Ayalew Sahlu, Confer Anthony W, Shrestha Binu, Wilson Amanda E, Montelongo Marie
Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma, USA.
Clin Vaccine Immunol. 2013 Feb;20(2):191-6. doi: 10.1128/CVI.00622-12. Epub 2012 Dec 12.
Mannheimia haemolytica, a major causative agent in bovine respiratory disease, inflicts extensive losses each year on cattle producers. Commercially available vaccines are only partially efficacious. Immunity to M. haemolytica requires antibodies to secreted toxins and outer membrane proteins (OMPs) of the bacterium. Gram-negative bacteria produce membrane blebs or vesicles, the membrane components of which are primarily derived from OMPs. Accordingly, vesicles have been used as immunogens with various degrees of success. This study characterized components of M. haemolytica vesicles and determined their immunogenicity in mice and cattle. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vesicles from this bacterium identified 226 proteins, of which 58 (25.6%) were OMPs and periplasmic and one (0.44%) was extracellular. Vesicles were used to vaccinate dairy calves and BALB/c mice. Analyses of sera from calves and mice by enzyme-linked immunosorbent assay (ELISA) showed that circulating antibodies against M. haemolytica whole cells and leukotoxin were significantly higher on days 21 and 28 (P < 0.05) than on day 0. For control calves and mice, there were no significant differences in serum anti-whole-cell and leukotoxin antibody levels from days 0 and 21 or 28, respectively. Lesion scores of lungs from vaccinated calves (15.95%) were significantly (P < 0.05) lower than those from nonvaccinated calves (42.65%). Sera from mice on day 28 and calves on day 21 showed 100% serum bactericidal activity. Sera from vesicle-vaccinated mice neutralized leukotoxin.
溶血曼氏杆菌是牛呼吸道疾病的主要病原体，每年给养牛户造成巨大损失。市售疫苗的效果并不理想。对溶血曼氏杆菌的免疫需要针对该细菌分泌毒素和外膜蛋白（OMP）的抗体。革兰氏阴性菌会产生膜泡或囊泡，其膜成分主要来源于外膜蛋白。因此，囊泡已被用作免疫原，并取得了不同程度的成功。本研究对溶血曼氏杆菌囊泡的成分进行了表征，并确定了它们在小鼠和牛体内的免疫原性。通过液相色谱 - 串联质谱（LC-MS/MS）分析该细菌的囊泡，鉴定出226种蛋白质，其中58种（25.6%）为外膜蛋白和周质蛋白，1种（0.44%）为胞外蛋白。用囊泡对奶牛犊牛和BALB/c小鼠进行免疫接种。通过酶联免疫吸附测定（ELISA）分析犊牛和小鼠的血清，结果显示，在第21天和第28天，针对溶血曼氏杆菌全细胞和白细胞毒素的循环抗体水平显著高于第0天（P < 0.05）。对于对照犊牛和小鼠，血清抗全细胞和白细胞毒素抗体水平在第0天和第21天或第28天之间分别没有显著差异。接种疫苗的犊牛肺部病变评分（15.95%）显著低于未接种疫苗的犊牛（42.65%）（P < 0.05）。第28天小鼠和第21天犊牛的血清显示出100%的血清杀菌活性。囊泡免疫小鼠的血清可中和白细胞毒素。
