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 the identification of a causative pathogen which requires at least 24 hours to obtain actionable results. In approximately 20% of cultures from clinical quarters, no bacterial growth can be determined potentially due to the cow’s immune response having already eliminated the pathogen, the pathogen not currently being shed in milk, or the presence of a pathogen that does not grow under aerobic conditions. Cytokines and chemokines are locally produced in the infected quarter and regulate immune cell activation, immune cell influx to the gland, and resolution of inflammation. Their production and secretion patterns are influenced by the invading pathogen and by the host’s immune system.

We hypothesize that cytokines and chemokines can serve as biomarkers for bovine mastitis.

Our goal is to associate cytokine and chemokine profiles in milk from mastitis quarters with infection status (presence or absence of bacterial growth in milk) and causative pathogens.

Aim 1 of this project will be to measure the concentrations of the cytokines IL-10 (regulatory cytokine, produced by mixed leukocytes), TNF-α (pro-inflammatory, produced by myeloid cells) and IFN-γ (pro-inflammatory, produced by lymphoid cells) in selected quarter milk samples using a previously established fluorescent bead-based multiplex assay. We will collect quarter milk samples identified with major mastitis pathogens including coliform bacteria (E. coli, Klebsiella spp.), bacteria from the Streptococcus family (S. uberis, S. dysgalactiea, and Lactococcus spp.), and S. aureus. We will also include samples from mastitic quarters without any bacterial growth after 24 h aerobic culture and samples from healthy quarters (no clinical sign, California mastitis test negative, no bacterial growth in aerobic culture). The obtained cytokine profiles will be associated with infection status and pathogen group determined in the samples.  Aim 2 of this project will be to develop a new chemokine multiplex assay for the detection of CCL2 and CCL5, which are highly up regulated during mastitis and responsible for attracting monocytes, lymphocytes, and granulocytes to the mammary gland. We will produce recombinant bovine CCL2 and CCL5 to immunize mice and generate monoclonal antibodies (mAbs) in hybridoma cell lines. Target specific mAbs will be coupled with fluorescent beads to develop a multiplex assay for the detection of CCL2 and CCL5 in quarter milk samples of the same category as used in aim 1.

Associating cytokine and chemokine profiles with different intramammary infection status and pathogen groups will deepen our understanding of host pathogen interactions in mastitis and explore diagnostic potential of these
inflammatory mediators. As potential diagnostic assay cytokine and chemokine profiles could be obtained faster
than aerobic culture results supporting timely management decisions. They also give crucial information on the host immune response and could be used to determine treatment protocols that are not only based on the pathogen but also account for the host’s immune status.