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

Authors: Wiarda, Jayne; Sarlo Davila, Kaitlyn; Trachsel, Julian; Loving, Crystal; Boggiatto, Paola; Lippolis, John; Putz, Ellie

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: The most prominent disease of dairy cows is mastitis infection usually by bacterial agents such as Staphylococcus aureus. Mastitis results in animal discomfort, serious health concerns, and loss of milk production collectively costing the US dairy industry over $2 billion annually. Understanding and characterizing the immune response of cells in the mammary gland is critical to the development of prevention and treatment of mastitis. In this work we characterize immune cells sourced from both blood and milk of lactating dairy cows infected with chronic S. aureus mastitis infections using cutting edge single cell RNA sequencing technology. Importantly we focus on granulocyte cells which are the first line of immune defense against bacterial pathogens in the mammary glad. We identify unique clusters of granulocytes identified in the milk, and explored their gene expression profiles. Highlights include gene signatures associated with immune cell migration, activation, and functional response to bacteria.

Technical Abstract: Mastitis remains the most prevalent and costly disease to dairy producers. Granulocytes are the primary host innate immune cell responders to mastitis infections. Here we examine three mid-lactation Holsteins challenged with ~150 CFU of Staphylococcus aureus (Newbould) that developed chronic mastitis infections as assessed by bacteria and somatic cell counts in a single quarter. Single-cell RNA sequencing (scRNA-seq) of blood and milk from these animals identified immune cell profiles and granulocyte populations of interest from both tissues, and proportional recovery of cell types recovered via scRNA-seq were highly similar to those that could also be recovered via flow cytometry. Granulocytes were the predominating cell type found in both blood and milk samples, and further inspection of granulocytes recovered via scRNA-seq revealed several cell clusters nearly exclusively comprised of milk-derived cells. Milk-enriched granulocyte clusters were further investigated to identify gene signatures indicative of the granulocyte-specific localized immune responses occurring in the mammary gland during chronic mastitis infection. Biological process enrichment analysis of gene signatures further articulated 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 identifies important granulocyte-specific features of the localized immune response occurring during chronic infection.