Mannheimia haemolytica serotype A2 is the principal cause of pneumonic mannheimiosis in ovine and caprine livestock; this disease is a consequence of immune suppression caused by stress and associated viruses and is responsible for significant economic losses in farm production worldwide. Gram-negative bacteria such as M. haemolytica produce outer membrane (OM)-derived spherical structures named outer membrane vesicles (OMVs) that contain leukotoxin and other biologically active virulence factors. In the present study, the relationship between M. haemolytica A2 and bovine lactoferrin (BLf) was studied. BLf is an 80 kDa glycoprotein that possesses bacteriostatic and bactericidal properties and is part of the mammalian innate immune system. Apo-BLf (iron-free) showed a bactericidal effect against M. haemolytica A2, with an observed minimal inhibitory concentration (MIC) of 16 µM. Sublethal doses (2–8 µM) of apo-BLf increased the release of OMVs, which were quantified by flow cytometry. Apo-BLf modified the normal structure of the OM and OMVs, as observed through transmission electron microscopy. Apo-BLf also induced lipopolysaccharide (LPS) release from bacteria, disrupting OM permeability and functionality, as measured by silver staining and SDS and polymyxin B cell permeability assays. Western blot results showed that apo-BLf increased the secretion of leukotoxin in M. haemolytica A2 culture supernatants, possibly through its iron-chelating activity. In contrast, holo-BLf (with iron) did not have this effect, possibly due to differences in the tertiary structure between these proteins. In summary, apo-BLf affected the levels of several M. haemolytica virulence factors and could be evaluated for use in animals as an adjuvant in the treatment of ovine mannheimiosis.

中文翻译：

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载脂乳铁蛋白对溶血曼海姆氏菌A2的白细胞毒素和外膜囊泡的影响。

溶血性曼氏痴呆A2血清型是绵羊和山羊家畜肺炎性甘露血症的主要原因。该病是压力和相关病毒引起的免疫抑制的结果，是造成全球农业生产重大经济损失的原因。革兰氏阴性细菌（如溶血支原体）会产生源自外膜（OM）的球形结构，称为外膜囊泡（OMV），其中包含白细胞毒素和其他生物活性毒力因子。在本研究中，溶血支原体A2和牛乳铁蛋白（BLf）之间的关系进行了研究。BLf是一种80 kDa的糖蛋白，具有抑菌和杀菌特性，是哺乳动物先天免疫系统的一部分。Apo-BLf（不含铁）对溶血支原体A2具有杀菌作用，观察到的最小抑菌浓度（MIC）为16 µM。亚致死剂量（2–8 µM）的apo-BLf增加了OMV的释放，可通过流式细胞仪对其进行定量。通过透射电子显微镜观察，Apo-BLf修饰了OM和OMV的正常结构。通过银染，SDS和多粘菌素B细胞通透性测定，Apo-BLf还诱导细菌从细菌释放脂多糖（LPS），破坏OM的通透性和功能。蛋白质印迹结果表明，apo-BLf可能通过其铁螯合活性增加了溶血支原体A2培养上清液中白细胞毒素的分泌。相反，hollo-BLf（含铁）没有这种作用，可能是由于这些蛋白质之间的三级结构不同。总之，apo-BLf影响了数个M的水平。