Abstract

A long-term study to characterize the immune systems of free-ranging dolphins is in progress in Sarasota Bay, Florida. This immunologic study is part of a larger program directed at defining potential associations between environmental contaminants and population health. The immune system is extremely sensitive to perturbation and, as such, is an excellent measure of animal health. The immune panel data included a complete blood cell count (CBC), lymphocyte subpopulation analysis using analytical flow cytometry, clinical chemistry, total immunoglobulin, fibrinogen, serum cortisol levels, lymphocyte stimulation (blastogenesis) assays and systemic levels of the acute phase protein, interleukin-6 (IL-6).

A normal range of immunologic values for free-ranging dolphins must be established before they can be applied to the assessment of individual animal health. While data collection and analysis are ongoing, preliminary baseline values have been established for multiple peripheral blood lymphocyte subpopulations using 46 samples derived from 41 animals of known age (two to 50 years) during 1999-2001 (some dolphins were sampled in multiple years). Blood samples were obtained immediately after capture. Data from animals with elevated fibrinogen (>400mg/dL) and/or IL-6 (>5U) were excluded from the analysis. Monoclonal antibodies specific for Tursiops CD2 (T cells; MAb #UCD-F21C), CD21 (B cells; MAb # UCD-F21F;) and CD45R (MAb #UCD-F21H) were used to identify and enumerate lymphocyte subpopulations using analytical flow cytometry. CD45R, was used in two-color analysis with the pan-T lymphocyte marker CD2 to differentiate naïve from memory T lymphocytes; naïve T lymphocytes express high cell surface densities of CD45R and moderate densities of CD2 while memory T cells express low densities of CD45R and high densities of CD2.

The range of values established for absolute numbers of lymphocytes and their subpopulations were as follows (cells/ µl ± 1 SD): total lymphocytes (2,461 ± 1,023), T lymphocytes (1,645 ± 608), naïve T lymphocytes (740 ± 424), memory T lymphocytes (887 ± 287) and B lymphocytes (CD21; 643 ± 467). Analysis of lymphocyte subsets as a function of age demonstrated younger animals had higher numbers of circulating lymphocytes as compared to older animals. Linear regression analysis identified negative correlations between age and absolute numbers of T (CD2) and B (CD21) lymphocytes (R=-0.60 and -0.68 respectively). Further breakdown of T lymphocyte subpopulations revealed a more pronounced correlation between age and the absolute numbers of naïve (R= -0.60) as opposed to memory T lymphocytes (R= -0.38). Increased animal numbers, combined with a division into multiple age groups, will better define associations between age and lymphocyte numbers. Similar age-related changes in peripheral blood lymphocyte populations have been reported in a wide variety of mammals and birds. This data underscores that interpretation of all immunologic data is better served with knowledge of the animals' ages.