Laboratory for Marine Mammal Immunology, School of Veterinary Medicine, Department of Pathology, Microbiology and Immunology, University of California
Davis, CA, USA
Abstract
Captive bottle-nosed dolphins (Tursiops truncatus) can become infected with a gram-positive, intracellular, encapsulated, rod shaped bacteria, Erysipelothrix rhusiopathiae, from their food source, and possibly from their environment. Erysipelas, the disease associated with the bacteria, can present as a septicemia in T. truncatus and develops within four hours following manifestation of clinical signs.1 The bacteria also causes disease in other animal species including turkeys, swine and humans; pathogenesis differs by species.
Immune responses established against a 65kD (p65) bacterial protein of E. rhusiopathiae are thought to impart immunity.2 Young pigs can develop chronic infections, resulting in substantial commercial losses due to arthritis and endocarditis3, prompting development of a vaccine (Pfizer) containing a semi-purified p65 bacterial protein. Based upon the efficacy of this product in swine, a vaccination program using the swine vaccine was initiated in T. truncatus.
In vitro assays were developed to assess dolphin cellular immune responses to p65. Peripheral blood mononuclear leukocytes from vaccinated dolphins were stimulated in vitro with the p65 bacterial antigen preparation used in the commercial Pfizer vaccine. Cytokine responses were measured by quantitative PCR using an intercalating fluorescent dye. Expression of IL-4, IL-10, IL-12, IL-13 and IFNγ mRNA's were quantitated at 48 hours and 72 hours post-stimulation.
Antigen (p65)-specific cytokine expression response patterns from 22 dolphins were measured at all three time points for which blood samples were available (T0 = time of 1st vaccination; T1 = 3 weeks post vaccination/day of booster; T2 = 1 week following the booster vaccination). Responses varied widely relative to the type and magnitude of pre-existing memory to p65 at the time of the 1st vaccination (T0). The percent of animals responding at T0 with transcription of IL-10, IL-4, IL-12, IL-13 and IFNγ mRNA's were 39%, 60%, 78%, 86%, and 91%, respectively. The strongest cytokine responses induced by p65 at T0 were determined to be IL-13 and IFNγ; IL-4 and IL-12 responses were comparatively moderate. Transcription levels of IFNγ and IL-13 were also the highest at T1 and T2. One animal, which had survived an acute infection, but had not been vaccinated, had, on average, the highest IFNγ responses of all animals at all 3 time points tested. Little to no antigen-specific IL-10 responses were observed.
Antigen specific cytokine responses induced by the vaccine were identified through comparison of values at T1 and T2, with those at T0. Statistically significant increases in IL-4 mRNA were recorded at both T1 and T2 (p < 0.05) while significant increases in IL-13 and IFNγ were recorded after the booster at T2 (p < 0.01 and p < 0.05, respectively).
Our hypothesis had been that there would be a vaccine-induced TH1 type polarization, due to the intracellular nature of the bacteria, that would be associated with protective immunity. The current data would support the presence of a combined TH type 1 and TH type 2 response following immunization; to date, no E. rhusiopathiae-associated deaths have occurred post-vaccination in these dolphins.
Acknowledgements
The authors would like to thank SeaWorld and the Office of Naval Research for funding this work.
References
1. Dierauf LA, Gulland MD. 2001. CRC Handbook of Marine Mammal Medicine. 2nd Ed. Boca Raton, Fl.
2. Timoney JF, Groschup MM. 1993. Properties of a protective antigen of Erysipelothrix rhusiopathiae. Vet Micro 37:381-387.
3. Wood RL. 1999. Erysipelas. In: Straw B.E., D'Allaire S., Mengeling W.L., and Taylor DJ (Eds.), Diseases of Swine. Iowa State University Press, Ames, Iowa, pp. 419-430.