1,25 DIHYDROXYVITAMIN D 3 -MEDIATED EFFECTS ON BOVINE INNATE IMMUNITY AND ON BIOFILM-FORMING Staphylococcus spp. ISOLATED FROM CATTLE WITH MASTITIS.

Mastitis is one the most widespread and serious diseases in dairy cattle. Recurrent and chronic infections are often attributable to certain pathogenicity mechanisms in mastitis-causing pathogens such as Staphylococcus spp. These include growing in biofilm and invading cells, both of which make it possible to resist or evade antimicrobial therapies and the host's immune system. This study tested the effects of active vitamin D3 (i.e., calcitriol or 1,25-dihydroxyvitamin D3 ) on the internalization and phagocytosis of biofilm-forming Staphylococcus spp. isolated from animals with mastitis. Two established bovine cell lines were used: MAC-T (mammary epithelial cells) and BoMac (macrophages). Calcitriol (0-200nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72h. Concentrations of 0-100mM for 24h upregulated the expression of 24-hydroxylase in MAC-T cells, but did not alter that of VDR. Pre-treatment of the cells with calcitriol for 24h decreased the internalization of S. aureus V329 into MAC-T cells (0-100nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0-10nM). Calcitriol and two conditioned media, obtained by treating the cells with 25-200nM of the metabolite for 24h, were also assessed in terms of their antimicrobial and antibiofilm activity. Neither calcitriol by itself nor the conditioned media affected staphylococcal growth or biofilm formation (0-200nM for 12 and 24h, respectively). In contrast, the conditioned media (0-100nM for 24h) decreased the biomass of preformed non-aureus staphylococcal biofilms and killed the bacteria within them, without affecting metabolic activity. These effects may be mediated by reactive oxygen species and proteins with antimicrobial and/or antibiofilm activity. In short, calcitriol could make pathogens more accessible to antimicrobial therapies and enhance bacterial clearance by professional phagocytes. Moreover, it may modulate the host's endogenous defenses in the bovine udder and help combat preformed non-aureus staphylococcal biofilms (S. chromogenes 40, S. xylosus 4913, and/or S. haemolyticus 6). The findings confirm calcitriol's potential as an adjuvant to prevent and/or treat intramammary infections caused by Staphylococcus spp., which would in turn contribute to reducing antibiotic use on dairy farms.