A Serologic Survey of Florida Manatees (Trichechus manatus latirostris) for West Nile Virus and Development of a Competitive Inhibition ELISA
IAAAM Archive
Martha Keller; Maureen T. Long; Ruth Francis-Floyd; Ramiro Isaza
Marine Animal Health Program, University of Florida, College of Veterinary Medicine
Gainesville, FL, USA


Marine mammals, especially pinnipeds, are at risk of exposure to arboviruses. A novel alphavirus, thought to be transmitted by the elephant seal louse (Lepidophthirus macrorhini), has recently been reported in Australia in Southern elephant seals (Mirounga leonina).3 More recently, several harbor seal deaths have been attributed to West Nile virus (WN virus).4 These animals showed clinical signs including anorexia, tremors and ataxia. West Nile virus is an arthropod-borne flavivirus recently introduced to the New World in 1999.1 It causes neurological disease in birds, horses, humans and a variety of wildlife species. Since its arrival, WN virus has spread across the country and as of December 2003 was present in 46 states.2 Because of the endangered status of the Florida manatee, it was important to learn the extent of their exposure to West Nile virus.

Currently, screening for WN virus in marine mammals is difficult. The mainstay of serologic testing in birds, horses and humans relies on the development of IgM and IgG responses in acute phase serum as tested by an IgM capture enzyme-linked immunosorbent assay (MAC-ELISA) and plaque reduction neutralization test (PRNT), respectively. The PRNT detects IgG and provides specificity for West Nile virus itself, but requires Biosafety level 3 containment. The use of live virus also limits volume of testing and is time consuming. It was our goal to develop a rapid IgG-based testing technique that could be applied for use in future zoological animal serology. This could be used clinically as a rapid screening test for a wide variety of species.

A total of 83 manatee serum samples were obtained, all but one of which were taken after West Nile's arrival into Florida in 2000. Fifteen samples came from captive manatees in the states of Florida and Ohio, all in good health. The remaining 68 samples were wild manatees caught in association with the Florida Fish and Wildlife service. All wild animals appeared clinically normal. A PRNT was performed by the Florida Department of Health on all serum samples. Duplicate samples were tested using our competitive inhibition ELISA (CI-ELISA). A 96-well Immulon 2HB plate was coated with WN antigen and allowed to incubate overnight. Plates were blocked with 5% powdered milk for one hour. During the incubation, the serum samples were diluted 1:2 and placed on a transfer plate along with an equal volume of diluted monoclonals. Three monoclonal antibodies against West Nile were chosen for this test because they act on different epitopes and thus provide superior neutralization. The serum/monoclonal combination was added to the plate where the antibodies competed for binding sites. Following incubation, conjugated antibody against WN was added and the plate read in a plate reader. Positive samples were determined by measuring the percent inhibition of the negative control. Greater than 35% inhibition was considered a positive sample.

All manatees tested negative for WN virus exposure by both the PRNT and the CI-ELISA. Due to their evolutionary relationship to manatees, we are concurrently performing a serosurvey of elephants in the state of Florida. We have identified several positive elephants via virus neutralization that we are using as positive controls. We are also using various other positive samples including alligator, tiger, goat, camel and lion. Further validation of the CI-ELISA is underway using positive horse, alligator, and bird samples. We will also validate the test for cetaceans and pinnipeds. Preliminary results indicate that this ELISA is able to screen for the presence of West Nile antibodies in numerous species.


The authors would like to acknowledge Drs. Elsa Haubold, Mark Sweat, Lucy Keith and the staff at Florida Marine Research Institute, and Robert Bonde at the United States Geologic Survey for sample collection from wild manatees. The authors would also like to acknowledge Dr. Maya Magdalena and the staff at Miami Seaquarium, as well as Dr. Mike Barrie and the staff at Columbus zoo for their assistance in providing us with samples. This work was performed under the Fish and Wildlife permit numbers MA791721-3 and MA067116-0.


1.  Anderson JF, Andreadis TG, Vossbrinck CR, Tirrell S, Wakem EM, French RA. 1999. Isolation of West Nile virus from mosquitoes, crows, and a Cooper's Hawk in Connecticut. Science. 286:2331-2333.

2.  CDC: West Nile Virus status and statistics. 2003. Retrieved December 5, 2003, from http://www.cdc.gov/ncidod/dvbid/westnile/Mapsactivity/surv&control03Maps.htm.

3.  Linn ML, Gardner J, Warrilow D. 2001. Arbovirus of Marine Mammals; a New Alphavirus Isolated from the Elephant Seal Louse, Lepidophthirus macrorhini. Journal of Virology. 4103-4109.

4.  Stremme DW, Duncan A. 2003. Clinical signs of West Nile flavivirus polioencephalomyelitis in a harbor seal (Phoca vitulina). Proceedings of the International Association for Aquatic Animal Medicine, 34th Annual Conference, Hawaii, May 9-14, 2003. Pp 34-38.

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Martha Keller

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