鸭疫里默氏杆菌贵州流行株蜂胶灭活疫苗制备

doi:10.16431/j.cnki.1671-7236.2022.08.032

摘要/Abstract

摘要： 【目的】制备鸭疫里默氏杆菌(Riemerella anatipestifer,RA)贵州流行株蜂胶灭活疫苗。【方法】选取RA血清2型贵州流行株(RA-SS-8株)为基础菌株,依次利用分光光度法和平板计数法测定细菌生长曲线,再进行灭活条件筛选,以蜂胶为佐剂制备RA贵州流行株蜂胶灭活疫苗,并进行无菌检验、安全性检验及免疫鸭攻毒保护性试验。【结果】分光光度法测定RA-SS-8株菌液D_{600 nm}值随培养时间变化显示,细菌在0~3 h时增殖缓慢,在3~10 h时增殖趋势明显加快,在10 h以后增殖逐渐趋于平缓,随着培养时间的延长,细菌最终进入衰亡期;平板计数法结果显示,RA-SS-8株D_{600 nm}值为0.1~0.8时,该菌处于对数生长期,且D_{600 nm}值与活菌数呈现良好的线性关系;RA-SS-8株最佳灭活条件为0.2%甲醛溶液、37 ℃灭活12 h;蜂胶灭活疫苗含菌量为3.8×10⁹ CFU/mL,蜂胶干物质含量为10 mg/mL;无菌检验巧克力琼脂培养基上未见菌落生长;安全性检验以2倍免疫剂量接种雏鸭在观察期内未表现出不良反应,大体病变观察未见明显病变;免疫鸭攻毒保护性试验显示,蜂胶灭活疫苗免疫组鸭对RA-SS-8株的攻击后保护率为70%,蜂胶灭活疫苗对试验鸭心脏、肝脏组织均具有良好的保护效果。【结论】本研究成功制备了RA贵州流行株蜂胶灭活疫苗,为蜂胶灭活疫苗制备和动物免疫试验奠定基础。

关键词: 鸭疫里默氏杆菌; 蜂胶灭活疫苗; 攻毒保护性试验

Abstract: 【Objective】 This study was aimed to prepare propolis inactivated vaccine of Riemerella anatipestifer (RA) epidemic strain in Guizhou.【Method】 The RA serotype 2 Guizhou epidemic strain (RA-SS-8 strain) was selected as the basic strain,and the bacterial growth curve was measured by spectrophotometry and plate counting method in turn,and then the inactivation conditions were screened.Propolis was used as an adjuvant to prepare RA Guizhou epidemic strain propolis inactivated vaccine,and conduct sterility test,safety test and immune duck challenge protection test were carried out.【Result】 Spectrophotometric determination of D₆₀₀_nm value of strain RA-SS-8 with culture time showed that the bacteria proliferated slowly in the period of 0-3 h,and the proliferation trend was obviously accelerated in the period of 3-10 h,and the proliferation gradually became flat after 10 h,with the extension of the culture time,the bacteria finally entered the decay stage.The results of plate counting method showed that when the D_{600 nm} value of the RA-SS-8 strain was 0.1-0.8,the bacteria was in the logarithmic growth phase,and the D_{600 nm} value and the number of viable bacteria showed a good linear relationship. The best inactivation conditions of RA-SS-8 were 0.2% formaldehyde solution and 37 ℃ for 12 h.The bacterial content of the propolis inactivated vaccine was 3.8×10⁹ CFU/mL,and the dry matter content of propolis was 10 mg/mL.No colony growth was found on the chocolate agar medium in the sterility test.Ducks inoculated with 2 times of the immunization dose did not show adverse reactions during the observation period,and no obvious lesions were observed in gross lesions.The protection test of immunized ducks showed that the protection rate of ducks in the immunized group against RA-SS-8 strain was 70%,the propolis inactivated vaccine had good protective effect on the heart and liver tissues of the experimental ducks.【Conclusion】 The propolis inactivated vaccine of RA epidemic strain in Guizhou was successfully prepared,which laid a foundation for the preparation of propolis inactivated vaccine and animal immunity test.

Key words: Riemerella anatipestifer; propolis inactivated vaccine; challenge protection test

中图分类号:

S852.61

马光强, 刘丽娟, 陈国权, 吴良涛, 杨均, 刘军泽, 李潇蒙, 潘成文, 周碧君. 鸭疫里默氏杆菌贵州流行株蜂胶灭活疫苗制备[J]. 中国畜牧兽医, 2022, 49(8): 3163-3170.

MA Guangqiang, LIU Lijuan, CHEN Guoquan, WU Liangtao, YANG Jun, LIU Junze, LI Xiaomeng, PAN Chengwen, ZHOU Bijun. Preparation of Propolis Inactivated Vaccine of Riemerella anatipestifer Epidemic Strain in Guizhou[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3163-3170.

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