Abstract

The geographic range of the Steller sea lion (SSL), Eumetopias jubatus, extends from Japan along the Pacific Rim, through the Okhotsk and Bering seas, the Gulf of Alaska, and down the east coast of Alaska and west coasts of Canada and the United States. The population is divided at the 144E west longitude, which runs parallel to the northern border between Alaska and Canada, into two segments (eastern and western). The western segment has a proposed division between the Aleutian Islands of Alaska and the Kuril Islands of Russia, with the far western segment being commonly referred to as the "Russian population". Over the past twenty-five years, the population of SSLs in the United States, located primarily in Alaska, has declined by about 75%. The National Marine Fisheries Service listed the Steller sea lion as "threatened" under the Endangered Species Act in April of 1990. In 1997, the western SSL population was reclassified as "endangered," while the eastern segment of the population remained classified as "threatened."² Hypothesized factors contributing to the decline of the species have included environmental changes, contamination and decline of the food supply, falling reproductive rates, increased pup mortality, predation, disease, anthropogenic activities, and impairment of immune function. Although some factors may weigh heavier on the population, the root of the decline is thought to be multi-factorial. The immune system is sensitive to environmental changes; therefore monitoring perturbations in the immune system of this species may provide some insight into factors involved in the decline of the species.

Evaluation of immunoglobulin isotype levels produced during the humoral immune response provides one measure of immunocompetence. Development of the capacity to perform such an evaluation requires identification, purification and biochemical characterization of immunoglobulins in the species of interest, followed by the identification of immunological reagents that can be used for quantitative assessments of the humoral immune response. The development/identification of such reagents for use in assays of SSL isotype levels is reported herein. Purification of IgG, IgM, and IgA isotypes from pooled SSL serum was achieved by the use of affinity chromatography columns: Protein G Sepharose 4 Fast Flow column (Pierce) for IgG, Immobilized Jacalin column (Pierce) for IgA, and Amino-link affinity column coupled to anti-canine IgM (Bethyl Laboratories, Inc.) for IgM. Purified SSL immunoglobulins were used to immunize mice for the production of species-specific monoclonal antibodies and as standards in a capture ELISA (enzyme linked immunosorbent assay). There are commercially available reagents for canines. And, being that canines are the closest evolutionary relative of pinnipeds, such reagents have been shown to react with some pinniped species.^1,3 For analysis of SSL IgG and IgM levels, cross-reactive canine reagents were used as both capture and indicator antibodies in an ELISA.

Approximately 700 samples were collected from SSLs, primarily pups, on rookeries from the entire geographic range over a four-year period (1999-2002). Blood samples were taken and the serum was shipped to the University of Southern Mississippi where they were further processed by centrifugation to remove large particulate matter, diluted in saline, and frozen for future analysis. Preliminary results for IgM indicate lower levels in the Russian segment of the population (4.92 mg/ml) compared to the eastern (8.8 mg/ml) and the western (6.86 mg/ml) segments. Currently, physical and physiological data for the samples is being collected. These data will be compared with immunoglobulin levels in the respective populations to determine if there are correlations relating to immunoglobulin levels. The development of an assay to evaluate immunoglobulin levels provides a tool that can be used to establish baseline values in a population and to investigate humoral immunodeficiencies in a population or an individual through routine monitoring.

References

1.  Barlough JE, ES Berry, EA Goodwin, RF Brown, RL Delong, AW Smith. 1987. Antibodies to Marine caliciviruses in the Steller sea lion (Eumetopias jubatus shreber). J. Wild. Dis. 23(1): 34-44.

2.  Calkins DG, DC McAllister, KW Pitcher. 1999. Steller sea lion status and trend in southeast Alaska: 1979-1997. Marine Mammal Sci. 15(2):462-477.

3.  Nash DR, JP Mach. 1971. Immunoglobulin classes in aquatic mammals. J. Immunol. 107(5):1424-1430.