Effect of an IgY-Based Immunomodulatory Combo on the Internalization of Staphylococcus aureus by HC11 Cells: Preliminary in vitro Studies II

Infectious mastitis is a common disease of cattle of economic significance. Clinical mastitis affects between 20 to 25% of dairy cows each year. Staphylococcus aureus (SA) causes an estimated 10-11.7% of bovine infectious mastitis cases in the US, reducing milk production, increasing morbidity, increasing antibiotic use and milk waste, and fueling premature culling. The SA mastitis cure rate varies from 1.1% (spontaneous) to up to 50% (with antibiotics). Hence a need to establish more effective treatments for this disease. SA is internalized by mammary epithelial cells, thus evading most antibiotics and the cellular and humoral immune systems. Once internalized, SA persists subclinically and chronically. Approaches addressing internalization are needed. This study explores the effect of a combination of IgY, vitamin D3, and a novel immunomodulatory peptide, RP185, on the internalization of SA by mouse mammary epithelial cells. HC11 cells were grown in three 96 well plates in RPMI medium with growth factors. The cells were exposed to a gentamicin protection assay, exposing them to SA and DMEM medium containing IgY (at 5 or 10 µg/ml), either by itself, or in combination with vitamin D3 at 20, 50 or 80 nM, or in a further combination of said vitamin D3 concentrations with the novel peptide RP185 (1, 10 or 50 µg/ml). All combinations very significantly reduced internalization of SA by HC11 cells, with the combinations of IgY (10 µg/ml) with vitamin D3 at 50 and 80nM, IgY (10 µg/ml), and with vitamin D3 at 50nM with 1 or 10µg/ml of RP185, resulted consistently in 0 internalized CFU of S. aureus. RP185 did not seem to have contributed to a further reduction in internalization. The impact of RP185 cannot be fully evaluated without an in-vivo study. Both IgY and vitamin D3 at 50nM and 80nM had a positive impact on internalization. These results warrant an in-vivo study of this combination therapy.