Journal of Microbiological Methods 100 (2014) 99–104
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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 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 b
a r t i c l e
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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
a b s t r a c t 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 detection 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. DNApositive clinical samples. To our knowledge, this is the ﬁrst report on the use of a real-time PCR assay for identiﬁcation 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, respectively (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 polymorphisms in nine distinct genetic loci (Whatmore et al., 2007). Within ⁎ 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: [email protected]
http://dx.doi.org/10.1016/j.mimet.2014.03.001 0167-7012/© 2014 Elsevier B.V. All rights reserved.
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 within 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
Q. Wu et al. / Journal of Microbiological Methods 100 (2014) 99–104
antibodies, but false-positive results due to cross reactions with unrelated 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 of Brucella spp. by targeting the IS711 gene and another for rapid identiﬁcation of Brucella genotype ST27. Clinical samples collected from stranded bottlenose dolphins along the coast of South Carolina, USA were analyzed using these assays. To identify false-negative results due to the presence of inhibitors in DNA extracts, we included synthetically 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 this study (Table 1). Non-Brucella pathogens (Leptospira interrogans serovar Hardjo, L. interrogans serovar Bratislava, L. interrogans serovar Pomona, L. interrogans serovar Canicola, L. interrogans serovar Grippotyphosa, L. interrogans serovar Copenhageni were obtained from the National Veterinary Services Laboratories, Ames, Iowa. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Cryptosporidium parvum, Giardia lamblia and Toxoplasma gondii) were used as negative controls as these non-Brucella pathogens have been detected previously in marine mammals (Fayer et al., 2004; Higgins, 2000) and were included in the experiments to test the speciﬁcity of real-time PCR assay for Brucella spp. We intended to develop and validate the real-time PCR assay for marine mammals and thus other terrestrial Brucella spp. and Brucella-closely related microorganisms were not included in the study.
Table 2 Positive detection rates of the IS711 and ST27 real-time PCRs in clinical samples from bottlenose dolphins. Parenthetical values are no. of positive results out of no. of samples tested. Sample
Lung Brain Urine Spleen Pulmonary lymph node Amniotic ﬂuid Mesenteric lymph node Brain stem Right ventricle Prescapular lymph node Blood Lung associated lymph node Thymus Adrenal Liver Stomach ﬂuid Testis Total
36% (28/78) 29% (19/66) 40% (2/5) 17% (1/6) 20% (1/5) 50% (1/2) 100% (1/1) 100% (1/1) 100% (1/1) 0 (0/3) 0 (0/2) 0 (0/2) 0 (0/2) 0 (0/1) 0 (0/1) 0 (0/1) 0 (0/1) 31% (55/178)
39% (11/28) 26% (5/19) 0 (0/1)a 0 (0/1) 100% (1/1) 0 (0/1) 100% (1/1) 0 (0/1) 0 (0/1)
One IS711 positive urine sample was not tested with the assay due to insufﬁcient sample volume.
2.4. DNA extraction
Tissues for screening were collected from 86 bottlenose dolphins that stranded in the coastal region of South Carolina, USA between 2010 and 2013. The carcasses were necropsied according to standard procedures described previously (McFee and Lipscomb, 2009). Brieﬂy, different tissue and body ﬂuid samples listed in Table 2 were collected from dead carcasses. Tissues from moderately to advanced decomposed carcasses were usually limited to brain and lung samples. A total of 178 tissue and body ﬂuid samples were aseptically collected and placed in 1.5 ml Nalgene tubes and stored at −80 °C until analysis.
Genomic DNA was extracted from Brucella cultures (Table 1) using the DNeasy Tissue Kit (Qiagen, Valencia, CA) or using a simple boil preparation method. When prepared with the boiling method, bacteria were grown by plating one loop (1 μl) of stock cell suspension on Trypticase soy agar with 5% deﬁbrinated sheep blood agar (BBL Microbiology Systems, Cockeysville, MD) and aerobically incubated for 1 to 2 days at 37 °C with 5% CO2. To yield a DNA template, a single colony was suspended in 200 μl of 10 mM Tris (pH 8.0) within a 1.5-ml Millipore 0.22-μm-pore-diameter ﬁlter unit (Millipore, Bedford, MA) and heated at 95 °C for 20 min. The ﬁlter unit was then centrifuged at 8000 ×g for 2 min to recover the ﬁltrate containing the DNA template. DNA from non-Brucella spp. and DNA from tissue and body ﬂuid samples except urine were extracted with the QIAamp DNA Mini Kit (Qiagen). Isolation of genomic DNA from urine was accomplished using the QIAamp viral RNA mini kit (Qiagen).
2.3. Detection of Brucella spp. in tissue samples by culturing
2.5. Design of PCR primers and TaqMan probes
Thirty-seven tissue samples were submitted to the National Veterinary Services Laboratories (NVSL, Ames, Iowa, USA) for isolation and identiﬁcation of Brucella spp. in tissue samples.
Sequences of the IS711 gene from B. ceti B1/94 (FJ376557), B. ceti Cudo (ACJD01000007), Brucella sp. B1/94 (AF242533), B. pinnipedialis B2/94 (CP002079) and Brucella sp. JM13/00 (AB126349) were aligned
2.2. Sample collection
Table 1 Results of IS711 and ST27 real-time PCRs and MLST in Brucella strains. Strain
Brucella abortus 544 B. ceti B14-94 B. ceti B1/94 B. ceti B202R B. pinnipedialis B2/94 B. pinnipedialis 17A1 B. ceti 03-0312 B. ceti M04-0174 B. ceti 021MMS B. ceti IFAW12-087Dd B. ceti SC1135 B. ceti LA002 B. ceti LA001
Bovine Common dolphin Harbor porpoise Minke whale Common seal Hooded seal Dolphin vertebrae Dolphin placenta Bottlenose dolphin Common short beard dolphin Bottlenose dolphin Bottlenose dolphin Bottlenose dolphin
Type strain Scotland Scotland Norway Scotland Norway USA USA USA USA USA USA USA
1 26 23 23 25 Not tested 27 27 28 27 27 28 27
+ + + + + + + + + + + + +
− − − − − − + + − + + − +
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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 an extensive panel of marine and terrestrial Brucella spp. (Cloeckaert et al., 2011). The primers and probes were designed using PrimerQuest (http://www.idtdna.com/Primerquest/Home/Index). The IS711 speciﬁc primers IS711F (5′-TACCGCTGCGAATAAAGCCAAC-3′) and IS711R (5′TGAGATTGCTGGCAATGAAGGC-3′) were used to amplify a 150 bp fragment of the IS711 gene, which was detected by the probe, IS711P (FAM-5′-ACCCGGCCATTATGGTGACTGTCCGCA-3′-BHQ1). The ST27 speciﬁc primers ST27F (5′-TCGTTCAACACGCGTCGATCAT-3′) and ST27R (5′-TGCTCACGGCTGTTCTCCTTTA-3′) were used to amplify a 169 bp fragment of a speciﬁc IS711 chromosomal location of ST27, which was detected by the probe, ST27P (Hex-5′-TGGATCAAAGTGCG CCTTGCCGCGT-3′-BHQ1). The primers and probes were synthesized by Integrated DNA Technologies (Coralville, IA). Primer and probe speciﬁcities of the IS711 gene in Brucella spp. were veriﬁed by BLASTN analysis against all GenBank entries and evaluated using DNA extracted from Brucella strains listed in Table 1.
2.8. Analytical sensitivity
2.6. Real-time PCR analyses
2.10. Multilocus sequence typing (MLST)
PCR was performed in a volume of 15 μl with primer and probe concentration of 300 nM and 150 nM respectively, 7.5 μl of TaqMan® Universal PCR master mix (Applied Biosystems, Foster City, CA) and 1.5 μl DNA template in an Eco™ PCR system (Illumina, San Diego, CA). The PCR conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. DNA from B. ceti B1/94 was used as the positive template control. Nuclease-free water (Qiagen) instead of DNA extract was used as the no template control or negative control. All clinical samples and both negative and positive controls were run in triplicate. Ampliﬁcation efﬁciency (E) was estimated by the formula E = 10−1/slope − 1 (Bustin et al., 2009).
Genotyping based on multi-locus sequence analysis (MLSA) of nine house-keeping genes was performed as described previously (Whatmore et al., 2007). Full-length amplicons of the nine housekeeping genes from the seven B. ceti ﬁeld strains tested in this study were sequenced, concatenated and analyzed with the 27 classical Brucella sequence types described by Whatmore et al. Contig assembly, sequence editing and gene concatenation were performed in the DNASTAR Lasergene 8 software suite before multiple sequence alignments were constructed (DNASTAR Inc., Madison, WI). Pair wise analysis was performed and neighbor-joining consensus trees inferred from 1000 bootstrap replicates were constructed using MEGA version 4.0.
2.7. Construction of internal ampliﬁcation control (IAC) DNA
2.11. Statistical analysis
To assess whether inhibitors from tissue and body ﬂuid samples were present in the extracted DNA and causing false-negative PCR results, we constructed an internal ampliﬁcation control (145 bp) consisting of a fragment of the phyB gene coding region of potato root (Nolan et al., 2006) ﬂanked by the sequence matching the IS711F and IS711R primers. An amplicon was constructed from an oligonucleotide of the phyB gene (synthesized by Integrated DNA Technologies) in a conventional PCR using overhanging primers (IS711IACF 5′-TACCGCTGCGAATAAAGCCAACAACTTGGCTTTAATGG ACCTCCA-3′ and IS711IACR 5′-TGAGATTGCTGGCAATGAAGGCACAT TCATCCTTACATGGCACCA-3′). PCR was performed in reaction volumes of 20 μl with 10 μl of 2 × AmpliTaq Gold® 360 Master Mix (Applied 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, followed by 35 cycles of denaturation for 15 s at 95 °C, annealing for 15 s at 69 °C, elongation for 30 s at 72 °C and a ﬁnal extension step of 72 °C for 7 min. The amplicon from the ﬁrst PCR was puriﬁed using a QIAquick PCR Puriﬁcation Kit (Qiagen) and diluted at 1:1000 with nuclease-free water. The diluted amplicon was reampliﬁed with primers IS711F and IS711R using the process described above with one modiﬁcation: the annealing temperature was 59 °C instead of 69 °C. The ﬁnal amplicon was puriﬁed using the QIAquick PCR Puriﬁcation Kit and analyzed by an Agilent 2100 Bioanalyzer (Santa Clara, CA) to conﬁrm the presence of a single amplicon. The synthetically created DNA was used as IAC in every reaction mixture. The IAC was detected by the real-time PCR assay using the probe SPUD-T (Hex-5′-TGCACAAGCTATGGAACACCACGT-3′-BHQ1) (Nolan et al., 2006).
Cohen's kappa value was used to estimate agreement between real-time PCR and culture results with a 95% conﬁdence interval (CI). The following ranges were considered for interpretation of the kappa value (Landis and Koch, 1977): poor agreement: b 0.00; slight agreement: 0.00–0.20; fair agreement: 0.21–0.40; moderate agreement: 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 tissue and body ﬂuid samples on the threshold cycle (Ct) value of the IAC. Statistical signiﬁcance was considered for p-values b 0.05. Carcasses were categorized into age classes based on carcass length: fetus/neonate — ≤ 132 cm (Fernandez and Hohn, 1998); subadult — N132 cm and b240 cm; adult ≥ 240 cm (Mcfee and Hopkins-Murphy, 2002). A chisquare test was used to compare the prevalence of positive detections among age classes.
Genomic DNA isolated from B. ceti B1/94 was used to determine the analytic sensitivity of the assay. The quantity of Brucella genomic DNA was estimated by measuring the absorbance of DNA using the Spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, DE). Assuming a genome size of 3.28 Mb, 3.6 fg DNA was considered to be equivalent to one genomic DNA copy. A standard curve was developed 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 were extracted using the QIAquick PCR Puriﬁcation Kit and then run by the Agilent 2100 Bioanalyzer to conﬁrm the presence of a single amplicon prior to sequencing by SeqWright Inc. (Houston, TX) with primers IS711F and IS711R, respectively. The sequences of the amplicons were analyzed using BLASTN to assess homologies with sequences in the GenBank database.
3. Results 3.1. Sensitivity and speciﬁcity of the real-time PCR assay for detection of Brucella spp. We identiﬁed the primers and probe concentrations that optimize the ampliﬁcation of the IS711 gene fragment. The ampliﬁcation efﬁciency of the assay was 0.947 with a correlation coefﬁcient of 0.999 (Fig. 1). The limit of detection of the PCR assay was determined by testing serial dilutions of genomic DNA from B. ceti B1/94 and determined to be 0.27 fg of genomic DNA per PCR volume (Fig. 1). All Brucella strains included in this study were positive using this IS711 assay (Table 1). IS711 real-time PCR on non-Brucella pathogens
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Table 4 Comparison of culture and real-time PCR results for 37 tissue samples from bottlenose dolphins.
y = -3.4554x + 35.875 R² = 0.9992
Threshold cycles (Ct)
30 Positive Negative Total
9 18 27
0 10 10
9 28 37
20 15 10 -1
3.3. Performance of internal ampliﬁcation control (IAC)
Log concentraion of DNA (fg) Fig. 1. Standard curve developed using 10-fold serial dilution of DNA from B. ceti B1/94 ranging from 0.27 to 5.4 × 105 fg of genomic DNA per PCR volume. The regression line was for data that were in the linear range. The graph shows the mean values from four replicates for each dilution.
(listed in the Materials and methods section) did not result in any ampliﬁcation products.
The optimal IAC concentration (400 copies/reaction) was established based on the criterion that an IAC amplicon was always detected in samples containing 0–36 fg of genomic DNA from B. ceti B1/94 per reaction. The Ct value (34.7 ± 0.57, mean ± SD) for the IAC was recorded for all 369 real-time PCR runs, testing 123 tissue and body ﬂuid samples where no ampliﬁcation of Brucella DNA was detected. These results were compared to the Ct value (34.57 ± 0.45) recorded for the 36 real-time PCR runs of the 12 no template controls (p = 0.185). IAC was ampliﬁed successfully in all negative samples, demonstrating that the real-time PCR was not inhibited in these samples.
3.4. Conﬁrmation of amplicon identity by DNA sequence analysis 3.2. Detection of Brucella in clinical samples One hundred seventy eight tissue and body ﬂuid samples from 86 bottlenose dolphins were tested with the IS711 real-time PCR assay for 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 and amniotic ﬂuid (Table 2), representing 41% of the individual bottlenose dolphins tested by real-time PCR (Table 3). Brucella DNA was detected in 59% of fetus/neonates, 27% of subadults, and 33% of adult carcasses (Table 3). The chi-square test of independence (p = 0.02) indicated a relationship between age-class and detection of Brucella DNA. Brucella DNA was detected in the four carcasses that were believed to be fetuses based on total body length b100 cm (Neuenhoff et al., 2011) and the test showing that the lungs from the fetal-carcasses did not ﬂoat in water. Three of the fetuses were recovered without the mother (i.e., aborted fetuses), but one was removed from the mother at necropsy; in this case, Brucella DNA was detected in tissues from both the mother and fetus. In comparison to the detection of Brucella spp. in tissue samples between our real-PCR assay and the culture method, we detected Brucella DNA in 73% (27/37) of the tissue samples by real-time PCR, versus 24% (9/37) by culture (Table 4). Fair agreement was observed between real-time PCR and culture of tissue samples with a kappa value of 0.21 (95% CI, 0.05–0.37).
Table 3 Positive detection rates of the IS711 and ST27 real-time PCRs in clinical samples from bottlenose 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 Age class Fetus/neonate Subadult Adult Gender Male Female Undetermined Total
59% (19/32) 27% (9/33) 33% (7/21)
74% (14/19) 0 (0/9) 0 (0/7)
40% (18/45) 38% (15/39) 100% (2/2) 41% (35/86)
39% (7/18) 47% (7/15) 0 (0/2) 40% (14/35)
IS711 PCR products from two positive tissue samples were analyzed by the Agilent 2100 Bioanalyzer and showed a single band. The puriﬁed IS711 PCR products were sequenced and the results showed the same sequence for these PCR products. The outputs were compared with other known sequences in the GenBank database using BLASTN. They aligned with 100% identity to the sequence of IS711 gene fragment in seven strains of B. ceti and B. pinnipedialis and one strain of Brucella species 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 and B. pinnipedialis reference and ﬁeld strains. The seven B. ceti ﬁeld strains tested in this study were predominantly ST27 and a new sequence type that we are designating as ST28. This new B. ceti sequence type carries three unique single nucleotide polymorphisms at positions 198 in the gap gene, 1141 in the aroA gene and 3572 in the cobQ gene. This new marine ST shares a single nucleotide polymorphism with ST26 at position 3810 in the omp25 gene. The B. ceti ﬁeld isolates were recovered from tissues of stranded bottlenose dolphins from various areas of the United States.
3.6. Identiﬁcation of Brucella ST27 using real-time PCR A second real-time PCR assay was developed for the identiﬁcation of Brucella ST27. All DNA samples of B. ceti strains identiﬁed as ST27 by MLST were positive using this real-time PCR assay (Table 1). Other Brucella strains included in this study were negative using this assay (Table 1). The ST27 real-time PCR assay was used to identify this genotype ST27 in 54 IS711 PCR positive samples from 35 bottlenose dolphins. ST27 was identiﬁed in 33% of the clinical samples (Table 2) and 40% of bottlenose dolphins (Table 3); all positive detections for ST27 were in samples from fetus/neonate carcasses (Table 3). One IS711 positive urine sample was not tested with the assay due to insufﬁcient sample volume.
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4. Discussion We developed a real-time PCR assay using primers and a TaqMan probe for detecting the IS711 gene, or IS6501, which was speciﬁc for the Brucella genus (Ocampo-Sosa and Garcia-Lobo, 2008). Each genome of marine mammal Brucella species contains multiple copies of IS711 and the marine spp. have a higher number of copies of this insertion sequence relative to the terrestrial Brucella spp. (Bricker et al., 2000; Cloeckaert et al., 2011), which could explain the improved sensitivity of the real-time PCR assay. Real-time PCR has been used to detect a wide number of microorganisms of clinical importance. Real-time PCR assays targeting the IS711 gene have been used for detection of Brucella in clinical samples from wild boars and dromedaries (Hinić et al., 2009; Tomaso et al., 2010). The realtime PCR targeting IS711 was more sensitive, speciﬁc, efﬁcient and reproducible than the assays for bcsp31 and per genes to detect Brucella spp. (Bounaadja et al., 2009). The real-time PCR assay developed in this study proved to be highly speciﬁc for Brucella spp., since the target gene sequence was ampliﬁed in all known laboratory Brucella strains tested, but not observed in non-Brucella pathogens included in the study. The amplicon sequences from two positive tissue samples identiﬁed by real-time PCR aligned with 100% identity to the sequence of IS711 gene fragment in seven strains of B. ceti and B. pinnipedialis and one strain of Brucella species in North Paciﬁc common minke whales, further indicating the assay speciﬁcity for Brucella species. The cell numbers of Brucella in tissues and body ﬂuids are frequently very low (Al Dahouk et al., 2003; Bricker, 2002) and thus a highly sensitive assay was required for detection of Brucella in samples from marine mammals. The limit of detection of our real-time PCR assay was 0.27 fg of B. ceti B1/94 genomic DNA per reaction. This level of sensitivity was lower than or comparable to those (0.2– 24 fg genomic DNA of per reaction) reported for Brucella species using other assays (Al Dahouk et al., 2007; Bounaadja et al., 2009; Sidor et al., 2013; Tomaso et al., 2010). All of the culture-positive samples were detected as positive by our PCR assay and we detected additional positive samples in which no isolation was made by culture. Culture-negative but PCR positive samples may be due to either a higher sensitivity of the PCR assay, Brucella that are present but no longer viable, or contamination, but results from other studies also indicate that PCR has a higher sensitivity than culture (Sidor et al., 2013). However, it should be noted that a positive real-time PCR result only indicates the presence of the Brucella DNA; this does not necessarily indicate an active infection. PCR is a rapid and sensitive method for detecting Brucella DNA in samples, but bacterial culture and isolation, which can take weeks, is still an important tool in evaluating Brucella infection because it provides information that PCR cannot. In particular, bacterial isolation is necessary to obtain sufﬁcient genetic material for identiﬁcation of strain type, and while PCR detects both viable and nonviable organisms, only viable organisms can be cultured. In the serologic surveys of a managed population of 64 bottlenose dolphins (Meegan et al., 2012), 18 (28.1%) were seronegative for antibodies against Brucella, 18 (28.1%) were seropositive, and 28 (43.8%) had results interpreted as suspect. Bottlenose dolphins between the ages of 17 and 25 yr were 6.8 times more likely to be Brucella antibody positive compared to those that were younger or older (p = 0.02). In this report, Brucella spp. DNA was detected in 31% of the clinical samples from stranded bottlenose dolphins. Increased detection of the IS711 gene fragment in individuals classiﬁed as fetus/neonate, suggests that these young dolphins are at a higher risk for Brucella infection. This is not unexpected given that in terrestrial species, Brucella is known to cause poor reproductive outcomes, including spontaneous abortion, stillbirth and weak calves (CFSPH, 2009) and cases of Brucella-associated abortion have also been reported in cetaceans (Miller et al., 1999).
One major limitation of PCR in clinical samples is the possibility of polymerase inhibition by the presence of inhibitory compounds and excessive host DNA in biological samples (Al Dahouk et al., 2007; Bricker, 2002). We addressed this by including an internal ampliﬁcation control in our PCR to assess the potential for PCR inhibition in false-negative samples. We found no signiﬁcant difference in Ct values between IS711 negative clinical samples and no template controls, indicating that PCR inhibition was not occurring in our assay. Brucella strain F5/99 from a bottlenose dolphin in the United States and all three isolates (isolate 02/611 from New Zealand and isolates 01A09163 and 85A05748 from Peru) from naturally acquired human infections with marine mammal Brucella species have been characterized as having the same genotype, ST27 (Whatmore et al., 2008). Brucella isolates from marine mammals were tested for their ability to infect human macrophage cells (Maquart et al., 2009). The human isolate 02/611 was effectively virulent in human THP-1 macrophage cells to the same extent as the virulent strains Brucella melitensis 16M and Brucella suis 1330 reference strains were (Maquart et al., 2009). These ﬁndings indicated the zoonotic potential associated with genotype ST27 in humans. We developed a real-time PCR assay for rapid identiﬁcation of Brucella genotype ST27 by targeting an IS711 chromosomal location for the genotype (Cloeckaert et al., 2011). All Brucella strains which had been identiﬁed as ST27 by MLST were positive by our ST27 real-time PCR and no ampliﬁcation was found for other Brucella strains included in this study, indicating the assay speciﬁcity for genotype ST27. To our knowledge, this report is the ﬁrst description of the use of realtime PCR for identiﬁcation of Brucella genotype ST27. Of the 178 clinical specimens tested from bottlenose dolphins stranded along the US, South Carolina coastal region between 2010 and 2013, 31% were IS711 positive and 33% of those IS711 positive samples were ST27 positive. All positive reactions for ST27 were observed in fetus or neonates, indicating that this age class may be more susceptible to genotype ST27 infections. This is the ﬁrst documentation of the detection of the marine mammal Brucella spp. ST27 in wild cetaceans on the eastern coast of the United States. 5. Conclusions Two real-time PCR assays were developed using primers and probes speciﬁc for Brucella species and Brucella genotype ST27, respectively. The results in this study demonstrate the use of real-time PCR for rapid detection of the Brucella species and the identiﬁcation of the marine mammal Brucella genotype ST27. Inclusion of an internal ampliﬁcation control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. Acknowledgments We would like to thank Brian Thompson, for providing non-Brucella bacteria. We would like to thank Christine Quance of the National Veterinary Services Laboratories in Ames, Iowa, USA for analyzing the samples with the culture methods. Funding for this research was provided by NOAA's Oceans and Human Health Initiative and the National Marine Fisheries Service Marine Mammal Health and Stranding Response Program. This publication does not constitute an endorsement of any commercial product or intend to be an opinion 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: serological 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 identiﬁcation of Brucella spp Clin. Chem. Lab. Med. 45, 1464–1470.
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