Bovine Beta-defensin 3

Digital dermatitis (DD), a common ulcerative disease of the bovine foot causing lameness and reducing productivity and animal welfare, is associated with infection by spirochete Treponema bacteria. Topical tetracycline, the most common treatment, has inconsistent cure rates; therefore, new therapeutic options are needed. We compared effects of topical oxytetracycline and vitamin D3 on innate immunity in DD-affected skin. Cows with active DD lesions were treated topically with oxytetracycline or vitamin D3 and skin biopsies were collected from lesions. Tissue samples were examined histologically, transcriptional expression of pro-inflammatory cytokines, Toll-like receptors (TLRs), and host defense peptides assessed, and the presence of specific treponeme species determined. Effects of treatments at a mechanistic level were studied in a human keratinocyte model of treponeme infection. Oxytetracycline promoted hyperplastic scab formation in ulcerated DD lesions and decreased transcriptional expression of Cxcl-8 (neutrophil chemoattractant). Oxytetracycline also reduced numbers of Treponema phagedenis and T. pedis and enhanced Tlr2 mRNA expression. Vitamin D3 did not modify expression of cytokines or Tlrs, or bacterial loads, but enhanced transcription of tracheal antimicrobial peptide (Tap), a key bovine β-defensin. Combing oxytetracycline and vitamin D3 provides complementary clinical benefits in controlling DD through a combination of antimicrobial, immunomodulatory, and pro-healing activities.

Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human β-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn't have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

Antimicrobial peptides are a common defense against bacterial infections in many species and a significant part of the innate immune response of the bovine mammary gland. The objective of this study was to investigate the influence of epigenetic factors on vitamin D and toll-like receptor-mediated induction of β-defensins in mammary epithelial cells. Primary bovine mammary epithelial cells were treated with lipopolysaccharide (LPS, 0 or 100 ng/mL), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 0 or 10 nM], and 5-aza-2'-deoxycytidine (5-Aza, inhibitor of DNA methyltransferase, 0 or 5 μM) or trichostatin A (TSA, inhibitor of histone deacetylase, 0 or 80 nM) in a factorial arrangement. Effects of treatments on β-defensin gene expression along with genes for cytokines and enzymes known to be induced by LPS or 1,25(OH)2D3 were evaluated by quantitative PCR. The LPS treatment induced expression of β-defensin (DEFB)3, DEFB5, DEFB7, DEFB10, enteric β-defensin (EBD), lingual antimicrobial peptide (LAP), and tracheal antimicrobial peptide (TAP); whereas, the 1,25(OH)2D3 treatment increased DEFB5 and DEFB7 expression and decreased LAP. The 5-Aza treatment increased expression of DEFB3, DEFB5, DEFB10, EBD, LAP, and TAP in the presence and absence of LPS. The TSA treatment increased expression of DEFB3, DEFB4, DEFB5, DEFB7, and DEFB10 in the absence of LPS but decreased LPS-induced expression of and LAP and TAP. Together these results indicate that β-defensin expression in bovine mammary epithelial cells is likely influenced by DNA methylation and histone acetylation. Investigation of environmental and nutritional factors that influence epigenetic control of β-defensins in the mammary gland may be beneficial for improving resistance to intramammary infections.