Flow cytometry is a powerful technology used in many fields of cell biology. It is also used as a routine method to count somatic cells in milk and to characterize bovine milk leukocytes. In this study, we used flow cytometry to simultaneously assess the viability, the percentage of the single subsets of leukocytes and to quantify the expression of CD11b, an immunological marker of cell activation status. Immunological markers were then related with on farm recorded parameters as milk electrical conductivity (MEC) and average milk flow rate (MFR). Composite milk samples were collected from 43 cows, nine of which had naturally infected udders and 34 of which had no infected udders. First, the milk samples were classified according to bacteriological test in positive and negative. The results showed that the negative samples to bacteriological test had: (i) significantly higher percentages of live lymphocytes; (ii) significantly lower percentages of CD11b+ leukocytes; (iii) significantly lower MEC and higher MFR values. Then, samples were classified in three groups according to somatic cell count (SCC): Group A (n = 15), samples with SCC ≤ 100,000 cells/mL, all negative to bacteriological analysis; Group B (n = 11), samples with 100,000 < SCC < 300,000 cells/m, with four samples positive to bacteriological analysis; Group C (n = 17), samples with SCC ≥ 300,000 cell/mL with five samples positive to bacteriological analysis. Multivariate discriminant analysis was used to verify which flow cytometry immunological markers and on farm recorded parameters could better discriminate among the different groups of SCCs. Linear discriminant analysis (LDA) indicated that 5 of the 10 parameters could best be used to reveal the differences between positive and negative samples among the considered groups of SCCs. Furthermore, the Canonical discriminant analysis (CDA) indicated that composite milk samples with different SCC and infection status were clearly separated from each other in a two-dimensional space. In conclusion, the study highlighted that: (1) the conventional flow cytometry analysis applied on milk samples is a useful tool to investigate immunological parameters as potential indicators of udder health; (2) the combined evaluation of live milk leukocytes and recorded farm parameters could be useful to assess udder health status in dairy cows. The results obtained from this pilot study on few data require new and larger trials to be confirmed.
Keywords: cell viability; electrical conductivity; flow cytometry; milk flow rate; milk somatic cells.