Tear Film Analysis and Localization of Immunoglobulin G within the Tear Producing Apparatuses in the Florida Manatee, Trichechus manatus latirostris
The field of marine mammal immunology is still relatively new with only a select number of species having been investigated and with limited antibodies available for research and diagnostics. Immune system monitoring is a valuable tool in the health assessment of wild animal populations as well as in captive managed animals. Its function as an indicator of aquatic ecosystem health has been gaining attention as research supports marine mammals as a sentinel species.4 The Florida manatee, for example, often inhabits murky, microbial filled riverine waters likely high in potential pathogens. Manatees also inhabit coastal marine ecosystems, which can pose threats to manatee health via increased boat traffic, accumulation of harmful algal blooms such as red tide, run off from the terrestrial environment, and loss of warm water sources.
For this study, mouse anti-manatee IgG was used to establish the presence of this immunoglobulin in lymphoid tissues with an emphasis on those associated with mucosal immunity. Manatees possess the thickest tear film (88 cPa) known to man thus far and as shown in other species, tears play a significant role in mucosal protection.1-3,5 Tear film as well as the tear producing apparatus of the Florida manatee was therefore investigated with regard to IgG presence and distribution.
The tear film used was collected from Florida manatees with documented elevated serum IgG. The tear film was analyzed using an adapted protocol for a competitive quantitative ELISA initially set up for measuring the amount of IgG in manatee serum. Immunohistochemistry was then used to localize IgG from the selected lymphatic tissues. It was found that the 1:40 dilution of the tear films gave the highest and most consistent concentrations of IgG for the manatees with elevated serum IgG, and a dilution of 1:100 was the best for the tear films from the manatee samples with normal serum IgG.
For the immunohistochemistry and localization of IgG in the positive control, it was found that the 1:50 and 1:100 dilutions gave effective staining results. Successful localization has been found in the lymph nodes, nasopharyngeal mucosa and eyelids with an apparent increase in activity with red tide exposure and a distinct decreased activity in cold stressed animals. Although there was a direct correlation of an increase level of IgG in the tear film when compared to the elevated serum IgG, the concentrations and the sample size were too small for a supportive conclusion at this time. With further development of this project's analysis protocols it is hoped that tear film evaluation will be a quick and non-invasive method to evaluate the mucosal immune response of the Florida manatee and provide a more conclusive reflection of health when coupled with serologic analysis.
The authors wish to thank the University of Florida Aquatic Animal Health Program, the USGS-Sirenia Project, and the Marine Mammal Pathobiology Laboratory. This work was supported by a grant from the Florida Fish and Wildlife Commission.
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