Abstract
The ability of neutrophils and macrophages to phagocytize potentially infectious microorganisms and destroy them via respiratory burst represents a critical component of the innate immune system, an organism's first line of defense against infection. Numerous facets of immune function respond to changes in an organism's environment and neurophysiological state. Thus, immune function assessment may serve as a sensitive and highly informative addition to standard health assessment tools to evaluate the impact of environmental and neurophysiological perturbations on animal health.
This study refined and validated flow cytometric assays developed in terrestrial mammals1-3 to simultaneously measure phagocytic and respiratory burst activity by neutrophils and monocytes in small volumes of whole blood from beluga whales (Delphinapterus leucas). Whole blood was simultaneously incubated with fluorescent propidium iodide-labeled Staphylococcus aureus and 2',7'-dicholorofluorescein diacetate, a cell-permeable non-fluorescent compound oxidized intracellularly to fluorescent 2',7'-dicholorofluorescein by respiratory burst products. A two-color flow cytometric method was used to detect fluorescent indicators of phagocytosis and/or respiratory burst in granulocyte and monocyte cell populations.
This methodology was used to assess the impact on the innate immune function of a temporary translocation of seven belugas from an exclusively indoor exhibit at Shedd Aquarium (Chicago, IL) to an exclusively outdoor exhibit housing three belugas at Mystic Aquarium (Mystic, CT). We hypothesized that phagocytic and respiratory burst activity would be a sensitive indicator of the environmental and social changes associated with the translocation. For the three Mystic Aquarium belugas and four of the Shedd belugas, the phagocytosis/respiratory burst assay was performed on blood samples obtained prior to transport (baseline), immediately post-arrival (arrival), and 2-6 months post-introduction (acclimation). Epinephrine, norepinephrine, and dopamine levels were determined via high performance liquid chromatography (HPLC) on the same blood samples. Overall, the four Shedd belugas exhibited an increase in phagocytic and oxidative burst activity between baseline and arrival and again between arrival and acclimation. Catecholamine levels in the Shedd belugas increased between baseline and arrival and then subsequently decreased between arrival and acclimation. The Mystic Aquarium belugas exhibited the same pattern in catecholamine response but not in immune response as the Shedd belugas. The Mystic Aquarium belugas displayed a decrease in phagocytic activity between baseline and arrival and then a subsequent rebound between arrival and acclimation. No significant change was detected in oxidative burst activity.
We hypothesize that the increase in immune activity in the Shedd belugas may have resulted from exposure to increased levels of microorganisms associated with moving from an indoor exhibit to an outdoor exhibit. Conversely, the patterns of immune response shown by the Mystic Aquarium belugas may reflect the stress associated with the introduction of additional belugas as well as the heightened level of exhibit activity surrounding their arrival. In addition to successfully adapting and validating this methodology for belugas, the phagocytosis/respiratory burst assay appears to be highly sensitive to environmental and neurophysiological changes and may serve as a valuable tool in health assessment programs.
References
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2. Perticarari S, Presani G, Banfi E 1994. A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J Imunol Methods 170:117-124.
3. Smits E, Burvenich R, Heyneman R 1997. Simultaneous flow cytometric measurement of phagocytic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood. Vet Immunol Immunopath 56:259-269.