Selected reaction monitoring mass spectrometry of mastitis milk reveals pathogen-specific regulation of bovine host response proteins

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 (SRN) 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 alpha-2 macroglobulin, alpha-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.