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.