绵羊源荚膜血清D型多杀性巴氏杆菌的生物学特性及2种候选疫苗的免疫效果评价

Biological characterization of Pasteurella multocida capsular serotype D from sheep and immune evaluation of two candidate vaccines

旨在研究新疆绵羊源荚膜血清D型多杀性巴氏杆菌(PmD)的生物学特性,探索其灭活疫苗与OmpH重组疫苗对小鼠的免疫保护效果,为防控新疆地区绵羊巴氏杆菌病提供参考。采用细菌分离鉴定、PCR鉴定、药敏试验、耐药与毒力基因检测、致病性试验等方法初步明确PmD的生物学特性;并通过最佳灭活条件和佐剂筛选、安全性试验、原核表达等方法制备PmD灭活疫苗与PmD-OmpH重组疫苗,采取小鼠攻毒保护试验评价2种疫苗的免疫效力。结果:从绵羊肺脏中分离出1株PmD,药敏试验显示,该菌为7重耐药菌,未检测到耐药基因;毒力基因检测显示,该菌具有12种毒力基因(exbB、exbD、hgbA、tonB、fimA、Oma87、OmpH、Psl、sodA、sodC、tbpA、toxA);致病性较强,对小鼠的最小致死菌量为2.2×105 CFU;灭活疫苗和重组疫苗对相同血清型菌株攻毒的保护率分别为80%和10%;灭活疫苗对血清型A、F和未知菌株攻毒保护率分别为0、20%和0,而重组疫苗的保护率则全为0。本研究发现1株强耐药、强毒力的绵羊源PmD,灭活疫苗对同种血清型菌株的保护性远高于重组疫苗,发病地区羊场可采用同种血清型灭活疫苗进行预防和控制绵羊巴氏杆菌病,对多价疫苗和新型疫苗的研制还有待进一步发掘。

This study was to identify the biological characteristics of Pasteurella multocida capsular serotype D(PmD)from sheep in the Xinjiang area,and to explor the immune protection effect of its inactivated vaccine and the OmpH recombinant vaccine in mice,in order to provide reference measures for the prevention and control of sheep pasteuriasis in Xinjiang.The biological characteristics of PmD were pre⁃liminarily clarified by bacterial isolation and identification,PCR identification,drug susceptibility test,drug resistance and virulence gene detection,and by pathogenicity test.PmD inactivated vaccine and PmD-OmpH recombinant vaccine were prepared using methods such as optimal inactivation conditions,adjuvant screening,safety test and prokaryotic expression.Then,mouse challenge protection test was used to evaluate the immune efficacy of both the vaccines.One strain of PmD was isolated from sheep lung,and drug susceptibility tests showed that the bacterium resisted against 7 resistant drugs at the same time,but no resistance gene was detected.The virulence gene test showed that the bacteriium had 12 virulence genes(exbB,exbD,hgbA,tonB,fimA,Oma87,OmpH,Psl,sodA,sodC,tbpA,and toxA).Its pathogenicity was strong,and the minimum lethal quantity in mice was 2.2×105 CFU.The protection rates of the inactivated vaccine and recombinant vac⁃cine against the same serotype strain were 80%and 10%,respectively.The protection rates against different serotypes(A,F,unknown)strains were 0,20%,0,0,0,and 0.In this study,a strong drug-resistant and virulent sheep-derived PmD was identified.The inactivated vaccine was much more protective against the same serotype strain than the recombinant vaccine.In sheep farms in the disease area,the inac⁃tivated vaccine could be used to prevent and control pasteulosis in sheep.The development of the multivalent vaccines and new vaccines still requires further exploring.