Journal of Microbiological Methods 100 (2014) 99–104

Contents lists available at ScienceDirect

Journal of Microbiological Methods

j ourna l homepage: www.e lsev ie r .com/ locate / jmicmeth

Real-time PCR assays for detection of Brucella spp. and the identificationof 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, USAb 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, USAc One Health Institute, School of Veterinary Medicine, University of California, Davis, CA 95616, USAd Zoonoses and Select Agent Laboratory, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA

⁎ Corresponding author at: NOAA National Centers forMarine Laboratory, 331 Fort Johnson Rd., Charleston, SC8940.

E-mail address: qingzhongwu@yahoo.com (Q. Wu).

http://dx.doi.org/10.1016/j.mimet.2014.03.0010167-7012/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 December 2013Received in revised form 5 March 2014Accepted 5 March 2014Available online 13 March 2014

Keywords:Bottlenose dolphinsIS711Real-time PCRBrucella spp.LungBrain

Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitivediagnosis 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 infectionandmay 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 thecoast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and thelimit of detection was 0.27 fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification wasdetected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samplestested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection ofBrucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification ofBrucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained forBrucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was foundfor 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 frommarinemammals in 1994,Brucella strains have been isolated from and detected in a variety offree-ranging marine mammals from most parts of the world (Nymoet al., 2011). Brucella ceti and Brucella pinnipedialis are the proposedtaxon names for the cetacean and pinniped Brucella isolates, respec-tively (Foster et al., 2007). B. ceti and B. pinnipedialis are largely hostspecific and exhibit characteristically different pathology in theirpreferred hosts. According to a review by Nymo et al., gross pathologyis mostly seen in cetaceans while the disease state in pinnipeds is largelyunknownproviding evidence that different strains have variable levels ofvirulence and zoonotic potential (Nymo et al., 2011).

Multilocus sequence typing (MLST) has proven to be a useful toolto characterize Brucella spp. by examining single nucleotide polymor-phisms in nine distinct genetic loci (Whatmore et al., 2007). Within

Coastal Ocean Science, Hollings29412, USA. Tel.: +1 843 762

the marine mammal Brucella spp., there are five documented sequencetypes (ST), three of which are predominantly associated with the B. cetispecies (ST23, ST26 and ST27) and two STs that aremost commonwith-in the B. pinnipedialis species (ST24 and ST25) (Whatmore et al., 2007).To date three human cases of naturally acquired infection and one caseof laboratory-acquired infection with marine mammal Brucella specieshave been reported (Brew et al., 1999; McDonald et al., 2006; Sohnet al., 2003; Whatmore et al., 2008). MLST analysis of over 160 Brucellaisolates showed that the three isolates from naturally acquiredhuman infections shared an identical genotype (ST27) with that ofstrain F5/99 isolated from an aborted bottlenose dolphin fetus fromthe United States Pacific waters and the isolate recovered from the1999 laboratory acquired case was determined to be ST23 (Whatmoreet al., 2007, 2008). These findings suggest a higher zoonotic risk withgenotype ST27 for infection of humans however marine-associatedbrucellosis in humans has not been documented in the United States.

Microbiologic culture is considered to be the “gold standard” fordefinitive diagnosis of brucellosis. However, culture methods aretime 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

Table 2Positive detection rates of the IS711 and ST27 real-time PCRs in clinical samples frombottlenose dolphins. Parenthetical values are no. of positive results out of no. of samplestested.

Sample IS711 ST27

Lung 36% (28/78) 39% (11/28)Brain 29% (19/66) 26% (5/19)Urine 40% (2/5) 0 (0/1)a

Spleen 17% (1/6) 0 (0/1)Pulmonary lymph node 20% (1/5) 100% (1/1)Amniotic fluid 50% (1/2) 0 (0/1)Mesenteric lymph node 100% (1/1) 100% (1/1)Brain stem 100% (1/1) 0 (0/1)Right ventricle 100% (1/1) 0 (0/1)Prescapular lymph node 0 (0/3)Blood 0 (0/2)Lung associated lymph node 0 (0/2)Thymus 0 (0/2)Adrenal 0 (0/1)Liver 0 (0/1)Stomach fluid 0 (0/1)Testis 0 (0/1)Total 31% (55/178) 33% (18/54)

a One IS711positive urine samplewas not testedwith the assay due to insufficient sam-ple volume.

100 Q. Wu et al. / Journal of Microbiological Methods 100 (2014) 99–104

antibodies, but false-positive results due to cross reactions with un-related Gram-negative bacteria have been reported (Delpino et al.,2004; Muňoz et al., 2005). As an alternative diagnostic approach,we developed two real-time PCR assays: one for the detection ofBrucella spp. by targeting the IS711 gene and another for rapid iden-tification of Brucella genotype ST27. Clinical samples collected fromstranded bottlenose dolphins along the coast of South Carolina, USAwere analyzed using these assays. To identify false-negative resultsdue to the presence of inhibitors in DNA extracts, we included synthet-ically created DNA as an internal amplification control.

2. Materials and methods

2.1. Reference strains

The reference and field strains of Brucella spp. were included in thisstudy (Table 1). Non-Brucella pathogens (Leptospira interrogans serovarHardjo, L. interrogans serovar Bratislava, L. interrogans serovar Pomona,L. interrogans serovar Canicola, L. interrogans serovar Grippotyphosa,L. interrogans serovar Copenhageni were obtained from the NationalVeterinary Services Laboratories, Ames, Iowa. Escherichia coli,Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis,Cryptosporidium parvum, Giardia lamblia and Toxoplasma gondii) wereused as negative controls as these non-Brucella pathogens have beendetected previously in marine mammals (Fayer et al., 2004; Higgins,2000) and were included in the experiments to test the specificity ofreal-time PCR assay for Brucella spp. We intended to develop and vali-date the real-time PCR assay for marine mammals and thus otherterrestrial Brucella spp. and Brucella-closely related microorganismswere not included in the study.

2.2. Sample collection

Tissues for screening were collected from 86 bottlenose dolphinsthat stranded in the coastal region of South Carolina, USA between2010 and 2013. The carcasses were necropsied according to standardprocedures described previously (McFee and Lipscomb, 2009). Briefly,different tissue and body fluid samples listed in Table 2 were collectedfrom dead carcasses. Tissues frommoderately to advanced decomposedcarcasses were usually limited to brain and lung samples. A total of 178tissue and body fluid samples were aseptically collected and placed in1.5 ml Nalgene tubes and stored at −80 °C until analysis.

2.3. Detection of Brucella spp. in tissue samples by culturing

Thirty-seven tissue samples were submitted to the NationalVeterinary Services Laboratories (NVSL, Ames, Iowa, USA) for isola-tion and identification of Brucella spp. in tissue samples.

Table 1Results of IS711 and ST27 real-time PCRs and MLST in Brucella strains.

Strain Host Or

Brucella abortus 544 Bovine TyB. ceti B14-94 Common dolphin ScB. ceti B1/94 Harbor porpoise ScB. ceti B202R Minke whale NoB. pinnipedialis B2/94 Common seal ScB. pinnipedialis 17A1 Hooded seal NoB. ceti 03-0312 Dolphin vertebrae USB. cetiM04-0174 Dolphin placenta USB. ceti 021MMS Bottlenose dolphin USB. ceti IFAW12-087Dd Common short beard dolphin USB. ceti SC1135 Bottlenose dolphin USB. ceti LA002 Bottlenose dolphin USB. ceti LA001 Bottlenose dolphin US

2.4. DNA extraction

Genomic DNA was extracted from Brucella cultures (Table 1)using the DNeasy Tissue Kit (Qiagen, Valencia, CA) or using a simpleboil preparation method. When prepared with the boiling method,bacteria were grown by plating one loop (1 μl) of stock cell suspen-sion on Trypticase soy agar with 5% defibrinated sheep blood agar(BBL Microbiology Systems, Cockeysville, MD) and aerobically incu-bated for 1 to 2 days at 37 °C with 5% CO2. To yield a DNA template, asingle colony was suspended in 200 μl of 10 mM Tris (pH 8.0) withina 1.5-ml Millipore 0.22-μm-pore-diameter filter unit (Millipore, Bed-ford, MA) and heated at 95 °C for 20min. The filter unit was then cen-trifuged at 8000 ×g for 2 min to recover the filtrate containing theDNA template. DNA from non-Brucella spp. and DNA from tissueand body fluid samples except urine were extracted with theQIAamp DNA Mini Kit (Qiagen). Isolation of genomic DNA fromurine was accomplished using the QIAamp viral RNA mini kit(Qiagen).

2.5. Design of PCR primers and TaqMan probes

Sequences of the IS711 gene from B. ceti B1/94 (FJ376557), B. cetiCudo (ACJD01000007), Brucella sp. B1/94 (AF242533), B. pinnipedialisB2/94 (CP002079) and Brucella sp. JM13/00 (AB126349) were aligned

igin MLST PCR

IS711 ST27

pe strain 1 + −otland 26 + −otland 23 + −rway 23 + −otland 25 + −rway Not tested + −A 27 + +A 27 + +A 28 + −A 27 + +A 27 + +A 28 + −A 27 + +

101Q. Wu et al. / Journal of Microbiological Methods 100 (2014) 99–104

and a consensus sequence was selected using ClustalW2 (Goujon et al.,2010; Larkin et al., 2007). A ST27 specific IS711 chromosomal locus(BCETI_7000072) has been previously identified and evaluated in anextensive panel of marine and terrestrial Brucella spp. (Cloeckaertet al., 2011). The primers and probes were designed using PrimerQuest(http://www.idtdna.com/Primerquest/Home/Index). The IS711 specificprimers IS711F (5′-TACCGCTGCGAATAAAGCCAAC-3′) and IS711R (5′-TGAGATTGCTGGCAATGAAGGC-3′) were used to amplify a 150 bpfragment of the IS711 gene, which was detected by the probe,IS711P (FAM-5′-ACCCGGCCATTATGGTGACTGTCCGCA-3′-BHQ1). TheST27 specific primers ST27F (5′-TCGTTCAACACGCGTCGATCAT-3′) andST27R (5′-TGCTCACGGCTGTTCTCCTTTA-3′) were used to amplify a169 bp fragment of a specific IS711 chromosomal location of ST27,which was detected by the probe, ST27P (Hex-5′-TGGATCAAAGTGCGCCTTGCCGCGT-3′-BHQ1). The primers and probes were synthesized byIntegrated DNA Technologies (Coralville, IA). Primer and probe specific-ities of the IS711 gene in Brucella spp. were verified by BLASTN analysisagainst all GenBank entries and evaluated usingDNAextracted from Bru-cella strains listed in Table 1.

2.6. Real-time PCR analyses

PCR was performed in a volume of 15 μl with primer and probeconcentration of 300 nM and 150 nM respectively, 7.5 μl of TaqMan®Universal PCR master mix (Applied Biosystems, Foster City, CA) and1.5 μl DNA template in an Eco™ PCR system (Illumina, San Diego, CA).The PCR conditionswere as follows: 10min at 95 °C, followed by 40 cy-cles of 10 s at 95 °C and 30 s at 60 °C. DNA from B. ceti B1/94was used asthe positive template control. Nuclease-free water (Qiagen) instead ofDNA extract was used as the no template control or negative control.All clinical samples and both negative and positive controls were runin triplicate. Amplification efficiency (E) was estimated by the formulaE = 10−1/slope − 1 (Bustin et al., 2009).

2.7. Construction of internal amplification control (IAC) DNA

To assess whether inhibitors from tissue and body fluid sampleswere present in the extracted DNA and causing false-negative PCRresults, we constructed an internal amplification control (145 bp)consisting of a fragment of the phyB gene coding region of potatoroot (Nolan et al., 2006) flanked by the sequence matching theIS711F and IS711R primers. An ampliconwas constructed from an ol-igonucleotide of the phyB gene (synthesized by Integrated DNATechnologies) in a conventional PCR using overhanging primers(IS711IACF 5′-TACCGCTGCGAATAAAGCCAACAACTTGGCTTTAATGGACCTCCA-3′ and IS711IACR 5′-TGAGATTGCTGGCAATGAAGGCACATTCATCCTTACATGGCACCA-3′). PCR was performed in reaction vol-umes of 20 μl with 10 μl of 2× AmpliTaq Gold® 360 Master Mix (Ap-plied Biosystems), 100 nM of each primer and 2 μl of 1 μM SPUD-A(Nolan et al., 2006) and conditions used were 5 min at 95 °C, follow-ed by 35 cycles of denaturation for 15 s at 95 °C, annealing for 15 s at69 °C, elongation for 30 s at 72 °C and a final extension step of 72 °Cfor 7 min. The amplicon from the first PCR was purified using aQIAquick PCR Purification Kit (Qiagen) and diluted at 1:1000 withnuclease-free water. The diluted amplicon was reamplified withprimers IS711F and IS711R using the process described above withone modification: the annealing temperature was 59 °C instead of69 °C. The final amplicon was purified using the QIAquick PCR Purifi-cation Kit and analyzed by an Agilent 2100 Bioanalyzer (Santa Clara,CA) to confirm the presence of a single amplicon. The syntheticallycreated DNA was used as IAC in every reaction mixture. The IACwas detected by the real-time PCR assay using the probe SPUD-T(Hex-5′-TGCACAAGCTATGGAACACCACGT-3′-BHQ1) (Nolan et al.,2006).

2.8. Analytical sensitivity

Genomic DNA isolated from B. ceti B1/94 was used to determinethe analytic sensitivity of the assay. The quantity of Brucella genomicDNA was estimated by measuring the absorbance of DNA using theSpectrophotometer ND-1000 (NanoDrop Technologies, Wilmington,DE). Assuming a genome size of 3.28 Mb, 3.6 fg DNA was consideredto be equivalent to one genomic DNA copy. A standard curve was de-veloped using a 10-fold serial dilution of DNA from B. ceti B1/94,ranging from 0.27 to 5.4 × 105 fg per PCR volume.

2.9. Sequencing of IS711 amplicons from PCR positive tissue samples

The IS711 amplicons from PCR positive tissue samples wereextracted using the QIAquick PCR Purification Kit and then run bythe Agilent 2100 Bioanalyzer to confirm the presence of a singleamplicon prior to sequencing by SeqWright Inc. (Houston, TX) withprimers IS711F and IS711R, respectively. The sequences of theamplicons were analyzed using BLASTN to assess homologies withsequences in the GenBank database.

2.10. Multilocus sequence typing (MLST)

Genotyping based on multi-locus sequence analysis (MLSA) ofnine house-keeping genes was performed as described previously(Whatmore et al., 2007). Full-length amplicons of the nine house-keeping genes from the seven B. ceti field strains tested in thisstudy were sequenced, concatenated and analyzed with the 27 clas-sical Brucella sequence types described byWhatmore et al. Contig as-sembly, sequence editing and gene concatenationwere performed inthe DNASTAR Lasergene 8 software suite before multiple sequencealignments were constructed (DNASTAR Inc., Madison, WI). Pairwise analysis was performed and neighbor-joining consensus treesinferred from 1000 bootstrap replicates were constructed usingMEGA version 4.0.

2.11. Statistical analysis

Cohen's kappa value was used to estimate agreement betweenreal-time PCR and culture results with a 95% confidence interval(CI). The following ranges were considered for interpretation of thekappa value (Landis and Koch, 1977): poor agreement: b0.00; slightagreement: 0.00–0.20; fair agreement: 0.21–0.40; moderate agree-ment: 0.41–0.60; substantial agreement: 0.61–0.80; almost perfect:0.81–1.00. A two sample t-test was used to compare the effect of tissueand body fluid samples on the threshold cycle (Ct) value of the IAC. Sta-tistical significance was considered for p-values b 0.05. Carcasses werecategorized into age classes based on carcass length: fetus/neonate —

≤132 cm (Fernandez and Hohn, 1998); subadult — N132 cm andb240 cm; adult ≥240 cm (Mcfee and Hopkins-Murphy, 2002). A chi-square test was used to compare the prevalence of positive detectionsamong age classes.

3. Results

3.1. Sensitivity and specificity of the real-time PCR assay for detection ofBrucella spp.

We identified the primers and probe concentrations that opti-mize the amplification of the IS711 gene fragment. The amplificationefficiency of the assay was 0.947 with a correlation coefficient of0.999 (Fig. 1). The limit of detection of the PCR assay was determinedby testing serial dilutions of genomic DNA from B. ceti B1/94 and de-termined to be 0.27 fg of genomic DNA per PCR volume (Fig. 1). AllBrucella strains included in this study were positive using thisIS711 assay (Table 1). IS711 real-time PCR on non-Brucella pathogens

y = -3.4554x + 35.875 R² = 0.9992

10

15

20

25

30

35

40

-1 0 1 2 3 4 5 6

Thr

esho

ld c

ycle

s (C

t)

Log concentraion of DNA (fg)

Fig. 1. Standard curve developed using 10-fold serial dilution of DNA from B. ceti B1/94ranging from 0.27 to 5.4 × 105 fg of genomic DNA per PCR volume. The regression linewas for data thatwere in the linear range. The graph shows themean values from four rep-licates for each dilution.

Table 4Comparison of culture and real-time PCR results for 37 tissue samples frombottlenose dol-phins.

Culture Real-time PCR Total

Positive Negative

Positive 9 0 9Negative 18 10 28Total 27 10 37

102 Q. Wu et al. / Journal of Microbiological Methods 100 (2014) 99–104

(listed in the Materials and methods section) did not result in anyamplification products.

3.2. Detection of Brucella in clinical samples

One hundred seventy eight tissue and body fluid samples from 86bottlenose dolphins were tested with the IS711 real-time PCR assayfor the detection of Brucella spp. Brucella DNA was detected in 31%of the clinical samples including lung, brain, brain stem, spleen,right ventricle, mesenteric and pulmonary lymph nodes, urine andamniotic fluid (Table 2), representing 41% of the individual bottlenosedolphins tested by real-time PCR (Table 3). Brucella DNA was detectedin 59% of fetus/neonates, 27% of subadults, and 33% of adult carcasses(Table 3). The chi-square test of independence (p = 0.02) indicated arelationship between age-class and detection of Brucella DNA. BrucellaDNAwas detected in the four carcasses that were believed to be fetusesbased on total body length b100 cm (Neuenhoff et al., 2011) and thetest showing that the lungs from the fetal-carcasses did not float inwater. Three of the fetuses were recovered without the mother(i.e., aborted fetuses), but one was removed from the mother at nec-ropsy; in this case, Brucella DNA was detected in tissues from boththe mother and fetus. In comparison to the detection of Brucellaspp. in tissue samples between our real-PCR assay and the culturemethod, we detected Brucella DNA in 73% (27/37) of the tissue sam-ples by real-time PCR, versus 24% (9/37) by culture (Table 4). Fairagreement was observed between real-time PCR and culture of tis-sue samples with a kappa value of 0.21 (95% CI, 0.05–0.37).

Table 3Positive detection rates of the IS711 and ST27 real-time PCRs in clinical samples frombottlenose dolphins based on the number of individuals of each age and gender tested.Parenthetical values are no. of positive results out of no. of samples tested.

IS711 ST27

Age classFetus/neonate 59% (19/32) 74% (14/19)Subadult 27% (9/33) 0 (0/9)Adult 33% (7/21) 0 (0/7)

GenderMale 40% (18/45) 39% (7/18)Female 38% (15/39) 47% (7/15)Undetermined 100% (2/2) 0 (0/2)

Total 41% (35/86) 40% (14/35)

3.3. Performance of internal amplification control (IAC)

The optimal IAC concentration (400 copies/reaction)was establishedbased on the criterion that an IAC amplicon was always detected insamples containing 0–36 fg of genomic DNA from B. ceti B1/94 perreaction. The Ct value (34.7 ± 0.57, mean ± SD) for the IAC was re-corded for all 369 real-time PCR runs, testing 123 tissue and bodyfluid samples where no amplification of Brucella DNA was detected.These results were compared to the Ct value (34.57 ± 0.45) recordedfor the 36 real-time PCR runs of the 12 no template controls (p =0.185). IAC was amplified successfully in all negative samples, demon-strating that the real-time PCR was not inhibited in these samples.

3.4. Confirmation of amplicon identity by DNA sequence analysis

IS711 PCR products from two positive tissue samples were analyzedby the Agilent 2100 Bioanalyzer and showed a single band. The purifiedIS711 PCR products were sequenced and the results showed the samesequence for these PCR products. The outputs were compared withother known sequences in the GenBank database using BLASTN. Theyaligned with 100% identity to the sequence of IS711 gene fragment inseven strains of B. ceti and B. pinnipedialis and one strain of Brucellaspecies in North Pacific common minke whales (ACJD01000007,AF242533, AF242534, CP006896/CP006897, CP006898/CP006899,AF242532, CP002078/CP002079 and AB126349).

3.5. Multilocus sequence typing

The IS711 and ST27 PCR assays were tested on a panel of B. ceti andB. pinnipedialis reference and field strains. The seven B. ceti field strainstested in this study were predominantly ST27 and a new sequence typethat we are designating as ST28. This new B. ceti sequence type carriesthree unique single nucleotide polymorphisms at positions 198 in thegap gene, 1141 in the aroA gene and 3572 in the cobQ gene. This newmarine ST shares a single nucleotide polymorphism with ST26 at posi-tion 3810 in the omp25 gene. The B. ceti field isolates were recoveredfrom tissues of stranded bottlenose dolphins from various areas of theUnited States.

3.6. Identification of Brucella ST27 using real-time PCR

A second real-time PCR assay was developed for the identificationof Brucella ST27. All DNA samples of B. ceti strains identified as ST27by MLST were positive using this real-time PCR assay (Table 1).Other Brucella strains included in this study were negative usingthis assay (Table 1). The ST27 real-time PCR assay was used to iden-tify this genotype ST27 in 54 IS711 PCR positive samples from 35bottlenose dolphins. ST27 was identified in 33% of the clinical sam-ples (Table 2) and 40% of bottlenose dolphins (Table 3); all positivedetections for ST27 were in samples from fetus/neonate carcasses(Table 3). One IS711 positive urine sample was not tested with theassay due to insufficient sample volume.

103Q. Wu et al. / Journal of Microbiological Methods 100 (2014) 99–104

4. Discussion

We developed a real-time PCR assay using primers and a TaqManprobe for detecting the IS711 gene, or IS6501, which was specific forthe Brucella genus (Ocampo-Sosa and Garcia-Lobo, 2008). Each ge-nome of marine mammal Brucella species contains multiple copiesof IS711 and the marine spp. have a higher number of copies of thisinsertion sequence relative to the terrestrial Brucella spp. (Brickeret al., 2000; Cloeckaert et al., 2011), which could explain the im-proved sensitivity of the real-time PCR assay. Real-time PCR hasbeen used to detect a wide number of microorganisms of clinical im-portance. Real-time PCR assays targeting the IS711 gene have beenused for detection of Brucella in clinical samples from wild boarsand dromedaries (Hinić et al., 2009; Tomaso et al., 2010). The real-time PCR targeting IS711 was more sensitive, specific, efficient andreproducible than the assays for bcsp31 and per genes to detectBrucella spp. (Bounaadja et al., 2009). The real-time PCR assay devel-oped in this study proved to be highly specific for Brucella spp., sincethe target gene sequence was amplified in all known laboratoryBrucella strains tested, but not observed in non-Brucella pathogensincluded in the study. The amplicon sequences from two positive tis-sue samples identified by real-time PCR aligned with 100% identityto the sequence of IS711 gene fragment in seven strains of B. cetiand B. pinnipedialis and one strain of Brucella species in North Pacificcommon minke whales, further indicating the assay specificity forBrucella species.

The cell numbers of Brucella in tissues and body fluids are frequentlyvery low (Al Dahouk et al., 2003; Bricker, 2002) and thus a highlysensitive assay was required for detection of Brucella in samplesfrom marine mammals. The limit of detection of our real-time PCRassay was 0.27 fg of B. ceti B1/94 genomic DNA per reaction. Thislevel of sensitivity was lower than or comparable to those (0.2–24 fg genomic DNA of per reaction) reported for Brucella speciesusing other assays (Al Dahouk et al., 2007; Bounaadja et al., 2009;Sidor et al., 2013; Tomaso et al., 2010). All of the culture-positivesamples were detected as positive by our PCR assay and we detectedadditional positive samples in which no isolation was made by cul-ture. Culture-negative but PCR positive samples may be due to eithera higher sensitivity of the PCR assay, Brucella that are present but nolonger viable, or contamination, but results from other studies alsoindicate that PCR has a higher sensitivity than culture (Sidor et al.,2013). However, it should be noted that a positive real-time PCR re-sult only indicates the presence of the Brucella DNA; this does notnecessarily indicate an active infection. PCR is a rapid and sensitivemethod for detecting Brucella DNA in samples, but bacterial cultureand isolation, which can take weeks, is still an important tool in eval-uating Brucella infection because it provides information that PCRcannot. In particular, bacterial isolation is necessary to obtain suffi-cient genetic material for identification of strain type, and whilePCR detects both viable and nonviable organisms, only viable organ-isms can be cultured.

In the serologic surveys of a managed population of 64 bottlenosedolphins (Meegan et al., 2012), 18 (28.1%) were seronegative forantibodies against Brucella, 18 (28.1%) were seropositive, and 28(43.8%) had results interpreted as suspect. Bottlenose dolphinsbetween the ages of 17 and 25 yr were 6.8 times more likely to beBrucella antibody positive compared to those that were younger orolder (p = 0.02). In this report, Brucella spp. DNA was detected in31% of the clinical samples from stranded bottlenose dolphins.Increased detection of the IS711 gene fragment in individuals classi-fied as fetus/neonate, suggests that these young dolphins are at ahigher risk for Brucella infection. This is not unexpected given thatin terrestrial species, Brucella is known to cause poor reproductiveoutcomes, including spontaneous abortion, stillbirth and weakcalves (CFSPH, 2009) and cases of Brucella-associated abortion havealso been reported in cetaceans (Miller et al., 1999).

One major limitation of PCR in clinical samples is the possibilityof polymerase inhibition by the presence of inhibitory compoundsand excessive host DNA in biological samples (Al Dahouk et al.,2007; Bricker, 2002). We addressed this by including an internalamplification control in our PCR to assess the potential for PCR inhi-bition in false-negative samples. We found no significant differencein Ct values between IS711 negative clinical samples and no tem-plate controls, indicating that PCR inhibition was not occurring inour assay.

Brucella strain F5/99 from a bottlenose dolphin in the United Statesand all three isolates (isolate 02/611 from New Zealand and isolates01A09163 and 85A05748 from Peru) from naturally acquired humaninfections with marine mammal Brucella species have been character-ized as having the samegenotype, ST27 (Whatmore et al., 2008). Brucel-la isolates from marine mammals were tested for their ability to infecthuman macrophage cells (Maquart et al., 2009). The human isolate02/611 was effectively virulent in human THP-1 macrophage cells tothe same extent as the virulent strains Brucella melitensis 16M andBrucella suis 1330 reference strains were (Maquart et al., 2009). Thesefindings indicated the zoonotic potential associated with genotypeST27 in humans. We developed a real-time PCR assay for rapid identifi-cation of Brucella genotype ST27 by targeting an IS711 chromosomal lo-cation for the genotype (Cloeckaert et al., 2011). All Brucella strainswhich had been identified as ST27 by MLST were positive by our ST27real-time PCR and no amplification was found for other Brucella strainsincluded in this study, indicating the assay specificity for genotype ST27.To our knowledge, this report is the first description of the use of real-time PCR for identification of Brucella genotype ST27. Of the 178 clinicalspecimens tested from bottlenose dolphins stranded along the US,South Carolina coastal region between 2010 and 2013, 31% were IS711positive and 33% of those IS711 positive samples were ST27 positive.All positive reactions for ST27 were observed in fetus or neonates, indi-cating that this age class may be more susceptible to genotype ST27 in-fections. This is the first documentation of the detection of the marinemammal Brucella spp. ST27 in wild cetaceans on the eastern coast ofthe United States.

5. Conclusions

Two real-time PCR assays were developed using primers and probesspecific for Brucella species and Brucella genotype ST27, respectively.The results in this study demonstrate the use of real-time PCR forrapid detection of the Brucella species and the identification of the ma-rinemammal Brucella genotype ST27. Inclusion of an internal amplifica-tion control in the real-time PCR assay showed no inhibitory effects inPCR negative samples.

Acknowledgments

Wewould like to thank Brian Thompson, for providing non-Brucellabacteria. We would like to thank Christine Quance of the NationalVeterinary Services Laboratories in Ames, Iowa, USA for analyzingthe samples with the culture methods. Funding for this researchwas provided by NOAA's Oceans and Human Health Initiative andthe National Marine Fisheries Service Marine Mammal Health andStranding Response Program. This publication does not constitutean endorsement of any commercial product or intend to be an opin-ion beyond scientific or other results obtained by the NOAA.

References

Al Dahouk, S., Tomaso, H., Nockler, K., Neubauer, H., Frangoulidis, D., 2003.Laboratory-based diagnosis of brucellosis—a review of the literature. Part II: se-rological tests for brucellosis. Clin. Lab. 49, 577–589.

Al Dahouk, S., Nockler, K., Scholz, H.C., Pfeffer, M., Neubauer, H., Tomaso, H., 2007.Evaluation of genus-pecific and species-pecific real-ime PCR assays for the iden-tification of Brucella spp Clin. Chem. Lab. Med. 45, 1464–1470.

104 Q. Wu et al. / Journal of Microbiological Methods 100 (2014) 99–104

Bounaadja, L., Albert, D., Chenais, B., Henault, S., Zygmunt, M.S., Poliak, S., Garin-Bastuji, B.,2009. Real-time PCR for identification of Brucella spp.: a comparative study of IS711,bcsp31 and per target genes. Vet. Microbiol. 137, 156–164.

Brew, S.D., Perrett, L.L., Stack, J.A., MacMillan, A.P., Staunton, N.J., 1999. Human exposureto Brucella recovered from a sea mammal. Vet. Rec. 144, 483.

Bricker, B.J., 2002. PCR as a diagnostic tool for brucellosis. Vet. Microbiol. 90, 435–446.Bricker, B.J., Ewalt, D.R., MacMillan, A.P., Foster, G., Brew, S., 2000.Molecular characterization

of Brucella strains isolated from marine mammals. J. Clin. Microbiol. 38, 1258–1262.Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan,

T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE guide-lines: minimum information for publication of quantitative real-time PCR experi-ments. Clin. Chem. 55, 611–622.

CFSPH, 2009. Brucellosis. http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis.pdf.Cloeckaert, A., Bernardet, N., Koylass, M.S., Whatmore, A.M., Zygmunt, M.S., 2011. Novel

IS711 chromosomal location useful for identification of marine mammal Brucella ge-notype ST27, which is associated with zoonotic infection. J. Clin. Microbiol. 49,3954–3959.

Delpino, M.V., Fossati, C.A., Baldi, P.C., 2004. Occurrence and potential diagnostic applica-tions of serological cross-reactivities between Brucella and other alpha-proteobacteria.Clin. Diagn. Lab. Immunol. 11, 868–873.

Fayer, R., Dubey, J.P., Lindsay, D.S., 2004. Zoonotic protozoa: from land to sea. TrendsParasitol. 20, 531–536.

Fernandez, S., Hohn, A.A., 1998. Age, growth, and calving season of bottlenose dolphins,Tursiops truncatus, off coastal Texas. Fish. Bull. 96, 357–365.

Foster, G., Osterman, B.S., Godfroid, J., Jacques, I., Cloeckaert, A., 2007. Brucella ceti sp. nov.and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as theirpreferred hosts. Int. J. Syst. Evol. Microbiol. 57, 2688–2693.

Goujon, M., McWilliam, H., Li, W., Valentin, F., Squizzato, S., Paern, J., Lopez, R., 2010. Anew bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38,W695–W699 (Suppl.).

Higgins, R., 2000. Bacteria and fungi of marine mammals: a review. Can. Vet. J. 41,105–116.

Hinić, V., Brodard, I., Thomann, A., Holub, M., Miserez, R., Abril, C., 2009. IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparisonwith bacterial isolation and serology. BMC Vet. Res. 5, 22.

Landis, J.R., Koch, G.G., 1977. The measurement of observer agreement for categoricaldata. Biometrics 33, 159–174.

Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam,H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J.,Higgins, D.G., 2007. Clustal W and Clustal X version 2. Bioinformatics 23,2947–2948.

Maquart, M., Zygmunt, M.S., Cloeckaert, A., 2009. Marine mammal Brucella isolates withdifferent genomic characteristics display a differential response when infectinghuman macrophages in culture. Microbes Infect. 11, 361–366.

McDonald, W.L., Jamaludin, R., Mackereth, G., Hansen, M., Humphrey, S., Short, P.,Taylor, T., Swingler, J., Dawson, C.E., Whatmore, A.M., Stubberfield, E., Perrett,

L.L., Simmons, G., 2006. Characterization of a Brucella sp. strain as a marine-mammal type despite isolation from a patient with spinal osteomyelitis in NewZealand. J. Clin. Microbiol. 44, 4363–4370.

Mcfee, W.E., Hopkins-Murphy, S.R., 2002. Bottlenose dolphin (Tursiops truncatus)strandings in South Carolina, 1992–1996. Fish. Bull. 100, 258–265.

McFee, W.E., Lipscomb, T.P., 2009. Major pathologic findings and probable causes of mor-tality in bottlenose dolphins stranded in South Carolina from 1993 to 2006. J. Wildl.Dis. 45, 575–593.

Meegan, J., Dunn, J.L., Venn-Watson, S.K., Smith, C.R., Sidor, I., Jensen, E.D., Van Bonn,W.G.,Pugh, R., Ficht, T., Adams, L.G., Nielsen, K., Romano, T.A., 2012. Serologic response inbottlenose dolphins Tursiops truncatus infected with Brucella sp. using a dolphin-specific indirect ELISA. Dis. Aquat. Org. 102, 73–85.

Miller, W.G., Adams, L.G., Ficht, T.A., Cheville, N.F., Payeur, J.P., Harley, D.R., House, C.,Ridgway, S.H., 1999. Brucella-induced abortions and infection in bottlenose dolphins(Tursiops truncatus). J. Zoo Wildl. Med. 30, 100–110.

Muňoz, P.M., Marín, C.M., Monreal, D., González, D., Garin-Bastuji, B., Díaz, R., Mainar-Jaime, R.C., Moriyón, I., Blasco, J.M., 2005. Efficacy of several serological tests and an-tigens for diagnosis of bovine brucellosis in the presence of false-positive serologicalresults due to Yersinia enterocolitica O:9. Clin. Diagn. Lab. Immunol. 12, 141–151.

Neuenhoff, R.D., Cowan, D.F., Whitehead, H., Marshall, C.D., 2011. Prenatal data impactscommon bottlenose dolphin (Tursiops truncatus) growth parameters estimated bylength-at-age curves. Mar. Mamm. Sci. 27, 195–216.

Nolan, T., Hands, R.E., Ogunkolade, W., Bustin, S.A., 2006. SPUD: a quantitative PCR assayfor the detection of inhibitors in nucleic acid preparations. Anal. Biochem. 351,308–310.

Nymo, I.H., Tryland, M., Godfroid, J., 2011. A review of Brucella infection in marine mam-mals, with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophoracristata). Vet. Res. 42, 93.

Ocampo-Sosa, A.A., Garcia-Lobo, J.M., 2008. Demonstration of IS711 transposition inBrucella ovis and Brucella pinnipedialis. BMC Microbiol. 8, 17.

Sidor, I.F., Dunn, J.L., Tsongalis, G.J., Carlson, J., Frasca Jr., S., 2013. A multiplex real-time po-lymerase chain reaction assay with two internal controls for the detection of Brucellaspecies in tissues, blood, and feces from marine mammals. J. Vet. Diagn. Investig. 25,72–81.

Sohn, A.H., Probert, W.S., Glaser, C.A., Gupta, N., Bollen, A.W., Wong, J.D., Grace, E.M.,McDonald,W.C., 2003. Human neurobrucellosis with intracerebral granuloma causedby a marine mammal Brucella spp Emerg. Infect. Dis. 9, 485–488.

Tomaso, H., Kattar, M., Eickhoff, M., Wernery, U., Al Dahouk, S., Straube, E., Neubauer,H., Scholz, H.C., 2010. Comparison of commercial DNA preparation kits for thedetection of Brucellae in tissue using quantitative real-time PCR. BMC Infect.Dis. 10, 100.

Whatmore, A.M., Perrett, L.L., MacMillan, A.P., 2007. Characterisation of the genetic diver-sity of Brucella by multilocus sequencing. BMC Microbiol. 7, 34.

Whatmore, A.M., Dawson, C.E., Groussaud, P., Koylass, M.S., King, A.C., Shankster, S.J.,Sohn, A.H., Probert, W.S., McDonald, W.L., 2008. Marine mammal Brucella genotypeassociated with zoonotic infection. Emerg. Infect. Dis. 14, 517–518.