Abstract

The Florida Department of Environmental Protection maintains a program to recover marine mammal carcasses, especially manatees, statewide. To accomplish this task the program includes 14 staff members located within 5 field locations around the state. Whenever practical, carcasses are transported to a central necropsy facility (Marine Mammal Pathobiology Laboratory) located in St. Petersburg. Data collected from this program is especially useful for management of manatees.

The State began its commitment to carcass recovery of marine mammals in 1985 when it accepted the responsibility for manatee carcass recovery from the United States Fish and Wildlife Service. Over the ensuing 11 years, the State has expanded the program to include 5 field stations and sufficient staff as well as a necropsy facility dedicated to manatees and other marine mammals. The MMPL is staffed with a pathologist and anatomist/functional morphologist along with support staff. Extensive data is collected and maintained in a number of computer based systems which coupled with the data collected by the USFWS represents over 22 years of manatee mortality information. Data collected on other marine mammal mortalities are archived and sent to the Southeastern Marine Mammal Stranding Network headquartered at Sea World in Orlando.

At the MMPL there are several long term collections of various marine mammal tissues. While the majority of the material is from manatees, there are samples collected from other species as well. As a matter of routine, we collect and freeze liver, kidney, and blubber from every mortality of appropriate body condition. These tissues are frozen in vacuum sealed heavy duty plastic bags and inventoried on computer. We also have a wet fixed tissues collection, trimmed tissues in cassettes (blocks) and slides (stained H&E). We maintain an osteo logical collection for voucher and research. During the last 8 years, we have established a microbiological database of clinical isolates from manatees and we have established a serum bank of sera collected from wild manatees from around the state. Over the last two years, we have established an epithelial and fibroblast cell lines from manatees and we are developing virus isolation and characterization capabilities.

Because we have developed the necropsy program over the last 11 years, we were in a better position to respond to a recent manatee epizootic this past winter. Indeed, this single event represented a challenge to the program which was already enduring the greatest level of manatee mortality experienced during any year.

From early March to mid-May 1996 at least 149 manatees died along approximately 80 miles of the southwest coast of Florida. At approximately the same time, there was a significant algal bloom largely composed of Gymnodinium brevis dinoflagellates in the same geographic area as the manatee epizootic. These algae produce a biotoxin known as breve toxin which is neurotoxic and can be fatal to fish, mammals and birds.

The appearance of the carcasses at necropsy suggested the animals were suffering a rapid death and there was a substantial consistency in the appearance of lung and upper respiratory lesions among the carcasses. A microscopic examination of tissues supported rapid death but overall there was a limited inflammatory response. Moderate brain lesions were found along with significant inflammation in the nasal mucosa. These findings were not compatible with the findings described from breve toxin intoxication of manatees in a previous event. Furthermore, during the previous manatee epizootic in the same area in 1982, many of the animals had ingested sea squirts (ascidians) which were believed to be the source of concentrated breve toxin. However during the current epizootic, very few manatees had ingested sea squirts. Comparable to the previous manatee epizootic, a red tide bloom of unprecedented proportions was occurring in the area. Algal cell counts in the 20s of millions of cells per liter were occurring in inshore waters where red tide does not usually appear. Other environmental conditions such as increased salinity and an elevated pH in the inshore waters were favorable to algal growth. After a relatively cold winter warm weather had begun, which causes manatees to leave their warm water refugia and pass directly through the area of concentrated breve toxin. As a result of conditions which appeared juxtaposed, we felt it was essential to establish the identity of the agent/s involved by pursuing those that appeared to be most likely involved. Appropriate tissues were sent to many collaborators to determine if there was evidence of an infectious agent, biotoxin, or toxicant involved in these mortalities.

We immediately launched into a simultaneous investigation for various possible causes of the mortalities. Within a relatively short time period and with extensive collaboration, we had determined that an infectious agent was not a likely cause of this event. This conclusion was supported by the progression of mortalities throughout the event. That is, carcasses appeared throughout the entire geographic range of the involved area throughout the entire time of the event. This strongly suggested that the mortalities did not spread as they might if an infectious agent was involved. What's more, the mortalities declined at the same approximate time as the red tide in the area subsided. These combined factors also ruled out toxicants as a cause of this event. Dr. Dan Baden and his staff at the University of Miami, examined stomach contents and various tissues for the presence of biotoxins. Because there are many potential biotoxins in Florida waters (approximately 30), analyses were conducted to assure that all potential biotoxins were tested. Dr. Baden isolated breve toxin in stomach contents that was at a concentration 10 fold higher than controls. He also found breve toxin in various tissues (liver, kidney, lung) and performed mouse bioassays utilizing extracts from affected manatees further confirming brevetoxin toxicity. Drs. Baden and Greg Bossart developed an immunoperoxidase assay that visualizes brevetoxin in fixed tissues. With this assay, breve toxin was revealed in brain tissue as well as all the other tissues. During the last month of the event (April) four live manatees were recovered that had clinical signs compatible with those described from breve toxin intoxication. All four animals survived and have been released back into the wild.

Through extensive analyses and collaboration along with a process of elimination it appears that breve toxicosis is a primary component of the manatee mortality event. There remain many questions. We know that manatees are exposed to brevetoxin repeatedly throughout their lives in southwest Florida. The significance of brevetoxin in tissues needs to be evaluated against tissues from previous mortalities in the same area at an earlier time. Investigation of the possible inhalation route of exposure to brevetoxin is necessary as this may represent a whole new avenue of exposure.

Press coverage of this event has brought an immense public attention to manatees. At the same time, we have gained a great deal of information about manatees in a shorter period of time largely as a result of the tremendous response of the scientific community. The level of cooperation and eagerness to assist in the investigation on the part of government and private agencies as well as the concern expressed by the public was responsible for the high level of response to this event.