Abstract

Proper function of the immune system is necessary to maintain health. Suppression or augmentation of that function can result in disease. Dive-related pathologies involving alterations of the immune system, observed in humans and other terrestrial animals, are uncommon in marine mammals that possess specific physiological and behavioral adaptations to diving. However, the response of marine mammal immune cells to challenges associated with diving, such as changes in pressure, remain unknown. In recent decades, pathologies resembling dive-related injuries have been reported in stranded marine mammals suspected to have been compromised by anthropogenic stressors. A compromised immune system may interrupt dive adaptation, leading to the development of injury. This study compares the function of beluga whale neutrophils at baseline versus stressor conditions which include a 30-minute out of water examination (OWE) and/or localized mild chronic inflammation of the fluke vessels (clinically determined). Blood samples were obtained during routine collection for monthly health assessments and under behavioral participation in order to avoid sampling stress. A stainless steel pressure cell was used to pressurize samples to either 2000 psi (1360 m) or 1000 psi (680 m) with 2-minute compression and decompression periods. Dive profiles consisted of a single 30-minute dive (profile 1), a single 5-minute dive (profile 2), and two repeated 5-minute dives with a 1-minute resting period (profile 3). Flow cytometry was used to measure phagocytosis of propidium iodide-labeled Staphylococcus aureus and neutrophil activation via binding of anti-canine CD11b. Measurements were obtained immediately after decompression and again after a 20-minute recovery period. For baseline and stressor conditions, changes in activity between non-pressurized and pressurized samples were represented as a percentage of non-pressurized values, and data were analyzed using a nonparametric Wilcoxon Sign Rank Test or Kruskal Wallis ANOVA (α = 0.05). Comparisons were made for exposure to 2000 psi between baseline and OWE and between baseline and inflammatory conditions. In addition, comparisons between whales with mild chronic inflammation versus without (normal) were made for exposure to 1000 psi. While no statistically significant differences were detected, a larger percent increase in phagocytosis was observed during the OWE as compared to baseline conditions. Recovery periods following dive profiles 1 and 2 to 2000 psi also showed a greater percent increase in phagocytosis associated with mild chronic inflammation compared to normal condition. However, when exposed to 1000 psi, whales with mild chronic inflammation generally showed a decrease in phagocytosis, whereas normal animals showed minimal change or an increase in phagocytosis. A different pattern of change in CD11b expression is also noted following dive profile 1 for all stressor conditions, and for dive profile 2 for the OWE. Whales with mild chronic inflammation appear to exhibit a greater increase in CD11b expression following a single 5-minute dive (profile 2) than during baseline conditions. Results indicate that overall health can impact an animal's ability to deal with dive-related challenges. Changes in immune function may alter the ability of compromised animals to fight infection, heal wounds, or resist dive-related pathologies.

Acknowledgements

The authors would like to acknowledge Dr. Cara Field for use of the pressure cell, and the research, veterinary and beluga whale husbandry staff at the Mystic Aquarium.