Single-cell RNA sequencing characterization of Holstein cattle blood and milk immune cells during a chronic Staphylococcus aureus mastitis infection.

Mastitis remains the most prevalent and costly disease to dairy producers. Granulocytes are the primary host innate immune cell responders during infectious mastitis. Here we examine three mid-lactation Holsteins challenged with ~ 150 CFU of Staphylococcus aureus (Newbould) that developed chronic mastitis as assessed by bacteria and somatic cell counts in a single quarter. Single-cell RNA-sequencing (scRNA-seq) of blood and milk cells identified immune cell populations of interest from both tissues, and the proportion of cell types recovered via scRNA-seq were highly similar to those recovered via flow cytometry. Granulocytes were the predominating cell type in both blood and milk samples; however granulocytes identified via scRNA-seq revealed several clusters comprised primarily of milk-derived cells. Milk-enriched granulocyte clusters were further investigated to identify gene signatures indicative of the granulocyte-specific localized immune responses in the mammary gland during chronic mastitis infection. Biological process enrichment analysis of gene signatures further revealed relevant networks such as granulocyte migration, myeloid cell differentiation, and inflammatory responses. In total, the work describes the immune landscape occurring at both peripheral and local sites of cattle with mastitis and identified important granulocyte-specific features of the localized immune response occurring during chronic infection.