Flow cytometric differential cell counts in milk for the evaluation of inflammatory reactions in clinically healthy and subclinically infected bovine mammary glands

Abstract

Somatic cell counts (SCC) are generally used as an indicator of udder health. In Germany, a cutoff value of 100,000 cells/mL is currently used to differentiate between healthy and diseased mammary glands. In addition to SCC, differential cell counts (DCC) can be applied for a more detailed evaluation of the udder health status. The aim of this study was to differentiate immune cells in milk of udder quarters classified as healthy based on SCC values of <100,000 cells/mL. Twenty cows were selected and 65 healthy udder quarters were compared with a control group of 15 diseased udder quarters (SCC>100,000 cells/mL). Cells were isolated from milk of all quarters to measure simultaneously percentages of lymphocytes, macrophages, and polymorphonuclear neutrophilic leukocytes (PMNL) by flow cytometric analysis. The bacteriological status of all 80 quarters was also determined. Differential cell count patterns of milk samples (n=15) with extreme low SCC values of ≤6,250 cells/mL revealed high lymphocyte proportions of up to 88%. Milk cell populations in samples (n=42) with SCC values from >6,250 to ≤25,000 cells/mL were also dominated by lymphocytes, whereas DCC patterns of 6 out of 41 milk samples with SCC values from ≥9,000 to ≤46,000 cells/mL indicated already inflammatory reactions based on the predominance of PMNL (56–75%). In 13 of 15 milk samples of the diseased udder quarters (SCC >100,000 cells/mL), PMNL were categorically found as dominant cell population with proportions of ≥49%. Macrophages were the second predominant cell population in almost all samples tested in relation to lymphocytes and PMNL. Further analysis of the data demonstrated significant differences of the cellular components between udder quarters infected by major pathogens (e.g., Staphylococcus aureus; n=5) and culture-negative udder quarters (n=56). Even the percentages of immune cells in milk from quarters infected by minor pathogens (e.g., coagulase-negative staphylococci; n=19) differed significantly from those in milk of culture-negative quarters. Our flow cytometric analysis of immune cells in milk of udder quarters classified as healthy by SCC <100,000 cells/mL revealed inflammatory reactions based on DCC.