Xinjiang Brown Cattle-Derived β-Defensin EBD Alleviates Enterococcus faecalis-Induced Mammary Inflammation in Mice

Bovine mastitis is a major infectious disease in dairy cattle, and the increasing prevalence of antimicrobial-resistant pathogens has created an urgent need for the development of effective antimicrobial agents. In this study, bovine enteric β-defensin (EBD) from Xinjiang Brown Cattle was cloned, expressed, and preliminarily evaluated for its antibacterial and protective effects against Enterococcus faecalis-induced mastitis. Bioinformatic analysis identified EBD as a secreted protein belonging to the bovine β-defensin family. The recombinant PCDH-EBD construct was successfully transfected into 293T cells, resulting in markedly increased EBD mRNA expression and detectable recombinant EBD protein expression, as confirmed by SDS-PAGE and Western blotting. In vitro agar diffusion assays showed that the EBD-containing preparation exhibited inhibitory activity against E. faecalis. In a murine mastitis model, EBD treatment significantly attenuated mammary histopathological lesions and reduced the expression of Mpo, Tnf, and Il6 compared with the mastitis group. 16S rRNA sequencing of mammary gland tissues showed that EBD treatment was associated with a reduced relative abundance of Enterococcus and partial restoration of microbial diversity. Transcriptomic analysis further revealed that EBD treatment was associated with alterations in NOD-like receptor signaling- and phagosome-related pathways, suggesting that these processes may contribute to its protective effects. Collectively, these findings indicate that enteric β-defensin EBD from Xinjiang Brown Cattle possesses antibacterial and anti-inflammatory potential, making it a candidate for developing antimicrobial agents against bovine mastitis.