Proteolytic and proteomic changes in milk at quarter level following infusion with Escherichia coli lipopolysaccharide

Mastitis is a major disease in dairy cattle, which causes significant economic losses due to decreased milk production, veterinary costs, and discarded milk. Escherichia coli is one of the most prevalent species of gram-negative bacteria that induce clinical mastitis. The objective of the present study was to characterize the proteolytic and proteomic changes in milk in response to infusion with lipopolysaccharide (LPS) at quarter level in a model mastitis system. One quarter of each of 2 cows was infused with 0.1 or 5μg of LPS. The somatic cell count of the infused quarters reached a peak 6h after infusion to a greater extent in the cow infused with 5μg of LPS and changes in plasmin activity in milk differed between the 2 animals. Urea-polyacrylamide gel electrophoretograms of milk samples of the cow infused with 5μg of LPS obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α _s1-casein during incubation of milk samples due to indigenous proteolytic activity. Two-dimensional gel electrophoretograms of milk at 0, 6, or 12h after infusion with LPS showed hydrolysis of α _s-casein and β-casein as well as the appearance of lower molecular weight products. Eleven fragments from proteolysis of the caseins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and, in addition, proteolysis patterns of casein by the indigenous bovine milk proteases plasmin and cathepsin D were studied in model studies using 2-dimensional gel electrophoretograms. Twelve hours after infusion, lower abundance markers of inflammation were identified, including serotransferrin, fibrinogen β chain, protein S100 A12, and the antimicrobial polypeptide cathelicidin.