Conclusion

1. 9.2 9 8.8 8.6 8.4 Yeast/Cell 8.2 8 7.8 7.6 7.4 7.2 High PUFA Low PUFA Diet The role of polyunsaturated fatty acids in immune function and disease progression in striped bass, Morone saxatilisGonsalves, L.1,2, Baya, A.3, Jacobs, J.4, and May, E. 1,21NOAA Living Marine Resources Cooperative Science Center2University of Maryland Eastern Shore3University of Maryland College Park4NOAA Cooperative Oxford Laboratory Results Fig. 4: LtB4 production by isolated leukocytes was measured following stimulation with 10 uM calcium ionophore in 0.5% DMSO for 20 minutes. Supernatants were analyzed via enzyme immunoassay kits. Concentrations are reported as pg/ml supernatant. Striped bass fed high PUFA diets produced significantly higher amounts of LtB4 following stimulation. Abstract There is increased concern that depletions in striped bass (Morone saxatilis) forage may lead to immune suppression, providing a mechanism for increases in mortality due to mycobacteriosis. Depletions in forage may lead to alterations in dietary intake of polyunsaturated fatty acids (PUFA). The objective of this experiment is to confirm whether striped bass feeding on variable levels of PUFA will demonstrate a diminished immune response that may lead to greater progression of mycobacteriosis. Aquaculture-reared striped bass fed a diet deficient in PUFA for 12 weeks were injected with either Mycobacterium marinum or phosphate buffer solution. Spleens were collected from these fish at 0,2, 4, 8, and 17 weeks. These tissues were used to isolate leukocytes in order to determine the phagocytitic capabilities, production of leukotriene B4, and to assess progression of disease by counting M. marinum colony forming units. Striped bass fed a low PUFA diet demonstrated a diminished immune response marked by decreases in their ability to phagocytise FITC-yeast, production of the leuktoriene B4, and increases in splenic bacterial load. Fig. 1: Internal manifestations of M. marinum infection at 4 weeks included (a) enlarged and granular spleen and head kidney, along with multiple small foci on the internal organs. (b) Advanced progression of the disease results in fusion and hardening of the viscera. * Introduction Efforts are underway to determine if changes in the amount of dietary polyunsaturated fatty acids (PUFA) effect the immune response and progression of mycobacteriosis in striped bass. The current study, conducted at the NOAA Cooperative Oxford Laboratory, seeks to elucidate the underlying mechanisms behind dietary PUFA intake and immune response. Common striped bass prey species such as Atlantic menhaden, Brevoortia tyrannus, are rich in PUFA. Depletions in the dietary intake of PUFA (ex. changes in predation patterns) may lead to immune suppression by altering the innate immune response and ecosanoid production. Ecosanoids regulate the response of leukocytes during infection and PUFA act as substrates during the enzyme-mediated production of these molecules1. The current study focuses on the ecosanoid leukotriene B4 (LtB4) which is released by activated macrophages and possesses chemotactic properties. During mycobacterial infection, leukocytes aggregate around bacteria and infected host cells in order to wall off the site of infection and minimize dessimination of the pathogen while clearing the infection2. It is important that infected striped bass maintain the ability to recruit immune cells to sites of infection by the release of leukotriene B4 and other lipid metabolites. It is equally imperative for these cells to phagocytise pathogens that they come into contact with in order to clear the host’s body of disease. A B Fig. 2: Striped bass fed a diet deficient in PUFA demonstrated a decrease in phagocytosis of FITC-labeled yeast (p<0.05). Fig. 4: M. marinum colony forming units were counted 2-3 weeks after each sampling period. The number of colony forming units increased over time and reached a maximum at eight weeks post-infection. Bacterial counts in fish fed the low PUFA diet remained higher than those of the high PUFA diet at all time periods. Phagocytosis vs. Diet * • Methods • 216 striped bass (avg. length= 211 mm) were fed either a high or low PUFA diet (Ziegler Bros. Inc.) for 10 weeks (Table 1). The experiment consisted of 16 tanks arranged into 8 flow-through systems. Each dietary group was randomly assigned to 4 systems/diet. Fish from 2 systems/diet were injected with either 1 X 106 CFU Mycobacteria marinum in 200 µl phosphate buffered solution or 200 µl phosphate buffered solution alone. • Fish were sampled at0, 2, 4, 8, and 17 weeks post injection. • Laboratory analysis: • Total leukocytes were isolated from spleens and assayed for their ability to phagocytise FITC-labeled yeast (data shown) and production of LtB4 following stimulation with 10 uM calcium ionophore in 0.5% DMSO. • Bacterial cultures were created from the spleen and head kidney to obtain M. marinum cell counts • A 2X2 factorial design using Statistix 9.0 was used to analyze the data from the phagocytosis assay. Conclusion • A diet deficient in PUFA significantly decreased phagocytosis in striped bass leukocytes. Reduction of phagocytosis may lead to immune deficiency in fish feeding on low PUFA diets in the wild. • Decreased phagocytosis in fish infected with M. marinum may cause these fish to become more susceptible to secondary infections from other pathogens. • Leukotriene B4 production decreased in fish fed a low PUFA diet. LtB4 is a potent chemo attractant for leukocytes in vertebrates. Decreases in production of LtB4 and other lipid metabolites can disrupt normal immune responses to infection and possible interrupt the formation of granulomas by immune cells. • Striped bass fed a low PUFA diet maintained higher counts of M. marinum in their spleen. It is likely that the inability of these fish to halt bacterial growth is correlated with their ability to phagocytise bacterial cells and recruit leukocytes to the site of infection. • Future work will focus on examining lipid profiles of wild fish in order to determine if striped bass showing signs of advanced mycobacteriosis also possess markers of PUFA deficiency (altered lipid composition, decreased LtB4 production and phagocytosis). Fig. 3: Phagocytosis was highest in fish injected with mycobacterium 2 weeks post-infection. There was a sharp decrease in phagocytosis during week 8 with some improvement during week 17. Table 1: Experimental diets were formulated using either marine oil (high PUFA) or olive oil (low PUFA). The lipid content of each diet was analyzed using gas chromatography/mass spectrometry at the NOAA NCCOS Laboratory (Charleston, SC). * * Diet % PUFA n-3:n-6 * • Acknowledgements • Maintenance of experimental aquaculture facilities was performed by Jimmy Councilman and Matthew Rhodes at NOAA Cooperative Oxford Laboratory (Oxford, MD). • Lipid analysis of experimental diets was performed by Gloria Seaborn and Joe Wade, NOAA NCCOS (Charleston, SC) References 1. Prete, A.D., Shao, W., Mitola, S., Santoro, G., Sozzani, S., and Haribabu, B. 2007. Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function. Immunobiology. Vol. 109: 626-631. 2. Davis, J.M., Clay, H., Lewis, J.L., Ghori, N., Herbomel, P., and Ramakrishnan, L. 2002. Real-time visualization of Mycobacterium-macrophage interactions leading to initiation of granulomas formation in zebrafish embryos. Immunity. Vol. 17: 693-702