WO2008137506A2 - Methods and kits for dog plaque disease - Google Patents

Abstract

The invention is directed to the identifying or predicting periodontal disease in a dog. The identifying the presence or absence of at least one micro-organism, from a sample from the mouth of a dog, wherein the micro-organism which is associated with periodontal disease in a dog is one or more disease-associated micro-organisms from: Peptostreptococcus sp., Synergistes sp., Clostridials sp., Eubacterium nodatum, Selenomonas sp., Bacteroidetes sp., 'Odoribacter denticanis ', Desulfomicrobium ovale, Moraxella sp., Bacteroides denticanoris, Fillif actor villosus, Porphyromonas canons, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa.

Description

METHODS AND KITS FOR DOG PLAQUE DISEASE

This application claims priority to UK Patent Application No. 0708424.7 filed May 1, 2007; UK Patent Application No. 0711125.5 filed June 8, 2007, UK Patent Application No . 0711127.1 filed June 8 , 2007 , UK Patent Application No . 0804427.3 filed March 10, 2008 which are incorporated in its entirety.

The present invention relates to methods and kits for determining periodontal disease in a dog, as well as to novel microbes which are associated with periodontal disease in a dog.

Oral disease is one of the most common health complications presenting in dogs visiting veterinary clinics in the USA. Periodontal disease is the most widespread oral disease in dogs with incidence increasing with age. The result is that 70-80% of dogs over the age of 3 years demonstrate signs of periodontal disease. The aetiological agent of periodontal disease is dental plaque.

Dental plaque comprises a biofilm of bacteria suspended in a matrix of bacterial exudate and secreted products. Enzymes secreted by plaque bacteria initiate activation of the host immune response, which involves the activation of host matrix metalloproteinases. These host proteases are the major cause of tissue damage and inflammation, which is observed clinically as red and swollen gums or gingivitis. Gingivitis is the initial stage of periodontal disease and without preventative treatment may progress to periodontitis, which is characterised by the destruction of the periodontal ligament and, eventually the supporting tissues including the bone. This phase of the disease is not reversible and tooth mobility and eventual loss will follow in the absence of treatment. The chronic inflammation associated with disease is likely to cause significant pain to the animal in the later stages of periodontitis.

The human oral cavity contains a diverse population of microbes with over 350 different taxa and at least 37 bacterial genera. Human models of dental plaque formation describe primary colonising species binding to the salivary pellicle on the tooth surface. These organisms provide specific attachment sites that can be exploited by secondary colonisers and bridging organisms. In turn these organisms increase the available binding sites for the tertiary colonisers present in mature plaque, which are often associated with disease. Plaque biofilms therefore comprise a complex ecological mixture of bacterial species that utilise nutrients from the environment and produce metabolites providing a nutrient source for neighbouring organisms.

Despite the relatively detailed knowledge regarding plaque formation and the microorganisms associated with disease in humans, the oral microflora in dogs is relatively undescribed. Several studies have assessed the cultivable species from the canine oral cavity and detected substantial differences in the oral flora of dogs compared to humans. One study described 84 phylotypes from 37 genera cultured from canine plaque and saliva and found only 28% of the organisms identified by 16S rRNA gene sequence were considered indigenous to the human oral flora. In addition, over half of the taxa cultured were novel species with no similar organisms represented in the GenBank database. The majority of organisms cultured were therefore not normally associated with the human oral cavity.

Assessment of the canine microflora has to date been limited to bacterial culture and/or has been targeted through the extrapolation of human studies. Both limitations impact seriously on the scope of research into the canine microflora and the elucidation of organisms associated with disease. This is especially true in light of the apparent differences between the cultivable flora in humans and in dogs, and with approximately 50% of the oral flora representing unculturable species.

The present invention aims to overcome the problems with the prior art and to provide methods and kits to identify or predict periodontal disease in a dog as well as novel microbial strains.

Accordingly, the present invention provides a method of identifying or predicting periodontal disease in a dog, the method comprising identifying the presence or absence of at least one micro-organism, from a sample from the mouth of a dog, wherein the micro-organism which is associated with periodontal disease in a dog is one or more disease associated micro-organism from: Peptostreptococcus sp., Synergistes sp., Clostridials sp., Eubacterium nodatum, Selenomonas sp., Bacteroidetes sp., "Odoribacter denticanis", Desulfomicroium orale, Moraxella sp., Bacteroides denticanoris, Fillifactor villosus, Porphyromonas canons, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa. The sample, preferably a plaque sample, can be taken from any area of the mouth of the dog in question, preferably from the tooth area. The sampling can be conducted by any method, including all those known in the art, in particular from a swab wiped in the dog's mouth. The sample is then placed in a suitable container, under conditions which allow the identification of the presence or absence of at least one micro-organism which is associated with periodontal disease. Preferably, all aspects of the invention identify the presence of a disease-associated micro-organism.

An example of suitable conditions is given in the example section herein. In the present invention, various novel species of micro-organism have been isolated. Since they are novel, they have not previously been characterised according to genus and/or species or published as valid species in the Journal of Systemic Microbiology and Evolution. In order to do so for the present application, taxonomic designations were assigned according to the latest acknowledged bacterial code of the International

Union of Microbiological Societies (the IUMS). Classification involved identification of microbes using 16S rRNA gene sequences including comparison to nearest known organisms, sequence alignment and phylogenetic tree construction for 16S rRNA sequences (Paster and Dewhirst, 1988). The neighbour-joining method (Saitou and Nei, 1987) was then used to construct a phylogenetic tree of the canine-derived organisms. The numerous organisms of the present invention are novel and the method used herein to classify them is constantly changing. In view of this, these micro-organisms could potentially be re-classified in the future. Thus, the name given to each micro-organism is not limiting, should the micro-organism be re-classified. Throughout the present text, reference is made to identity with 16S rRNA gene sequences, given as sequence references 1 to 16. While reference is made to the sequences being those of 16S rRNA, they are, in fact, the DNA coding sequences which are given. Thus, the 16S rRNA sequences given contain the nucleotide T, rather than the nucleotide U. Reference in this text to the 16S rRNA sequences should, more correctly, be described as DNA encoding the 16S rRNA sequences or sequences which are the 16S rRNA sequence when the T bases are replaced with U bases. Thus, reference in this text to "a 16S rRNA gene sequence more than x % identical to sequence reference..." should read "a 16S rRNA gene sequence more than x % identical to sequence reference... when the T bases are replaced with U bases".

In particular, the micro-organism which is associated with periodontal disease in a dog may be one or more micro-organisms comprising: a 16S rRNA gene sequence more than 98% identical to sequence reference:1, a 16S rRNA gene sequence more than 96% identical to sequence reference:2, a 16S rRNA gene sequence more than 94% identical to sequence reference:3, a 16S rRNA gene sequence more than 99% identical to sequence reference:4, a 16S rRNA gene sequence more than 98% identical to sequence reference:5, a 16S rRNA gene sequence more than 92% identical to sequence reference:6, a 16S rRNA gene sequence more than 96% identical to sequence reference:7, a 16S rRNA gene sequence more than 87% identical to sequence reference:8, a 16S rRNA gene sequence more than 99% identical to sequence reference:9, a 16S rRNA gene sequence more than 92% identical to sequence reference: 10, a 16S rRNA gene sequence more than 99% identical to sequence reference: 11, a 16S rRNA gene sequence more than 99% identical to sequence reference: 12, a 16S rRNA gene sequence more than 98% identical to sequence reference: 13, a 16S rRNA gene sequence more than 99% identical to sequence reference: 14, a 16S rRNA gene sequence more than 99% identical to sequence reference: 15 or a 16S rRNA gene sequence more than 98% identical to sequence reference: 16.

The method of the first aspect of the invention includes identifying the presence or absence of the micro-organism which is associated with periodontal disease by one or more of bacterial culture or by a detector for one or more of nucleic acid, peptide, carbohydrate or lipid or by amplification of nucleic acid of the micro-organism or by biochemical or phenotypic (including microscopic examination) profiling. Any one or more of these identifying steps may be preceded by culturing of the microorganism. Such techniques are standard techniques known in the art, including methods described in the references listed herein. Typically, a detector for nucleic acid is another hybridising nucleic acid sequence, including probe sequences which are described herein as the fourth aspect of the invention. When a nucleic acid detector is utilised, there may be amplification of nucleic acid of the micro-organism before identification with a nucleic acid detector.

Detection of nucleic acid sequences can also be carried out by binding partners, such as peptides and antibodies, which are able to recognise and identify the nucleic acid sequences. Alternatively, a detector which recognises and identifies a disease-associated micro-organism of the invention can be a peptide, carbohydrate or lipid detector or indeed can be a detector to identify the micro-organism, by any other means. Such methods are again, known in the art, in particular including binding partners, such as antibodies (especially monoclonal or polyclonal antibodies) or recognition peptides (Sambrook et al. 1989).

Most preferably, the first aspect of the invention includes identifying the presence or absence of any one, two, three, four or five micro-organisms from table 1 in particular identifying the presence or absence of any one, two, three, four or five micro-organisms from Table 1 having the references 1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

According to the present invention, the term "dog" preferably means the domesticated or "pet" dog (Cams domesticus).

A second aspect of the invention provides a micro-organism (preferably isolated) which is associated with periodontal disease in a dog and which comprises a 16S rRNA gene sequence which is more than 98% identical to sequence reference: 1 or a 16S rRNA gene sequence which is more than 96% identical to sequence referenced or a 16S rRNA gene sequence which is more than 94% identical to sequence referenced or a 16S rRNA gene sequence which is more than 99% identical to sequence referenced or a 16S rRNA gene sequence which is more than 98% identical to sequence referenced or a 16S rRNA gene sequence which is more than 92% identical to sequence referenced or a 16S rRNA gene sequence which is more than 76% identical to sequence referenced or a 16S rRNA gene sequence which is more than 87% identical to sequence referenced or a 16S rRNA gene sequence which is more than 99% identical to sequence referenced or a 16S rRNA genesequence which is more than 92% identical to sequence reference: 10, or a 16S rRNA gene sequence which is more than 99% identical to sequence reference: 11, or a 16S rRNA gene sequence which is more than 99% identical to sequence reference: 12 or a 16S rRNA gene sequence which is more than 98% identical to sequence reference: 16.

The percentage identity for any aspect of the invention is across the complete 16S rRNA sequence and is determined by lining up sequences for maximum similarity. The percentage identity is determined over the length that the two sequences have similarity. Any method to determine the % identity can be used, such as the BLAST alignment method.

The method of the first aspect of the invention preferably includes identifying the presence or absence of one or more of the micro-organisms of the third aspects of the invention. Such identification is preferably by using a nucleic acid detector of a nucleic acid probe.

A third aspect of the invention provides any probe which comprises at least 80% or at least 90% identity with at least 10 sequential residues (or at least 11, 12, 13, 14, 15, 16 residues in length) from any one of the sequences in Table A or from any of the probe sequences in Appendix 2, not including the poly T tail which have a given reference number or a probe of at least 10 residues (or at least 11, 12, 13, 14, 15 or 16 residues) which hybridizes to any of the sequences in Table A or any of the probe sequences in Appendix 2, not including the poly T tail, which have a given reference number under the hybridization conditions of for example, prewashing solution 5 x SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0) and hybridization conditions of 40-75°C, 5 x SSC overnight. A higher stringency involves the same hybridisation conditions at 50-75°C. The probes may be nucleic acid or any other probe which have the per cent identity defined above or are capable of hybridising to a nucleic acid.

Table A

The probe sequences according to the third aspect of the invention are referred to herein by reference numbers. The reference number corresponds to the species which the probe identifies and thus the reference numbers for the probe sequences correspond to the reference numbers used later herein as part of Table 1. For example, micro-organism Peptostreptococcus sp. is reference number 1 in Table 1. The probe sequences which can be used to identify this micro-organism are also probe sequences numbered 1.

Determining percentage sequence identity with the probe sequences given can be carried out by aligning sequences for maximum similarity (as described earlier herein) using programmes such as the blast align programme

(http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2,cgi). Preferably the identity is across the full length of the probe sequence given according to the third aspect.

A fourth aspect of the invention relates to the use of a probe, according to the third aspect of the invention, for identifying or predicting periodontal disease in a dog.

Preferably, one, two, three, four, five or six different probe sequences are used. Most preferably, these one, two, three, four, five or six probe sequences are selected to each identify a different micro-organism.

The probe sequences are usually used simultaneously to identify the presence of one or more micro-organisms associated with disease. The fourth aspect of the invention is used preferably in accordance with the various embodiments of the first aspect of the invention in order to identify or predict periodontal disease in a dog. A fifth aspect of the invention relates to a kit for identifying or predicting periodontal disease in a dog, the kit comprising an agent to determine the presence or absence of at least one micro-organism, from a sample from the mouth of the dog, which micro-organism is associated with periodontal disease. The kit will comprise one or more agents.

In order to determine the presence or absence of the micro-organism such an agent is likely to be a detector for nucleic acid, peptide, carbohydrate or lipid, as described above. Preferably, the agent is one, two, three, four, five or six probes of the third aspect of the invention. The kit will preferably include packaging and/or instructions for use of the agent to determine the presence or absence of the micro-organism(s). The agent of the kit is designed to identify a micro-organism which is associated with periodontal disease in a dog being one or more disease-associated micro-organisms from: Peptostreptococcus sp., Synergistes, Clostridials sp., Eubacterium nodatum, Selenomonas sp., Bacteriodetes, "Odoribacter denticanis" , Desulfomicrobium ovale,

Moraxella sp., Bacteroides denticanoris, Fillif actor villosus, Porphyromonas canons, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa.

Preferably, the micro-organism which is associated with periodontal disease is one or more micro-organisms comprising: a 16S rRNA gene sequence more than 98% identical to sequence reference:1, a 16S rRNA gene sequence more than 96% identical to sequence reference:2, a 16S rRNA gene sequence more than 94% identical to sequence reference:3, a 16S rRNA gene sequence more than 99% identical to sequence reference:4, a 16S rRNA gene sequence more than 98% identical to sequence reference:5, a 16S rRNA gene sequence more than 92% identical to sequence reference:6, or a 16S rRNA gene sequence more than 96% identical to sequence reference:7 or a 16S rRNA gene sequence more than 87% identical to sequence reference:8, a 16S rRNA gene sequence more than 99% identical to sequence reference:9, a 16S rRNA gene sequence more than 92% identical to sequence reference: 10, a 16S rRNA gene sequence more than 99% identical to sequence reference: 11, a 16S rRNA gene sequence more than 99% identical to sequence reference: 12, a 16S rRNA gene sequence more than 98% identical to sequence reference: 13, a 16S rRNA gene sequence more than 99% identical to sequence reference: 14, a 16S rRNA gene sequence more than 99% identical to sequence reference: 15 or a 16S rRNA gene sequence more than 98% identical to sequence reference: 16.

In accordance with the fifth aspect of the invention, the kit may comprise an agent to determine the presence or absence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or 16 of the micro-organisms from the list.

The methods, kits and probes according to the present invention can all be utilised such that, in addition to the micro-organisms set out above, any one or more micro-organisms from the following genuses can also be identified: Treponema sp., Moraxella sp., Granulicatella sp., Eubacterium sp., Peptostreptococcus sp., Porphyromonas gulae, Porphyromonas cangingivalis or Porphyromonas cams.

The enclosed text includes a list of 16 sequences which are 16S rRNA sequences of disease-associated micro-organisms. According to the classification of these micro-organisms, the micro-organisms from which the 16S rRNA sequences were obtained, as follows:

Sequence Reference: 1 Peptostreptococcus sp.

Sequence Reference:2 Synergistes sp.

Sequence Reference:3 Clostridales sp. Sequence Reference:4 Eubacterium nodatum

Sequence Reference:5 Selenomonas sp.

Sequence Reference:6 Bacteroidetes sp.

Sequence Reference:7 "Odoribacter denticanis"

Sequence Reference:8 Synergistes sp. Sequence Reference:9 Desulfomicrobium orale

Sequence Reference: 10 Moraxella sp.

Sequence Reference: 11 Bacteroides denticanoris Sequence reference: 12 Fillif actor villosus Sequence Reference: 13 Porphyromonas canons Sequence Reference: 14 Porphyromonas gulae Sequence Reference: 15 Treponema denticola Sequence Reference: 16 Porphyromonas salivosa

Sequence Reference: 17 Treponema.

In the present text, reference to Porphyromonas salivosa should be read to include a Porphyromonas species which is, or is more than or less than 1.5% divergent from Porphorymonas salivosa as defined on the NCBI genbank database.

It is pointed out here that all preferred embodiments of each aspect and within aspects of the invention apply to each other mutatis mutandis. Throughout the present text, reference to "more than" in terms of a percentage identify means higher than the number specified. In all cases the identity includes a percentage identity of more than the number given, up to 100% identity in one percent rises, e.g. more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% up to 100% identity.

The example section of the text which follows also describes the benefits of the present invention in terms of the quantification of the periodontal health or disease of a dog. In accordance with the description of the odds ratios as herein follow, the present invention preferably provides a method, test or kit for a single disease-associated micro-organism or for a combination of disease-associated micro-organisms, wherein the odds ratio is at least 10, at least 20, at least 30, at least

40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100.

The present invention is described with reference to the following, non-limiting, examples. Examples

Example 1

Methodology

Animals and inclusion criteria

Two distinct sampling sites were utilised for sample collection during the study. Clinically healthy dogs and dogs suffering periodontal disease were recruited. The study was approved by the necessary ethical review committee, in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. The dogs were a UK population.

For inclusion in the disease group, dogs were required to have a minimum of four sites displaying periodontal disease of at least stage 2; equivalent to approximately

25% attachment loss or approximately 5mm gingival recession (see Appendix 1). The majority of the animals in the disease group however displayed scores of 3 or 4 (greater than 25% attachment loss).

Clinically healthy gingiva were required for inclusion in the healthy group with no or only low levels of localised gingivitis. Where such gingivitis was present this was at sites away from the sampling sites.

Initially samples from 10 diseased and 10 healthy dogs were collected for the cloning and sequencing study phase. Although exact breed matching was not possible, healthy dogs and sampling sites were selected to reflect those taken from the disease group. A further 60 healthy and 60 diseased animals were scored according to Appendix 1 and sampled.

Sampling procedure

Periodontally diseased dogs were sampled for subgingival plaque at 4 diseased sites during their normal periodontal treatment. Healthy dogs were sampled at 8 sites (4 canines, 2 lower 1st molars and 2 upper 4th premolars) to target sites most often affected in the disease group and increase plaque volumes in the absence of periodontal pockets. Healthy animals were sampled under sedation which was reversed at the end of the sampling.

Initially, supra-gingival and gingival margin plaque and calculus were removed using a sterile Gracey curette to prevent contamination of the sub-gingival sample. A sterile periodontal probe was then used to remove plaque from the sub-gingival pockets. The resulting subgingival sample was suspended in 350ul of 50mM Tris (pH 7.6), ImM EDTA (pH 8.0), 0.5% Tween 20 and was immediately stored at -20°C prior to

DNA extraction, quantification and amplification of the 16S rRNA gene (according to Paster et al, 1998).

Bacterial gene library production and analysis Chemicals were obtained from Sigma unless stated otherwise whilst bacterial growth media were obtained from Oxoid Ltd.

DNA purification: DNA was extracted from 20 plaque samples (10 healthy and 10 diseased) using MasterPure™ DNA Purification Kits (Epicentre, Madison Wisconsin) following the manufacturer's standard protocol.

DNA amplification: The 16S rRNA genes from the total plaque bacterial population were amplified from each individual sample using universal primers D88 (7-27 forward, 5'-GAG AGT TTG ATY MGG CTC AG-3') and E94 (1525-1541 reverse, 5'-GAA GGA GGT GWT CCA DCC-3'). Diseased samples were also amplified with the D88 forward primer paired with the Bacteroidetes selective reverse primer F01 (1487-, 5'-CCT TGT TAC GAC TTA GCC C-3'), or with the Spirochete selective reverse primer C90 (1483-1503, 5'-GTT ACG ACT TCA CCC TCC T-3'). A standard PCR cycle was performed with denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds and amplicon elongation at 72°C for 90 seconds. Following amplification for 30 cycles a final 15 min elongation step was included at 72°C. Cloning: PCR amplicons were subject to electrophoresis on a 1% (w/v) agarose gel and the appropriate size band was extracted and purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, California) according to manufacturer's instructions. The purified amplicons were cloned using TOPO TA Cloning Kits (Invitrogen, Carlsbad, California) using the manufacturer's standard protocol. Clone selection was performed on LB medium containing 50μg/ml kanamycin. Clones were collected into 40μl 50mM Tris (pH 7.6), ImM EDTA (pH 8.0), 0.5% Tween 20. The size of cloned genes (approximately 1500bp) was determined by PCR using flanking vector primers (M 13 forward and reverse; cycles as above) followed by electrophoresis on 1% (w/v) agarose gel. Clones with full size inserts were selected for sequencing.

Sequencing: Amplified DNA was treated with ExoSap to remove primer contamination (USB, Cleveland, Ohio) and DNA sequencing was performed using an

ABI Prism cycle-sequencing kit (BigDye Terminator Cycle Sequencing kit 3.1).

Initially, primer Y31 TTACCGCGGCTGCTG was used to obtain 500 bases of sequence information. Full sequences were then obtained for a reference clone representative of each taxonomic group based on the sequence from Y31 (approximately bases 550-1050). Sequencing reactions were performed using an ABI

3100 DNA Sequencer.

Identification and phylo genetic analysis: Sequences were compared to GenBank entries. Sequence alignment and phylogenetic tree construction for 16S rRNA sequences was performed using a series of BASIC programmes (Paster and Dewhirst,

1988). The neighbour-joining method (Saitou and Nei, 1987) was used to construct a phylogenetic tree of the canine organisms identified in the study.

Species selection and probe generation The number of clones of each species/phylotype in the libraries from healthy and periodontally diseased dogs, as well as the proportion of the animals possessing the phylotype in each group were analysed to facilitate selection of the key bacteria associated with health and disease.

Candidate sequences were examined for melting temperature and secondary structure formation. Finally, candidate probe sequences were checked against all entries in the

Ribosomal Database Project using the online Probe_Match program

(http://rdp.cme.msu.edu/probematch/search.jsp). Where sequences had the potential to cross-react with alternative species they were discarded. The probes were validated against parent E. coli clones of all probe sequences generated (40-50 phylotypes) to yield strong bands and no cross reactivity against any other canine microbial species.

Where probes failed the validation process alternatives were designed in an iterative development process.

Checkerboard hybridisation

Reverse capture checkerboard hybridisation methodology was developed previously for the analysis of human oral species (Socransky et al., 2004, Paster et al., 1998). The technique involved fixation of DNA probes against bacterial 16S rRNA genes on a positively charged nylon membrane using uv irradiation and a poly-thymidine 5' tail in a horizontal orientation (Minislot 30; Immunetics Inc, USA). Amplified 16S rDNA from the total plaque bacterial population, labelled with Digoxigenin on the 5' primer, were then applied in the vertical orientation across all immobilised probes using a Miniblotter 45 (Immunetics Inc, USA). Following washing to remove unbound probe, hybridised DNA was detected using an anti-DIG-AP antibody conjugate (Roche), chemiluminescent detection with CDP-Star (Roche) and exposure to X-ray film according to manufacturers instructions.

Statistical analysis of bacterial associations with periodontal health and disease Checkerboard hybridisation data were transformed by visual assessment from X-ray film illuminated using a light box into both presence/absence (score of 0 or 1) equating to levels above and below the detection limit. Numerical scores (0, 1, 2, 3) for each reaction based on the intensity of the hybridisation reaction were also recorded. In addition, hybridisation intensity was assessed on a continuous scale using a densitometer and Quantity One software (BioRad).

Differences between the reactivity of each probe with (or prevalance of species in) the healthy or disease group were assessed using the Fishers Exact test on binary data scores (presence/absence; http://www.matforsk.no/ola/fisher.htm). Differences in the intensity of the hybridisation reactions with each probe between healthy and diseased populations were assessed using Mann Whitney (Statgraphics Plus V4.1). Probe health scores were calculated from the proportion of the diseased population in which the species was detected, minus the proportion of the healthy population testing positive for the species. This gave a measure of the relatedness of the probe or species to disease. Scores were normalised to a scale of 0-1 by adding the minimum theoretical value. Binary logistic regression analysis (LRA; Minitab V12) was used to define odds ratios for the individual probes and to develop combinations of probes (models) best able to distinguish between the healthy and the diseased population. Probes were included sequentially in LRA models based on their ranking in the univariate analysis. Models were assessed for discriminative ability using P values, odds ratios (individual and combined) and classification accuracy.

Results

Bacterial associations with health and disease

Probes were developed and validated against the 16S rRNA gene sequence from the 37 most prevalent discriminatory taxa in subgingival plaque from the healthy and diseased groups. Following quantification of labelled amplicons representing the total bacterial content of canine subgingival plaque from 60 healthy and 60 periodontally diseased dogs, levels of the target species present were assessed by checkerboard hybridisation. The original disease samples used for generation of the disease libraries were included as positive controls. Of the 120 subgingival plaque samples analysed, 114 produced standardised detection levels with the universal probes and were therefore considered suitable for comparative analysis. The 6 unsuitable samples were obtained from periodontally diseased dogs. Data were transformed into presence (above detection limit) and absence (below detectable levels) and the incidence of each species within the 60 healthy and 54 diseased animals were assessed to indicate associations with the healthy or diseased state. Nine species found to be associated with the disease state are shown in Table 1, which lists individual species presence/absence data in terms of the % of the periodontally healthy or diseased population displaying presence or absence with associated Odds Ratios and consequent predictive (likelihood) ability to determine disease in the presence of the organism. Table 1

Table 1 Describes the percentage of the healthy and diseased populations in which the microorganisms were present or absent as well as the P value (significance level), odds ratio and multiplication factor of each organism's indication of disease. The reference number relates to the micro-organism identified (under the Taxon designation header).

* The reference number used in this table refers to the micro-organism listed.

In Table 1, the micro-organism reference number relates to the micro-organism identified (under the Taxon designation header). In the remainder of this text throughout, the micro-organism references numbers refer to the taxa as indicated in Table 1.

From the tests, the nine organisms in Table 1 were above detection levels in a greater percentage of the diseased population compared with the healthy population and hence showed a statistically significant association with the diseased state in the canine oral cavity. Logistic regression analysis was utilised to determine the odds ratio, an indication of the ability of the species (or probes) to predict the disease status of the animal. The reciprocal of the odds ratios for disease associated species was determined to give a measure of the likelihood of the animal being diseased based on the presence of the taxa. Odds ratios for the organisms identified as bacterial indicators of disease ranged between 2.15 and 29.91 giving predictions of disease of between 29.91 and 2.15 times greater than if the taxon were absent.

Logistic regression analysis using species or probes in combination of two or more to determine the likelihood of an animals' periodontal or oral health status further improved the strength of the prediction (Table 2). Dual species combinations (Table 2; A-L) resulted in predictive strengths ('combined likelihood of health') between 10.89 and 285.71, describing animals as between 10.89 and 285.71 fold more likely to be healthy if the relevant organisms are detected. Furthermore organisms used in combinations of 3 (Table 2; M-Q) again enhanced the detection or prediction of health resulting in levels of certainty between 60-819 times greater than if these taxa were not detected.

Table 2 lists the predictive ability of disease indicator organisms in combinations of two or more taxa enhancing the Odds Ratios and hence the ability to determine disease in the presence of the organism (likelihood of disease). The reference number refers to a probe used to identify a micro-organism having the reference number in table 1 above.

Table 2

Discussion and Conclusions

Associations with canine periodontal health and disease

Checkerboard hybridisation of subgingival samples from the 114 dogs uncovered various organisms enriched in both the healthy and diseased state. Presence or absence under the detection levels inherent in the checkerboard hybridisation methodology was most often used to distinguish between healthy and disease associated species. The results are shown below.

Indicators of disease were identified. Novel species were also associated with the disease state including a novel Peptostreptococcus species and a novel species from the phylum Synergistes, both of which were indicators of disease. Other disease indicators, despite sometimes being lower in prevalence within the disease population, were often specific for the disease state and hence were particularly useful for discriminating between disease and health on the basis of presence or absence respectively. Examples of this included a canine E. nodatum strain, which was present in only 5% of healthy, but 26% of diseased animals and novel species from the phyla Bacteriodetes and Synergistes which were absent in the healthy population tested, yet present in 11 % and 17% of the periodontitis samples studied respectively.

Analysis of hybridisation intensity (not shown) indicated an approximation of the bacterial content within any given taxon or species and indicated that a smaller number of species showed a numerical association with disease compared to presence or absence.

Maximum intensity scores for Desulfomicrobium ovale were also associated with disease, suggesting higher levels of these organisms in the disease state.

Examples 2 and 3

A previous study (described above) in UK dogs described the subgingival microflora in a cohort of 114 dogs and through checkerboard hybridisation highlighted a number of target species of interest due to their association with periodontal status or close relationships with organisms linked to the health or disease state.

Novel Peptostreptococcus and Synergistes species as well as Odoribacter denticanis were found to be strongly associated with periodontal disease, while other species were more weakly indicative of disease. In addition to guiding the development of diagnostic and monitoring kits, this information was of interest for facilitating the development of technologies for measurement of the effect of oral health actives on dental plaque.

These examples confirm or clarify associations identified in the previous study.

These examples investigate further the opportunities posed by the initial UK study through confirmation of the associations resulting from the original study in independent dog populations. This was conducted through 2 studies. The first was a study involving sample collections from a smaller population of dogs presented to a referral clinic in the UK. This study aimed to assess bacterial species in the subgingival plaque of animals from a population within the UK and discount any potential associations enriched due to geographical isolation of the healthy animals in the previous study. The second involved sample collections from a population of dogs from the USA visiting veterinary hospitals, and aimed to assess the relevance of the UK associations in USA dogs.

Methodology

Animals and inclusion criteria UK study (Example 2)

Twenty dogs suffering periodontal disease were recruited from a veterinary dental referral clinic and were sampled during the course of their normal treatment for the condition. For inclusion in the disease group, dogs were required to have a minimum of 4 sites displaying periodontal disease of at least stage 3; equivalent to between 25% and 50% attachment loss (see Appendix 1). These minimum requirements allowed consistency with the samples achieved in the initial phase where the majority of the animals sampled were periodontal disease stages 3 and 4. Animals under the age of 2 were not sampled.

Due to the number of animals involved in the study an upper limit of 3 diseased animals from any single predisposed breed was adhered to in order to ensure that the study focus was maintained on severe chronic periodontal disease and not early onset or aggressive periodontitis.

USA study (Example 3)

Animals were recruited to the study from 9 veterinary hospitals in the USA. A single sampling technician collected gingival margin and supragingival plaque samples from 40 periodontally diseased dogs.

Sampling procedures

UK study

Periodontally diseased dogs were sampled for subgingival, supragingival and gingival margin plaque from at least 4 diseased sites during treatment for periodontal disease.

Initially, supra-gingival and gingival margin plaque were collected using a sterile Gracey curette to prevent sample cross-contamination. A sterile periodontal probe was then inserted under the gingival margin and swept along the tooth surface. The resulting subgingival sample was suspended in 350ul of 50mM Tris (pH 7.6), ImM EDTA (pH 8.0), 0.5% Tween 20 and was immediately stored at -20°C. Chemicals were obtained from Sigma chemical company unless stated otherwise.

USA study

All dogs were sampled while conscious and hence only supragingival and gingival margin plaque samples were collected. Sterile plastic disposable periodontal probes were utilised to remove plaque from the tooth surface. As in the UK study, 4 sites were targeted in the periodontally diseased group. However since the sampling areas were larger compared to previous subgingival collections, where more than 2 affected sites were present, samples were collected for the disease group. Supragingival plaque was removed from the buccal surface of the tooth from the tip of the crown to 2mm above the gingival margin and gingival margin plaque was collected at the level of gingival attachment to the tooth and not at the centmento-enamel junction (where these points differed). Again samples were collected into 350ul of 50mM Tris (pH 7.6), ImM EDTA (pH 8.0), 0.5% Tween 20 and stored immediately at -20°C.

Sample preparation DNA was extracted from all plaque samples using Qiagen DNeasy Tissue Kit (Qiagen Ltd UK) following the manufacturer' s standard protocol for isolation of genomic DNA from Gram-positive bacteria. Amplification of total bacterial 16S rDNA was conducted by polymerase chain reaction (PCR) from the extracted genomic DNA using a standard volume of 25ul (approximate DNA concentration 10ng/ul) and 16S rDNA universal primers (Forward; 5'-Digoxigenin-AGAGTTTGATYMTGGC-3' and reverse 5'-GYTACCTTGTTACGACTT-3'). AmpliTaq Gold DNA Polymerase (Applied Biosystems, CA USA) was utilised to enhance sequence integrity of the 16S rRNA amplicons. PCR was performed through 30 cycles of denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds and elongation at 72°C for 90 seconds with an additional 5 seconds added for each cycle. Amplification yields were assessed by agarose gel electrophoresis and where yields were low the amplification step was repeated using 50ul genomic DNA. Reaction products were stored at -20°C until checkerboard hybridisation was performed.

DNA checkerboard hybridisation analysis

This was carried out as described for example 1. The results are shown below.

USA Study (Example 3)

USA Study (Example 3)

UK External Study Example 2

UK External Study (Example 2)

Statistical analysis of bacterial associations with periodontal disease Checkerboard hybridisation data were transformed by visual assessment from X-ray film illuminated using a light box. Data points were converted into absence and presence scores (0 and 1 respectively) equating to levels above and below the detection limit. Differences between the detection of each species (probe reactivity) with the healthy or disease samples were assessed using the Fishers Exact test (http://www.physjcs.csbsju.edu/stats/flsher.form, html)

Comparisons between the UK and USA data and of these studies to the initial UK dataset were made by visual assessment and binary logistic regression analysis (LRA; Minitab V12) of the reactivity of the dogs from diseased groups to each individual species (probe) and including study as a factor (blocking for study).

Results

Bacterial associations in an independent population of UK dogs Dogs recruited to the study were from a total of 22 breeds including Labrador, Greyhound, King Charles Cocker Spaniel, West Highland white terrier, Old English sheep dog, Border collie, Yorkshire terrier, miniature Poodle, Doberman, Weimeraner, Tibetan terrier, German shepherd dog, Staffordshire bull terrier, American bull dog, Chinese crested, Golden retriever Belgian Shepherd and miniature Schnauser as well as 3 cross breeds. Mean age 10 years (stdev 3.4). Mean periodontal score in the diseased group was 3.63 (stdev 0.47). All of the dogs were privately owned animals from Surrey and the surrounding area.

Probes previously showing some ability to differentiate between healthy and periodontally diseased animals or specific for species of potential interest were utilised in the checkerboard hybridisation technique. Several additional probes were included in the analysis compared to the previously reported study, these were designed for the detection of Porphyromonas canons (reference 13 in the present invention); Bacteroides denticanoris (reference 11 in the present invention) and an unspeciated Treponema having 16S RNA sequence as set out in sequence reference: 17. Presence or absence of the target species was achieved within subgingival plaque samples from 19 of the animals. Control samples combined plaque from healthy and disease dogs taken from each of the 3 sites (control samples; subgingival, gingival margin and supra-gingival sites from animals 1 & 2 Health and 1 & 2 Periodontal disease) in order to ensure maximum numbers of reactive species for comparison of signal strength between blots. The 3 most similar blots were used for data transformation.

Data were transformed into presence (1; above detection limit) and absence (0; below detectable levels) and the incidence of each species within the 20 healthy and 20 diseased animals were assessed to indicate associations with the healthy or diseased state, Table 2 as follows:

Subgingival bacterial profiles were used to assess the species associated with periodontal health status since organisms present in these biofilms are in direct contact with the gingiva and hence are considered most directly linked with the initiation of periodontal disease. Subgingival populations are also most relevant for comparative purposes since previous studies have highlighted subgingival species associated with periodontal health and disease.

A total of 11 organisms were significantly associated with the diseased state. Odoribacter "denticanis" was the most highly associated organism with periodontal disease with 17 of the 19 diseased and only 1 of the healthy dogs showing reactivity to this probe (P=1.94x10^-7; Table 2). The novel Peptostreptococcus species (reference 1) detected was the second most highly associated with 14 dogs from the disease group showing evidence of this species compared to 2 healthy animals (P=0.0002). Porphyomonas salivosa (P. macacae) and the Synergistes species detectable by probes 2a to 2f in Table A and/or comprising a 16S rRNA sequence more than 96% identical to sequence referenced both showed similar levels of disease association with 3 of the healthy animals and 14 (P=0.0008) or 13 (P=0.0025) of the diseased group showing detectable levels of these organisms respectively. A second novel Synergistes species detectable by probes 8a to 8j in table A and/or comprising a 16S rRNA sequence more than 87% identical to sequence reference: 8 was absent in all of the samples from healthy dogs but present in 8 diseased dogs (P=0.0031). Several other organisms were also associated significantly (but less strongly) with disease including Fillifactor villosus; Treponema denticola; Porphyromonas canoris; a novel Clostridials species; Desulfomicrobium orale and Bacteroides denticanoris.

Bacterial associations with health and disease in USA dogs

The USA population in the healthy and diseased dogs sampled totalled 82 dogs and comprised pure bred dogs from 26 different breeds as well as 25 mixed breed animals, 12 in the disease and 13 in the healthy group. The mean age was 5.4 years (stdev 3.08) with a mean of 7.2 years (stdev 2.6) in the disease group and 3.7 years (stdev 2.43) in the healthy group. Even localised gingivitis was not observed in the healthy dogs with periodontal scores of 0 throughout, mean periodontal score in the diseased group was 3.3 (stdev 0.57). The majority (29) of the 39 diseased dogs had a periodontal health score of 3, with 8 animals scoring 4 and only 2 with the most severe level of periodontal disease (score of 5; for periodontal scoring definitions see Appendix 1). Since the USA scoring system used requires an adjustment of 1 point for scores of 1+ in order to make comparisons with the UK system, the comparative score for the disease group resulted in a mean periodontal disease score of 2.31 (stdev 0.57). The mean score of 0 in the healthy group was unaffected by this adjustment.

Following sample preparation, checkerboard hybridisation analysis of the supragingival and gingival margin samples collected was used to detect the same target species analysed in the UK population (Appendix 2). Again data were transformed into presence (above detection levels; score of 1) or absence (below detection levels; score of 0) by visual assessment of back-lit X-ray film. Supra-gingival plaque samples appeared to show lower incidence levels of certain organisms of interest including "Odoribacter denticanis" (data not shown) therefore the profiling data used was that from the gingival margin plaque. Comparisons between blots were made by analysis of signal strength produced from pooled standards (controls; supra-gingival samples from animals 1 Health, 1 Gingivitis and 1 Periodontal disease, as well gingival margin samples from animals 2 Health, 1 Gingivitis and 1 Periodontal disease).

Of the target organisms analysed, 16 were significantly associated with the diseased periodontum in the USA population (Table 4).

The probe most strongly indicative of disease was specific for a novel Synergistes species detectable by probes 8a to 8j in table A and/or comprising a 16S rRNA sequence more than 87% identical to sequence reference:8 present in only 2.4% of the healthy animals tested and 84.6% of those suffering disease (P=3.37x10^-15). "Odoribacter denticanis " was the second most strongly associated organism with the disease being detected in 46.2% of the diseased compared to 2.2% of the healthy samples. The novel Peptostreptococcus species detectable by probes 1a to 1h in table A and/or comprising a 16S rRNA sequence more than 98% identical to sequence reference: 1 was also significantly associated with disease and was present in 28.4% of the healthy population and 81.3% of the diseased animals. The novel Moraxella (reference 2), enhanced within the disease population was found in only 21.8% of healthy and in 66.7% of diseased dogs within the US population studied. Eubacterium nodatum, an organism implicated in periodontitis in humans was also strongly linked to the diseased group of dogs being below detectable levels in the healthy population but detected in 30.8% of dogs suffering periodontitis. Other organisms significantly associated with the disease group included canine Porphyromonas species P. canons; P. salivosa and P. gulae as well as T. denticola, D. ovale, B. denticanoris, F. villosus and novel undescribed species from the taxa Synergistes, Clostridales, Selenomonas and Bacteroidetes.

Comparison of disease associated species in geographically distinct populations (Comparison of Examples 1, 2 and 3 above).

The completed studies in which bacterial populations in plaque samples from healthy populations had been compared to those from diseased dogs by checkerboard hybridisation were compared to highlight any differences between the studies or geographical populations (Table 4). This enabled the comparison of 25 bacterial populations across three studies, the first being Example 1 above, which compared healthy WCPN animals to an external UK diseased population. The second and third datasets being from studies of UK animals external to WCPN and that in USA dogs reported here (Examples 2 and 3 above). Table 5. Statistical comparison of data from three checkerboard hybridisation analyses for detection of the target species in healthy and diseased dogs from distinct populations. P values are given for significant associations (p<0.05j or are shown in brackets where P=<0.2. Example 1 (n=120), Example 2 (n=40) and Example 3 (n=80). LRA = Logistic regression analysis.

Logistic regression analysis was utilised with study included as a factor and showed no significant differences between the data sets from Example 1 and Example 2 (Table 5). However, 4 bacterial species showed differences in their associations with disease between the USA population analysed (Example 3) and the initial UK study (Example 1). These included Porphyromonas salivosa (reference 16); "Odoribacter denticanis" (reference 7) and the Synergistes spp. references 2 and 8. Comparison of the external UK study (Example 2) and USA dogs (Example 3) showed one significant difference in the datasets with the Synergistes species (reference 8) again found to differ significantly between studies. The organisms "Odoribacter denticanis" (reference 7) and Synergistes sp. (reference 2) were also approaching significance and hence were highlighted as potentially differing between geographical locations. These differences may be utilised to enhance the assessment of health status in geographically diverse populations of animals by assessment of species most relevant or descriptive within the particular population under assessment. P. canons (reference 13) and B. denticanoris (reference 11) were not assessed in the phase 1 UK study (Example 1), and thus were not able to be included in the analysis of the total results of all three studies.

Associations observed in Example 2 and Example 3

Analysis of the bacterial species from the Example 2 highlighted 11 organisms significantly associated with disease. In Example 3, however, 16 organisms were linked with disease. It is unclear whether the differences in population numbers between the studies affected the numbers or strength of the associations observed. Several of the disease associations identified in the USA dogs but not the UK (Example 2) study appear to result from a complete absence in detection in the healthy population and hence may be potential statistical artefacts Eubacterium nodatum (reference 4); Bacteroidetes sp. (reference 6) and Selenomonas sp. (reference 5). Others such as P. gulae (reference 14) and the Moraxella sp. (reference 10) showed trends towards increased incidence in the diseased or healthy populations respectively in Example 2 and therefore may have been significant on assessment of a larger population.

Inter-study consistency of bacterial associations with disease

The association of both P. canons (reference 13) and Bacteroides denticanoris (reference 11) with disease was consistent in Example 2 and Example 3 studies but was not previously assessed in Example 1. Hence these represent novel and seemingly geographically independent associations with the diseased state.

Statistical analyses between the datasets from the 3 studies represented the product of the extent of the incidence in the total population as well as the difference in the association with (incidence in) the healthy and diseased groups. Although no statistically significant differences were identified between the datasets from Examples 1 and 2, several potential differences were observed. These may have been masked in the statistical analysis due to the differences in the numbers of animals included in the studies. Visual comparison of Examples 1 and 2 showed variation in the incidence and associations of Filifactor villosus (reference 12); Moraxella sp. (reference 10); Treponema denticola (reference 15); P. salivosa (reference 16) and P. gulae (reference 14). Therefore these data would not be combined for assessment of a 'total' association across Examples 1 and 2. In each of these cases the incidence of the bacterial species in the healthy population was enhanced in Example 1 compared to levels in Example 2. It seems therefore that these organisms may be present at higher incidence in the healthy population used in Example 1 compared to the general UK population and as such represent enriched WCPN populations, consequently appearing as health associations in Example 1 where the healthy study group comprised only WCPN animals (or masking disease associations observed in examples 2 or 3).

It is interesting however that several of the populations described above including P. salivosa; T denticola; P. gulae I and Fillifactor villosus are indeed associated with the diseased state (by comparison between all 3 studies) despite a lack of clinical signs of disease in the WCPN dogs involved in Example 1. Since multiple breeds were represented in the population this is unlikely to be due to genetic factors and may therefore suggest that oral care regimes such as regular toothbrushing and veterinary management of oral health as employed at WCPN may prevent the onset of clinical signs of disease despite the presence of disease associated populations. P. salivosa is a particularly good example of this phenomenon with the organism observed at high incidence (>60%) but at low bacterial levels (numerical score 1; Example 1) in the clinically healthy WCPN dogs compared to high incidence and higher bacterial loads in the diseased animals. In latter studies however the organism was detected at lower incidences in the healthy compared to diseased groups, uncovering the actual association of this organism with disease.

Across each of the 3 examples, although the majority of associations detected were conserved to at least the same periodontal status, several associations were altered. Several additional disease associations were identified in the latter studies compared to the Example 1 in which 8 species were linked with periodontitis. In the Example 2, 3 additional associations were identified while 8 additional species showed higher incidence in diseased animals in Example 3.

Such differences would make combining the study data into a single data set invalid since Example 1 represented a substantial portion (50%) of the total animals tested, and seemed to show several differences compared to Examples 2 and 3. Due to these differences, which as discussed are likely to be at least in part due to geographical isolation of the health group recruited to Example 1, geographical differences in the bacterial associations with periodontal disease were assessed with the emphasis on the latter two studies (Examples 2 and 3).

Summary of bacterial associations within differing geographical locations Comparison of Example 2 with that from Example 3 showed that all of the associations were maintained while some were strengthened. Significant statistical differences were found in only a small number of cases between Examples 2 and 3, including Synergistes sp. (reference 8; P= 0.015). Although some evidence of potential (non- significant) differences between the data sets was found for "Odorbicater denticanis" (reference 7) and Synergistes sp. (reference 2), these appear to be based on the incidence within the population and not on the association with the diseased state.

Subgingival plaque collections for the UK disease samples (Examples 1 and 2) were undertaken from cases visiting a veterinary dental referral clinic for treatment. This lead to a mean periodontal score of 3.63 in the disease group, while the disease group in Example 3 had a mean periodontal score of 3.3 corrected to 2.31 after allowing for differences between the USA and UK periodontal scoring system (Appendix 1). The USA collections from veterinary hospitals, endorsing wellness programmes and collections in animals visiting the hospitals for non-referral based treatment may have impacted on the cases available for collection and hence resulted in the observed sampling of less severe disease cases compared to those in the UK studies. These differences in the disease status may have impacted on the bacterial populations present. A further possible source of differences between studies is the sampling site with subgingival plaque samples used in Examples 1 and 2 and gingival margin samples in Example 3. However sampling site between the supragingival, gingival margin and subgingival plaque has previously been described to result in consistent detection of the majority of the target species with the Treponema sp. detected by probe (reference 15) being the only organism identified as differing significantly between sites (not reported).

Results: Summary Tables

Table 5 (UK phase 2 data)

Table 6 (USA data)

Table 7 (combined data)

Table 8 Predictive Combinations

Table_.9

Example of method of assessing likelihood of health or risk of disease from exact bacterial profile present. Where risk is less than 1.0 the status indicated is periodontal health. Where risk is greater than 1.0 Periodontal disease is indicated.

References

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Appendix 1. Periodontal scoring system employed for assessment of health and disease

Scoring system summary:

Appendix 2. Probes for checkerboard hybridisation analysis of canine subgingival plaque

O

In the sequences below, references to a, b, c etc. are optional alternate sequences.

Sequence Reference :1 GAGTTTGATTATGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATG CAAGTCGAGCGCGGTTGTGCTTAGTATTGAGTGTTTTATTGATATAAAAC ATTGAATTCTATGCACAACTGAGCGGCGGACGGGTGAGTAACGCGTGG GTAACCTGCCCTATACACATGGATAACATACTGAAAAGTTTACTAATACAT GATAAAATAGTTTTTCGGCATCGAAGAATTATCAAAGTGTTTGCGGTATA GGATGGACCCGCGTCTGATTAGCTAGTTGGTGAGATAACTGCCCACCAA GGCGACGATCAGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGAAC TGAGACACGGTCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGC ACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTGAACGATGAAGGT CTTCGGATCGTAAAGTTCTGTTGCAGGGGAAGATAATGACGGTACCCTG TGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG GGGGCTAGCGTTATCCGGATTTACTGGGCGTAAAGGGTGCGTAGGTGG TCTTTCAAGTCGGTGGTTAAAGGCTACGGCTCAACCGTAGTAAGCCTCC GAAACGGTTAGACTTGAGTGCAGGAGAGGAAAGTGGAATTCCCAGTGTA GCGGTGAAATGCGTAGATATTGGGAGGAACACCAGTAGCGAAGGCGGC TTTCTGGACTGCAACTGACACTGAGGCACGAAAGCGTGGGTAGCAAACA GGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTACTAGGTGT CGGGGGTTACCCCCCTCGGTGCCGCAGCTAACGCATTAAGTACTCCGC CTGGGGAGTACGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGAC CCGCACAGGTAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAAC CTTACCTAAGCTTGACATCCCTCGGACCGGTGTTTAATCACACCTTTCCT TCGGGACTGAGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCG TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTT GCCATCATTAAGTTGGGCACTCTAGAGAGACTGCCAGGGACAACCTGGA GGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGCTTAGGGCTA CACACGTGCTACAATGGGTGGTACAGAGGGTTGCCAAACCGTGAGGTG GAGCCAATCCCTTAAAGCCACTCTCAGTTCGGATTGTAGGCTGAAACTC GCCTACATGAAGCTGGAGTTACTAGTAATCGCAGATCAGAATGCTGCGG TGAATGCGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGT CGGAAGCACCCGAAGCCGATTATCTAACCGCAAGGAGGAGATCGTCGA AGGTGGCGTCGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCG GAAGGTGCGGCTGGATCACCTCC

Sequence Referenced

GAGTTTGATTATGGCTCAGGACGAACGCTGGCGGCGTGCGTAACACATG CAAGTTGAACGACGGTGTCATGAAGTGGTAACACGGAGTGGCATACGGA GTAGCGGACGGGTGAGTACAACATGAGAAGCTGTCCAATAGCGGGGGA TAACGTTCGGAAACGGACCCTAATACCCCATAGGCCTTTTGGTTAAAGCA GCGATGCGCTATTGGAGGTGCTCGTGTCCTATCAGCTGGTTGGTGAGGT AACGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGTCC GGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCA GTGGGGAATATTGGGCAATGGGAGAAATCCTGACCCAGCGACGCCGCG TGAACGAAGAAGGCCTTCGGGTCGTAAAGTTCTTTTATGTGGGAAGAAG GAAGTGACGGTACCACATGAATAAGCCCCGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGGGGCGAGCGTTGTCCGGAATTACTGGGCGTA AAGGGCACGCAGGCTGTGCTTCAAGTCAGCTGTTAAAGGATGCGGCTTA ACCGTGTTATGCAGTTGAGACTGAGGTGCTGGAGTGCCGTAGAGGCAAG TGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATATTGGGAAGAACATC GGTGGCGAAGGCGACTTGCTGGACGGTAACTGACGCTGAGGTGCGAAA GCCAGGGGAGCAAACGGGATTAGATACCCCGGTAGTCCTGGCTGTAAA CGATGAATGCTAGGTGTGCGAGCAGAGATGTTGGTGTGCCGCAGTTAAC GCGATAAGCATTCCGCCTGGGGAGTACGGTCGCAAGATTGAAACTCAAA GGAATTGACGGGGGCCCGCACAAGCGGTGGAGCACGTGGTTTAATTCG ATGCAAACCGAAGAACCTTACCTGGGTTTGACATGTACGTGGTAGGAGT CTGAAAGGATGACGACGCTGCCTTCGGGCAGTGAGCGTACACAGGTGC TGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCCTGCGATTAGTTGCCAGCGATTCGGTCGGGCACTC TAATGGGACTGCCATCGACAAGATGGAGGAAGGTGGGGACGACGTCAA GTCATCATGGCCCTTATGTCCAGGGCGACACACGTGCTACAATGGTCGG TACAGAGGGAAGCGAGGTTGTGAGGCCGAGCGGATCCCGAAAAGCCGA TCTCAGTTCGGATTGAAGTCTGCAATTCGACTTCATGAAGCTGGAATCGC TAGTAATCGCAGATCAGCCAAGCTGCGGTGAATACGTTCCCGGGCCTTG TACACACCGCCCGTCACACCACCCGAGCCGGGTGTACCCGAAGCCGGT GGCCTAACCTTATGGGAGGAGCCGTCGAAGGTGTGTCTGGTGAGGGGG GTGAAGTCGTAAC

Sequence Reference: 3

GAGTTTGATTCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATG CAAGTCGAACGGAGCACCTCGGATCAAGTTTTCGGACGAGAGAAGAGAC TGCTTAGTGGCGGACGGGTGAGTAACGCGTGAGGAACCTGCCTTGGAG AGGGGAATAACACAAGGAAACTTGTGCTAATACCGCATGAAGCATAGCT ATCGCATGGTAACTATGCCAAAGATTTATCGCTCTGAGATGGCCTCGCGT CTGATTAGCTAGTAGGTGGGGTAACGGCCTACCTAGGCGACGATCAGTA GCCGGACTGAGAGGTTGACCGGCCACATTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTGGGGAATATTGGGCAATGGGCGCAAG CCTGACCCAGCAACGCCGCGTGAAGGAAGAAGGCTTTCGGGTTGTAAA CTTCTTTTATTCGGGACGAAAGAGATGACGGTACTGAATGAATAAGCCAC GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGTT ATCCGGATTTACTGGGTGTAAAGGGCGTGTAGGCGGGACAGCAAGTCA GGTGTGAGATCCCAGGGCTCAACCCTGGACGTGCACTTGAAACTGTAGT TCTTGAGTGATGGAGAGGCAGGCGGAATTCCGTGTGTAGCGGTGAAATG CGTAGATATACGGAGGAACACCAGTGGCGAAGGCGGCCTGCTGGACAT TAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATA CCCTGGTAGTCCACGCTGTAAACGATGGATACTAGGTGTGGGGGGACTG ACCCCTTCCGTGCCGCAGTTAACACAATAAGTATCCCACCTGGGGAGTA CGATCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC GGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGG CTTGACATGGAGATGACCGATGTAGAGATACATCCTCCCTTCGGGGCAT CTCACACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG GGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCTACGCAAG AGCACTCTAGCGAGACTGCCGTTGACAAAACGGAGGAAGGTGGGGACG ACGTCAAATCATCATGCCCCTTATGTCCTGGGCTACACACGTACTACAAT GGCGGCGAACAGAGGGAAGCAAGACCGCGAGGTGGAGCAAATCCCTAA AAGCCGTCCCAGTTCGGATTGCAGGCTGAAACCCGCCTGTATGAAGTTG GAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGG GCCTTGTACACACCGCCCGTCACACCACGAAAGTCGGGAACACCCGAA GTCCGTAGCCTAACAGCAATGAGGGCGCGGCCGAAGGTGGGCTTGATA ATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGG AACACC

Sequence Referenced

GAGTTTGATTCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATG CAAGTTGAGCGAGAAATCACTTAAAGAAGCTTCGGTAGACTTAAGAGATG GAGAGCAGCGGACGGGTGAGTAACGCGTGGGAAACCTGCCCTTGACAG GAGGATAGCCGAGAGAAATTTCGATTAATACTTCATAAAGCAGAGCATTC GCATGGATGAACTGCCAAAGAATTATCGGTCAAGGATGGTCCCGCGTCT GATTAGCTGGTTGGTAAGGTAGCGGCTTACCAAGGCGACGATCAGTAGC CGGCCTGAGAGGGTGAACGGCCACATTGGAACTGAGACACGGTCCAGA CTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCC TGATGCAGCAACGCCGCGTGAAGGAAGAAGGCTTTCGAGTCGTAAACTT CTGTCCAAAGGGAAGAATAATGACGGTACCTTTGAAGAAAGCCCCGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTATCC GGAATTACTGGGCGTAAAGAGTATGTAGGTGGTTAAGTAAGCGTAGGGT TTAAGGCGACAGCCCAACTGTCGTATGCCCCGCGAACTGTTTAACTTGA GTACAGGAGGGGAAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAG ATATTAGGAGGAACACCAGTGGCGAAGGCGGCCTTCTGGACTGTAACTG ACACTGAGATACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGAGCACTAGGTGTCGGGCTCGCAAGAGTTC GGTGCCGGAGCAAACGCATTAAGTGCTCCGCCTGGGGAGTACGCACGC AAGTGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAG CATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGACTTGACAT CCCACTGAAAGCTCGGGTAAAGCTGAGCCCTTCTTCGGAACAGTGGAGA CAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA GTCCCGCAACGAGCGCAACCCTTGCCGTTAGTTGCCATCATTAAGTTGG GCACTCTAATGGGACTGCCGGGGAGAACCCGGAGGAAGGCGGGGATG ACGTCAAATCATCATGCCCCTTATGTTCTGGGCTACACACGTGCTACAAT GGCCGTCACAGAGGGAAGCGAGAGAGCGATCTTAAGCGAAACCAAAAA GGCGGTCCCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGCCG GAGTTGCTAGTAATCGCGGATCAGAATGTCGCGGTGAATGCGTTCCCGG GTCTTGTACACACCGCCCGTCACACCATGGAAGTTGGGGGTGCCCAAAG TCGGTCAAGAAAAGGTCGCCTAAGGCAAAACCAATGACTGGGGTGAAGT CGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTT

Sequence Reference: 5

GAGTTTGATTCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATG CAAGTCGAACGGAGCTGTTTATTTCGGTAGATAGCTTAGTGGCAAACGG GTGAGTAACGCGTAGGCAACCTGCCCTTAGGATGGGGACAACGGCCCG AAAGGACCGCTAATACCGAATGCACTCTAACTTCCGCATGGAAGAAAGA GGAAAGATGGTGCAAGCCATCGCCGAAGGAAGGGCCTGCGTCTGATTA GCCAGTTGGTGAGGTAACGGCTCACCAAAGCGACGATCAGTAGCCGGT CTGAGAGGATGAACGGCCACAATGGGACTGAGACACGGCCCATACTCC TACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGGCGAAAGCCTGAC GGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAGCTCTGT TGTTGGGGACGAAAGGAAAAGGGAGGAAATGCCCTTTTTAAGACGGTAC CCGACGAGCAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATAC GTAGGTGGCGAGCGTTGTCCGGAATGATTGGGCGTAAAGGGAGCGCAG GTGGGACGGTAAGTCCTTCTTAAAAGCGTGGGGCTCAGCCCCATGAAGG GAAGGAAACTATCGATCTTGAGTGCCGGAGAGGAAAGCGGAATTCCCAG TGTAGCGGTGAAATGCGTAGATATTGGGAAGAACACCAGTGGCGAAGGC GGCTTTCTGGACGGCAACTGACACTGAGGCTCGAAAGCCAGGGGAGCG AACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAG GTGTAGGAGGTATCGACCCCTTCTGTGCCGGAGTTAACGCAATAAGTAT CCCGCCTGGGGAGTACGGTCGCAAGATTGAAACTCAAAGGAATTGACG GGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGACGCAACGCG AAGAACCTTACCAGGGCTTGACATTGAGTGAAAGGCGTAGAGATACGTC CCTTATCTTCGGATAACACGAAAACAGGTGGTGCATGGCTGTCGTCAGC TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTAT GTTCTGTTGCCAGCGCGTAAAGGTGGGCACTCAGAAGAGACTGCCGCG GAGAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGCCCCTTA TGACCTGGGCTACACACGTACTACAATGGGATGTACAGAGGGCAGCGAA CGGGCGACCGGAAGCGAACCCCAGAAAACATCTCCCAGTTCGGATTGC AGGCTGAAACCCGCCTGCATGAAGATGGAATCGCTAGTAATCGCAGGTC AGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA CACCACGGAAGTCATTCACACCCGAAGCCGGAGGAAATCCGTCGAAGG TGGGGGCGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAA GGTGCGGCTGGATCACCTCCTT

Sequence Reference:6

GAGTTTGATTCTGGCTCAGGATGAACGCTAGCGGCAGGCCTAATACATG CAAGTCGAACGGGATCGGGGCCTCCGGGCCCTGTGAGAGTGGCGCAC GGGTGCGTAACGCGTATGCAACCCACCCCGACCTGCGGGAGAGCCGGT GGAAACGCCGGGTGATACCGCATGAGGCCGCGTCCGGGCATCCGGGT GCGGCGAAAGCAGCGATGCGGGCCGGGACGGGCATGCGTTCCATTAG CTAGTCGGCGGGGTGACGGCCCACCGAGGCCACGATGGATAGGGGAT CTGAGAGGACGGTCCCCCACACTGGTACTGAGACACGGACCAGACTCC TGCGGGAGGCAGCAGTAAGGGATATTGGGCAATGGGGGGAACCCTGAC CCAGCCATGCCGCGTGAGGGAGCGAGGCCCTACGGGTCGTGAACCTCT TTTGGAGGGGGATAAGGCGGGGCGCTAGACGCCCTGTTGCAGGTACCC TCCGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGA GGATGCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGCAGGCGG CCCGGCAAGTCAGTGGTGAAAGGCAGGGGCTCAACCCCGGCAGTGCCG TTGATACTGTTGGGCTGGAATGCGGTCGAGGCGGGCGGAATGTGGCGT GTAGCGGTGAAATGCATAGATATGCCACAGAACGCCGATAGCGGAGGC AGCTCGCCAGGCCTGCATTGACGCTCGGGCACGAAAGCGTGGGTATCG AACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGGACGCTCG TCGTCGGCGGCAGACGGTCGGCGGCCAAGCGAAAGTGATAAGCGTCCC ACCTGGGGAGTACGGTCGCAAGGCTGAAACTCAAAGGAATTGACGGGG GCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGGA ACCTTACCCGGGCTTGAACGTGCGCGGACGCCTTCCGGAAACGGGAGT CTCCCTTCGGGGCCGCGTGCGAGGTGCTGCATGGTTGTCGTCAGCTCG TGCCGTGAGGTGTCGGGTCAAGTCCCATAACGAGCGCAACCCCTGCCC CCAGTTGCAAACGGTCCGGCCGTGCACTCTGCGGGGACTGCCTGCGCA AGCAGCGAGGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTTACGT CCGGGGCGACACACGTGCTACAATGGCCGGCACAGAGGGAGGCCACC CTGCGAGGGGGCGCGGATCCCGAAAGCCGGTCCCAGTCCGGATCGGA GTCTGCAACCCGACTCCGTGAAGCTGGAATCGCTAGTAATCGCGCATCA GCCATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC AAGCCATGGAAGCCGTGGGCGCCTGAAGTCGGCGTTAGCCGCCTAGGG CGAATTCGGTGACTGGGGCTAAGTCGTAACAAGGTCC

Sequence Reference:7

GAGTTTGATTCTGGCTCAGGATGAACGCTAGCGATAGGCTTAACACATG CAAGTCGAGGGGTAACAGGCGGTAGCAATACTGTGCTGACGACCGGCG GATGGGTGAGTAACGCGTATGCAATCTACCTTTTACCCGGGGATAGCCC ATGGAAACGTGGATTAATACCCGATGCATTTCTTTTGTGGCCTCATGAAG GGAATAAAGATTTATCGGTAAGAGATGAGCATGCGTTCCATTAGGAAGTT GGTAAGGTAACGGCTTACCAATCCGATGATGGATAGGGGTTCTGAGAGG AAGGTCCCCCACACTGGAATTGAGAAACGGTCCAGACTCCTACGGGAG GCAGCAGTGAGGAATATTGGTCAATGGTCGTAAGACTGAACCAGCCAAG TCGCGTGAAGGAAACTGCCCTATGGGTTTTCAACTTCTTTTGTCAGGGAA GAATAAGGAGGATTCAATTCTCCGATGCCGGTACCTGACGAATAAGGAT CGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGT TATCCGGATTTATTGGGTTTAAAGGGTGCTTAGGCGGCAAATTAAGTTAG TGGTTAAATAGTTCGGCTCAACCGGATTTCGCCATTAAAACTGATATGCT AGAGATTAAACGAGGTAGGCGGAATAAGTTAAGTAGCGGTGAAATGCAT AGATATAACTTAGAACACCGATAGCGAAGGCAGCTTACCAGGCTATATCT GACGCTGAATCACGAGAGCGTGGGTATCGAACAGGATTAGATACCCTGG TAGTCCACGCCGTAAACGATGCTCACCGGCTCTATGCGATAAGACAGTA TGGGGCTAATAGAAATAATTAAGTGAGCCACCTGGGGAGTACGTCGGCA ACGATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAAC ATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGTTTAAATGT ATGTTGCATTATGTAGAAATACGTATTTTCTTCGGAACTGCATACAAGGTG CTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGGTTAAGTCCCA TAACGAGCGCAACCCTTATGATTAGTTGCTAACGGTTCAAGCCGAGCACT CTATTCACACTGCCACCGTAAGGTGCGAGGAAGGAGGGGATGATGTCAA ATCAGCACGGCCCTTATATCCGGGGCTACACACGTGTTACAATGGTCGG TACAGCGGGTTGCATTTACGTGAGTAACAGCTAATCCCAAAAATCGGTCT CAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTTGGATTCGCTA GTAATCGCACATCAGCCATGGTGCGGTGAATACGTTCCCGGGCCTTGTA CACACCGCCCGTCAAGCCATGGGAGCTGAGGGTGCCTGAAGTTCGTAA CCGCGAGGAGCGGCCTAGGGCAAACTTGGTAACTGGGGCTAAGTCGTA ACAAGGTAGCTGTACCGGAAGGTGCGGCTGGAACACCTCTT

Sequence Reference:8

GAGTTTGATTATGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATG CAAGTCGAACGGGGTTATATTGGTTAGTTTACTAAGCGATATGACTTAGT GGCGGACGGGTGAGTAACGCGTGAGGACCTATCCCAAGCTGGGGGACA ACAGCTGGAAACGGTTGCTAATACCGCATAAGCGCATAATCTGCGTAAA AGGAGAGATCCGGCATGGGAGGGGCTCGCGTCCTATCAGCTAGTAGGT GGGGTAAAGGCCTACCTAGGCGATGACGGGTAGCCGGCCTGAGAGGG CGCACGGCCACACTGGGACTGAGATACGGCCCAGACTCCTACGGGAGG CAGCAGTGGGGAATATTGGGCAATGGGGGGAACCCTGACCCAGCGACG CCGCGTGAGTGAAGAAGTCCTTCGGGACGTAAAGCTCTGTTGTGTGGGA AGAAGAGGATGACGGTACCACACGAGGAAGCCCCGGCAAACTACGTGC CAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTGTCCGGAATTACTG GGCGTAAAGGGCACGCAGGCGGAATGGTAAGTCAGTTGTGAAAGGCTG TGGCTCAACCACAGGGAGACGACTGATACTGTCAGTCTAGAGTATGTGA GAGGGAAGTGGAATTCCCGGTGTAGCGGTGAAATGCGTAGATATCGGG AGGAACACCAGTGGCGAAGGCGGCTTCATGGCACATAACTGACGCTCAT GTGCGAAAGCTAGGGTAGCGAACGGAATTAGATACTCCGGTAGTCCTAG CCGTAAACGATGGATACTAGGTGTGGGTGTCGCAGGGCATCCGTGCCG GAGTCAACGCGTTAAGTATCCCGCCTGGGGACTACGGCCGCAAGGCTG AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCACGTGG TTTAATTCGATGCAAACCGAAGAACCTTACCTGGGTTTGACATGTATATG TTAGAGAAGCGAGAGCGGATCGACCACAGTTTACTGTGGAGTATACACA GGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT CCCGCAACGAGCGCAACCCCTGTTGTCAGTTGCTAACGGTTAGAGCCGA GCACTCTGGCGAGACTGCCGTCGACAAGACGGAGGAAGGTGGGGACGA CGTCAAGTCATCATGGCCTTTATGTCCAGGGCGACACACGTGCTACAAT GGTATGCACAGAGGGAAGCGAAGTGGTGACATGGAGCGGATCCAAGGA AGCATATCTCAGTTCGGATTGTAGTCTGCAACTCGGCTACATGAAGCCG GAATCGCTAGTAATCGCAGATCAGCCAAGCTGCGGTGAATACGTTCCCG GGCCTTGTACACACCGCCCGTCACACCACTCGAGTTGTCTGCACCCGAA GCCAGTGGCATAACCGCAAGGGATGAGCTGTCTAAGGTGTGGGGGGTA AGGGGGGTGAAGTCGTAAC

Sequence Referenced

GAGTTTGATTATGGCTCAGAATGAACGCTGGCGGCGTGCCTAACACATG CAAGTCGAACGAGAAAGTTCCTCCGGGAATGAGTAGAGTGGCGCACGG GTGAGTAACGCGTGGGTAATCTGCCCTTGGATTTGGGATAACTTCGCGA AAGTGGAGCTAATACCGGATAGTCTGGCTTTGTAAAAGGAGTCGGTAAA GGATGCCTCTGCATATGCATTCGTCCGAGGATGAGCCCGCGTCTCATTA GCTAGTTGGTAGGGTAACGGCCTACCAAGGCGACGATGAGTAGCTGGT CTGAGAGGATGATCAGCCACACTGGGACTGAAACACGGCCCAGACTCCT GCGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGAAAGCCTGTCG CAGCAACGCCGTGTGAGGGATGAAGGTTTTCGGATCGTAAACCTCTGTC TGAAGGGAAGAAGTGATGCGGGTCCAATAGGCCCGCATTTTGACGGTAC CTTCAAAGGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATAC GGAGGGTGCAAGCGTTATTCGGAATTACTGGGCGTAAAGCGCACGTAG GCGGCCTTGTAAGTCAGGTGTGAAATCCCACGGCTCAACCGTGGAACTG CACTTGAAACTGCGGGGCTCGAATCCTGGAGAGGGCGGCGGAATTCCT GGTGTAGGAGTGAAATCCGTAGATATCAGGAGGAACACCGGTGGCGAA GGCGGCCGCCTGGACAGGTATTGACGCTGAGGTGCGAAAGTGTGGGGA GCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGGATAC TAGGTGTCGGGGACTTGATCCTCGGTGCCGCAGTTAACGCGTTAAGTAT CCCGCCTGGGGAGTACGGTCGCAAGGCTGAAACTCAAAGAAATTGACA GGGGCCCGCATAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGA AGAACCTTACCTGGGCTTGACATCCTGGGAATTCCGCAGAGATGCGGAA GTGCCCTTCGGGGAATCCAGAGACAGGTGCTGCATGGCTGTCGTCAGC TCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTAT CCTCAGTTGCCAGCAGGTAAAGCTGGGCACTCTGTGGAGACTGCCCGG GTTAACCGGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTA CGCCCAGGGCTACACACGTACTACAATGGTGGGCACAAAGGGCAGCGA AACCGCAAGGTCCAGCCAATCCCAAAAAACCCATCATAGTCCGGATTGC AGTCTGCAACTCGACTGCAAGAAGTTGGAATCGCTAGTAATCCCGGATC AGCATGCCGGGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC ACACCACGAAAGTCGGTTCTACCCGAAGCCGCCAGGCTAACCCGCAAG GGATGCAGGCGTCTACGGTAGGGCTGGTAATTGGGGTGAAGTCGTAAC AAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTAGGGCGAA TCGCGGCGCTAATTCATTC

Sequence Referencer10

GAGTTTGATTATGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATG CAAGTCGAACGGAAGYTACTAGCTTGCTAGTAACTTTAGTGGCGAACGG GTGAGTAATATTTAGGAATCTGCCTCATAACGGGGGATAGCTCGGGGAA ACTCGAATTAATACCGCATACACCCTACGGGGGAAAGGTGGCGCAAGCT GCCGATATGAGATGAGCCTAAATCAGATTAGCTAGATGGTGGGGTAAAG GCCTACCATGGCGACGATCTGTAACTGGTCTGAGAGGATGATCAGTCAC ACCGGAACTGAGACACGGTCCGGACTCCTACGGGAGGCAGCAGTGGGG AATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGA AGAAGGCCATAAGGTTGTAAAGCACTTTAAGCAGGGAGGAAGAACTTTT AGTTAATAGCTAAAAGTGGTGACGTTACCTGCAGAATAAGCACCGGCTAA CTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGG AATTACTGGGCGTAAAGCGAGCGTAGGTGGTTGATTAAGTCAGCTGTGA AATCCCCGGGCTTAACCTGGGAAAGTCAGCTGATACTGGTTAACTAGAG TATGTGAGAGGAAAGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAG ATCTGGAGGAATACCGATGGCGAAGGCAGCTTTCTGGCATAATACTGAC ACTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAG TCCACGCCGTAAACGATGTCTACTAGCCGTTGGTTACCAAGAGTAGCAA GTGGCGCAGCAAACGCGATAAGTAGACCGCCTGGGGAGTACGGCCGCA AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGC ATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATA TCTAGAATCCTACAGAGATGTGGGAGTGCCTTCGGGAATTAGAATACAG GTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTATCCTTAGTTACCATCAAGTGAAGTTGGG GACTCTAAGGAGACTGCCAGTGACAAACTGGAGGAAGGCGGGGACGAC GTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATG GTAGGTACAGAGGGTCGCTACACTGCGAAGTGATGCCAATCTCAAAAAG CCTATCGTAGTCCAGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGA ATCGCTAGTAATCGCGGATCAGAATGTCGCGGTGAATACGTTCCCGGGC CTTGTACACACCGCCCGTCACACCATGGGAGTCTATTGCACCAGAAGTA GTTAGCCTAACGCAAGAGGGCGATTACCACGGTGTGGTCGATGACTGG GGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGAACAC CTCCTT

Sequence Reference: 11

(a)

GAGTTTGATTATGGCTCAGGATGAACGCTAGCTACAGGCTTAACACATGC AAGTCGAGGGGCAGCATGAATATTGGCTTGCCAATATTTGATGGCGACC GGCGCACGGGTGAGTAACACGTATCCAACCTTCCGGTTACTCGGGGATA GGCTTTCGAAAGAAAGATTAATACCCGATGTTGTGTATCTTTCTCCTGAA AGATACGCCAAAGGATTCCGGTAACCGATGGGGATGCGTTCCATTAGGC AGTTGGCGGGGTAACGGCCCACCAAACCTTCGATGGATAGGGGTTCTGA GAGGAAGGTCCCCCACATTGGAACTGAGACACGGTCCAAACTCCTACGG GAGGCAGCAGTGAGGAATATTGGTCAATGGACGGAAGTCTGAACCAGC CAAGTAGCGTGAAGGATGACTGCCCTCTGGGTTGTAAACTTCTTTTATAC GGGAATAACATGAGGTACGGGTACCTTATTGCATGTACCGTTATGAATAA GCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGA GCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGTGGGATATTAA GTCAGCTGTGAAAGTTTGGGGCTCAACCTTAAAATTGCAGTTGATACTGG TTTTCTTGAGTACGGTATAGGTGGGCGGAATTCGTGGTGTAGCGGTGAA ATGCTTAGATATCACGAAGAACTCCTATTGCGAAGGCAGCTCACTGGAC CGGCACTGACACTGATGCTCGAAAGTGCGGGTATCAAACAGGATTAGAT ACCCTGGTAGTCCGCACAGTAAACGATGAATACTCGCTGTTTGCGATACA CGGTAAGCGGCCAAGCGAAAGCGTTAAGTATTCCACCTGGGGAGTACG CCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG AGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCT TAAATTGCGCTGGCTTTTACCGGAAACGGTATTTTCTTCGGACCAGCGTG AAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAA GTGCCATAACGAGCGCAACCCTTATCTTTAGTTGCCATCAGGTTTTGCTG GGGACTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATG ACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAA TGGGGAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAA AACTCCTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTG GATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATACGTTCCCG GGCCTTGTACACACCGCCCGTCAAGCCATGAAAGCCGGGGGTACCTGA AGTACGTAACCGCAAGGATCGTCCTAGGGTAAACCTGGTGATTGGGGCT AAGTCGTAACAAGGTAGCCGTTCCGGAAGGTGCGGCTGGATCACCTCC

(b)

GAGTTTGATTATGGCTCAGGATGAACGCTAGCTACAGGCTTAACACATGC

AAGTCGAGGGGCAGCATTATCTTAGCTTGCTAAGATAGATGGCGACCGG CGCACGGGTGAGTAACACGTATCCAACCTTCCGGTTACTCGGGGATAGG CTTTCGAAAGAAAGATTAATACCCGATGTTGCGTATCTTTCTCCTGAAAG ATACGCCAAAGGATTCCGGTAACCGATGGGGATGCGTTCCATTAGGCAG TTGGCGGGGTAACGGCCCACCAAACCTTCGATGGATAGGGGTTCTGAGA GGAAGGTCCCCCACATTGGAACTGAGACACGGTCCAAACTCCTACGGGA GGCAGCAGTGAGGAATATTGGTCAATGGACGGAAGTCTGAACCAGCCAA GTAGCGTGAAGGATGACTGCCCTCTGGGTTGTAAACTTCTTTTATACGGG AATAACATGAGGTACGCGTACCTTATTGCATGTACCGTTATGAATAAGCA TCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCG TTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGTGGGATATTAAGTCA GCTGTGAAAGTTTGGGGCTCAACCTTAAAATTGCAGTTGATACTGGTTTC CTTGAGTACGGTACAGGTGGGCGGAATTCGTGGTGTAGCGGTGAAATGC TTAGATATCACGAAGAACTCCGATCGCGAAGGCAGCTCACCGGGCCGG AACTGACACTGATGCTCGAAAGTGCGGGTATCAAACAGGATTAGATACC CTGGTAGTCCGCACAGTAAACGATGAATACTCGCTGTTTGCGATACACTG TAAGCGGCCAAGCGAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCG GCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGG AACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTAAA TTGCGCTGGCTTTTACCGGAAACGGTATTTTCTTCGGACCAGCGTGAAG GTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTG CCATAACGAGCGCAACCCTTATCTTTAGTTACTAACAGTTTTGCTGAGGA CTCTAAAGAGACTGCCGTCGTAAGATGCGAGGAAGGTGGGGATGACGT CAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGG GAGCACAGCAGGTTGCTACACGGCGACGTGATGCCAATCCGTAAAACTC CTCTCAGTTCGGATCGAAGTCTGCAACCCGACTTCGTGAAGCTGGACTC GCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATACGTTCCCGGGCC TTGTACACACCGCCCGTCAAGCCATGAAAGCCGGGGGTACCTGAAGTAC GTAACCGCGAGGATCGTCCTAGGGTAAACCTGGTGATTGGGGCTAAGTC GTAACAAGGTCC

Sequence Reference: 12

GAGTTTGATCATGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATG CAAGTCGAACGGGCGAATTATATCGGAGGTCTTCGGGCCGAAGAGATAA TAAGCTAGTGGCGGACGGGTGCGTAACGTGTGGGTAATCTGCCTTTGTC ATAGGAATAACTGCTTGAAAAAGTAGCTAATACCAAATAACATATCGTATA GGCATCTATAAGATATCAAAGAGAAATCGGACAAAGATGAGCCCGCATC TGATTAGCTGGTTGGTAGGGTAAAAGCCTACCAAGGCGACGATCAGTAG CCGGCCTGAGAGGGTGAACGGCCACATTGGAACTGAGACACGGTCCAA ACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGGAACC CTGATGCAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGAGTCGTAAAAC TCTGTTGTAAGGGAAGATAATGACGGTACCTTAAAAGAAAGCCCCGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCC GGAATAACTGGGCGTAAAGGGTGCGCAGGTGGTCTGTCAAGTTAGTGGT GAAAGGCATAGGCTCAACCAATGTAAGCCATTAAAACTGACGGACTTGA GTGCAGGAGAGGAAAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGA TATTAGGAGGAATACCGGTGGCGAAGGCGACTTTCTGGACTGTAACTGA CACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGAGTACTAGGTGTCGGGAGGAATCTCGGTG CCGAAGTTAACACATTAAGTACTCCGCCTGGGGAGTACGCTCGCAAGAG TAAAACCCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGT GGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAACTTGACATACCGA TGCCGATTCGGTAATGCGAATTTTCCTTTCGGGGACATTGGATACAGGTG GTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG CAACGAGCGCAACCCTTGTCAATAGTTGCCAGCATATAAGGTGGGGACT CTATTGAGACAGCTAAGGACAACTTAGAGGAAGGTGGGGATGACGTCAA ATCATCATGCCCCTTATGTTTAGGGCTACACACGTGCTACAATGGTCATT ACAGAGGGAAGCGAGATTGTGAAATGGAGCAAACCCCAAAAAGATGATC TAAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGTTGGAGTTGCTA GTAATCGCAAATCAGAATGTTGCGGTGAATGCGTTCCCGGGTCTTGTAC ACACCGCCCGTCACACCATGGGAGTTCGGGGGGCCCAAAGTCAGTGAG CAAACCGCGAGGGTGCAGCTGCCTAAGGCAAAACGAATGACTGGGGTG AAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCT T

Sequence Reference:13

(a)

GAGTTTGATTCTGGCTCAGGATGAACGCTAGCGATAGGCTTAACACATG CAAGTCGAGGGGCAGCATAAAGTCAGCTTGCTGATTTGGATGGCGACCG GCGGATGGGTGCGTAACGCGTATGCAACTTACCTGTCAGAGGGGGATA ACCCGGAGAAATTCGGACTAATACCGCATACACTTGAGATACTGCATGG TATTTCAAGGAAATATTTATAGCTGACAGATAGGCATGCGTTCCATCAGC TGGTAGGTGAGGTAACGGCTCACCTAGGCGACGATGGATAGGGGAACT GAGAGGTTAAACCCCCACACTGGTACTGAGACACGGACCAGACTCCTAC GGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCA GCCAATTCGCGTGAAGGATGACTGCCCTATGGGTTGTAAACTTCTTTAGT AGGGGATTAAAGTTTGCCTTGCGAGGCAATTTGCAAGTACCCTAAGAATA AGTATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATACG AGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGTTGCTTTTTAA GTCAGTGGTGAAAAGCTGTGGCTCAACCATAGTCTTGCCGTTGAAACTG AGGAGCTTGAGTGTAGATGCTGTAGGCGGAACGCGTAGTGTAGCGGTG AAATGCATAGATATTACGCAGAACTCCGATTGCGAAGGCAGCTTACAAAG TTACAACTGACACTGAAGCACGAGAGCGTGGGTATCAAACAGGATTAGA TACCCTGGTAGTCCACGCCGTAAACGATGATTACTAAGAGTATGCGATAT AGTGTATGTTCTACAGCGAAAGCGTTAAGTAATCCACCTGGGGAGTACG TCGGCAACGATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG AGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGAT TGAAATGTGCATGACGTAGACTGGAGACGGTTTATACCCTTCGGGGCAT GTATGTAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGG CTTAAGTGCCATAACGAGCGCAACCCACATTGTCAGTTACTAACAGGTAA AGCTGAGGACTCTGGCGAGACTGCCGGCGTAAGCCGTGAGGAAGGTGT GGATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCGACACACGTG TTACAATGGTGAGGACAAAGGGCAGCTACCTGGCGACAGGGTGCGAAT CTCCAAACCTCATCTCAGTTCGGATCGGAGTCTGCAACTCGACTCTGTGA AGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGT TCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGTCGGGAGTA CCTGAAGTACGTCACCGCGAGGATCGTACTAGGGTAATACCGGTAACTG GGGCTAAGTCGTAACAAGGTAGCTGTACCGGAAGGTGCGGCTGGAACA CCTCCTT

(b)

GAGTTTGATCCTGGCTCAGGATGAACGCTAGCGATAGGCTTAACACATG CAAGTCGAGGGGCAGCACGTATTCAGCTTGCTGAATATGGTGGCGACCG GCGGATGGGTGCGTAACGCGTATGCAACTTACCTGTCAGAGGGGGATA ACCCGGAGAAATTCGGACTAATACCGCATACACTTGAGATACTGCATGG TATTTCAAGGAAATATTTATAGCTGACAGATAGGCATGCGTTCCATTAGCT GGTAGGTGAGGTAACGGCTCACCTAGGCGACGATGGATAGGGGAACTG AGAGGTTAAACCCCCACACTGGTACTGAGACACGGACCAGACTCCTACG GGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAG CCAATTCGCGTGAAGGATGACTGCCCTATGGGTTGTAAACTTCTTTAGTA GGGGATTAAAGTTTGCCTTGCGAGGCAATTTGCAAGTACCCTAAGAATAA GTATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATACGA GCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGTTGCTTTTTAAG TCAGTGGTGAAAAGCTGTGGCTCAACCATAGTCTTGCCGTTGAAACTGA GGAGCTTGAGTGTAGATGCTGTAGGCGGAACGCGTAGTGTAGCGGTGA AATGCATAGATATTACGCAGAACTCCGATTGCGAAGGCAGCTTACAAAGT TACAACTGACACTGAAGCACGAGAGCGTGGGTATCAAACAGGATTAGAT ACCCTGGTAGTCCACGCCGTAAACGATGATTACTAAGAGTATGCGATATA ATGTATGTTCTACAGCGAAAGCGTTAAGTAATCCACCTGGGGAGTACGTC GGCAACGATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAG GAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGATTG AAATGTGCATGACGTAAATTGGAGACAGTTTATACCCTTCGGGGCATGTA TGTAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTT AAGTGCCATAACGAGCGCAACCCACATTGTCAGTTACTAACAGGTAGAG CTGAGGACTCTGGCGAGACTGCCGGCGTAAGCCGCGAGGAAGGTGTGG ATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCGACACACGTGTTA CAATGGTGAGGACAAAGGGCAGCTACCTGGCGACAGGGTGCGAATCTC CAAACCTCATCTCAGTTCGGATCGGAGTCTGCAACTCGACTCTGTGAAG CTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTC CCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGTCGGGAGTACC TGAAGTACGTCACCGCGAGGATCGTACTAGGGTAATACCGGTAACTGGG GCTAAGTCGTAACAAGGTCCA

(C)

GAGTTTGATCATGGCTCAGGATGAACGCTAGCGATAGGCTTAACACATG

CAAGTCGAGGGGCAGCATAGAGTCAGCTTGCTGATTTAGATGGCGACCG

GCGGATGGGTGCGTAACGCGTATGCAACTTACCTGTCAGAGGGGGATA ACCCGGAGAAATTCGAACTAATACCGCATATACTTGAGATGCTGCATGGT ATTTCAAGGAAATATTTATAGCTGACAGATAGGCATGCGATCCATTAGCT AGTAGGTGAGGTAACGGCTCACCTAGGCGACGATGGATAGGGGAACTG AGAGGTTAAACCCCCACACTGGTACTGAGACACGGACCAGACTCCTACG GGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAG CCAATTCGCGTGAAGGACGACTGCCCTATGGGTTGTAAACTTCTTTAGTA GGGGATTAAAGTTTGCCTTGCGAGGCAATTTGCAAGTACCCTAAGAATAA GTATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATACGA GCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGTGGCTTTTTAAG TCAGTGGTGAAAAGCTGTGGCTCAACCATAGTCTTGCCGTTGAAACTGA GGAGCTTGAGTGTAGATGCTGTAGGCGGAACGCGTAGTGTAGCGGTGA AATGCATAGATATTACGCAGAACTCCGATTGCGAAGGCAGCTTACAAAGT TACAACTGACACTGAAGCACGAGAGCGTGGGTATCAAACAGGATTAGAT ACCCTGGTAGTCCACGCCGTAAACGATGATTACTAAGAGTATGCGATATA ATGTATGTTCTACAGCGAAAGCGTTAAGTAATCCACCTGGGGAGTACGTC GGCAACGATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAG GAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGATTG AAATGTGCATGACGTAAATTGGAGACAGTTTATACCCTTCGGGGCATGTA TGTAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTT AAGTGCCATAACGAGCGCAACCCACATTGTCAGTTACTAACAGGTAGAG CTGAGGACTCTGGCGAGACTGCCGGCGTAAGCCGTGAGGAAGGTGTGG ATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCGACACACGTGTTA CAATGGTGAGGACAAAGGGCAGCTACCTGGCGACAGGGTGCGAATCTC CAAACCTCATCTCAGTTCGGATCGGAGTCTGCAACTCGACTCTGTGAAG CTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTC CCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGTCGGGAGTACC TGAAGTACGTCACCGCGAGGATCGTACTAGGGTAATACCGGTAACTGGG GCTAAGTCGTAACAAGGTCC Sequence Referenced 4

GAGTTTGATTATGGCTCAGGATGAACGCTAGCGATAGGCTTAACACATG CAAGTCGAGGGGCAGCATGAACTTAGCTTGCTAAGTTTGATGGCGACCG GCGCACGGGTGCGTAACGCGTATGCAACTTGCCTTACAGAGGGGGATA ACCCGTTGAAAGACGGACTAATACCGCATACACTTGCTTGGTTGCATGAT CGGGCAAGGAAATATTTATAGCTGTAAGATAGGCATGCGTCCCATTAGCT GGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGGGTAGGGGAACTG AGAGGTTTATCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACG GGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAG CCAAGTCGCGTGAAGGATGACTGTCCTAAGGATTGTAAACTTCTTTTATA CGGGAATAACGGGCGATACGTGTATTGCTGTGAATGTACCGTAAGAATA AGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCG AGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGTTGTTCGGTAA GTCAGCGGTGAAAACTGAGCGCTCAACGTTCAGCGTGCCGTTGAAACTG CCGGGCTTGAGTTCAGTGGCGGCAGGCGGAATTCGTGGTGTAGCGGTG AAATGCATAGATATCACGAGGAACTCCGATTGCGAAGGCAGCTTGCCAT ACTGCGACTGACACTGAAGCACGAAGGCGTGGGTATCAAACAGGATTAG ATACCCTGGTAGTCCACGCAGTAAACGATGATTACTAGGAGTTTGCGATA TACCGTCAAGCTTCCACAGCGAAAGCGTTAAGTAATCCACCTGGGGAGT ACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAG CGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGG GATTGAAATGTAGACGACGGATGGTGAAAGCCGTCTTCCCTTCGGGGCG TCTATGTAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCG GCTTAAGTGCCATAACGAGCGCAACCCACATCGGTAGTTGCTAACAGGT TTAGCTGAGGACTCTACCGAGACTGCCGTCGTAAGGCGTGAGGAAGGT GTGGATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCTACACACGT GTTACAATGGGAGGGACAAAGGGCAGCTACCGGGCGACCGGATGCGAA TCTCGAAACCCTTCCCCAGTTCGGATCGGAGTCTGCAACTCGACTCCGT GAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATAC GTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGGAGTCGGGGG TACCTGAAGGGCGTAACCGCAAGGGGCGCACTAGGGTAATACCGGTGA CTGGGGCTAAGTCGTAACAAGGTCC

Sequence Reference :15

(a)

GAGTTTGATTCTGGCTCAGAACGAACGCTGGCGGCGCGTCTTAAGCATG CAAGTCGAACGGTAAGGGAGAGCTTGCTCTCCCCTAGAGTGGCGGACT GGTGAGTAACGCGTGGGTGACCTGCCCTGAAGATGGGGATAGCTAGTG GAAATATTAGATAATACCGAATGTGCTCATTTACATAAAGGTAAATGAGG AAAGGAGCTACGGCTCCGCTTCAGGATGGGCCCGCGTCCCATTAGCTG GTTGGTGAGGTAAAGGCCCACCAAGGCAACGATGGGTATCCGGCCTGA GAGGGTGAACGGACACATTGGGACTGAGATACGGCCCAAACTCCTACG GGAGGCAGCAGCTAAGAATCTTCCGCAATGGACGAAAGTCTGACGGAG CGACGCCGTGTGAATGAAGAAGGCCGAAAGGTTGTAAAATTCTTTTGCA GATGAAGAATAAACACAGGAGGGAATGCCTGTGAGATGACGGTAGTCAT GCGAATAAGCCCCGGCTAATTACGTGCCAGCAGCCGCGGTAACACGTAA GGGGCGAGCGTTGTTCGGAATTATTGGGCGTAAAGGGTATGTAGGCGG TTAGGTAAGCCCGGTGTGAAATCTACGAGCTCAACTCGTAAACTGCATTG GGTACTGCTTGACTTGAATCACGGAGGGGAAACCGGAATTCCAAGTGTA GGGGTGGAATCTGTAGATATTTGGAAGAACACCGGTGGCGAAGGCGGG TTTCTGGCCGATGATTGACGCTGATATACGAAGGTGCGGGGAGCAAACA GGATTAGATACCCTGGTAGTCCGCACAGTAAACGATGTACACTAGGTGT CGGGGCAAGAGCTTCGGTGCCGACGCAAACGCATTAAGTGTACCGCCT GGGAAGTATGCCCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCC ACACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGAAACCT TACCTGGGTTTGACATCAAGAGCAATGACATAGAGATATGGCAGCGTAG CAATACGGCTCTTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGT GAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTACTGCCAGTTA CTAACAGGTAAAGCTGAGGACTCTGGCGGAACTGCCGATGACAAATCGG AGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGTCCAGGGCT ACACACGTGCTACAATGGTTGCTACAAATCGAAGCGACACCGCGAGGTC AAGCAAAACGCAAAAAAGCAATCGTAGTCCGGATTGAAGTCTGAAACTC GACTTCATGAAGTTGGAATCGCTAGTAATCGCACATCAGCACGGTGCGG TGAATACGTTCCTGGGCCTTGTACACACCGCCCGTCACACCATCCGAGT CGAGGGTACCCGAAGTCGCTAGTCTAACCCGTAAGGGAGGACGGTGCC GAAGGTACGTTTGGTAAGGAGGGTGAAGTCGTAAC

(b)

GAGTTTGATTCTGGCTCAGAACGAACGCTGGCGGCGCGTCTTAAGCATG CAAGTCGAACGGTAAGGGAGAGCTTGCTCTCCCCTAGAGTGGCGGACT GGTGAGTAACGCGTGGGTGACCTGCCCTGAAGCTGGGGATAGCTAGTA GAAATATTAGATAATACCGAATGTGCTCATTTACATAAAGGTAAATGAGG AAAGGAGCTACGGCTCCGCTTCAGGATGGGCCCGCGTCCCATTAGCTA GTTGGTGAGGTAAAGGCCCACCAAGGCAACGATGGGTATCCGGCCTGA GAGGGTGAACGGACACATTGGGACTGAGATACGGCCCAAACTCCTACG GGAGGCAGCAGCTAAGAATCTTCCGCAATGGACGAAAGTCTGACGGAG CGACGCCGTGTGAATGAAGAAGGCCGAAAGGTTGTAAAATTCTTTTGCA GATGAAGAATAAACACAGGAGGGAATGCCTGTGAGATGACGGTAGTCAT GCGAATAAGCCCCGGCTAATTACGTGCCAGCAGCCGCGGTAACACGTAA GGGGCGAGCGTTGTTCGGAATTATTGGGCGTAAAGGGTATGTAGGCGG TTGGGTAAGCCCGGTGTGAAATCTACGAGCTCAACTCGTAAACTGCATT GGGTACTGCTTGACTTGAATCACGGAGGGGAAACCGGAATTCCAAGTGT AGGGGTGGAATCTGTAGATATTTGGAAGAACACCGGTGGCGAAGGCGG GTTTCTGGCCGATGATTGACGCTGATATACGAAGGTGCGGGGAGCAAAC AGGATTAGATACCCTGGTAGTCCGCACAGTAAACGATGTACACTAGGTG TCGGGGCAAGAGCTTCGGTGCCGGCGCAAACGCATTAAGTGTACCACCT GGGAAGTATGCCCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCC ACACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGAAACCT TACCTGGGTTTGACATCAAGAGCAATGACATAGAGATATGGCAGCGTAG CAATACGGCTCTTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGT GAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTACTGCCAGTTA CTAACAGGTAAAGCTGAGGACTCTGGCGGAACTGCCGATGACAAATCGG AGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGTCCAGGGCT ACACACGTGCTACAATGGTTGCTACAAATCGAAGCGACACCGCGAGGTC AAGCAAAACGCAAAAAAGCAATCGTAGTCCGGATTGAAGTCTGAAACTC GACTTCATGAAGTTGGAATCGCTAGTAATCGCACATCAGCACGGTGCGG TGAATACGTTCCTGGGCCTTGTACACACCGCCCGTCACACCATCCGAGT CGAGGGTACCCGAAGTCGCTAGTCTAACCCGTAAGGGAGGACGGTGCC GAAGGTACGTTTGGTAAGGAGGGTGAAGTCGTAAC

Sequence Reference:16 (a)

GAGTTTGATCATGGCTCAGGATGAACGCTAGCGATAGGCTTAACACATG CAAGTCGAGGGGCAGCATGGTCTTAGCTTGCTAAGACTGATGGCGACCG GCGCACGGGTGCGTAACGCGTATGTAACTTGCCTGATAGAAAGGGATAA CCCGGTGAAAGTCGGACTAATACCTTATGGTCTTGGGTTATTGCATGATG ATTCAAGTAAAGATTAATTGCTATCAGATAGGCATGCGTTCCATTAGTTAG TTGGTGAGGTAACGGCTCACCAAGGCGACGATGGATAGGGGAACTGAG AGGTTTATCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGG AGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAGCC ACGTCGCGTGAAGGAAGACGGTTCTATGGATTGTAAACTTCTTTTATAGT GGATTAAAGTTATCCACGCGTGGATATTTGCAAGTACCCTATGAATAAGC ATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGC GTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGTGGCCTGATAAGTC GGGGGTGAAAAGCTGTGGCTCAACCACAGTCTTGCCTTCGAAACTGTTT GGCTTGAGTATAGATGAAGTAGGCGGAATTTGTGGTGTAGCGGTGAAAT GCATAGATATCACGAGGAACTCCGATTGCGAAGGCAGCTTACTAAGTTAT GACTGACACTGAAGCACGAAAGCGTGGGTATCAAACAGGATTAGATACC CTGGTAGTCCACGCAGTAAACGATGATAACTGGGCGTATGCGATATACA GTATGCTCCTGAGCGAAAGCGTTAAGTTATCCACCTGGGGAGTACGCCG GCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGG AACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGATTGAA ATTTAGCGGACTATGTATGAAAGTACATATCCTGTCACAAGGCCGCTAAG TAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAA GTGCCATAACGAGCGCAACCCACGTTGTCAGTTACTATCGGGTAAAGCC GAGGACTCTGACAAGACTGCCGTCGTAAGGCGCGAGGAAGGTGTGGAT GACGTCAAATCAGCACGGCCCTTACATCCGGGGCGACACACGTGTTACA ATGGTGGGGACAAAGGGCAGCTACTTGGTGACAAGATGCGAATCTCCAA ACCCCATCCCAGTTCGGATCGTAGTCTGCAACTCGACTATGTGAAGCTG GATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTCCCG GGCCTTGTACACACCGCCCGTCAAGCCATGGGAGTCGGGAGTACCTAA AGCACGTAACCGCGAGGGGCGTGTTAGGGTAATACCGGTGACTGGGGC TAAGTCGTAACAAGGTCC (b)

TGAGAGTTTGATTCTGGCTCAGGATGAACGCTAGCGATAGGCTTAACACA TGCAAGTCGAGGGGCAGCATGGTCTTAGCTTGCTAAGACTGATGGCGACC GGCGCACGGGTGCGTAACGCGTATGTAACTTGCCTGATAGAAAGGGATAA CCCGGTGAAAGTCGGACTAATACCTTATGGTCTTGGGTTATTGCATGATGA TTCAAGTAAAGATTAATTGCTATCAGATAGGCATGCGTTCCATTAGTTAGT TGGTGAGGTAACGGCTCACCAAGGCGACGATGGATAGGGGAACTGAGAG GTTTATCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGG CAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAGCCACGTC GCGTGAAGGAAGACGGTTCTATGGATTGTAAACTTCTTTTATAGGGGATT AAAGTTATCCACGCGTGGATATTTGCAAGTACCCTATGAATAAGCATCGG CTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCGTTATCC GGATTTATTGGGTTTAAAGGGTGCGTAGGTGGCCTGATAAGTCGGGGGTG AAAAGCTGTGGCTCAACCACAGTCTTGCCTTCGAAACTGTTTGGCTTGAGT ATAGATGAAGTAGGCGGAATTTGTGGTGTAGCGGTGAA ATGC ATAGATAT CACGAGGAACTCCGATTGCGAAGGCAGCTTACTAAGTTATGACTGACACT GAAGCACGAAAGCGTGGGTATCAAACAGGATTAGATACCCTGGTAGTCCA CGCAGTAAACGATGATAACTGGGCGTATGCGATATACAGTATGCTCCTGA GCGAAAGCGTTAAGTTATCCACCTGGGGAGTACGCCGGCAACGGTGAAAC TCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAAT TCGATGATACGCGAGGAACCTTACCCGGGATTGAAATTTAGCGGACTATG TATGAAAGTACATATCCTGTCACAAGGCCGCTAAGTAGGTGCTGCATGGT TGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGC AACCCACGTTGTCAGTTACTATCGGGTAAAGCCGAGGACTCTGACaAGACT GCCGTCGTAAGGCGCGAGGAAGGTGTGGATGACGTCAAATCAGCACGGC CCTTACATCCGGGGCGACACACGTGTTACAATGGTGGGGACAAAGGGCAG CTACTTGGTGACAAGATGCGAATCTCCAAACCCCATCCCAGTTCGGATCGT AGTCTGCAACTCGACTATGTGAAGCTGGATTCGCTAGTAATCGCGCATCA GCCATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAA GCCATGGGAGTCGGGAGTACCTAAAGCACGTAACCGCGAGGGGCGTGTTA GGGTAATACCGGTGACTGGGGCTAAGTCGtAACAAGGTAGCCGTACCGGA AGGTGCGGCTGGAACACCTCCTTAA

Sequence Reference:17

GAGTTTGATTCTGGCTCAGAACGAACGCTGGCGGCGCGTCTTAAACATG CAAGTCGAACGGCAAGAGAGAAGCTTGCTTTTCTCCTAGAGTGGCGGAC TGGTGAGTAACACGTGGGTGACATACCTTTTGGCTGGGGATAGCTGTTA GAAATAGCAGGTAATACCGAATGTGACCGCATCTGTTAGAGAGGTGCGA GGAAAGGAGCTAAGGCTCCGCCAGAAGAATGGCTCGCGGCCCATTAGC TTGTTGGTGAGGTAACGGCCCACCAAGGCAATGATGGGTATCCGGCCTG AGAGGGTGAACGGACACATTGGGACTGAGATACGGCCCAGACTTCTAC GGGAGGCAGCAGTTAAGAATATTCCGCAATGGACGAAAGTCTGACGGAG CGACGCCGCGTGGATGAAGAATGCCGAAAGGTTGTAAAATCCTTTTAAG CCTGAGGAATAAGCGGAGGAGGGAGTGCCTCTGCGGTGACTGTAGGGC TTGAATAAGCAACGGCTAATTACGTGCCAGCAGCCGCGGTAACACGTAA GTTGCGAGCGTTGTTCGGAATTATTGGGCGTAAAGGGCATGTAGGCGGA TATGCAAGCTTGGTGTGAAATACTGCAGCTTAACTGCGGAACTGCATTGA GAACTGCGCATCTTGAATTACTGAAGGGTAACCAGAATTCCACGTGTAG GGGTGAAATCTGTAGATATGTGGAAGAATACCAATGGCGAAGGCAGGTT ACCGGCAGATAATTGACGCTGAGGTGCGAAAGTGCGGGGAGCGAACAG GATTAGATACCCTGGTAGTCCGCACCGTAAACGATGTACACTAGGTGTC CGGCGTTGAAGCTGGGTGCCAAAGCAAACGTGATAAGTGTACCGCCTG GGGAGTATGCCCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCCG CACAAGCGGTGGAGCATGTGGTTTAATTCGATGGTACGCGAGGAACCTT ACCTGGGTTTGACATAGTATCTGATGCCGTAGAGATACGGCAGCGTAGC AATACGAGGTACAACAGGTGCTGCATGGCTGCCGTCAGCTCGTGCCGTG AGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTACTGCCAGTTAC TAACAGGTAAAGCTGAGGACTCTGGCGGAACTGCCGGTGACAAACCGG AGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTATGTCCAGGGCT ACACACGTGCTACAATGGTAGGAACAAAGTGAGGCGAAGCTGTAAAGCG GAGCAAAACGCAAAAAAACTATCGTAGTCCGGATTGGAGTCTGAAACTC GACTCCATGAAGTTGGAATCGCTAGTAATCGCGCATCAGCACGGCGCGG TGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATCCGAGT AGAGGGTACCCGAAGTCGGTAGTCTAACCGCAAGGAGGACGCTGCCGA AGGTATGTTTTGTAAGGGGGGTGAAGTCGTAAC.

Claims

Claims

1. A method of identifying or predicting periodontal disease in a dog, the method comprising identifying the presence or absence of at least one micro-organism, from a sample from the mouth of a dog, wherein the micro-organism which is associated with periodontal disease in a dog is one or more disease-associated micro-organisms from: Peptostreptococcus sp., Synergistes sp., Clostridials sp., Eubacterium nodatum, Selenomonas sp., Bacteroidetes sp., "Odoribacter denticanis", Desulfomicrobium ovale, Moraxella sp., Bacteroides denticanoris, Fillif actor villosus, Porphyromonas canons, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa.

2. The method as claimed in claim 1, wherein the micro-organism associated with periodontal disease is one or more micro-organisms comprising: a 16S rRNA gene sequence more than 98% identical to sequence reference:1, a 16S rRNA gene sequence more than 96% identical to sequence reference:2, a 16S rRNA gene sequence more than 94% identical to sequence reference:3, a 16S rRNA gene sequence more than 99% identical to sequence reference:4, a 16S rRNA gene sequence more than 98% identical to sequence reference:5, a 16S rRNA gene sequence more than 92% identical to sequence reference:6 or a 16S rRNA gene sequence more than 96% identical to sequence reference:7 or a 16S rRNA gene sequence more than 87% identical to sequence reference:8 or a 16S rRNA gene sequence more than 99% identical to sequence reference:9, or a 16S rRNA gene sequence more than 92% identical to sequence reference: 10 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 11 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 12 or a 16S rRNA gene sequence more than 98% identical to sequence reference: 13 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 14 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 15 or a 16S rRNA gene sequence more than 98% identical to sequence reference: 16.

3. The method, as claimed in claim 1 or claim 2, wherein the presence or absence of the micro-organism which is associated with periodontal disease is identified by one or more of bacterial culture or by a detector for one or more of nucleic acid, peptide, carbohydrate or lipid or by amplification of nucleic acid of the microorganism or by biochemical or phenotypic profiling.

4. The method, as claimed in claim 3, wherein amplication of nucleic acid of the micro-organism is carried out before identification with a nucleic acid detector.

5. A method, as claimed in any one of claims 1 to 4, wherein the method comprises identifying the presence or absence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 micro-organisms from the list.

6. A micro-organism which is associated with periodontal disease in a dog and which comprises a 16S rRNA gene sequence which is more than 98% identical to sequence reference: 1 or a 16S rRNA gene sequence which is more than 96% identical to sequence referenced or a 16S rRNA gene sequence which is more than 94% identical to sequence reference:3 or a 16S rRNA gene sequence which is more than 99% identical to sequence referenced or a 16S rRNA gene sequence which is more than 98% identical to sequence reference:5 or a 16S rRNA gene sequence which is more than 92% identical to sequence referenced or a 16S rRNA gene sequence which is more than 96% identical to sequence reference:7 or a 16S rRNA gene sequence which is more than 87% identical to sequence reference:8 or a 16S rRNA gene sequence which is more than 99% identical to sequence referenced or a 16S rRNA gene sequence which is more than 92% identical to sequence reference: 10 or a 16S rRNA gene sequence which is more than 99% identical to sequence reference: 11 or a 16S rRNA gene sequence which is more than 99% identical to sequence reference: 12 or a 16S rRNA gene sequence which is more than 98% identical to sequence reference: 16.

7. A method as claimed in claim 1, the method comprising identifying one or more micro-organisms as claimed in claim 6.

8. A probe comprising at least 80% identity with at least 10 sequential residues from any of the sequences in the table below or any of the probe sequences in Appendix 2, not including the poly T tail, which have a given reference number, or a probe of at least 10 residues which hybridises to any sequence in the table below or any of the probe sequences in Appendix 2, not including the poly T tail, which have a given reference number, under the hybridisation conditions of pre- washing solution 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridisation conditions of 40-75°C, 5 x SSC overnight.

Table A

9. Use of a probe, as claimed in claim 8, for identifying or predicting periodontal disease in a dog.

10. A kit for identifying or predicting periodontal disease in a dog, the kit comprising an agent to determine the presence or absence of at least one micro-organism, from a sample from the mouth of the dog, wherein the micro-organism which is associated with periodontal disease in a dog is one or more disease-associated micro-organisms from Peptostreptococcus sp., Synergistes sp., Clostridials sp., Eubacterium nodatum, Selenomonas sp., Bacteroidetes sp., " Odoribacter denticanis" ,r Desulfomicrobium ovale, Moraxella sp., Bacteroides denticanoris, Fillifactor villosus, Porphyromonas canoris, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa.

11. A kit, as claimed in claim 10, wherein the micro-organism which is associated with periodontal disease is one or more micro-organisms comprising a 16S rRNA gene sequence more than 98% identical to sequence reference:1, a 16S rRNA gene sequence more than 96% identical to sequence reference:2, a 16S rRNA gene sequence more than 94% identical to sequence reference:3, a 16S rRNA gene sequence more than 99% identical to sequence reference:4, a 16S rRNA gene sequence more than 98% identical to sequence reference:5, a 16S rRNA gene sequence more than 92% identical to sequence reference:6 or a 16S rRNA gene sequence more than 96% identical to sequence reference:7 or a 16S rRNA gene sequence more than 87 % identical to sequence reference:8, a 16S rRNA gene sequence more than 99% identical to sequence reference:9, or a 16S rRNA gene sequence more than 92% identical to sequence reference: 10 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 11 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 12 or a 16S rRNA gene sequence more than 98% identical to sequence reference: 13 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 14 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 15 or a 16S rRNA gene sequence more than 98% identical to sequence reference: 16.

12. A kit, as claimed in claim 10 or claim 11, wherein the kit comprises an agent to determine the presence or absence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of the micro-organisms from the list.