Deviation of bovine milk leukocyte functions against Escherichia coli and Staphylococcus aureus after co-stimulation with Weissella paramesenteroides

Weissella paramesenteroides (WP) is a lactic acid bacterium found in the bovine udder microbiota, and some strains have been proposed as probiotics in the food industry. However, its immunomodulatory effects, particularly in the presence of mastitis pathogens, remain poorly understood. This study evaluated the effects of two W. paramesenteroides isolates (WPI1 and WPI2) on bovine milk white blood cell (mWBC) functions following co-stimulation with Escherichia coli (EC) or Staphylococcus aureus (SA). mWBC were isolated from 12 quarters of 12 cows (one quarter per cow; CMT = 2) in two independent experiments. Milk-derived mWBC were isolated and stimulated with EC, SA, WPI1, WPI2, or their co-stimulations. Leukocyte migration, phagocytosis, bacterial killing activity, and neutrophil extracellular trap (NET) formation were assessed, and data were analyzed using repeated linear mixed models ( p < 0.05). Both WP isolates induced stronger migration and phagocytosis than the mastitis pathogens alone. WPI1 maintained these responses during co-stimulation and demonstrated enhanced bacterial killing activity against S. aureus . In contrast, WPI2 showed reduced migration and phagocytosis in co-stimulation. Although both strains stimulated extracellular DNA release individually, WPI2 co-stimulation with S. aureus induced the greatest NET release, whereas WPI1 co-stimulations achieved superior bacterial killing with lower NET production. These findings support further investigation of selected W. paramesenteroides strains as potential adjunctive candidates for mastitis-control strategies.