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  1. 1. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2006, p. 4256–4263 Vol. 72, No. 60099-2240/06/$08.00ϩ0 doi:10.1128/AEM.02706-05Copyright © 2006, American Society for Microbiology. All Rights Reserved. Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and vapA Genotyping of Rhodococcus equi† ´guez-Lazaro,1 Deborah A. Lewis,1 Alain A. Ocampo-Sosa,1,3 Ursula Fogarty,3 David Rodrı ´ Laszlo Makrai,4 Jesus Navas,5 Mariela Scortti,1,2 Marta Hernandez,1 ´ ´ ´ ´ and Jose A. Vazquez-Boland1,2* ´ ´ Bacterial Molecular Pathogenesis Group, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford, United Kingdom1; Facultad de Veterinaria, Universidad de Leon, Leon, Spain2; Irish Equine Centre, Johnstown, Naas, Ireland3; Department of ´ ´ Microbiology and Infectious Diseases, Faculty of Veterinary Science, Budapest, Hungary4; and Departamento de Biologı Molecular, Facultad de Medicina, Universidad de Cantabria, Santander, Spain5 ´a Received 15 November 2005/Accepted 11 April 2006 We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA؉ and vapA-negative (including vapB؉) plasmid genotypes. The vapA؉ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoal- veolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to Ϸ10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA؉) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA؉ “horse- pathogenic” genotype determination. Rhodococcus equi is a soil-dwelling actinomycete of the my- mediastinal pyogranulomatous lymphadenitis (6) and in pigscolata group that causes pyogranulomatous infections in the from submaxillary lymph nodes (18).lungs and other different body locations in a variety of animal Horse isolates of R. equi typically harbor an 85- to 90-kbhosts. This facultative intracellular parasite is well known in virulence plasmid, of which an example has been fully se-veterinary medicine as the causal agent of foal pneumonia, a quenced (11, 32). This plasmid encodes virulence-associatedsevere purulent bronchopneumonic infection with high case- protein A or VapA, a 17.4-kDa surface lipoprotein presumedfatality rates. The disease is recognized in many countries as to be involved in pathogenesis but whose role in the infectiousthe leading cause of mortality in foals and is a cause of serious process remains unknown (10, 14). VapA is encoded by theconcern to the equine industry as it can become endemic in vapA gene, which is present in a plasmidic, 27.5-kb pathoge-stud farms and there is no effective vaccine for its prevention nicity island together with six other vapA homologues (32). In(9, 23). In recent years R. equi has emerged as an opportunistic nonhorse R. equi isolates, including human isolates, the VapAhuman pathogen, especially in individuals infected with human protein/vapA gene is much less frequently found than a variantimmunodeficiency virus. In the human host the infection pre- protein/allele designated VapB/vapB. The VapB antigen issents usually as tuberculosis-like cavitary pneumonia or bacte- structurally and immunologically closely related to VapA but isremia (2, 36). R. equi is also being increasingly reported in larger (18.2 to 20 kDa as detected by sodium dodecyl sulfate-other animal species, mainly associated with extrapulmonary, polyacrylamide gel electrophoresis immunoblotting) and is en-purulent, caseating infections (5, 33). In cattle the organism is coded by plasmids of various sizes (79 to 100 kb), not yettypically isolated from chronic retropharyngeal, bronchial, or characterized genetically. The vapB plasmids are not found in equine isolates, suggesting that vapBϩ strains are not patho- genic for the horse (22, 31, 34). Except for soil isolates from * Corresponding author. Mailing address: Veterinary Molecular Mi- horse breeding farms, in which vapA-type plasmids are com-crobiology Section, Faculty of Medical and Veterinary Sciences, Uni- mon, environmental isolates of R. equi do not usually carryversity of Bristol, Langford BS40 5DU, United Kingdom. Phone: 44 plasmids, or if they do, these are smaller in size and most often(0) 117 928 9615. Fax: 44 (0) 117 928 9505. E-mail: v.boland@bristol.ac.uk. vapA and vapB negative (31). † Supplemental material for this article may be found at http://aem Laboratory diagnosis of rhodoccocal infections currently re-.asm.org/. lies on classical bacteriological methods involving the isolation 4256
  2. 2. VOL. 72, 2006 choE- AND vapA-TARGETED RHODOCOCCUS EQUI Q-PCR 4257of the organism from clinical samples or postmortem material scribed conventional choE-PCR test (15). R. equi isolate 103S from J. Prescott(23). However, these culture-based procedures are lengthy and (University of Guelph, Canada), deposited as PAM 1126 in our collection, was used as the reference strain. This strain was originally isolated from a case of foalsometimes lack adequate sensitivity due either to prior antibi- pneumonia and is currently being used for the determination of the completeotic treatments or, in the case of respiratory specimens (typi- genome sequence of R. equi by the International R. equi Genome Consortiumcally bronchoalveolar lavage [BAL] aspirate or sputum), to the (www.sanger.ac.uk/Projects/R_equi). A detailed list of R. equi isolates is availablepresence of multiple bacterial contaminants (28). An added in the supplemental material as Table S1. The non-R. equi isolates are listed in Table S2 in the supplemental material. Bacteria were maintained at Ϫ80°C in aproblem is the difficulties posed by the identification due to the medium containing 2% tryptone, 4% skimmed milk, and 16% glycerol. Rhodo-micro- and macroscopic morphological variability exhibited by coccus spp. were grown at 30°C in brain heart infusion (BHI) and non-Rhodo-these bacteria and the lack of accuracy of biochemical tests for coccus isolates at 37°C in YME medium (0.4% yeast extract, 1% malt extract,R. equi species determination (8, 15, 29, 36). There is therefore 0.4% glucose), supplemented with 1.5% agar for plate cultures. All media wereconsiderable interest in developing new, simpler tests for the purchased from Oxoid (Hampshire, United Kingdom), except BHI (from Difco- BD, Detroit, MI).rapid and reliable detection and identification of R. equi for DNA isolation and quantification. Bacterial genomic DNA was isolated fromuse in both veterinary and medical clinical microbiology labo- overnight cultures on solid medium using a cetyltrimethylammonium bromideratories. (CTAB)-based protocol. Bacterial colonies from half a petri dish were collected Several molecular methods for R. equi have been described with a loop, suspended in 1 ml phosphate-buffered saline, pelleted at 4,000 ϫ gbased on amplification of DNA sequences by conventional for 10 min, and incubated for 1 h at 37°C after resuspension in 567 ␮l Tris-EDTA buffer and 3 ␮l 100-mg/ml (30,000 units) lysozyme (Sigma) solution. Subse-PCR (1, 3, 12, 15, 22, 28, 30). Although comparatively faster quently, 30 ␮l 10% sodium dodecyl sulfate and 3 ␮l 20-mg/ml (1.8 units) pro-than culture-based methods, conventional PCR, however, pro- teinase K (Sigma) was added and the mixture was incubated again for 1 h atvides only qualitative results and requires post-PCR proce- 37°C. Then, 170 ␮l 5 M NaCl, 80 ␮l CTAB-NaCl solution (10% CTAB in 0.7 Mdures. The “open” post-PCR processing of massive amounts of NaCl), and 5 ␮l 100-mg/ml (50 Kunitz units) RNase A (Sigma) were added followed by a 30-min incubation at 65°C. After cooling to room temperature, theamplicon increases the risk of false-positive results due to mixture was extracted with phenol-chloroform and chloroform-isoamyl alcoholsample cross-contamination. This risk is minimized in the real- followed by DNA precipitation with isopropanol and washing with 70% ethanoltime PCR technique as the reaction and fluorescent probe- (27). DNA was resuspended in 100 ␮l 10 mM Tris-HCl, pH 8.0, and its amountbased amplicon detection are brought about simultaneously in and quality were determined spectrophotometrically by calculating the ratio ofa closed tube. Real-time monitoring of the amplification curve optical density at 260 nm to that at 280 nm and visually by agarose gel electro- phoresis.via fluorescence emission permits also a much more sensitive Oligonucleotides. The oligonucleotide primers and TaqMan probes used indetection of positive reactions and at the same time, impor- this study were designed using Primer Express 2.0 software (Applied Biosystems,tantly, an accurate quantification of the target DNA present in Foster City, CA) and purchased from Metabion AG (Martinsried, Germany).the sample (35). However, to date only one quantitative real- They are listed in Table 1. The IAC probe was labeled with HEX (6-carboxy- 2Ј,4,4Ј,5Ј,7,7Ј-hexachlorofluorescein) and the choE probe with 6-carboxyfluores-time PCR (Q-PCR) assay has been reported for R. equi (13). cein (FAM).This assay targets the vapA gene and therefore detects only IAC construction. The IAC consisted of a 100-bp chimerical DNA containingstrains carrying vapAϩ plasmids, which as mentioned above are a portion of the listeriolysin (hly) gene from Listeria monocytogenes (GenBankrarely found in human and most other nonhorse R. equi iso- accession no. M24199), which we previously validated as a Q-PCR probe targetlates, thus limiting its applicability to the field of equine med- (24), flanked by the R. equi-specific choE gene sequences targeted by reqF and reqR primers (Table 1). This chimeric DNA molecule was generated by twoicine. Moreover, the quantification accuracy of this assay can rounds of PCR as previously described (26). The first PCR used 1 ng L. mono-be compromised by strain-to-strain differences in plasmid copy cytogenes DNA template and primers riacF and riacR (Table 1), which containednumber or plasmid DNA extraction efficiency. the corresponding hly target sequences plus a 5Ј tail with the reqF or reqR primer We recently identified the R. equi cholesterol oxidase gene sequence. The second PCR used the purified first-round PCR product (diluted 1:1,000) as a template and the reqF and reqR primers. PCR conditions were aschoE and demonstrated that this chromosomal locus is univer- previously described (26). The IAC PCR product was purified using the QIAquicksally conserved in these bacteria (21) and is a suitable target for gel extraction kit (QIAGEN, Hilden, Germany), quantified, and diluted to thetheir specific and sensitive detection by conventional PCR appropriate concentration in 10 mM Tris-HCl, pH 8.0, in the presence of 500(15). Here we report the design and development of a novel ng/ml of acetylated bovine serum albumin as a blocking agent to minimizedual-reaction Q-PCR method that allows both the species- binding of the negatively charged IAC DNA to the plastic microtubes. With the exception of its target sequence in the L. monocytogenes hly gene (nucleotidespecific quantification of R. equi and determination of its positions 114 to 177), the IAC did not display significant similarity to any DNA“horse-associated” subtype via detection of choE and vapA sequence deposited in public databases, as determined by BLAST-N searchessequences, respectively. The method includes an internal am- (National Center for Biotechnology Information, Bethesda, Md.; http://wwwplification control (IAC) for monitoring the occurrence of .ncbi.nlm.nih.gov). The IAC amplicon, 100 bp in size, was longer than the 68-bpfalse-negative results due to PCR failure or inhibition. choE-specific amplicon, facilitating the differentiation of the two PCR products by gel electrophoresis. Q-PCR. The assays were performed essentially as described previously (24) in 20-␮l reaction volumes containing 1ϫ PCR buffer II; 6 mM MgCl2; 200 ␮M MATERIALS AND METHODS dATP, dCTP, and dGTP; 400 ␮M dUTP; 300 nM specific primers; 150 nM probe Bacterial strains, culture media, and growth conditions. A total of 255 bac- (for the duplex choE-IAC system, 100 nM of IAC probe was added); 1 unit ofterial strains were used in this study: 197 were Rhodococcus spp. (178 R. equi and AmpliTaq Gold DNA polymerase (Applied Biosystems-Roche Molecular Sys-19 non-R. equi isolates) and 58 belonged to different actinomycete genera, tems Inc., Branchburg, N.J.); 0.2 units of AmpErase uracil N-glycosylase; and 5including cholesterol oxidase-producing species. The R. equi strains included ␮l of the target DNA solution. Reactions were run on an iCycler IQ platformhorse (n ϭ 81), human (n ϭ 35), pig (n ϭ 30), bovine (n ϭ 8), soil (n ϭ 13), and (Bio-Rad Laboratories Inc., Hercules, CA) with the following program: 2 min atancillary (n ϭ 11, from sheep, goat, dog, cat, pheasant, primate, iguana, and 50°C, 10 min at 95°C, and 50 cycles of 15 s at 95°C and 1 min at 60°C. Q-PCRunknown origin) isolates from 14 countries (Argentina, Australia, Brazil, Can- results were analyzed using the Optical System Software v3.0a (Bio-Rad Labo-ada, China, Dominican Republic, Germany, France, Hungary, Ireland, Japan, ratories Inc., Hercules, CA). Quantification was obtained by interpolation in aSlovenia, Spain, and the United Kingdom). All were confirmed as R. equi by standard regression curve of cycle threshold (CT) values generated from samplesanalysis of colony morphology, API Coryne biochemical profiling, synergistic of known DNA concentrations. One molecule of R. equi DNA, or genomehemolysis (CAMP-like) test with Listeria ivanovii (21), and our previously de- equivalent, corresponds to approximately 5.5 fg of DNA considering a genome
  3. 3. 4258 ´ ´ RODRIGUEZ-LAZARO ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Oligonucleotides used in this study Oligonucleotide Tm a G-CTarget Application Sequence Reference(s) name (°C) (%)choE reqF Q-PCR forward primer 5Ј-CGA CAA GCG CTC GAT GTG-3Ј 59 61 This study reqR Q-PCR reverse primer 5Ј-TGC CGA AGC CCA TGA AGT-3Ј 59 56 This study reqP TaqMan probe 5Ј-FAM-TGG CCG ACA AGA CCG ATC AGC 69 64 This study C-TAMRAb-3Ј COX-F PCR forward primer 5Ј-GTC AAC AAC ATC GAC CAG GCG-3Ј 62.3 57.1 15 COX-R PCR reverse primer 5Ј-CGA GCC GTC CAC GAC GTA CAG-3Ј 64.7 66.7 15vapA RvapA114F Q-PCR forward primer 5Ј-CAG CAG TGC GAT TCT CAA TAG TG-3Ј 59 48 This study RvapA188R Q-PCR reverse primer 5Ј-GAA GTC GTC GAG CTG TCA TAG CT-3Ј 59 52 This study RvapA140P TaqMan probe 5Ј-FAM-CAG AAC CGA CAA TGC CAC TGC 69 58 This study CTG-TAMRA-3Ј IP1 PCR forward primer 5Ј-AC TCT TCA CAA GAC GGT-3Ј 46 50 22, 30 IP2 PCR reverse primer 5Ј-TAG GCG TTG TGC CAG CTA-3Ј 55.1 55.6 22, 30vapB H1 PCR forward primer 5Ј-TGA TGA AGG CTC TTC ATA A-3Ј 47.6 36.8 22 H2 PCR reverse primer 5Ј-TTA TGC AAC CTC CCA GTT G-3Ј 53.2 47.4 22IAC IACP TaqMan probe 5Ј-HEX-CGC CTG CAA GTC CTA AGA CGC 68 61 24 CA-TAMRA-3Јhly riacF Forward primer IAC 5Ј-CGA CAA GCG CTC GAT GTG CAT GGC 81 63 This study construction ACC ACC-3Ј riacR Reverse primer IAC 5Ј-CGA CAA GCG CTC GAT GTG ATC CGC 78 57 This study construction GTG TTT-3Ј a Theoretical melting temperature. b TAMRA, 6-carboxytetramethylrhodamine.size of 5.2 Mb as determined for PAM 1126, according to the following equation: gene sequences deposited in public databases using theDNA amount in fg ϭ bp ϫ 660 Da/bp ϫ 1.6 ϫ 10Ϫ27 kg/Da ϫ 1 ϫ 10Ϫ18 fg/kg CLUSTALW multiple-alignment tool (European Bioinfor-(25). Q-PCRs with CT values of Ͼ50 were considered negative. The 95% con-fidence interval was calculated for every serial dilution according to a binomial matics Institute, EMBL; www.ebi.ac.uk). The new choE prim-distribution (with the statistical software SPSS 12.0S for Windows v8.0 [SPSS ers, reqF and reqR (Table 1), amplify a 100% specific, con-Inc., Chicago, Ill.]) (25, 26). Unless otherwise stated, all reactions were per- served 68-bp DNA fragment corresponding to positions 938 toformed in triplicate. 1005 of the coding sequence deposited in GenBank under Quantitative detection of R. equi in BAL fluid. An overnight culture in BHI of accession no. AJ242746 (21).R. equi PAM 1126 was centrifuged for 3 min at 3,000 ϫ g, the bacterial pellet wasresuspended in sterile phosphate-buffered saline, and the suspension was serially Gene regions suitable for vapA-specific Q-PCR oligonucle-10-fold diluted in BAL fluid obtained from a healthy adult horse with the use of otides were selected by visual inspection of CLUSTALW mul-0.9% NaCl intravenous infusion solution (Baxter Healthcare Corp.) as vehicle. tiple alignments of all known sequences of the vap multigeneThe concentration of R. equi in the BAL fluid dilutions was determined by family. These include, in addition to vapA, six other vap genesstandard plate counting. DNA was extracted from R. equi-contaminated BALdilutions as follows: 1-ml samples were transferred to clean 1.5-ml microtubes (vapC to -H, identified in vapAϩ virulence plasmids fromand centrifuged for 5 min at 10,000 ϫ g at 4°C; the pellets were resuspended in strains ATCC 33701 and 103) (32) and vapB identified in100 ␮l Instagene Matrix suspension (Bio-Rad Laboratories, Hercules, CA) by plasmids from “nonequine” R. equi isolates (22). Primer pairvortexing, and the suspensions were incubated at 56°C for 20 min; after 10 s of RvapA114F-RvapA188R amplifies a vapA-specific, conservedvigorous vortexing, these were incubated at 100°C for 8 min and then placed onice and centrifuged for 5 min at 14,000 ϫ g at 4°C; finally, 50 ␮l of the super- 75-bp DNA fragment corresponding to positions 114 to 188 ofnatants was transferred to a fresh microtube and stored at Ϫ20°C until use. Each the gene sequence deposited in GenBank with accession no.PCR used 5 ␮l of DNA preparation. NC002576 (32). The BLAST-N tool v.2.2.12 was used (with default settings and low-complexity filter off) to confirm in silico that none of RESULTS the selected oligonucleotides recognized any registered DNA Design and optimization of choE- and vapA-specific Q-PCR sequence other than the target sequence. Primers, TaqManassays. To specifically identify R. equi, we used the conserved probes, and MgCl2 concentrations were optimized for Q-PCRchoE gene (21) as a target. A previously developed conven- assays by using 1 ng of template DNA from R. equi strain PAMtional PCR assay based on detection of choE sequences was 1126. The minimum primer and probe concentrations that100% specific and sensitive for R. equi taking as a positive gave the lowest CT value and the highest fluorescence intensityresult the expected 959-bp amplicon (15). Using this PCR were retained as the standard optimal conditions (see Materi-assay, smaller products are occasionally observed with other als and Methods).rhodococcal species (reference 12 and our unpublished obser- Optimization of duplex choE-IAC Q-PCR assay. The opti-vations). To minimize the risk of unspecific reactions, we iden- mal IAC probe concentration (i.e., the minimum concentrationtified a new choE target region suitable for Q-PCR primers not resulting in an increase of CT) (26), 100 nM, was experi-and probe design by careful analysis of all cholesterol oxidase mentally determined by performing Q-PCRs in the presence of
  4. 4. VOL. 72, 2006 choE- AND vapA-TARGETED RHODOCOCCUS EQUI Q-PCR 42591,000 IAC molecules, no R. equi DNA, 150 nM FAM-labeledchoE probe, and increasing amounts (from 25 to 250 nM) ofHEX-labeled IAC probe. Since an excess of IAC may inhibitthe target-specific reaction, Q-PCRs were also carried out inthe presence of various IAC amounts (10,000, 1,000, 100, and10 molecules per reaction) and a fixed amount (30 genomeequivalents) of R. equi PAM 1126 DNA. The maximum IACamount with no inhibitory effect on the choE-specific FAMsignal was 100 copies. Specificity and sensitivity of the choE-IAC Q-PCR. The ca-pacity of the choE Q-PCR assay to discriminate between targetand nontarget bacteria was assessed using 1 ng of genomicDNA from 178 R. equi strains from a variety of sources (in-cluding clinical isolates from different animal species and en-vironmental isolates), 19 non-R. equi rhodococcal speciesstrains, and 58 strains from 18 different non-Rhodococcusactinomycete genera. The choE Q-PCR assay was 100% sen-sitive and 100% specific as all 178 R. equi strains tested gave a FIG. 1. Distribution of the vapA allele according to isolate origin.positive choE signal whereas none of the 77 nontarget bacteria The number of isolates within each category is indicated above the bars; within the bars are the percentages of vapAϩ (gray section) anddid (detailed results in Table S1 in the supplemental material). vapA-negative (empty section) isolates. “Other” includes sheep, goat,Rhodococcus fascians, reported by others (12) to give an un- dog, cat, pheasant, primate, iguana, and unknown origin. Most soilspecific (smaller) amplicon by conventional PCR using our isolates are from equine-related environments, explaining the rela-previously described primers (15), did not give any significant tively high percentage of vapAϩ isolates (31).signal in the choE Q-PCR assay. All the reactions generated apositive IAC (HEX) signal, ruling out that the absence of choE(FAM) signal observed in non-R. equi isolates was due to curves obtained with each Q-PCR assay; Table 3 shows thefailure of the PCR. All the nonactinomycete bacteria that we mean CT values for a total of nine replicates in three indepen-have tested to date, including a variety of common gram neg- dent experiments. The two Q-PCR assays yielded similar re-atives and gram positives, have yielded negative results in the sults in terms of absolute detection values. Positive amplifica-choE Q-PCR assay (not shown). tion in all nine replicates of each DNA dilution was achieved Specificity and sensitivity of the vapA Q-PCR. As with the when 10 or more target molecules were present, and as few aschoE PCR, none of the 77 nontarget bacteria (non-Rhodococ- one target molecule could be detected with 67 to 78% proba-cus strains and non-R. equi rhodococci) gave a positive ampli- bility for choE- and vapA-based Q-PCR assays, respectivelyfication signal with the vapA Q-PCR. However, this assay de- (Table 3). The slopes of the linear regression curves calculatedtected the target sequence in only 48% of R. equi isolates (85 over a 6-log range were similar to the theoretical optimum ofout of 178), as expected from the varied composition of the Ϫ3.32 (26) (choE, Ϫ3.337; choE-IAC duplex reaction, Ϫ3.335;strain panel tested, which contained only a proportion of vapA, Ϫ3.379) and showed that the amplifications were veryhorse-derived bacteria (see above and Table S1 in the supple- efficient (E ϭ 0.994 Ϯ 0.001, 0.995 Ϯ 0.002, and 0.977 Ϯ 0.002mental material). The distribution of vapAϩ isolates per ani- for choE-, choE-IAC-, and vapA-based Q-PCR assays, respec-mal species is shown in Fig. 1. tively). Moreover, R2 values were above the optimal 0.995 The discrimination capacity of the vapA Q-PCR assay was (0.998 for choE and choE-IAC reactions, 0.999 for vapA reac-assessed on a selection of 60 R. equi strains using as reference tion), indicating that the Q-PCRs that we developed are ap-method a previously described dual-reaction conventional propriately linear. The confidence intervals based on the stan-PCR system that differentiates vapAϩ isolates from vapB-neg- dard deviations of CT values did not overlap each other downative or vapA- and vapB-negative isolates using two pairs of to 10 target molecules, indicating that reliable quantificationprimers (22). Prior to applying this method, we confirmed its was possible above this limit.100% efficacy on a representative sample of R. equi strains with Quantitative detection of R. equi in BAL fluid. We assessedknown vapA/B genotypes (17, 18). As shown in Table 2, there the applicability of the choE-IAC Q-PCR for the quantitativewas a perfect concordance between the results obtained by the detection of R. equi bacteria in artificially contaminated BALtwo techniques. As all the strains tested positive with the choE- fluid. Taking into consideration that 100 ␮l of DNA was ob-IAC system, these results indicated that our vapA Q-PCR assay tained from the processing of 1 ml of BAL fluid, and that 5 ␮lis 100% specific and 100% sensitive. of DNA preparation was used for the PCR, our choE-IAC Detection and quantification limits of the choE-IAC and assay consistently detected down to approximately 100 R. equivapA Q-PCR assays. The detection and quantification limits of cells/ml or 5 genomic units per reaction (Table 4). Quantitativethe developed PCR assays were determined by using R. equi amplification parameters were optimal, with linearity (R2)PAM 1126 genomic DNA. Amplification reactions were per- above 0.99 down to 1 ϫ 103 R. equi CFU/ml and overall PCRformed with a range of DNA concentrations equivalent to efficiency of 0.94. Relative accuracy values (24, 27) rangedapproximately 1 ϫ 106, 1 ϫ 105, 1 ϫ 104, 1 ϫ 103, 1 ϫ 102, 10, between 88.95% and 113.53%, indicating a high degree ofand 1 target molecule. Figure S1 in the supplemental material correspondence between the quantitative results obtainedillustrates typical amplification profiles and the regression by the reference method (number of R. equi CFU/ml as
  5. 5. 4260 ´ ´ RODRIGUEZ-LAZARO ET AL. APPL. ENVIRON. MICROBIOL. determined by plate counting) and the results obtained by TABLE 2. Specificity and sensitivity of vapA Q-PCRa the choE Q-PCR method (Table 4). Conventional Q-PCR Strain Origin PCR resultb result Concordancec DISCUSSION vapA vapB for vapA We describe here a Q-PCR method that permits the sensi-PAM 1126 Horse ϩ Ϫ ϩ ϩ tive and specific, accurate quantitative detection of the patho-PAM 1286 Iguana Ϫ Ϫ Ϫ ϩPAM 1335 Horse ϩ Ϫ ϩ ϩ genic actinomycete R. equi. This is achieved by targeting se-PAM 1340 Horse ϩ Ϫ ϩ ϩ quences from the chromosomal choE gene, previouslyPAM 1346 Horse ϩ Ϫ ϩ ϩ identified in our laboratory and shown to provide a usefulPAM 1348 Soil Ϫ Ϫ Ϫ ϩ marker for the molecular detection and identification of R.PAM 1350 Soil ϩ Ϫ ϩ ϩPAM 1351 Soil ϩ Ϫ ϩ ϩ equi (15, 21). A previous Q-PCR assay for R. equi, recentlyPAM 1358 Horse ϩ Ϫ ϩ ϩ reported by others (14), targets the plasmidic gene vapA andPAM 1365 Horse ϩ Ϫ ϩ ϩ thus detects only R. equi bacteria carrying this allelic variant.PAM 1367 Horse ϩ Ϫ ϩ ϩ vapAϩ strains are associated with infections in the horse andPAM 1371 Horse ϩ Ϫ ϩ ϩ hence are predominantly found in equine-associated speci-PAM 1374 Horse ϩ Ϫ ϩ ϩPAM 1376 Human Ϫ ϩ Ϫ ϩ mens. However, R. equi can be isolated from a variety of otherPAM 1387 Unknown Ϫ Ϫ Ϫ ϩ animal species in which, with few exceptions, vapAϩ strains arePAM 1404 Horse ϩ Ϫ ϩ ϩ rarely found (17, 19, 22, 34) (Fig. 1). Indeed, our data showPAM 1406 Human Ϫ ϩ Ϫ ϩ that only a small proportion (20%) of human clinical isolatesPAM 1408 Horse ϩ Ϫ ϩ ϩPAM 1410 Horse ϩ Ϫ ϩ ϩ are vapAϩ, consistent with previously reported figures on thePAM 1413 Human Ϫ ϩ Ϫ ϩ prevalence of VapAϩ/vapAϩ R. equi bacteria in human speci-PAM 1414 Human Ϫ ϩ Ϫ ϩ mens (12, 22, 34). The vapAϩ plasmid genotype appears to bePAM 1415 Human Ϫ Ϫ Ϫ ϩ particularly rare among bovine and pig isolates, as shown byPAM 1416 Horse ϩ Ϫ ϩ ϩ our data (0% positives; Fig. 1) and other studies (6, 17). Over-PAM 1418 Horse ϩ Ϫ ϩ ϩPAM 1422 Horse ϩ Ϫ ϩ ϩ all, more than 50% of the isolates tested in this study werePAM 1424 Horse ϩ Ϫ ϩ ϩ vapA negative (Fig. 1), clearly showing that a vapA-only-basedPAM 1425 Horse ϩ Ϫ ϩ ϩ detection assay misses a very significant proportion of commonPAM 1427 Horse ϩ Ϫ ϩ ϩ R. equi strains. Importantly, 15% of the horse clinical isolatesPAM 1430 Horse ϩ Ϫ ϩ ϩPAM 1431 Horse ϩ Ϫ ϩ ϩ included in our study were vapA negative (Fig. 1), question-PAM 1436 Soil ϩ Ϫ ϩ ϩ ing the value of vapA as a sensitive molecular diagnosticPAM 1437 Horse ϩ Ϫ ϩ ϩ marker for R. equi even if its application is restricted toPAM 1441 Soil Ϫ Ϫ Ϫ ϩ equine specimens.PAM 1447 Pig Ϫ ϩ Ϫ ϩ Besides being universally highly conserved in R. equi, thePAM 1448 Human Ϫ ϩ Ϫ ϩPAM 1453 Horse ϩ Ϫ ϩ ϩ choE gene offers also the advantage that it is present on thePAM 1463 Human Ϫ Ϫ Ϫ ϩ chromosome in monocopy (21), thus permitting an accuratePAM 1467 Pig Ϫ ϩ Ϫ ϩ quantification of the genomic units present in a sample. InPAM 1468 Pig Ϫ Ϫ Ϫ ϩ contrast, the detection (and quantification in terms of genomePAM 1469 Pig Ϫ ϩ Ϫ ϩPAM 1473 Pig Ϫ ϩ Ϫ ϩ equivalents) of a plasmidic gene, as is vapA, relies on thePAM 1474 Pig Ϫ ϩ Ϫ ϩ efficiency of plasmid DNA extraction and, critically, also on thePAM 1475 Pig Ϫ ϩ Ϫ ϩ number of copies of the plasmid carried by each individualPAM 1479 Pig Ϫ ϩ Ϫ ϩ strain. From the CT values obtained for choE (control monocopyPAM 1480 Pig Ϫ ϩ Ϫ ϩ gene) and vapA we have estimated that 39% of the isolatesPAM 1483 Pig Ϫ Ϫ Ϫ ϩPAM 1485 Pig Ϫ Ϫ Ϫ ϩ contained two or more plasmid copies per genome (Table 5),PAM 1487 Pig Ϫ Ϫ Ϫ ϩ indicating that Q-PCR data based on vapA would overestimatePAM 1488 Pig Ϫ Ϫ Ϫ ϩ on a significant number of occasions the R. equi bacterial loadPAM 1493 Pig Ϫ ϩ Ϫ ϩ present in the sample by at least a factor of two.PAM 1495 Pig Ϫ ϩ Ϫ ϩPAM 1499 Pig Ϫ Ϫ Ϫ ϩ Although vapA is clearly unsuitable as a target for the spe-PAM 1500 Pig Ϫ ϩ Ϫ ϩ cies-specific quantitative detection of R. equi by Q-PCR, it isPAM 1504 Pig Ϫ Ϫ Ϫ ϩ indisputable that this gene has diagnostic value as a predictorPAM 1518 Pig Ϫ Ϫ Ϫ ϩ of horse pathogenicity (31). Moreover, horse isolates are quan-PAM 1533 Pig Ϫ Ϫ Ϫ ϩ titatively the most significant component of R. equi epidemiol-PAM 1547 Pig Ϫ Ϫ Ϫ ϩPAM 1549 Pig Ϫ Ϫ Ϫ ϩ ogy. Indeed, this fully justifies the incorporation of vapA de-PAM 1550 Pig Ϫ Ϫ Ϫ ϩ tection capabilities in any diagnostic method targeting R. equi.PAM 1563 Bovine Ϫ Ϫ Ϫ ϩ Bearing this in mind, we designed our Q-PCR method for R. a Note that vapAϩ and vapBϩ scores are mutually exclusive, indicating that equi as a dual-reaction system with independent assays forvapB is most likely an allelic variant of vapA. ϩ, positive; Ϫ, negative. choE and vapA, the former as an entry-level, primary test b Reference method (primers, PCR setup, and conditions as described in aiming at the quantitative detection of R. equi species, and thereferences 22 and 30). c Concordance between results of vapA conventional PCR and our vapA Q-PCR. latter as a complementary, optional test for vapAϩ genotype determination. This modular design provides full flexibility as
  6. 6. VOL. 72, 2006 choE- AND vapA-TARGETED RHODOCOCCUS EQUI Q-PCR 4261 TABLE 3. Determination of the detection and quantification limits of R. equi choE, choE-IAC, and vapA Q-PCR assaysa Approx no. of Confidence interval limitb Signal ratio for assay c: CT for assay d:genome eq/reaction Lower Upper choE choE-IAC vapA choE choE-IAC vapA 1 ϫ 10 6 997,600 1,003,300 9/9 9/9 9/9 19.83 Ϯ 0.02 19.90 Ϯ 0.04 18.80 Ϯ 0.02 1 ϫ 105 99,643 100,358 9/9 9/9 9/9 23.32 Ϯ 0.05 23.40 Ϯ 0.06 22.25 Ϯ 0.06 1 ϫ 104 9,887 10,113 9/9 9/9 9/9 26.05 Ϯ 0.02 26.01 Ϯ 0.03 25.60 Ϯ 0.03 1 ϫ 103 964 1,036 9/9 9/9 9/9 29.50 Ϯ 0.02 29.60 Ϯ 0.04 29.00 Ϯ 0.03 1 ϫ 102 89 111 9/9 9/9 9/9 32.88 Ϯ 0.03 32.95 Ϯ 0.05 32.20 Ϯ 0.04 1 ϫ 101 7 14 9/9 9/9 9/9 36.77 Ϯ 0.15 36.80 Ϯ 0.14 35.80 Ϯ 0.12 1 0 2 6/9 6/9 7/9 38.38 Ϯ 0.41 38.70 Ϯ 0.34 38.20 Ϯ 0.29 a Nontemplate controls were negative in the three Q-PCR assays (CT values of Ͼ50 in all the replicates). b Calculated for the expected number of template molecules at each dilution at 95% confidence level. c Number of positive results out of nine reactions. d Cycle number at which fluorescence intensity equals a fixed threshold. Mean values Ϯ standard errors of the means were calculated with a prefixed threshold of200. Differences in CT values were statistically significant with P Ͻ 0.05.the vapA Q-PCR assay will not always be needed, in particular detection/quantification of the target DNA and assessment ofin medical (human) microbiology laboratories, due to its spe- PCR efficiency. The inclusion of an IAC did not have anycific relevance for the horse. It also helps avoid possible prob- significant impact on the performance of the choE Q-PCRlems of loss of analytical performance, as is sometimes seen in assay (Table 3).multiplex PCR assays (7, 16). This is important as the primary A critical aspect in the design of molecular diagnostic meth-choE Q-PCR test includes an IAC, thus being already de facto ods for microbial pathogens is achieving low detection anda duplex-format assay. quantification limits. This goal is of particular interest in the A major limitation to the application of Q-PCR-based tests case of R. equi. Indeed, rhodococcal foal pneumonia initiallyin diagnostic laboratories is the relatively common occurrence follows an insidious course (9, 23), and it has been suggestedof false-negative results due to the presence of PCR inhibitors that accurate detection (and quantification) of low levels of R.in the sample. To tackle this problem, we included an IAC in equi in respiratory specimens during the “silent” phase of in-our choE Q-PCR assay. An IAC consists of a nontarget DNA fection, before the development of gross lesions in the lungsfragment that is coamplified with the target sequence, prefer- and the manifestation of clinical symptoms, could be of diag-ably with the same primers used for the test reaction (4, 26). To nostic value (13, 20). On purified DNA, our choE Q-PCR assayachieve this, we constructed the IAC by fusing the forward and could detect approximately one target genome equivalent in atreverse choE target sequences to both ends of an unrelated least 67% of the replicates and 10 genome equivalents in allDNA fragment to which a second fluorescent probe (the IAC cases. Accurate quantification, with excellent linearity (R2 ϭprobe) hybridized. The use of two differently labeled fluores- 0.998) and efficiency (E ϭ 0.994 without IAC and 0.995 withcent probes in the same reaction permitted the simultaneous IAC), was possible down to Ϸ10 R. equi genome equivalentsTABLE 4. Quantitative detection of R. equi bacteria in BAL fluida TABLE 5. Numbers of copies of the vapA gene in R. equi isolatesaApprox Approx no. of Avg no. of copies of vapA for value type Signal RelativeR. equi R. equi genome CT d % of ratioc accuracy e Exptl continuous TheoreticalCFU/ml eq/reactionb isolates range discrete1 ϫ 107 5 ϫ 105 9/9 20.67 Ϯ 0.10 100.051 ϫ 106 5 ϫ 104 9/9 24.10 Ϯ 0.10 103.59 0.57b–1.49 1 61.21 ϫ 105 5 ϫ 103 9/9 27.82 Ϯ 0.04 88.95 1.50–2.49 2 25.91 ϫ 104 5 ϫ 102 9/9 30.93 Ϯ 0.13 113.53 2.50–3.49 3 5.91 ϫ 103 5 ϫ 101 9/9 34.68 Ϯ 0.20 95.57 3.50–4.49 4 01 ϫ 102 5 ϫ 100 9/9 37.90 Ϯ 0.21 NAf 4.50–5.49 5 1.21 ϫ 101 5 ϫ 10Ϫ1 0/9 Ͼ50 NA 5.50–6.49 6 1.21 5 ϫ 10Ϫ2 0/9 Ͼ50 NA 6.50–7.49 7 2.40g 0 0/9 Ͼ50 NA 7.50–8.49 8 0 8.50–9.49 9 1.2 a Results from three independent experiments with three replicates each.Overall efficiency E is 0.94, and linearity R2 is 0.99. Total 100 b Estimated number of R. equi genome equivalents in each PCR run assuming100% DNA extraction efficiency (each reaction mixture contained 5 ␮l of a DNA a ϩ Estimated from Q-PCR results for choE and vapA in vapA isolates (at leastpreparation of 100 ␮l extracted from 1 ml BAL fluid). three replicates per reaction per isolate) using 1 ng of DNA and the following c Number of positive results out of nine reactions. formula: number of vapA copies ϭ 2Ϫ⌬CT, where ⌬CT ϭ CTvapA Ϫ CTchoE, d As defined in Table 3, footnote d. assuming 100% plasmid DNA extraction efficiency relative to chromosomal e Degree of correspondence between the quantitative results obtained by stan- DNA. This calculation is possible because the PCR efficiencies for both targets,dard plate counting (R. equi CFU/ml) and the results obtained by the choE 0.977 and 0.995, were close to the optimal value E ϭ 1, meaning that duplicationQ-PCR method (27, 29). of each amplicon occurs in each cycle across a wide linear range. The coefficient f NA, not applicable. of variance was 7% and 11% for choE and vapA Q-PCR systems, respectively. g Noncontaminated BAL fluid. b Minimum value obtained (see Table S1 in the supplemental material).
  7. 7. 4262 ´ ´ RODRIGUEZ-LAZARO ET AL. APPL. ENVIRON. MICROBIOL.per reaction. A similar performance was achieved when the 10. Giguere, S., M. K. Hondalus, J. A. Yager, P. Darrah, D. M. Mosser, and J. F. ` Prescott. 1999. Role of the 85-kilobase plasmid and plasmid-encoded viru-technique was applied to the enumeration of bacterial cells in lence-associated protein A in intracellular survival and virulence of Rhodo-BAL fluid, a specimen commonly used in the clinical diagnosis coccus equi. Infect. Immun. 67:3548–3557.of R. equi pneumonia (9, 28). Here, due to the DNA extraction 11. Haites, R. E., G. Muscatello, A. P. Begg, and G. F. Browning. 1997. Preva- lence of the virulence-associated gene of Rhodococcus equi in isolates fromprotocol used, which involved processing of 1-ml BAL fluid infected foals. J. Clin. Microbiol. 35:1642–1644.samples, and the inclusion per reaction of a fraction of DNA 12. Halbert, N. D., R. A. Reitzel, R. J. Martens, and N. D. Cohen. 2005. Eval-extract (5 out of 100 ␮l), the practical detection limit for R. uation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene. Am. J. Vet. Res. 66:1380–equi bacteria was 100 CFU/ml (or 5 genome equivalents per 1385.reaction). It should be possible to lower this detection limit by 13. Harrington, J. R., M. C. Golding, R. J. Martens, N. D. Halbert, and N. D. Cohen. 2005. Evaluation of a real-time quantitative polymerase chain reac-refining the sample processing so that DNA is extracted from tion assay for detection and quantitation of virulent Rhodococcus equi.larger specimen amounts and recovered in smaller volumes. Am. J. Vet. Res. 66:755–761.Moreover, accurate determination of horse-pathogenic (i.e., 14. Jain, S., B. R. Bloom, and M. K. Hondalus. 2003. Deletion of vapA encoding virulence associated protein A attenuates the intracellular actinomycetevapAϩ) R. equi numbers in environmental samples or in fecal Rhodococcus equi. Mol. Microbiol. 50:115–128.or nasal specimens could also provide a predictive tool to 15. Ladron, N., M. Fernandez, J. Aguero, B. Gonzalez Zorn, J. A. Vazquez- ´ ´ ´ ´assess the risk of R. equi clinical infection in stud farms where Boland, and J. Navas. 2003. Rapid identification of Rhodococcus equi by a PCR assay targeting the choE gene. J. Clin. Microbiol. 41:3241–3245.the organism is endemic. The optimal performance parameters 16. Mackay, I. M. 2004. Real-time PCR in the microbiology laboratory. Clin.of our vapA Q-PCR (R2 ϭ 0.999, E ϭ 0.977) indicate that this Microbiol. Infect. 10:190–212.assay is suitable for this purpose. 17. Makrai, L., S. Takai, M. Tamura, A. Tsukamoto, R. Sekimoto, Y. Sasaki, T. Kakuda, S. Tsubaki, J. Varga, L. Fodor, N. Solymosi, and A. Major. 2002. In conclusion, the dual-reaction Q-PCR method that we Characterization of virulence plasmid types in Rhodococcus equi isolateshave developed for the rapid (results in less than 2 h), species- from foals, pigs, humans and soil in Hungary. Vet. Microbiol. 88:3777–3784. 18. Makrai, L., S. Takayama, B. Denes, I. Hajtos, Y. Sasaki, T. Kakuda, S.specific quantitative monitoring and determination of the Tsubaki, A. Major, L. Fodor, J. Varga, and S. Takai. 2005. CharacterizationvapAϩ (equine) subtype of R. equi provides a diagnostic tool of virulence plasmids and serotyping of Rhodococcus equi isolates frompotentially very useful in both medical and veterinary diagnos- submaxillary lymph nodes of pigs in Hungary. J. Clin. Microbiol. 43:1246– 1250.tic laboratories. 19. Morton, A. C., A. P. Begg, G. A. Anderson, S. Takai, C. Lammler, and G. F. ¨ Browning. 2001. Epidemiology of Rhodococcus equi strains on thoroughbred horse farms. Appl. Environ. Microbiol. 67:2167–2175. ACKNOWLEDGMENTS 20. Muscatello, G., and G. F. Browning. 2004. Identification and differentiation of avirulent and virulent Rhodococcus equi using selective media and colony We thank all those who kindly provided us with the R. equi strains blotting DNA hybridization to determine their concentrations in the envi-used in this study (J. Aguero, Spain; T. Buckley, Ireland; R. Callejo, ¨ ronment. Vet. Microbiol. 100:121–127.Argentina; A. Caterino-de-Araujo, Brazil; T. Chakraborty, Germany; 21. Navas, J., B. Gonzalez-Zorn, N. Ladron, P. Garrido, and J. A. Vazquez- ´V. Garcı Dominican Republic; J. M. Garcı ´a, ´a-Arenzana, Spain; A. Boland. 2001. Identification and mutagenesis by allelic exchange of choE,Enrı´quez, Spain; A. Kodjo, France; J. L. Hernandez, Spain; P. Martin- ´ encoding a cholesterol oxidase from the intracellular pathogen RhodococcusRabadan, Spain; A. Martı ´nchez, Spain; A. Morton, Australia; ´ ´n-Sa equi. J. Bacteriol. 183:4796–4805.M. Pate, Slovenia; S. Petry, France; J. F. Prescott, Canada; S. Ricketts, 22. Oldfield, C., H. Bonella, L. Renwick, H. I. Dodson, G. Alderson, and M.United Kingdom; F. Quigley, Ireland; I. Simarro, Spain; S. Takai, Goodfellow. 2004. Rapid determination of vapA/vapB genotype in Rhodo- coccus equi using a differential polymerase chain reaction method. AntonieJapan). Thanks are also due to D. Leadon for stimulating and constant Leeuwenhoek 85:317–326.cooperation, C. Helps for help and advice on the iCycler machine, and 23. Prescott, J. F. 1991. Rhodococcus equi: an animal and human pathogen. Clin.A.-L. Cohen for help in the identification and curation of R. equi Microbiol. Rev. 4:20–34.isolates. 24. Rodrı ´guez-Lazaro, D., M. Hernandez, M. Scortti, T. Esteve, J. A. Vazquez- ´ ´ ´ This work was supported by a Leverhulme grant from the Institute Boland, and M. Pla. 2004. Quantitative detection of Listeria monocytogenesof Advanced Studies, University of Bristol. 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