Incorporation of the bacterial lipopolysaccharide from Aeromonas salmonicida into liposomes and the effects on antibody responses in rainbow trout (Oncorhynchus mykiss)

Nakhla, N. Anthony (1995) Incorporation of the bacterial lipopolysaccharide from Aeromonas salmonicida into liposomes and the effects on antibody responses in rainbow trout (Oncorhynchus mykiss). Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The Gram negative bacterial fish pathogen Aeromonas salmonicida is the causative agent of furunculosis in salmonid fishes. The lipopolysaccharide (LPS) from A. salmonicida, a major factor in determining bacterial virulence, was incorporated into liposomes to determine the ability of these formulations to function as delivery vehicles and immunological adjuvants in rainbow trout (Oncorhynchus mykiss). Several aspects were considered important in examining the potential of liposomal LPS formulations as vehicles and adjuvants. -- 1H-Nuclear Magnetic Resonance (1H-NMR), mass spectroscopic (MS), and polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that the LPS from A. salmonicida was consistent with the structures previously proposed for the O- antigen and the core-oligosaccharide moieties using conventional chemical analyses. These studies also suggested that an unusual resonance at 0 ppm in the 1H-NMR spectrum of the LPS may relate to both the LPS and a component of the antifoaming agent, Antifoam A™. -- Incorporation studies demonstrated that LPS and PS from A. salmonicida can be incorporated into multi- and unilamellar vesicles composed of both positively and negatively charged lipid to form liposomes of varying incorporation ratios, sizes and lamellarities, -- Biodistribution studies indicated that the uptake of LPS, liposomes, and liposomally-incorporated LPS administered via the IV, IP and IM routes was primarily by the kidney and spleen in rainbow trout. Although oral administration of the antigens resulted in the lowest overall uptake, an enhanced uptake by the liver was observed as compared to the other tissues and organs examined. These results propose that liposomes may serve as efficient delivery vehicles for LPS to the primary hemopoietic organs of rainbow trout. -- Immunization experiments demonstrated that multi- and unilamellar vesicles composed of both positively and negatively charged lipid are capable of prolonging the humoral immune response to LPS from A. salmonicida in rainbow trout when compared to the non-incorporated LPS. -- ABx chromatography is a practical procedure for the purification of rainbow trout immunoglobulins from serum. It is likely that this protocol may be adapted for the isolation of IgM from other fish species. -- Our data suggests that liposomally-incorporated LPS can safely be administered to rainbow trout and function as delivery vehicles and immunological adjuvants. These results propose that liposomal LPS formulations are worthy of farther investigation in terms of potential and application in rainbow trout.

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