WO2007102007A2 - Diagnostic methods and reagents for detection and quantification of rhodococcus equi based on choe and vapa genes - Google Patents

Abstract

A two-stage assay for detecting Rhodococcus equi in a DMA containing sample, said method comprising in a first stage, detecting in said sample, the presence or absence of a nucleic acid which is characteristic of a first gene from Rhodococcus equi (eg. ChoE) , which is conserved in all strains of R. equi, and, if this is found to be present, subjecting the sample to a second stage in which the presence or absence of a nucleic acid which is characteristic of a second gene Rhodococcus equi (eg. VapA) which is specific to a specific host, is detected. The first nucleic acid is suitably quantitated to provide a useful means for determining the amounts of R. equi in the sample. ChoE is present as monocopy on the chromosome, in contrast to VapA which is present in variable gene copy number in different bacterial cells. Kits for use in the assay are also described and claimed.

Description

Diagnostic methods and reagents

The present invention relates to method of detection of Rhodococcus equi, and/or for the diagnosis of Rhodococcus equi infection in humans and animals, in particular equines, as well as to reagents and kits including amplification primers and probes useful in these methods.

Rhodococcus equi is a soil-dwelling actinomycete of the mycolata group that causes pyogranulomatous infections in the lungs and other different body locations in a variety of animal hosts. This facultative intracellular parasite is well known in veterinary medicine as the causal agent of foal pneumonia, a severe purulent bronchopneumonic infection with high case-fatality rates. The disease is recognized in many countries as the leading cause of neonatal (0-5 months) mortality in horses and is a cause of serious concern to the equine industry as it can become endemic in stud farms and there is no effective vaccine for its prevention. In recent years R.equi has emerged as a significant opportunistic human pathogen, especially in individuals infected with human immunodeficiency virus. In the human host the infection presents usually as tuberculosis-like cavitary pneumonia or bacteremia. R. equi is also being increasingly reported in other animal species, mainly associated with extrapulmonary, purulent or caseating infections. In cattle the organism is typically isolated from chronic retropharyngeal, bronchial or mediastinal pyogranulomatous lymphadenitis, and in pigs from submaxillary lymph nodes.

Horse isolates of R. equi typically harbor an 85- to 90-Kb virulence plasmid of which an example has been fully sequenced recently. This plasmid encodes virulence-associated protein A or VapA, a 17.4-kDa surface lipoprotein presumed to be involved in pathogenesis but whose role in the infectious process remains unknown. VapA is encoded by the vapA gene, which is present in a plasmidic 27.5-Kb island together with six other vapA homologues. In non-horse R. equi isolates, including human isolates, the VapA protein/vapA gene is much less frequently found than a variant protein/allele designated VapB/vapB. The VapB antigen is structurally and immunologically closely related to VapA but is larger (18.2 kDa, 20 kDa as detected by SDS-PAGE immunoblotting) and is encoded by plasmids of varying size (79 to 100 Kb) not yet characterized genetically. The vapB plasmids are not found in equine isolates, suggesting that vapB* strains are not pathogenic for .the horse (7) . Except soil isolates from horse-breeding farms, in which the vapA-type plasmid is common, environmental isolates of R. equi do not usually carry plasmids or, if they do, these are smaller in size and most often vapA^~/B^~.

Laboratory diagnosis of rhodoccocal infections currently relies on classical bacteriological methods involving the isolation of the organism from clinical samples or postmortem material.

However, these culture-based procedures are lengthy and sometimes lack of adequate sensitivity due either to prior antibiotic treatments or, in the case of respiratory specimens (typically tracheobronchial aspirate fluid or sputum) , to the presence of multiple bacterial contaminants (10) . An added problem is the difficulties posed by the identification due to the micro- and macroscopic morphological variability exhibited by these bacteria and the lack of accuracy of biochemical tests for R. equi species determination (5, 11) . There is therefore a considerable interest in developing new, simpler tests for the rapid and reliable detection and identification of R, equi for use in both veterinary and medical clinical microbiology laboratories.

Several molecular methods have been described for R. equi based on amplification of DNA sequences by conventional PCR (1, 2, 3, 5, 7, 10, 13) . Although comparatively faster than culture-based methods, conventional PCR, however, only provides qualitative results and requires post-PCR processing. Being PCR an exquisitely sensitive technique, the "open" post-PCR processing of massive amounts of amplicon increases the risk of false- positive results due to sample cross-contamination. This risk is minimized in the real-time PCR technique as the reaction and fluorescent probe-based amplicon detection are brought about simultaneously in a closed tube.

Real-time monitoring of the amplification curve via fluorescence emission permits also a much more sensitive detection of positive reactions and at the same time, importantly, an accurate quantification of the target DNA present in the sample (12) . To date, however, only one quantitative real-time PCR (Q-PCR) assay has been reported for R. equi (4). This assay targets the vapA gene and therefore only detects strains carrying vapA-type plasmids, which as mentioned above are rarely found in human and most other non-horse R. equi isolates, thus limiting its applicability to the field of equine medicine. Moreover, the quantification accuracy of this assay can be compromised by strain-to-strain differences in plasmid copy number or plasmid DNA extraction efficiency.

The R. egui cholesterol oxidase gene choE (6) has been identified and the usefulness of this chromosomal locus as a target for the specific and sensitive detection of the organism by conventional PCR (5) has been determined.

The applicants have designed and developed a novel dual-reaction Q-PCR method that allows both the species-specific quantification of R. equi and determination of its "horse-associated" subtype.

According to the present invention there is provided a two-stage assay for detecting Rhodococcus egui in a DNA containing sample, said method comprising in a first stage or reaction, detecting in said sample, the presence or absence of a nucleic acid which is characteristic of a first gene from Rhodococcus equi, which is conserved in all strains of R. equi, and, (for example if this is found to be present, ) subjecting the sample to a second stage or reaction in which the presence or absence of a nucleic acid which is characteristic of a second Rhodococcus equi gene which is specific to a specific host, and in particular horses is detected. The assay is suitably carried out in such a way that at least one of the first or second nucleic acids is quantified so as to provide information about the amount of R. equi DNA in the sample .

This assay is particularly useful in diagnosing a specific form of disease in a particular specimen. By detecting the two genes individually rather than together in a multiplex assay, the assay is kept relatively simple, in the complex multiplex assays for multiple analytes is avoided. However, multiplex assay formats, where both sequences are detected concurrently in the same reaction, may be utilised where there is the option of conducting these in a manner in which at least one of the nucleic acids is quantitated. Thus as used herein, the expression "two-stage assay" refers to a procedure in which two reactions are conducted.

DNA containing samples may be obtained from a variety of sources, including clinical, pathological and environmental samples, in particular soil samples. Suitable clinical samples may include blood, lavage, sputum, nasal lavages or aspirates, bronchial lavage or, in the case of horses, horse breath samples. Suitable pathological samples include lung tissue, lymphatic ganglia, intestine, abscesses, and in general any other tissue where Rhodococcus may be found as part of an infectious process. The nature of the sample will depend upon the nature of the animal being treated, and will be determined by the clinician. Clinical samples may be obtained from humans or other animals who are subject to R. equi infection, such as equines (such as horses) , ruminants including bovines such as cows, and ovines such as sheep and goats, pigs, felines such as cats, birds such as pheasants .

DNA is then extracted from these samples using conventional methods, as are known in the art, including for example, extraction into extraction solutions and buffers etc. The precise nature of the extraction procedure will depend upon the nature of the sample, and will be either known in the art, or could be determined using conventional methods.

Suitably the first gene is a gene from a chromosomal locus, which is universally conserved, in R. equl. In particular, the first gene from Rhodococcus equi is the R. equi cholesterol oxidase gene choE, but other conserved genes, in particular those present in monocopy or other known numbers of copies, may be selected. In this instance, quantification of the DNA in the sample, as described below, may provide an indicator of the level of infection or the stage of the disease. This is because quantitative data of say a monocopy gene such as choE will always perfectly correlate with the number of bacteria (or genome equivalents; each bacterium contains one genome and each genome carries one copy of the choE gene) . This may be particularly helpful in clinical diagnosis, for example in the early "silent" stages of a disease, where an animal may not show clear symptoms of illness. Where genes of higher known copy numbers are utilised, the calculation of the relative amounts of nucleic acid will be readily determinable using a simple calculation.

Suitably the second gene is present in only certain strains of the bacteria, for example in the strains found in horse isolates. This then allows the precise risk to a specific animal, or to animals in the vicinity to be assessed. If, for example, a soil sample is positive for R. equi in the first stage, it will only be a risk to for example horses grazing or housed on the land from which the soil sample is taken if it is a strain which infects horses, and this can be determined in the second stage of the assay. Similarly, if a DNA containing sample obtained from a horse isolate tests positive in the first stage, the risk or presence of disease can be determined to the horse can be determined during the second stage, and appropriate treatment administered.

Suitably the second gene from Rhodococcus equi is the virulence- associated protein A or VapA gene, carried on a virulence plasmid associated with horses, but again other genes, and in particular plasmid genes may be selected. In this case, the copy number of the gene may be widely variable, and if this is the case, though quantification of the amount of nucleic acid in the sample will be possible, quantitation may not necessarily reflect the number of bacteria (genome equivalents) present in the said sample, because the plasmid can be present in each bacterial cell in various copies, each copy carrying a vapA gene. However, where this is the case, the quantitative information can be based upon the results of the first stage of the assay, in particular where the gene utilised is a monocopy gene.

Detection is suitably carried out using a nucleic acid amplification method, and in particular the polymerase chain reaction (PCR) . In a particular embodiment, the progress of each PCR is monitored in "real-time", for instance using the TaqMan™ detection system, but other real-time detection systems such as those described for example in EP-A-512334 , WO 99/28500, WO 99/66071, WO2004/033726, European Patent Application No.0912760 the content of which is incorporated herein by reference, may also be utilised.

By monitoring the rise in the amount of amplification product during the repeated amplification cycles, it is possible to quantitate the amount of nucleic acid present in the sample. As outlined above, this may be particularly useful, especially in the case of the nucleic acid detected in the first stage of the assay, in order to determine the level of infection, or the stage of the disease.

Preferably one or both of the PCRs is carried out in the presence of an internal control sequence, which is of generally similar length to the target sequence, and which is also detected during the PCR. This can be achieved using for instance, differently labelled TaqMan probes, one specific for the target sequence and one specific for the internal control sequence, as well as adding pairs of primers able to amplify both the target and the control sequence, and detecting the individual labels during the course of the reaction.

Suitable internal control sequences may be derived from a wide variety of organisms, but the applicants have found that an available Listeria gene sequence may provide a suitable internal control, at least in the first stage of the assay.

The method can be used to diagnose the presence of disease in animals, in particular horses, including mares and foals, who are not showing clinical symptoms, either to assess the risk, or to provide assurance that the animal is free of the disease. This may be particularly useful, for example, in stud farms and the like, where very valuable breeding stock is kept. Early diagnosis, even where clinical symptoms are not apparent, can enhance the therapeutic or containment prognosis of the disease.

Therefore, in a particular embodiment, the invention provides a method for detecting R.egui infection in equines, said method comprising subjecting a sample obtained from an equine, to the following steps:

(i) detecting the presence or absence in the sample, and quantifying the amount in said sample, of a nucleic acid of the R. equi cholesterol oxidase gene choE using a real-time PCR method, conducted in the presence of an internal control/ (ii) detecting the presence or absence in the sample, and optionally quantifying the amount in said sample, of a nucleic acid of the virulence-associated protein A gene using a real-time PCR method, and relating the results of steps (i) and (ii) to the presence or absence of R. equi infection

Suitably, the first and second stages or reactions described above, and in particular steps (i) and (ii) are carried out sequentially, on separate aliquots of the sample, so that only samples which are positive in the first stage or in step (i) are progressed to the second stage or step (ii) respectively.

Alternatively, the stages may be conducted concurrently if rapid results are required. In this case, where the detection method used is PCR, the PCR may be carried out in a multiplex format. In this instance, different labels will be attached to the probes used for the first and second nucleic acids sequences, so that the signal from each is distinguishable, and so at least one of the amplification reactions can be monitored throughout to allow for quantitation of the sequence. A further advantage of the use of such a format is that the chances of detecting R. equi in the sample is maximised, even if one of the reactions fails for any reason. Therefore the risk of false negatives is reduced.

Particular examples of nucleic acid sequences from the genes which may be targeted are suitably sequences found in the gene of between 50 and 200 base pairs in length. Particular examples are nucleic acid sequences which are amplified using the primers and probes listed hereinafter in Table 2. Novel probes and primers from within this Table, as well as their use in the methods described above form a further aspect of the invention.

Kits containing reagents required for detecting the first and second nucleic acids, and in particular probes and primers suitable for amplifying the first and second nucleic acid sequences form and in particular for quantifying at least the first nucleic acid sequence form yet a further aspect of the invention. Such kits may comprise additional reagents such as polymerase enzymes, buffers, magnesium salts etc. which may be required for conducting an amplification reaction such as PCR.

In particular the kits will comprise fluorescently labelled probes or fluorescent DNA binding agents, whose fluorescence changes when bound to double stranded DNA as compared to when free in solution, which allow the progress of the amplification of at least the first nucleic to be monitored in real-time so as to allow for quantification of the amount of DNA in the sample.

Typical labelled probes will be dual-labelled probes known as TAQMAN™ probes which are used in the well known quantitative assay. These probes are digested during the amplification reaction, thereby allowing the two labels to separate so that fluorescent energy is no longer transferred between them, which results in a detectable change in fluorescence.

However, other forms of quantitative PCR are known, including for instance, methods which use fluorescent DNA binding agents whose fluorescence changes when bound to double stranded DNA as compared to when free in solution and so the increase in the amount of total amount of DNA present in the solution (see EP-A- 512334) as well as a range of methods which utilise hybridisation of probes as a signalling system, either because on hybridisation, for example to a target, the configuration of the probe is changed so that the fluorescent labels interact differently (probes known as molecular beacons, or Scorpion™ or probes or LUX probes) , or, where single labelled probes are used, these ^' interact either with other single labelled probes hybridised adjacent to them on the target, or with a DNA binding agent.

Any suitable labelled probe and/or DNA binding agent suitable for use in these methods may form additional components of the kit.

The invention will now be particularly described by way of example, with reference to the accompanying diagrammatic drawings in which:

Figure 1 shows the Distribution of the vapA allele according to origin of isolate. Above the bars are indicated the number of isolates within each category, inside the bars the percentage of vapA* (gray section) and vapA^" (empty section) isolates. "Other" include sheep, goat, dog, cat, pheasant, primate, iguana and unknown. Most soil isolates are derived from a equine-related environment, explaining the relatively high percentage of vapA⁺ (34); and

Figure 2 illustrates representative amplification plots for choE- IAC (A) and vapA (B) Q-PCRs obtained in the experiments shown in Table 4. Each reaction contained decreasing amounts of R. equi PAM 1126 genomic DNA, equivalent to IxIO⁶ (•) , IxIO⁵ (■) , IxIO⁴ (A), IXIO³ (♦), IxIO² (▼) , 10 (no symbol). RFU, relative fluorescence units. Insets show representative standard plots of log genomic equivalents vs C_τ values.

In a particular embodiment, a novel real-time (Q-) PCR method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen was developed. Species- specific quantification is achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q- PCR includes an internal amplification control (IAC) for identification of false-negatives due to PCR failure. A second Q- PCR targets the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from humans and other sources. The choE-IΑC and vapA assays were 100% sensitive and specific as determined using 178 R. equi and 77 non-target bacteria (including 19 non-egui Rhodococcus spp. strains) , and a panel of 60 R. equi isolates with known vapA⁺ and vapA^~ (including vapB⁺) plasmid genotypes. The frequency of vapA⁺ according to isolate' s origin was: horse 85%, human 20%, bovine and pig 0%, others 27 %. The choE-IAC Q-PCR could detect one genome equivalent in 66% of the replicates, and quantification was possible over a 6-log dynamic range down to «10 target molecules, with excellent PCR efficiency (E = 0.995) and linearity [R² = 0.998). The vapA assay had similar performance but appeared unsuitable for accurate {vapA⁺) R. equi quantification due to variability in target gene/plasmid copy number (1 to 9) . The dual-reaction Q-PCR system here reported, complemented with ad hoc DNA extraction methods, 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.

Here we report the design and development of a novel dual- reaction Q-PCR method that allows both the species-specific quantification of R. equi and determination of its "horse- associated" subtype via detection of choE and vapA sequences, respectively. The method of this embodiment includes an internal amplification control (IAC) for monitoring the occurrence of false-negative results due to PCR inhibition.

MATERIALS AND METHODS

Bacterial strains, culture media and growth conditions. A total of 255 bacterial strains were used in this study: 197 were Rhodococcus spp. (178 R. equi and 19 non-egui isolates) and 58 belonged to different actinomycete genera, including cholesterol oxidase-producing species. The R. equi strains included horse (n = 81), human (n - 35), pig (n = 30), bovine (n = 8), soil (n = 13) and ancillary {n = 11, from sheep, goat, dog, cat, pheasant, primate, iguana and unknown origin) isolates from 14 different countries (Argentina, Australia, Brazil, Canada, China, Dominican Republic, Germany, France, Hungary, Ireland, Japan, Slovenia, Spain, United Kingdom) . All were confirmed as R. equi by analysis of colony morphology, API Coryne biochemical profiling, synergistic hemolysis (CAMP-like) test with Listeria ivanovii (6) , and our previously described choE-PCR test (5) . R. equi isolate 103S from J. Prescott (University of Guelph, Canada) , deposited as PAM 1126 in our collection, was used as reference strain. This strain was originally isolated from a case of foal pneumonia and is currently being used for the determination of the complete genome sequence of R. equi by the "R. equi genome consortium" (www. Sanger. ac.uk/Projects/R equi) . A detailed list of R. equi isolates is available as Supplementary Material and the non-J?. equi isolates are listed in Table 1. Bacteria were maintained at -80⁰C in a medium containing 2% tryptone, 4% skimmed milk and 16% glycerol. Rhodococcus spp. were grown at 3O⁰C in brain heart infusion (BHI) and non-Rhodococcus isolates at 37⁰C in YME medium (0.4% yeast extract, 1% malt extract, 0.4% glucose), supplemented with the appropriate amount of agar for plate cultures. All media were purchased from Oxoid (Hampshire, UK), except BHI (Difco, Detroit, MI, USA).

DNA isolation and quantification. Bacterial genomic DNA was isolated from overnight cultures using a CTAB-based protocol. The content of half Petri dish was transferred into a 1.5-mL tube containing 1 mL PBS, pelleted at 4,000 x g for 10 min and incubated for 1 h at 37 ⁰C after resuspension in 567 μL TE buffer and 3 μL 100 mg/itiL lysozyme solution. Subsequently, 30 μL 10% SDS and 3 μL 20 mg/mL proteinase K was added and the mixture incubated again for 1 h at 37°C. Then, 170 μL 5 M NaCl, 80 μL CTAB/NaCl solution (10% CTAB in 0.7 M NaCl) and 5 μL 5 mg/mL

RNase was added followed by a 30-min incubation at 65⁰C. After cooling to room temperature, the mixture was subject to extraction with phenol-chloroform and chloroform-isoamyl alcohol followed by DNA precipitation with isopropanol (30) . After washing with 70 % ethanol, the DNA was resuspended in 100 μL 10 roM Tris-HCl pH 8.0. The amount of DNA was determined spectrophotometrically and the quality was assessed by calculating the OD₂6o_/28o ratio and visually by agarose gel electrophoresis .

Oligonucleotides. The oligonucleotide primers and TaqMan probes used in this study were designed using Primer Express™ 2.0 software (Applied Biosystems, Foster City, CA, USA) and purchased from Metabion AG (Martinsried, Germany) . They are listed in Table 2.

IAC construction. The IAC consisted of a 100-bp chimerical DNA containing a portion of the listeriolysin (hly) gene from Listeria monocytogenes (GenBank ace. no. M24199) flanked by the R. egui-specific choE gene sequences targeted by reqF/R primers (Table 2) . This chimeric DNA fragment was generated by two rounds of PCR as described previously (7) . The first PCR round used 1 ng Listeria monocytogenes DNA template and primers riacF and riacR (Table 2), which contained the corresponding L. monocytogenes Λ2y-target sequences plus a 5' tail with the reqF/R primer sequences. The second PCR round used the purified first-round PCR product (diluted 1:1,000) as a template and the reqF/R primers. PCR conditions were as previously described (7) . The IAC PCR product was purified, quantified and diluted to the appropriate concentration in 10 mM Tris-HCl pH 8.0 in the presence of 500 ng/mL of acetylated BSA as blocking agent to minimise binding of the negatively charged IAC DNA to the plastic microtubes. With the exception of its target sequence in the L. monocytogenes hly gene (nucleotide positions 114-177), the IAC did not display significant similarity to any DNA sequence deposited in public

DNA databases, as determined by BLAST-N searches (National Center for Biotechnology Information, Bethesda, USA (http: //www.ncbi .nlm.nih. gov) .

Q-PCR. The assays were performed essentially as described previously (8) in 20-μL reaction volumes containing Ix PCR buffer II, 6 mM MgCl₂, 200 μM dATP, dCTP and dGTP, 400 μM dUTP, 300 nM specific primers, 150 nM probe (for the duplex αho£-IAC system, 100 nM of IAC probe was added), 1 unit of AmpliTag Gold^® DNA polymerase (Applied Biosystems-Roche Molecular Systems Inc.,

Branchburg, Germany), 0.2 units of AmpErase^® uracil N-glycosylase (UNG), and 5 μL of the target DNA solution. Reactions were run on an iCycler^™ IQ platform (Bio-Rad Laboratories Inc., Hercules, CA) with the following program: 2 min at 5O⁰C, 10 min at 95⁰C, and 50 cycles of 15 s at 95⁰C and 1 min at 60⁰C. Q-PCR results were analyzed using the Optical System Software v3.0a (Bio-Rad Laboratories Inc., Hercules, CA). Quantification was performed by interpolation in a standard regression curve of C_τ values generated from samples of known concentrations. Negative values or lack of amplification was considered for Q-PCRs with threshold C_τ value >50. Unless otherwise stated, all reactions were performed in triplicate. The 95% confidence interval was calculated for every serial dilution according to a binomial distribution with the statistical software SPSS 12.0S for Windows v8.0 (SPSS Inc, Chicago, 111, USA).

RESULTS

Design and optimization of choE- and vapA-specific Q-PCR assays.

To specifically identify R. equi, the choE gene was used as the target. This chromosomal locus is universally conserved in R. equi and the activity of its product, a cholesterol oxidase, constitutes a very useful species-specific phenotypic marker for identification of R. equi isolates via a CAMP-like hemolytic reaction using sphingomyelinase C-producing indicator bacteria (e.g. L. ivanovii) (6). A previously developed conventional PCR assay based on detection of choE sequences was 100% specific and sensitive for R. equi taking as positive result the expected 959- bp amplicon (5) . Only very occasional smaller-size products are observed with other rhodococcal species in conventional PCR (ref . 3 and our unpublished observations) . To minimize the risk of unspecific reactions a new choE region suitable as target for Q- PCR primers and probe was identified by careful analysis of multiple alignments (CLUSTALW multiple-alignment tool, European Bioinformatics Institute, EMBL, www.ebi .ac.uk) . The new choE target primers, reqF and reqR (Table 2), amplify a 68-bp DNA fragment corresponding to positions 938 to 1,005 of the coding sequence deposited in GenBank under ace. no. AJ242746 (6).

Gene regions suitable for vapA-specific Q-PCR oligonucleotides were identified in a multiple alignment of all known sequences of the vap multigene family. These include the six other vap genes present in the vapA locus identified in strains ATCC 33701 and 103 plasmids, designated vapC to H, and the vapB gene present in plasmids from "non-equine" R. equi isolates (7). Similarity indexes (Pairwise Sequence Alignment tool, MegAlign, DNASTAR Inc., Madison, WI, USA) between vapA and vapB to H are, respectively: 88.2, 54.1, 44.3, 19.5, 33.7, 59.4 and 43.9. Primer pair RvapA114F-RvapA188R was designed, which amplifies a vapA- specific 75-bp DNA fragment corresponding to positions 114 to 188 of the gene sequence deposited in GenBank with ace. no. NC002576.

The BLAST-N tool was used to confirm in silico that none of the selected oligonucleotides recognized any registered DNA sequence other than the target sequence. Primers, TaqMan probes, and MgCl₂ concentrations were optimized for TaqMan Q-PCR assays by using as a template 1 ng of DNA from R. equi strain PAM 1126. Optimal conditions (described in Materials and Methods) were the minimum primer and probe concentrations necessary to give the lowest C_τ value and the highest fluorescence intensity (27-29) .

Optimization of duplex choE-IAC Q-PCR assay. A major limitation to the application of Q-PCR in diagnostic laboratories is the relatively common occurrence of false-negative results due to the presence of PCR inhibitors in the sample. It is therefore preferable to include an internal amplification control IAC in any PCR-based test. An IAC consist of a non-target DNA fragment that is coamplified with the target sequence, ideally with the same primers used for the test reaction (9) . A common strategy for construction of an IAC for Q-PCR is to fuse the forward and reverse target sequences to both ends of an unrelated DNA fragment to which a second fluorescent probe (the IAC probe) hybridizes. The use of two differently labeled fluorescent probes in the same reaction permits the simultaneous detection/quantification of the target DNA and assessment of PCR efficiency. The absence of positive signals for both the target and the IAC DNAs indicates failure of the PCR.

For the IAC, a previously assessed Q-PCR probe sequence that recognized a fragment of the listerial hly gene (8) was used. This IAC probe was labeled with HEX whereas that of the choE- targeted probe with FAM (Table 2) . The IAC amplicon, 100 bp in size, was longer than the 68-bp choS-specific amplicon, facilitating distinction between the two PCR products by gel electrophoresis. The optimal IAC probe concentration (i.e. the minimum concentration not resulting in an increase of C_τ) (9), 100 nM, was determined by performing Q-PCRs in the presence of 1,000 IAC molecules, no R. equi DNA, 150 nM FAM-labeled choE probe, and increasing amounts (from 25 to 250 nM) of the HEX- labeled IAC probe. Since an excess of IAC may inhibit the target- specific reaction, Q-PCRs were also carried out in the presence of various IAC amounts (10,000, 1,000, 100, and 10 molecules per reaction) and a fixed amount of R. equi PAM 1126 DNA (30 genome equivalents) . The maximum IAC amount with no-inhibitory effect on the αhoE-specific FAM signal was 100 copies.

Specificity and sensitivity of the choE-IAC Q-PCR. The capacity of the choE Q-PCR assay to discriminate between target and non- target bacteria was assessed using 1 ng of genomic DNA from 178 R. equi strains from a variety of sources (including clinical isolates from different animal species and environmental isolates) , 19 non-egui rhodococcal spp. strains, and 58 strains from 18 different non-Rhodococcus actinomycete genera. The choE Q-PCR assay was 100% sensitive and 100% specific as all 178 R. equi strains tested gave a positive choE signal whereas none of the 77 non-target bacteria did (detailed results in Supplementary Material table) . Rhodococcus fascians, reported by others (13) to give an unspecific (smaller-size) amplicon by conventional PCR using our previously described primers, did not give any significant signal in the choE Q-PCR assay. All the reactions generated a positive IAC (HEX) signal, ruling out that the absence of choE (FAM) signal observed in non-f?. egui isolates was due to failure of the PCR. All the non-actinomycete bacteria tested to date, including a variety of common Gram-negatives and Gram-positives, have yielded negative results in the ChOiS-Q-PCR assay (not shown) .

Specificity and sensitivity of the vapA Q-PCR. The results with the non-Rhodococcus strains were entirely consistent with those obtained in the choE Q-PCR assay as none of the 77 isolates in this panel gave a positive amplification signal. Significantly different results were obtained when the R. equi isolates were tested. Here, as expected from the varied composition of the R. equi isolate panel, which only contained a proportion of horse- derived isolates (see above and Supplementary Material table) , the vapA Q-PCR assay only detected the target sequence in 48% (85 out of 178) of the R. equi isolates. The distribution of vapA* isolates per origin animal species is shown in Fig. 1.

The discrimination capacity of the vapA Q-PCR assay was assessed on a selection of 60 R. equi strains using as reference method a previously described dual-reaction conventional PCR system that differentiates vapA⁺ isolates from vap∑T or vapA^~/vapB^~ isolates using two pairs of primers (7) . Prior to applying this method its 100% efficacy on a representative sample of R. equi strains was confirmed with known vapA/B genotypes. As shown in Table 3, there was a perfect concordance between the results obtained by both techniques. As all the strains tested positive with the choE-IΑC system, these results indicated that our vapA Q-PCR assay is 100% specific and 100% sensitive.

Detection and quantification limits of the choE-IAC and vapA Q- PCR assays. The detection and quantification limits of the PCR assays were determined by using R. equi PAM 1126 genomic DNA. Amplification reactions were performed with a range of DNA concentrations equivalent to approx. IxIO⁶, IxIO⁵, IxIO⁴, IxIO³, IxIO², 10 and 1 target molecules. One molecule of R. equi DNA corresponds to approx. 4.75 fg of DNA taking into consideration a genome size of 5.2 Mb as determined for PAM 1126. Figure 2 illustrates typical amplification profiles and the regression curves obtained with each Q-PCR assay; Table 4 shows the mean C_τ values for a total of nine replicates in three independent experiments. The two Q-PCR assays yielded similar results in terms of absolute detection values. Positive amplification in all nine replicates of each DNA dilution was achieved when 10 or more target molecules were present, and as few as 1 target molecule could be detected with 67 to 78% probability for choE- and vapA- based Q-PCR assays, respectively (Table 4) . The slopes of the linear regression curves calculated over a 6-log range were similar to the theoretical optimum of -3.32 (9) {choE, -3.337; choE-IAC duplex reaction, -3.335; vapA, -3.379) and showed that the amplifications were very efficient (E = 0.994, 0.995 and 0.977 for choE-, choE-lAC- and vapΛ-based Q-PCR assays, respectively) . Moreover, R² values were above the optimal of 0.995 (0.998 for choE and cho£-IAC reactions, 0.999 for vapA reaction) , indicating that the Q-PCRs described herein are highly linear. The confidence intervals based on the standard deviations of C_τ values did not overlap each other down to 10 target molecules, indicating that reliable quantification was possible above this limit.

DISCUSSION

The method described herein represents the first Q-PCR method that permits the sensitive and specific, accurate quantitative detection of the pathogenic actinomycete R. equi. In this embodiment, this is achieved by targeting sequences from the chromosomal choE gene, previously identified and shown to provide a useful marker for the detection and identification of R. equi by molecular and functional tests (5, 6) . A previous Q- PCR assay for R. equi, recently reported (4), targets the plasmidic gene vapA and thus only detects R. equi bacteria carrying this allelic variant. vapA⁺ strains are associated with infections in the horse and hence are predominantly found in equine-related specimens. However, R. equi is isolated from a variety of other animal species and, with few exceptions, vapA* plasmids are rarely found in these (Fig. 1) . Indeed, the data shows that only a small proportion (20%) of human clinical isolates are vapA* (Fig. 1), consistent with previously reported figures on the prevalence of VapA⁺/vapA⁺ R. equi bacteria in human specimens (7) . The vapA* plasmid genotype appears to be particularly rare among bovine and pig isolates, as shown by our data (0% positives; Fig. 1) and other studies. Overall, more than 50% of the isolates tested in this study were vapA^~ (Fig. 1), clearly showing that a vapΛ-only-based assay misses a very significant proportion of common R. equi strains. Importantly, 15% of the horse clinical isolates included in our study were vapA^~ (Fig. 1), questioning the value of vapA as sensitive molecular diagnostic target even if its application is restricted to equine specimens.

Besides being universally and highly conserved in R. equi, the choE gene offers also the advantage that it is present on the chromosome in monocopy (6), thus permitting an accurate quantification of the genomic units present in a sample. In contrast, the detection (and quantification in terms of genome equivalents) of a plasmidic gene, as is vapA, relies on the efficiency of plasmid DNA extraction and, critically, also in the number of copies of the plasmid carried by each individual strain. From the C_τ values obtained for choE (control monocopy gene) and vapA we have estimated that 36% of the isolates contained two or more plasmid copies per genome (Table 5), indicating that quantitative Q-PCR data based on vapA, as in the method recently described by Harrington et al. (4), will overestimate on a significant number of occasions the number of R. equi bacteria present in the sample by at least a factor of two.

Although vapA is clearly unsuitable as a target for the species- specific quantitative detection of R. equi by Q-PCR, this gene has diagnostic value as predictor of horse pathogenicity. Moreover, horse isolates are quantitatively the most significant component of R. equi casuistry. Indeed, this fully justifies the incorporation of vapA-detection capabilities in any diagnostic method targeting R. equi. The Q-PCR method for R. equi described herein is a dual-reaction system with independent assays for choE and vapA, the former as primary reaction aiming at the quantitative detection of R. equi species, and the latter as complementary, optional reaction for vapA⁺ genotype determination. This modular design provides full flexibility as the vapA Q-PCR assay will not always be needed, in particular in medical (human) microbiology laboratories, due to its specific relevance for the horse. It also helps avoiding possible problems in terms of loss of analytical performance, as sometimes seen in multiplex PCR assays (3) . This is particularly important as the primary choE Q-PCR test includes an IAC, thus being already de facto a duplex-format assay. The inclusion of an IAC endows our method with false-negative-discrimination capabilities, which are lacking in the vapΛ-based R. equi Q-PCR assay recently reported by others (4) . This IAC did not have any significant impact on the performance of the choE Q-PCR assay (Table 4) .

An important aspect in the design of molecular diagnostic methods for microbial pathogens is achieving low detection and quantification limits. This goal is of particular interest in the case of R. equi. Foal pneumonia follows initially an insidious course, with fulminating clinical symptoms manifesting only after gross lesions have developed in the infected lungs. It is therefore presumed that accurate detection (and quantification) of low levels of R. equi exhaled by the infected animals could be of diagnostic importance during the "silent" phase of infection (14, 22). Also, accurate determination of horse-pathogenic (i.e. vapA⁺) R. equi numbers in environmental samples or in fecal or nasal specimens may provide a predictive tool to assess the risk of R. equi clinical infection in endemic studs. The choE Q-PCR assay could detect approx. one target genome equivalent in at least 66% of the experiments, and 10 genome equivalents in all cases. The quantification capacity of Q-PCR relies on linearity and amplification efficiency values. These are calculated by the R² coefficient and the slope of the regression curve relating G₁ and initial target concentration of known standards (9) . Accurate quantification of R. equi using the choE Q-PCR assay was possible down to «10 genome equivalents per reaction, as demonstrated by excellent R² (0.998) and efficiency E values (0.994 without IAC and 0.995 with IAC). Similarly optimal performance was shown by our vapA Q-PCR assay (R² = 0.999, E - 0.977) . These performances are similar to those of other Q-PCR assays reported for other bacteria or eukaryotic organisms.

In conclusion, the dual-reaction Q-PCR method described herein provides for the rapid (results in less than 2 hours) , species- specific quantitative monitoring and determination of vapA⁺ (equine) subtype of R. equi and so provides a useful diagnostic tool for both medical and veterinary diagnostic laboratories.

REFERENCES

1. Arriaga, J. M., N. D. Cohen, J. N. Dβrr, M. K. Chaffin, and R. J. Martens. 2002. J. Vet. Diagn. Investig. 14:347-353.

2. Bell, K. S., J.C. Philp, N. Christofi, and D.W. Aw. 1996. Lett. Appl. Microbiol. 23:72-74. 3. Halbert, N.D., R.Λ. Reitzel, R.J. Martens, and N.D. Cohen.

2005 Am. J. Vet. Res. 66:1380-1385.

4. Harrington, J.R. , M.C. Gold±ng, R.J. Martens, N.D. Halbert, and N.D. Cohen. 2005 Am. J. Vet. Res. 66: 755-761.

5. Ladrόn, N., M. Fernandez, J. Aguero, B. Gonzalez Zorn, J.A. Vazquez-Boland, and J. Navas. 2003. J. Clin. Microbiol. 41:

3241-3245.

6. Navas, J., B. Gonzalez-Zorn, N. Ladrόn, P. Garrido, and J. A. Vazquez-Boland. 2001. J. Bacteriol. 183:4796-4805.

7. Oldfield, C, H. Bonella, L. Renwick, H.I. Dodson, G. Alderson, and M. Goodfellow. 2004. Antonie Van Leeuwenhoek 85: 317-326.

8. Rodriguez-Lazaro, D., M. Hernandez, M. Scortti, T. Esteve, J.A. Vazquez-Boland, and M. PIa. 2004. Appl. Environ.

Microbiol. 70: 1366-1377. 9. Rodriguez-Lazaro, D., M. PIa, M. Scortti, H.J. Monzό, and J.A. Vazquez-Boland. 2005. Appl. Environ. Microbiol. 71: In press. lO.Sβllon, D.C. , T.E. Besser, S. L. Vivrette and R. S. McConnico.

2001. J. Clin. Microbiol. 39: 1289-1293. ll.Soto, A., J. Zapardiel, and F. Soriano. 1994. J. Clin. Pathol.

47: 756-759.

12.Walker, N. 2002. Science 296: 557-559. 13.Sβllon D. 1997, Am. J. Vet. Res. 58, 1232-1237

Table 1: Non-i?. equi strains used in this study. All isolates listed were negative in the choE and vapA Q-PCRs and positive for IAC ^a.

Species Strains Other designation(s)

Rhodococcus erythropolis DSMZ 43066 CECT 3008 ATCC 11048 CECT 3013 ATCC 25544 CECT 4066 DSMZ 1069

Rhodococcus fascians CECT 3001 Rhodococcus marinonascens CECT 4621 ATCC 35653 Rhodococcus rhodochrous ATCC 999 CECT 3042 ATCC 6846 CECT 3046 ATCC 4273, DMSZ 43269 CECT 4806 CECT 5044 ATCC 53968 CECT 5749 ATCC 13808, DSMZ 4321

Rhodococcus roseus ATCC 4004 Rhodococcus coprophilus CECT 5751 Rhodococcus rhodnii CECT 5759 ATCC 35071, DMSZ 43336 Rhodococcus sputi CECT 3012 CECT 3014 CECT 3048

Rhodococcus spp. ATCC 13258 Actinomadura hibisca ATCC 53557 Actinoplanes globisporus DSMZ 43857 Actinoplanes missouriensis ATCC 23342 A ctinosynnema pretiosum ATCC 31281 Amycolatopsis lactamdurans NRRL 3802 Catenuloplanes japonicus ATCC 700014 Dactylosporangium vescum ATCC 39499 Kibdelosporangium aridum ATCC 39323 Lenzea albidocapillata ATCC 51859 Micromonospora carbonacea ATCC 25486 Micromonospora rosea ATCC 33326 Micromonospora rosaria ATCC 29337 Microtetraspora glauca ATCC 23057 Nocardia asteroides ATCC 9969 Nocardiafarcinica ATCC 6846 Nocardia rubra ATCC 13778 Species Strains Other designation^)

Nocardia sylvodorifera ATCC 4919 Nocardia uniformis ATCC 21806 Nocardia spp. NRRL 5646 ATCC 53695

Nonomuraea fastidiosa ATCC 33516 Nonomuraea ferruginea ATCC 35575 Planobispora venezuelensis ATCC 23865 Pseudonocardia autotrophica ATCC 35203 DSMZ 43103 DSMZ 43098

Pseudonocardia compacta ATCC 35407 Pseudonocardia halophobica DSMZ 43089 Pseudonocardia orientalis NRRL 2450 Pseudonocardia nitriβcans DSMZ 46012 Pseudonocardia petroleophila DSMZ 43193 Pseudonocardia thermophila ATCC 19285 Saccharopolyspora spinosa NRRL 18395 Saccharothrix NRRL B3298 aerocolonigenes Saccharothrix flava DSMZ 43885

Streptomyces albidoflavus ATCC 25422 Streptomyces ambofaciens ATCC 23877 Streptomyces antibioticus ATCC 11891 ATCC 8663

Streptomyces aeurofaciens NRRL 1287 Streptomyces canescens ATCC 19736 Streptomyces coelicolor ATCC 23899 Streptomyces chrysomallus ATCC 11523 Streptomyces cyaneus ATCC 23899 Streptomyces diastaticus ATCC 3315 Streptomyces fradiae DSMZ 41757 Streptomyces griseinus DSMZ 40047 Streptomyces griseus ATCC 6855 Streptomyces hygroscopicus ATCC 53110 Streptomyces lavendulae ATCC 14159 Streptomyces lividans ATCC 19844 Streptomyces peucetius NRRL B3826 Streptomyces platensis ATCC 13865 Streptomyces setonii ATCC 39116 Streptomyces thermotolerans ATCC 11416 Streptomyces virginiae ATCC 13161 Streptomyces spp. ATCC 53770 Streptosporangium vulgar e ATCC 33329

ATCC, American Type Culture Collection, USA; CECT, Spanish Type Culture Collection, Spain; DSMZ, German Collection of Microorganisms, Germany; NRRL, ARS Culture Collection, USA. Table 2: Oligonucleotides used in this study. ^a Theoretical melting temperature ^b see sequence listing attached

Oiigonucieot

Target ide Tm ^a G-C

Application Sequence name/SEQ (⁰C) ID NO.^b

Q-PCR forward reqF/1 5'- CGA CAA GCG CTC GAT GTG -3' 59 61 primer Q-PCR reverse reqR/2 5'- TGC CGA AGC CCA TGAAGT -3' 59 56 primer choE reqP/3 TaqMan Probe 5'- FAM -TGG CCG ACA AGA CCG 69 64 ATCAGC C -TAMRA- 3'

PCR forward 5-GTC AAC AAC ATC GAC CAG

COX-F/4 62.3 57.1 primer GCG-3'

PCR reverse 5'-CGA GCC GTC CAC GAC GTA

COX-R/5 64.7 66.7 primer CAG-3'

RvapA114F Q-PCR forward 5'-CAG CAGTGC GATTCT CAA TAG 59 48

/6 primer TG-3' RvapA188 Q-PCR reverse 5'-GAA GTC GTC GAG CTGTCA 59 52

R/7 primer TAGCT-3' vapA RvapAMOP TaqMan Probe 5'-FAM- CAG AAC CGA CAATGC 69 58

Io CAC TGC CTG -TAMRA-3'

PCR forward

IP 1/9 5- AC TCT TCA CAA GAC GGT-3 ' 46 50 primer PCR reverse IP2/10 5'-TAG GCG TTG TGC CAG CTA-3' 55.1 55.6 primer

PCR forward

Hl/11 5'-TGA TGA AGG CTC TTC ATA A-3' 47.6 36.8 vapB primer PCR reverse H2/12 5'-TTA TGC AAC CTC CCA GTT G-3' 53.2 47.4 primer

5'- HEX - CGC CTG CAA GTC CTA

IAC IACP/13 TaqMan Probe 68 61 AGA CGC CA -TAMRA -3 '

Forward primer 5'- CGA CAA GCG CTC GAT GTG riacF/14 81 63

My IAC construction CAT GGC ACC ACC -3' Reverse primer 5'- CGA CAA GCG CTC GAT GTG riacR/15 78 57 IAC construction ATC CGC GTGTTT -3'

Table 3. Specificity and sensitivity of yap A Q-PCR. Note that vapA⁺ and vapB⁺ scores were mutually exclusive, indicating that vapB is most likely an allelic variant of vapA.

Conventional

Q-PCR

Strain Origin PCR ^a Concordance b vapΛ vapB vapA

PAM 1126 horse + + +

PAM 1286 iguana — — — +

PAM 1335 horse + + +

PAM 1340 horse + + +

PAM 1346 horse + + +

PAM 1348 soil +

PAM 1350 soil + + +

PAM 1351 soil + + +

PAM 1358 horse + + +

PAM 1365 horse + — + +

PAM 1367 horse + + +

PAM 1371 horse + + +

PAM 1374 horse + + +

PAM 1376 human + +

PAM 1387 unknown +

PAM 1404 horse + + +

PAM 1406 human + +

PAM 1408 horse + + +

PAM 1410 horse + + +

PAM 1413 human + +

PAM 1414 human + +

PAM 1415 human +

PAM 1416 horse + + +

PAM 1418 horse + + +

PAM 1422 horse + + +

PAM 1424 horse + + +

PAM 1425 horse + + +

PAM 1427 horse + — + +

PAM 1430 horse + + +

PAM 1431 horse + + +

PAM 1436 soil + + +

PAM 1437 horse + + +

PAM 1441 soil +

PAM 1447 Pig + +

PAM 1448 human + +

PAM 1453 horse + + +

PAM 1463 human +

PAM 1467 Pig + + Conventional

Q-PCR Concordance

Strain Origin PCR ^a b vapA vapJB vapA

PAM 1468 Pig +

PAM 1469 Pig +

PAM 1473 Pig + +

PAM 1474 Pig + +

PAM 1475 Pig + +

PAM 1479 pig + +

PAM 1480 Pig + +

PAM 1483 Pig +

PAM 1485 Pig +

PAM 1487 Pig +

PAM 1488 Pig +

PAM 1493 Pig + +

PAM 1495 Pig + +

PAM 1499 Pig +

PAM 1500 Pig + +

PAM 1504 Pig +

PAM 1518 Pig +

PAM 1533 Pig +

PAM 1547 Pig +

PAM 1549 Pig +

PAM 1550 Pig +

PAM 1563 bovine + ^a Reference method, using the primers described in refs. 25 and 33. ^b Concordance between results of yap A conventional PCR and our vapA Q-

PCR.

Table 4. Determination of the detection and quantification limits of R. equi choE choE-lAC and vapA Q-PCR assays.

choE

Confidence interval

Approx. genome limit ⁸ Signal

C_τ ^c equivalents/reaction ratio ^b

Lower Upper

19.83 ±

I x IO⁶ 9/9

997600 1003300 0.05

23.32 ±

1 x lO" 9/9

99643 100358 0.15

26.05 ±

I x IO⁴ 9/9

9887 10113 0.05 Confidence interval

Approx. genome limit³ Signal equivalents/reaction - ratio ^b CT ^£

Lower Upper

29.50 ±

IxIO³ 9/9

964 1036 0.06

32.88 ±

IxIO² 9/9

89 111 0.10

36.77 ±

IxIO¹ 9/9

7 14 0.45

38.38 ±

1 6/9

O 2 1.22

choE-ΪAC

Confidence interval

Approx. genome limit Signal

C_x equivalents/reaction - ratio

Lower Upper

19.90 ±

IxIO⁶ 9/9

997600 1003300 0.10

23.40 ±

IxIO⁵ 9/9

99643 100358 0.17

26.01 ±

IxIO⁴ 9/9

9887 10113 0.09

29.60 ±

IxIO³ 9/9

964 1036 0.12

32.95 ±

IxIO² 9/9

89 111 0.16

36.80 ±

IxIO¹ 9/9

7 14 0.50

38.70 ±

1 6/9

0 2 1.12 vapA

Confidence interval

Approx. genome limit Signal equivalents/reaction - ratio C_x

Lower Upper

I x IO⁶ 997600 1003300 9/9 18.80 ± 0.07

1 x lO⁵ 99643 100358 9/9 22.25 ± 0.17

I x IO⁴ 9887 10113 9/9 25.60 ± 0.10

I x IO³ 964 1036 9/9 29.00 ± 0.09

I x IO² 89 111 9/9 32.20 ± 0.12

1 x lO¹ 7 14 9/9 35.80 ± 0.34

1 0 2 7/9 38.20 ± 0.87

^a Calculated for the expected number of template molecules at each dilution at 95% confidence level.

' Positive results out of 9 reactions.

Cycle number at which fluorescence intensity equals a fixed threshold. Mean value ± standard deviation as calculated with a prefixed threshold of 200. The experimental results were statistically significant (P<0.05), taking into consideration the unavoidable error associated with the serial dilutions.

Table 5. Number of copies of the vapA gene in R. equi isolates ^a.

No. of copies of vapA % isolates

1 64

2 18

3 10

4 3

5 1

6 2

7 1

9 1

100

^a Estimated from Q-PCR results for choE and yap A in vapA⁺ isolates using the formula: no. of vapA copies = 2^~ΔCT, where ΔC_T = C_x ^vapA - C_x ^choE, assuming 100% plasmid DNA extraction efficiency relative to chromosomal DNA. This calculation is possible because the PCR efficiencies for both targets, 0.977 and 0.995, were close to the optimal value E = I, meaning that duplication of each amplicon occurs in each cycle across a wide linear range.

Supplementary Material Table. Rhodococcus equi isolates used in this study and PCR results ^a.

Original Q-PCR ^b

Strain Origin designation cho . _E vapA

PAM 294 Pira human + -

PAM 296 12 Oct human + -

PAM 531 RE2 horse + -

PAM 533 H7623G human + -

PAM 535 T3717F human + +

PAM 538 CC human + -

PAM 541 C 1933/88 horse + +

PAM 1016 1512 horse + +

PAM 1017 1513 horse + +

PAM 1126 103S ^c horse + +

PAM 1202 292 unknown + +

PAM 1203 168 human + +

PAM 1205 222 horse + +

PAM 1206 299 horse + +

PAM 1210 5 dog + -

PAM 1211 6 horse + +

PAM 1212 275 bovine + -

PAM 1213 226 horse + +

PAM 1214 221 pheasant + +

PAM 1215 102 cat + +

PAM 1216 223 horse + +

PAM 1217 OHP393 horse + +

PAM 1218 OHP435 horse + +

PAM 1220 Pig-2 Pig + -

PAM 1221 AIDS 8 human + -

PAM 1222 China soil + -

PAM 1223 Japan 2 horse + +

PAM 1224 Japan 1 soil + -

PAM 1225 OHP353 horse + +

PAM 1226 OHP356 horse + +

PAM 1227 OHP409 horse + +

PAM 1228 OHP418 horse + +

PAM 1229 CECT 5273 horse + +

PAM 1230 CECT 4443 horse + +

PAM 1231 CECT 4568 horse + +

PAM 1232 134 bovine + -

PAM 1235 E-405 human + -

PAM 1236 H4788J human + -

PAM 1237 8604 J human +

PAM 1238 407413 human + +

PAM 1239 412/91 horse + +

PAM 1240 1898/88 goat + -

PAM 1241 1917/88 sheep + -

PAM 1242 9500/86 horse + +

PAM 1243 58 human + -

PAM 1244 LR horse + -

PAM 1245 VM horse + -

PAM 1246 2 horse + +

PAM 1247 9 horse + +

PAM 1248 11 horse + +

PAM 1249 118 horse + +

PAM 1250 1064 human + -

PAM 1251 1095 human + -

PAM 1252 1335 human + -

PAM 1253 1333 human + +

PAM 1254 1365 human + +

PAM 1255 Elda human + -

PAM 1256 San Carlos human + -

PAM 1257 Burgos human + -

PAM 1258 Trias human + -

PAM 1259 RE62 human + -

PAM 1260 960131 human + +

PAM 1261 96280 human + -

PAM 1262 13MD horse + -

PAM 1263 144 horse + +

PAM 1264 19NT soil + +

PAM 1265 LI lMD horse + -

PAM 1266 EIa horse + -

PAM 1267 E2a soil + -

PAM 1268 P4CI2 horse + +

PAM 1269 CICIl horse + +

PAM 1270 VMD soil + -

PAM 1271 ATCC 33701 horse + +

PAM 1273 87121 human + -

PAM 1274 377 horse + -

PAM 1275 378 horse + -

PAM 1276 480 horse + +

PAM 1277 1 soil + +

PAM 1278 2 horse + +

PAM 1281 125 goat + -

PAM 1282 170 horse + -

PAM 1283 173 horse + +

PAM 1284 225 horse + +

PAM 1285 280 goat + -

PAM 1286 291 iguana + -

PAM 1287 293 primate + -

PAM 1289 03P266 horse + +

PAM 1290 03P297 horse + +

PAM 1291 03P356 horse + +

PAM 1292 03P372 horse + +

PAM 1293 03P282 horse + +

PAM 1294 03P432 horse + PAM 1295 03P491 horse + + PAM 1298 HIM 97011099 human + + PAM 1300 15 horse + + PAM 1301 24 horse + + PAM 1302 33 horse + + PAM 1303 45 horse + + PAM 1304 60 horse + + PAM 1308 544 horse + + PAM 1309 145 horse + + PAM 1326 B 18 A.1 horse + PAM 1327 B 18 A.2 horse + + PAM 1328 B 18 A.3 horse + + PAM 1329 B 18 A.4 horse +

201 autopsy

PAM 1335 horse + +

(1998)

210 autopsy PAM 1340 horse + +

(2004)

AFSSA Doz. 03- PAM 1346 horse + +

06-04

AFSSA Doz. 07- PAM 1348 soil +

01-98

AFSSA Doz. 24- PAM 1349 soil +

02-98

AFSSA Doz. 15- PAM 1350 soil + +

07-98

AFSSA Doz. 30-

PAM 1351 soil + +

07-98

PAM 1355 236 Pig + PAM 1358 IAL 1999 horse + PAM 1365 IAL 2020 horse + + PAM 1367 IAL 2022 horse + + PAM 1371 IAL 2026 horse + + PAM 1374 IAL 2051 horse + + PAM 1376 IAL 2040 human + PAM 1387 IAL 2042 unknown +

PAM 1406 21 human + -

PAM 1408 24 horse + +

PAM 1410 26 horse + +

PAM 1413 31 human + -

PAM 1414 32 . human + -

PAM 1415 33 human + -

PAM 1416 34 horse + +

PAM 1418 38 horse + +

PAM 1422 45 horse + +

PAM 1424 49 horse + +

PAM 1425 50 horse + +

PAM 1427 56 horse + +

PAM 1430 62 horse + +

PAM 1431 65 horse + +

PAM 1432 66 soil + +

PAM 1436 81 soil + +

PAM 1437 93 horse + +

PAM 1441 110 soil + -

PAM 1444 123 Pig + -

PAM 1447 130 Pig + -

PAM 1448 131 human + -

PAM 1453 162 horse + +

PAM 1463 201 human + -

PAM 1466 246 pig + -

PAM 1467 248 pig + -

PAM 1468 249 Pig + -

PAM 1469 250 pig + -

PAM 1471 255 pig + -

PAM 1472 257 pig + -

PAM 1473 259 pig + -

PAM 1474 262 pig + -

PAM 1475 263 Pig + -

PAM 1479 277 pig + -

PAM 1480 294 pig + -

PAM 1483 300 Pig + -

PAM 1485 303 pig + -

PAM 1486 305 pig + -

PAM 1487 312 pig + -

PAM 1488 314 pig + -

PAM 1493 344 pig + -

PAM 1495 356 Pig + -

PAM 1499 392 Pig + -

PAM 1500 396 pig + -

PAM 1504 52/00 Pⁱg +

PAM 1518 158/00 Pig +

PAM 1533 187/00 Pig +

PAM 1547 60/01 Pig +

PAM 1549 95/02 Pig +

PAM 1550 56/03 Pig +

PAM 1556 TB05-003436 bovine +

PAM 1557 TB05-003434 bovine +

PAM 1563 TB05-004439 bovine +

PAM 1569 TB05-004659 bovine +

PAM 1571 TB05-004634 bovine +

PAM 1572 TB05-004641 bovine +

PAM 1673 128/05 human +

PAM 1674 127/05 human + ^a PAM, collection of Bacterial Molecular Pathogenesis Group, Veterinary Molecular Microbiology Section, University of Bristol, Bristol, UK. CECT, Spanish Type Culture Collection, Valencia, Spain. ^b Qualitative results of Q-PCR (C_τ threshold > 50): +, positive; -, negative. All reactions were positive for IAC. ^c Strain whose genome is currently being sequenced by the "Rhodococcus equi Genome Consortium".

Claims

Claims

1. A two-stage assay for detecting the amount of Rhodococcus equi in a DNA containing sample, said method comprising in a first stage, detecting in said sample, the presence or absence of a nucleic acid which is characteristic of a first gene from Rhodococcus equi, which is conserved in all strains of R. equi, and subjecting the sample to a second stage in which the presence or absence of a nucleic acid which is characteristic of a second gene Rhodococcus equi which is specific to a specific host, is detected, wherein at least one of the first or second nucleic acid is quantitated and the results used to determine the amount of R. equi present in the sample.

2. An assay according to claim 1 wherein the nucleic acid quantitated is the first nucleic acid, which is characteristic of a gene which is present in R. equi in a known copy number.

3. An assay according to claim 2 wherein the first nucleic acid is characteristic of a gene which is present in R. equi in monocopy.

4. An assay according to any one of the preceding claims wherein the first and second stages are conducted concurrently.

5. An assay according to any one of claims 1 to 3 wherein the second stage of the assay is carried out only if the first stage shows that the first nucleic acid is present in the sample .

6. An assay according to any one of the preceding claims wherein the sample is a DNA containing sample extracted from a clinical, pathological or environmental sample.

7. An assay according to any one of the preceding claims wherein the first gene from Rhodococcus equi is the R. equi cholesterol oxidase gene choE.

8. An assay according to any one of the preceding claims wherein the second gene is the virulence-associated protein A gene (VapA gene) .

9. An assay according to any one of the preceding claims wherein the nucleic acid sequences are detected using a nucleic acid amplification reaction.

10. An assay according to claim 9wherein the amplification reaction is polymerase chain reaction (PCR) .

11 A method for detecting R. equi infection in equines, said method comprising subjecting a sample obtained from an equine, to the following steps:

(i) detecting the presence or absence in the sample, and quantifying the amount in said sample, of a nucleic acid of the R. equi cholesterol oxidase gene choE using a real-time PCR method, conducted in the presence of an internal control; (ii) detecting the presence or absence in the sample, and optionally quantifying the amount in said sample, of a nucleic acid of the virulence-associated protein A gene using a realtime PCR method, and relating the results of steps (i) and (ii) to the presence or absence of R. equi infection.

12 A novel primer or probe, specific for R. equi as shown in Table 2 herein.

13. A kit for carrying out a method according to any one of the claims 1 to 11, said kit comprising means for detecting said first and second nucleic acids, and for quantifying at least said first nucleic acid.

14. A kit according to claim 13 wherein said means comprises at least one oligonucleotide, capable of acting as an amplification primer for one of said nucleic acid sequences, or a detection probe.

15. A kit according to claim 14 wherein the detection probe is a dual-labelled TAQMAN™ probe, which is specific for the first nucleic acid sequence.