Proposal for Phase II NGI
Immuno-physiological Assessment of Fish from the Gulf of Mexico:
Effects of the Deepwater Horizon Oil Spill
Principal Investigator: Dr. Lora Petrie-Hanson (Dept. Basic Sciences, CVM)
Co-Investigators: Dr. Peter Allen (Dept. of Wildlife, Fisheries, and Aquaculture), Dr.
Jan Chambers (Dept. Basic Sciences, CVM), Dr. Stephen Pruett (Dept. Basic Sciences,
Contact information:
Lora Petrie-Hanson, [email protected], 325-1291
Stephen Pruett, [email protected], 662-325-1130
Background: Fish population declines began in the year following the Exxon Valdez Oil Spill
(EVOS) (1) and increased occurrence of fish diseases occurred in the years following the EVOS
(2). Carls demonstrated that laboratory exposures of oil resulted in decreased inflammatory
cells, leading to immunosuppression (2). Following the Deepwater Horizon oil spill, we can
examine the functions of fish immune cells and define how crude oil and contaminant aromatic
hydrocarbons affect the immune system, resulting in increased disease susceptibility in fish.
Objectives and Methods: Alligator gar (Atractosteus spatula) from affected sites along the Gulf
of Mexico have been sampled via near-shore and estuarine sampling. Oil-exposed red snapper
(Lutjanus campechanus) have been sampled via offshore research vessels in cooperation with
the National Oceanic and Atmospheric Administration (NOAA). Future sample collection trips
have been scheduled, but examining these fish without established baseline data could result in
invalid interpretations of data. To validate collected samples with baseline data, we propose
additional controlled studies using alligator gar as an in vivo model for controlled parametric
studies of the effects of crude oil exposure on immune system functions and the metabolomic
response of fish. We will investigate the correlations between immunological and physiological
responses to crude oil exposure. This will allow us to evaluate its’ potential to pre-dispose fish to
infectious disease.
Dr. Peter Allen has obtained an agreement from BP to provide samples of 3 different types of
crude oil. Oil will be added to tanks with fish at several concentrations to establish doseresponse effects. Dr. Allen will cannulate alligator gar through the caudal vasculature enabling
us to obtain blood samples at regular time intervals for immunological and metabolomic
analyses (3)(4). These samples will be analyzed by flow cytometry, and lymphoid tissues will be
stained for cytochemical immune profiles (5)(6)(7) to monitor the time course of immunological
and physiological changes. Liver samples will be measured for enzyme activity (liver
ethoxyresorufin O-deethylase [EROD]). Terminal blood and tissue samples will be measured for
metabolomic profile and plasma ion concentrations (vapor-pressure osmometer) and lymphoid
tissues will be stained for cytochemical immune profiles (5)(6).
The flow cytometer purchased with funding from an NSF RAPID grant will be used for functional
assays to determine immune system status. These include phagocytosis and production of
reactive oxygen species. Lymphocyte activation in response to concanavalin A will be assessed
by increased size of cells and increased cells entering the cell cycle (determined by propidium
iodide staining of fixed and permeabilized cells). Blood smears will be obtained for differential
counts. Cell killing assays will evaluate the ability of isolated cells to kill heterologous non-self
cells for target cells. The protocols of Bottley (8) and Hogan (9) will be used as guidelines.
Killing of target cells will be assessed using flow cytometry based on pre-staining of target cells
with cell tracker orange and identification of killed cells with 7 amino-actinomycin D (10).
Red snapper exposed to offshore oil will be collected in cooperation with NOAA. Peripheral
blood and lymphoid tissues will be collected. Blood will be preserved and later analyzed by flow
cytometry. Lymphoid tissues will be stained for cytochemical and histopathological analyses.
Control red snapper will be sampled in cooperation with the Gulf Coast Research Laboratory.
Expected Results and Significance: We expect fish from exposed areas to exhibit
immunosuppression compared to fish from unexposed or minimally exposed areas. However, if
we find no differences, this will indicate that the effects of this warm water spill are different from
those reported after the Exxon Valdez spill, which caused disease outbreaks in fish. By
measuring polycyclic aromatic hydrocarbons (PAH), which are found in petroleum and persist
and bioaccumulate in fish, we will be able to estimate exposure history. A correlation between
immunological functions and PAH concentration will suggest the oil spill as the cause of the
immunological dysfunction.
References Cited
Thorne, R., and G. Thomas. 2008. Herring and the 'Exxon Valdez'oil spill: an
investigation into historical data conflicts. J Marine Sci 65:44-50.
Carls, M., G. Marty, T. Meyers, R. Thomas, and S. Rice. 1998. Expression of viral
hemorrhagic septicemia virus in pre-spawning Pacific herring (Clupea pallasi)exposed to
weathered crude oil. Can J Fish Aquat Sci 55:2300-2309.
Allen, P.J., C. C. Barth, S. J. Peake, M. V. Abrahams and W. G. Anderson. (2009a).
“Cohesive social behaviour shortens the stress response: the effects of conspecifics on
the stress response in lake sturgeon Acipenser fulvescens.” J Fish Biol 74: 90-104.
Allen, P. J., J. J, Cech, Jr., and D. Kultz. (2009b). “Mechanisms of seawater acclimation
in a primitive anadromous fish, the green sturgeon”. J Comp Phys B 179 (7): 903-920.
Petrie-Hanson, L. and A. J. Ainsworth (2000). "Differential cytochemical staining
characteristics of channel catfish leukocytes identify cell populations in lymphoid
organs." Veterinary Immunology and Immunopathology 73(2): 129-144.
Petrie-Hanson, L. and A. E. Peterman (2005). "American paddlefish leukocytes
demonstrate mammalian-like cytochemical staining characteristics in lymphoid tissues."
Journal of Fish Biology 66: 1101-1115.
Petrie-Hanson, L., C. Hohn and L. Hanson (2009). "Characterization of rag1 mutant
zebrafish leukocytes." BMC Immunology 10(8)
Bottley, G., G. Cook, and G. Blair. 2007. A Flow Cytometric Assay for Analysis of
Natural-Killer Cell-Mediated Cytolysis of Adenovirus-Transformed Cells. In Adenovirus
Methods and Protocols: Ad Proteins, RNA, Lifecycle, Host Interactions, and
Phylogenetics, Second ed. W. A. T. Wold, ed. Humana Press, Inc., Totowa, NJ. 221230.
Hogan, R., T. Stuge, L. Clem, N. Miller, and V. Chinchar. 1996. Anti-viral cytotoxic cells
in the chanel catfish (lctalurus punctatus). Developmental and Comparative Immunology
Kim, G. G., V. S. Donnenberg, A. D. Donnenberg, W. Gooding, and T. L. Whiteside.
2007. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously
immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay. J Immunol
Methods 325:51-66.
The NSF RAPID grant was used to purchase the major instrumentation needed for this project,
a FACSAria III flow cytometer and cell sorter. Matching funds were required and these were
provided by the Department of Basic Sciences, CVM Office of Research and Graduate Studies,
MAFES, the Office of the Vice President for the Division of Agriculture Forestry and Veterinary
Medicine, and NGI. The funds requested include the work outlined in the NSF RAPID grant
Supplies for immunological analyses of gar and snapper
Gar: fish holding and surgeries, oil exposures, metabolomic analyses
Travel for alligator gar pick-up and analyses
Toxicology, tissue samples and controlled exposure analyses
Travel and related expenses for red snapper sampling
Travel to present results at a national meeting
Publication costs