2308、M28、S1330、16M四株布鲁氏菌灭活参数研究

Comparison of the inactivation parameter of four Brucella strains B. abortus 2308, B. melitensis M28, B. suis S1330 and B. melitensis 16M

【目的】比较不同灭活方法对布鲁氏菌灭活的效果,确定灭活参数,为制备布鲁氏菌灭活抗原提供参考。【方法】将4株布鲁氏菌参考强毒株2308(牛种)、M28(羊种)、S1330(猪种)以及16M(羊种)分别经大豆酶消化蛋白胨(TSA)培养基培养繁殖后,用生理盐水制成(4-8)×1010 CFU/m L的菌悬液,分成等份于80 oC灭活不同时间,另将同样浓度的菌悬液分别用不同浓度甲醛于37 oC灭活不同时间,通过灭活检验,确定灭活效果。取经甲醛和热灭活的16M抗原,分别以1×1010 CFU/只剂量皮下注射1.5-2.0 kg家兔2只,免疫6周内,每周采血用虎红平板凝集试验(RBT)和试管凝集试验(SAT)测定抗体效价。【结果】80 oC、5 min可灭活2308、S1330和16M三种菌株,80 oC、10 min可灭活全部4种菌株。0.2%甲醛灭活7 d,4种试验菌株均不能被彻底灭活;0.4%甲醛在12 h内只能灭活16M,72 h可灭活M28;0.4%甲醛灭活2308和S1330两次试验结果差异较大。0.6%甲醛可在72 h内灭活4种试验菌。不同方法灭活的16M抗原免疫家兔后,其血清抗体虎红平板凝集和试管凝集效价消长趋势基本一致,甲醛灭活的抗原免疫原性略高于热灭活抗原。【结论】80 oC热灭活和0.6%甲醛灭活均可用于对布鲁氏菌的灭活,且不影响布鲁氏菌的免疫原性。

[Objective] To define and compare the parameters for the heat and formaldehyde inactivation of Brucella strains, and provide information for preparation of Brucella inactivated antigen. [Methods] The four Brucella virulent reference strains 2308 （B. abortus）, M28 （B. melitensis）, S1330 （B. suis） and 16M （B. melitensis） were cultivated in TSA media, and the harvested culture were resuspended to a concentration of （4-8）× 10^10 CFU/mL by saline. Then the suspension was divided into the equal two parts for heat inactivation at 80 ℃ for different time or formaldehyde inactivation at different concentration and for different time at 37 ℃, and the inactivation effects for the two methods were compared. The rabbits of 1.5-2.0 kg were subcutaneously inoculated with the heat or formaldehyde inactivated 16M antigen at the dosage of 1× 10^10 CFU/mL. The serum antibody titer was measured weekly during six weeks post inoculation by Rose Bengal test （RBT） and Serum agglutination test （SAT）. [Results] It takes 5 min to inactivate Brucella strains 2308, S1330 and 16M by heating at 80℃, and 10 min for all four strains. The four Brucella strains could not be completely inactivated while incubated in 0.2% formaldehyde even for 7 days. The strains 16M and M28 could be inactivated thoroughly via incubation in 0.4% formaldehyde for 12 or 72 hours, respectively. Although the effect varied for the inactivation of strains 2308 and S1330 in two independent experiments, the four strains could be entirely inactivated by incubation in 0.6% formaldehyde for 72 hours. The fluctuation trends were almost consistent for the stimulated serum antibodies against the two heat or formaldehyde inactivated 16M antigens. Moreover, the antibody titer against the formaldehyde inactivated antigen was slightly better than that against the heat inactivated antigen. [Conclusion] Heat inactivation at 80 ℃ and inactivation in 0.6% formaldehyde could be used to prepare Brucella inactivated antigen and did not change the immunogenicity of Brucella.