WO2008137541A2 - Dog plaque health - Google Patents

Abstract

The invention is directed to the identifying or predicting periodontal health 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 is associated with periodontal health.

Description

This application claims priority to UK Patent Application No. 0708424.7 filed May 1, 2007; UK Patent Application No. 0711127.1 filed June 8, 2007; and 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 the periodontal health of a dog, as well as to novel microbes which are associated with the periodontal health of 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 health in a dog as well as novel microbial strains.

Accordingly, the present invention provides a method of identifying or predicting periodontal health 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 is associated with periodontal health. 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 health.

An example of suitable conditions is given in the example section herein. The method may identify the micro-organism which is associated with periodontal health in a dog being one or more health-associated micro-organisms from: Moraxella sp., Frigovirgula sp., Neisseria sp., Pasturellaceae sp., or Helcococcus sp.

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 26. 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 health in a dog may be one or more micro-organisms comprising: a 16S rRNA gene sequence more than 92% identical or more than 96% identical to sequence referenced, a 16S rRNA gene sequence more than 89% identical to sequence referenced, a 16S rRNA gene sequence more than 95% identical to sequence reference:7, a 16S rRNA gene sequence more than 94% identical to sequence referenced, or a 16S rRNA gene sequence more than 93% identical to sequence reference: 10.

The method of the first aspect of the invention includes identifying the presence or absence of the micro-organism which is associated with periodontal health 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 health-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 one, two, three, four or five micro-organisms from table 1 having the references 2, 6, 7, 8 or 10. For all aspects of the invention, identification of the presence of the health-associated micro-organism Moraxella sp. , in particular having a 16S rRNA gene sequence more than 92% identity or more than 96% identity with sequence referenced, is useful for UK and/or European populations of dogs.

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

A second aspect of the invention relates to a method of identifying a micro-organism which reflects or predicts periodontal health in a dog, the method comprising identifying the presence of a particular micro-organism in the mouth of a dog and also identifying that the dog has good periodontal health or that its healthy periodontium/good periodontal health will be maintained or will improve.

A third aspect of the invention provides a micro-organism (preferably isolated) which is associated with periodontal health in a dog and which comprises a 16S rRNA gene sequene which is more than 92% identical or more than 96% identical to sequence referenced or a 16S rRNA gene sequence which is more than 89% identical to sequence referenced or a 16S rRNA gene sequence which is more than 95% identical to sequence reference:7 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 93% identical to sequence reference: 10.

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 fourth 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 one of the probe sequences in Appendix 2, not including the poly T tail, with a reference number 2, 6, 7 or 8 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 the probe sequences in Appendix 2, not including the poly T tail, with a reference number 2, 6, 7 or 8 under the hybridization conditions of for example, prewashing solution 5 x SSC, 0.5% SDS, 1.OmM EDTA (pH 8.0) and hybridization conditions of 40-75°C, 5 x SSC overnight. A higher stringency involves the same hybridisation conditions at 50-75⁰C. 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.

The probe sequences according to the fourth 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 Frigovirgula sp. is reference number 6 in Table 1. The probe sequences which can be used to identify this micro-organism are also probe sequences numbered 6.

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

Preferably, one, two, three, four or five different probe sequences are used. Most preferably, these one, two, three, four or five 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 health. The fifth 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 the periodontal health in a dog.

A sixth aspect of the invention relates to a kit for identifying or predicting periodontal health 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 health. 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 or five probes of the fourth 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 preferably designed to identify a micro-organism which is associated with periodontal health in a dog being one or more health-associated micro-organisms from: Moraxella sp., Frigovirgula sp., Neisseria sp., Pasturellaceae sp. or Helcococcus sp.. Most preferably, the agent is designed to identify a micro-organism which is associated with periodontal health being one or more micro-organisms comprising: a 16S rRNA gene sequence more than 92% identical or more than 96% identical to sequence referenced, a 16S rRNA gene sequence more than 89% identical to sequence referenced, a 16S rRNA gene sequence more than 95% identical to sequence reference:7, a 16S rRNA gene sequence more than 94% identical to sequence reference:8, or a 16S rRNA gene sequence more than 93% identical to sequence reference: 10.

A seventh aspect of the invention provides a method which provides information on the periodontal health status of 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 is associated with periodontal health and comprising identifying the presence or absence of one or more micro-organisms from a sample from the mouth of said dog, wherein the micro-organism is associated with periodontal disease. In such a method the presence or absence of at least one micro-organism associated with health is identified and the presence or absence of at least one micro-organism associated with disease is determined.

In accordance with the seventh aspect of the invention, the method may seek the identification of one micro-organism associated with health, together with one, two, three, four, five or more micro-organisms associated with disease. Alternatively, the method may seek the identification of two micro-organisms associated with health, together with one, two, three, four or more micro-organisms associated with disease. Alternatively the method may seek the identification of three micro-organisms associated with health, together with one, two, three or more micro-organisms associated with disease. Alternately, the method may seek the identification of four micro-organisms associated with health, together with one, two or more micro-organisms associated with disease. Alternatively, the method may seek the identification of five micro-organisms associated with health, together with one or more micro-organisms associated with disease.

Preferably, the micro-organism associated with periodontal health is one or more health-associated micro-organisms from: Moraxella sp., Frigovirgula sp., Neisseria sp., Pasturellaceae sp. or Helcococcus sp.. Preferably, the micro-organism associated with periodontal disease is one or more disease-associated micro-organisms from: Peptostreptococcus sp., Synergistes, Clostridials sp., Eubacterium nodatum, Selenomonas sp., Bacteroidetes, "Odoribacter denticanis", Desulfomicrobium orale, Moraxella sp., Bacteroides denticanoris, Fillifactor villosus, Porphyromonas canoris, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa.

Even more preferably, the micro-organism which is associated with periodontal health is one or more micro-organism comprising: a 16S rRNA gene sequence more than 92% identical or more than 96% identical to sequence referenced, a 16S rRNA gene sequence more than 89% identical to sequence reference:6, a 16S rRNA gene sequence more than 95% identical to sequence reference:7, or a 16S rRNA gene sequence more than 94% identical to sequence referenced or a 16S rRNA gene sequence more than 93% identical to sequence reference: 10 and/or wherein the micro-organisms associated with periodontal disease is one or more micro-organisms comprising: a 16S rRNA gene sequence more than 98% identical to sequence reference:l l, a 16S rRNA gene sequence more than 96% identical to sequence reference: 12, a 16S rRNA gene sequence more than 94% 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 98% identical to sequence reference: 15, a 16S rRNA gene sequence more than 92% identical to sequence reference: 16 or a 16S rRNA gene sequence more than 96% identical to sequence reference: 17 or a 16S rRNA gene sequence more than 87% identical to sequence reference: 18 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 19 or a 16S rRNA gene sequence more than 92% identical to sequence reference:20 or a 16S rRNA gene sequence more than 99% identical to sequence referenced 1 or a 16S rRNA gene sequence more than 99% identical to sequence reference :22 or a 16S rRNA gene sequence more than 98% identical to sequence reference:23 or a 16S rRNA gene sequence more than 99% identical to sequence reference:24 or a 16S rRNA gene sequence more than 99% identical to sequence reference:25 or a 16S rRNA gene sequence more than 98% identical to sequence reference:26.

The method of the seventh aspect of the invention includes identifying the presence or absence of the micro-organism which is associated with periodontal health and periodental 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 micro-organism. 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 health-associated micro-organism or a disease-associated micro-organism 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).

In accordance with identifying micro-organisms associated with disease, it is useful to have probe sequences which can be used to identify particular microbes and/or particular strains or sub-types. The following Table B and Appendix 2, probe references 11 to 26 inclusive provides probe sequences which can be used to identify the micro-organisms indicated, which micro-organisms or strains are associated with periodontal disease in a dog.

The probes comprises at least 80% or at least 90% identity with at least 10 sequential residues (or at least 11, 12, 13, 14, 15, 16 residue in length) from any one of the sequences in Table B or from any one of the probe sequences in Appendix 2 with references 11 to 26 inclusive, without the poly T tail 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 B or the probe sequences in Appendix 2, not including the poly T tail, with a reference number 11 to 26 inclusive under the hybridization conditions of for example, prewashing solution 5 x SSC, 0.5% SDS, 1.OmM EDTA (pH 8.0) and hybridization conditions of 40-75°C, 5 x SSC overnight. A higher stringency involves the same hybridisation conditions at 50-75⁰C. The probes may be nucleic acid or any other probes which have the per cent identity defined above or are capable of binding to nucleic acid.

Table B

As for Table A above, the reference numbers used correspond to the species which the probe identifies.

Any one or more of the probe sequences described herein can be used in accordance with any aspect of the invention.

In accordance with the seventh aspect of the invention the method may comprise identifying the presence or absence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 micro-organisms from the list.

Preferably, the seventh aspect of the invention includes the identification of one, two, three, four or five micro-organisms from table 1 having references 2, 6, 7, 8 and/or 10 and includes the identification of one, two, three, four, five, six or seven disease-associated microbes from the list of Desulfomicrobium orale, Eubacterium nodatum, Clostridiales sp., "Odoribacter denticanis ", Synergistes sp., Selenomonas sp., Peptostreptococcus sp., Bacteroidetes sp., Moraxella sp., Bacteroides denticanoris, Fillifactor villosus, Porphyromonas canons, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa.

An eighth aspect of the invention provides a kit suitable for providing information on the periodontal health status of 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 a dog, wherein the micro-organism is associated with periodontal health and comprising an agent to determine the presence or absence of at least one micro-organism from a sample from the mouth of said dog, wherein the micro-organism is associated with periodontal disease. All aspects of the kit according to the sixth aspect of invention apply to this kit according to the eighth aspect of the invention. In particular, the micro-organism associated with periodontal health may be one or more health-associated micro-organisms from: Moraxella sp., Frigovirgula sp., Neisseria sp., Pasturellaceae sp., or Helcococcus sp. Furthermore, the micro-organism associated with periodontal disease is one or more disease-associated micro-organisms from: Peptostreptococcus sp., Synergistes, Clostridiales sp., Eubacterium nodatum, Selenomonas sp., Bacteroidetes, "Odoribacter denticanis", Desulfomicrobium orale, Moraxella sp., Bacteroides denticanoris, Fillifactor villosus, Porphyromonas canoris, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa.

More preferably the micro-organism which is associated with periodontal health is one or more micro-organism comprising: a 16S rRNA gene sequence more than 92% identical or more than 96% identical to sequence referenced, a 16S rRNA gene sequence more than 89% identical to sequence reference :6, a 16S rRNA gene sequence more than 95% identical to sequence reference:7, a 16S rRNA gene sequence more than 94% identical to sequence referenced, or a 16S rRNA gene sequence more than 93% identical to sequence reference: 10 and/or the micro-organism associated with periodontal disease may be one or more micro-organisms comprising: a 16S rRNA gene sequence more than 98% identical to sequence reference:l l, a 16S rRNA gene sequence more than 96% identical to sequence reference: 12, a 16S rRNA gene sequence more than 94% 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 98% identical to sequence reference: 15, a 16S rRNA gene sequence more than 92% identical to sequence reference: 16, a 16S rRNA gene sequence more than 96% identical to sequence reference: 17, a 16S rRNA gene sequence more than 87% identical to sequence reference: 18, a 16S rRNA gene sequence more than 99% identical to sequence reference: 19, a 16S rRNA gene sequence more than 92% identical to sequence reference:20, a 16S rRNA gene sequence more than 99% identical to sequence referenced 1, a 16S rRNA gene sequence more than 99% identical to sequence reference:22, a 16S rRNA gene sequence more than 98% identical to sequence reference:23, a 16S rRNA gene sequence more than 99% identical to sequence reference:24, a 16S rRNA gene sequence more than 99% identical to sequence reference:25 or a 16S rRNA gene sequence more than 98% identical to sequence reference:26.

In accordance with the eighth 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, 16, 17, 18, 19, 20 or 21 of the micro-organisms from the list.

In accordance with the method and kit according to the seventh and eighth aspects of the invention, the probes as listed in Table B or in Appendix 2 (with references 11 to 26 inclusive) with or without the poly T tails may be used.

In accordance with the present invention there is also disclosed a ninth aspect of the invention which provides methods, kits and probes which identify or predict periodontal health in a dog, by identifying the presence or absence of one, two, three, four, five or more micro-organisms associated with periodontal disease. The method, kit and probe details disclosed herein in relation to micro-organisms associated with periodontal disease of the ninth aspect can be applied to identification of periodontal disease in the absence (optionally) of also identifying the presence or absence of identifying micro-organisms associated with periodontal health. In particular, the probe sequences given in Table B or in Appendix 2 (with references 11 to 26 inclusive) with or without the poly T tails can be used to identify the presence or absence of micro-organisms associated with periodontal disease (optionally in the absence of identifying micro-organisms associated with periodontal health). The probes may be used individually or in a combination of two, three, four, five or six. The methods, kits and probes according to the present invention can all be utilised such that, in addition to the micro-organisms listed in the claims, any one or more micro-organisms for the following genuses can also be identified: Treponema sp., Moraxella sp., Granulicatella sp., Eubacterium sp., Peptostreptococcus sp., Porphyromonas gulae, Porphyromonas cangingivalis or Porphyromonas canis.

The enclosed text includes a list of 26 sequences, of which 5 are 16S rRNA sequences of health-associated micro-organisms (sequences 2, 6, 7, 8 and 10) and 16 of which are disease-associated micro-organisms (sequences 11 to 26). According to the classification of these micro-organisms, the micro-organisms from which the 16S rRNA sequences were obtained, as follows:

sequence reference: 1 Eubacterium sp. sequence reference: 2 Moraxella sp. sequence reference: 3 Moraxella sp. sequence reference: 4 Peptostreptococcaceae sp. sequence reference: 5 Granulicatella sp. sequence reference: 6 Frigovirgula sp. sequence reference: 7 Neisseria sp. sequence reference: 8 Pasteurellaceae sp. sequence reference: 9 Porphyromonas gulae sequence reference: 10 Helcococcus sp. sequence reference: 11 Peptostreptococcus sp. sequence reference: 12 Synergistes sp. sequence reference: 13 Clostridales sp. sequence reference: 14 Eubacterium nodatum sequence reference: 15 Selenomonas sp. sequence reference: 16 Bacteroidetes sp. sequence reference: 17 Odoribacter denticanis sequence reference: 18 Synergistes sp. sequence reference: 19 Desulfomicrobium orale sequence reference :20 Moraxella sp. sequence reference :21 Bacteroides denticanoris sequence reference:22 Fillifactor villosus sequence reference:23 Porphyromonas canons sequence reference:24 Porphyromonas gulae sequence reference :25 Treponema denticola sequence reference:26 Porphyromonas salivosa

In addition, this text contains a sequence reference:27 which is the 16S rRNA sequence sequence from a Treponema species.

In the present text, reference to Porphyromonas saliva should be read to include a Porphyromonas species which is or is more than or less than 1.5% divergence from Porphrymonas 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 health-associated micro-organism or for a combination of micro-organisms (including at least one health-associated micro-organism, 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 from the Waltham Centre for Pet Nutrition (WCPN).

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 5OmM Tris (pH 7.6), ImM EDTA (pH 8.0), 0.5% Tween 20 and was immediately stored at -2O⁰C 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 FOl (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 94⁰C for 45 seconds, annealing at 5O⁰C for 45 seconds and amplicon elongation at 72⁰C for 90 seconds. Following amplification for 30 cycles a final 15 min elongation step was included at

72⁰C.

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 5OmM 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 (Ml 3 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 phylogenetic 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.

The following probes (Table C) can also be used to identify the presence or absence of the micro-organisms listed in Table 1 (with the reference numbers used in Table C below corresponding to the reference numbers used in Table 1 :

Table C

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. Ten species found to be associated with the healthy 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 health in the presence of the organism.

Table 1

* 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 ten organisms in Table 1 were above detection levels in a greater percentage of the healthy population compared with the disease population and hence showed a statistically significant association with the healthy 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 health status of the animal. The reciprocal of the odds ratios for health associated species was determined to give a measure of the likelihood of the animal being healthy based on the presence of the taxa. Odds ratios for the organisms identified as bacterial indicators of health ranged between 0.03 and 0.38 giving predictions of health of between 2.63 and 33.33 times greater than if the taxon were absent.

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.

A Granulicatella species was strongly associated with the healthy state. This represents the first demonstration of the Gram-positive Streptococcus-like Granulicatella sp. being associated with the healthy periodontum. The genus Frigovirgula is largely undescribed, with only a single known species within the genus. The type species is a catalase-negative obligate anaerobe and hence it seems that not all subgingival anaerobes are involved in or even indicative of the disease process. A Neisseria species was also an indicator of health. This species is known to colonise the upper respiratory tract in dogs. However, this organism has not previously been associated with oral health. The novel Eubacterium species identified provides an interesting comparison to human periodontal disease where Eubacterium species have been linked to periodontitis. Other organisms associated with health were also identified, with a species from the family Pasteurellaceae an example of an organism previously cultured from the oral microflora of cats and dogs, but not previously recognised as playing a role in periodontal health.

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 orale were also associated with disease, suggesting higher levels of these organisms in the disease state.

Examples 2 and 3

A previous study in UK dogs (Example 1) 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. Of the bacterial species associated with health, seven including Granulicatella and Frigovirgula species were strongly indicative of a healthy oral cavity and five were more weakly associated with periodontal health.

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. Twenty healthy animals were recruited from the same veterinary practise during treatment for other veterinary procedures requiring anaesthetic including extractions of fractured teeth and orthopaedic treatments. 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. For inclusion in the healthy group clinically healthy gingiva were required with no or only low levels of localised gingivitis. Where such gingivitis was present this was at locations away from the sampling sites. Animals under the age of 2 were not sampled in order to avoid exaggerating the age bias between healthy and diseased groups, a common problem in studies of diseases in which incidence and severity progresses with age.

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. Although breed matching was not possible, healthy dogs and sampling sites were selected to reflect those taken from the disease group. USA study (Example 3)

Animals were recruited from 9 veterinary hospitals in the USA. A single sampling technician collected gingival margin and supragingival plaque samples from 40 healthy and 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. Dogs with clinically healthy gingiva were sampled similarly, however plaque was collected from 8 sites (in the majority of cases 4 canines, 2 lower 1st molars and 2 upper 4th premolars depending on gingival score) to target sites most often affected in the disease group and allow sufficient plaque to be collected in the absence of periodontal pockets.

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 5OmM 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 and 8 in the healthy 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 5OmM 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.

p g C

ntitis Positive Animals (Diseased) o >a

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. 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.physics.csbsju.edu/stats/fisher.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 V 12) of the reactivity of the dogs from healthy and 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 was 7.4 years (stdev 4.0) with a disease group mean of 10 years (stdev 3.4) and a mean of 4.6 years (stdev 2.5) in the healthy animals. Mean periodontal score in the diseased group was 3.63 (stdev 0.47), with all healthy dogs scoring 0. 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 23 in the present invention); Bacteroides denticanoris (reference 21 in the present invention) and an unspeciated Treponema having 16S RNA sequence as set out in sequence reference:27.

Presence or absence of the target species was achieved within subgingival plaque samples from 19 of the animals in each group. 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 while 2 were associated with health. 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=1.94xlO^"7; Table 2). The novel Peptostreptococcus species (reference 11) 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=O.0002). Porphyomonas salivosa (P. macacae) and the Synergistes species detectable by probes 12a to 12f in Table B and/or comprising a 16S rRNA sequence more than 96% identical to sequence reference: 12 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 18a to 18j in table B and/or comprising a 16S rRNA sequence more than 87% identical to sequence reference: 18 was absent in all of the samples from healthy dogs but present in 8 diseased dogs (P=O.0031). Several other organisms were also associated significantly (but less strongly) with disease including Fillifactor villosus; Treponema denticola; Porphyromonas canons; a novel Clostridials species; Desulfomicrobium orale and Bacteroides denticanoris. The unspeciated Frigovirgula sp. was significantly associated with health being present in 6 of the subgingival plaque samples from healthy animals and showing an absence of detection in disease (P=0.0197). Likewise the novel Pasteurellaceae was also associated with the healthy periodontum (P=0.0217). 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 28 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. Supragingival 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 28 target organisms analysed, 16 were significantly associated with the diseased periodontum in the USA population while 3 were linked to the healthy state (Table 3).

The probe most strongly indicative of disease was specific for a novel Synergistes species detectable by probes 18a to 18 j in table B and/or comprising a 16S rRNA sequence more than 87% identical to sequence reference: 18 present in only 2.4% of the healthy animals tested and 84.6% of those suffering disease (P=3.37xlO^"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 11a to Hh in table B and/or comprising a 16S rRNA sequence more than 98% identical to sequence reference: 11 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 20), 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 .

The organism most strongly associated with the healthy population in the USA dogs was the Neisseria canis-like organism detectable by probes 7a to 7j in Table A and/or comprising a 16S rRNA sequence more than 95% identical to sequence referenced. This organism showed the second strongest association determined within the USA population studied being present in 85.7% of the healthy and 33.3% of the diseased population (P=I .4xlO^"6). A novel Frigovirgula species and organisms from the family Pasteurellaceae were also found in higher frequency in the healthy USA dogs, being present in 83.3% and 81% of healthy animals compared to 53.8% and 59% of diseased dogs respectively. Comparison of health and 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 4. Statistical comparison of data from three checkerboard hybridisation analyses for detection of the 28 target species in healthy and diseased dogs from distinct populations. P values are given for significant associations (p<0.05) 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 4). However, 5 bacterial species showed differences in their associations with health or disease between the USA population analysed (Example 3) and the initial UK study (Example 1). These included Frigovirgulα sp. (reference 6); Porphyromonαs sαlivosα (reference 26); " Odoribαcter denticαnis" (reference 17) and the Synergistes spp. references 12 and 18. Comparison of the external UK study (Example 2) and USA dogs (Example 3) showed 2 significant differences in the datasets with the Frigovirgulα sp. (reference 6) and Synergistes species (reference 18) again found to differ significantly between studies. The organisms "Odoribαcter denticαnis" (reference 17) and Synergistes sp. (reference 12) were also approaching significance and hence were highlighted as potentially differing between geographical locations. Helcococcus sp. was also found to have a slight difference in indication in different populations. 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. cαnoris (reference 23) and B. denticαnoris (reference 21) 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 2 organisms significantly associated with the healthy periodontum and 11 organisms significantly associated with disease. In Example 3, however, 3 health associations were identified and 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 14); Bacteroidetes sp. (reference 16) and Selenomonas sp. (reference 15). Others such as P. gulae (reference 24) and the Moraxella sp. (reference 20) as well as the health associated Neisseria sp. (reference 7) 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 health and disease The association of both P. canons (reference 23) and Bacteroides denticanoris (reference 21) 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 Filif actor villosus (reference 22); Moraxella sp. (reference 20); Helcococcus sp. (reference 10) and Peptostreptococcus sp (reference 4); Treponema denticola (reference 25); P. salivosa (reference 26); P. gulae (reference 24) and Frigovirgula sp. (reference 6). 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. These included those initially found linking the novel Eubacterium sp. (reference 1), Moraxella sp. (reference 3) and Granulicatella sp. (reference 5) with the healthy periodontum. Bacterial species such as these which do not show consistent associations between studies are not appropriate as markers of bacterial combinations for prediction of periodontal disease. Several additional disease associations were identified in the latter studies compared to Example 1 , in which 8 species were linked with periodontitis. In 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 health and 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 the Frigovirgula sp. (reference 6; P=0.011) and Synergistes sp. (reference 18; P= 0.015). Although some evidence of potential (non-significant) differences between the data sets was found for "Odorbicater denticanis" (reference 17) and Synergistes sp. (reference 12), these appear to be based on the incidence within the population and not on the association with the diseased state.

The Helcococcus species were more prevalent in the healthy population in USA dogs (Example 3) and were slightly but importantly not significantly more prevalent in the disease population in Example 2. 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 and particularly in considering those such as the novel Frigovirgula sp. (reference 6) which was consistently low (<10%) in incidence in the disease cases from the UK but was present in over 50% of the USA periodontitis cases. 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 28 target species with the Treponema sp. detected by probe (reference 25) being the only organism identified as differing significantly between sites (not reported).

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Socransky S. S., A. D. Haffajee, C. Smith, L. Martin, J. A. Haffajee, N. G. Uzel, J.M.

Goodson (2004) Use of checkerboard DNA-DNA hybridization to study complex microbial ecosystems. Oral Microbiol Immunol 19(6): 352-362.

Socransky S.S., A.D. Haffajee, M.A. Cugini, C. Smith, R.L. Jr. Kent (1998) Microbial complexes in subgingival plaque. J Clin Periodontol 25(2): 134-144.

Syed S. A., M. Svanberg, G. Svanberg (1981) The predominant cultivable dental plaque flora of beagle dogs with periodontitis. J Clin Periodontol 8(1): 45-56.

Wunder J.A., W.W. Briner, G.P. Calkins (1976) Identification of the cultivable bacteria in dental plaque from the beagle dog. J Dent Res 55(6): 1097-102. Appendix 1. Periodontal scoring system employed for assessment of health and disease

Scoring system summary:

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

Sequence Reference: 1

GAGTTTGATTATGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATG

CAAGTCGAACGAAGAATTTTTTGGAAGTTTTCGGATGGAAGAAAAATTAC

TTAGTGGCGGACGGGTGAGTAACGCGTAAGTAATCAACCTACAACACAC

GAATAACTAAGAGAAATCTTAGCTAATACGAGATGAAGTAAGTATATCGG

GAGATATAAATATGAAAGCTGAGGCGGTTGTAGACGAGCTTGCGTCTGA

TTAGCTAGTTGGCAGGGTAAGAGCCTACCAAGGCGACGATCAGTAGCCG

GCCTGAGAGGGTGAACGGCCACATTGGAACTGAGAGACGGTCCAAACT

CCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGGCGAAAGCCTG

ATGCAGCAACGCCGCGTGAGCGAAGAAGGCTTTCGAGTCGTAAAGCTCT

GTCGTATGGGAAGAAAAAAATGACAGTACCATACAAGAAAGCCCCGGCT

AACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCC

GGAATTACTGGGCGTAAAGGGTGCGTAGGCGGCTTATTAAGTCAAGGTT

AAAAGGCAGTAGCTCAACTACTGTTCGGCCTTGAAACTAATGAGCTTGAG

TATAGGAGAGGAAAGTGGAATTCCCAGTGTAGCGGTGAAATGCGTAGAT

ATTGGGAGGAATACCGGTGGCGAAGGCGACTTTCTGGACTAAAACTGAC

GCTGAGGCACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTA

GTCCACGCCGTAAACGATGAACACTAGGTGTCGGGAGGAATCTCGGTG

CCGGCGCAAACGCAATAAGTGTTCCGCCTGGGGAGTACGTTCGCAAGA

ATAAAACTCAAAGGAATTGACGGGGACCCGCACAAGTAGCGGAGCATGT

GGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAGCTTGACATGGGG

ATGAAAAATAATGTAAAGTTATAATATCACTTCGGTGAACATCCCACACAG

GTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC

CCGCAACGAGCGCAACCCCTATTAATAGTTGCCAGTGAGTAAAGTCAGG

CACTCTAAAAAGACAGTTAGGGATAACCTAAAGGAAGGTGGGGATGACG

TCAAATCATCATGCCCCTTATGCTTAGGGCTACACACGTGCTACAATGGC

CATAACAAAGAGAAGCGAGATCGTAAGATGGAGCAAACCTGAAAAAAAT

GGTCCAAGTTCGGATTGTGGGCTGAAACTCGCCCACATGAAGTCGGAGT

TACTAGTAATCGCAAATCAGAATGTTGCGGTGAATGCGTTCCCGGGTCTT

GTACACACCGCCCGTCACACCATGGGAGCTGAGGGCACCCGAAGTCAG

TGATCCAACGAAAGAGGAAGCTGCCTAAGGTGAACTTAGTAACTGGGGT

GAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTC

CTT

Sequence Referenced

GAGTTTGATTATGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATG

CAAGTCGAACGATGATTACTAGCTTGCTAGTATGATTAGTGGCGAACGG

GTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTCCGAA

AGGAATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTT

TGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGG

GTAAAGGCCTACCAAGGCGACGATCTGTAGCTGGTCTGAGAGGATGATC AGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA

GTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCG

TGTGTGAAGAAGGCCTTTTGGTTGTAAAGCACTTTAAGTGGGGAGGAAA

AGCTAATAATTAATACCTATTAGCCCTGACGTTACCCACAGAATAAGCAC

CGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGT

TAATCGGAATTACTGGGCGTAAAGCGAGCGTAGGTGGTTACTTAAGTCA

GATGTGAAAGCCCCGGGCTTAACCTGGGAACTGCATCTGATACTGGGTA

ACTAGAGTAGGTGAGAGGGAAGTAGAATTCCAGGTGTAGCGGTGAAATG

CGTAGAGACCTGGAGGAATACCGATGGCGAAGGCAGCTTCCTGGCATC

ATACTGACACTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATAC

CCTGGTAGTCCACGCCGTAAACGATGTCTACCAGTCGTTGGGTCTTTTAA

AGACTTAGTGACGCAGTTAACGCAATAAGTAGACCGCCTGGGGAGTACG

GCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGG

TGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTT

GACATAGTAGGAATCCTGCAGAGATGCGGGAGTGCCTTCGGGAATCTAC

ATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT

TAAGTCCCGCAACGAGCGCAACCCTTTTCCTTAGTTACCAGCGGGTTAT

GCCGGGAACTCTAAGGATACTGCCAGTGACAAACTGGAGGAAGGCGGG

GACGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCT

ACAATGGTAGGTACAAAGGGTTGCTACACAGCGATGTGATGCTAATCTC

AAAAAGCCTATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAA

GTCGGTATCGCTAGTAATCGCAGATCAGAATGCTGCGGTGAATACGTTC

CCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGATCTCACCA

GAAGTGGGTAGCCTAACGCAAGAGGGCGCTCACCACGGTGGGGTCGAT

GACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTG

GATCACCTCCTT

Sequence Referenced

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 Referenced

GAGTTTGATTCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATG

CAAGTCGAACGTGATTTTAGTGGAAGGGTCTTCGGAAACGGAAGCTAAA

TGAAAGTGGCGAACGGGTGAGTAACACGTGAGCAACCTGCCTTACACAG

AGGGATAGCCGTTGGAAACGGTGATTAATACCTCATGAGACCACAGTAT

CGCATGGTATAGGGGTTAAAGATTTATCGGTGTAAGATGGGCTCGCGTC

TGATTAGCTAGTTGGGAGGGTAAAGGCCTACCAAGGCGACGATCAGTAG

CCGGTCTGAGAGGATGAACGGCCACACTGGAACTGAGACACGGTCCAG

ACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACC

CTGATGCAGCGACGCCGCGTGAGCGAAGAAGGCTTTCGAGTCGTAAAG

CTCTGTCCTATGAGAAGATAATGACGGTATCATAGGAGGAAGCCCCGGC

TAAATACGTGCCAGCAGCCGCGGTAATACGTATGGGGCGAGCGTTGTCC

GGAATTATTGGGCGTAAAGGGTACGTAGGCGGCCTTTTAAGTCAGGTGT

GAAAGCGTGAGGCTTAACCTCATTAAGCACTTGAAACTGGAAGGCTTGA

GTGAAGGAGAGGAAAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGA

TATTAGGAGGAATACCGGTGGCGAAGGCGACTTTCTGGACTTTTACTGA

CGCTCAGGTACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTA

GTCCACGCCGTAAACGATGAATGCTAGGTGTTGGGTGTCAAAGCTCGGT

GCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTGGCAACA

CTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGTAGCGGAGCATGT

GGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGGCTTGACATATAGT

TGAGTCATTGAGAAACCAATGAGTCCCTCGGGACAGCTATACAGGTGGT

GCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA

ACGAGCGCAACCCTTATCTTCAGTTACCAGCATTTTGGATGGGGACTCTG

GAGAGACTGCCGATGATAAATCGGAGGAAGGTGGGGATGACGTCAAAT

CATCATGCCCTTTATGTCTTGGGCCACACACGTGCTACAATGGTTGGTAC

AACGGGCAGCGACACAGTGATGTGAAGCAAATCCCTAAAAGCCAATCTC

AGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAGTTACTAG

TAATCGCGGATCAGAACGCCGCGGTGAATGCGTTCCCGGGTCTTGTACA CACCGCCCGTCACACCATGGGAGTTGGCAATACCCGAAGCCGCCGATC TAACCGCAAGGAGGAAGGCGTCGAAGGTAGGGTTAATGACTGGGGTGA AGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTT

Sequence Reference:5

GAGTTTGATCATGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATG

CAAGTCGAACGAACGGAGAAAGAAGCTTGCTTTTTTCAAAGTTAGTGGC

GAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAGGGGGATAAC

ATTCGGAAACGGATGCTAAAACCGCATAGGTTCCTTAGTTGCATGACTGA

GGAAGGAAAAGAGGCGTAAGCTTCTGCTGGTGGATGGACCCGCGGTGC

ATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGATGCATAGCC

GACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAAC

TCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGCAAGTCT

GACGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTC

TGTTGTTAGAGAAGAATAAGCATAATAGTAACTGATTATGCCGTGACGGT

ATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA

CGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCA

GGCGGTTCCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGG

GTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCC

ATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAA

GGCGACTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGTA

GCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGC

TAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGTTAACGCATTAAG

CACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGA

CGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACG

CGAAGAACCTTACCAAGTCTTGACATCCTTTGACCACTCTAGAGATAGAG

CTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCT

CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATT

ACTAGTTGCCAGCATTCAGTTGGGCACTCTAGTGAGACTGCCGGTGACA

AACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACT

TGGGCTACACACGTGCTACAATGGATGGTACAACGAGCAGCGAACTCGC

GAGGGTAAGCGAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTG

CAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACG

CCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCAC

GAGAGTTTGTAACACCCAAAGTCGGTGAGGTAACCTATTGGAGCCAGCC

GCCTAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGT

ATCGGAAGGTGCGGCTGGATCACCTCCTT

Sequence Referenced

(a)

GAGTTTGATCATGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATG

CAAGTCGAGCGGAGTCATAGTAGAAAGATCTTCGGAAATGGAAATTATGT

ACTTAGCGGCGGACGGGTGCGTAACGCGTGGGTAATCTGCCCTTATCAT

CGGGATAACACAACGAAAGATGTGCTAATACCGAATAACATATTCATTTG

GCATCGAGAGGATATCAAAGCGAAAGCGGATAAGGATGAGCCTGCGTCT GATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGC

CGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGA

CTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCC

TGATGCAGCAACGCCGCGTGAGCGATGAAGGTCTTCGGATCGTAAAGCT

CTGTCGCATGGGAAGATAATGACGGTACCATGTGAGGAAGCCCCGGCTA

ACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCG

GAATTACTGGGCGTAAAGGGTGCGTAGGTGGCCTGACAAGTCAGAGGT

CAAAGGCAACGGCTCAACCGTTGTAAGCCTTTGAAACTGTCGGGCTTGA

GTTCTGGAGGGGAAATCGGAATTCCTAGTGTAGCGGTGAAACGCGTAGA

TATTAGGAGGAGCACCGGTGGCGAAGGCGGATTTCTGGACAGATACTGA

CACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT

AGTCCACGCCGTAAACGATGAGTACTAGGTGTTGGCCCGTTAGGGTCGG

TGCCGCAGTTAACACATTAAGTACTCCGCCTGGGATGTACGCTCGCAAG

AGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGTAGCGGAGCAT

GTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAGCTTGACATCCC

TTTGACCGCAGGGTAATGCCTGCTTTCCCTTCGGGGACAGAGGAGACAG

GTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC

CCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCAGGTTATGCTGGG

CACTCTAGAGAGACTGCCGAGGACAACTCGGAGGAAGGTGGGGATGAC

GTCAAATCATCATGCCCCTTATGCTTAGGGCTACACACGTGCTACAATGG

CTGTTACAAAGGGTAGCCAAACCGTGAGGTGGAGCCAATCCCATAAAAA

CAGTCTAAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCTGGAG

TTACTAGTAATCGCAGATCAGCATGCTGCGGTGAATGCGTTCCCGGGTC

TTGTACACACCGCCCGTCACACCATGGGAGCTGGGGGGGCCCAAAGTC

AACGTTCTAACTGCAAAGAGGAAGTTGCCTAAGGCAAAACCAGTGACTG

GGGTGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGTGGCTGGATCA

CCTCCTT

(b)

GAGTTTGATCATGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATG

CAAGTCGAGCGGAGTCATAGTAGAAAGATCTTCGGAAATGGAAATTATGT

ACTTAGCGGCGGACGGGTGCGTAACGCGTGGGTAATCTGCCCTTATCAT

CGGGATAACACAACGAAAGATGTGCTAATACCGAATAACATGCATTTTCG

GCATCGAAGATGTATCAAAGTGTTAGCGGATAAGGATGAGCCTGCGTCT

GATTAGCTAGTTGGTGGGGTAATGGCCTACCAAGGCGACGATCAGTAGC

CGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGA

CTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCC

TGATGCAGCAACGCCGCGTGAGCGATGAAGGTCTTCGGATCGTAAAGCT

CTGTCGCATGGGAAGATAATGACGGTACCATGTGAGGAAGCCCCGGCTA

ACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCG

GAATTACTGGGCGTAAAGGGTGCGTAGGTGGCCTGACAAGTCAGGGGT

CAAAGGCAACGGCTCAACCGTTGTAAGCCTTTGAAACTGTCGGGCTTGA

GTTCTGGAGGGGAAATCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGA

TATTAGGAGGAACACCGGTGGCGAAGGCGGATTTCTGGACAGATACTGA

CACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGAGTACTAGGTGTTGGCCCGTTAGGGTCGG

TGCCGCAGTTAACACATTAAGTACTCCGCCTGGGAAGTACGCTCGCAAG

AGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGTAGCGGAGCAT

GTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAGCTTGACATCCC

TTTGACCGCAGGGTAATGCCTGCTTTCCCTTCGGGGACAGAGGAGACAG

GTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC

CCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCAGGTTATGCTGGG

CACTCTAGAGAGACTGCCGAGGACAACTCGGAGGAAGGTGGGGATGAC

GTCAAATCATCATGCCCCTTATGCTTAGGGCTACACACGTGCTACAATGG

CTGTTACAAAGGGTAGCCAAACCGTGAGGTGGAGCCAATCCCATAAAAA

CAGTCTAAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCTGGAG

TTACTAGTAATCGCAGATCAGCATGCTGCGGTGAATGCGTTCCCGGGTC

TTGTACACACCGCCCGTCACACCATGGGAGCTGGGGGGGCCCAAAGTC

AACTATCTAACTGCAAAGAGGAAGTTGCCTAAGGCAAAACCAGTGACTG

GGGTGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGTGGCTGGATCA

CCTCCTT

Sequence Reference:7

GAGTTTGATTATGGCTCAGATTGAACGCTGGCGGCATGCTTTACACATGC

AAGTCGAACGGCAGCACGGAAGAGCTTGCTCTTTTGGTGGCGAGTGGC

GAACGGGTGAGTAACGCATCGGAACGTACCGAGTAGTGGGGGATAACT

GTCCGAAAGGATGGCTAATACCGCATATTCTCTGAGGAGGAAAGCAGGG

GACCTTCGGGCCTTGCGCTATTTGAGCGGCCGATGTCTGATTAGCTAGT

TGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCGGGTCTGAGA

GGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG

AGGCAGCAGTGGGGAATTTTGGACAATGGGGGGAACCCTGATCCAGCC

ATGCCGCGTGTCTGAAGAAGGCCTTCGGGTTGTAAAGGACTTTTGTCAG

GGAAGAAAAGCTTGAGGTTAATACCCTTRAGTGATGACGGTACCTGAAG

AATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGT

GCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGGGCGCAGACGGTTA

CTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCGTTTGA

AACTGGGTGACTAGAGTATGTCAGAGGGGGGTAGAATTCCACGTGTAGC

AGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCC

CCTGGGATAATACTGACGTTCATGCCCGAAAGCGTGGGTAGCAAACGGG

ATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAATTAGCTGTTGG

GGTACTTGATGCCTTAGTAGCGTAGCTAACGCGTGAAATTGACCGCCTG

GGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCG

CACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAGAACCTT

ACCTGGTCTTGACATGTACGGAATCCTCCAGAGACGGAGGAGTGCCTTC

GGGAACCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGA

GATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCC

ATCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAGCCGGAGGA

AGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGACCAGGGCTTCAC

ACGTCATACAATGGTCGGTACAGAGGGTAGCCAAGCCGCGAGGTGGAG

CCAATCCCAAAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAG TGCATGAAGTCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGA

ATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGG

GGATACCAGAAGTAGGTAGGGTAACCGTAAGGAGCCCGCTTACCACGG

TATGCTTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAAC

CTGCGGCTGGATCACCTCC

Sequence Referenced

GAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATG

CAAGTCGAACGGTAGCAGGAGAAAGCTTGCTTTCTTGCTGACGAGTGGC

GGACGGGTGAGTAATGCTTGGGAATCTGGCTTATGGAGGGGGATAACTA

CTGGAAACGGTAGCTAATACCGCGTAATATCGAGAGATTAAAGACTGGG

ACCTACGGGCCAGTTGCCATAAGATGAGCCCAAGTGGGATTAGGTAGTT

GGTGGGGTAAAGGCCTACCAAGCCCGCGATCTCTAGCTGGTCTGAGAG

GATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGA

GGCAGCAGTGGGGAATATTGCACAATGGGGGGAACCCTGATGCAGCCA

TGCCGCGTGAATGAAGAAGGCCTTCGGGTTGTAAAGTTCTTTCGGTGGT

GAGGAAGGTATCAACTTTAATAGAGTTGGTAATTGACGTTATCCACAGAA

GAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTG

CGAGCGTTAATCGGAATGACTGGGCGTAAAGGGCACGCAGGCGGATGC

TTAAGTGAGATGTGAAAGCCCCGGGCTTAACCTGGGAATTGCATTTCAG

ACTGGGCATCTAGAGTATTTTAGGGAGGGGTAGAATTCCACGTGTAGCG

GTGAAATGCGTAGAGATGTGGAGGAATACCGAAGGCGAAGGCAGCCCC

TTGGGAATATACTGACGCTCATGTGCAAAAGCGTGGGGAGCAAACAGGA

TTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGGGATTG

GGCTATAAGCTTGGTGCCCGAAGCTAACGTGATAAATCGACCGCCTGGG

GAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCA

CAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTAC

CTACTCTTGACATCCATGGAAGTTCGCAGAGATGTGAATGTGCCTTCGG

GAACCATGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAA

TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAG

CGATTTGGTCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAA

GGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACA

CGTGCTACAATGGTGCATACAGAGGGAAGCAATATGGCGACATGGAGCA

AATCTCACAAAGTGCATCTAAGTCCGGATTGGAGTCTGCAACTCGACTCC

ATGAAGTCGGAATCGCTAGTAATCGCAAATCAGAATGTTGCGGTGAATAC

GTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGT

ACCAGAAGTAGATAGCTTAACCGCAAGGGGGGCGTTTACCACGGTATGA

TTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCG

GTTGGATCACCTCCTT Sequence Referenced

GAGTTTGATTATGGCTCAGGATGAACGCTAGCGATAGGCTTAACACATG

CAAGTCGAGGGGCAGCATGAACTTAGCTTGCTAAGTTTGATGGCGACCG

GCGCACGGGTGCGTAACGCGTATGCAACTTGCCTTACAGAGGGGGATA

ACCCGTTGAAAGACGGACTAATACCGCATATACTTATTCGGTTGCATGAC

CGGGCAAGGAAATATTTATAGCTGTAAGATAGGCATGCGTCCCATTAGCT

GGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGGGTAGGGGAACTG

AGAGGTTTATCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACG

GGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAG

CCAAGTCGCGTGAAGGATGACTGTCCTAAGGATTGTAAACTTCTTTTATA

CGGGAATAACGGGCGATACGCGTATTGCTATGAATGTACCGTAAGAATA

AGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCG

AGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGTTGTTCGGTAA

GTCAGCGGTGAAAACTGAGCGCTCAACGTTCAGCGTGCCGTTGAAACTG

CCGGGCTTGAGTTCAGTGGCGGCAGGCGGAATTCGTGGTGTAGCGGTG

AAATGCATAGATATCACGAGGAACTCCGATTGCGAAGGCAGCTTGCCAT

ACTGCGACTGACACTGAAGCACGAAGGCGTGGGTATCAAACAGGATTAG

ATACCCTGGTAGTCCACGCAGTAAACGATGATTACTAGGAGTTTGCGATA

TACCGTCAAGCTTCCACAGCGAAAGCGTTAAGTAATCCACCTGGGGAGT

ACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAG

CGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGG

GATTGAAATGTAGACGACGGATGGTGAAAGCCGTCTTCCCTTCGGGGCG

TCTATGTAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCG

GCTTAAGTGCCATAACGAGCGCAACCCACATCGGTAGTTGCTAACAGGT

TTAGCTGAGGACTCTACCGAGACTGCCGTCGTAAGGCGCGAGGAAGGT

GTGGATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCGACACACG

TGTTACAATGGGAGGGACAAAGGGCAGCTACCGGGCGACCGGATGCGA

ATCTCGAAACCCTTCCCCAGTTCGGATCGGAGTCTGCAACTCGACTCCG

TGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATA

CGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGGAGTCGGGG

GTACCTGAAGGGCGTAACCGCAAGGGGCGCACTAGGGTAATACCGGTG

ACTGGGGCTAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGG

AACACCTCCTT

Sequence Reference: 10

GAGTTTGATCATGGCTCAGGACAAACGCTGGCGGCGTGCATAACACATG

CAAGTCGAACGAGATCTAAAAGATTTAGTGGCGAACGGGTGAGTAACAC

GTGAGAAACCTGCCTCATACAGAGGGATAGCCGTTGGAAACGACGATTA

ATACCTCATGAGACTACATTATCGCATGATAAAGTGGTTAAAGAAAATCG

GTATGAGATGGTCTTGCGTCTGATTAGCTAGTTGGTGGGATAACGGCCT

ACCAAGGCGACGATCAGTAGCCGATCTGAGAGGGTGGACGGCCACACT

GGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAAT

TTTGCACAATGGACGAAAGTCTGATGCAGCGACGCCGCGTGAACGAAGA

AGGCCTTCGGGTTGTAAAGTTCTGTCCTAGGTGAAGATAATGACAGTAAC CTAGAAGAAAGCCCCGGCTAAATACGTGCCAGCAGCCGCGGTAATACGT

ATGGGGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGGGTACGTAGGC

GGTTAATTAAGTCCATATTAAAAGGCATTGGCTTAACCAATGTAAGGTGT

GGAAACTGAATAACTTGAGTAGATGAGGGGAAAGTGGAATTCCAAGTGT

AGCGGTGAAATGCATAGATATTTGGAGGAATACCGGTGGCGAAGGCGAC

TTTCTGGAATCTAACTGACGCTGAGGTACGAAAGCGTGGGGAGCAAACA

GGATTAGATACCCTGGTAGTCCACGCAGTAAACGATGAGTGCTAGGTGT

TGGGGGTCAAACCTCGGCGCCGCCGCTAACGCATTAAGCACTCCGCCT

GGGGAGTACGTACGCAAGTATGAAACTCAAAGGAATTGACGGGGACCC

GCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT

TACCAAGGCTTGACATATACAGGAATAAGCTGGAGACAGCTTAGTCTCTT

CGGAGACTTGTATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTG

AGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCCTATCTTTAGTTACC

AACAGTACGGCTGGGGACTCTAGAGAGACTGCCGGTGATAAACCGGAG

GAAGGTGGGGATGACGTCAAATCATCATGCCCTTTATGTCTTGGGCTAC

ACACGTGCTACAATGGTCTGAACAAAGTGCAGCAAGCTTGTGAAAGTAA

GCAAATCACAGAAAACAGATCTCAGTTCGGATTGTAGGCTGCAACTCGC

CTACATGAAGTCGGAGTTGCTAGTAATCGTGAATCAGAATGTCACGGTG

AATGCGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTTG

GCAATACCCGAAGTCGTCGAGCTAACCGCAAGGAGGCAGACGCCGAGG

GTAGGGTTAATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAA

GGTGCGGCTGGATCACCTCCTT

Sequence Reference: 11

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 Reference: 12

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: 13

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 Reference: 14

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:15

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: 16

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: 17

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: 18

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 Reference: 19

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 Reference:20

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:21

(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:22

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:23

(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 Reference:24

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:25

(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:26

(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

ATAGATGAAGTAGGCGGAATTTGTGGTGTAGCGGTGAAATGCATAGATAT

CACGAGGAACTCCGATTGCGAAGGCAGCTTACTAAGTTATGACTGACACT

GAAGCACGAAAGCGTGGGTATCAAACAGGATTAGATACCCTGGTAGTCCA

CGCAGTAAACGATGATAACTGGGCGTATGCGATATACAGTATGCTCCTGA

GCGAAAGCGTTAAGTTATCCACCTGGGGAGTACGCCGGCAACGGTGAAAC

TCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAAT

TCGATGATACGCGAGGAACCTTACCCGGGATTGAAATTTAGCGGACTATG

TATGAAAGTACATATCCTGTCACAAGGCCGCTAAGTAGGTGCTGCATGGT

TGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATAACGAGCGC

AACCCACGTTGTCAGTTACTATCGGGTAAAGCCGAGGACTCTGACaAGACT

GCCGTCGTAAGGCGCGAGGAAGGTGTGGATGACGTCAAATCAGCACGGC

CCTTACATCCGGGGCGACACACGTGTTACAATGGTGGGGACAAAGGGCAG

CTACTTGGTGACAAGATGCGAATCTCCAAACCCCATCCCAGTTCGGATCGT

AGTCTGCAACTCGACTATGTGAAGCTGGATTCGCTAGTAATCGCGCATCA

GCCATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAA GCCATGGGAGTCGGGAGTACCTAAAGCACGTAACCGCGAGGGGCGTGTTA

GGGTAATACCGGTGACTGGGGCTAAGTCGtAACAAGGTAGCCGTACCGGA

AGGTGCGGCTGGAACACCTCCTTAA

Sequence Reference:27

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 health 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 is associated with periodontal health.

2. The method, as claimed in claim 1, wherein the micro-organism which is associated with periodontal health in a dog is one or more health-associated micro-organisms from: Moraxella sp., Frigovirgula sp., Neisseria sp., Pasteurellaceae sp., or Helcococcus sp.

3. The method, as claimed in claim 1 or claim 2, wherein the micro-organism which is associated with periodontal health in a dog is one or more micro-organisms comprising: a 16S rRNA gene sequence more than 92% identical to sequence reference:2, a 16S rRNA gene sequence more than 89% identical to sequence reference:6, a 16S rRNA gene sequence more than 95% identical to sequence reference:7, a 16S rRNA gene sequence more than 94% identical to sequence reference:8 or a 16S rRNA gene sequence more than 93% identical to sequence reference:10.

4. The method, as claimed in any one of claims 1 to 3, wherein the presence or absence of the micro-organism which is associated with periodontal health 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 micro-organism or by biochemical or phenotypic profiling.

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

6. A method of identifying a micro-organism which reflects or predicts periodontal health in a dog, the method comprising identifying the presence of a particular micro-organism in the mouth of a dog and also identifying that the dog has good periodontal health or that its healthy periodontium will be maintained or will improve.

7. A micro-organism which is associated with periodontal health in a dog and which comprises a 16S rRNA gene sequence which is more than 92% identical to sequence referenced or a 16S rRNA gene sequence which is more than 89% identical to sequence referenced or a 16S rRNA gene sequence which is more than 95% identical to sequence reference :7 or a 16S rRNA gene sequence which is more than 94% identical to sequence reference:8 or a 16S rRNA gene sequence which is more than 93% identical to sequence reference: 10.

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

9. A probe comprising at least 80% identity with at least the 10 sequential residues from any of the sequences in the table below or from Appendix 2, without the poly T tail, with reference 2, 6, 7 or 8 or a probe of at least 10 residues which hybridises to any sequence in the table below or from Appendix 2, without the poly T tail, with reference 2, 6, 7 or 8 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 40-75⁰C, 5 x SSC overnight:

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

11. A kit for identifying or predicting periodontal health 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 is associated with periodontal health.

12. A kit, as claimed in claim 11, wherein the micro-organism which is associated with periodontal health in a dog is one or more health-associated micro-organisms from: Moraxella sp., Frigovirgula sp., Neisseria sp., Pasteurellaceae sp., or Helcococcus sp.

13. A kit as claimed in claim 12, wherein the micro-organism which is associated with periodontal health is one or more micro-organisms comprising: a 16S rRNA gene sequene more than 92% identical to sequence referenced or a 16S rRNA gene sequence more than 89% identical to sequence referenced, a 16S rRNA gene sequence more than 95% identical to sequence reference:?, a 16S rRNA gene sequence more than 94% identical to sequence referenced or a 16S rRNA gene sequence more than 93% identical to sequence reference: 10.

14. A method which provides information on the periodontal health status of 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 is associated with periodontal health and comprising identifying the presence or absence of one or more micro-organisms from a sample from the mouth of said dog, wherein the micro-organism is associated with periodontal disease.

15. The method as claimed in claim 14, wherein the micro-organism associated with periodontal health is one or more health-associated micro-organisms from: Moraxella sp., Frigovirgula sp., Neisseria sp., Pasteurellaceae sp., ox Helcococcus sp.

16. The method as claimed in claim 14 or claim 15, wherein the micro-organism associated with periodontal disease is one or more disease-associated micro-organisms from: Peptostreptococcus sp., Synergistes sp., Clostridiales sp., Eubacterium nodatum, Selenomonas sp., Bacteroidetes sp., "Odoribacter denticanis", Moraxella sp., Bacteroides denticanoris, Fillif actor villosus, Porphyromonas canons, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa, Desulfomicrobium or ale.

17. The method as claimed in any one of claims 14 to 16, wherein the micro-organism which is associated with periodontal health in one or more micro-organism comprising: a 16S rRNA gene sequence more than 92% identical to sequence referenced, a 16S rRNA gene sequence more than 89% identical to sequence referenced, a 16S rRNA gene sequence more than 95% identical to sequence reference:7, or a 16S rRNA gene sequence more than 94% identical to sequence referenced or a 16S rRNA gene sequence more than 93% identical to sequence reference: 10.

18. The method as claimed in any one of claims 14 to 17, wherein the micro-organisms associated with periodontal disease is one or more micro-organisms comprising: a 16S rRNA gene sequence more than 98% identical to sequence reference: 11, a 16S rRNA gene sequence more than 96% identical to sequence reference: 12, a 16S rRNA gene sequence more than 94% 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 98% identical to sequence reference: 15, a 16S rRNA gene sequence more than 92% identical to sequence reference: 16 or a 16S rRNA gene sequence more than 96% identical to sequence reference: 17 or a 16S rRNA gene sequence more than 87% identical to sequence reference: 18 or a 16S rRNA gene sequence more than 99% identical to sequence reference: 19, a 16S rRNA gene sequence more than 92% identical to sequence reference :20, a 16S rRNA gene sequence more than 99% identical to sequence reference :21, a 16S rRNA gene sequence more than 99% identical to sequence reference :22, a 16S rRNA gene sequence more than 98% identical to sequence reference:23, a 16S rRNA gene sequence more than 99% identical to sequence reference: 24, a 16S rRNA gene sequence more than 99% identical to sequence reference:25 or a 16S rRNA gene sequence more than 98% identical to sequence reference:26.

19. A method, as claimed in any one of claims 14 to 18, wherein the presence or absence of the micro-organism which is associated with periodental health or with periodental 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 micro-organism or by biochemical or phenotypic profiling.

20. A method as claimed in claim 19, wherein amplification of nucleic acid of the micro-organism is carried out before identification with a nucleic acid detector.

21. A method, as claimed in any one of claims 14 to 20, 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, 16, 17, 18, 19, 20 or 21 micro-organisms from the list.

22. A kit suitable for providing information on the periodontal health status of 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 a dog, wherein the micro-organism is associated with periodontal health and comprising an agent to determine the presence or absence of at least one micro-organism from a sample from the mouth of said dog, wherein the micro-organism is associated with periodontal disease.

23. A kit as claimed in claim 22, wherein the micro-organism associated with periodontal health is one or more health-associated micro-organisms from: Moraxella sp., Fήgovirgula sp., Neisseria sp., Pasteurellaceae sp., or Helcococcus sp.

24. A kit, as claimed in claim 22 or claim 23, wherein the micro-organism associated with periodontal disease is one or more disease-associated micro-organisms from: Peptostreptococcus sp., Synergistes, Clostridials sp., Eubacterium nodatum, Selenomonas sp., Bacteroidetes, "Odoribacter denticanis", Desulfomicrobium orale, Moraxella sp., Bacteroides denticanoris, Fillifactor villosus, Porphyromonas canoris, Porphrymonas gulae, Treponema denticola or Porphrymonas salivosa.

25. A kit, as claimed in any one of claims 22 to 24, wherein the micro-organism which is associated with periodontal health is one or more micro-organism comprising: a 16s rrna gene sequence more than 92% identical to sequence reference:2, a 16S rRNA gene sequence more than 89% identical to sequence reference:6, a 16S rRNA gene sequence more than 95% identical to sequence reference:7, or a 16S rRNA gene sequence more than 94% identical to sequence reference:8 or a 16S rRNA gene sequence more than 93% identical to sequence reference: 10.

26. A kit, as claimed in any one of claims 22 to 25, 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: 12, a 16S rRNA gene sequence more than 94% 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 98% identical to sequence reference: 15, a 16S rRNA gene sequence more than 92% identical to sequence reference: 16 or a 16S rRNA gene sequence more than 96% identical to sequence reference: 17 or a 16S rRNA gene sequence more than 87% identical to sequence reference: 18, a 16S rRNA gene sequence more than 99% identical to sequence reference: 19, a 16S rRNA gene sequence more than 92% identical to sequence reference:20, a 16S rRNA gene sequence more than 99% identical to sequence reference:21, a 16S rRNA gene sequence more than 99% identical to sequence reference:22, a 16S rRNA gene sequence more than 98% identical to sequence reference:23, a 16S rRNA gene sequence more than 99% identical to sequence reference :24, a 16S rRNA gene sequence more than 99% identical to sequence reference :25 or a 16S rRNA gene sequence more than 98% identical to sequence reference:26.

27. A kit, as claimed in any one of claims 22 to 26, 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, 16, 17, 18, 19, 20 or 21 of the micro-organisms from the list.