| Objective The primary aim of this study was to explore the optimal dose of CT plus ESA of Toxoplasma gondii for intranasal immunization in BALB/c mice and the mucosal and systemic immune response induced by intranasal immunization mice, and determines the role of anti-T. gondii infection. The purpose is to provide the experimental basis for the development of T. gondii vaccine.Methods This study included three parts. The first part was to study the mucosal adjuvant effect induced by intranasal immunization with different doses of CT plus ESA of T. gondii and the optimal dose of CT for intranasal immunization in BALB/c mice. Sixty 5-to 6-week-old BALB/c mice were randomly divided into five groups,12 mice per group. The mice were immunized intranasally two times at two-week intervals with 0μg,0.5μg,1.0μg,1.5μg or 2.0μg CT plus ESA 20μg, respectively. The health and survival rate of mice was observed. Venous bloods of all animals were collected and then were killed on day 30 after the last immunization. Lymphocytes of spleen, Peyer's patch, mesenteric lymphocyte code (MLN) were counted. The level of sIgA antibody in feces and IgG in the sera were detected by ELISA.The second part was to study the immune responses in mice after intranasal immunization with cholera toxin and ESA of T. gondii. BALB/c mice were randomly divided into three groups, 16 mice per group. Mice were intranasally immunized with PBS 20μl, ESA 20μg or 1.0μg CT+ 20μg ESA per mouse twice at an internal of two weeks. Mice were sacrificed on day 14 after the last immunization. IgA antibodies in feces and nasopharynx washes were detected by ELISA. Lymphocytes in PP, IEL, NALT and NC were isolated and counted. Percentage of CD4+ and CD8+ T cells was determined by immunocytochemistry.The third part was to study the effects of intranasal immunization with ESA and CT adjuvant on protection of mice against T. gondii infection and determine the optimal procedure for intranasal immunization. BALB/c mice were divided into 3 groups, and mice were intranasally immunized with 20μl PBS or 1.0μg CT+20μg ESA per mouse, twice at 2 weeks internal, respectively. All mice were challenged intragastrically with 4×104 tachyzoites per mouse on day 14 after last immunization. The condition of mice about health and death were observed after challenge. IgG in serum and slgA in feces were detected by ELISA on the 30th day after challenge. The tachyzoites of the liver and brain were counted, and lymphocytes in spleen and PP were counted.Results The higher level immunologic responses of MLN, PP and splenic lymphocytes were induced by intranasal immunization with CT adjuvant and ESA, showed a dose effect. In compared with the 0μg group, immunologic responses in 1.0μg,1.5μg and 2.0μg groups were increased significantly (P<0.05), but not statistically significant in this three groups (P>0.05).1.0μg CT adjuvant and ESA induced higher level mucosal immune response and system immune response, CT can acts as the mucosal adjuvant of ESA and 1.0μg is the optimal dose.IgA antibodies in feces and nasopharynx washes of mice immunized in CT group were higher than the PBS group after immunity (P<0.05). Lymphocytes in NALT significantly increased after immunity in CT group (P<0.05). The CD4+ and CD8+ T cells were both higher than that of the PBS group (P<0.01), while the ratio of CD4+ and CD8+ T cells was decreased (P<0.05). Lymphocytes in NC and PP increased (P<0.01) with CD4+ T cells mainly increasing (P<0.01). For IEL, there was a predominant number of CD8+T cells increasing significantly (P<0.01), and the ratio of CD4+ and CD8+T cells was reversed with significance (P<0.05).The health status of mice in CT adjuvant and ESA group were better than that of PBS group, survival rate (95%) was higher than that of PBS group (55%). Compared with PBS group, tachyzoites in liver and brain tissues of CT+ESA group were decreased 80.19%(P<0.001) and 78.24%(P<0.005).Conclusion Intranasal immunization with cholera toxin and ESA of Toxoplasma gondii can effectively induce mucosal reaction and systemic immune responses which protect mice against T. gondii infection.
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