Broad-spectrum activity against bacterial mastitis pathogens and activation of mammary epithelial cells support a protective role of neutrophil cathelicidins in bovine mastitis

Abstract

Cathelicidins are peptide components of the innate immune system of mammals. Apart from exerting a direct antibiotic activity, they can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied, but there is a lack of published information on their expression and activities in the context of mastitis. The aims of this study were to investigate the expression of the bovine cathelicidins BMAP-27, BMAP-28, Bac5, and indolicidin in healthy and infected mammary tissue and in lipopolysaccharide (LPS)-treated cells, to determine their activities against bacteria isolated from bovine mastitis, and to examine their potentials to trigger defense responses in bovine mammary cells. The genes were found to be upregulated in LPS-stimulated neutrophils, but not in infected quarters or epithelial cells. All peptides showed a variably broad spectrum of activity against 28 bacterial isolates from bovine mastitis (MIC values, 0.5 to 32 microM), some of which were antibiotic resistant. The activity of each peptide was significantly enhanced when it was pairwise tested with the other peptides, reaching the synergy threshold when indolicidin was present. The bactericidal activity was sensitive to milk components; BMAP-27 and -28 were highly effective in mastitic bovine milk and inhibited in milk from healthy cows. Both peptides were also active in whey and in blood serum and triggered the expression of tumor necrosis factor alpha (TNF-alpha) in bovine mammary epithelial cells. Our results indicate multiple roles for the bovine cathelicidins in mastitis, with complementary and mutually enhanced antimicrobial activities against causative pathogens and the capacity to activate host cells.

FIG. 1.

(a) Expression levels of the LAP gene in mammary glands. Tissue samples were grouped based on SCC in milk. The arithmetic mean for each group is indicated by a horizontal line. (b) Expression levels of antimicrobial peptide genes in bovine neutrophils incubated in vitro for 20 h in the absence (open bars) and presence of E. coli LPS at 0.1 μg/ml (hatched bars) or 1 μg/ml (solid bars). The gene expression levels were evaluated using qPCR and are reported as the normalized fold change compared to the expression level in healthy tissue (a) or in unstimulated cells (b). The data in panel b are the means plus standard deviations (SD) of three independent experiments. *, P < 0.05, and ***, P < 0.001 versus unstimulated cells, as assessed by one-way analysis of variance and the Bonferroni posthoc test.

FIG. 2.

Killing of E. coli ATCC 25922 in milk collected from cases of clinical mastitis. Bacteria (4 × 10⁴ to 7 × 10⁴ CFU/ml) were incubated in mastitic milk samples (a to f) at 37°C for 60 min in the absence and presence of BMAP-27, BMAP-28, Bac5, or indolicidin at concentrations corresponding to 8× MIC detected in MH broth. The bacteria were diluted in ice-cold physiological salt solution and plated on MH agar. The petri dishes were incubated at 37°C for 16 to 18 h to allow colony counts. The results are reported as percent survival relative to untreated bacteria and are representative of two experiments with similar results.

FIG. 3.

Induction of the TNF-α gene in BME-UV1 cells. The cells were incubated for 8 h in the presence of increasing concentrations of BMAP-27 or BMAP-28. The TNF-α gene expression levels were evaluated using qPCR and are reported as the normalized fold change compared to the expression level in unstimulated cells. The data are the means ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus unstimulated cells, as assessed by one-way analysis of variance and the Bonferroni posthoc test.