Abstract

In marine mammal health science we are confronted with a number of threatened and declining populations. There is an urgent need to investigate the contribution of disease and loss of genetic diversity to these declines. Unfortunately, efforts to address the health status are often impaired by inability to access biological samples. Hawaiian Monk Seals have been on the endangered species list since 1975, yet like many species, knowledge about them is limited. A group of 10 seals were awaiting release from rehabilitation when some developed ocular lesions and became blind; the cause is still unknown. These animals were considered unfit for release and are currently housed at Sea World of San Antonio. Here they help to educate the public about their fragile status. But can we better utilize this precious resource?

With a goal of assay development focusing on genetics and health assessment, a concerted and collaborative effort was initiated to maximize the scientific information obtained utilizing samples collected from these 10 captive seals during routine health evaluation in February, 2000. Assay parameters were evaluated while simultaneously gaining knowledge about monk seal genetics, disease exposure and basic and immunologic health.

The genes of the Major Histocompatibility Complex (MHC class I and II) were chosen for monk seal genotyping. The variability and immunological importance of the genes in this complex makes these ideal candidates for identifying parental lineage, evaluating genetic diversity and predicting susceptibility to specific pathogens. Preliminary data was generated from a cDNA library which was constructed from peripheral blood mononuclear leukocyte RNA. Full-length MHC genes were sequenced and primers developed for rapid genotyping using a technique that combines polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and direct automated sequencing. These data lay the foundation to access immunologically pertinent genetic information on both archived and fresh samples.

Assay development related to health assessment was multifaceted. Efforts were made to evaluate immunologic health via leukocyte phenotyping and lymphocyte function. Leukocyte phenotyping was approached utilizing analytical flow cytometry and a variety of reagents developed for other species. By identifying cross-reactive reagents, we were able to establish absolute numbers of peripheral blood T and B lymphocytes, thus generating hematologic data that will augment traditional measurements. Evaluation of lymphocyte function was approached with a lymphocyte blastogenesis (stimulation) assay, as it can be adapted to all species. Cryopreserved monk seal mononuclear leukocytes collected during the health assessment will be employed in the titration of mitogen concentrations and incubation times in order to adapt a mitogen-based blastogenesis approach that we developed for identification of cetacean immune system dysfunction.

Information about past disease exposure was gather by employing ELISA technology. An anti-gray seal IgG monoclonal antibody, shown to bind harbor seal, elephant seal and monk seal IgG with high affinity, was used as a secondary antibody. Previous exposure to herpesvirus was evaluated by looking for cross-reacting serum antibodies to Phocine herpesvirus-1 (PHV-1). One animal was found to react in this assay and suggests exposure to a PHV-1 related organism. Nasal swabs collected in February, 2000 are being analyzed by PCR for evidence of current viral shedding.

Serum from 8 of the 10 seals during the health assessment was used in a B9 bioassay to look for evidence of interleukin-6 (IL-6) production. This cytokine increases dramatically during the initial stages of acute inflammation and appears earlier in the process than other inflammatory mediators, such as fibrinogen. No serum IL-6 was detected, nor were any of the classical mediators of inflammation. The IL-6 bioassay is being further validated for use in the Monk seals using cryo-preserved leukocytes as a source of cytokine.

Development/identification of many useful reagents, optimization and validation of a variety of assays and acquisition of baseline data will assist in management of both captive and wild populations, while reducing response time in addressing problems. In conclusion, the scientific insight gained from these blind animals should remind us of the importance of maximizing sampling opportunities for this and other endangered species.