Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece; Proteomics Research Unit, Biomedical Research Foundation of Academy of Athens, 11527 Athens, Greece.
Proteomics Research Unit, Biomedical Research Foundation of Academy of Athens, 11527 Athens, Greece.
J Proteomics. 2019 Aug 15;205:103393. doi: 10.1016/j.jprot.2019.103393. Epub 2019 May 31.
Objective was the differential quantitative proteomics study of ovine mastitis induced by Mannheimia haemolytica; clinical, microbiological, cytological and histopathological methods were employed for confirmation and monitoring. Proteins were separated by two-dimensional gel electrophoresis (2-DE) for all samples and differentially abundant proteins were identified by mass spectrometry; comparisons were performed with pre- (blood, milk) and post- (milk of contralateral gland) inoculation findings. Animals developed mastitis, confirmed by isolation of challenge strain and increase of neutrophils in milk and by histopathological evidence. In blood plasma, 33 differentially abundant proteins (compared to findings before challenge) were identified: 6 with decrease, 13 with new appearance and 14 with varying abundance. In a post-challenge milk whey protein reference map, 65 proteins were identified; actin cytoplasmic-1, beta-lactoglobulin-1/B, cathelicidin-1 predominated. Further, 89 differentially abundant proteins (compared to findings before challenge) were identified: 18 with decrease, 53 with new appearance, 3 with increase and 15 with varying abundance; 15 proteins showed status changes in blood plasma and milk whey. Differential abundance from inoculated and contralateral glands revealed 74 proteins only from the inoculated gland. Most differentially abundant proteins in milk whey were involved in cell organisation and biogenesis (n = 17) or in inflammatory and defence response (n = 13). SIGNIFICANCE: The proteomes of blood and milk from ewes with experimental mastitis caused by Mannheimia haemolytica and the differential proteomics in sequential samples after challenge are presented for the first time. This is the first detailed proteomics study in M. haemolytica-associated mastitis in ewes. An experimental model fully simulating natural mastitis has been used. Use of experimentally induced mastitis minimised potential variations and allowed consistency of results. The study included evaluation of changes in blood plasma and milk whey. Protein patterns have been studied, indicating with great accuracy changes that had occurred as part of the disease process and development, during the acute phase of infection. Relevant protein-protein interactions were studied. The entirety of proteomics findings has suggested that affected ewes had mounted a defence response that had been regulated by many proteins (e.g., cathelicidins, haptoglobin, serum amyloid A) and through various pathways (e.g., acute phase response, binding and transporting significant ions and molecules); these were interdependent at various points. Potential biomarkers have been indicated for use in diagnostic assays of mastitis.
目的是对绵羊乳腺炎的曼海姆菌诱导进行差异定量蛋白质组学研究；采用临床、微生物学、细胞学和组织病理学方法进行确认和监测。所有样本的蛋白质均通过二维凝胶电泳（2-DE）分离，并通过质谱鉴定差异丰度蛋白；比较接种前后（血液、奶）的发现。动物通过分离挑战株和增加牛奶中的中性粒细胞以及组织病理学证据而发生乳腺炎。在血浆中，与挑战前的发现相比，鉴定出 33 种差异丰度蛋白：6 种减少，13 种新出现，14 种丰度变化。在接种后牛奶乳清蛋白参考图谱中，鉴定出 65 种蛋白质；细胞溶质-1 肌动蛋白、β-乳球蛋白-1/B、抗菌肽-1 为主。此外，与挑战前的发现相比，鉴定出 89 种差异丰度蛋白：18 种减少，53 种新出现，3 种增加，15 种丰度变化；15 种蛋白质在血浆和牛奶乳清中表现出状态变化。接种和对侧腺体的差异丰度仅从接种腺体中鉴定出 74 种蛋白质。牛奶乳清中大多数差异丰度蛋白参与细胞组织和生物发生（n=17）或炎症和防御反应（n=13）。意义：首次呈现了绵羊乳腺炎曼海姆菌引起的实验性乳腺炎的绵羊血液和牛奶的蛋白质组学和接种后序贯样本的差异蛋白质组学。这是绵羊乳腺炎中曼海姆菌相关蛋白质组学的首次详细研究。使用了完全模拟自然乳腺炎的实验模型。使用实验性诱导的乳腺炎最大限度地减少了潜在的变化，并使结果具有一致性。该研究包括评估血浆和牛奶乳清的变化。研究了蛋白质图谱，表明在感染的急性期，作为疾病过程和发展的一部分发生的变化具有很高的准确性。研究了相关的蛋白质-蛋白质相互作用。蛋白质组学的全部发现表明，受影响的绵羊已经启动了防御反应，该反应受到许多蛋白质（例如，抗菌肽、触珠蛋白、血清淀粉样蛋白 A）和各种途径（例如，急性期反应、结合和转运重要离子和分子）的调节；这些在不同点相互依存。已经为乳腺炎诊断检测指出了潜在的生物标志物。
