Contribution of Macrophages to Proteolysis and Plasmin Activity in Ewe Bulk Milk

Abstract

A total of 225 bulk sheep milk samples were collected from 5 intensively managed flocks during early, mid, and late lactation to assess the contribution of macrophages to the regulation of the plasmin-plasminogen system. Samples were analyzed for composition, somatic cell counts, milk renneting characteristics, and for plasmin (PL), plasminogen (PG), and plasminogen activators (PA) activities. Isolation of macrophages from milk was performed using a magnetic positive separation and mouse antiovine macrophage antibody; separated cells were lysed by several freeze-thaw cycles, and activity of urokinase PA (u-PA) was determined. Plasmin activity decreased during lactation (42.06±0.66, early; 31.29±0.66, mid; 28.19±0.66 U/mL, late). The reduction in PL activity recorded in the mid and late lactation milk matched the increase in PG:PL ratio. The activity of PA increased throughout lactation; the highest value being recorded in the late lactation milk (260.20±8.66 U/mL). Counts of isolated and concentrated macrophages were higher in early and mid lactation milk (3.89±0.08 and 3.98±0.08 log10 cells/mL, respectively) than in late lactation milk (3.42±0.08 log10 cells/mL). Stage of lactation did not influence the activity of u-PA detected in isolated macrophages. The activity of u-PA associated with isolated milk macrophages only minimally contributed to total PA activity detected in milk. Proteolytic enzymes, associated with isolated macrophages, act on α-casein hydrolysis, as shown by urea-PAGE electrophoresis analysis. Somatic cell counts did not exceed 600,000 cells/mL, and this threshold can be considered a good index of health status of the flock and of the ability of milk to being processed. Our results lend support to the hypothesis that macrophages in ewe bulk milk from healthy flocks only slightly contribute to the activation of the PL-PG system.