Abstract

California sea lions (Zalophus californianus, CSL) are common inhabitants of the Pacific coast waters and as such comprise a significant proportion of the sick and injured animals treated at rehabilitation centers along the west coast. In addition, CSLs are excellent sentinels for disturbances in marine and coastal ecosystems, since they feed high on the marine food web. This species is also recognized as a highly valuable partner to mankind in several open-ocean work tasks and in performance environments. Unfortunately there is a shortage of reagents and tools that can be used for health assessment in this species, particularly in comparison to those available for the Pacific harbor seal (Phoca vitulina).

The aim of this project was to develop immunologic reagents that can be employed in a wide range of diagnostic and research assays, and thereby enhance our ability to study CSL in health and disease. Since infectious disease is an important cause of morbidity and mortality in many of the pinniped species treated at rehabilitation centers, we decided to develop reagents that could be used to study health in captive and free-ranging populations. Monoclonal antibodies against CSL immunoglobulins (Ig) could be employed to develop a number of diagnostic and research assays. These assays could have broad application, ranging from population-based epidemiologic studies in large populations, to evaluating pathogen-specific immune responses in individual animals.

The antigen for producing these monoclonal antibodies was purified CSL serum immunoglobulin. The CSL immunoglobulin was used to immunize mice and therefore induce the production of anti-CSL Ig producing B cells. Several weeks after vaccination spleen cells were collected from the mice, and all healthy splenic B cells were immortalized by fusing them with a commercially available myeloma cell line. To identify and isolate the B cells that produced anti-CSL Ig antibodies a series of cell expansions and clonings were performed. The cell culture supernatants were evaluated for the presence of anti-CSL Ig antibodies by sequential analysis in the following assays: 1) ELISA using purified Ig as the antigen coated onto the plates and 2) ELISA using sera from leptospira seropositive CSLs bound to leptospira proteins. Using this approach several anti-CSL Ig antibody-producing B cell lines were purified. The specificity of these cell lines is currently being confirmed by 1) immunoprecipitation of the target antigen for determination of molecular weight and 2) western blot for identification of subunit (heavy or light chain) specificity and molecular weight of the target antigen. Once these anti-CSL Ig monoclonal antibodies have been characterized, their utility will be examined by employing them in a number of serologic assays in both captive and free-ranging populations.