P. heterotremus was found to be closer to P. skrjabini and quite a distance from P. westermani in the phylogenetic tree. Conclusion All studied populations can be regarded as different strains of P. skrjabini; P. szechuanensis is not a separate species, but possibly a geographical strain of P. skrjabini. Also found was that P. veocularis and P. miyazakii may be the synonyms of P. skrjabini. As a separate species, P. heterotremus was found to be closer to P. skrjabini and quite a distance from P. westermani in the genetic relationship.


Prokaryotic Expression of Gene Encoding Schistosoma japonicum SjE16 and its Potential Application in Immunodiagnosis

WANGZhao-jun;HUWei;SHENDa-kang;WUXiao-wen;WANGJu-jun;HANZe-guang;XUEChun-Hang

2003, 21(2):  4-79. 

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　Objective To sub-clone and express the gene encoding Schistosoma japonicum calcium-binding protein (SJE16) and study its immunological response. Methods The specific primers were designed according to the expressed sequence tags(ESTs) sequence, which was used for amplification of the encoding sequence from the cDNA clone containing SJE16. The gene was subcloned into pGEX4T-l plasmid and expressed. The rSjE16 was tested for its immunological response by ELISA. Results The gene encoding Schistosoma japonicum SJE16 was cloned and expressed successfully. The immuno-genicity and diagnostic potential of rSjE16 were investigated. It was demonstrated by immunoassay in rabbits that the specificity and sensitivity of the test were 94.1% (16/17) and 88. 2% (15/17), respectively, and the level of antibody liter of the untreated group reached a peak at 9 - 11 wk post infection and maintained high at least for 21 wk post infection, while the antibody level in the treated group rapidly decreased to pre-infection level in 11 wk after treatment. In human, the specificity of the test was 98.3% (57/58); the sensitivity of acute and chronic patient serum assay was 85.5% (53/62) and 70.2% (40/57), respectively. Conclusion The recombinant protein of SjE16 (rSjE16) was acquired. It can be recognized by the sera from schistosomiasis patients, and the level of antibodies decreased quickly after treatment in experimental rabbits, which implicates the potential value for the evaluation of chemotherapy and detection of active infection.


Protective Antibody Response to Recombinant Fragments of Plasmodium falciparum Apical Membrane Antigen 1

LIXun;XUECai-fang;LIUZhong-xiang;WANGXian-feng;DINGJin;LEIJun-chuan;ZHENRong-fen

2003, 21(2):  5-83. 

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　Objective To determine the role of putative apical membrane antigen (AMA)l domains in inducing protective immunity and to provide basis for selection of vaccine applicable segments. Methods Encoding gene segments of AMA1 were amplified and cloned into pET prokaryotic expression vectors. Recombinant proteins were expressed and purified. Groups of BALB/c mice were immunized by using recombinant protein in Freund's adjuvant, and the IgG liter and specificity of the immune sera were analyzed by IFA and Western blotting. Efficiency of the immune sera in inhibiting Plasmodium falciparum in vitro growth was evaluated. Results Recombinant AMA1 fragments including the entire ectodomain E and subdomain Ⅰ + Ⅱ, Ⅰ, Ⅱ and Ⅲ were successfully expressed and purified. Different levels of antibody were induced in mice by individual proteins and all the immune sera recognized native antigen in the parasites. Sera from protein E and Ⅰ + Ⅱ immunized mice inhibited the growth of parasites. Conclusion The integrality of the ectodomain of AMA1 determines the conformation of the protective antibody epitopes, and these protective epitopes distribute mainly in the subdomain I.


Study on the Relationship between Intracellular Free Calcium and Melanization in Oocysts of Plasmodium yoelii

ZHANGXi-lin;XUWen-yue;WANGYing;DUANJian-hua;KUANGMing-shu

2003, 21(2):  6-86. 

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　Objective To study the change of intracellular free Ca2+ in the oocyst when it melanized and to find out the relationship between the melanized oocyst and its intracellular level of free Ca2+ in a Plasmodium-refractory strain of Anopheles dirus. Methods The distribution and experimental condition of the intracellular free Ca2+ in oocyst of Plasmodium yoelii was measured with Ca2+ sensitive dye Fluo-3/AM and Plutonic F-127 under confocal laser scanning microscope (CLSM) at different time. Results The best load condition was that the oocysts were incubated in 3 μmol/ml Fluo-3/AM adding 1 μl/ml 25% Pluronic F-127 for 60 min at 37 ℃ . Fluorescent imaging of oocysts was affected by an increase or decrease of the concentration of Fluo-3/Am and incubation time. The distribution of intracellular free Ca2+ was heterogeneous in the oocysts. The mean value of Ca2 + in the mature oocysts was (137.15 ±7.02) nmol/L(X±S) but was (18.44± 1.75) nmol/L in melanized oocysts with Ca2+ sedimentation in the wall of oocyst. Conclusion The results suggest that the level of the intracellular free Ca2+ in oocyst decreased and excreted during its melanization in a Plasmodium-reiractory anopheline mosquito species.


Immunological Screening of Toxoplasma Tachyzoite cDNA Expression Libraries with Serum from Infected Rats

JIANGLi-ping;WUXiang;CAILi-ting;WANGDan-jing;SHUHeng-ping*

2003, 21(2):  7-89. 

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　Objective To screen and identify the potential candidates for the development of toxoplasmosis vaccine. Methods Rats were infected with Taxoplasma gondii ( T. gortdii) RH strain and their sera were used as a probe to screen T. gondii tachyzoite cDNA expression libraries. The positive clones were analyzed by PCR amplification and DNA sequencing. Results Thirteen positive clones were obtained from about 4 × 105 phage plagues after three rounds of screening. The size of the inserts ranged from 0.45 kb to 2.4 kb. A BLAST search of all available sequence databases using the partial sequences from four positive clones (LI, L2, L4, L5) showed that the-sequence of L2 clone was identical with T. gondii P24 major antigen gene (TgP24). Clone L4 had a high homology with Saccharum officinarum pyruvate orthophosphate dikinase. There is no significant hit of any sequences to clone L1, suggesting that L1 could be a novel gene(GenBank accession number AY180109), named T. g-R1, which encodes a non-transmembrane protein with 134 amino acid open reading frame. PROSCAN analysis of the T. g-R1 amino acid sequence showed that this gene product contains two protein kinase C phosphorylation site, two casein kinase Ⅱ phosphorylation site, one N-myristoylation site and one microbodies C-terminal targeting signal. Clone L5 was a small partial fragment. Conclusion The identification of positive clones provides a possible way for the development of toxoplasmosis vaccine.


The Correlation between DDVP Resistance of Culex pipiens pattens and Esterase Activity

WANGXin-guo;ZHENTian-min;TANWen-bin;WANGHuai-wei;GONGMao-qing;SUNChuan-hong;ZHAOYu-qiang

2003, 21(2):  8-92. 

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　Objective To detect the resistance index and esterase activity of each generation of DDVP-resistant Culex mosquitoes and analyze the relationship between insecticide resistance and esterase. Methods WHO bioassay and micro-plate measurement were used for the detection. Results The resistance index increased to 12.17 after 43 generations' insecticide selection compared to 1.00 as sensitive isolate. The nonspecific esterase(NSE) activity of the mosquitoes became strengthened with the extension of the generations, and the individual frequency of those with OD values no less than 0.9 increased gradually, consistent basically to the bioassay. The AChE average inhibition rate decreased with the extended generation and increased resistance, and the individual frequency of those with inhibition rate less than 30% became strengthened with the extension of generations, showing a positive correlation. Conclusion The activity of NSE and AChE shows a correlation with DDVP resistance.


Construction and Expression of the Recombinant Plasmid pcDNA3.1/Ts87 of Trichinella spiralis

YANGYa-ping;ZHUXin-ping*;YANGJing;ZHANGLu-ping;XUXu-na;HUANGSong;PascalBoireau

2003, 21(2):  9-95. 

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　Objective To construct and express DNA plasmid of Trichinella spiralis. Methods The BamHI and Hindlll enzyme sites and KOZAK sequences were introduced at both ends of Ts87 gene by PCR. Ts87 gene was ligated into pcDNAS. 1( + ) vector with T4 ligase. The recombinant plasmid pcDNAS. 1/Ts87 and plasmid pcDNAS. 1 were transfected into an eukaryotic cell line GOS7 through Lipofectamine, respectively. The BALB/c mice were immunized with the purified plasmid DNA pcDNAS. 1/Ts87 through two routes: intramuscular injection and gene-gun injection. Results and Conclusion The pcDNAS. 1/Ts87 was expressed both in COS7 and in the BALB/c mice.


Investigation on the Impact of Imported Cases on Filariasis Elimination Program in Shandong Province

FUBin;LIGui-ling;HUYing-xin;CAOXin-chun;SUNChuan-hong;LIHuai-ju

2003, 21(2):  10-98. 

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　Objective To study the impact of imported filariasis cases on the elimination program in different areas of Shandong Province. Methods Dezhou was selected as former low endemic area and Yicheng as former high endemic area. Blood examination was carried out for both mobile population and local people for microfilariae(Mf). Mosquitoes were caught in field and dissected to count the ratio of those having laid eggs and the natural filarial infection rate. Mosquitoes reared at different temperatures were fed with Mf-positive blood and dissected after certain time period to observe the development of the larvae. The vectorial capacity and case transmission quantity were calculated and compared with those from different areas. Results The Mf positive rate of inflow population was 3.18% in average. No case was detected from 9 411 local residents after blood exam in Dezhou while 2 out of 692 local residents were found Mf positive in Yicheng. Mosquitoes'natural infection rate was 3.81% but no third stage larva was found. The shortest time period needed for the larva to develop into an infective stage was 16 days in Dezhou and 11 days in Yicheng. The time period from blood meal to egg-laying on average was 4. 95 days in Dezhou and 4.33 days in Yicheng. The ratio of vectorial capacity and case transmission quantity was 1:4.41 and 1:5. 82 respectively in Dezhou and Yicheng. Conclusion Filarial transmission seems unlikely in Dezhou for its low vectorial capacity and low transmission quantity resulted from low and evidently fluctuating temperature in the north. A low level filarial transmission may be possible in former high-endemic area such as Yicheng if there are as many imported cases as in Dezhou.


Determination of Free Thiols in the Chimeric Protein PfCP-2.9 of Plasmodium faldparum

QIANFeng;PANWei-qing

2003, 21(2):  11-101. 

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　Objective To determine the free thiols in the chimeric protein PfCP-2. 9 of Plasmodium falciparum expressed by Pichia pastoris. Methods Two experiments of reverse phase HPLC and Ellman' s reaction were applied to the PfCP-2.9 for the determination of its free thiols. For RP-HPLC analysis, three kinds of samples were tested: PfCP-2. 9, dithiothreitol-reduced PfCP-2.9 and indoacetic acid-alkylated PfCP-2.9. Results Both experiments showed that there were no any free thiols present in the PfCP-2. 9. Conclution The disulfide bonds between cysteine residues of PfCP-2. 9 were formed completely.


Isoenzyme Analysis on Different Isolates of Trichomonas vaginalis

YUANLi-jie;GAOXing-zheng

2003, 21(2):  12-105. 

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Objective To study the biological types on the seven isolates of Trichomonas vaginalis from Beijing, Hebei-Tangshan, Hebei-Chengde and Jiangxi-Jiujiang in the mainland of China. Methods The samples were analyzed by PAGE, isoenzyme stain and cluster analysis. Results The isoenzyme systems used in the study included MDH, LDH, G-6-PD, PGI and PGM. No difference in the isoenzyme patterns of G-6-PD and PGI was found among the seven isolates. The MDH and LDH patterns of Beijing 1, Beijing 2, Jiujiang 3 strains were identical, while they were distinguishable from those of Chengde, Tangshan, Jiujiang 1, Jiujiang 2 isolates. The PGM pattern of Beijing 1 and Beijing 2 isolates were same but was different from that of the remainders. Gene tree was constructed according to the isoenzyme profiles. The results showed that there are differences in the patterns of the five isoenzymes between the isolates of Beijing 1, Beijing 2, Jiujiang 3 and other four isolates, and Jiujiang 3 was different from Beijing 1, Beijing 2 slightly. Conclusion It seems reasonable to assume that there are at least three different biological types of Trichomanas vaginalis in China.


Construction of the Plant Expression Vectors Containing the Multiepitope Gene of Toxoplasma gondii

ZHOUXiao-hong;CHENXiao-guang;ZHANGXiao-dong;YANGPei-liang;XIJia-fei;HUJian-jun;WANGYa-nan;LILin;SHENShu-man

2003, 21(2):  13-109. 

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　Objective To construct the plant expression vectors containing the multiepitope gene of Tazoplasma gondii (TGMG). Methods ① TGMG was subcloned into pBAC55 vector to construct the intermediate plasmid pB35MG. The E35S/TGMG/NOS3' fragment was cleaved from pB35MG and ligated into the plant binary vector pCAMBIA2300 to construct the plant expression vector pC35MG. ② Tomato fruit-specific E81. 1 promoter was introduced to pB35MG to construct pB35E!MG vector. The E35SE81.1/TGMG/NOS3' fragment was subcloned into pCAMBIA2300 to construct the plant expression vector pCSSElMG. ③ Tomato fruit-specific E82.2 promoter was inserted to pB35MG to construct pBE2MG vector. The E82.2/TGMG/NOS3' fragment was subcloned into pCAMBIA2300 to construct the plant expression vector pCEZMG. The insert gene TGMG in the vectors pB35hKjs pCSSElMG and pCE2MG were confirmed by sequencing. ④ pC35MG, pCSSElMG and pCE2MG were introduced into Agrobacterium tumefaciens strain LBA4404 competent cell. Results Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments. And the sequencing results were confirmed correct. Conclusion The TGMG intermediate vectors pB35MG, pBSSElMG and pBE2MG and the plant expression vectors pC35MG, pCSSElMG and pCE2MG were constructed successfully, and the three plant expression vectors were introduced into Agrribacterium tumefaciens.


流行病学调查

Epidemiological Study on Group Infection of Angiostrongylus cantonensis in Changle City

LINJin-xiang;LIYou-song;ZHUKai;CHENBao-jian;CHENGYou-zhu;LINJin-cai;CAOYi;CHENRi-zhong

2003, 21(2):  14-112. 

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　Objective To make etiological and epidemiological investigation on the infection of Angiostrongylus canto-nensis in 8 pupils in Changle City. Methods (1) CSF of patients was examined with the conventional method to detect pathogens and eosinophiles. (2) The fecal samples of wild rodents were collected from the spot and examined microscopically to discover the first stage larvae of A. cantonensis. (3) Snails (Pila gigas) were collected in the spot. The smashed head tissue was examined for the third stage larvae of A. cantonensis. (4) The patient's clinical symptoms and physical signs were recorded with an emphasis on central nervous system. Results (1) Two larvae of the third stage of A. cantonensis were found in CSF of one patient. Eosinophiles occupied 68% of the cell number in average (ranged from 47% to 83%) in CSF of the 8 patients. (2) The infection rate of the first stage larvae of A. cantonensis was 39.3% (44/112) in feces of the rodents. (3) The infection rate of the third stage larvae of A. cantonensis was 40. 0% (82/205) in the snails. (4) Major clinical manifestations in the 8 patients included: severe headache(8/8), dizziness(8/8), nausea(8/8), vomiting(8/8), lethargy(7/8), lower limb hypody-namia(7/8). Conclusion The confirmation of severe infection of A. cantonensis in 8 child patients demonstrated that a natural nidus of angiostrongyliasis is present in Chengle City.


临床研究

Clinical Observation on the Efficacy of Ivermectin in the Treatment of Intestinal Nematode Infections

WENLi-yong;LISi-wen;WULing-Juan;YANGJi-shun;YANXiao-Ian;YANG.Ming-Jin;LOULei-jun;ZHANFu-chu;WANGXu-yang;LITu-rong;SHAOJian-qiang;ZHENGJin-chun;FUMei-hua

2003, 21(2):  15-115. 

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　Objective To observe the efficacy and side effects of ivermectin in the treatment of intestinal nematode infections. Methods A single dose of ivermectin 0.1, 0.2, 0.2 and 0.2 mg/kg was orally administered to cases with infection of Ascaris, hookworm, Trichuris and Enterobius respectively. A single dose of albendazole 400 mg was used as control. Results The egg negative conversion rates of ivermectin and albendazole were both 100% (34/34) for Ascaris infection, 17.6% (6/34)and 76.5% (26/34)respectively for hookworm infection, 67.6% (23/34)and 47.1 % (16/34)respectively for Trichuris infection,58.8% (20/34) and 100% (34/34)respectively for Enterobius infection. The worm discharge reached a peak in 1 - 2 days after treatment. The side effect of ivermectin was mild and transient showing no adverse effect on blood picture, liver function, renal function or ECG. Conclusion Ivermectin shows similar effect on Ascaris with albendazole, better effect on Trichuris and poorer effect on hookworm and Enterobius than albendazole.


试验研究

Study on Morphology of Blastocystis hominis in Culture and from Diarrhea Patients

ZHANGXu;QIAOJi-ying;DONGXiao-hui;LIYa-qing;LIXiao-qi;LIChen

2003, 21(2):  16-119. 

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　Objective To study morphology of different stage Blastocystis haminis (B.h) for establishing a base in the research of life cycle and pathogenicity of B. h and providing information for clinical laboratory. Methods B. h from diar-rheal patients was continuously cultured in LES medium, and morphology of B.h was studied with iodine and iron hematoxylin staining under light microscope. Results The vacuolar, granular, amoeboid and cyst forms of B. h and transformation among the forms were observed microscopically. Conclusion Among different forms, the vacuolar and granular forms were often seen clinically and the vacuolar form can transform to cysts.