Abstract

The immune system, which protects the body from potential infections, is critical for health maintenance and survival. Polymorphonuclear neutrophils (PMN) are an important part of the innate immune system of mammals and play a key role in defense against bacterial infections. Two important functions of PMNs are phagocytosis and respiratory burst. Phagocytosis is the ingestion and digestion of microbes, other cells and foreign particles. Respiratory burst is the metabolic activity that occurs during oxidative killing following phagocytosis.

Classical methods and prior flow cytometry methods for measuring phagocytosis and respiratory burst required two separate assays to be performed, relatively large amounts of blood, and time-consuming cell isolation procedures. The overall objectives of this study were to: 1) develop a functional assay which simultaneously measures phagocytosis and respiratory burst in bottlenose dolphins utilizing minimal amounts of whole blood and to 2) acquire baseline values for bottlenose dolphins (Tursiops truncatus).

Blood was collected from 16 bottlenose dolphins, 12 males (6-34 years) and 4 females (11-30 years) maintained by the U.S. Navy Marine Mammal Program. Two fluorophores were used to simultaneously measure phagocytosis and respiratory burst. Propidium iodide-labeled Staphylococcus aureus (ATCC 14923) (wavelength of 585nm) was used to measure phagocytized bacteria, and 2',7'-dichlorofluorescein diacetate (DCFDA), a non-fluorescent cell permeable dye which upon oxidation becomes fluorescent (wavelength of 525nm) was used to measure respiratory burst activity in both neutrophils and monocytes. Percent phagocytosis and mean fluorescence intensity (MFI) for respiratory burst activity were measured in whole bottlenose dolphin blood after optimization of the assay including bacteria-to-cell ratio, concentration of DCFDA, and length of time exposure to the bacteria. Utilizing a bacteria-to-cell ratio of 25:1, a DCFDA concentration of 50 µm, and 20 minutes exposure to bacteria, 32.2% ± 11.1% of neutrophils and 21.4% ±10.6% monocytes from fresh dolphin blood were shown to contain propidium iodide-labeled Staphylococcus aureus. Respiratory burst activity after 20 minutes of incubation showed a mean fluorescence intensity of 58.5 ± 34.7 in neutrophils and 42.2 ± 26.6 from monocytes.

This assay provides for both independent and comparative values for phagocytosis and respiratory burst from a small volume of whole blood in bottlenose dolphins. The use of whole blood is advantageous over isolated cells since it uses the animal's own serum to opsonize the bacteria without adding steps and reduces changes in function caused by purification procedures. This assay will be extremely useful in evaluating these immune functions in marine mammals in which using minimal amounts of blood is necessary such as in live capture health assessments, stranded animals, and those kept under human care. Additional females, different age grouped animals as well as ill and pregnant animals are being evaluated as samples become opportunistically available.