Animals and sampling
The present study used 48 udder quarters from 18 clinically healthy Holstein dairy cows, collected at different lactation stages from a commercial herd. From these dairy cows, we selected 20 C. bovis-infected quarters from 10 dairy cows and 28 culture-negative control quarters from 12 dairy cows with no abnormal secretions in the strip cup test and a quarter SCC lower than 1×105 cells/mL, as the threshold for SCC described by Bansal et al.  for uninfected quarters.
First, the strip cup test was carried out to detect potential clinical mastitis cases. Then, a single milk quarter sample (about 4 mL) was aseptically collected for bacteriological analysis as recommended by National Mastitis Council . Furthermore, quarter milk samples for SCC measurements (40 mL) were taken in sterile tubes containing micropellets of Bronopol (2-bromo-2-nitroprane-1,3-diol). Finally, 1 L of milk samples for the evaluation of monocyte/macrophage function and lymphocyte profile were collected. Until milk samples arrived at the laboratory, they were maintained at 4°C. Subsequently, quarter milk samples for bacteriological examination wrer retained at -20°C until the analysis.
Afterwards, all samples were randomized and codified, and milk analyses were carried out without knowledge of the status of the udder quarter. All methods were carried out in accordance with relevant guidelines and regulations.
The bacterial analysis was carried out by culturing 0.01 mL of each milk quarter sample on 5% ovine blood agar plates. The plates were incubated for 72 hours at 37°C, followed by Gram staining, observation of colony morphology, and biochemical testing . A milk sample was considered culture positive when the growth of > 4 pure C. bovis colonies was detected . A sample was considered culture negative if there was no growth (no colony from a 0.01 mL sample; < 100 colony-forming units per mL).
Determination of the somatic cell count
The SCC was determined with the automated somatic cell counter Somacount 300 (Bentley Instruments, Chaska, MN, USA).
Separation of milk cells
Milk cells were separated as previously described by Blagitz et al. . Briefly, 1 L of milk was diluted in 1 L of phosphate-buffered saline solution (PBS). Centrifugation at 1000×g was performed for 15 min, and the fat layer and the supernatant were discarded. The cell pellet was then washed again with 30 mL of PBS solution and centrifuged at 400 × g for 10 min. This cell pellet was resuspended in 1 mL of RPMI-1640 cell culture medium (R7638, Sigma Aldrich, USA) supplemented with 10% fetal bovine serum (Cultilab, Brazil), and then the cells were counted in a Neubauer chamber. Cell viability was initially assessed by exclusion using Trypan blue. The cells present in the milk were then resuspended in cell culture medium containing 10% fetal bovine serum at a concentration of 2 × 106 mL-1 viable cells.
Enumeration of lymphocyte subpopulations
The enumeration of lymphocyte subsets was performed as previously described  with some slight modifications. Briefly, the cells were washed with PBS and stained with a combination of CD3, CD4, and CD8 (tube 1) and for CD21 (tube 2) for 30 min at room temperature. The lymphocyte subpopulations were identified based upon their cytoplasmatic granularity and mean fluorescence intensity following two-step fluorescence immunolabeling with primary anti-bovine monoclonal antibodies (mAbs) and the secondary antibody (Ab) coupled to the long-wavelength fluorescent probes (Supplemental Material 2). After washing with PBS, the cells were incubated for 30 min at room temperature with the secondary Abs. The cells were subsequently washed with PBS and quickly evaluated by flow cytometry (FACSCalibur, BD Bioscience, San Jose, CA, USA). Here, 20,000 milk cells, excluding most of the cell debris, were examined in each quarter milk sample. A single-stained, fluorochrome-conjugated secondary Ab control and unstained control milk samples were also prepared as compensation controls. FlowJo software (TreeStar Inc., Ashland, OR, USA) was used to analyze the data.
Identification of milk macrophages
The identification of macrophages was carried out as previously described . Initially, the cells were incubated with 1 μL of mouse IgG1 mAb against bovine CD14 (cat. n. MM61A, VMRD, Pullman, WA, USA) for 30 min at room temperature. Immediately after, 1 mL of PBS was added to the specific cytometry tube, and the samples were centrifuged for 8 min at 400 × g. Next, 1 μL of allophycocyanin-conjugated goat anti-mouse IgG1 secondary antibody (cat. n. A10541, Invitrogen, Carlsbad, CA, USA) was added to the samples, which were incubated for 30 min at room temperature. Then, PBS solution (1 mL) was added to the cell suspension, which was centrifuged for 8 min at 400 × g. Lastly, PBS (300 μL) was added to the samples, which were examined by flow cytometry (FACSCalibur, BD Bioscience, San Jose, CA, USA). A single-stained, fluorochrome-conjugated secondary Ab control and unstained control milk samples were also prepared as compensation controls. FlowJo software (TreeStar Inc., Ashland, OR, USA) was used to analyze the data.
Preparation of Staphylococcus aureus stained with propidium iodide
The staining of Staphylococcus aureus (ATCC 25923) with propidium iodide (PI) was done as proposed [47, 48]
Intracellular production of reactive oxygen and nitrogen species
The intracellular production of reactive oxygen and nitrogen species (RONS) was measured by flow cytometry as described [30, 41, 48]. Briefly, 2×105 viable milk cells were incubated with 200 µL of 2′,7′-dichlorofluorescein diacetate (DCFH2-DA, 0.3 mM, cat. n. D6883, Sigma Aldrich, St. Louis, USA) for 30 min at 37°C. Various types of RONS (hydrogen peroxide, peroxynitrite, nitric oxide, hydroxyl radicals, and peroxyl) oxidize DCFH2-DA into 2’,7’-dichlorofluorescein (DCF), which is fluorescent and can be detected in a flow cytometer equipped with a set of standard filters for fluorescein green . After the incubation in DCFH2-DA, 2 mL of 3 mM EDTA was added. Macrophages were identified using the CD14 mAb as described above. Finally, the samples were centrifuged at 400×g for 10 min, and the leukocytes were resuspended in 300 µL of PBS and analyzed by flow cytometry.
In the present study, 20,000 cells per sample were examined – most cellular debris was excluded. The readings of the samples were performed in a flow cytometer with argon (excitation 488 nm) and diode lasers (excitation 635 nm) (FACSCalibur, BD Bioscience, San Jose, CA, USA). FlowJo software (TreeStar Inc., Ashland, OR, USA) was used to examine the data. The data are presented as the percentage of macrophages (CD14+ cells) that produced RONS (percentage of fluorescent cells), and the geometric mean fluorescence intensity (GMFI) indicated the intensity of RONS production of each cell. The results were corrected for autofluorescence content using nonstained milk cells from milk samples from the same udder quarter.
The phagocytosis assay was performed by flow cytometry using S. aureus conjugated with propidium iodide (PI) as previously described [41, 46, 48]. Briefly, 2×105 viable milk cells were incubated with 100 µL of PI-conjugated S. aureus for 30 min at 37°C and 900 µL of PBS. Then 2 mL of 3 mM EDTA was added to drastically reduce the number of bacteria adhering to the cell membrane that could be mistakenly considered phagocytized [47, 50]. The macrophages were identified using the CD14 mAb as described above. Finally, the samples were centrifuged at 400×g for 10 min, and the leukocytes were resuspended in 300 µL of PBS and analyzed by flow cytometry.
As above, 20,000 cells per sample were examined, and most cellular debris was excluded. FlowJo software (TreeStar Inc., Ashland, OR, USA) was used to examine the data. The data are presented as the percentage of macrophages (CD14+ cells) that phagocytized PI-stained bacteria (percentage of fluorescent cells), and the GMFI indicates the number of bacteria phagocytized by macrophages that phagocytosed S. aureus by measuring the fluorescence intensity, which was correlated with the number of phagocytized bacteria per cell. The results were corrected for autofluorescence content using nonstained milk cells from milk samples from the same udder quarter.
Detection of apoptosis by flow cytometry
Apoptosis of milk macrophages was determined by double staining with annexin-V conjugated to fluorescein isothiocyanate (FITC) and propidium iodide (PI) by flow cytometry analysis using a commercial kit (cat. n. K2350, APOPTEST-FITC, DakoCytomation, Netherlands), as previously described [30, 41, 48]. Initially, 2×105 milk cells were resuspended in 100 µL of binding buffer (10 mM HEPES, 150 mM NaCl, 1 mM MgCl2 and 1.8 mM CaCl2) containing annexin-V FITC and incubated at room temperature for 20 min in the dark. The macrophages were identified using the CD14 mAb as described above. Immediately before the flow cytometry analysis, 5 μL of a PI solution (250 μg mL-1) was added. Cells negative for FITC-stained annexin-V and for PI were considered alive. Cells that were reactive to FITC-stained annexin-V but negative to PI were classified as apoptotic. Again 20,000 cells were examined per sample, and most cellular debris were excluded. FlowJo software (TreeStar Inc., Ashland, OR, USA) was used to examine the data.
The distributions of all variables were analyzed using normal probability plots obtained from the Shapiro-Wilk test. As all data presented high coefficient of variation, we carried out a logarithmic transformation (Log10). First, interclass correlation at the cow and quarter levels was calculated to determine the strength of clustering, as previously described by McGraw and Wong . The data were analyzed ANOVA following by the pos-hoc Student-Newnan-Keuls test was applied. The model of mammary quarters and cows nested within cows was considered . Statistical analyses were performed using the statistical software InfoStat (Cordoba, Argentina). The results are presented as the mean ± standard error. The level of significance was set at P ≤ 0.05.