Mastitis is a major challenge to bovine health. The detection of sensitive markers for mastitis in dairy herds is of great demand. Suitable biomarkers should be measurable in milk and should report pathogen-specific changes at an early stage to support earlier diagnosis and more efficient treatment. However, the identification of sensitive biomarkers in milk has remained a challenge, in part due to their relatively low concentration in milk. In the present study, we used a selected reaction monitoring (SRM) mass spectrometry approach, which allowed the absolute quantitation of 13 host response proteins in milk for the first time. These proteins were measured over a 54-h period upon an in vivo challenge with cell wall components from either gram-negative (lipopolysaccharide from Escherichia coli; LPS) or gram-positive bacteria (peptidoglycan from Staphylococcus aureus; PGN). Whereas our data clearly demonstrate that all challenged animals have consistent upregulation of innate immune response proteins after both LPS and PGN challenge, the data also reveal clearly that LPS challenge unleashes faster and shows a more intense host response compared with PGN challenge. Biomarker candidates that may distinguish between gram-negative and gram-positive bacteria include α-2 macroglobulin, α-1 antitrypsin, haptoglobin, serum amyloid A3, cluster of differentiation 14, calgranulin B, cathepsin C, vanin-1, galectin 1, galectin 3, and IL-8. Our approach can support further studies of large cohorts of animals with natural occurring mastitis, to validate the relevance of these suggested biomarkers in dairy production.

乳腺炎是对牛健康的重大挑战。奶牛群中乳腺炎敏感标志物的检测非常有必要。合适的生物标记应在牛奶中可测量，并应在早期报告病原体特异性变化，以支持早期诊断和更有效的治疗。然而，牛奶中敏感生物标志物的鉴定仍然是一个挑战，部分原因是它们在牛奶中的浓度相对较低。在本研究中，我们使用了一种选择的反应监测（SRM）质谱方法，该方法首次允许对牛奶中的13种宿主反应蛋白进行绝对定量。这些蛋白质是在体内用来自革兰氏阴性菌（大肠杆菌的脂多糖）的细胞壁成分进行体内攻击后，在54小时内进行测量的。; LPS）或革兰氏阳性细菌（金黄色葡萄球菌的肽聚糖; PGN）。尽管我们的数据清楚地表明，LPS和PGN攻击后所有受攻击的动物均具有一致的先天免疫应答蛋白上调，但数据也清楚地表明，与PGN攻击相比，LPS攻击释放得更快，并显示出更强烈的宿主反应。可以区分革兰氏阴性菌和革兰氏阳性菌的生物标志物候选物包括α-2巨球蛋白，α-1抗胰蛋白酶，触珠蛋白，血清淀粉样蛋白A3，分化簇14，钙粒蛋白B，组织蛋白酶C，vanin-1，半乳糖凝集素1，半乳糖凝集素3，IL-8。我们的方法可以支持对患有自然乳腺炎的大型动物群的进一步研究，以验证这些建议的生物标志物在乳制品生产中的相关性。