Abstract
Brucella is a known bacterial pathogen in terrestrial animals, currently having 6 recognized species.12,7 Over the last decade, an uncharacterized Brucella species has been identified in multiple marine mammal species; however, its prevalence and severity are uncertain due to unreliable techniques for detecting infection in these animals.3,6,8,9,10 Serologic tests previously used to detect infection in marine mammals were developed from antigens of terrestrial Brucella strains; therefore, the results received from these tests may not be specific for marine mammal species.
A new indirect enzyme-linked immunosorbent assay (iELISA) was developed at Texas A & M University using a whole-cell antigen from a Brucella organism isolated from an aborted bottlenose dolphin fetus. This iELISA was specifically designed to screen dolphin sera for specific antibodies to the marine mammal Brucella sp. The iELISA was examined with other existing serologic tests to compare results in order to determine whether the new assay will be a beneficial diagnostic tool for marine mammal brucellosis.
A total of 106 serum samples originally were collected from 65 Atlantic bottlenose dolphins (Tursiops truncatus) at the U.S. Navy Marine Mammal Program (NMMP--San Diego, CA) to perform a herd analysis that evaluated the potential prevalence of exposure to the marine mammal Brucella sp. These samples were tested using the iELISA method at the NMMP. The same serum samples were separated by control thaw and shipped to the Canadian Food Inspection Agency for a blind study using fluorescence polarization assay (FPA) and competitive enzyme-linked immunosorbant assay (cELISA) methods.2,5,11 52 of 106 samples (49%) were positive with the iELISA method; 37 of 106 samples (34.9%) using the cELISA test; and 32 of 106 samples (30.2%) tested positive with the FPA.
The dolphin-specific iELISA test yielded a higher overall prevalence of antibodies to Brucella sp. compared to the cELISA and FPA test methods. Evidence from molecular and biological characterization studies strongly suggests that there is at least one separate Brucella sp. from the 6 previously categorized species in marine mammals.1,4,8 These findings, as well as the higher number of positive values derived from the iELISA, suggest that this assay allows improved detection of antibodies specific to the antigens of the marine mammal Brucella sp. Alternatively, the higher percentage of positive values with the iELISA may be related to the nature of the antigen developed for the assay. The antigen used for the iELISA is a whole-cell lysate, in contrast to the cELISA and FPA test methods that involve only the components of the lipopolysaccharide chain (LPS) of the B. abortus antigen. Thus, the whole-cell lysate developed for the iELISA potentially may lead to false positive values. This outcome could be due to cross-reaction with antibodies specific to other gram-negative organisms or nonspecific binding of unknown origin on the assay. This possibility warrants further development of this assay using only the LPS portion of the antigen rather than a whole-cell antigen.
The results from this comparative study suggest that the indirect ELISA may be more specific for detecting Brucella sp. antibodies in marine mammals than the previously validated test methods using terrestrial Brucella strains.
Acknowledgements
I would like to thank the U.S. Navy Marine Mammal Program and staff for their support and use of their facilities and equipment. The authors also thank Dr. Klaus Nielsen and the research staff at the Canadian Food Inspection Agency, and the Animal Diseases Research Institute for their serologic testing using the cELISA and FPA methods. Thanks to A.P. MacMillan, Darla Ewalt, and Claire Dawson for consultation during this study.
References
1. Bricker BJ, DR Ewalt, AP MacMillan, G Foster, S Brew. 2000. Molecular characterization of Brucella strains isolated from marine mammals. Journal of Clinical Microbiology 38(3): 1258-1262.
2. Canadian Food Inspection Agency, Animal Diseases Research Institute. 2001. Protocol for a competitive enzyme immunoassay for detection of serum antibody to Brucella abortus.
3. Clavareau C, V Weelemans, K Walravens, M Tryland, J Verger, M Grayon, A Cloeckaert, J Letesson, J Godfroid. 1998. Phenotypic and molecular characterization of Brucella strain isolated from a minke whale (Balaenoptera acutorostrata). Microbiology 44: 3267-3273.
4. Ewalt DR, JB Payeur, BM Martin, DR Cummins, WG Miller. 1994. Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus). Journal Veterinary Diagnostic Investigation 6: 448-452.
5. Gall D, K Nielsen, L Forbes, D Davis, P Elzer, S Olsen, S Balsevicius, L Kelly, P Smith, S Tan, D Joly. 2000. Validation of the fluorescence polarization assay and comparison to other serologic assays for the detection of serum antibodies to Brucella abortus in bison. Journal of Wildlife Diseases 36(3): 469-476.
6. Gonzalez L, IA Patterson, RJ Reid, G Foster, M Barberan, JM Blasco, S Kennedy, FE Howie, J Godfroid, AP MacMillan, A Shock, D Buxton. Chronic meningoencephalitis associated with Brucella sp. infection in live-stranded striped dolphins (Stenella coeruleoalba). 2002. Journal Comparative Pathology 126: 147-152.
7. Hirsh DC, YC Zee. 1999. Brucella. In: Hirsh, D.C. and Y.C. Zee. Veterinary Microbiology. Blackwell Science, New York. Pp. 196-202.
8. Jahans KL, G Foster, ES Broughton. 1997. The characterization of Brucella strains isolated from marine mammals. Veterinary Microbiology 57: 373-382.
9. Jepson PD, S Brew, AP MacMillan, JR Baker, J Barnett, JK Kirkwood, T Kuiken, Robinson IR, VR Simpson. 1997. Antibodies to Brucella in marine mammals around the coast of England and Wales. The Veterinary Record 141: 513-515.
10. Miller GW, LG Adams, TA Ficht, NF Cheville, JP Payeur, DR Harley, C House, SH Ridgeway. 1999. Brucella-induced abortions and infection in bottlenose dolphins (Tursiops truncatus). Journal of Zoo and Wildlife Medicine 30(1): 100-110.
11. Nielsen K, M Lin, D Gall, M Jolley. 2000. Fluorescence polarization immunoassay: detection of antibody to Brucella abortus. Methods 22: 71-76.
12. Quinn PJ, BK Markey, ME Carter, WJC Donnelly, FC Leonard. 2002. Brucella species. In: Quinn, P.J., B.K. Markey, M.E. Carter, W.J.C. Donnelly, and F.C. Leonard. Veterinary Microbiology and Microbial Disease. Blackwell Science, Massachusetts. Pp.162-167.