abstract

Bottlenosed dolphins (Tursiops truncatus) are susceptible to infection with a gram-positive, intracellular bacterium, Erysipelothrix rhusiopathiae via contaminated food and/or environmental sources. Erysipelas, the associated disease, can present as a septicemia within hours following manifestation of clinical signs.¹ Immune responses specific for a 65kD (p65) bacterial protein of E. rhusiopathiae are thought to convene immunity.³

Substantial erysipelas-related commercial losses in swine from arthritis and endocarditis⁴ prompted development of a preventative vaccine (Pfizer) containing the semi-purified p65 bacterial protein. Based upon product efficacy in swine, and a shift of disease pathogenesis from dermatologic to predominantly septicemic,² in cetaceans, a vaccination program was initiated in T. truncatus.

In vitro assays were developed to assess dolphin cellular immune responses to the E. rhusiopathiae p65 protein. Peripheral blood mononuclear leukocytes from dolphins (pre- and post-vaccination) were stimulated in vitro with the p65 bacterial antigen preparation used in the commercial Pfizer vaccine. Antigen (p65)-specific cytokine expression response patterns from 22 dolphins in three oceanariums were measured at three time points (T0 = time of 1st vaccination; T1 = three weeks post vaccination/day of booster; T2 = one week following booster vaccination). Cytokine responses measured by quantitative PCR included both T_H2 (IL-4, IL-10, and IL-13) and T_H1 (IL-12 and IFNγ) cytokines.

Pre-vaccination (T=0) T cell memory was recorded in a number of animals with IL-13 (p=0.0311) and IFNγ (p=0.0052) showing the strongest antigen-induced expression. One animal which had recently survived an acute infection, consistently demonstrated high IFNγ responses compared with all principals at all time points in this study. Vaccination induced increases in lymphocyte memory responses were recorded between T0 and T1 for IFNγ (p=0.0037) and between T0 and T2 for IFNγ (p=0.0065) and IL-13 (p=0.0465). No significant changes in expression were noted between T1 and T2.

Distinct expression patterns were recorded for each park, suggesting immune responses could be environmentally influenced. As E. rhusiopathiae is an intracellular bacterium, we had hypothesized that a T_H1 polarization would be evident, however, no obvious polarization towards either a T_H1 or T_H2 response was observed, consistent with a balanced immune response. Such responses correlate with protective immunity as no cases of erysipelas have been documented since the vaccination program was initiated in 2003. Sustained protection from erysipelas would support continued vaccination. However, the potential to develop vaccine-induced hypersensitivities should be of concern and continued monitoring of immune responses is warranted.

Four years into the erysipelas vaccination program, memory responses of dolphins on a six month booster schedule were compared to those on a one to two year booster schedule. Though preliminary in nature, data demonstrates significantly higher memory responses in animals on the six-month schedule (IL-4, p=0.0276; IL-10, p=0.0101; IFNγ, p=0.0386). While less in magnitude, memory responses were still present in animals 1-2 years after the last booster.

acknowledgements

The authors would like to thank the Office of Naval Research and Sea World Adventure Parks for supporting this work.

References

1.  Dierauf LA, Gulland FMD. 2001. CRC Handbook of Marine Mammal Medicine. 2nd Ed. Boca Raton, FL. Pp 316-318.

2.  McBain J. Sea World Adventure Parks, San Diego, CA. Personal Communication.

3.  Timoney JF, Groschup MM. 1993. Properties of a protective antigen of Erysipelothrix rhusiopathiae. Vet Micro 37:381-387.

4.  Wood RL. 1992. Erysipelas in: Leman, et al. (Eds.), Diseases of Swine, Iowa State University Press, Ames, Iowa. Pp 475-486.