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Objective To study the system of streptococcal C5a peptidase (ScpB) and the specif-ic antibody levels in serum, lung, vagina and recta after subcutaneous and intranasal immunization with dif-ferent doses of C5a peptide. Methods Recombinant protein C5a peptide was expressed in E. coli strain BL21 and purified by affinity chromatography. The expressed product was identified by SDS-PAGE and pep-tide mass fingerprinting (PMF). BALB/c mice were subcutaneously and intranasally injected with different doses of ScpB. Antibody titer was tested by ELISA. Opsonophagocytosis assay was used to test the function of antibody. Results ScpB protein was successfully expressed and purified. The probability based mouse score of ScpB was 175 by PMF analysis. ELISA data showed that both subcutaneous and intranasal immtmi-zation could elicit significantly higher levels of IgG in immunized mice serum than that of control group (P ＜0.01), 30 μg group waa better than 5 μg and 10 μg group. Intranasal immunization could elicit higher lev-els of IgA in lung, vagina and rectum (P ＜0.001) while system immunization could not. Opsenophagocyto-sis tests indicated that anti-serum of ScpB had opsenophagocytic activity than that of control (P ＜ 0. 05).Conclusion The results demonstrated that intranasal immunization with ScpB could induce significantly higher levels of lgG and IgA, and its anti-serum had better opsenic activity.
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Objective To establish mice model that induces mucosal immunity by orally infected tachyzoites of Toxoplasma gondii. Methods Respectively, 5?103, 5?104, 5?105, 5?106 tachyzoites of RH strain were inoculated to BALB/c mice by stomach delivery. The control group was given PBS solution. Symptoms and pathological changes of mice were observed. Secretory im-munoglobulin A (SIgA) was assayed. The lymphocytes from Peyer's patches (PP) and intraepithe-lial lymphocyte (IEL) were observed and the changes of CD4+ and CD8+ T cells assayed by SABC immunohistochemistry. Results Inoculation of 5?104 tachyzoites of Toxoplasma gondii caused symptoms and pathological changes in mice. The titre of SIgA increased in intestine, and CD4+ T subset of the mucosal inductive sites and CD8+ T subset of the mucosal effectors' sites increased. Conclusion Mucosal immunity may be induced by oral infection of 5?104 tachyzoites of T. gondii RH strain in BALB/c mice.
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Objective To study the mucosal and systemic immune response after intranasal immunization with mucosal complex vaccine for Toxoplasma gondii,and to observe the protective effect on mice. Methods The mucosal complex vaccine was made of soluble tachyzoite antigen (STAg) and cholera toxin (CT),which were mixed and dissolved in PBS (1 ml PBS containing 1 mg STAg and 50 ?g CT). Fifty-two BALB/c mice were randomly divided into two groups: immunized group and control. Mice were intranasally immunized with 20 ?l mucosal complex vaccine (20 ?g STAg and 1?g CT) per mouse twice at an interval of two weeks,while the control mice were given PBS solution instead. Six mice of each group were killed by dislocation of cervical vertebra on day 14 after the last immunization. The specific IgG antibodies in serum and IgA in feces were detected by ELISA. Lymphocytes in spleen,Peyer's patches (PP) and intestinal intraepithelial lymphocyte(IEL) were isolated and counted. Percentage of CD4+ and CD8+ T cells was determined by immunocytochemistry. Other mice were challenged intragastrically each with 4?104 tachyzoites of RH strain Toxoplasma gondii on day 14 after the last immunization. Their health condition was observed and the number of tachyzoites in liver and brain was determined microscopically on the 30 th day after challenge. Results IgG antibodies in serum and IgA antibodies in feces of immunized mice were higher than the control (P
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Objective To study the invasion and proliferation in IEC-6 cells of Toxoplasma gondii RH strain tachyzoites in vitro.Methods T.gondii tachyzoites of RH strain were co-cultured with IEC-6 cells in vitro,the process of cell adhesion,invasion and proliferation by tachyzoites was observed consecutively with inverted microscope.At 5 min,10 min,20 min,30 min,1,2,4,6,12,24 and 48 h after co-culture,the tachyzoite invasion to IEC-6 and intra-cellular proliferation were observed with Giemsa-Wright's staining,respectively.The invasive rate of tachyzoites to IEC-6 was counted.Results T.gondii tachyzoites invaded the IEC-6 cells 5 min after culture,thenceforth the invasive rate increased gradually.The invasive rate was about 55.0% at the first hour after culture with 1-5 tachyzoites in one cell.In the second hour after culture,the rate reached highest with 81.8 % and there were many pseudocysts emerging.At the same time,tachyzoites invaded the cell nucleus and proliferated in the nucleus.At the 4th hour after culture,the invasive rate began to decrease(80.8?9.2)%,the pseudocysts began to break and tachyzoites were released to cluster.The clustering tachyzoites increased significantly at the 6th hour.At the 12th hour the clustering tachyzoites decreased and most tachyzoites were free,the number of complete cells decreased obviously.There were only a few cells and pseudocysts left at the 24th hour,and a great quantity of free tachyzoites existed out of the IEC-6 cells.There were plenty of mobile tachyzoites while none of IEC-6 cells existed after 48 h culture.Conclusion IEC-6 cell may be the suitable target cell of Toxoplasma gondii tachyzoite.The tachyzoites can invade the IEC-6 cells quickly in vitro and proliferate in the plasma and nucleus with a reproductive cycle of about 6 to 12 hrs.
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Objective To observe dynamically the location and time of attachment and invasion of Toxoplasma gondii tachyzoites to murine intestinal mucosa by chromogenic in situ hybridization targeting SAG2 mRNA. Methods Thirty 7- to 8-week-old BALB/c mice were randomly divided into experiment group(24 mice)and control group(6 mice). Each animal in the experiment group was given 2?104 tachyzoites of RH stain in 0.2 ml PBS by intragastric administration and that in the control group was given 0.2 ml PBS. Four mice in the experiment group and one in the control group were sacrificed at 15 min,30 min,1 h,2 h,4 h and 8 h after infection,respectively,and paraffin sections of duodenum,jejunum and ileum were prepared to perform the in situ hybridization with Dig-labeled oligonucleotide probe complementary to SAG2 mRNA of T. gondii. Results Tachyzoites were found on the striated border of small intestine epithelial cells (absorptive cells,goblet cells and endocrine cells),in or between two absorptive cells or in the lamina propria. At 15 min-2 h after infection,there was significant difference in the number of attachment on jejunum and ileum (P