WO2010031127A1

WO2010031127A1 - Detection of treponema infection

Info

Publication number: WO2010031127A1
Application number: PCT/AU2009/001234
Authority: WO
Inventors: Eric Charles Reynolds; Neil Martin O'brien-Simpson; Paul David Veith; Stuart Geoffrey Dashper
Original assignee: Oral Health Australia Pty Ltd
Priority date: 2008-09-17
Filing date: 2009-09-17
Publication date: 2010-03-25

Abstract

The present invention relates to methods for detecting Treponema, in particular Treponema denticola, infections and related pathologies such as periodontal disease.

Description

Detection of Treponema infection

Field of the invention

The invention relates to detecting Treponema, in particular Treponema denticola, infections and related pathologies.

Background of the invention

Treponemes are Gram-negative, motile, asaccharolytic, anaerobic spirochaetes with characteristic helical morphology, some of which are associated with chronic diseases of humans. Treponema denticola (T. denticola) is a part of the normal microbiota of the oral cavity and has been implicated as an aetiological agent of chronic (adult) periodontitis. Treponema socranskii, Treponema vincentii and Treponema medium are also reported to play, as yet, poorly defined roles in chronic periodontitis. T. denticola along with Porphyromonas gingivalis and Tannerella forsythia (formerly Bacteroides forsythus) have been shown to form a bacterial consortium called the Red Complex that is strongly associated with the clinical progression of chronic periodontitis. Chronic diseases such as periodontitis and syphilis are characterized by an inflammatory immune response to the pathogen followed by extensive host tissue degradation. Cell surface components, especially proteins, are largely the mediators of the host response, as such cell surface exposed proteins offer targets for the development of immunodiagnostics and novel therapeutics. T. denticola is a relatively poorly studied bacterium to date.

Treponemes are evolutionarily quite separate from the majority of Gram-negative eubacteria and have evolved a number of highly unique characteristics, such as the form of glycocongugate in the outer leaflet of the outer sheath and the presence of periplasmic flagella. Unlike lipopolysaccharide (LPS) of other Gram-negative bacteria the outer membrane sheath of T. denticola lacks 3-deoxy-D-manno-octulosonic acids, heptoses and β-hydroxy fatty acids and the cytoplasmic membrane and outer membrane sheath have similar fatty acyl chain composition (anteiso-pentadecanoic acid, palmitic acid and iso-palmitic acid) indicating that the membrane fluidity of T. denticola is similar to the membranes containing lipoteichoic acid of Gram-positive bacteria. T. denticola lipid does form normal bilayer membranes with a distinct phase transition typical of LPS, but the phase transition of the outer sheath lipid is broader and the temperature lower (22°C) than for the phase transition of LPS (35.5°C). The periplasmic location of the T. denticola flagella, and the helically-shaped cell cylinder of T. denticola, like all spirochaetes, result in a cork screw-like locomotion, which aids movement in highly viscous environments and has been linked to tissue penetration. The flagellum of T. denticola is composed of a basal body, rod, hook and a filament. The flagellar filament consists of three core proteins FIaBI , FlaB2 and FlaB3 and a major sheath protein FIaA.

Treponemes share many characteristics with Gram-positive bacteria and recent phylogenetic studies have shown that spirochaetes form a single cluster distinct from both Gram-positive and Gram-negative bacteria. It is highly likely therefore that the treponemes as a group will also have evolved a range of other unique characteristics. The divergence of T. denticola from a common treponeme ancestor was an, ancient event indicating that in addition to having a range of characteristics unique to treponemes T. denticola will have evolved many virulence factors unique at a, species level. Of the predicted 2,786 protein encoding genes 734 (26%) encode putative proteins that have no significant similarity to any other sequenced protein.

One of the challenges of researching treponemes is the difficulty in cultivating them. T. denticola will not grow on the surface of agar plates and has a long generation time in liquid media. No proteomic studies have been conducted on this bacterium and only a small number of proteins have been analysed from this important pathogen. Consequently, at the time of this invention it was not known which proteins from the predicted over 2,500 proteins expressed by T. denticola would give rise to an antigenic response and therefore be useful as targets for disease or condition diagnosis.

There is a need for an assay for detecting Treponema related organisms such as T. denticola and especially those organisms involved in Treponema related disease, such as periodontal disease. Summary of the invention

In one embodiment the invention provides a method for determining whether an individual has a Treponema infection including:

- selecting an individual;

-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 2 or 3 herein, wherein detection of said protein, peptide or fragment thereof determines that the individual has a Treponema infection,

thereby determining whether the individual has a Treponema infection.

In one embodiment the invention provides a method for determining whether an individual is susceptible to periodontal disease, likely to develop periodontal disease or screening for early stage periodontal disease including:

- selecting an individual;

^detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 2 or 3 herein, wherein detection of said protein, peptide or fragment thereof determines that the individual is susceptible to periodontal disease, likely to develop periodontal disease or has early stage periodontal disease,

thereby determining whether the individual is susceptible to periodontal disease, likely to develop periodontal disease or has early stage periodontal disease.

In one embodiment the invention provides a method for monitoring Treponema infection in an individual receiving treatment for periodontal disease including:

- selecting an individual who is undergoing or who has undergone treatment for periodontal disease; -detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 2 or 3 herein, wherein detection of said protein, peptide or fragment thereof determines the individuals response to treatment for periodontal disease,

thereby determining whether the individual is responding to treatment for periodontal disease.

In one embodiment the invention provides a method for determining the risk of an individual developing periodontal disease including:

- selecting an individual;

-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 2 or 3 herein, wherein detection of said protein, peptide or fragment thereof determines the risk of an individual developing periodontal disease,

thereby determining the risk of an individual developing periodontal disease.

In one embodiment the invention provides a method for determining the risk of periodontal disease progression in an individual including:

- selecting an individual;

-detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 2 or 3 herein, wherein detection of said protein, peptide or fragment thereof determines the risk of periodontal disease progression in an individual;

thereby determining the risk of periodontal disease progression in an individual.

In certain embodiments, the methods of the invention are used for determining whether a periodontal site has a Treponema infection, susceptible to periodontal disease, likely to develop periodontal disease, screening for early stage periodontal disease, for monitoring Treponema infection in a periodontal site receiving treatment for periodontal disease, for determining risk of a periodontal site developing periodontal disease and/or for determining the risk of periodontal disease progression at a periodontal site.

In other embodiments there is provided a use of an antibody specific for a protein, peptide or fragment thereof described in Table 2 or 3 herein for determining whether an individual has a Treponema infection.

In another embodiment there is provided a use of a polynucleotide or fragment thereof for detecting a nucleic acid that encodes or controls the expression of a protein described in Table 2 or 3 herein for determining whether an individual has a Treponema infection.

In further embodiments there is provided a kit for determining whether an individual has a Treponema infection including:

- an antibody specific for a protein, peptide or fragment thereof described in Table 2 or 3 herein; or

- a polynucleotide or fragment thereof capable of detecting a nucleic acid encoding or controlling the expression of a protein, peptide or fragment thereof described in Table 2 or 3 herein.

As described further below, the kit may further include one or more Treponema proteins, peptides or fragment thereof for use as a control and written instructions for use of the kit in a method as described herein.

In one embodiment there is provided a device or apparatus for the detection of Treponema infection including:

- a protein, peptide or fragment thereof described in Table 2 or 3 herein;

wherein the protein, peptide or fragment thereof is arranged on a solid phase to permit capture of an antibody in a sample taken from a person for whom Treponema infection is to be detected. In another embodiment there is provided a device or apparatus for the detection of Treponema infection including:

- an antibody directed to a protein, peptide or fragment thereof described in Table 2 or 3 herein;

wherein the antibody is arranged on a solid phase to permit capture of a protein, peptide or fragment thereof in a sample taken from a person for whom Treponema infection is to be detected.

In one embodiment the invention provides a substantially non-glycosylated protein, peptide or fragment thereof described in Table 2 or 3 herein. Preferably, the protein, peptide or fragment thereof described in Table 2 or 3 herein does not contain any glycosylated amino acids. Preferably, the substantially non-glycosylated protein is a non-glycosylated form of a protein selected from the group consisting of OppA (TDE1071), Msp (TDE0405), PrcA (TDE0761), FIaB (TDE1004), and FIaBI (TDE1477). Preferably, the protein, peptide or fragment thereof described in Table 2 or 3 herein that does not contain any glycosylated amino acids is selected from the group, consisting of OppA (TDE1071), Msp (TDE0405), PrcA (TDE0761 ), FIaB (TDE1004), and FIaBI (TDE1477).

Typically, the protein, peptide or fragment thereof of the invention is in a composition. The composition further comprises a carrier.

"Substantially non-glycosylated" protein, peptide or fragments thereof are described further herein.

In one embodiment there is provided an antiserum raised against a protein, peptide or fragment thereof described in Table 2 or 3 herein. In one embodiment, the antiserum has specificities that react with non-glycosylated protein, peptide or fragments thereof described in Table 2 or 3 herein. In one embodiment the antiserum is polyclonal. In another embodiment the antiserum is monoclonal. In one embodiment there is provided a use of a substantially non-glycosylated protein, peptide or fragment thereof described in Table 2 or 3 herein in the preparation of a medicament for the treatment or prevention of a Treponema infection (and/or the other conditions identified herein as suitable for treatment). In one embodiment the medicament is for the treatment or prevention of periodontal disease.

In one embodiment there is provided a substantially non-glycosylated protein, peptide or fragment thereof described in Table 2 or 3 herein for use in the treatment or prevention of a Treponema infection (and/or the other conditions identified herein as suitable for treatment).

In another embodiment the invention provides a composition for the treatment or prevention of periodontal disease (and/or the other conditions identified herein as suitable for treatment) comprising as an active ingredient a substantially non- glycosylated protein, peptide or fragment thereof described in Table 2 or 3 herein.

Also provided is a use of an antigen in the form of:

- a protein, peptide or fragment thereof that is expressed in a Treponema or;

- a protein, peptide or fragment thereof described in Table 2 or 3 herein

in the manufacture of means for determining whether an individual is infected with a Treponema.

In another embodiment there is provided is a use of a non-glycosylated antigen in the form of:

- a protein, peptide or fragment thereof that is expressed in a Treponema or;

- a protein, peptide or fragment thereof described in Table 2 or 3 herein

in the manufacture of means for determining whether an individual is infected with a Treponema. In other embodiments there is provided a use of an antibody specific for:

- a protein, peptide or fragment thereof that is expressed in a Treponema or;

- a protein, peptide or fragment thereof described in Table 2 or 3 herein

for determining whether an individual is infected with a Treponema.

In another embodiment there is provided a use of a polynucleotide for detecting a nucleic acid that encodes or controls the expression of:

- a protein, peptide or fragment thereof that is expressed in a Treponema or;

- a protein, peptide or fragment thereof described in Table 2 or 3 herein

In further embodiments there is provided a kit for determining whether an individual is infected with a Treponema including:

- an antigen being a protein, peptide or fragment thereof that is expressed in a Treponema; or

- an antigen being a protein, peptide or fragment thereof described in Table 2 or 3 herein; or

- an anti- Treponema antibody specific for at least one of the above described antigens; or

- an antibody specific for an idiotype of an above described anti- Treponema antibody; or

- a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of at least one of the above described antigens. In one embodiment there is provided a method of treating a Treponema infection in an individual including

- detecting a Treponema infection in an individual according to a method of the invention described herein; and

- applying a therapeutic agent to treat the Treponema infection.

A kit of the invention may further include written instructions for use of the kit in a method as described herein.

In further embodiments there is provided an immune complex including an antigen being a protein, peptide or fragment thereof described in Table 2 or 3 herein bound to an antibody specific for said protein, peptide or fragment thereof.

Brief description of the drawings

Figure 1: Cell density of T. denticola growing in continuous culture in a C-30 BioFlo chemostat with OBGM media and the medium flow rate set to give a mean generation time of 15.75 h as determined by absorbance at 650 nm.

Figure 2: Relationship of T. denticola cell density as determined by flow cytometry to absorbance of the cell suspensions measured at a wavelength of 650 nm.

Figure 3: 2D gel of (A) French Pressure cell membrane fraction (FPP) and (B) TritonX- 114 extract (TX) showing identity of selected spots. (A) 1-CheA (TDE1491), 2-Lipoprotein (TDE1072), 3-Tp92 Omp (TDE2601), 4-Dentilisin (TDE0762), 5-OppA (TDE1071) & TDE0985, 6-Cytoplasmic filament protein A (TDE0842), 7-Msp (TDE0405), 8-MgIB (TDE2217), 9-TmpC (TDE1950), 10-Tpn38b (TDE0951), 11-FIaA (TDE1712), 12-FIaB (TDE1475), 13-FIaB (TDE1477), 14-FIaB (TDE1004), 15-TmpB (TDE2433), 16-FIaA (TDE1408), 17-LysM domain protein (TDE0018), 18-FIaA (TDE1409). (B) 1 -Lipoprotein (TDE1072), 2-Dentilisin (TDE0762), 3-TDE0985, 4-OppA (TDE1071), 5-Urocanate hydratase (TDE2606), 6-ABC-type peptide binding protein (TDE1273), 7-Msp (TDE0405), 8- GrdC (TDE0240), 9-Tyrosine phenol-lyase (TDE1118), 10-Tryptophanase (TDE0251), 11- GndB (TDE2119), 12-LpdA (TDE1629), 13-Glycine cleavage system P1 subunit (TDE1625), 14-Glycine cleavage system P2 subunit, 15-Methylaspartate ammonia lyase (TDE2235), 16- Methylaspartate mutase E subunit (TDE2236), 17-Hemolysin (TDE1669), 18-Glycine cleavage system T protein (TDE1627), 19-MgIB (TDE2217), 20-TmpC (TDE1950), 21- Ornithine carbamoyltransferase (TDE0929), 22-ABC-type iron-binding protein (TDE2234), 23-Glutamate formiminotransferase (TDE0296), 24-GrdE2 (TDE2120), 25-GrdE2 (TDE2120), 26-OmpH (TDE2602), 27-Glycine cleavage system H protein (TDE1626).

Figure 4: Identification of TDE0296 and TDE0947 from a single spot.

A. MALDI-TOF MS spectrum obtained from a 2D gel spot. The Mascot PMF search (inset table) yielded a positive match to TDE0296. Boxed peptides were successfully identified by MS/MS analysis. B. LIFT MS/MS spectrum of m/z 1504.70 showing matched y-ion series to a TDE0296 peptide (LLDYESDKDHNR). C. LIFT MS/MS spectrum of m/z 1106.67 showing matched y-ion series and other significant ions to a TDE0947 peptide (WLAIEPLPR).

Figure 5: 2D gel (A) and Western blot (B) of Triton X-114 extract of T. denticola showing identity of antigenic spots. The Western blot was probed with antisera raised in BALB/c mice that were immunized twice with formallin-killed T. denticola cells. The antigenic spots were compared to the 2D gel and identified on the basis of pi and MW.

Figure 6: Amino acid metabolic pathways of T. denticola. Amino acids are shown in grey boxes, and enzyme numbers are TDE accession numbers (see Table 2). The fermentation of glycine to acetate mostly involves members of the glycine cleavage system and glycine reductase complex whose TDE accessions are shown. Conversion of ornithine to glutamate and mesaconate to pyruvate are only speculative. The enzymes required for these conversions have not yet been found at the genome or protein level. The pathways shown are suggested by the enzymes identified, further work is required to demonstrate that these pathways are actually followed. Many of the reactions catalysed can also operate in the reverse direction of that shown. TDE0933 (acetate kinase) was not identified in this study, but is included to demonstrate the location of ATP formation in this pathway. Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

The inventors have identified the major proteins and antigenic proteins of T. denticola. Importantly, these proteins can be used to detect Treponema infection in individuals and provide a means for diagnosing periodontitis, periodontal disease or related pathology caused, initiated or potentiated by Treponema bacteria in an individual.

Thus in one embodiment there is provided a method for determining whether an individual has a Treponema infection including:

-selecting an individual;

- detecting whether the selected individual contains a protein, peptide or fragment thereof described in Table 2 or 3 in the individual, wherein detection of said protein determines that the individual has a Treponema infection.

thereby determining whether the individual has a Treponema infection.

Typically detection of the protein, peptide or fragment thereof in the individual is assessed by the steps of:

-obtaining a sample from the individual;

- measuring for the expression or presence of the target protein in the sample; and

- comparing the measured level with an uninfected control that describes the level of expression of the target as observed in a sample from a subject that has been determined as not having a Treponema infection.

A Treponema infection is an infection that is characterised by invasion or contamination of tissues or cells by a Treponeme. A Treponema infection may have clinical or subclinical manifestations including acute or chronic inflammation of the gingiva. The hallmarks of acute inflammation may be present including an increased movement of plasma and leukocytes from the blood into the injured tissues. Clinical signs of acute infection of the gingiva may also be present including rubor (redness), calor (increased heat), tumor (swelling), dolor (pain), and functio laesa (loss of function). Chronic inflammation may be characterised by leukocyte cell (monocytes, macrophages, lymphocytes, plasma cells) infiltration. Tissue and bone loss may be observed. A Treponema infection may also be characterised by an increased level of Treponema bacteria, particular T. denticola, above a normal range observed in individuals without a Treponema infection. For example, a Treponema infection may be manifested in an individual at a periodontal site that has greater than approximately 10³ to 10⁵ Treponema cells/mg of subgingival plaque. A Treponema infection may be manifested in an individual at a periodontal site that has greater than approximately 10⁴ Treponema cells/mg of subgingival plaque.

An "uninfected control" sample is further defined herein.

The "measuring for the expression" of a target protein in a sample is the detection of the degree or level or amount or concentration of a protein, peptide or fragment thereof described in Table 2 or 3 herein.

An individual that is selected and from whom a sample is obtained may be an individual that has a previous history of Treponema infection or periodontal disease or a person already diagnosed with periodontal disease. Alternatively, the individual may not have had a prior Treponema infection or experienced periodontal disease previously.

In certain embodiments, the target protein is a protein, peptide or fragment thereof described in Table 2 or 3.

Detection of a protein, peptide or fragment thereof indicates the presence of a Treponema bacteria, in particular T. denticola. The detection of a level or degree of expression of a protein, peptide or fragment thereof may indicate the level of Treponema bacteria, in particular T. denticola, in a sample which in turn may indicate the occurrence of, or degree of, or progression of a Treponema infection or periodontal disease. For example, a strong level of expression, increased amount or concentration of a protein, peptide or fragment thereof in a sample taken from an individual compared to an uninfected control may indicate a high level of Treponema in the sample. The detection of the presence or level or degree of expression of a protein, peptide or fragment thereof may correlate with the risk of developing a Treponema infection or periodontal disease.

A "Treponema infection" in an individual or an individual "infected with a Treponema" generally refers to:

(1) an elevated level of Treponema in a sample taken from the individual compared to an uninfected control sample;

(2) an increased proportion of Treponema bacteria in a sample taken from the individual compared to the total level of bacteria in an uninfected control sample;

(3) an increased proportion of Treponema bacteria relative to one or more other bacteria species in a sample taken from the individual when compared to an uninfected control sample; or

(4) the presence of Treponema bacteria in a sample compared to an uninfected control sample when Treponema is undetectable in the uninfected control.

In any embodiment of the invention the Treponema may be Treponema denticola.

In one embodiment the sample obtained from the individual is a body fluid or fraction thereof. The body fluid or fraction thereof from the individual may be an oral fluid taken from the oral cavity. In particular, an oral fluid may be saliva, gingival crevicular fluid or blood. It is recognized that oral fluids, for example saliva, are a combination of secretions from a number of sources such as partiod, submandibular, sublingual, accessory glands, gingival mucosa and buccal mucosa and the term oral fluid includes the secretion of each of these sources individually or in combination. The saliva may be stimulated or in a preferred embodiment unstimulated. Stimulation of the saliva in the individual may occur by allowing the individual to chew on sugar-free gum, a piece of paraffin film or tart candy. Unstimulated saliva means that the subject will expectorate into a collection vessel without stimulation of salivary flow. Saliva specimens for testing can be collected following various methods known in the art, for example, stimulated or unstimulated saliva can be sampled by the individual expectorating into a collection vessel or using a swab or syringe to extract the saliva. Other ways for obtaining unstimulated saliva are known in the art. (Nazaresh and Christiansen, J. Dent. Res. 61 : 1158- 1162 (1982)). Methods and devices for collecting saliva have also been described. (See also, U. S. Patent No. 5,910, 122).

It is contemplated that the methods of the present invention can also be practiced by analyzing stimulated saliva.

Furthermore, the methods of the present invention are not limited to performing salivary analysis immediately after collection of the sample. In certain embodiments, salivary analysis following the methods of the present invention can be performed on a stored saliva sample. The saliva sample for testing can be preserved using methods and apparatuses known in the art. (See e.g., U. S. Patent No. 5,968, 746).

It is also contemplated that the methods of the present invention be used to perform salivary analysis on saliva samples that have been treated to reduce its viscosity.

The viscous nature of saliva, due to the nature of mucopolysaccharides, makes testing of these fluids difficult. In order to prepare saliva for any laboratory testing procedure, the saliva may be rendered sufficiently fluid (i.e.viscosity must be reduced) and free from debris. Techniques used to remove debris include centrifugation and filtration. The viscosity of saliva can also be reduced by mixing a saliva sample with a cationic quaternary ammonium reagent. (See, U. S. Patent No. 5,112, 758).

In another embodiment, the sample from an individual may be taken from the crypts of the dorsum of the tongue.

The sample from the individual may be taken from a specific periodontal site. The sample may be taken from a periodontal site that exhibits clinical signs of periodontal disease or Treponema infection. A method, use, protein or composition of the invention could then be used to determine whether the periodontal site is infected with Treponema, or at risk of periodontal disease progression. In another embodiment, the sample from an individual may be a sample of a tissue. The tissue or part thereof may be from the oral cavity. In certain embodiments the tissue is gingival. The gingival tissue may be from various sites around a tooth including disto- buccal, mid-buccal, mesio-buccal, mesio-palatal, mid-palatal and disto-palatal and disto- lingual, mid-lingual and mesio-lingual. The tissue may be obtained by normal biopsy or may be obtained from an extracted tooth.

In another embodiment the sample from the individual may be dental plaque. The plaque may be subgingival or supragingival. Subgingival plaque may be sampled using a sterile curette or paper point. Supragingival plaque may be removed using standard techniques known in the art. The subgingival plaque may be collected from various sites around a tooth including disto-buccal, mid-buccal, mesio-buccal, mesio-palatal, mid- palatal and disto-palatal and disto-lingual, mid-lingual and mesio-lingual periodontal sites. The subgingival plaque samples may be obtained during the normal dental examination provided by a qualified dentist or periodontist. The plaque sample may be analysed as is or treated to extract the protein, peptide or fragment thereof of interest using an extraction buffer. An extraction buffer could contain a pH buffer (e.g. phosphate, HEPES, etc), salts (e.g. NaCI) to maintain ionic strength and protein solubilising agents [e.g. detergents (SDS, Triton X100, etc), reducing agents (e.g. dithiothreitol, cysteineHCI) and/or chaotropic agents (e.g. urea, guanidinium chloride, lithium perchlorate).

An "uninfected control" is a sample from an individual or represents a set of parameters previously defined from individuals that do not have all the attributes of an individual with a Treponema infection. For example, the individual from whom the "uninfected control" sample is derived generally does not have inflammation of the gums, antibodies directed against Treponema in their blood, have an amount of Treponema above approximately 10³ to 10⁵ cells/mg of subgingival plaque and/or have an amount of Treponema above approximately 10³ cells/ml of saliva. The "uninfected control" sample may be taken from the oral cavity of a subject that, but for an absence of a Treponema infection, is generally the same or very similar to the individual selected for determination of whether they have a Treponema infection (the latter otherwise known as the "test sample"). The measurement of the level of expression of the target in the sample from the oral cavity of the subject for deriving the uninfected control is generally done using the same assay format that is used for measurement of the target protein in the test sample. It will be appreciated that the control sample may also be taken from the same individual from which the test sample is taken, but at a different time-point, in order to determine the risk of, or progression of the Treponema infection or periodontal disease.

It is contemplated that individuals with a healthy oral cavity may contain a low level of Treponema present, in particular T. denticola. This low level or normal level of bacterial colonisation may be sampled and is also within the scope of "uninfected control". When using such an uninfected control and comparing it to a test sample, determination of whether an individual has a Treponema infection or periodontia! disease includes (1) an elevated level of Treponema in a sample taken from the individual compared to the uninfected control sample, or (2) an increased proportion of Treponema bacteria in a sample taken from the individual compared to the total level of bacteria in the uninfected control sample, or (3) an increased proportion of Treponema bacteria relative to one or more other bacteria species in a sample taken from the individual when compared with the uninfected control sample.

In certain embodiments, the method includes measuring the expression of the target in the uninfected control to compare the measured level in the test sample with the level in the uninfected control.

In other embodiments, an internal standard is applied. This may be used to ensure that the method operates within accepted decision limit quality control criteria. The assay then provides a value/number/result or other output for each test sample. That output is then deemed to represent infection or non infection based on independent data derived from frequency distributions of results from an uninfected control. This control may describe distributions of results obtained from more than one uninfected subject, for example, from an uninfected population which may be of the same species, geographic origin, age, sex as the test sample. A positive -negative cut -point for the method is determined from these distributions to provide defined levels of diagnostic sensitivity and specificity for the method. The internal standard or reference may obviate the need to physically provide an uninfected control in the form of uninfected cells or otherwise to physically measure the level of the target in an uninfected control. Where the internal standard or reference is used, the level of expression in an uninfected control has been predetermined and may be provided for example in the form of written information that is supplied with a diagnostic kit.

The measurement of the level of expression of the target in the subject for deriving the uninfected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied.

Generally the subject from which the uninfected control is derived and the individual selected for determination of whether they have an infection are of the same family. For example, where the individual for determination of whether they have a Treponema infection is a human, the negative control is generally derived from the measurement of the level of expression of the target in a human. In one embodiment, the individual selected for determination of whether they have an infection and the subject from which the negative control is to be derived are from the same species. It may not be necessary that they be of similar age or sex or have been exposed to similar environmental influence.

In another embodiment, the detection of expression of the protein in the individual is assessed by the steps of:

-obtaining a sample from the oral cavity of the individual;

- measuring for the level of expression or presence of the target protein in the sample; and

- comparing the measured level with an infected control that describes the level of expression of the target as observed in a sample from the oral cavity of a subject that has been determined as having a Treponema infection; thereby determining whether the individual has a Treponema infection.

An "infected control" is a sample from an individual or represents a set of parameters previously defined from individuals that do have all the attributes of an individual with a Treponema infection. For example, the individual from whom the "infected control" sample is derived generally does have subgingival plaque, inflammation of the gums, antibodies directed against Treponema in their blood, have an amount of Treponema at a level above approximately 10³ to 10⁵ cells/mg of subgingival plaque and/or have an amount of Treponema at a level above approximately 10³ cells/ml of saliva. The "infected control" sample may be taken from the oral cavity of a subject that, but for the presence of a Treponema infection, is generally the same or very similar to the individual selected for determination of whether they have a Treponema infection (the latter otherwise known as the "test sample"). The measurement of the level of expression of the target in the sample from the oral cavity of the subject for deriving the infected control is generally done using the same assay format that is used for measurement of the target protein in the test sample. It will be appreciated that the control sample may also be taken from the same individual from which the test sample is taken, but at a different time-point, in order to determine progression of the Treponema infection or periodontal disease.

In certain embodiments, the method includes measuring the level of expression of the target in the infected control to compare the measured level in the test sample with the level in the infected control. However, again an internal standard may be applied that obviates the need to provide an infected control or otherwise to measure the level of the target in an infected control.

The measurement of the level of expression of the target in the tissue of the subject for deriving the infected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, again it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied. Generally the subject from which the infected control is derived and the individual selected for determination of infection are of the same family. For example, where the individual for determination of whether they have a Treponema infection is a human, the infected control is generally derived from the measurement of the level of expression of the target in a human. In one embodiment, the individual selected for determination of whether they have an infection and the subject from whom the positive control is to be derived are from the same species. It may not be necessary that they be of similar age or sex or have been exposed to similar environmental influence.

In one embodiment the method includes

- measuring the level of expression of the target protein in a sample from the oral cavity of the individual; and

- comparing the measured level with an uninfected control and an infected control.

In one embodiment, the measurement of the level of one or more Treponema protein, peptide or fragments thereof in the "test sample" may be compared with standard measurements of the same protein, peptide or fragments thereof associated with a series of known treponeme levels, for example, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or 10⁹ cells grown in the laboratory and cell numbers verified using FACS or real time PCR. The antibodies used to capture and detect the protein, peptide or fragment thereof will be used at dilutions to ensure that any chairside test for the bacteria in a sample (e.g. saliva and/or subgingival plaque sample) will give a positive reaction (e.g. a noticeable colour) only for concentrations above certain amounts of cells, e.g. 10⁴ treponeme cells per mg of subginigval plaque sample. The intensity of the colour reaction will be a guide to the clinician on the level of the treponeme at the periodontal site. This will be provided as a colour guide with a kit of the invention described further herein. The positive reading will be taken by the clinician to indicate a treponeme infection at that periodontal site. Hence that site will be treated (e.g. debridement of the tooth root to remove all subgingival plaque) and the patient, depending on the severity of the infection, may be prescribed a course of antibiotic therapy. Typically the target protein is directly detected to determine whether the individual has a Treponema infection. This is otherwise known as a "direct detection" of the target to measure the level of expression of the target. In these embodiments, the target protein, peptide or fragment thereof described in Table 2 or 3 herein may be detected.

In certain embodiments, the level of expression of a molecule, the expression of which is modulated in accordance with the expression of the target is measured. This is otherwise known as an "indirect detection" of the target to measure the level of expression of the target.

In one embodiment, a nucleic acid contained in the individual selected for determination of whether they have a Treponema infection that encodes a target protein, or that is complementary to a nucleic acid that encodes a target protein, is measured. In this embodiment, the nucleic acid may be one which can be used to determine the presence of a given protein, or level of expression of a given protein in an individual as per a molecular genetic approach. One example is where a polynucleotide that is complementary to a nucleic acid (DNA, RNA, cDNA) that encodes a target protein is hybridised to the nucleic acid and hybridisation is detected. One example is quantitative

PCR. Others include quantitative Northern and Southern blotting, and microarray.

In another embodiment, the method includes the step of detecting a target protein, or peptide or fragment thereof in a sample from the individual to assess the level of expression of the target in the individual. The presence of a given protein, or level of expression of a given protein in an individual can be detected by any number of assays. Examples include immunoassays, chromatography and mass spectrometry. One example of an immunoassay that is particular preferred is FACS.

Immunoassays, i.e. assays involving an element of the immune system are particularly preferred. These assays may generally be classified into one of:

(i) assays in which purified antigen (for example, an antigen that is expressed in a Treponema) is used to detect an antibody in host serum. For example, purified antigen is bound to solid phase by adsorption or indirectly through another molecule and serum from an individual is applied followed by another antibody for detecting presence or absence of host antibody;

(ii) assays in which purified antigen (for example, an antigen that is expressed in a

Treponema) is used to detect immune cells, such as T and B lymphocytes. For example, peripheral white cells are purified from an individual and cultured with purified antigen. The presence or absence of one or factors indicating immunity are then detected. Other examples include assays that measure cell proliferation (lymphocyte proliferation or transformation assays) following exposure to purified antigen, and assays that measure cell death (including apoptosis) following exposure to purified antigen;

(iii) assays in which purified antibody specific for an antigen (for example, an antigen that is expressed in a Treponema) is used to detect an antigen in the individual. For example, purified antibody is bound to solid phase, sample from an individual is then applied followed by another antibody specific for the antigen to be detected. There are many examples of this approach including ELISA, RIA;

(iv) assays in which a purified anti-idiotypic antibody is used to detect an antibody from an individual. For example, anti-idiotypic antibody is adsorbed to solid phase, serum from an individual is added and anti-Fc antibody is added to bind to any antibodies from the individual having been bound by the anti-idiotypic antibody.

It will be understood that the level of expression of the target protein may be measured by obtaining a sample from an individual selected for assessment and determining the level of expression of the target in the sample. Alternatively, the level of expression could be determined in vivo, for example by providing labelled antibodies to the individual which can be visualised in vivo.

Various assays that can be used to detect the presence of a target protein in a sample include:

Enzyme linked immunosorbent assay (ELISA): This method involves fixation of a sample, for example saliva, containing a target protein, peptide or fragment thereof to a surface such as a well of a microtiter plate. A target protein specific antibody coupled to an enzyme is applied and allowed to bind to the target protein, peptide or fragment thereof. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of target protein, peptide or fragment thereof present in the sample is proportional to the amount of color produced. A target protein, peptide or fragment thereof standard is generally employed to improve quantitative accuracy.

Western blot: This method involves separation of a target protein, peptide or fragment thereof from other protein by means of an acrylamide gel followed by transfer of the protein, peptide or fragment thereof to a membrane (e.g., nylon or PVDF). Presence of the target protein, peptide or fragment thereof is then detected by antibodies specific to the target protein, peptide or fragment thereof, which are in turn detected by antibody binding reagents. Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of target protein, peptide or fragment thereof and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.

Radio-immunoassay (RIA): In one version, this method involves precipitation of the desired target protein, peptide or fragment thereof with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labelled with I¹²⁵) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of target protein, peptide or fragment thereof.

In an alternate version of the RIA, a labelled target protein, peptide or fragment thereof and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of a target protein, peptide or fragment thereof is added in varying amounts. The decrease in precipitated counts from the labelled target protein, peptide or fragment thereof is proportional to the amount of target protein, peptide or fragment thereof in the added sample.

Fluorescence activated cell sorting (FACS): This method involves detection of a target protein, peptide or fragment thereof in situ in cells by target protein, peptide or fragment thereof specific antibodies. The target protein, peptide or fragment thereof specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.

lmmunohistochemical analysis: This method involves detection of a target protein, peptide or fragment thereof in situ in fixed cells by target protein, peptide or fragment thereof specific antibodies. The target protein, peptide or fragment thereof specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or

Giemsa stain.

In situ activity assay: According to this method, a chromogenic substrate is applied on the cells containing an active enzyme and the enzyme catalyzes a reaction in which the substrate is decomposed to produce a chromogenic product visible by a light or a fluorescent microscope.

In vitro activity assays: In these methods the activity of a particular enzyme is measured in a protein mixture extracted from the cells. The activity can be measured in a spectrophotometer well using colorimetric methods or can be measured in a non- denaturing acrylamide gel (i.e., activity gel). Following electrophoresis the gel is soaked in a solution containing a substrate and colorimetric reagents. The resulting stained band corresponds to the enzymatic activity of the protein of interest. If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of colour produced. An enzyme standard is generally employed to improve quantitative accuracy. In a preferred embodiment, the assay is a point-of-care or point-of-use diagnostic test. Point-of-care testing (POCT) is defined as diagnostic testing at or near the site of patient care or tissue or body fluid or plaque sampling. The motivation behind POCT is to bring the test conveniently and immediately to the patient, which in turn increases the likelihood that the patient will receive the results in a timely manner. Therefore, treatment can immediately follow diagnosis.

A non-limiting example of a point-of-care test is a lateral flow test. Lateral flow tests, also known as lateral flow immunochromatographic assays are a simple device intended to detect the presence (or absence) of a target protein, peptide or fragment thereof in sample. Lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate, for example paper strip, via capillary action. After the sample is applied to the test it encounters a coloured reagent which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with an antibody or antigen. Depending upon the analytes present in the sample the coloured reagent can become bound at the test line or zone. One result of the antibody-antigen binding is that it can release some material that has been pre- bound to the antibody, such as gold or colloid nanoparticles. The colloid in turn may produce a visible line on the substrate which can be detected either by the naked eye or imaging device such as silicon photodiode or CCD device.

Lateral flow tests can operate as either competitive or sandwich assays. In principle any coloured particle can be used, however in a preferred embodiment either latex (blue colour) or nanometer sized particles of gold (red colour) are used. The gold particles are red in colour due to localised surface plasmon resonance. Fluorescent or magnetic labelled particles can also be used in combination with an electronic reader to assess the test result. In the case of sandwich assay format the sample first encounters coloured particles which are labelled with antibodies raised to the target protein, peptide or fragment thereof. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the target protein, peptide or fragment thereof. The test line will show as a coloured band in positive samples. However, for competitive assays the sample first encounters coloured particles which are labelled with the target protein, peptide or fragment thereof or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled target protein, peptide or fragment thereof in the sample will block the binding sites on the antibodies preventing uptake of the coloured particles and the test line will show as a coloured band in negative samples.

Different versions of the methods and kits of the present invention can be used for various applications.

For example, one version could be used to assess the risk of future periodontal disease development in an individual (e.g., high, medium or low risk for future periodontal disease development).

Another test version could be used to quantitate periodontal disease risk leading to the prediction of periodontal disease experience at subsequent ages. This test might be administered in a dentist's office where appropriate countermeasures could be initiated. Yet another test version would be diagnostic and used with medically compromised patients, such as those suffering from diabetes or AIDS. Still another test version would feature multiple sample, high throughput characteristics. The use of this test version would be targeted to screening populations of samples, for example saliva samples, such as those used for epidemiological surveys.

It will be appreciated that the tools necessary for detecting the presence of a target protein, peptide or fragment thereof in a sample from an individual may be provided as a kit, which may contain one or more unit dosage form containing an active ingredient for detection of the target protein, peptide or fragment thereof.

Alternatively, the kit may comprise means for collecting the sample and specific detection means packaged separately. The kit may be accompanied by instructions for use.

For example, the kit may include devices such as a dipstick or a cartridge, (optionally comprised in a housing) which the individual or clinician places into the oral cavity or sample obtained from the individual. The device may comprise any agent capable of specifically detecting the target proteins, peptides or fragments thereof. For example, the device may comprise one or a combination of monoclonal and polyclonal antibody reagents or fragments thereof and an indicator for detecting binding. Antibody supports are known in the art. In an embodiment of this invention, antibody supports are absorbent pads to which the antibodies are removably or fixedly attached.

According to a preferred embodiment, the device of the invention is a lateral flow device comprising an inlet means for flowing a fluid, for example body fluid, into contact with one or more agents, for example a polyclonal or monoclonal antibody or fragment thereof, capable of detecting the proteins, peptides or fragments thereof of the present invention. The test device can also include a flow control means for assuring that the test is properly operating. Such flow control means can include control proteins, peptides or fragments thereof bound to a support which capture detection antibodies as a means of confirming proper flow of sample fluid through the test device. Alternatively, the flow control means can include capture antibodies in the control region which capture the detection antibodies, again indicating that proper flow is taking place within the device. In one embodiment, the kit comprises a monoclonal target protein, peptide or fragment thereof coloured conjugate and polyclonal anti-target protein coated on a membrane test area. By capillary action, the sample migrates over the test area and reacts with the impregnated reagents to form visible coloured bands in the test window. The presence of the target protein, peptide or fragment thereof in concentrations above normal will result in the formation of a distinct coloured band in the test area thus indicating a positive result for the for a Treponema infection or risk of progression of periodontal disease or any other outcome described herein. Conversely, if no line appears in the test area, the test is negative.

It will be understood that in certain embodiments the bacteria may be a species or subspecies of Treponema including Treponema socranskii, Treponema vincentii, Treponema medium or Treponema denticola. Preferably the Treponema is of the species denticola (Treponema denticola).

In certain embodiments, the test for a Treponema, particularly T. denticola, may be used in combination with a method detection of other bacteria known to effect periodontal disease initiation or progression. For example, the bacteria may be Porphyromonas gingivalis or Tannerella forsythia.

The methods of the present invention may be performed at the same time as analysis of clinical parameters. Such clinical parameters include modified gingival index (Lobene et al. Clin Rev Dent 1986: 8:3-6), plaque index (Silness et al. Acta Odontol Scand 1964: 22 121-135), pocket depth, recession, clinical attachment level, bleeding on probing and suppuration.

In certain embodiments, the method may be useful for assessing a response of an individual to administration of a protein or substance representing part or all of a Treponema. In these embodiments, the protein or substance is administered to an individual and the expression of a target protein described in Table 2 or 3 herein is assessed to determine a response to the Treponema protein or substance.

In a particular embodiment, the individual is a human. The human may be either male or female. The age of the human can be between 18 and 35 years old, between 2 and 45 years old; between 2 and 80 years old or above; or between 15 and 60 years old or above. It is not intended that the methods of the invention are limited to determining Treponema infection in individuals within a particular age group.

In another embodiment, the individual may be an animal including a domestic animal. Preferably the animal is a cat, dog, sheep, cow or horse.

In one embodiment the invention provides a substantially non-glycosylated protein, peptide or fragment thereof described in Table 2 or 3 herein. Preferably, the protein, peptide or fragment thereof described in Table 2 or 3 herein does not contain any glycosylated amino acids. Preferably, the substantially non-glycosylated protein is a non-glycosylated form of OppA (TDE1071), Msp (TDE0405), PrcA (TDE0761 ), FIaB (TDE1004), and FIaBI (TDE1477). The amino acid sequences of these proteins, and others, of the invention are listed in Table 4 and referenced by the SEQ ID NO: shown therein. In one embodiment the invention provides an antigenic region of a substantially non- glycosylated protein of the invention. An antigenic region of a protein may be determined using various algorithms including EMBOSS Antigenic (Kolaskar,AS and Tongaonkar.PC (1990). A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Letters 276: 172-174) or Antigenicity Plot (Hopp.T.P. and Woods.K.R. (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci USA 86:152-156).

In one embodiment, the fragment of a substantially non-glycosylated protein of the invention is a region of the protein that is exposed on the surface of the bacteria when the protein is in its native conformation in the bacteria.

Treponema have an outer membrane and outer membrane proteins are those which are embedded in or protrude from the outer membrane.

In other embodiments, a protein, peptide or fragment thereof of the invention has a sequence that has 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% amino acid identity to a protein, peptide or fragment thereof described in Table 2 or 3 herein. In certain embodiments, the protein, peptide or fragment thereof is substantially non- glycosylated.

In one embodiment, a protein, peptide or fragment thereof of the invention has a sequence that has 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% amino acid identity to OppA (TDE1071), Msp (TDE0405), PrcA (TDE0761 ), FIaB (TDE1004), and FIaBI (TDE1477). In certain embodiments, the protein, peptide or fragment thereof is substantially non-glycosylated.

Typically, the protein, peptide or fragment thereof is in a composition. In one embodiment, substantially all of the protein in a composition of the invention is substantially non-glycosylated. In another embodiment, all of the protein in a composition of the invention is substantially non-glycosylated.

In one embodiment the composition consists solely of protein. In another embodiment, the composition of the invention may include, further to protein, other cellular components such as fragments or parts of a membrane or cell wall. In other words, the composition may include lipids, carbohydrates and nucleic acids.

A composition of the invention may be used to generate antibodies in an animal, for example a mouse, rat, rabbit, sheep or human. The composition may be used to generate polyclonal or monoclonal antibodies. The antibodies have a number of utilities including detection of an immune response in an individual generated against a Treponema or other in vitro or in vivo applications.

A protein or composition of the invention may also be used to detect an immune response in an individual generated against a Treponema.

The composition further comprises a carrier, diluent preservative or other component that could be used to modify the immune response in an animal having received the composition. The carrier, diluent, preservative or other component is particularly useful for increasing the likelihood of generating an antibody response in an animal to a protein, peptide or fragment thereof or composition of the invention.

In another embodiment, a composition of the invention comprises a substantially non- glycosylated protein, peptide or fragment thereof differing from a protein, peptide or fragment thereof described in Table 2 or 3 by conservative amino acid substitutions.

Whilst the concept of conservative substitution is well understood by the person skilled in the art, for the sake of clarity conservative substitutions are those set out below.

GIy, Ala, VaI, lie, Leu, Met;

Asp, GIu, Ser;

Asn, GIn ;

Ser, Thr;

Lys, Arg, His; Phe, Tyr, Tip, His; and

Pro, Nα-alkalamino acids.

Accordingly, without being bound by any theory, or mode of action, it is believed that a non-glycosylated protein, peptide or fragment thereof described in Table 2 or 3 herein when used to generate antibodies will only generate antibodies to the protein, peptide or fragment thereof and not to any glycan moiety.

"Substantially non-glycosylated" with respect to a protein, peptide or fragment thereof described in Table 2 or 3 herein, is defined as less than half the maximum level of glycosylation of the protein, peptide or fragment thereof predicted by any available computer software anaylsis, for example on http://www.exDasv.ch/tools/ or http://www.cbs.dtu.dk/services/. "Substantially non-glycosylated" in certain embodiments is also defined as less than half the maximum level of glycosylation of the protein, peptide or fragment thereof as determined experimentally. "Substantially non- glycosylated" may refer to a reduced number of amino acids per protein, peptide or fragment thereof that are glycosylated or to reduced number of saccharides on each glycosylated amino acid. "Substantially non-glycosylated" may also refer to a protein, peptide or fragment thereof that has been treated with one or more glycosidases or may refer to a protein, peptide or fragment that has a glycosylation profile that is the same or similar to the glycosylation profile of a protein, peptide or fragment thereof that has been treated with a glycosidase.

"Percent (%) amino acid identity" or " percent (%) identical" with respect to a protein, peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific protein, peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples described below) needed to achieve maximal alignment over the full-length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity = X/Y^*100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.

In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. MoI. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673- 4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non- limiting examples of a software program useful for analysis of ClustalW alignments is GENEDOC™ or JalView (http://www.jalview.org/). GENEDOC™ allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

In one embodiment the invention provides a system for determining whether -an individual has a Treponema infection including:

- selecting an individual;

thereby determining whether the individual has a Treponema infection.

The invention is further illustrated by the following Examples which are included by way of exemplification and not limitation of the invention.

EXAMPLES

Bacterial strains and growth conditions

The inventors have developed methods for the cultivation of this bacterium in continuous culture. Continuous culture has the advantages of allowing the generation time and environmental factors to be rigourously controlled and has been demonstrated to provide highly reproducible conditions for transcriptomic and proteomic bacterial analyses (Shockley, K. R. et al. 2OO5.Applied and environmental microbiology 71 :5572- 5576). The conditions for the cultivation of T. denticola developed by the inventors mimic the environment of a site affected by periodontal disease in an oral cavity.

T. denticola ATCC 35405 (Makinen, K. K. et al. 1996. Infection and immunity 64:702- 708) was grown in oral bacterial growth medium (OBGM), a modified version of NOS (Leschine, S. B., and E. Canale-Parola. 1980. J Clin Microbiol 12:792-795) and GM-1 (Kesavalu, L., S. G. et al. 1997. Infection and immunity 65:5096-5102) media containing; brain heart infusion (12.5 g/L), trypticase (10 g/L), yeast extract (7.5 g/L), sodium thioglycolate (0.5 g/L), asparagine (0.25 g/L) D-glucose (2 g/L), ascorbic acid (2 g/L), pyruvic acid (1 g/L) and sodium chloride (2 g/L). The medium was supplemented with cysteine (1 g/L), ammonium sulfate (2 g/L), thiamine pyrophosphate (6 mg/L), sodium hydrogen carbonate (2 g/L), heat inactivated rabbit serum (2% vol/vol), haemin (5 mg/L), menadione (1 mg/L) and volatile fatty acid mix (0.5% vol/vol). The volatile fatty acid mix was 0.1 M potassium hydroxide containing isobutyric acid (0.5% vol/vol), DL-2-methylbutyric acid (0.5% vol/vol), isovaleric acid (0.5% vol/vol), valeric acid (0.5% vol/vol). All chemicals were supplied by Sigma, and the growth media was supplied by Oxoid.

T. denticola was grown in continuous culture using either a model C-30 BioFlo chemostat (New Brunswick Scientific, USA) with a working volume of 365 mL or a BioFlo 110 Modular Benchtop Fermentor (New Brunswick Scientific) with a working volume of 900 mL. The bacteria were grown at 37oC with constant agitation (30 rpm) under a constant stream of anaerobic gas (5% CO₂ and 4% H₂ in N₂). The dilution rate was 0.044 h^~1 giving a mean generation time of 15.75 h and the pH was maintained at 7.4 ± 0.1. The conditions in which the bacteria were grown ensure that the generation time is similar to the generation time of bacteria in an oral cavity suffering from periodontal disease. Bacterial cell density was monitored using spectrophotometry at a wavelength of 650 nm (AU_6So; Novaspec III, Amersham Biosciences). Culture purity was determined daily by Gram stain. Bacterial cell culture samples were collected aseptically from the chemostat overflow. Flow Cytometry

Bacterial cells were enumerated using the Molecular Probes Bacteria Counting Kit using the manufacturer's protocol where dilutions of the bacterial cells were performed in TSBN (5% w/v trypticase soy broth in 0.15 M NaCI). A standard curve was created using T. denticola cultures of various cell densities as determined by measuring absorbance at a wavelength of 650 nm. Cell cultures were diluted so that the highest final cell density of bacteria in the assay was ^ x IO⁶ cells/mL Aliquots of 10 μL of these dilutions were mixed with 990 μL of TSBN. Modifications to the manufacturer's protocol included the addition of 10 μL of a microsphere suspension, to give a final concentration of 1 x 10⁶ microspheres/mL in the assay, to each sample before the addition of the provided SYTO BC bacterial stain. The SYTO BC was also diluted 1 in 2 in TSBN prior to addition. Controls used in this experiment were samples containing bacterial cells with SYTO BC stain but no microspheres, bacterial cells with microspheres but no SYTO BC stain, and microspheres with SYTO BC stain but no bacteria. Flow cytometry analysis was carried out on a Beckman Coulter Cytomics FC 500 cytometer equipped with a Uniphase Argon ion laser set at 488 nm with a 20 mW output.

Triton X-114 treatment of T. denticola

To isolate T. denticola outer sheath proteins a Triton-X extraction (TX-114) similar to that described previously was used (Pathirana, R. D. et al. Microbiology

152:2381-2394). T. denticola cells grown in continuous culture were harvested by centrifugation (6,000 g, 4⁰C, 30 min). The pellet was washed twice in TC 50 buffer (50 mM sodium chloride, 50 mM Tris, 5 mM calcium chloride, pH 7.4). The pellet was then suspended in TC 50 buffer and 0.5% TX-114 (v/v) added, which was followed by gentle agitation at room temperature for 60 min. The solution was centrifuged (10,000 g, 30 min) and the supernatant collected and immediately stored at -2O⁰C until used.

French Pressure preparation of T. denticola

T. denticola was harvested, pelleted by centrifugation (7,000 g, 4⁰C, 30 min) and washed in TC50 buffer. Cell suspensions were passed three times through the French Pressure Cell Press (SLM Instruments) at 100 MPa. The suspension was pelleted by centrifugation (7,000 g, 30 min) and the supernatant was retained, which was then centrifuged at 40,000 g for 30 min. The resulting supernatant was labelled "FPS", while the pellet containing membranes was washed twice with TC50 buffer (40,000 g, 30 min), and labelled "FPP". Both samples were stored at -2O⁰C.

2D-gel electrophoresis

TX-114 extracted T. denticola protein and the FPS sample was precipitated with 1/10 dilution of TCA on ice for 20 min, followed by three washes in cold acetone. After precipitation, all protein samples were treated in the same way. Each T. denticola preparation was solubilized for 2 h in 500 μL of immobilized pH gradient (IPG) sample solution [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% (w/v) Triton X-100, 50 mM dithiothreitol (DTT), 0.4% (v/v) carrier ampholytes (Pharmalytes 3-10), 40 mM Tris, 5 mM EDTA, 0.005% Bromophenol Blue] and applied to 17 cm long pH 4-10 linear IPG strips (Bio-Rad, Hercules, CA, USA) for rehydration of gel with sample. Isoelectric focussing was performed using a Protein IEF Cell (BioRad) at 2O⁰C under paraffin oil for 24 h at 70 kVh. IPG strips were prepared for the second dimension by reduction with DDT and alkylation with iodoacetamide in equilibration solution [50 mM Tris-HCI (pH 6.8), 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS]. SDS/PAGE was conducted using the Protean Il xl system (Bio-Rad) with 1 mm spacers.

Fluorescent imaging and Spot Picking of 2D gels

After thorough equilibration of the gel in water, the SyproRuby stained gel was placed in an SP gel frame of cut-out size 175^155 mm which was further placed in a FLA multi-tray adapted to fit the SP gel frame in portrait orientation. The gel was then imaged in an FLA-5100 laser scanner (Fujifilm, Japan) at 100 μm resolution using the blue (473 nm) laser with a Y510 long pass filter and after scanning immediately transferred within the SP gel frame to the Proteineer SP spot picker (Bruker Daltonics) where it was submerged under water. The fluorescent gel image was exported in TIFF format and analyzed using Proteomweaver software (Biorad) to enable spot detection and gel calibration with respect to pi and MW. The spotlist was transferred to the spot picker and a three point calibration using three corners of the SP gel frame was applied to match the fluorescent image to the visible image produced by the spot picker. Spot picking was performed using the preset "pick pick" method for non-backed gels, and the gel plugs were transferred to 96 well digest adaptors (Bruker Daltonics).

Automated digestion and MALDI target preparation After spot picking, the digest adaptors containing the gel plugs were transferred to the Proteineer DP robot (Bruker Daltonics) for automated digestion and MALDI target preparation. In brief, the plugs were alternately washed with 15 μl of 8% acetonitrile in 20 mM ammonium bicarbonate (ABC) and 50% acetonitrile in ABC, dehydrated twice in 100% acetonitrile, swelled for 15 min with 3 μl of trypsin (10 ng/μl in ABC with 1 mM CaCb) and digested for 4 h at 30°C after replacement of excess trypsin solution with 8% acetonitrile in ABC (3 μl). Peptides were extracted with 1 % TFA v/v at 20⁰C for a minimum of 30 min. This digestion procedure was programmed to start during the night so that the target preparation could be done fresh in the morning. A 600 μm anchorchip MALDI target was manually prepared with HCCA matrix using the thinlayer technique according to the anchorchip manual (Bruker Daltonics) just prior to sample deposition. The robot spotted 4 μl of each sample onto the target and after 5 min adsorption the samples were washed by the addition and immediate removal of 7 μl of 1 % TFA. A tip off-set of +2 mm in the Z-direction was used for sample deposition which helped to localize the sample to its anchor.

Automated TOF/TOF MS

All MS related hardware and software in this section are from Bruker Daltonics. Automated TOF/TOF was performed on anchorchip targets using an Ultraflex TOF/TOF (Suckau, D., A. et al. 2003. Analytical and bioanalytical chemistry 376:952-965) upgraded to a PAN source and Lift II, and controlled by FlexControl v2.4 software. MS was performed using a 25 kV positive reflectron method which was calibrated using a peptide mix applied to the centre of the target prior to automated analysis. MS spectra were then automatically acquired for each sample with the laser power set to a narrow range and under fuzzy logic control. 300 spectra were accumulated for each sample in sets of 15 spectra, with each set having to exceed a S/N of 2 and resolution of 5000 in order to be included. Peaks within a mass range of 1200-2500 were used for this evaluation. At most, 75 spectra were recorded at each raster position. Peak detection and subsequent export to the ProteinScape database were achieved using FlexAnalysis v2.4. Monoisotopic peaks were automatically labeled using the SNAP algorithm within the range 800-4000 Da with a S/N threshold of 6, a quality factor of 50 and a maximum of 100 peaks. A second, extended peaklist was generated after smoothing of the spectra and applying a S/N threshold of 8. MS/MS spectra were acquired using the Lift method on parent ions selected by ProteinScape (see below). A single set of 50 spectra were accumulated for the parent ion with a S/N threshold of 3 at a fixed laser power followed by 14 sets of 50 fragmentation spectra at a laser power elevated by 30%, each set being acquired at a different raster position. The MS/MS spectra were smoothed, baseline subtracted and peaks were detected as above except that the quality factor threshold was reduced to 30. Peaklists were automatically sent to ProteinScape as above.

Protein identification ProteinScape v1.2 was used for internal calibration of MS spectra, removal of calibrants and contaminants from the MS peaklists and submission of peaklists to Mascot 2.1 (Matrix Science) for both PMF and MS/MS ions searches (Chamberlain, N. R. et al. 1989. Infection and immunity 57:2872-2877). All searches were against the T. denticola sequence database obtained from TIGR (http://www.tigr.org) in May 2005 and limited to fully tryptic peptides with carbamidomethyl-Cys and Met-Oxidation set as fixed and variable modifications, respectively. Two peptide mass fingerprinting (PMF) searches were conducted for each MS spectrum, the first with a large mass tolerance of 300 ppm and zero partial cleavages allowed, and the second with 150 ppm and one partial cleavage allowed. Internal calibration using trypsin autolysis products and common contaminants such as keratins was applied for the second PMF search only. A third PMF search was conducted on positively identified MS spectra to enable further proteins to be identified. In this case internal calibration was applied using the peptides already identified and a mass tolerance of 20 ppm and zero partial cleavages were allowed. After the first two PMF searches, data dependent MS/MS acquisition was triggered by ProteinScape. Providing that peaks had a "goodness for MS/MS" value greater than 200, up to two peaks were chosen to verify PMF results, a further two to identify additional proteins and two if no protein was identified from PMF. MS/MS ions searches were conducted with a mass tolerance of 300 ppm on the parent and 0.8 Da on fragments. One missed cleavage was allowed. PMF and MS/MS searches were deemed correct if Mascot score was greater than 60 (p<0.005) or 25 (p<0.01) respectively.

Formalin killing of bacteria

Bacterial cell cultures were grown to a cell density of ~7 x 10⁸ cells/mL and harvested by centrifugation (7,000 g, 20 min, 4°C). The cells were then washed once in TC150 (50 mM Tris, 150 mM NaCI, 5 mM CaCI₂, pH 7.4) and suspended in a 2-5% vol. of formal saline (0.5% v/v formaldehyde in saline, 150 mM NaCI). Cells were incubated overnight at room temperature under agitation, then washed and suspended in TC150. After preparation, formalin-killed cells were stored at 4⁰C.

Preparation of antisera

25 BALB/c female mice (6-8 weeks old) were immunized twice, one week apart with 2 x 10⁹ formalin-killed T. denticola cells per experimental animal in a total volume of 100 μl_

(50 μL of adjuvant); the first was an intraperiotoneal injection with Complete Fruend's

Adjuvant (CFA) and the second a subcutaneous injection with Incomplete Fruend's

Adjuvant 30 days later (O'Brien-Simpson, N. M. et al. 2001. Infection and immunity

69:7527-7534 and O'Brien-Simpson, N. M. et al. 2005. J Immunol 175:3980-3989). Sera were collected by eye bleed two weeks after the second immunization and immediately stored at -2O⁰C. Experiments were approved by the University of

Melbourne Ethics Committee for Animal Experimentation.

Electrophoretic transfer onto PVDF membranes

Immediately after the second dimension of the 2D gel electrophoresis procedure, the gels were equilibrated for 15 min in transfer buffer (25 mM Tris, 192 mM glycine pH

8.3, 2% v/v methanol) before being electroblotted onto a PVDF membrane prewetted with methanol. Electroblotting was conducted for 2 h at 70 V using a Transblott Cell

(Bio-Rad) filled with transfer buffer at 4⁰C. Western blotting

Western blots were performed according to Dashper et al. 1998. Aust. Dent. J. 43: 99-104. The primary antibody used was pooled sera collected from mice as described above at a 1/100 dilution, and goat anti-mouse horseradish peroxidase conjugate was used as a secondary antibody at a dilution of 1/10,000. The membranes were developed using SuperSignal® West Pico Chemiluminescent Substrate (Pierce). The antigenic proteins determined by Western blot analysis were compared with the corresponding 2D gel and identified on the basis of pi and MW.

Continuous Culture of T. denticola T. denticola was grown under steady state conditions in continuous culture.

Growth was considered to be at steady state when the cell density (AUβso) remained stable for a minimum of 10 bacterial generations (157.5 h or 6.5 days). Following inoculation of the chemostat, the cell density stabilized after 9 days, with an average AU₆₅o of 0.225 ± 0.011 (Fig 1) which corresponded to 7.35 ± 0.33 x 10⁸ cells/mL as determined by flow cytometry (Fig 2).

2D-PAGE and MS analyses of T. denticola fractions

In order to identify the major proteins of T. denticola from both membrane and soluble fractions, three preparations were analysed; (i) an outer sheath enriched sample (TX) prepared by gentle agitation in Triton X-114 detergent; (ii) soluble proteins (FPS) prepared by disrupting the cells in a French Press and removing the membranes by centrifugation; and (iii) the membrane pellet (FPP). Multiple 2D gels of each preparation were performed and subjected to robotic spot picking and in-gel digestion followed by automated MS and MS/MS analysis using a MALDI-TOF/TOF instrument. From the set of nine 2D gels that were analysed extensively, five were analysed with sufficient success to identify well over 200 spots per gel and up to 125 non-redundant proteins for a particular gel (Table 1 ). In total, more than 1700 spots were identified corresponding to 216 non-redundant proteins (p<0.005) (Table 2). The TX and FPS samples resulted in 2D gel patterns that contained a large number of membrane-associated proteins such as Msp and OppA, but also a large number of cytoplasmic enzymes such as glycine reductase (Grd) (Fig 3B, Table 2). The FPP samples in contrast, were very rich in flagellar filament proteins and membrane-associated proteins while containing few cytoplasmic enzymes (Fig 3A, Table 2).

Due to the several levels of redundancy afforded in this analysis, most proteins were identified with a very high Mascot PMF score (Table 2). The MS/MS score distribution showed only a slight correlation with PMF scores, with several low scoring

PMF hits scoring highly at the MS/MS level (Data not shown). Approximately half of the proteins identified were confirmed by MS/MS (Table 2).

As an example of the usefulness of MS/MS in this study and the automated protein identification process, the analysis of a single spot of moderately low intensity is described. PMF of this spot resulted in the positive identification of TDE0296, a putative formiminotransferase with a moderately low Mascot score of 66 (Fig 4A). This identification was confirmed by MS/MS of a peak at m/z 1504.7 with a Mascot score of 42 (Fig 4B). Searching the unmatched peaks in an attempt to identify additional proteins using PMF was not successful, however, MS/MS of two strong peaks that did not match TDE0296 resulted in the identification of a C-terminal fragment of TDE0947 (translation elongation factor G) with Mascot scores of 37 and 40 (Fig 4C; data not shown). Hence automated MS/MS allowed the confident assignment of two proteins to a spot that was not easily identified by PMF.

Western blot analyses

T. denticola TX-114 protein extracts were separated by 2D gel electrophoresis, transblotted onto PVDF membranes and probed with antisera raised against formalin- killed T. denticola cells. Alignment of the Western blot to the corresponding Coomassie Blue stained gel enabled several antigens to be putatively identified (Fig 5A &B). These proteins included Msp, PrcA, OppA, MgIB, OppA10, FIaB, FIaBI , FlaB3 and others (eg TmpC) (Table 3). Several spots apparent on the Western blots could not be assigned to a specific protein due to there being multiple proteins identified in these regions. The proteins identified

Various T. denticola identified in this study is shown in Figure 3. These include Msp (TDE0405), dentilisin (TDE0762) and associated PrcA proteins (TDE0761 ), OppA (TDE1071), hemolysin (cystalysin) (TDE1669), hemin-binding proteins a and b (TDE2056, TDE2055), OpdB (TDE2140), and the chemotactic proteins CheA, CheX, CheW, and CheY (TDE1492-1494). In addition to the 78 kDa OpdB (TDE2140) that was identified from several spots and from multiple gels, a second putative OpdB (TDE1195) of the same size and sharing 45% sequence similarity (28% identity) was also identified.

Peptide and Sugar SBPs

Of the eight ABC-type peptide uptake systems predicted from the T. denticola genome, five substrate-binding proteins (SBPs) of these systems were identified demonstrating the importance of peptide capture and transport for this organism. Three of these, TDE1071 , TDE0985 and TDE1273 that were present in the Triton extract sample as well as the membrane and soluble protein preparations were very abundant (Fig 3) while two further proteins, TDE0398 and TDE2049 were also identified, but from less intense spots. These SBPs belong to Family 5 of the bacterial extracellular solute- binding proteins, which primarily includes oligopeptide uptake systems. Apart from TDE2049, each of these SBPs contain an N-terminal signal sequence consistent with Type Il signal peptidase processing and the acylation of the resultant N-terminal Cys residue to form a lipoprotein. In this regard, these T. denticola SBPs are more similar to SBPs of Gram-positive bacteria than to the non-acylated periplasmic SBPs of non- spirochaete Gram negative bacteria. TDE1071 (OppA) is as a major 70 kDa protein that has been purified from Triton X-114 cell surface extracts using preparative gel electrophoresis, and was localized to the cell surface by immunogold electron microscopy. A knock-out mutant of oppA was not observed to adversely affect the growth of T. denticola which suggests the presence of redundant peptide uptake systems. This study shows the presence of multiple OppA-like proteins at high expression levels. Despite the close proximity of TDE0985 and TDE1071 on the 2D gels, the Western Blot analysis strongly suggests that OppA (TDE1071) and OppAIO (TDE1273) are antigenic, but that TDE0985 is not (Fig 5). This may relate to the stronger sequence similarity between TDE1071 and TDE1273 compared to TDE0985.

Unlike the multiple systems for peptide uptake, only a few ABC-type systems are predicted to take up other nutrients. The SBP of a glucose/galactose transport system, the MgIB lipoprotein (TDE2217) was also identified as an abundant protein (Fig 3), present in all samples, but especially in the membrane preparation. A second, less abundant SBP (TDE1283) that may be involved in sugar transport was also identified.

Iron/Haem transport

Similarly to the peptide SBPs, multiple SBPs believed to be involved in the uptake of iron compounds were also identified, namely TDE0386, TDE0748, TDE0758 and TDE2234. However unlike the SBPs involved in peptide uptake, most of these lack a lipoprotein-type signal sequence, and therefore are most likely to be typical Gram- negative periplasmic binding proteins. They do however appear to bind to membranes or associate with membrane proteins, as each is present in the membrane preparation in significant quantities (Table 2).

Flagellar and Cytoplasmic filaments

Of the ~35 structural and biosynthetic proteins predicted to be associated with flagella, all three predicted filament core (FIaB) proteins (TDE1004, TDE1475 and TDE1477) and all three predicted filament outer layer (FIaA) proteins (TDE1408, TDE1409 and TDE1712) were identified, being particularly abundant in the membrane fraction (Fig 3A). Apart from these and the hook-associated protein (TDE2353), no other flagellar proteins were identified. According to gel stain intensities it appears that T. denticola has a major FIaA protein in TDE1712 that is at least ten times more abundant than the other two FIaA proteins (TDE1408 & TDE1409) that are predicted to be organised in an operon (www.microbesonline.org). In other spirochaetes, the FIaA proteins have been shown to form a sheath around the filament core comprised of FIaB proteins. FIaA proteins have been shown to affect both the pitch and diameter of the filament helix. In addition to the flagella filament, the cytoplasmic filament protein CfpA (TDE0842), which is required for proper cell division was found to be very abundant, especially in the membrane fraction (Fig 3A) which is consistent with its attachment to the inside surface of the cytoplasmic membrane.

Possible antigens and outer membrane proteins

In addition to the outer sheath-associated proteins already mentioned, three OmpA family proteins were identified (TDE0664, TDE 1663 and TDE2028). TDE0664 is a homolog of Tpn50 from T. pallidum, which is thought to be equivalent to OmpA. Several identified proteins were found to be named after antigens identified from T. pallidum due to sequence similarity. These include a predicted lipoprotein (TDE1511 ), a basic membrane protein (TDE1658) that may have peptidyl-prolyl cis-trans isomerase activity and was the most abundant protein evident at the extreme basic end of the gel, TmpC membrane lipoproteins (TDE1950 and TDE0951 (Tpn38b)) (Fig 3A), TmpB antigen (TDE 2433) identified only from the membrane fraction (Fig 3A), and Tp92 Omp (TDE2601 ) (Fig 3A). TDE2699 is similar to an antigen identified from Borrelia burgdorferi. A T. denticola specific antigenic lipoprotein (TDE2242) was also identified along with many other putative lipoproteins of unknown function, some of which may be associated with the outer sheath.

Of the antigens characterised in other species, only the major TmpC lipoprotein (TDE1950) was found to be antigenic in this study (Fig 5). Other lipoproteins found to be antigenic were MgIB, PrcA, OppA and OppAIO (Fig 5). Of particular interest is the strong antigenicity of Msp, OppA and PrcA as these have been demonstrated to various degrees to be exposed to the extracellular environment, and hence are suitable candidates for a vaccine or for the development of a diagnostic. Flagellar filament proteins were also found to be antigenic.

Major metabolic enzymes In keeping with the importance of protein as a nutrient source for this organism, many proteinases, peptidases and enzymes related to the degradation of amino acids were identified, particularly from the soluble fraction and TritonX-114 fraction (Fig 3B). These include all five members of the glycine cleavage system (TDE1624-1629) which may combine with the glycine reductase (Grd) complex in a Stickland-type reaction to form 1 mol ATP for each mole of glycine utilised (Fig 6). The glycine cleavage system oxidises glycine providing reduction potential for the Grd complex. Three Grd components, GrdB (TDE2119), GrdC (TDE0240) and GrdE2 (TDE2120) were identified together with thioredoxin (TDE0744) and thioredoxin reductase (TDE0743) which are required by Grd for electron donation. The T. denticola genome contains two divergent copies each of grdB and grdE the protein products of which are proposed to dictate substrate specificity of the Grd complex. Enzymes predicted to be involved in the degradation of arginine (TDE0451 and TDE0929), proline (TDE2776 and 2754), histidine (TDE0588, TDE2606, TDE0047 and TDE0296 or TDE0046), methionine (TDE2200), tyrosine (TDE1118), tryptophan (TDE0251) and serine (TDE2668) were identified (Fig 6). Methylaspartate ammonia lyase (TDE2235) and methylaspartate mutase (TDE2236), two enzymes involved in the degradation of glutamate to acetate and pyruvate via the mesaconate pathway (methylaspartate pathway) were also abundant, suggesting that this may be the major fermentation pathway for the glutamate family of amino acids in T. denticola (Fig 6). In regards to sugar metabolism, about half of the enzymes required for glycolysis/gluconeogenesis were identified.

It should be understood that while the invention has been described in detail herein, the examples are for illustrative purposes only. Other modifications of the embodiments of the present invention that are obvious to those skilled in the art of molecular biology, dental diagnostics and related disciplines are intended to be within the scope of the invention.

Table 1. Comparison of the number of spots and proteins identified in various 2D gels of TritonX-114 and French Pressure cell preparations. The FPP#2 gel was analysed manually. All other gels were analysed using robots and automated MS as described in the methods.

Gel name Spots Nonredundant

Identified proteins

TX#1 293 90

TX#2 157 64

TX#3 78 49

TX#4 101 69

FPS #1 247 109

FPS #2 303 124

FPS #3 303 125

FPP#1 235 64

FPP#2 49 28

Table 2: MS Identification data for 216 non-redundant proteins identified

Accession Protein Definition Identified In ³PMF ⁴MS/MS 'Surface

[kDa] p<0.005 p<0.01 exposure TX FPS FPP

TDE0011 alkyl hydroperoxide 24.2 94 25 reductase/peroxiredoxin

TDE0017 conserved hypothetical protein 38.2 72

TDE0018 LysM domain protein 18.7 134 38

TDE0019 formate-tetrahydrofolate ligase (fhs) 59.5 266 61

TDE0042 phosphate acetyltransferase (pta) 35.7 118 109

TDE0046 form im inotransferase- 22.8 152 94 cyclodeaminase family protein

TDE0047 imidazolonepropionase (hutl) 45.1 88 79

TDE0048 hypothetical protein 24.3 81

TDE0051 alcohol dehydrogenase, iron- 41.6 64 containing

TDE0068 peptidase, M20/M25/M40 family 45.3 59

TDE0102 cyclic nucleotide-binding protein 49.6 215

TDE0117 lipoprotein, putative 34.8 109

TDE0139 hypothetical protein 91.9 145 31 L₁R

TDE0153 coenzyme A disulfide reductase, 61.6 246 putative

TDE0167 ABC transporter, ATP-binding 26.2 125 47 protein

TDE0182 ABC transporter, ATP-binding 62.4 105 protein

TDE0186 hypothetical protein 49.0 296 67

TDE0231 DNA polymerase III, beta subunit 41.7 270 47

(dnaN)

TDE0240 glycine reductase complex protein 54.8 182

GrdC (grdC)

TDE0249 flavoredoxin, putative 20.4 70 87

TDE0251 tryptophanase (tnaA) 51.6 316 58

TDE0296 formiminotransferase, putative 33.3 254 104

TDE0300 cytosol aminopeptidase family 52.1 167 38 protein

TDE0311 thymidylate synthase- 30.8 80 complementing family protein

TDE0313 TrkA domain protein 25.3 212

TDE0325 hypothetical protein 22.9 107

TDE0337 glucosam ine-6-phosphate 30.5 95 37 isomerase (nagB)

TDE0340 fructose-bisphosphate aldolase, 32.8 212 110 class-l

TDE0351 L-lactate dehydrogenase (Idh) 33.6 61

TDE0354 general stress protein 14 20.8 81

TDE0386 ABC transporter, periplasmic 36.6 234 44 substrate-binding protein

TDE0389 (R)-2-hydroxyglutaryl-CoA 46.4 131 dehydratase, beta subunit, putative

TDE0398 oligopeptide/dipeptide ABC 58.9 151 91 transporter, periplasmic peptide- binding protein

TDE0405 major outer sheath protein 58.2 314 153 TDE0407 glutamate synthase (NADPH), 55.1 97 homotetrameric (gltA)

TDE0434 rubrerythrin 21.5 77 110

TDE0444 glutamine amidotransferase class-l 26.8 129 domain protein

TDE0449 ferritin, putative 18.4 107 54

TDE0451 arginine deiminase (arcA) 46.1 60

TDE0456 pyridoxine biosynthesis protein 30.3 99

TDE0463 purine nucleoside phosphorylase 25.6 79

(deoD)

TDE0467 hypothetical protein 53.2 187 31 S₁O₁I

TDE0525 hypothetical protein 20.0 106 62

TDE0576 glutamyl-tRNA(Gln) 52.8 107 amidotransferase, A subunit (gatA)

TDE0585 hypothetical protein 72.5 75

TDE0588 histidine ammonia-lyase (hutH) 54.0 196

TDE0603 conserved hypothetical protein 34.6 150 84

TDE0610 3-hydroxyacyl-CoA dehydrogenase, 33.5 133 putative

TDE0628 chaperone protein DnaK (dnaK) 69.7 275 130

TDE0648 protein-glutamate methylesterase 40.3 117 45

(cheB)

TDE0664 OmpA family protein 48.8 114 34

TDE0665 pyruvate ferredoxin/flavodoxin 130.6 227 106 oxidoreductase family protein

TDE0677 conserved hypothetical protein 21.4 60

TDE0679 aminotransferase, class V 41.2 91

TDE0704 SPFH domain/Band 7 family protein 34.2 oyon

TDE0731 hypothetical protein 24.5 111

TDE0743 thioredoxin reductase (trxB) 34.2 147 33

TDE0744 thioredoxin (trxA) 11.9 141 42

TDE0748 iron compound ABC transporter, 43.7 166 32 periplasmic iron compound-binding protein, putative

TDE0754 hypothetical protein 23.0 141 93

TDE0758 iron compound ABC transporter, 37.4 103 32 periplasmic iron compound-binding protein, putative

TDE0761 protease complex-associated 69.7 221 95 S₁L₁I polypeptide (prcA)

TDE0765 translation elongation factor Tu (tuf) 43.8 230 147

TDE0816 peptidase, M20/M25/M40 family 43.4 195 81

TDE0823 (3R)-hydroxymyristoyl-(acyl-carrier- 15.9 81 40 protein) dehydratase, putative

TDE0829 aspartyl aminopeptidase, putative 47.3 323 58

TDE0842 cytoplasmic filament protein A (cfpA) 78.5 442 104

TDE0845 conserved hypothetical protein 23.7 146 60

TIGR00266

TDE0855 DNA-binding response regulator 21.2 72

TDE0911 type Il restriction endonuclease 29.6 80

Tdelll (tdelllR)

TDE0925 peptidase T (pepT) 44.8 75

TDE0929 ornithine carbamoyltransferase 38.2 186 100

(argF)

TDE0939 lipoprotein, putative 35.4 66 31

TDE0947 translation elongation factor G, 75.7 221 40 putative

TDE0949 enolase (eno) 47.4 135 47 TDE0951 lipoprotein, putative 37.9 184 S₁L₁I

TDE0985 oligopeptide/dipeptide ABC 75.2 393 110 transporter, periplasmic peptide- binding protein, putative

TDE1000 3-hydroxyacid dehydrogenase family 31.5 175 protein

TDE1001 orotate phosphoribosyltransferase 25.7 80 30

(pyrE)

TDE1004 flagellar filament core protein 31.5 158 114

TDE1041 polyribonucleotide 76.7 147 nucleotidyltransferase (pnp)

TDE1049 translation elongation factor G (fusA- 77.2 148

TDE1050 hypothetical protein 25.0 95

TDE1071 peptide ABC transporter, peptide- 66.7 262 139 binding protein OppA (oppA)

TDE1072 lipoprotein, putative 95.5 346 60

TDE1078 metallo-beta-lactamase family 46.5 129 protein

TDE1090 threonyl-tRNA synthetase (thrS) 66.9 63

TDE1118 tyrosine phenol-lyase (tpl) 51.8 389 100

TDE1127 TPR domain protein 81.3 71 R₁I

TDE1149 hypothetical protein 35.4 270

TDE1175 chaperonin, 60 kDa (groEL) 58.0 233 47

TDE1195 prolyl endopeptidase 77.2 79

TDE1231 hypothetical protein 64.7 174 S,O,l

TDE1236 triosephosphate isomerase (tpiA) 27.0 133

TDE1237 hypothetical protein 14.0 41

TDE1246 lipoprotein, putative 89.6 123

TDE1247 hypothetical protein 25.4 73

TDE1252 lipoprotein, putative 23.9 69

TDE1273 oligopeptide/dipeptide ABC 60.3 314 90 S₁L₁I transporter, peptide-binding protein

TDE1283 extracellular solute-binding 48.7 69 S₁L lipoprotein, putative

TDE1292 TldD/PmbA family protein 47.3 113 38

TDE1301 DNA repair protein RecN (recN) 62.6 70

TDE1308 transketolase (tkt) 73.2 67

TDE1310 modulator of DNA gyrase family 48.6 107 protein

TDE1356 lipoprotein, putative 36.3 113

TDE1357 aldose 1-epimerase (galM) 39.1 226

TDE1371 RNB-like family protein 70.5 67

TDE1372 hypothetical protein 63.3 65

TDE1398 conserved hypothetical protein 80.4 94

TDE1408 flagellar filament outer layer protein 27.0 247 74

FIaA, putative

TDE1409 flagellar filament outer layer protein 27.8 112

FIaA, putative

TDE1413 cytidylyltransferase/phosphoenolpyr 48.4 112 72 uvate phosphomutase, putative

TDE1415 nucleotidyl 68.9 61 41 transferase/aminotransferase, class \ V_/

TDE1426 aminotransferase, 41.6 125

DegT/DnrJ/EryC1/StrS family

TDE1440 glucose-1 -phosphate 32.3 112 thymidylyltransferase (rfbA) TDE1475 flagellar filament core protein 30.9 157 41

TDE1477 flagellar filament core protein 31.3 165 126

TDE1482 peptidase, M24 family protein 65.5 309 109

TDE1488 glyceraldehyde-3-phosphate 38.0 196 55 dehydrogenase, type I (gap)

TDE1491 chemotaxis protein CheA (cheA) 88.2 141

TDE1492 chemotaxis protein CheW (cheW-1 ) 49.7 60

TDE1493 chemotaxis protein CheX (cheX) 16.8 75

TDE1494 chemotaxis protein CheY (cheY) 16.0 114 38

TDE1499 adenylosuccinate lyase, putative 53.8 147

TDE1511 pathogen-specific surface antigen, 22.7 122 L₁O₁I putative

TDE1520 hydro-lyase, tartrate/fumarate family, 30.8 123 42 alpha subunit

TDE1558 YD repeat protein 377.7 65 C₁R₁I

TDE1584 lipoprotein, putative 27.7 116 61

TDE1589 purine-binding chemotaxis protein 19.0 84

(cheW-2)

TDE1598 ABC transporter, ATP-binding 56.1 74 protein

TDE1624 glycine cleavage system P protein, 53.4 166 79 subunit 2 (gcvP2)

TDE1625 glycine cleavage system P protein, 47.2 228 72 subunit 1 (gcvP1)

TDE1626 glycine cleavage system H protein 13.6 70 106

(gcvH)

TDE1627 glycine cleavage system T protein 40.0 157 28

(gcvT)

TDE1629 dihydrolipoamide dehydrogenase 48.4 269 90

(IpdA)

TDE1631 citrate lyase, alpha subunit (citF) 53.8 123

TDE1632 citrate lyase, beta subunit (citE) 31.9 87 35

TDE1642 conserved hypothetical protein 26.9 112 40

TDE1658 basic membrane protein, putative 40.4 240 154

TDE1663 OmpA family protein 19.2 68

TDE1664 conserved domain protein 37.9 238 104 S.R.I

TDE1669 hemolysin 46.1 136 98

TDE1671 trigger factor (tig) 52.1 140

TDE1682 V-type ATPase, B subunit (atpB) 47.3 91

TDE1697 phosphoglycerate mutase (gpm) 28.6 112 61

TDE1712 flagellar filament outer layer protein 39.3 198 84

(flaA)

TDE1715 phosphoglycerate kinase (pgk) 45.4 175 63

TDE1717 hypothetical protein 23.2 146 S.O.I

TDE1727 conserved hypothetical protein 14.9 108

TDE1728 hypothetical protein 76.6 242 34

TDE1754 desulfoferrodoxin/neelaredoxin 13.9 117 58

TDE1848 hypothetical protein 53.8 113 60

TDE1857 conserved hypothetical protein 19.6 64

TDE1862 conserved domain protein 35.8 82

TDE1915 alcohol dehydrogenase, iron- 42.9 123 containing

TDE1950 membrane lipoprotein TmpC, 38.4 219 126 putative

TDE2028 OmpA family protein 146.6 94 67

TDE2049 bacterial extracellular solute-binding 60.4 91 S₁I proteins, family 5

TDE2055 hemin-binding protein B (hbpB) 44.8 86 L₁I TDE2056 outer membrane hemin-binding 45.2 182 protein A

TDE2058 conserved hypothetical protein 30.1 164 133

TDE2069 endoribonuclease L-PSP, putative 13.3 93 85

TDE2085 amino acid kinase family protein 25.2 63

TDE2104 hypothetical protein 94.6 127 L₁O

TDE2120 glycine reductase complex 46.0 142 112 proprotein GrdE2 (grdE-2)

TDE2132 cobalt ABC transporter, ATP-binding 28.8 62 protein, putative

TDE2140 protease Il (ptrB) 78.4 312 49 C₁F

TDE2164 hypothetical protein 25.7 148

TDE2188 hypothetical protein 58.6 225

TDE2194 8-amino-7-oxononanoate synthase, 43.0 114 putative

TDE2200 methionine gamma-lyase (megL) 43.5 168 87

TDE2211 hypothetical protein 22.0 93

TDE2217 galactose/glucose-binding 42.7 179 103 lipoprotein (mglB)

TDE2234 iron compound ABC transporter, 33.5 79 46 periplasm ic iron compound-binding protein, putative

TDE2235 methylaspartate ammonia-lyase 45.6 265 121

TDE2236 methylaspartate mutase, E subunit 53.1 156 71

(glmE)

TDE2242 antigen, putative 90.9 84

TDE2257 5-nucleotidase family protein 57.8 244 64

TDE2290 transcriptional regulator, putative 23.7 64

TDE2300 trypsin domain/PDZ domain protein 53.4 171 66

TDE2315 conserved hypothetical protein 31.5 130

TIGR00044

TDE2337 aminopeptidase 45.9 220 65

TDE2353 flagellar hook-associated protein 3 45.9 69

TDE2369 conserved domain protein 40.7 241 76

TDE2390 hypothetical protein 39.2 81 114

TDE2391 peptidyl-prolyl cis-trans isomerase 35.2 120 75

TDE2392 hypothetical protein 25.6 71 51

TDE2405 conserved hypothetical protein 49.5 126

TDE2406 TldD/PmbA family protein 53.6 107 62

TDE2422 ribosomal protein L7/L12 (rplL) 13.2 83

TDE2433 treponemal membrane protein, 26.8 66 putative

TDE2439 conserved hypothetical protein 39.8 61

TDE2480 chaperone protein HtpG (htpG) 73.5 84

TDE2489 peptide chain release factor 1 (prfA) 41.3 80

TDE2508 hypothetical protein 50.7 185 45

TDE2540 lipoprotein, putative 105.6 62 S,L,O,I

TDE2567 hypothetical protein 32.2 241

TDE2584 dipeptidase 54.9 131

TDE2589 aminopeptidase, putative 51.8 154 85

TDE2601 surface antigen, putative 93.8 185 S₁I

TDE2602 outer membrane protein, putative 20.4 132 104

TDE2606 urocanate hydratase (hutU) 75.7 220 49

TDE2639 oligoendopeptidase F (pepF) 71.3 208 89

TDE2647 lipoyltransferase and lipoate-protein 38.0 158 ligase family protein

TDE2665 inosine-5-monophosphate 55.6 239 49 dehydrogenase (guaB) TDE2668 serine hydroxymethyltransferase 55.8 202 116

(giyA)

TDE2693 ankyrin repeat protein 102.0 169 S₁R₁I

TDE2699 antigen, putative 61.7 218 R, I

TDE2712 hypothetical protein 20.7 102 71

TDE2716 HAD-superfamily hydrolase, 23.9 66 subfamily IA

TDE2730 hydrolase, TatD family 29.0 87 65

TDE2734 hypothetical protein 25.4 117

TDE2738 oligoendopeptidase F, putative 66.6 275 61

TDE2754 ornithine cyclodeaminase (arcB) 37.8 140 73

TDE2776 proline iminopeptidase (pip) 35.7 127

TDE2779 hypothetical protein 29.6 81

1. Accessions and definitions from TIGR (now JCVI, www.tiqr.org). Definitions are from TIGR's automated annotation of the genome.

2. Sequence MW as automatically generated from each protein entry. Many proteins contain signal peptides and therefore their mature MW is less than shown.

3. PMF score generated from Mascot 2.1. A score of 60 approximately corresponds to a p value of 0.005

4. MS/MS ions search score generated from Mascot 2.1. A score of 25 approximately corresponds to a p value of 0.01.

5. Protein likely to be surface exposed as reported in Table 6 of Seshadri et al., 2004 PNAS, 101 : 5646-5651. S =

10 signal peptide; L = Lipoprotein; O = Omp; C = Choline binding; F = fibrinogen binding; R = protein repeat; I = Iterative DNA

Table 3: Identities of antigenic proteins from TX-114 extract of T. denticola.

Gene ID Protein Definition Predicted Cellular Demonstrated Cellular Location

Location

TDE1004 FIaB Flagellar filament Periplasm Located within the T. denticola core protein periplasm, as determined by Western blot analysis.

TDE1477 FIaBI Flagellar filament Periplasm Located within the T. denticola core protein periplasm, as determined by Western blot analysis.

TDE1475 FlaB3 Flagellar filament Periplasm Located within the T. denticola core protein periplasm, as determined by Western blot analysis.

TDE0761 PrcA Protease complex- Outer membrane, Associated with the surface- associated periplasm expressed dentilisn by Western polypeptide (prcA) blot analysis.

TDE1950 TmpC Membrane Periplasm, cytoplasm, Location determined by Western lipoprotein TmpC, extracellular blot of the lipidated cell envelope putative fraction of T. pallidum.

TDE2217 MgIB Galactose/glucose- Periplasm binding lipoprotein

(mglB)

TDE1712 FIaA Flagellar filament Periplasm Located within the T. denticola outer layer protein periplasm, as determined by Western blot analysis

TDE0405 Msp Major outer sheath Outer membrane, Demonstrated to be located on protein extracellular the cell-sruface of T. denticola by immunogold electron microscopy , and in the periplasm by immunogold electron microscopy following whole-cell sectioning

TDE1071 OppA Peptide ABC Outer membrane Demonstrated to be located on transporter, peptide- the cell-sruface of T. denticola by binding protein immunogold electron microscopy

OppA TDE1273 OppAIO Oligopeptide/dipepti Periplasm, outer de ABC transporter, membrane, peptide-binding protein 1. Predicted cellular location as reported by LANL (www.oralqen.lanl.gov)

Table 4: Amino acid sequences

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for determining whether an individual or periodontal site has a Treponema infection including:

- selecting an individual or periodontal site;

-detecting whether the selected individual or site contains a protein, peptide or fragment thereof described in Table 2 or 3 herein, wherein detection of said protein, peptide or fragment thereof determines that the individual or site has a Treponema infection,

thereby determining whether the individual or site has a Treponema infection.

2. A method for determining risk of periodontal disease progression in an individual or periodontal site including:

- selecting an individual or periodontal site;

-detecting whether the selected individual or site contains a protein, peptide or fragment thereof described in Table 2 or 3 herein, wherein detection of said protein, peptide or fragment thereof determines the risk of periodontal disease progression in an individual or site;

thereby determining the risk of periodontal disease progression in an individual or site.

3. A method according to claim 1 or 2, wherein the protein, peptide or fragment thereof is involved in peptide and sugar transport, iron/haem transport or metabolism.

4. A method according to claim 1 or 2, wherein the protein, peptide or fragment thereof is an outer membrane protein.

5. A method according to claim 1 or 2, wherein the protein, peptide or fragment thereof is a lipoprotein.

6. A method according to claim 1 or 2, wherein the protein, peptide or fragment thereof is selected from the group consisting of OppAIO (TDE1273), Msp (TDE0405), PrcA (TDE0761), MgIB (TDE2217), TmpB (TDE2433), TDE1511 , FIaBI (TDE1477) and FIaB (TDEI 004).

7. A method according to claim 6, wherein the protein, peptide or fragment thereof is selected from the group consisting of OppA (TDE1071 ), Msp (TDE0405), PrcA (TDE0761), FIaB (TDE1004), and FIaBI (TDE1477).

8. A method according to any one of claims 1 to 7, wherein the Treponema is Treponema denticola.

9. Use of a method of any one of claims 1 to 8 in any one of the following applications:

- determining whether an individual or periodontal site is susceptible to periodontal disease;

-determining whether an individual or periodontal site is likely to develop periodontal disease;

- screening for early stage periodontal disease;

- monitoring Treponema infection in an individual or periodontal site receiving treatment for periodontal disease; or

- determining the risk of an individual or periodontal site developing periodontal disease.

10. A substantially non-glycosylated protein, peptide or fragment thereof described in Table 2 or 3 herein.

11. A substantially non-glycosylated protein, peptide or fragment according to claim 10, wherein the protein, peptide or fragment thereof is selected from the group consisting of OppA (TDE1071), Msp (TDE0405), PrcA (TDE0761 ), FIaB (TDE1004), and FIaBI (TDE1477).

12. A composition comprising a substantially non-glycosylated protein, peptide or fragment thereof described in Table 2 or 3 herein and a carrier.

13. A composition according to claim 12, wherein the protein, peptide or fragment thereof is selected from the group consisting of OppAIO (TDE1273), Msp (TDE0405), PrcA (TDE0761 ), MgIB (TDE2217), TmpB (TDE2433), TDE1511 , FIaBI (TDE1477) and FIaB (TDEI 004).

14. A composition according to claim 12 or 13, wherein the protein, peptide or fragment thereof is selected from the group consisting of OppA (TDE1071), Msp (TDE0405), PrcA (TDE0761), FIaB (TDE1004), and FIaBI (TDE1477).

15. A use of an antibody specific for a protein, peptide or fragment thereof described in Table 2 or 3 herein for determining whether an individual has a Treponema infection.

16. A use according to claim 15, wherein the antibody is specific for OppA (TDE1071 ), Msp (TDE0405), PrcA (TDE0761), FIaB (TDE1004) or FIaBI (TDE1477).

17. A use of a polynucleotide for detecting a nucleic acid that encodes or controls the expression of a protein, peptide or fragment thereof described in Table 2 or 3 herein for determining whether an individual or periodontal site has a Treponema infection.

18. A kit for determining whether an individual or periodontal site has a Treponema infection including:

- a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of a protein, peptide or fragment thereof described in Table 2 or 3 herein.

19. A kit according to claim 18 wherein the antibody is specific for a protein selected from the group consisting of OppAIO (TDE1273), Msp (TDE0405), PrcA (TDE0761), MgIB (TDE2217), TmpB (TDE2433), TDE1511 , FIaBI (TDE1477) and FIaB (TDE1004).

20. A use of an antigen in the form of:

- a protein, peptide or fragment thereof that is expressed in a Treponema or;

- a protein, peptide or fragment thereof described in Table 2 or 3 herein

in the manufacture of means for determining whether an individual or periodontal site is infected with a Treponema.

21. A use of an antibody specific for:

- a protein, peptide or fragment thereof that is expressed in a Treponema or;

- a protein, peptide or fragment thereof described in Table 2 or 3 herein

for determining whether an individual or periodontal site is infected with a Treponema.

22. A use of a polynucleotide for detecting a nucleic acid that encodes or controls the expression of:

- a protein, peptide or fragment thereof that is expressed in a Treponema or;

- a protein, peptide or fragment thereof described in Table 2 or 3 herein

23. A kit for determining whether an individual or periodontal site is infected with a Treponema including:

- an antigen being a protein, peptide or fragment thereof described in Table 2 or 3 herein; or - an anti- Treponema antibody specific for at least one of the above described antigens; or

- a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of at least one of the above described antigens.

24. An immune complex including an antigen being a protein, peptide or fragment thereof described in Table 2 or 3 herein bound to an antibody specific for said protein, peptide or fragment thereof.