BLASTOGENESIS AND INTERLEUKIN-2 RECEPTOR EXPRESSION ASSAYS IN THE HARBOR SEAL (PHOCA VITULINA)

Two in vitro functional assays were developed to evaluate mitogen induced responses of peripheral blood mononuclear leukocytes (PBML) from free-ranging harbor seals, Phoca vitulina. Lymphocyte proliferation was measured by a standard blastogenesis assay following optimization of culture conditions including mitogen concentration, cell density, and incubation time. These optimized parameters, with the exception of incubation time, were subsequently employed to measure lymphocyte activation by analytical flow cytometry using fluorochrome-based identification of cell surface interleukin-2 receptor (IL-2r) expression. Baseline values established for free-ranging harbor seals had extensive animal variability; there was evidence that the samples were derived from a group of animals with a normal distribution. Positive correlations were observed between blastogenesis assays, and between blastogenesis and activation assays, when using pokeweed or concanavalin A as the stimulus. However, no relationship was found in the expression of the IL-2r induced by these mitogens. This result supports the contention that the two mitogens stimulate different lymphocyte subpopulations. This was observed only with the IL-2r expression assay because of its unique ability to measure the number of T lymphocytes initially activated rather than the ultimate number of progeny cells identified by blastogenesis. Both assays, used concurrently, should provide a more comprehensive representation of lymphocyte competence and serve as a measure of animal health.