AU2003264590B2 - Determining virulence of bacteria - Google Patents

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AUSTRALIA

Patents Act 1990 COMPLETE

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PATENT

Invention Title: Determining virulence of bacteria The following statement is a full description of this invention, including the best method of performing it known to us: Freehills Carter Smith Beadle Sydney\004541 525 Printed 27 November 2OO~ (14:41) page Freehills Carter Smith Beadle, Sydney\004541525 Printed 27 November 2003 (14:41) page 2

TITLE

DETERMINING VIRULENCE OF BACTERIA TECHNICAL FIELD OF THE INVENTION The invention relates to detecting virulent D. nodusus strains and to genetic markers associated with bacterial virulence.

BACKGROUND OF THE INVENTION The principal causative agent of ovine footrot is Dichelobacter nodosus nodusus) Different strains of D. nodusus cause disease of varying severity, ranging from benign to virulent.

The virulence of D. nodusus is typically determined by determining the thermostability of proteases produced by the bacterium. The gelatin gel test is an example of an assay for determining whether a bacterium produces thermostable or thermolabile proteases.

According to the gelatin gel test, protease produced by the bacterium is heated to a temperature for destroying thermolabile protease produced by the bacterium, and the functional activity of the heated enzyme, and accordingly, the thermostability of the enzyme, is then determined by determining the extent to which the heated enzyme is capable of digesting gelatin gel. Thermostable enzymes are those enzymes that are capable of digesting (or.in other words "clearing") a gelatin gel to a certain extent.

D. nodusus strains which produce thermostable proteases are considered as "gel stable" and are recognised as virulent strains. Strains which are observed to.produce 3 thermolabile protease in the gelatin gel test (i.e.

proteases which do not clear a gelatin gel, or which clear a gel to a lesser extent than a known thermostable protease) are considered to be "gel unstable" and are recognised as non virulent, or in other words, benign.

A problem with assays for determining protease thermostability, such as the gelatin gel test, is that some strains that are observed to produce thermostable proteases strains that are gel stable) and accordingly, that are recognised as being virulent, are, in fact, benign. Accordingly, a significant limitation applies to determining virulence of D. nodusus strains by reference to protease thermostability.

SUMMARY OF THE INVENTION The invention seeks to at least minimise the above identified limitation and in a first aspect provides a method for determining whether a D. nodusus strain is virulent. The method comprises the following steps: determining the thermostability of at least one protease produced by the strain; and determining whether the strain comprises an intA gene.

In a second aspect, the invention provides a method for determining that a D. nodusus strain is virulent. The method comprises the following steps: determining that the strain produces at least one thermostable protease; and determining that the strain comprises an intA gene.

In a third aspect, the invention provides a nucleic acid molecule suitable for use in the methods of the first and second aspects of the invention.

4 In a fourth aspect, the invention provides a peptide for use in the methods of the first and second aspects of the invention.

In a fifth aspect, the invention provides an antibody for use in the methods of the first and second aspects of the invention.

In a sixth aspect, the invention provides a kit or composition, comprising a nucleic acid molecule, peptide or antibody of the third, fourth or fifth aspects of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Southern-blot analysis of genomic DNA from gelstable, field benign strains and control gel-stable, field virulent strain digested with EcoRI and probed with pDT20(10). Strains are: Lane 1 238, 2- 239, 3- 245, .4- 250, 5-251, 6- 269, 7- 272, 8- 277, 9-280, 10-283, 11- 1311. The sizes in kb of the lambda-HindIII standard markers are shown on the left-hand side.

Figure 2. Southern blot analysis of genomic DNA from virulent, intermediate and benign strains digested with EcoRI and probed with pDT20(10). Strains are: Lane 1 A198, 2- C305, 3- 1311, 4- 1311A, 5-AC3577, 6- B1006, 7- G1220, 8- H1204, 9-H1215, 10-819, 11- 1169, 12- 2483, 13- 1493, 14- 3138, 15-1469. The sizes in kb of the lambda- HindIII standard markers are shown on the left-hand side.

Figure 3. Southern blot analysis of genomic DNA from gelstable, field benign strains and control gel-stable, field virulent strain digested with EcoRI and probed with pDT17(1). Strains are: Lane 1 238, 2- 239, 3- 245, 4- 250, 5-251, 6- 269, 7- 272, 8- 277, 9-280, 10-283, 11- 1311. The sizes in kb of the lambda-HindIII standard markers are shown on the left-hand side.

5 DETAILED DESCRIPTION OF THE INVENTION As described herein, the inventors analysed more than D. nodusus strains, each of a particular virulence. Some of the strains the subject of the analysis, more particularly, strains 238, 239, 245 ,251 ,269 ,272, 277, 280 and 283, are strains which produce thermostable protease and which are benign. The inventors found that each of these strains is distinguished from virulent strains such as A198, 1311, B1006, G1220 H1215 and D1172, because strains A198, 1311, B1006, G1220 H1215 and D1172 comprise an intA gene, whereas strains 238, 239, 245, 251, 269 ,272, 277, 280 and 283 do not. This is a significant finding and with real utility in the diagnosis of footrot because it provides the basis for a diagnostic test that has sufficient fidelity for distinguishing benign D.

nodusus strains that produce thermostable proteases from virulent strains.

The intA gene is a genetic element identified as part of GenBank accession no. L31763, more specifically, nucleotides 418-1621. The nucleotide sequence of intA is shown in Figure 4 The inventors recognise that some D. nodusus strains may comprise a gene that has one or more nucleotides, or one or more regions of nucleotide sequence, that differ from the sequence shown in Figure 4. For example, in some strains, a gene which has the function of the intA gene may contain one or more nucleotide insertions, deletions or substitutions, that are not found in the sequence shown in Figure 4. Notwithstanding such nucleotide differences, or such regions of nucleotide sequence difference, such a gene is considered to be an intA gene, provided that the gene possesses regions of 100 nucleotides or more with or more nucleotide sequence identity with intA.

6 Accordingly, it follows, and it will be understood, that the virulence of a D. nodusus strain can be determined according to the method of the first or second aspects by determining whether the strain comprises a gene that has the aforementioned nucleotide sequence identity to the intA gene, notwithstanding whether the gene also comprises one or more nucleotides, or one or more regions of nucleotide sequence, that differ from the sequence shown in Figure 4.

The inventors have also found that some benign strains that produce thermolabile proteases, such as H1204, may also comprise an intA gene. Accordingly, the inventors recognise that a determination of whether a D. nodusus strain comprises an intA gene is not a sufficient determination in itself for determining the virulence of the strain. Notwithstanding this point, the inventors recognise that it is not essential that a determination on whether the D. nodusus strain comprises an intA gene be made after determining the.thermostability of protease produced by the strain, for permitting the virulence of the strain to be determined. Thus in accordance with the method of the first or second aspect, the virulence of the D. nodusus strain may be determined by determining whether the strain comprises an intA gene and thereafter, determining the thermostability of at least one protease produced by the strain.

The inventors recognise that the determination on whether the D. nodusus strain comprises an intA gene may be made by detecting a nucleic acid of the strain. The nucleic acid that is detected for the purpose of determining whether the strain comprises an intA gene may be DNA, RNA or cDNA. The nucleic acid may comprise the intA gene.

Examples of such nucleic acids are pDT20(10), which plasmid contains a Rsal fragment corresponding to nucleotides 924-1423 from the sequence identified by 7 GenBank accession number L31763, and Pdtl9(6), which plasmid contains an Alul fragment corresponding to nucleotides 1138-1417 from the sequence identified by GenBank Accession number L31763. Alternatively, the nucleic acid that is detected for the purpose of determining whether the strain comprises an intA gene may be in linkage with the intA gene. Examples of such nucleic acids are pGW34.1,which plasmid contains a RsaI/FspI corresponding to nucleotides 1819-2171 from the sequence identified by GenBank accession number L31763, and pDT3.4, which plasmid contains an NruI/XbaI fragment comprising nucleotides 2102-3314 from the sequence identified by GenBank Accession number L31763. It will be understood that the nucleic acid that is detected for the purpose of determining whether the strain comprises an intA gene may be detected on the basis of a particular nucleotide, region or conformation of the nucleic acid, as described below.

In one embodiment, the method of the first or second aspect comprises sequencing a portion of nucleic acid of the strain to determine whether the strain comprises an intA gene. The portion may be one which comprises the intA gene, or one which comprises a nucleotide, or region of nucleotide sequence, in linkage with the intA gene.

In one embodiment, the method of the first or second aspect comprises cleaving a nucleic acid of the strain with a restriction endonuclease for detecting the intA gene to determine whether the strain comprises an intA gene. The endonuclease may be one capable of cleaving within the intA gene. Alternatively, the endonuclease may be one capable of cleaving within a region in linkage with the intA gene. As described herein, an example of a suitable enzyme is HindIII.

In another embodiment, the method of the first or second

I

aspect comprises hybridising a nucleic acid molecule capable of detecting intA with a nucleic acid of the strain to determine whether the strain comprises an intA gene. The nucleic acid molecule may be one capable of hybridising to the intA gene. Alternatively, the nucleic acid molecule may be one capable of hybridising to a region in linkage with the intA gene. As described herein, an example of a suitable nucleic acid molecule is that comprising nucleotides 924 to 1423 of the sequence identified by GenBank accession no. L31763.

The inventors also recognise that the determination on whether the D. nodusus strain comprises an intA gene may be made by detecting a peptide to determine whether the strain comprises an intA gene. The peptide may be a gene product of the intA gene, including for example, a peptide translated from RNA that is transcribed from the intA gene. Alternatively, the gene product may be a product produced by the action of a peptide translated from RNA that is transcribed from the intA gene. Further, the peptide may be produced by a gene or open reading frame that is linked with the intA gene.

In one embodiment, the method comprises contacting a peptide of the strain with an antibody for selectively binding to the peptide in conditions for permitting the antibody to selectively bind to the peptide to determine whether the strain comprises an intA gene.

The inventors recognise that a number of methods could be used for determining whether the D. nodusus strain produces thermostable protease. The gelatin gel test is one method. Another method is the Hide Powder Azure Method. Examples of methods that could be used for determining whether the D. nodusus strain produces thermostable protease are described in Stewart, D.J. and Claxton, P.D. (1993) Ovine footrot. Clinical Diagnosis 9 and Bacteriology. In: Corner, L.A. and Bagust, T.J., (Eds.) Australian Standard Diagnostic Techniques for Animal Diseases, pp. 3-27. Melbourne: CSIRO.

In one embodiment, the method comprises heating the at least one protease to a temperature for destroying the enzymatic activity of a thermolabile protease, and determining the functional activity of the at least one protease, to determine whether the strain produces at least one thermostable protease.

Typically, the determination on whether the D. nodusus strain produces thermostable protease is made according to the gelatin gel test described in Stewart, D.J. and Claxton, P.D. (1993) supra. Accordingly, the functional activity of the at least one protease is typically determined by contacting.the heated enzyme with a gelatin gel in conditions for permitting a thermostabile protease to digest the gelatin gel and determining digestion of the gelatin gel.

It will be understood that the strain for determination in the method of the invention may be provided in a sample from an animal, for example a sample derived from an animal hoof.

As the inventors have found that the virulence of more than 20 different strains of D. nodusus, including benign strains that produce thermostable protease, and benign strains that produce thermolabile protease, can be determined according to the invention, the inventors recognise that the D. nodusus strain for determination in the method may be any strain of D. nodusus, including those capable of causing ovine footrot.

10

EXAMPLES

Aim To develop an improved laboratory test for distinguishing gel-stable, field benign D. nodusus strains from gelstable, field-virulent strains.

Materials and Methods 1. D. nodosus strains and media.

The D. nodusus benign strains 819, 1169, and 2483, and virulent strain 1311, were obtained from Regional Veterinary Laboratory, NSW Agriculture, Wagga, NSW.

Virulent strains A198, B1006, G1220, H1215, D1172, intermediate strain AC3577 and benign strains C305, H1204, AC390, 1493, 3138, 1469 were obtained from Department of Microbiology, Monash University, Clayton, Victoria, Australia. D. nodusus strain 1311A was derived from parent strain 1311 during routine laboratory growth in.

this work. Gel-stable, field benign strains 238, 239, 245, 251, 269, 272, 277, 280, 283, together with benign strain 250, were obtained from Regional Veterinary Laboratory, NSW Agriculture, Orange, NSW. All D. nodusus strains were cultured on Blood Eugon agar (45 mg/ml Eugonagar, 2 mg/ml yeast extract, 5% defibrinated horseblood) or in Eugonbroth (30 mg/ml Eugonbroth powder, 2 mg/ml yeast extract [Skerman, T.M. (1975) J.Gen.Microbiol. 87, 107-119] for 3 to 7 days at 37 0

C

under anaerobic conditions in an atmosphere of 80% (v/v)

N

2 10% H2 and 10% CO 2 2. Preparation of D. nodusus genomic DNA.

D. nodusus genomic DNA was prepared from 150 ml liquid cultures. 1 ml of Eugonbroth was added to a plate culture of D. nodusus, and cells were resuspended in the Eugonbroth by gently scraping the bacteria off the solid medium using a sterile glass spreader. Using a sterile pipette, the 1 ml cell suspension was then transferred into 150 ml of Eugonbroth and grown under anaerobic conditions at 37 0 C for 48 to 72 hours, depending upon the 11 strain of D. nodusus. D. nodusus genomic DNA was prepared using the method described by Anderson, et al.

(1984) J.Bacteriol. 160, 748-754.

3. Preparation of plasmid DNA.

To prepare plasmid DNA for probe preparation, a method modified from that described by Holmes, D.S. and Quigley, M. (1981) Anal.Biochem. 114, 1.93-197 was used. recombinant cells from a 10 ml overnight culture were centrifuged for 10 mins at 1 500 g in an MSE benchtop centrifuge. To remove chromosomal DNA, proteins and cellular debris, the pellet was resuspended in 500 il of STET (50 mM Tris pH 8.0, 50 mM EDTA, 8% sucrose, Triton-X100), after which 40 pl of Lysozyme (10 mg/ml in mM Tris pH 8.0) was added. The sample was then boiled for secs, cooled on ice, and centrifuged (Clements Orbital 100) at 12 000 g for 10 mins. The resulting gelatinous precipitate was discarded and the plasmid DNA precipitated by adding 500 pl of isopropanol to the supernatant, which was subsequently held at -20 0 C. After 20 mins the plasmid DNA was collected by centrifugation at 12 000 g for mins, washed with 70% ethanol, dried under vacuum, and resuspended in 100 pl of TE buffer. One half-volume of phenol and a half-volume of chloroform/isoamyl alcohol (24:1 v/v) were added. The sample was vortexed and centrifuged (Clements Orbital 100) at 12 000 g for 5 mins.

To the supernatant, an equal volume of chloroform/isoamyl alcohol was added, mixed and again subjected to centrifugation at 12 000 g for 1 min. The supernatant was collected and a one-tenth volume of 3 M sodium acetate pH and 2.5 volumes of 100% ethanol added, and then held at -20 0 C for 20 mins to precipitate the DNA. The DNA was then collected by centrifugation at 12 000 g and 4 0 C for mins. The pellet was washed with 70% ethanol, dried under vacuum and resuspended in the desired volume of TE buffer. DNA for probe preparation was linearised with the restriction enzyme EcoRI before labelling.

12 4. Probe preparation and Southern blotting.

The Prime-a-gene labelling kit (Promega) was used to label ng of DNA template with 300 Ci/mmol of [a- 32 P]-dATP or [y- 32 P]-dATP in accordance with the manufacturer's instructions. Following the labelling reactions unincorporated nucleotides were removed from the labelled probe using Sephadex G-50 columns. A hole was punched in the bottom of a 0.5 ml microfuge tube, which was then plugged with silanised glass wool [Ausubel, et al.

(1989) Current Protocols in Molecular Biology, New York: John Wiley and Sons]. The 0.5 ml microfuge tube was then transferred to a 1.5 ml microfuge tube, and 0.6 ml of Sephadex G-50 Sephadex G-50, 20 ml Spun Column Stop Buffer [10 mM Tris-HCl, 1 mM EDTA, 0.2% SDS]) was added and then centrifuged at 3 500 g (Clements Orbital 100) for 2 to 3 seconds. The column was subsequently washed with spun-column stop buffer, and centrifuged at 3 500 g (Clements Orbital 100). 50 pl of spun column stop buffer was added to the radiolabelled probe mixture, and.

then the sample was transferred to the top of the column.

A further 50 1l of spun column stop buffer was used to wash out the reaction tube, and then added to the top of the column, which was then centrifuged for 2 to 3 seconds.

The eluent contains labelled probe molecules whilst unincorporated nucleotides remain in the column, which is discarded. Percent incorporation was determined by comparing the counts per second of the eluent to the column.

D. nodusus genomic DNA (1-2 pg) was digested with HindIII overnight. The digested DNA was size-fractionated on a 1% cm x 20 cm) agarose gel (Bio-Rad) at a constant 35 V for 16 hrs. Pretreatment of the DNA involved partial hydrolysis using 0.25 M HCl for 30 mins, followed by denaturation by soaking in 0.5 M NaOH/1 M NaC1 for mins, and neutralisation in 0.5 M Tris-HC1/1.5 M NaCl for 13 a further 30 mins. The DNA was then transferred to a nylon membrane (Amersham or Boehringer-Mannheim) via capillary action using the method described by Southern, E.M. (1975) J.Mol.Biol. 98, 50.3-517. After blotting for 4 hrs, the nylon membrane was baked at 65 0 C overnight to fix the DNA to the membrane.

Membrane-bound DNA was prehybridised with 2.5 ml of hybridisation solution (50% formamide, 4 x SSC diluted from 20 x SSC stock [0.3 M sodium citrate, 3 M sodium chloride], 0.5% blotto from 10% Blotto stock [10% (w/v) Skim milk powder in water, with sodium azide 0.001%], 1% SDS, 0.3 mg/ml sheared salmon sperm DNA) per 10 cm 2 for two to six hours at 37 0 C. The membrane was then hybridised with 50 pV of 32 P-labelled probe added to 1 ml of hybridisation solution per 10 cm 2 of membrane for 16 hours at 37 0 C. After hybridisation probes were decanted and stored at -20 0 C for re-use. The membranes were rinsed in 2X SSC, then washed in 2 x SSC for 30 mins, 2 x SSC/0.1% SDS for 30 mins and 0.1 x SSC/0.1% SDS for 15 mins. All stringency washes were performed at 65 0 C. Blots were wrapped in cling wrap, and exposed to X-ray film for between 2 hours and 4 days.

5. Gelatin gel tests.

Gelatin gel tests were performed at the Regional Veterinary Laboratory, NSW Agriculture, Forest Road, Orange, NSW, 2351 using the Australian Standard Method [Stewart, D.J. and Claxton, P.D. (1993) Ovine footrot.

Clinical Diagnosis and Bacteriology. In: Corner, L.A. and Bagust, (Eds.) Australian Standard Diagnostic Techniques for Animal Diseases, pp. 3-27. Melbourne: CSIRO]. Supernatants from D. nodusus cultures were heated at 67 0 C for 0, 8 and 16 minutes, then aliquoted into wells of a 1.2% agarose gel containing 7.5 mg/ml gelatin. The diameter of the zone of clearing around the well due to proteolysis was measured after eighteen hours, following 14 precipitation of the gelatin with a saturated solution of ammonium sulphate. Culture supernatants from virulent strains produce much larger zones of clearing than culture supernatants from benign strains.

Results and Discussion Genomic DNA was prepared from gel-stable, field benign strains (238, 239, 245, 251, 269, 272, 277, 280 and 283), gel-stable, field virulent strains (A198, 1311, B1006, G1220, H1215), gel-stable intermediate strain AC3577, gelunstable, field-benign strains (C305, 250, 819, 1169, 2483, 1493, 3138 and 1469) and strain 1311A, which is produces unstable proteases, but is of unknown virulence in the field. The DNA was digested with HindIII, transferred to a membrane and hybridised with the probe pDT20(10). This plasmid contains a RsaI fragment corresponding to nucleotides 924-1423 from the sequence identified by GenBank accession number L31763. These nucleotides form part of the intA coding region, which spans nucleotides 418-1621.

The results from this experiment are shown in Figs 1 and 2. Fig. 1, lanes 1-3 and 5-10 shows that there was no hybridisation between DNA from any of the gel-stable, field benign strains with this probe. By contrast, DNA from all of the gel-stable, field virulent strains hybridised to this probe (Fig. 1, lane 11, and Fig. 2, lanes 1, 3, 6, 7, DNA from most of the benign strains tested did not hybridise with this probe (Fig. 1, lane 4, Fig. 2, lanes 2, 10-15) with the exception of DNA from benign strain H1204 (lane 8) To confirm that DNA from the gel-stable, field-benign samples was indeed present on the membrane, the same membrane was hybridised with the probe pDT17(1), which contains an XhoI/Nrul fragment corresponding to nucleotides 239-897 from the sequence identified by 15 GenBank accession number Y15939. This contains part of the genes askA and glpA from D. nodusus, which are thought to be present in all strains. This probe hybridised to all of the gel-stable, field benign strains (Fig. 3), confirming that the lack of hybridisation to the intA probe was not due to the absence of DTA on the membrane.

Thus, these results show that gel-stable, field-virulent strains contain intA, or part thereof, while gel-stable, field-benign strains do not. Hybridisation to this probe could thus be used as a diagnostic test to distinguish gel-stable, field benign isolates from gel-stable, field virulent isolates. Hybridisation to this probe by the gel-unstable, field benign isolate H1204 suggests that this test would only be useful to distinguish virulent isolates from benign isolates after they were shown to be gel-stable.

Prolonged exposure of the X-ray films shows faint bands in the lanes containing gel-stable, field virulent strains (data not shown). This may be due to cross-hybridisation with other integrase genes, or may indicate that a small portion of the intA gene, within the region detected by the probe pDT20(10) is present in these strains.

This Southern blot procedure provides a means of identifying gel-stable, field benign strains. In addition to the Southern blot procedure, a PCR-based test could be developed using primers spanning part of the intA gene.

Alternatively, an antibody prepared could be prepared against IntA, and tested for suitability in a western blot assay.

Claims (9)

1. A method for determining whether a D. nodusus strain is virulent, the method comprising the following steps: determining the thermostability of at least one protease produced by the strain; and determining whether the strain comprises an intA gene.

2. A method according to claim 1 comprising determining whether the strain comprises a gene having a nucleotide sequence shown in Figure 4 to determine whether the strain comprises an intA gene.

3. A method according to claim 1 comprising determining whether the strain comprises a fragment of a gene having a nucleotide sequence shown in Figure 4, to determine whether the strain comprises an intA gene.

4. A method according to claim 1 comprising determining whether the strain comprises a gene having at least 95% nucleotide identity with an intA gene to determine whether the strain comprises an intA gene.

A method according to claim 1 comprising determining whether the strain comprises a nucleic acid that is in linkage with the intA gene, to determine whether the strain comprises an intA gene.

6. A method according to any one of the preceding claims comprising detecting DNA, RNA, cDNA or a peptide to determine whether the strain comprises an intA gene.

7. A method according to any one of the preceding claims wherein the thermostability of the at least one protease is determined by the gelatin gel test.

8. A method according to any one of the preceding claims wherein step follows step

9. A method for determining that a D. nodusus strain is virulent, the method comprising the following steps: 004868929 -17- t determining that the strain produces at least one thermostable protease; and 0 determining that the strain comprises an intA gene. A method for determining whether a D. nodusus strain is virulent, the method being substantially as described herein with reference to the Examples. tV 5 Dated 27 June 2005 c Freehills Patent Trade Mark Attorneys c Patent Trade Mark Attorneys for the Applicant: N The University of New England