Abstract

The Florida manatee (Trichechus manatus latirostris) presently has a population slightly greater than 3000 with the cows producing a single calf every 2.5 to 5 years. With the present death rate of approximately 400 animals each year, the population may continue to decline until it can no longer survive in its Florida-based ecosystem. A leading factor responsible for manatee declination in Florida waters is the exposure to red tide brevetoxins, produced by the dinoflagellate organism, Karenia brevis.¹ Further research regarding the effects of brevetoxins on manatee health and survival is greatly needed. In the present study, tissue samples collected from manatees that have died in conjunction with a red tide event have been examined for the presence of K. brevis using immunohistochemistry (IHC). These samples include portions of gastrointestinal, pulmonary, and immune systems. Similar samples were evaluated from animals that died from other causes. Our initial goal was to demonstrate exposure of red tide in the Florida manatee by revealing the presence of the dinoflagellate within portions of the different organ systems of the Florida manatee by IHC. From this study we will examine specimens of all manatees that have been determined histopathologically to have died from red tide exposure. Specimens from other manatees collected from the same regions but determined to have died from other causes will be similarly analyzed. We hope to reveal possible associations that red tide exposure may have had with regard to increased mortality of the Florida manatee.

Tissue sections were made from formalin-preserved paraffin-embedded manatee tissue blocks associated with the pulmonary tract and the gastrointestinal tract as well as primary and secondary lymphoid tissues. Specimens from five animals that had been collected over a four year period were used in this study. Specifically specimens included specimens from stomach, small and large intestines, liver, lungs, and lymph nodes (mandibular, axillary, retropharyngeal, tracheobronchial, T-septal, cranial mesenteric, caudal mesenteric, superficial inguinal, and anorectal) as well as the spleen and the muscosal-associated lymphoid tissue (MALT) of the respiratory tract (including the nasopharyngeal mucosa), conjunctiva, and small intestine. Following incubation with the primary K. brevis antibody (polyclonal goat) overnight as well as no antibody as a negative control, the tissue sections were incubated with biotinylated donkey anti-sheep IgG, streptavidin ABC-AP, and chromagen AEC for 10 minutes each. The tissue slides were then examined for traces of K. brevis, which appeared red on the sections.

In animals that were determined to have died from red tide exposure, the organism itself was infrequently found in nasal passage as well as within the gut. No reaction was found in the liver or lungs. Instead, the organism was observed largely in macrophages, being most concentrated in the mandibular, axillary and retropharyngeal lymph nodes, as well as elements of the mucous-associated lymphoid tissues (MALT) (Figures 1&2).

Click on an image to see a larger view.

	Figure 1. IHC localization of K. brevis in the mandibular lymph node.
	Figure 2. IHC localization of K. brevis in a Peyer's Patch of the small intestine.

To date, we have found localization of the organism in both the respiratory and gastrointestinal systems of the same animal. We have also been able to demonstrate the presence K. brevis by IHC localization in all five individuals that had died from red tide exposure. The localization was found, in part, to be similar to that previously observed for brevetoxin.² It is hoped that an intensity distribution of the organism within these immune components can be established in order to shed light further on the impact of red tide on the Florida manatee.

Acknowledgements

This work was supported by a grant from the Florida Fish and Wildlife Commission.

References

1.  Baden DG. 1998. Brevetoxins: unique polyether dinoflagellate toxins. In Federation of American Societies for Experimental Biology Journal. 3: 1807-1817.

2.  Bossart, GD, DG Baden, RY Ewing, B Roberts, S Wright. 1998. Brevetoxicosis in Manatees (Trichechus Manatus Latirostris) from the 1996 Epizootic: Gross, Histologic, and Immunohistochemical Features. In Toxicology Pathology. 26.2: 276-282.