The distribution of lymphocyte subpopulations from dry secretions, colostrum and blood from 10 healthy adult Hostein-Fresian cows was studied using the TH21A and B26A mouse monoclonal antibodies (MAb) to adult bovine B and T lymphocytes, respectively. The mammary gland lymphocytes (MGL) were isolated from composite sample of all four quarters by density centrifugation over discontinuous gradient of ficoll-diatrizoate. The peripheral blood lymphocytes (PBL) were purified using the ficoll-thrombin method. Isolated PBL and MGL were analyzed using the two fluorochromes method (TFM) and laser flow cytometry (LFC). The mean viability of isolated PBL and MGL from dry secretions and colostrum after the TFM and LFC were 92.4%±3.2%, 91.4%±6.0% and 87.1% ± 6.1%, respectively. There was a good correlation between the two MAbs and the percentage of surface immunoglobulin (SIg) positive cells in the peripheral blood using the TFM. The PBL yielded a mean percentage of 21.2% B cells, 66.4% T cells and 9.4% "Null cells" (TH21A+; SIg-). The TFM on MGL from dry secretions and colostrum indicated two distinct patterns (group I and II) of SIg and reactivity to MAb markers (p<0.001). The MGL data included in group I and group II were gathered from both colostral and dry secretions. In comparison to the distribution of lymphocyte subsets within peripheral blood the mean percentages of B cells, T cells and "Null cells" in the mammary gland were respectively, 2.8%, 88.1% and 5.4% for group I and 3.5%, 89.0% and 15.1% for group II. In the mammary secretions, the use of SIg alone was not considered to be a good marker for B cells; in four animals a mean percentage of 15.6% ( 13.9 89.0 × 100) of the mammary gland T lymphocytes were also SIg+. Of the TH21A+ MGL, only 18.8% were SIg+ in group II compared with 34.1% for MGL from group I and 69.3% for the PBL. Marked differences in cell size distribution and cell surface antigen density were found when PBL and MGL from dry secretions were compared by LFC using the B26A MAb. The results of this study demonstrate a difference in the percentages of peripheral blood and mammary gland B and T lymphocytes and confirm previous findings in which the T lymphocytes were found to represent the major subpopulation of lymphocytes in bovine mammary secretions. This may represent an essential event in the adoptive transfer of cellular immunity through the colostrum in cattle.
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