Chitosan nanoparticles as potential immunomodulatory agents for bovine mastitis prevention

Abstract

Bovine mastitis is an inflammatory disease of the mammary gland tissue, primarily caused by bacterial infections. This condition is highly relevant in the dairy industry due to its impact on animal welfare and the significant economic losses associated with decreased milk yield and quality. Although antibiotic therapies are still the primary strategy for treating bovine mastitis, the increasing concern about antibiotic-resistant pathogens necessitates reduced antimicrobial use, prompting the search for alternative treatments, especially those derived from natural products. Given that dairy cattle with enhanced and balanced immune responses exhibit a lower incidence of the disease, immunostimulant treatments emerge as a promising alternative. Our research focuses on chitosan, a biocompatible, bioactive, and non-toxic polymer with well-documented antimicrobial and immunostimulant properties. Specifically, we developed chitosan nanoparticles prepared using the reverse micelles method, which demonstrated enhanced microbicidal effects against Staphylococcus aureus strains isolated from cows with mastitis, compared to the native polymer. When assessing their effects on the immune system, RAW 264.7 macrophages pretreated with chitosan nanoparticles showed increased production of reactive oxygen species after infection with S. aureus V329, as measured by flow cytometry using the DCFDA probe. Additionally, treated macrophages exhibited upregulation of pro-inflammatory cytokine mRNA, such as IL-1β and IL-6, as evaluated by qPCR, and increased expression of MHCII, CD86, CD11b, and TLR2 markers, as evidenced by flow cytometry. Importantly, the treatment was non- cytotoxic to both murine macrophages and bovine epithelial cells (MAC-T), as determined by the MTT assay. Overall, our findings highlight the potential of chitosan nanoparticles as a novel therapeutic approach to enhance the immune response of the bovine mammary gland and effectively prevent mastitis development following pathogen infections.