Table of Content

30 April 2005, Volume 23 Issue 2

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论著

Boost Effect of Recombinant IL-4 on Protection of Schistosomajaponicum Cathepsin B DNA Vaccine in Mice Against the Parasite

CHENYu-xiao;WANGLin-xian;TANGLian-fei;ZHANGShun-ke;ZHANGJie;ZENGXian-fang;YIXin-yuan

2005, 23(2):  1-68. 

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Objective To investigate the enhancement effect of IL-4 expression plasmid on cathepsin B DNA vaccine of Schistosoma japonicum (Sj) in mice. Methods The recombinant IL-4 plasmid constructed by cloning PCR amplified product of murine IL-4 gene into eukaryotic expression vector pcDNA3.1 was co-injected intramuscularly with Sj cathepsin B expression plasmid DNA to mice as the test group. The other three groups of mice were set up as control including IL-4 expression plasmid, Sj cathepsin B expression plasmid and two vacant vector plasmids. The expression of IL-4 and cathepsin B was visualized by immunohistochemistry. Challenge infection in mice was carried out 3 weeks after the last vaccination and immune protection was assessed by worm and egg reduction rates. Results The recombinant mIL-4 plasmid and cathepsin B DNA vaccine were expressed in muscular cells of the vaccinated mice. Immunization with cathepsin B DNA plus recombinant mIL-4 plasmid yielded a 43.2 % of worm reduction rate and a 76.6% of egg reduction rate, showing a significant difference（P<0.01, P<0.05） compared with that of cathepsin B DNA vaccine alone. Conclusion As an adjuvant, IL-4 DNA can improve the protective effect of cathepsin B DNA vaccine in mice against S. japonicum infection.

Discovery of a New Species of the Pentastomid Genus Porocephalus（Humboldt，1811）from Taiwan, China and its Pathogenic Features

QIUMing-hua;MAKuo-chun;FANPing-chin;LUSen-shi

2005, 23(2):  2-72. 

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Objective To describe the morphological characteristics of Porocephalus taiwana sp. nov., discuss its pathogenic features and the method of etiological diagnosis of the new disease. Methods Fecal sedimentation concentration was used to collect nymphs from the patient’s watery stool for species identification. Clinical information was collected for determining the pathogenic features of the new infection. Results A new pathogenic pentastomid Porocephalus taiwana sp. nov. is discovered and a new disease-porocephaliasis taiwana-is nominated. With the findings from this case it is proposed that the traditional visceral pentastomiasis should be divided into two subtypes, Encystic and Excystic. According to the pathological features, this case belongs to the excystic visceral pentastomiasis. Conclusion Porocephalus taiwana sp. nov. is a new pathogenic pentastomid infecting humans. Porocephaliais taiwana belongs to a novel type (excystic) of visceral pentastomiasis.

Preliminary Study on Congenital Transmission of Trichinella spiralis in Mice

WANGZhong-quan;HANHua-min;CUIJing

2005, 23(2):  3-77. 

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Objective To study congenital transmission of Trichinella spiralis in mice and observe the protection of anti-Trichinella antibodies from the infected dams to challenge infection. Methods According to the gestation (fertilization), the Kunming mice were divided into two groups: the infected group after gestation and the gestated group after infection. New-born mice were cut into small pieces to separate the larvae within 1 day after birth. One-day-old offspring born to normal dams were nursed by the infected dams, slaughtered after 21 days and examined for the larvae. Serum anti-Trichinella antibody level in offspring born to the infected dams was assayed by ELISA at different time after birth, and its immune protection against challenge infection was studied. Results Out of 6 offspring born to the dams infected at 7 days after fertilization, two were found to be infected. Among other female mice which were first infected with T. spiralis and then gestated, only the offspring born to the dams fertilized at 8 and 22 days after infection were found to be infected, the infection rate of offspring was 20% (2/10) and 25%(2/8) respectively. All larvae recovered from the young were non-encapsulated. The cross-fostering experiment showed that none of 30 offspring born to normal dams were found to be infected. The serum antibody positive rate in 27 offspring born to the infected dams at 1, 7, 24, and 40 days after birth was 100%, 100%, 77.8% and 14.8%, respectively. The worm reduction rate in the offspring 40 days after birth was 62.0% after challenge infection. The worm reduction rate in mice in which sera from the offspring born to the infected dams were passively transferred was 55.7%, there was a significant difference (P<0.01) compared to the control group. Conclusion A transplacental transmission of T. spiralis is revealed in mice. Anti-Trichinella antibodies from the infected dams may partially protect the young from challenge infection.

Comparison of PCR and Isoenzyme Analysisin Identification of Anopheles minimus A and C

ZHENGBin;TANGLin-hua;MAYa-jun;WANGXue-zhong;ZHOUShui-sen;SHIWen-qi

2005, 23(2):  4-81. 

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Objective To compare the PCR and isoenzyme methods in identification of Anopheles minimus A and C. Methods Samples of An. minimus collected in the field were identified from An. aconitus and An. jeyporiensis by PCR-RFLP after morphological examination, then classified by PCR-ASA. The mosquitoes identified by this method were further analyzed for isoenzyme to observe the difference, which was previously used as a technique to classify An. minimus A and C. Results PCR method can differentiate An.minimus A and C simply and quickly. For Isoenzyme method, only EST allozyme pattern displayed difference. Conclusion PCR method is better than isoenzyme analysis in differentiating An. minimus A and C.

Protective immunity Induced by Multivalent DNA Vaccine of Schistosoma japonicum Mr23×103 Membrane Antigen and IL-12 in Mice

BULing-yi;SHIYou-en;GANYan;ZHUXiao-hua;NINGChang-xiu;ZHUHong-gang

2005, 23(2):  5-85. 

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Objective To develop multivalent DNA vaccine PV-IL12-Sj23 which co-expresses Sj23 and cytokine IL-12, and investigate its protective efficacy in BALB/c mice against challenge infection. Methods On the basis of the reconstructed plasmid PV-IL12-Sj23 and plasmid PV-IL12, blank plasmid PV and plasmid PV-Sj23 only expressing Sj23 were constructed. Fifty BALB/c male mice were divided into five groups, which were immunized intramuscularly with multivalent DNA vaccine PV-IL12-Sj23, plasmid PV-Sj23 expressing Sj23, plasmid PV-IL12 expressing cytokine IL-12, blank plasmid PV and saline, respectively. Each mouse was immunized with 100 μg DNA only once. All the mice were challenged with 40 cercariae at week 4, killed and perfused for collection of worms at week10. The number of recovered worms and eggs in the liver were counted. Results Blank plasmid PV and plasmid PV-Sj23 expressing Sj23 were successfully constructed. The worm reduction rate in PV-IL12-Sj23 group and PV-Sj23 group was 45.5% and 27.2% (P<0.05) respectively. The number of eggs in liver tissue was reduced by 58.4% and 33.9% respectively. Conclusion Multivalent DNA vaccine PV-IL12-Sj23 can induce protective immunity against Schistosoma japonicum in BALB/c mice significantly, with a better protective efficacy than the monovalent DNA vaccine PV-Sj23.

Field Trial on Rapid Detection of Echinococcosis by Dot Immunogold Filtration Assay (DIGFA) with Whole Blood Sample

CHENXin-hua;WENHao;ZHANGZhao-xia;FENGXiao-hui;ZHANGJing-ping;ZHANGJin-hui;MAXu-dong;ZHENGShu-seng

2005, 23(2):  7-92. 

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Objective To establish a rapid, simple and reliable assay with samples of whole blood for diagnosis and epidemiological study on hydatidosis. Methods The dot immunogold filtration assay kit was developed and potato agglutinin was applied to blot blood quickly. Results Among 1 678 persons from prevalent area, the positive rate of DIGFA was 8.46% while that of image examination was 3.04%. Both DIGFA and image technique showed positive results in 43 cases. 8 cases with positive image but negative DIGFA were followed up for 16 months, which turned out that 3 cases with necrotic hydatid cysts， 2 cases with calcified hydatid cysts and 2 cases with benign hepatic cysts. 99 cases with positive DIGFA but negative image were also followed up for 16 months, 3 pulmonary hydatid cases were confirmed. Among 38 cases proved by operation and histopathology, the positive rate of DIGFA was 89.5%. 52 samples from non-prevalent area all showed negative DIGFA. Another 40 non-hydatidosis cases (10 samples of hepatic hemangioma, 10 of non-parasitic cysts of liver, 10 of primary hepatic carcinoma, 6 of pulmonary tuberculosis, 4 of lung cancer) also showed negative DIGFA. 190 samples were selected randomly and detected blindly by DIGFA with whole blood, DIGFA with serum and ELISA with serum to evaluate their diagnostic effect with no statistical difference（P＞0.05）. Conclusion The DIGFA kit is rapid, simple and reliable in epidemiological study of hydatid disease, with an advantage of using whole blood sample instead of serum.

DNA/MVA Combined Immunization: Antibody Response to Plasmodium falciparum Merozoite Surface Protein 1 in Mice

LIShu-mei;LIXun;XUECai-fang;MIAOJun;LEIJun-chuan;LIUZhong-xiang;WANGXian-feng

2005, 23(2):  8-96. 

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Objective To explore the effect of DNA/MVA combined immunization in enhancing antibody response to MSP1. Methods DNA vaccine and recombined MVA were constructed based on synthesized MSP1 gene (3D7). BALB/c mice were primed with DNA solely or together with GM-CSF expressing plasmid and then boosted with rMVA/190. Serum IgG and subtype IgG1 and IgG2a were assayed by ELISA. All mice were challenged with allelic replaced Plasmodium berghei. Results Antibodies to MSP1-190 were detected after DNA immunization with an end-point dilution titer of 1:2500. When GM-CSF plasmid was added, the antibody end-point dilution titer reached 1:11150, with an increase of 53 and 10 times respectively after MVA boosting. Among them anti-19 000 antibodies were prominent, 1/4-1/3 of total IgG in serum. However, when the mice were challenged with Pb-PfM19 no prolonged survival was observed（P＞0.05）. Conclusion High titer antibodies can be elicited in mice by using codon optimized MSP1 gene and DNA/MVA combined immunization. The specificity and protection of these antibodies is being further investigated.

Protective Immunity of the Recombinant Schistosoma japonicum Specific Very Low Density Lipoprotein Binding Protein as a Vaccine Candidate

GANXiao-xian;ZENGXiao-peng;WANGYue;DINGJian-zu;SHENHui-ying;SHENLi-ying;FANJin-jiang

2005, 23(2):  9-99,1. 

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Objective To investigate the protective immunity against Schistosoma japonicum in mice immunized with recombinant specific very low density lipoprotein binding protein (SVLBP) and its potential as vaccine candidate. Methods Recombinant SVLBP antigen was over-expressed under IPTG induction and purified by Ni-NTA affinity chromatography. C57BL/6 mice were immunized three times with purified reSVLBP complexed with Freund's adjuvant, at biweekly intervals. Then 35±1 cercariae of S. japonicum were given to each mouse by abdominal skin 10 days after the 3rd immunization. 45 days later, all mice were sacrificed to collect adult worms and count liver eggs. serum samples were collected before immunization and after challenge respectively, and were probed the antigen-specific antibodies using a panel of ELISAs. Results The worm burden and the egg deposition in liver tissue were reduced by 33.4% and 47.6% respectively in the immunized group, in comparison with the adjuvant control group (P<0.05). Higher titer (>1:6 400) of total IgG was observed after challenge infection. The vaccinated mice developed significantly higher levels of IgG2a, IgG2b,IgG1 than those of control mice. Conclusion The recombinant tegumental SVLBP antigen could induce partial protection against S. japonicum infection. These data demonstrate the potential of SVLBP as a schistosome vaccine candidate.

Sequence Analysis and Expression of GRA7 Gene of Toxoplasma gondii Isolates in Escherichia coli

ZHENGBin;ZHENGHuan-qin;HEAi;LIZhuo-ya;CAOAi-lian;ZHANGRui-Lin;ZHANXi-mei

2005, 23(2):  10-105. 

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Objective To analyze the difference of GRA7 gene of Toxoplasma gondii different isolated strains and express GRA7 in Escherichia coli. Methods The GRA7 gene was amplified from genomes of T.gondii isolates by PCR and was cloned into pGEX-4T-1. The recombinant plasmid was transformed into JM109 and sent to be sequenced. The sequence was analyzed with CLUSTALW (an internet tool). The recombinant plasmid was induced by IPTG to express the fusion protein，which was identified by SDS-PAGE and Western blot with positive sera. The protein was purified and used as a diagnostic antigen for ELISA to test serum samples. Results There was no difference among the sequences of T.gondii GRA7 gene from different isolates. The recombinant plasmid pGEX-4T-1/GRA7 induced by IPTG was expressed in E.coli. It was a GST fusion protein and could react with human and rabbit positive sera analyzed by Western blot. Conclusion The GRA7 gene of T.gondii isolates is highly conservative. The GRA7 is expressed as a recombinant protein in Escherichia coli, which shows an immunoreactivity.

实验报道

Survival of the Third Stage Larvae of Anisakis simplexin Various Conditions and Experimental Infection in Rats

HUANGWeiyi

2005, 23(2):  11-109. 

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ObjectiveTo observe the characteristics of biology and pathogenicity of the third stage Anisakis simplex larvae (L₃).MethodsThe survival time of the L₃ in various conditions was observed and the pathological change after experimental infection in rats was examined.ResultsThe results showed that the L₃ frozen at -20 ℃ for 10-12 h can be killed. In the temperature range of 4-10 ℃, the L₃ can survive for over 8 months. The L₃ was very active at 37 ℃， and was killed at the high temperature over 40℃ in a very short time. The ingredients for sashimi cannot kill the L₃. The experiment of rats infected by the L3 showed that about 15%-25% of the L₃ penetrated into the gastrointestinal wall or migrated into the peritoneal cavity in 2 days. After 3d the L₄ was not infectious, and died automatically in 7-10 days and could not develop into adults. The animals can be easily infected when the stomach was empty. The pathological study showed that the primary infection was a kind of reaction to foreign body, while that of the re-infection was allergic.ConclusionThe L₃ has a strong resistance to low temperature and to ingredients, it can be killed by freezing at 20℃ in 24 hours. The L₃ can not mature in the body of terrestrial mammals but causes pathological change in the stomach and allergy.

Development and Identification of Monoclonal Antibodies against the Recombinant P38 Antigen of Schistosoma japonicum

WUJin-ya;ZHOUXiao-hong;CHENXiao-guang

2005, 23(2):  12-113. 

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Objective To express P38 of Schistosoma japonicum in Escherichia coli BL21 and develop the monoclonal antibodies (McAb) against rSjP38. Methods The recombinant plasmid pET32(a)-P38 was transformed into E.coli BL21(DE3). 1 mmol/L IPTG (isopropyl-beta D-thiogalactopyranoside) was used to induce the expression of the recombinant rSjP38. The rSjP38 was soluble in supernatant after sonication and further purified by His-Ni chromatography. BALB/c mice were immunized with the purified rSjP38 and hybridomas were generated with traditional technique. McAbs were screened by ELISA with limited dilution. The subtype and specificity of McAb were identified by kit and Western blot respectively. Results Eight hybridoma cell lines secreting monoclonal antibodies to rSjP38 were obtained. The subtype of all the 8 McAbs are IgG1. Western blotting showed that the 8 McAbs reacted strongly and specifically with native antigen (P38) of Schistosoma japonicum. Conclusions Eight hybridoma cell lines secreting highly specific McAbs against P38 have been established.

防治经验

Survey and Treatment of Pulmonary Acariasis among the Workers Involving inTraditional Chinese Medicinal Materials

XIAHui;HUShou-feng;CHENXing-bao;RUXiu-ying;SHAOXing-yi

2005, 23(2):  13-116. 

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Objective To investigate the prevalence of pulmonary acariasis among the employees working on traditional Chinese medicinal materials and observe the effect of treatment. Methods History inquiry, detection of mites in sputum, blood examination for eosinophils and specific antibodies, x-ray chest film were carried out for 327 workers involving in traditional Chinese medicinal materials. Mites were found in sputum in 121 persons who were then treated with metronidazole, twice a day with a daily dosage of 0.8g for seven days as a course of treatment. Two courses were conducted with an interval of 7-10 day. Prevalence and morbidity in different groups of occupation, age, and sex were analyzed. Results The overall infection rate of mites in sputum was 37.0% (121/327) with an average morbidity of 12.5% (41/327). Among the four types of worker investigated, the highest infection rate (51.8%), and morbidity (18.6%) were in those working in transfer warehouse; the second highest infection rate (40.7%) and morbidity (15.7%) were in employees in factory of Chinese traditional medicine. Both groups showed a significant difference with others(χ²_inf=11.36，P<0.01; χ²_inc=11.36，P<0.01). Higher morbidity was found in the middle-aged ones and those with more years of service. No difference was found between males and females(χ²=0.31，P>0.05). After treatment with metronidazole, 88.4% showed negative in sputum examination for mites and the efficacy of the treatment for pulmonary acariasis was 92.3%. Conclusions Employees engaged in traditional Chinese medicinal materials are one of the groups at the highest risk of pulmonary acariasis. Metronidazole is effective in treating the infection.