Measuring cytokines in quarter milk samples with a fluorescent bead-based multiplex assay

Mastitis is a highly prevalent, costly disease affecting well-being and productivity in dairy cows. Management of intramammary infections typically relies on aerobic culture for identification of a causative pathogen. Cytokines regulate inflammatory processes in the infected quarter. Their production and secretion patterns are influenced by the invading pathogen and by the host's immune system. A previously validated fluorescent bead-based multiplex assay was used for the quantification of bovine IL-10, TNF-α, and IFN-γ. Our goal was to demonstrate that this assay can be used for milk samples and to measure concentrations of the 3 analytes in quarter milk samples with different bacteriological culture results. We enrolled quarter milk samples that were submitted for routine mastitis diagnostics by farm personnel from commercial dairies (n = 17) in central New York. We selected samples identified with major mastitis pathogens, including coliform bacteria (n = 51; termed the coliform group, comprising Escherichia coli and Klebsiella spp.), bacteria from the Streptococcus family (n = 53; Strep group: Streptococcus uberis, Streptococcus dysgalactiae, and Lactococcus spp.), Staphylococcus aureus (n = 36; S. aureus group), samples from mastitic quarters without bacterial growth after 48-h aerobic culture (n = 44; no growth), and samples from healthy quarters (n = 55; healthy). When comparing cytokine concentrations for different bacteriological culture results, we found that the coliform group had the highest IL-10 and TNF-α concentrations compared with all other bacteriological culture results. The Strep group had higher IFN-γ and TNF-α concentrations compared with S. aureus, no growth, and healthy samples, and higher IL-10 concentrations compared with no growth and healthy quarters. No significant differences were found between S. aureus, no growth, and healthy samples, or between no growth and healthy samples. In this study we observed differences in cytokine concentration patterns across the selected bacteriological culture results. We demonstrated that the bovine multiplex assay can be used to measure IL-10, TNF-α, and IFN-γ in milk samples and could provide a useful tool for screening milk samples and furthering our understanding of inflammation dynamics in mastitis. Although differences were found between cytokine concentrations and bacteriological culture results, they showed high individual variation, and future studies should include additional information on enrolled samples such as occurrence of clinical signs, treatment, and resolution of infection.