Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus) Qingzhong Wu a, ⁎, Wayne E. McFee b , Tracey Goldstein c , Rebekah V. Tiller d , Lori Schwacke a a Hollings Marine Laboratory, National Centers for Coastal Ocean Science, National Ocean Service, National Oceanic Atmospheric Administration, Charleston, SC 29412, USA b Center for Coastal Environmental Health and Biomolecular Research, National Centers for Coastal Ocean Science, National Ocean Service, National Oceanic and Atmospheric Administration, Charleston, SC 29412, USA c One Health Institute, School of Veterinary Medicine, University of California, Davis, CA 95616, USA d Zoonoses and Select Agent Laboratory, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA abstract article info Article history: Received 19 December 2013 Received in revised form 5 March 2014 Accepted 5 March 2014 Available online 13 March 2014 Keywords: Bottlenose dolphins IS711 Real-time PCR Brucella spp. Lung Brain Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the de- tection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27 fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA- positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identi- fication of Brucella genotype ST27 in marine mammals. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Since the first isolation of Brucella from marine mammals in 1994, Brucella strains have been isolated from and detected in a variety of free-ranging marine mammals from most parts of the world (Nymo et al., 2011). Brucella ceti and Brucella pinnipedialis are the proposed taxon names for the cetacean and pinniped Brucella isolates, respec- tively (Foster et al., 2007). B. ceti and B. pinnipedialis are largely host specific and exhibit characteristically different pathology in their preferred hosts. According to a review by Nymo et al., gross pathology is mostly seen in cetaceans while the disease state in pinnipeds is largely unknown providing evidence that different strains have variable levels of virulence and zoonotic potential (Nymo et al., 2011). Multilocus sequence typing (MLST) has proven to be a useful tool to characterize Brucella spp. by examining single nucleotide polymor- phisms in nine distinct genetic loci (Whatmore et al., 2007). Within the marine mammal Brucella spp., there are five documented sequence types (ST), three of which are predominantly associated with the B. ceti species (ST23, ST26 and ST27) and two STs that are most common with- in the B. pinnipedialis species (ST24 and ST25) (Whatmore et al., 2007). To date three human cases of naturally acquired infection and one case of laboratory-acquired infection with marine mammal Brucella species have been reported (Brew et al., 1999; McDonald et al., 2006; Sohn et al., 2003; Whatmore et al., 2008). MLST analysis of over 160 Brucella isolates showed that the three isolates from naturally acquired human infections shared an identical genotype (ST27) with that of strain F5/99 isolated from an aborted bottlenose dolphin fetus from the United States Pacific waters and the isolate recovered from the 1999 laboratory acquired case was determined to be ST23 (Whatmore et al., 2007, 2008). These findings suggest a higher zoonotic risk with genotype ST27 for infection of humans however marine-associated brucellosis in humans has not been documented in the United States. Microbiologic culture is considered to be the “gold standard” for definitive diagnosis of brucellosis. However, culture methods are time consuming (can take up to 2 weeks for definitive diagnosis), have low sensitivity and can be hazardous to laboratory personnel. Serologic assays are rapid, sensitive and useful for the detection of Journal of Microbiological Methods 100 (2014) 99–104 ⁎ Corresponding author at: NOAA National Centers for Coastal Ocean Science, Hollings Marine Laboratory, 331 Fort Johnson Rd., Charleston, SC 29412, USA. Tel.: +1 843 762 8940. E-mail address: qingzhongwu@yahoo.com (Q. Wu). http://dx.doi.org/10.1016/j.mimet.2014.03.001 0167-7012/© 2014 Elsevier B.V. All rights reserved. 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