Transfer factor alleviates bovine mastitis and protects mammary epithelial barrier via the TAK1/NF-κB/MLCK signaling axis.

Bovine mastitis is a prevalent and economically devastating disease in the global dairy industry. Antibiotic overuse leads to increased antimicrobial resistance and reduced milk quality, becoming major bottlenecks in clinical treatment. Transfer factor (TF), a safe, low-cost, and readily available immunomodulator that enhances cell-mediated immunity, has emerged as a promising antibiotic alternative. This study aimed to investigate TF's alleviative effect on bovine mastitis and its underlying molecular mechanisms. We found that clinical mastitis cows had significantly higher mRNA levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) and markedly lower expression of tight junction (TJ) genes (nectin cell adhesion molecule 4 [NECTIN4], tight junction protein 1 [ZO-1], occludin) in mammary tissue and milk somatic cells compared to healthy controls. In vitro experiments, TF pretreatment inhibited the secretion of pro-inflammatory cytokines (IL-1β and IL-6) and the expression of TJ genes and proteins (NECTIN4, occludin, ZO-1) in a graded manner across the tested concentrations (up to 180 µg/mL) in lipopolysaccharide (LPS)-induced bovine mammary epithelial cells (MAC-T). Mechanistically, TF alleviated TJ protein inhibition by regulating myosin light chain kinase (MLCK) activity, mimicking the effect of MLCK inhibitor ML-7. It also mitigated LPS-induced changes via inhibiting the nuclear factor κB (NF-κB) pathway, similar to NF-κB inhibitor Bay 11-7082, and blocked NF-κB activation by inhibiting the transforming growth factor-β-activated kinase 1 (TAK1) pathway, comparable to TAK1 inhibitor Takinib. In vivo, intramammary infusion of TF via the teat canal into the infected quarter of cows with subclinical mastitis downregulated IL-1β/IL-6 mRNA, upregulated TJ genes, and reduced both MLCK expression and the phosphorylation of myosin light chain (MLC), p65, and TAK1. By day 5, TF group's (n = 17) California mastitis test (CMT) negative conversion rate reached 64.7% (vs. 13.3% in the normal saline (NS) group, n = 15) with a significantly lower somatic cell count (SCC). Our findings demonstrate that TF, by concurrently eliciting anti-inflammatory and barrier-repair effects via inhibition of the TAK1/NF-κB/MLCK axis, effectively alleviates bovine mastitis. This study furnishes a robust molecular framework for deploying TF as a non-antibiotic tool in the sustainable control of mastitis.