157 Mesenchymal stem cells as a regenerative therapy for the prevention of subclinical mastitis in cattle

Mastitis in cattle refers to inflammation of the mammary gland caused by bacteria, which reduces milk production and results in huge economic losses to dairy farmers worldwide. Currently, antibiotics are used to cure mastitis but they do not regenerate the mammary glandular tissue; rather, they lead to an increase in antibiotic resistance in animals, and antibiotic residues can be found in milk, which may cause human health hazards. Therefore, there is interest in developing alternative therapies using mesenchymal stem cells and extracellular vesicles (EV), which are reported to have anti-inflammatory and immunomodulatory effects and repair damaged tissue. The aim of the present study was to explore the use of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) to prevent subclinical mastitis in cattle. UCB-MSCs were isolated from umbilical cord blood from a cow after the delivery of calf, in vitro cultured and characterised by alkaline phosphatase staining, real-time PCR, immunostaining, colony-forming unit assay, and directed differentiation into adipogenic, osteogenic, and chondrogenic lineages. The MSCs were cryopreserved into liquid nitrogen and revived later in in vitro culture for treatment. The first safety trial was given to the cows using allogenic UCB-MSCs (fresh and cryopreserved), 5 × 10⁶ cells, and EV in 2 mL of Dulbecco’s PBS was injected on Day 0 and Day 7 locally and intravenously (IV) to four healthy cows in each group. Blood samples were taken on Days 0, 1, 7, and 15 for serum isolation and haemogram determination. Ten subclinical mastitis cows were taken in each group antibiotic (control), fresh MSCs, cryopreserved MSCs (5 × 10⁶ cells), and EV injected locally and IV on Day 0 and 7. Blood samples were taken on Day 0, 1, 7, and 15 for total RNA isolation for the gene expression study. In the safety trial of the healthy cow, UCB-MSCs and EV did not result in any significant (P > 0.05) change in haematological parameters or IgA, IgG, and IgM levels. Somatic cell count (SCC) in mastitic milk decreased significantly (P < 0.05) in all MSC and EV treatment groups compared to the control group. The gene expression study showed significant (P < 0.05) increases in anti-inflammatory cytokine IL-10, cathelicidin, cystatin, lipocalin 2, and angiopoietin and decreases in the proinflammatory cytokine IL-6 in treatment groups compared to the control group. We can conclude that UCB-MSCs and EV do not show any immune reaction in healthy cows. MSC therapy significantly reduced the SCC in mastitic milk in cows and enhanced gene expression compared with the control group. All MSC-treated cows were cured permanently within 15 days. MSCs can be used as an alternative therapy for the prevention of subclinical mastitis in cattle.