Abstract

The proposed project is designed to identify potential impact(s) of the recent oil spill on dolphins in the Gulf of Mexico by application of immunologic probes on blood samples collected from: 1) captive animals in facilities bordering the Gulf prior to and following the spill, 2) free-ranging dolphins captured as part of routine monitoring programs prior to and following the spill and 3) free-ranging dolphins that strand in the Gulf region following the spill. Specialized blood collection tubes (PAXgene vacutainers) will be employed that allow blood to be collected and stored in standard freezers for extended periods of time prior to transport to and/or processing at the University of California's Marine Mammal Immunology Laboratory for recovery of mRNA.

Background

A grant from the California Department of Fish and Game's Office of Oil Spill Prevention and Response Program provided initial funding for developing an animal model to study the long-term effects of chronic exposure to low levels of Bunker C fuel oil using ranch-raised mink. This study established a framework for evaluating the effects of environmental petroleum oil exposure on the immune system of captive and free-ranging marine mammals. Systemic oil-induced perturbations in erythrocyte homeostasis, hepatic metabolism, adrenal physiology and the immune system were identified. Immunologic changes included increased numbers of circulating neutrophils, lymphocytes and monocytes, elevation of cell-surface levels of functionally significant leukocyte differentiation antigens and heightened mitogen-induced mononuclear cell proliferative responses. Taken together, the data supported the conclusion that chronic exposure of mink to low levels of petroleum impacted both the innate and adaptive immune systems resulting in a pro-inflammatory or adjuvant-like effect. This data was consistent with cellular activation described in laboratory animal models of polycyclic aromatic hydrocarbon exposure.

A number of species-specific probes for quantifying perturbations in gene transcription in peripheral blood leukocytes from mink, sea otters, manatees, porpoises and dolphins have been developed. A total of 23 genes, representing 10 different physiological systems, were used to evaluate the above-described mink experiments. A variety of petroleum-induced elevations in gene transcripts encoding cytochrome P4501A1 (p450) and a variety of immunologically relevant genes in blood leukocytes were identified. Taken together, the results were consistent with the presence of endocrine-disrupting and immune system-modifying compounds in fuel oil. This approach to identify effects of petroleum contamination on marine mammals is now being successfully applied to captive and free-ranging dolphins and sea otters.

Approach

Our current approach to address the long-term impacts of the Gulf of Mexico oil spill of 2010 is to use quantitative reverse transcriptase polymerase chain reaction (Q-PCR) to measure well-defined inducible genes in peripheral blood leukocytes that will permit association with specific insults, specifically oil. Application of this molecular approach will facilitate identifying petroleum-induced transcriptional signatures in peripheral blood leukocytes of bottlenose dolphins that will assist in establishing both the acute and long-term impact of oil contamination in captive, stranded and free-ranging dolphins. Accurate documentation of baseline values prior to oil-exposure will be critical in establishing the long-term impacts of the contamination on dolphin health; an extensive collection of cryopreserved blood leukocytes previously collected from captive dolphins living in Gulf sea pens, and free-ranging dolphins in the Sarasota Bay area, are currently available to facilitate establishment of pre-spill profiles. A similar approach is currently being successfully used to document long-term effects of the Valdez spill on free-ranging sea otters.

PAXgene vacutainer tubes have been distributed to many facilities in the Gulf region for collection of blood from captive resident dolphins shortly after the oil spill, with the realization that some may already have been exposed to some level via natural sea pen-housing, aerosols and pumping water from the gulf into animal; future collections from these dolphin populations are planned. In addition, long-term collections from the Sarasota Bay free-ranging dolphin population will be pursued, as will collection of blood samples from dolphins stranding on Gulf shores. From a laboratory analysis perspective, 11 Tursiops truncatus-specific gene probes have been selected to analyze immunologically relevant genes; additional probes are being developed for measurement of aryl hydrocarbon receptor (AHR), p450 and a tumor promoting gene; these three later genes have been demonstrated to be induced by oil exposure in mink and sea otters.

Acknowledgements

Initial funding for this project has been generously provided by a grant from Siegfried & Roy's Secret Garden and Dolphin Habitat, the Mirage, Las Vegas, NV, USA.

References

1.  Schwartz JA, Aldridge BM, Stott JL, Mohr FC. Immunophenotypic and functional effects of bunker C fuel oil on the immune system of American mink (Mustela vison). Veterinary Immunology and Immunopathology 2004; 101(3–4):179–190.

2.  Schwartz JA, Aldridge BM, Lasley BL, Snyder PW, Stott JL, Mohr FC. Chronic fuel oil toxicity in American mink (Mustela vison): systemic and hematological effects of ingestion of a low-concentration of bunker C fuel oil. Toxicology and Applied Pharmacology 2004; 200(2):146–158.

3.  Bowen L, Riva F, Mohr C, Aldridge B, Schwartz J, Miles, AK, Stott JL. Differential gene expression induced by exposure of captive mink to fuel oil: a model for the sea otter. EcoHealth 2007; 4:298–309.