Yak defensin plasmid reduces mastitis inflammation in cell and mouse models

A plasmid expressing the yak antimicrobial peptide DEFB127 reduced inflammatory responses and tissue injury in laboratory models of Staphylococcus aureus mastitis, according to a study in bovine mammary epithelial cells and lactating mice. Researchers reported that mammary-gland-specific delivery of the construct, called pLF-DEFB127, produced effects broadly comparable to lincomycin in the models. Treatment was also associated with less disruption of intestinal microbial diversity than lincomycin, although the study did not directly measure functional DEFB127 protein or establish whether microbiome changes contributed to the improvement.

The work was presented as a potential antibiotic-sparing strategy for bovine mastitis, a disease that can impair dairy-cow health, milk production and milk quality. The authors cited a reported prevalence of 36.23% for S. aureus-induced bovine mastitis in China and annual mastitis-related economic losses estimated to reach €300 per cow in Spain. They also described increasing resistance among S. aureus isolates to commonly used β-lactams, macrolides and tetracyclines, alongside concerns about antibiotic-associated disruption of cattle microbiota and residues in dairy products. These background observations motivated evaluation of a biological alternative, but the experiments were conducted in cultured cells and mice rather than dairy cattle.

DEFB127 is a 99-amino-acid β-defensin whose coding sequence was amplified from yak spleen RNA. The investigators inserted the sequence into a mammary-specific plasmid containing the lactotransferrin promoter, producing pLF-DEFB127. Bioinformatics analysis found 18 positively charged and nine negatively charged residues, while hydrophobic amino acids made up 33% of the peptide. The authors said these features were consistent with the membrane-targeting characteristics of cationic antimicrobial peptides. A predicted tertiary structure was predominantly α-helical, and a Ramachandran analysis placed most residues in preferred or additionally allowed regions. These computational findings supported subsequent functional evaluation but did not demonstrate that DEFB127 directly killed S. aureus in the reported experiments.

For the cell experiment, investigators transfected MAC-T bovine mammary epithelial cells with pLF-DEFB127 and used reverse-transcription quantitative PCR to confirm increased DEFB127 messenger RNA. Cells were then exposed to S. aureus at a multiplicity of infection of 10 for four hours. Uninfected cells, infected cells without treatment and infected cells treated with penicillin-streptomycin served as comparison groups. Infection significantly increased inflammatory gene expression in the MAC-T model. Cells expressing DEFB127 had significantly lower interleukin-6 and interleukin-1β expression than infected cells without the plasmid, with P less than 0.01 for both comparisons. The study did not report direct measurements of bacterial viability, protein abundance or the intracellular signaling pathways responsible for the transcriptional changes.

The in vivo experiment used 8-week-old, specific-pathogen-free female ICR mice, with six animals assigned to each of four groups: normal control, S. aureus infection, lincomycin treatment and pLF-DEFB127 treatment. Seven days after giving birth, mice received sterile saline or an S. aureus suspension containing 1 × 10^7 colony-forming units per milliliter in each of two mammary glands. At 24 hours after infection, mice in the antibiotic group received 20 μL of lincomycin intramammary, while the plasmid group received a 1:1 volume mixture of pLF-DEFB127 and Pluronic L64, with a plasmid concentration of 1,000 ng/μL and a final L64 concentration of 0.1%. Mammary glands and intestinal contents were collected 48 hours after treatment, or 72 hours after infection. The procedures therefore modeled infection and subsequent treatment in mice rather than naturally occurring bovine mastitis.

Gross examination found that infected mammary glands were dark red, swollen and marked by hemorrhagic spots and erosion, whereas normal-control glands were cream colored and uniform. Lincomycin-treated glands had minor hemorrhagic spots and were slightly darker than normal tissue. Glands in the pLF-DEFB127 group retained localized hemorrhagic spots and milk-like secretions and were somewhat redder than controls. Histology showed acinar dilation, epithelial degeneration and occasional necrosis in infected animals, together with luminal secretions and inflammatory-cell accumulation. Mice receiving pLF-DEFB127 had less epithelial necrosis and no significant inflammatory-cell infiltration relative to the infection group, although some dilation and degeneration remained. Body weight declined in all infected groups, with the greatest loss among untreated infected mice, followed by the pLF-DEFB127 and lincomycin groups.

Molecular and protein measurements in mammary tissue showed a consistent pattern. Reverse-transcription PCR found significantly lower tumor necrosis factor-alpha, interleukin-6 and interleukin-1β messenger RNA in pLF-DEFB127-treated glands than in infected glands, with P less than 0.01 for all three comparisons. Enzyme-linked immunosorbent assays also detected lower cytokine concentrations in the plasmid-treated group: interleukin-1β showed the largest reduction, at P less than 0.01; tumor necrosis factor-alpha declined significantly at P less than 0.05; and interleukin-6 showed the smallest reduction. These observations support an anti-inflammatory effect in the experimental models, but the investigators did not test candidate signaling routes such as NF-κB, MAPK or the NLRP3 inflammasome, nor did they characterize immune-cell recruitment, bacterial clearance, apoptosis or oxidative stress in mammary tissue.

To compare the intestinal microbiome effects of plasmid treatment and conventional antibiotic treatment, the team sequenced the V3–V4 regions of 16S rRNA genes from intestinal luminal contents. Operational taxonomic unit counts were 827 in normal controls, 869 in infected mice, 860 after lincomycin and 797 after pLF-DEFB127. Lincomycin produced 667 unique operational taxonomic units, more than any other group, and its animals had significantly lower Shannon diversity with tightly clustered samples. Principal-component analysis based on Bray-Curtis dissimilarity separated the lincomycin group from the normal, infected and plasmid-treated groups, which clustered more closely together. By contrast, pLF-DEFB127 treatment was associated with diversity comparable to the normal control, although slightly lower, and with increased relative abundance of Bacteroidia and Bacilli and decreased Gammaproteobacteria relative to the normal and infected groups. Lincomycin was associated with reduced diversity and a distinct community structure, whereas the plasmid group more closely resembled the untreated control.

Taxonomic and computational analyses identified additional group-associated patterns. Linear discriminant analysis effect size indicated enrichment of Prevotellaceae and Erysipelotrichia-related taxa in the pLF-DEFB127 group, while infected animals were enriched for Enterobacteriales, Enterobacteriaceae and Escherichia-Shigella. PICRUSt2 predictions suggested differences in pathways including sphingolipid and glycosphingolipid biosynthesis and immune signaling, leading the authors to propose that DEFB127 might influence the microbiota through these routes. Those predictions were generated from 16S rRNA sequence data and reference databases rather than direct measurements of microbial metabolism, gene expression or pathway activity. The experiment also included no fecal microbiota transplantation, germ-free animal experiments or microbiota-depletion procedures, so it cannot establish that the observed microbiome changes caused the improvement in mammary inflammation. The changes could instead have been secondary consequences of reduced disease, leaving the proposed gut–mammary immune relationship as a hypothesis.

The central limitation is that the researchers verified only transcriptional induction of DEFB127, not the abundance, secretion, localization or biological activity of the peptide itself. Messenger RNA does not by itself demonstrate production of functional DEFB127, the form expected to influence inflammation or bacterial membranes. The models also used transfected cells, a deliberately infected mouse model and a single plasmid administration regimen, with no direct bacterial-killing assay or clinical outcome measured in cattle. Within those constraints, the study provides a preliminary signal: pLF-DEFB127 was associated with lower mammary cytokine levels and less tissue injury than untreated infection, while its intestinal microbiome profile differed from the pronounced reduction in diversity and community separation observed after lincomycin. Protein-level confirmation and mechanistic experiments would be needed to determine the peptide’s direct role and the causal significance of the microbiome observations.

Subject of Research: Experimental evaluation of plasmid-mediated yak DEFB127 expression in cell and mouse models of S. aureus-induced mastitis.

Article Title: In situ expression of yak DEFB127 via plasmid-mediated delivery alleviates bovine Staphylococcus aureus-induced mastitis through anti-inflammatory mechanisms

Article References: Lu, D., Wang, Z., Dai, W., Zhou, Z., Yue, S., Zhang, X., Li, B., Jiang, M., & Liu, Y. (2026). In situ expression of yak DEFB127 via plasmid-mediated delivery alleviates bovine Staphylococcus aureus-induced mastitis through anti-inflammatory mechanisms. Frontiers in Veterinary Science, 13, Article 1910975. https://doi.org/10.3389/fvets.2026.1910975

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1910975

Keywords: yak DEFB127, bovine mastitis, Staphylococcus aureus, plasmid delivery, mammary inflammation, antimicrobial peptide, mouse model, MAC-T cells, intestinal microbiota, lincomycin, anti-inflammatory response, veterinary medicine