Abstract
Serologic assays available for detection of terrestrial-origin Brucella have been shown to be unreliable when employed against marine-origin Brucella, as there has been a lack of agreement between serologic results derived from these tests and microbial isolations in both cetaceans and pinnipeds.2,3,4
Two recently developed enzyme-linked immunosorbant assays (ELISA) were specifically designed to screen marine mammal sera to detect antibodies for marine Brucella. These assays were used to determine the seroprevalence of Brucella in 48 wild bottlenose dolphins and compared with terrestrial Brucella tests with the intent of determining the efficacy and applicability of these diagnostic methods for marine Brucella.
The marine-origin assays utilized include an indirect ELISA, developed using a whole-cell Brucella antigen from an aborted bottlenose dolphin fetus; and a competitive ELISA developed using a whole-cell Brucella antigen isolated from a harbor seal.1,3,5 The commercial terrestrial-origin Brucella tests used in this study are specific for Brucella abortus and include a Rivanol agglutination test, card agglutination test, and a buffered acidified plate antigen test (BAPA).6
The seroprevalence utilizing the terrestrial-origin Rivanol, Card, and BAPA tests was found to be 25%, 39.5%, and 37.5% respectively. Seroprevalence was reduced to 21% when using the criteria that a true positive must be positive on all 3 tests. The seroprevalence using the marine-origin indirect ELISA was 50%. The competitive ELISA revealed a similar seroprevalence at 60%. All samples that tested positive on the indirect ELISA were positive on the competitive ELISA, with the exception of one sample.
Overall, the marine-origin ELISAs reported significantly higher seroprevalence than the terrestrial-origin assays. The lower seroprevalence with the terrestrial-origin tests suggests that they underestimate the prevalence of Brucella in marine mammals. This may be due to the fact that these tests were derived from terrestrial Brucella antigen, and/or to the subjective nature by which the results are determined. The higher seroprevalence with the marine-origin ELISAs may be an overestimation due to false positives. With the antigen on both tests being a whole-cell, non-specific binding and cross reactivity with antibodies against similar gram-negative organisms is possible.
However, because both marine ELISAs utilize a marine-specific antigen, and report similar test results, the marine-origin ELISA methods appear to be more sensitive for antibody detection in marine mammals than the terrestrial-origin assays. In addition, previous studies using bottlenose dolphin serum also reported a higher seroprevalence with the marine-origin indirect ELISA when compared with classical terrestrial-origin assays.3 In 2 cases of bottlenose dolphins with active Brucella infections confirmed by culture, the marine-origin indirect ELISA correctly identified these as positives, whereas, the terrestrial tests reported false negative results.3
In conclusion, the marine-origin ELISAs are effective screening tools for Brucella exposure or infection in marine mammals and improve diagnostic success when used in combination with other test modalities.
Further application of these serologic test methods is currently being done using sera from multiple marine mammal species and at-risk human populations to determine their efficacy and to better document the prevalence of marine-origin brucellosis.
Acknowledgements
The Brucella Project is funded by NOAA Oceans and Human Health Initiative Grant #NA04OAR4600209 and received previous support from NOAA Fisheries Prescott Grant Award # NA03NMF4390408. We acknowledge Drs. Patricia Fair, Gregory Bossart and Randall Wells for providing the wild bottlenose dolphin serum samples. The bottlenose dolphin blood samples were collected under the National Marine Fisheries Service Scientific Research Permit Nos. 522-1569 and 522-1785 issued to Dr. Randall Wells of the Sarasota Dolphin Research Program in Sarasota Bay, Florida, USA; and Nos. 998-1678 issued to Dr. Greg Bossart as part of the Health and Risk Assessment of Bottlenose Dolphin Project (HERA) conducted in the Indian River Lagoon; Florida, USA; and the coastal waters of Charleston, South Carolina, USA. Funding was partially provided by the Florida Project Wild Dolphins license plate, NOAA, National Marine Fisheries Service, Dolphin Quest, and Disney's Animal Programs. We also acknowledge Drs. Sylvain De Guise and Salvatore Frasca from the University of Connecticut for their advice during this project.
References
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