Distinct strains of Brucella have been recognized in marine mammals worldwide since 1994. The distribution and potential health effects of these Brucella species on marine mammals and humans are under study, but progress is slowed by unreliability of traditional microbiologic and serologic diagnostic tests. Real-time, or quantitative, polymerase chain reaction (qPCR) assays are currently being employed in diagnosis of medical and veterinary cases of brucellosis, and this approach shows promise in the marine mammal context. To this end, a multiplex qPCR assay has been developed to screen tissues or blood from a wide range of marine mammals for the presence of Brucella spp. bacteria. This Taqman probe-based assay targets a 150 bp amplicon from an outer membrane protein gene (bscp31), which has been rigorously tested and reported in the literature as specific to the genus Brucella.1,2 The triplex assay also includes two internal controls: a conserved eukaryotic mitochondrial gene target for DNA quality control, and a plasmid-based internal control that detects endogenous inhibitors of PCR. Tests of the assay against a panel of common aquatic bacterial isolates demonstrated 100% specificity for Brucella. Assays of DNA extracted from pinniped and cetacean origin Brucella isolates show a limit of detection at or below three bacteria. Preliminary results testing field-collected harbor seal (Phoca vitulina) tissues show increased sensitivity of this assay compared to culture. Because this assay can be used with both antemortem or postmortem samples, it shows promise as a useful screening tool for detection of marine mammal brucellosis.
The authors thank the institutions and individuals that have provided samples and assistance with this research, including Frances Gulland, Dyanna Lambourn, Amy Smith, Andrew Draghi, Guillermo Risatti, and the U.S. Navy Marine Mammal Program. Funding was provided by the NOAA Oceans and Human Health Initiative (NA04OAR4600209).
1. Casanas, M.C., M.I. Queipo-Ortuno, A. Rodriguez-Torres, A. Orduna, J.D. Colmenero, and P. Morata. 2001. Specificity of a polymerase chain reaction assay of a target sequence on the 31-kilodalton Brucella antigen DNA used to diagnose human brucellosis. Eur. J. Clin. Microbiol. Infect. Dis. 20: 127–131.
2. Probert, W.S., K.N. Schrader, N.Y. Khuong, S.L. Bystrom, and M.H. Graves. 2004. Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J. of Clin. Microbiol. 42(3): 1290–1293.