牛溶血性曼氏杆菌及牛荚膜A型多杀性巴氏杆菌灭活疫苗对小鼠的保护性研究

doi:10.11843/j.issn.0366-6964.2021.09.021

摘要/Abstract

摘要： 牛溶血性曼氏杆菌及牛荚膜A型多杀性巴氏杆菌是导致牛呼吸道疾病（bovine respiratory disease，BRD）的重要细菌性病原，每年给养牛业带来巨大的经济损失，目前对其疫苗研究仍显不足。本研究选用牛溶血性曼氏杆菌（Mannheimia haemolytica，Mh） Mh422株和牛荚膜A型多杀性巴氏杆菌（Pasteurella multocida，Pm） PmCQ2株作为疫苗菌株，分别制备了2种菌体浓度的Mh和Pm单价灭活菌苗及3种菌量配比（1∶1、2∶1和3∶1）的Mh-Pm二联灭活苗，以小鼠为模型，皮下多点免疫（0.2 mL），加强免疫2次，免疫剂量均为首免的一半。首免后第7天及其后每隔5 d，小鼠尾静脉采血分离血清，ELISA方法检测抗体效价，三免后第20天，分别以Mh422或PmCQ2进行腹腔攻毒测定免疫保护效果。结果显示，所有小鼠接种疫苗均无不良反应，二免后第10天抗体达较高水平，三免后抗体水平持续升高，第15天到达高峰，其后25 d维持高水平，后缓慢下降。Mh单菌苗的2种免疫剂量对Mh422株攻毒的免疫保护率均为0，而Pm单菌苗的2种免疫剂量对PmCQ2株攻毒的免疫保护率全为100%；Mh和Pm间无交叉免疫保护作用；3种菌量配比的Mh-Pm二联疫苗对Mh422株和PmCQ2株攻毒的各自免疫保护率分别为53%～71%和100%。该研究结果表明，所制备的Mh422单菌苗对同型攻毒无免疫保护作用，在诱导机体抗体产生方面，Mh和Pm间无相互抑制作用，PmCQ2株具有促进Mh422株灭活疫苗对Mh422的免疫保护作用，这为牛溶血性曼氏杆菌和牛多杀性巴氏杆菌二联疫苗的进一步研究提供了理论基础。

关键词: 牛溶血性曼氏杆菌, 牛荚膜A型多杀性巴氏杆菌, 灭活疫苗, 免疫保护

Abstract: Mannheimia haemolytica and Pasteurella multocida capsular serotype A both are Gram-negative, bipolarity staining cocobacilli, the main bacterial pathogens that lead to the bovine respiratory disease, resulting in significant economic losses to the cattle industry every year. At present, the lack of vaccines is still the key points of non-effective control to both pathogens. Here, bovine M. haemolytica (Mh) strain Mh422 and bovine P. multocida capsular serotype A (Pm) strain PmCQ2 isolated from calves were used as strains for inactivated bacterins. Overnight culture of Mh422 or PmCQ2 was seeded in Martin broth medium, and incubated at 37℃ for 12 h with shaking at 220 r·min^-1, the bacterial cells were collected and inactivated with 0.4% formalin, the monovalent inactivated bacterins with two concentration of Mh and Pm were prepared by the routine method, and Mh-Pm combined inactivated vaccines were made by using the mixture of Mh and Pm monovalent inactivated bacterin with a ratio of 1:1, 2:1 and 3:1. The mice were inoculated subcutaneously with monovalent and combined vaccines at a dose of 0.2 mL, respectively, the control group mice were inoculated with PBS emulsifier, and the mice had no adverse reactions after immunization with all inactivated vaccines. Booster immunizations were conducted with a half dose of the primary immunization on the 7th and 17th day, blood was sampled via tail vein on the 7th day and every 5 days thereafter and serum antibody titers were detected by ELISA, the specific antibodies were generated to high levels at the 10th day after the first booster and continue increase to the peak at the 15th day after the second booster and maintained 25 d then decreased slowly. Mice were challenged intraperitoneally with Mh422 and PmCQ2 on the 20th day after the third immunization, the results showed that the immune protective rates of Pm monovalent and combined inactivated vaccines against PmCQ2 strain were all 100%, but the immune protection rates of Mh monovalent inactivated vaccines against Mh422 were 0%, there were no cross-immune protections between Mh and Pm. Interesting here, the immune protection rates of Mh-Pm combined inactivated vaccines against Mh422 strain were up to 53%-71%. The results indicate that there is no mutual inhibition between Mh and Pm in its special antibody production inducing and PmCQ2 can promote the immune protection of Mh inactivated vaccines against Mh422. This study provides a theoretical basis for the further research on the combined vaccine of bovine M. haemolytica and bovine P. multocida.

Key words: bovine Mannheimia haemolytica, bovine Pasteurella multocida, inactivated bacterin, immune protection

中图分类号:

S858.235.12

李甜, 杨洋, 谢黎卿, 王远兰, 李攀, 彭远义, 李能章. 牛溶血性曼氏杆菌及牛荚膜A型多杀性巴氏杆菌灭活疫苗对小鼠的保护性研究[J]. 畜牧兽医学报, 2021, 52(9): 2579-2588.

LI Tian, YANG Yang, XIE Liqing, WANG Yuanlan, LI Pan, PENG Yuanyi, LI Nengzhang. Study on the Immunoprotection of Inactivated Vaccine of Bovine Mannheimia haemolytica and Bovine Pasteurella multocida Capsular Serotype A in Mouse Model[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2579-2588.

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