文章摘要
韦超,杨文慧,侯雪新,杨慧盈,孙丽娜,周冬生,李振军.布鲁氏菌104M液体气溶胶免疫BALB/c小鼠的有效性评价和安全性研究[J].中华流行病学杂志,2020,41(7):1103-1109
布鲁氏菌104M液体气溶胶免疫BALB/c小鼠的有效性评价和安全性研究
Immunization efficacy and safety of Brucella 104M against aerosol challenge in BALB/c mice
收稿日期:2019-09-11  出版日期:2020-07-15
DOI:10.3760/cma.j.cn112338-20190911-00665
中文关键词: 布鲁氏菌;气溶胶;免疫;有效性
英文关键词: Brucella;Aerosol;Immunization;Efficacy
基金项目:国家科技重大专项(2017ZX10303401,2018ZX10734401,2018ZX10734404);生物安全重点专项(2017YFC1200303)
作者单位E-mail
韦超 首都医科大学, 北京 100069  
杨文慧 军事医学研究院, 北京 100071 fionyoung@163.com 
侯雪新 中国疾病预防控制中心, 北京 102206  
杨慧盈 军事医学研究院, 北京 100071  
孙丽娜 中国疾病预防控制中心, 北京 102206  
周冬生 军事医学研究院, 北京 100071  
李振军 中国疾病预防控制中心, 北京 102206 lizhenjun@icdc.com 
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中文摘要:
      目的 评价布鲁氏菌104M液体气溶胶肺递送途径免疫BALB/c小鼠有效性和安全性。方法 随机选取6~8周龄BALB/c雌鼠,分别经肺递送、滴鼻和皮下注射3种途径免疫接种布鲁氏菌104M,于免疫后第4、8、16、24周观察并记录小鼠的症状、检测小鼠体重、脾重、脾脏载菌量及肺匀浆、血清的抗体和细胞因子。待鼠脾脏载菌完全清除,用布鲁氏菌A19液体气溶胶肺递送途径攻毒。结果 各组实验小鼠均未见异常症状;体重无显著下降;攻毒前,脾重没有明显变化;攻毒后,免疫组小鼠脾重显著低于空白对照组(P<0.05):液体气溶胶肺递送:实验组(0.26±0.16)g<空白对照组(0.40±0.19)g,滴鼻:实验组(0.21±0.11)g<空白对照组(0.28±0.19)g,皮下注射:实验组(0.14±0.02)g<空白对照组(0.30±0.18)g。随着免疫时间的增长,免疫组小鼠脾脏载菌量呈下降趋势,第20周完全清除。攻毒后2周(免疫24周),所有小鼠脾脏载菌均显著增加,各免疫组脾载菌量均显著低于空白对照组(P<0.05):脾载菌量以log10菌落形成单位(colony-forming units,CFU)/g计数并统计分析,液体气溶胶肺递送:实验组(4.49±0.13)<空白对照组(6.90±0.46);滴鼻:实验组(3.59±1.06)<空白对照组(7.08±0.14);皮下注射:实验组(3.00±2.03)<空白对照组(6.81±0.34)。布鲁氏菌104M激发了BALB/c小鼠细胞免疫和体液免疫反应。在免疫后第4周,检测到104M特异性抗体IgG、IgM、IgA,第8周达到高峰,攻毒后再次显著上升。各免疫组血清和肺匀浆中IFN-γ和IL-18浓度在攻毒前,均显著高于空白对照组(P<0.05),攻毒后,各免疫组血清IFN-γ和IL-18浓度均低于空白对照组(P<0.05),而肺匀浆细胞因子浓度在攻毒前后均持续高于空白对照组(P<0.05)。结论 液体气溶胶肺递送途径是一种有效的免疫途径,表现出有效的保护作用;104M未引起小鼠体重减轻,相对安全,但在小鼠体内存活时间较长,引起小鼠轻度脾脏肿大,有一定的残余毒力。
英文摘要:
      Objective To evaluate the protective efficacy and safety of Brucella 104M against aerosol challenge in BALB/c mice and characterize its immunological effects. Methods Female mice of 6-8 weeks old were immunized with Brucella abortus strain 104M by intratracheal aerosol delivery or intranasal instillation or subcutaneous injection route. Six mice of each group were sacrificed at 4, 8, 16, 24 weeks after immunization. At each time point, the clinical manifestations of mice were investigated, the serum, spleen and lung samples of mice were collected, body weight, spleen weight, bacteria loads in spleens, the anti-Brucella antibodies titers in serum and the cytokines concentrations of IFN-γ, IL-18 in serum or lung homogenate of the mice were detected. Twenty two weeks after immunization, all the mice were challenged with Brucella A19 through intratracheal aerosol delivery. Results Compared with the control group, neither abnormal clinical symptoms nor significant changes in body weight were found in 104M immunization groups, at each time point when immunized through either nose dropping route, subcutaneous injection or aerosol routes; and the spleen weight of immunization groups were lower than control group after challenge (P<0.05):*M1 (0.26±0.16)g*M3(0.21±0.11)g*M5(0.14±0.02)gP<0.05):*M1(4.49±0.13)log10 (Colony-Forming Units) CFU/g10CFU/g; *M3(3.59±1.06)log10CFU/g10CFU/g; *M5(3.00±2.03)log10CFU/g10CFU/g. The high titer of Brucella 104M specific antibodies were detected at week 4, and the peak was reached at week 8 of immunization groups. At each time point, the serum antibody titers IgG 2a was higher than that of IgG 1; before challenge, the cytokines concentrations of IFN-γ and IL-18 in all immunization groups were significantly higher than that in control group (P<0.05), after challenge, cytokines concentrations of IFN-γ and IL-18 in the serum were lower in all immunization groups than in control group (P<0.05). At each time point, IFN-γ, IL-1βand IL-18 concentrations in lung homogenate were higher in immunization groups than in control group (P<0.05). There were no significant differences among the three immunization routes in all sample detection indexes (P<0.05). Conclusions Intratracheal aerosol delivery is a safe and effective immunization route in BALB/c mice. Both humoral immunity and cellular immunity were stimulated by Brucella 104M, showing obvious protective efficacy against aerosol challenge in BALB/c mice. No significant weight loss was detected. However, the colonizing of Brucella 104M in the spleen of mice was too long (20 weeks), and slighter spleen swelling of the mice were detected, which illustrated that the attenuated strain 104M has residual virulence as a vaccine strain in mice.
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