J Med Microbiol 57 (2008), 1223-1227; DOI: 10.1099/jmm.0.2008/002337-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Development of a PCR assay for the identification of Salmonella enterica serovar Brandenburg
Kalyani Perera
and
Alan Murray
Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, Private Bag 11 222, New Zealand
Correspondence
Kalyani Perera
kalyani.perera{at}agresearch.co.nz
Received 7 April 2008
Accepted 30 June 2008
Currently, Salmonella enterica serovar Brandenburg is identified serologically on the basis of two surface antigens, somatic (O) polysaccharide and flagellar (H) proteins. This procedure is time-consuming and requires expensive typing reagents. To overcome these problems, a PCR method was developed and validated for the identification of S. Brandenburg. Portions of the invA, rfbJ(B), fliC and fljB genes were targeted for amplification using four pairs of oligonucleotide primers. To validate the assay, genomic DNA from an array of 72 Salmonella strains representing 28 serotypes and 5 non-Salmonella strains from 4 different genera was subjected to PCR. The four targeted genes were correctly amplified only from S. Brandenburg. These results indicate that this PCR assay is a simple, rapid, reliable and reproducible method for the identification of S. Brandenburg that will aid in surveillance, prevention and control of this pathogen.
Present address: AgResearch, National Centre for Biosecurity and Infectious Disease–Wallaceville, Upper Hutt, New Zealand. 
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INTRODUCTION
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Since 1996, Salmonella enterica serovar Brandenburg has been a major cause of ovine abortions and mortality, leading to significant economic impact to farmers in New Zealand (Clark, 2000; Clark et al., 2004; Roe, 1999; Smart, 2000). Sheep that have recovered from clinical disease may become carriers and excrete organisms in faeces. S. Brandenburg has also been isolated from humans and other animals, including horses, cattle, goats, deer, pigs and dogs. In New Zealand, human cases occurred through contact with infected animals and not through consumption of animal products (Clark et al., 2004). Therefore, rapid detection of S. Brandenburg would aid in controlling the spread of this pathogen to both animals and humans.
The number of Salmonella serotypes reported by 2002 was 2541 (CDC, 2007). The somatic O-antigen, together with phase-1 (H1) and phase-2 (H2) flagellar antigens, forms the basis for Salmonella serotyping. Each Salmonella serotype has a unique combination of O, H1 and H2 antigens. Laboratory methods for the identification of S. Brandenburg include serotyping, which is performed by the identification of O, H1 and H2 antigens according to the Kauffmann–White scheme (Popoff & Le Minor, 2001). This procedure is time-consuming, laborious and costly. Rapid and inexpensive PCR assays for the detection of Salmonella at the genus level have been developed (Rahn et al., 1992), but the number of assays to determine the Salmonella serotype is limited and includes those to detect serotypes Enteriditis (Lampel et al., 1996), Gallinarum (Shah et al., 2005), Typhi (Farrell et al., 2005; Kumar et al., 2006) and Typhimurium (Leon-Velarde et al., 2004). To date, there is no serotype-specific PCR assay for the detection of S. Brandenburg.
Somatic O-antigens are polymers of the O-subunit, and the rfb gene clusters are responsible for much of its variation. Variability of O-antigens among serogroups is due to differences in the type and arrangement of sugars in the O-subunit (Fitzgerald et al., 2003; Gillespie et al., 2003). The filament of flagella is a polymer of flagellin monomers that are composed of conserved terminal regions and a central variable region (Joys, 1985; Wei & Joys, 1985). The latter is responsible for flagellar antigenic specificity among serotypes (Newton et al., 1991). S. Brandenburg carries serogroup B-specific O : 4 antigen, H1 antigens l, v, and H2 antigens e, n, z15. These are encoded by rfbJ(B), the l and v alleles of fliC and the e, n and z15 alleles of fljB genes, respectively. Presence of invA is specific to Salmonella, and the combination of rfbJ(B), fliC (l, v) and fljB (e, n, z15) is specific to S. Brandenburg. Therefore, the amplification of invA will differentiate Salmonella from non-Salmonella isolates, and amplification of rfbJ(B), fliC (l, v) and fljB (e, n, z15) will differentiate S. Brandenburg from other Salmonella serotypes. We describe here the development of a PCR method targeting invA, rfbJ(B), fliC and fljB genes for the detection of S. Brandenburg from suspected Salmonella colonies.
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METHODS
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Bacterial strains.
A total of 72 Salmonella strains belonging to 28 different serotypes (Table 1
) and 5 non-Salmonella strains belonging to 4 different genera (Table 2) were used in this study. Salmonella serotypes carrying different O- and H-antigens were used. Salmonella serotypes Potsdam and Livingstone were obtained from the New Zealand Reference Culture Collection, Medical Section, Institute of Environmental Science & Research, Porirua, Wellington, New Zealand. Professor Peter R. Reeves (Department of Microbiology, University of Sydney, Australia) kindly provided genomic DNA of Salmonella strains Abortusovis, Azteca, Ball, Bredeney, Budapest, Jos, Gloucester, Mono, Togo and Wien. All other Salmonella and non-Salmonella strains were obtained from the Institutes of Veterinary, Animal & Biomedical Sciences (IVABS) and Molecular Biosciences (IMBS) of Massey University, Palmerston North, New Zealand.
DNA extraction.
Genomic DNA was extracted from cell suspensions of bacteria grown overnight on xylose-lysine-deoxycholate (XLD) agar at 37 °C. A single colony was resuspended in 30 µl distilled water and boiled at 100 °C for 10 min. The sample was immediately cooled on ice for 5 min, and centrifuged at 13 000 g at 4 °C for 10 min. The supernatant, containing DNA, was used as the template for PCR amplification.
PCR primers.
The target genes selected in this study were invA (Salmonella-specific), rfbJ(B) (serogroup B), fliC (l, v) and fljB (e, n, z15). Oligonucleotide primers (Table 3) used for the amplification of invA, rfbJ(B) and fljB genes were from Rahn et al. (1992), Luk et al. (1993) and Echeita et al. (2002), respectively; the pair used for amplification of fliC was designed using the sequence of region IV of the variable domain of S. Brandenburg fliC gene (GenBank accession number AY935580). All primers were synthesized by Life Technologies in the desalted form.
DNA amplification and detection.
The invA, fliC and fljB genes were amplified in a multiplex PCR and the rfbJ(B) gene was amplified in a separate reaction. Multiplex PCR amplification was performed in 0.2 ml thin-walled PCR tubes in a reaction volume of 50 µl, consisting 1 µl genomic DNA, 200 nM each primer invAF, invAR, fliCF, fliCR, fljBF and fljBR, 200 µM each of dCTP, dGTP, dATP and dTTP, 2 mM MgCl2, 1x PCR buffer [20 mM Tris/HCl (pH 8.4), 50 mM KCl], 1.25 U of Platinum Taq DNA Polymerase (Life Technologies) and sterile distilled water. The reactions were subjected to a single cycle of denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation (95 °C for 1 min), annealing (60 °C for 30 s), elongation (72 °C for 30 s) and a final elongation (72 °C for 7 min) in a GeneAmp 9600 thermocycler (Perkin Elmer). The rfbJ(B) gene was amplified separately in a 50 µl reaction volume containing the same PCR reagents as the multiplex reaction, replacing the primers with rfbJ(B)F and rfbJ(B)R. The thermocycler parameters were as above, except that annealing was done at 55 °C for 1 min and elongation was at 72 °C for 1 min. A negative control containing sterile distilled water in place of genomic DNA was included in each PCR assay. Reproducibility of the assay was confirmed by testing each strain in duplicate three times. The completed reactions were held at 4 °C for direct use or stored at –20 °C.
Multiplex PCR-amplified sample (10 µl) was mixed with 10 µl of the single reaction and electrophoresed on 2.5 % (w/v) agarose gels in 1x TAE buffer. One-kb plus DNA ladder marker (Life Technologies) was used as a size reference. Following electrophoresis, gels were stained with 0.5 µg ethidium bromide ml–1 and visualized on a UV transilluminator. The four PCR products obtained from S. Brandenburg amplification were excised from the agarose gel and purified using the QIAquick gel extraction kit (Qiagen). Products were sequenced by the Sequencing Facility of Massey University with the ABI Prism BigDye Terminator Sequencing Reaction kit on a Capillary ABI3730 DNA analyser (Applied Biosystems). DNA sequences were edited using Chromas software and analysed with the BLASTN network server at http://www.ncbi.nlm.nih.gov/blast/.
Validation.
Genomic DNA extracted from a panel of Salmonella strains representing 28 different serotypes and non-Salmonella strains from 4 genera were PCR-amplified to evaluate the validity of the PCR assay.
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RESULTS AND DISCUSSION
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Preliminary experiments to amplify invA, fliC, fljB and rfbJ(B) in a multiplex PCR did not yield consistent results with the rfbJ(B) gene. Therefore, this segment was amplified in a separate reaction. Fig. 1 shows the amplification products from a representative number of Salmonella strains used in the study. The invA gene was amplified in all Salmonella isolates but the rfbJ(B) gene was only amplified in serogroup B isolates. While fliC primers amplified only the Salmonella isolates carrying l, v alleles, fljB primers amplified only those carrying e, n, z15 alleles (Table 1
). The non-Salmonella strains did not produce amplification products under these conditions (Table 2). Only S. Brandenburg strains gave all four amplification products of the expected sizes of 883 bp (rfbJB), 285 bp (invA), 222 bp (fliC) and 135 bp (fljB). The primer pair sets did not show any cross-reactivity with Salmonella strains harbouring other somatic and flagellin gene groups. Sequencing and BLASTN analysis of the amplicons of S. Brandenburg confirmed that only the correct gene fragments had been amplified. All 25 S. Brandenburg isolates used in this study amplified all 4 genes. Repeated PCRs gave identical results.
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Fig. 1. PCR amplification of invA, rfbJ(B), fliC (l, v) and fljB (e, n, z15) genes. PCR products were electrophoresed on a 2.5 % (w/v) agarose gel, stained with ethidium bromide and photographed under UV light. Lanes: M, One-kb plus DNA ladder marker; 1–5, S. Brandenburg; 6, S. Azteca; 7, S. Bredeney; 8, S. Jos; 9, S. Budapest; 10, S. Abortusovis; 11, S. Wien; 12, S. Ball; 13, S. Gloucester; 14, S. Mono; 15, S. Togo; 16, S. Havana; 17, S. Newington; 18, Klebsiella edwardsii.
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The PCR assay developed in this study was based on the amplification of invA, rfbJ(B), fliC and fljB genes directly from a single Salmonella colony. invA was targeted for the diagnosis of Salmonella organisms at the genus level, and the combination of three polymorphic genes, rfbJ(B), fliC and fljB, was targeted for the identification of S. Brandenburg at the serotype level. invA, located on the pathogenicity island 1 of Salmonella species, is essential for invasion of epithelial cells (Collazo & Galán, 1997). It is present in all invasive strains of Salmonella (Galán, 1996) and absent from closely related genera such as Escherichia (Bäumler et al., 1998). The invA gene was amplified from all 72 Salmonella strains, while it was not amplified from non-Salmonella strains belonging to the 4 genera. These results agree with Rahn et al. (1992), who designed and established the specificity of this widely used pair of primers. They showed that 630 Salmonella strains representing over 100 serotypes yielded the target band of 285 bp, whereas two strains from each of the serotypes Litchfield and Senftenberg were negative. The strains that were shown to lack invA sequences were from environmental samples and were not specifically associated with disease (Ginocchio et al., 1997). Furthermore, the invA primer pair was also used in a European research project in the validation and standardization of PCR for the detection of Salmonella species in food (Hoorfar, 1999). Malorny et al. (2003) showed that one S. Saintpaul strain did not give the target band. It is speculated that invA is absent in these strains, which are not invasive, or that they might be using other invasive mechanisms. However, absence of invA in Salmonella seems to be rare (Malorny et al., 2003).
The lipopolysaccharide O-antigen together with H1 and H2 flagellar antigens forms the basis for Salmonella serotyping (Popoff & Le Minor, 2001). The O-antigen specificity is based on the sugar composition and arrangement in the O-subunit (Fitzgerald et al., 2003). The O-subunit of serogroup B salmonellae is an oligosaccharide of four sugar residues. Of these four sugars, three form a mannosyl–rhamnosyl–galactose backbone common to some other serogroups such as A and D. The fourth sugar is a dideoxyhexose substituted on the mannosyl residue. While the dideoxyhexose sugar present in the O-subunit of serogroups A and D is paratose and tyvelose, respectively, that of both serogroups B and C2 is abequose. Abequose confers on serogroup B strains their specific O : 4 antigen (Wyk & Reeves, 1989). The rfb gene cluster encodes the enzymes for biosynthesis of O-antigens. The rfbJ gene encodes abequose synthase, which is responsible for the final specific step in the synthesis of abequose. There is a 44 % difference between the nucleotide sequence of rfbJ gene of serogroup B [rfbJ(B)] and that of serogroup C2 [rfbJ(C2)] (Luk et al., 1993; Wyk & Reeves, 1989). Luk et al. (1993) designed a pair of primers targeting the serogroup B specific rfbJ gene that was used in this study and as expected, it amplified only the rfbJ(B) gene. In S. Brandenburg, H1 and H2 flagellins belong to the L- (l, v) and E-complex (e, n, z15), respectively. Region IV of Salmonella flagellin is the most variable, and is believed to carry the major serotype-specific epitopes of the flagellar antigens (Newton et al., 1991; Wei & Joys, 1985). Some of the flagellar antigens are composed of a single antigenic factor (b, c, d, i, r) while others are composed of multiple antigenic factors (l, v; l, w; e, h; e, n, x; e, n, z15; 1, 2). Flagellar antigens are grouped into complexes depending on the antigenic factors that they share. Examples are the E-complex, which contains antigenic factor e (e, h; e, n, x; e, n, z15) and the L-complex, which contains antigenic factor l (l, v; l, w; l, z13, l, z28) (Popoff & Le Minor, 2001). The two primer pairs used to amplify fliC and fljB (Echeita et al., 2002) were from region IV of the variable region and did not amplify other E- and L-complex genes of the Salmonella strains used in this study. This shows the specificity of these two pairs of primers for l, v and e, n, z15 alleles of fliC and fljB genes, respectively.
All of the S. Brandenburg strains in this study were obtained from clinical samples (uterine swabs, faeces, intestinal contents and meat) collected from sheep, cattle and pigs, and sheep yard dust. Pure cultures of S. Brandenburg were used for the initial evaluation of the assay because PCR assays are prone to inhibition by substances in the samples (Iijima et al., 2004). All 25 S. Brandenburg cultures that were confirmed by serotyping were positive in the PCR, with no false-negative reactions. The PCR described here detected the combination of three genes [rfbJ(B), fliC (l, v) and fljB (e, n, z15)] that is unique to S. Brandenburg. A combination of enrichment, subcultures, biochemical tests and serotyping is currently used for the identification of S. Brandenburg and the whole process normally takes approximately 7 days. With the PCR assay described in this study, identification of pure cultures of Salmonella to the serotype Brandenburg could be done in approximately 8 h. In total, the time taken from receipt of the clinical sample to confirming the identity of the organism by this PCR assay will be approximately 4 days. Since this assay requires only genomic DNA, it does not depend on the expression of antigens, thus making the need for time-consuming phase reversal of flagellin antigens unnecessary.
The PCR assay described here will be a useful complement to traditional culture and serotyping techniques for the identification of S. Brandenburg in clinical samples.
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ACKNOWLEDGEMENTS
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The authors wish to thank IVABS at Massey University for providing financial support of this project through the Postgraduate Research Fund and a Lewis Fitch Grant. We also wish to thank Associate Professor Stanley Fenwick at Murdoch University, Australia, for his input, Professor Peter R. Reeves and Gordon Stevenson at University of Sydney for providing us with genomic DNA from some of the Salmonella isolates, and Barbara Asmundson at Massey University for her help with some Salmonella cultures. Kalyani Perera is grateful to Massey University for the Massey Doctoral Scholarship.
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INTRODUCTION
METHODS
