A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows

Abstract

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription–polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.

Milk Immune Cell Composition in Dromedary Camels With Subclinical Mastitis

“…The present study divided camel milk leukocytes based on their differential expression of the cell surface molecules CD172a and CD14 into a dominant CD172a + /CD14 low /SSC high granulocyte population followed by a smaller fraction of CD172a + /CD14l high /SSC high macrophages and a minor CD172a − /CD14 − /SSC low lymphocytes population. The higher proportion of granulocytes in camel milk, compared to other species like bovine ( 17 , 28 ), could be a result of their dominance in peripheral blood ( 49 ). The results of the present study, however, largely correspond to data reported for goats regarding the differential composition of milk cells ( 19 , 20 ).…”

“…The milk somatic cell count and the CMT are widely accepted tools for the evaluation of the mammary gland health status ( 17 , 28 ). In camels, elevated SCC values were observed in milk samples from infected mammary glands with values ranging from 1 × 10 5 to 10 × 10 6 cells/ml milk ( 8 , 25 ).…”

“…Macrophages and neutrophils are key effector innate immune cells of the mammary gland with an essential role during the early immune response to mastitis pathogens ( 17 , 40 ). In the present work, the observed shape-change of granulocytes and macrophages from SCM milk with higher forward scatter values, which correlates with the cell size, and lower side scatter values of granulocytes, which is indicative of cell degranulation ( 66 , 67 ), indicate the activation status of these phagocytes in the infected mammary gland.…”

“…The contribution of the different cell types to the cellular compartment of milk differs according to species. While macrophages are the predominant cells in healthy human and bovine milk ( 17 , 18 ), the majority of cells in sow and goat milk have been identified as milk granulocytes ( 19 – 21 ). The assessment of the cellular composition of milk, including the absolute counting of somatic cells and the differential proportions of immune cell subpopulations is widely accepted as a valuable tool for the evaluation of the health status of the mammary gland ( 22 – 24 ).…”

“…Milk phagocytes, including macrophages and neutrophils, are the primary effector cells of the mammary gland innate immune system with a key role during mammary gland infections ( 17 ). They contribute to the early elimination of bacterial pathogens by several antimicrobial functions, including phagocytosis, production of reactive oxygen and nitrogen species, and formation of extracellular traps ( 40 ).…”