Identification of Three Brucella ceti Genotypes in Bottlenose Dolphins (Tursiops truncatus) Using a Multiplex SYBR Green Real-Time PCR

Abstract: Three sequence types (ST) of Brucella ceti (ST23, ST26, and ST27) in marine mammals have been identified using multilocus sequence analysis and multilocus variable number tandem repeat analysis. This study reports a multiplex SYBR green real-time PCR assay and melting curve analysis for rapid identification of these B. ceti strains and for application to test clinical samples from 272 bottlenose dolphins (Tursiops truncatus) stranded in the coastal region of northern Florida, South Carolina, and Virginia in the United States. The multiplex real-time PCR assay detected all B. ceti ST23, ST26, and ST27 strains and field isolates, and none of the other Brucella spp. and non-Brucella pathogens tested. The limit of detection was 15 genome copies from B. ceti B1/94 (ST23), B. ceti B14-94 (ST26), and B. ceti SC1135 (ST27) per PCR volume. Brucella DNA fragments specific for ST26 and ST27 were found in 15% (41/272) and 7% (20/272) of dolphin samples, respectively. No specific fragment of Brucella DNA for ST23 was detected in these samples. The presence of the gene fragments specific for ST26 and ST27 in positive samples observed with multiplex real-time PCR was further confirmed by conventional PCR, consisting of a set of six specific PCRs, targeting IS711-specific chromosomal locations for Brucella in marine mammals. To our knowledge, this study is the first report on identification of B. ceti genotypes ST26 and ST27 in dolphins using a multiplex real-time PCR assay. The results in this study indicate that the assay may be used as a fast and reliable alternative approach for identification of B. ceti in samples from dolphins.
Key Words: real-time PCR, multiplex, Brucella spp., bottlenose dolphins, Tursiops truncatus
Document Type: Research Article
Page Numbers: 335-343

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