Methods of identifying a companion animal at risk of oral disease

Abstract

Disclosed herein are methods of identifying a companion animal as being at risk of oral disease or condition comprising obtaining a plaque sample from a tooth of the companion animal; determining a relative abundance of each of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, and positive effect phyla in the first plaque sample; and calculating a Tooth Microbiome Gingival Index (TMGI) score. Also disclosed herein are methods of determining whether a pet food composition improves the oral health of a companion animal, methods for preventing or treating an oral disease or condition in a companion animal, and kits for identifying a companion animal as being at risk of an oral disease or condition.

Description

METHODS OF IDENTIFYING A COMPANION ANIMAL AT RISK OF ORAL DISEASECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 708,950, filed 18 October 2024, the contents of which are hereby incorporated herein by reference in its entirety.BACKGROUND

[0002] Humans and non-human mammals, including companion animals, are susceptible to periodontal disease and other afflictions and conditions of the oral cavity. Periodontal disease develops, in pail, as a consequence of the adherence of plaque, a mixture of oral bacteria and saliva components, to the surface of the teeth. This adherent plaque hardens to form tartar (calculus), which can lead to inflammation, swelling, and infection of the gums.

[0003] Periodontal disease is the most widespread oral disease in dogs, with an estimated prevalence of at least 80% of dogs over 3 years of age having oral disease. The prevalence of oral disease is higher in small breed canines compared to medium, large, and giant breeds. Evolutionarily, several hypotheses have been proposed regarding the etiology of periodontitis, specifically how plaque, a sticky mass of microbes, causes gingival inflammation, known as gingivitis, which in turn leads to periodontitis.

[0004] In the 1970s, specific oral pathogenic species were identified as associated with necrotizing ulcerative gingivitis, leading to the development of a specific plaque hypothesis. In 1994, a new hypothesis was formulated, the “ecological plaque hypothesis,” suggesting that an overall microbial imbalance caused by ecological stress results in enriching certain disease-related microbes. In 2012, based on the launch of the 10-year Human Microbiome Project launched by the National Institute of Health, the “keystone pathogen hypothesis” was proposed, assuming that certain low abundant microbes, such as Porphyromonas gingivalis, impact the host immune system, which remodels the microbiota to cause inflammation.

[0005] Recent research on the oral microbiome has explored causative factors, identifying a hypothesis called “Inflammation mediated polymicrobial emergence and dysbiotic exacerbation” (IMPEDE). This hypothesis suggests that the host’s innate immune system response plays a role in maintaining oral health or restoring symbiosis at the onset of gingivitis. If the host isunsuccessful in restoring or maintaining a symbiotic or balanced state in the oral microbiome, then potential disease-causing microbes, or pathobionts, can tip the balance and lead to periodontitis. In other words, this hypothesis suggests that both the host immune system and the plaque microbiome disbalance, by specifically enriching pathobionts, can cause gingivitis and periodontitis.

[0006] The recent advancement of sequencing technology allows studying of the oral microbiome in detail. The oral microbiome comprises different niche microbiomes of oral mucosa, tongue, saliva, supragingival, and subgingival plaque.

[0007] Plaque microbiome, and in particular plaque biofilm, may play an important role in the etiology of periodontal disease development. Plaque maturation and subsequent accumulation of specific pathogenic microbial species can contribute to activating the host inflammatory response. As the plaque buildup extends from the gingival margin to the subgingival sulcus (subgingival plaque), the anaerobic environment helps to proliferate anaerobic bacteria, including pathobionts, which are also considered late colonizers. The persistence of pathobionts can trigger inflammation in gingival tissue (i.e., gingivitis) and impact the host immune response, which further disturbs the plaque microbiome balance and eventually leads to gingival recession, the formation of pockets, attachment loss, and finally tooth loss. Preventative measures against gingivitis, such as prognostic tools like prediction modeling, are therefore of clinical relevance.

[0008] There remains, therefore, a need for new or alternative methods and compositions for treating or preventing oral diseases in companion animals, as well as accurate methods for classifying a companion animal as at risk for oral disease versus not at risk for oral disease based on the companion animal’s oral microbiome profile.BRIEF SUMMARY

[0009] This summary is intended merely to introduce a simplified summary of some aspects of one or more implementations of the present disclosure. Further areas of applicability of the present disclosure will become apparent from the detailed description provided hereinafter. This summary is not an extensive overview, nor is it intended to identify key or critical elements of the present teachings, nor to delineate the scope of the disclosure. Rather, its purpose is merely to present one or more concepts in simplified form as a prelude to the detailed description below.

[0010] Aspects of the disclosure are directed to methods of identifying companion animals (e.g., canines) as being at risk of oral disease or condition. In certain embodiments, the methods disclosed herein comprise (1) obtaining a first plaque sample from a tooth of the companion animal, (2) determining from that plaque sample a relative abundance of positive effect microbiotic species, a relative abundance of negative effect microbiotic species, a relative abundance of early colonizers, a relative abundance of late colonizers, and a relative abundance of positive effect phyla in the first plaque sample; and (3) calculating a first Tooth Microbiome Gingival Index (TMGI) score, wherein the companion animal is identified as being at risk of oral disease or condition if the first TMGI score is less than about -0.12.

[0011] The TMGI score may be calculated as follows:

[0012] According to another aspect, disclosed herein is a method of determining whether a pet food composition improves the oral health of a companion animal, the method comprising (1) identifying a companion animal as being at risk of an oral disease or condition according to the methods disclosed herein; optionally administering a dental prophylaxis or cleaning to the companion animal; (3) administering the pet food composition to the companion animal for at least about 7 days; (4) obtaining a second plaque sample from a tooth of the companion animal after administering the pet food composition; (5) determining a second relative abundance of each of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, and positive effect phyla in the second plaque sample; and (5) calculating a second TMGI score, wherein the second TMGI score can be calculated as follows:wherein the pet food composition improves the oral health of the companion animal if the second TMGI score is higher than the first TMGI score.

[0013] In certain embodiments, the second TMGI score is greater than about -0.12, and in certain embodiments the pet food composition is administered for at least about 14 days, such as at least about 21 days or at least about 28 days. In certain embodiments, the companion animal is administered a dental prophylaxis cleaning prior to administering the pet food composition.

[0014] In accordance with a further aspect of the disclosure, the companion animal is a canine, such as a small breed canine. In certain embodiments of the methods disclosed herein, the oraldisease or condition is periodontitis, dental plaque, calculus, or gingivitis, preferably gingivitis. In certain embodiments, the first plaque sample is a subgingival plaque sample, and in certain embodiments, the second plaque sample is a subgingival plaque sample. According to certain embodiments, the positive effect microbiotic species and negative effect microbiotic species are determined by next generation sequencing technology, preferably by 16s rRNA amplicon sequencing and metagenomics, targeted multiplex qPCR technology, or customized microarray technology.

[0015] In certain embodiments, the positive effect microbiotic species are selected from at least one of Moraxella osloensis, Acinetobacter johnsonii, Moraxellaceae spp., Haemophilus spp., Bergeyella HMT 22, Streptococcus spp., and Solobacterium moorei. In certain embodiments, the negative effect microbiotic species are selected from at least one of Treponema denticola, Fusobacterium nucleatum_subsp_vincentii, Treponema HMT 490, Ruminococcaceae spp., Mollicutres_[G-2] bacterium HMT 906, Peptostreptococcaceae_[XI][G-8] bacterium HMT 382, Treponema HMT 239, Escherichia coli, Treponema HMT 238, Fastidio sipila sanguinis, and Mycoplasma lipophilum.

[0016] According to certain embodiments, the early colonizers are selected from at least one of Moraxella nonliquifaciens, Bergeyella HMT 422, Capnocytophaga spp., Escherichia coli, Mycoplasma hominis, Neisseria weaveri, Abiotrophia defectiva, Streptococcus spp., Haemophilus spp., Moraxella osloensis, Bergeyalla spp., and Mycoplasma lipophilum, and according to certain embodiments, the late colonizers are selected from at least one of Treponema HMT 249, Filifactor cdocis, Porphyromonas HMT 285, Treponema parvum, Treponema HMT 490, Prevotella intermedia, Treponema HMT 951, Treponema HMT 257, Treponema HMT 227, Treponema HMT 234, Treponema HMT 238, Treponema HMT 237, Fusobacterium nucleatum vincentii, Tannerella forsythia, Treponema denticola, Porphyromonas gingivalis, Treponema HMT 235, Treponema HMT 239, Treponema HMT 258, Campylobacter rectus, and Treponema spp.

[0017] In certain embodiments, the positive effect phyla are Actinobacteria and Proteobacteria. In certain embodiments, the methods disclosed herein further comprise determining a relative abundance of negative effect phyla, such as a first relative abundance of negative effect phyla and a second relative abundance of negative effect phyla, and in certain embodiments, the negative effect phylum are chosen from at least one of Spirochaetes and Firmicute.

[0018] Also disclosed herein is a method for preventing or treating oral disease or an oral condition in a companion animal comprising identifying a companion animal at risk for an oral disease or condition according to the methods disclosed herein, and administering to the companion animal an oral-disease mitigating pet food composition.

[0019] Also disclosed herein is a kit for identifying a companion animal a being at risk of an oral disease or condition, the kit comprising a device for collecting a plaque sample from a tooth of the companion animal; and components for detecting a relative abundance of each of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, positive effect phyla, and negative effect phyla in the plaque sample.

[0020] In certain embodiments of the kits disclosed herein, the positive effect microbiotic species are Moraxella osloensis, Acinetobacter johnsonii, Moraxellaceae unclassified, Haemophilus unclassified, Bergeyella HMT 22, Streptococcus unclassified, and Solobacterium moorei', the negative effect microbiotic species are Treponema denticola, Fusobacterium nucleatum_subsp_vincentii, Treponema HMT 490, Ruminococcaceae unclassified, Mollicutres_[G-2] bacterium HMT 906, Peptostreptococcaceae_[XI][G-8] bacterium HMT 382, Treponema HMT 239, Escherichia coli, Treponema HMT 238, Fastidiosipila sanguinis, and Mycoplasma lipophilum', the early colonizers are Moraxella nonliquifaciens, Bergeyella HMT 422, Capnocytophaga unclassified, Escherichia coli, Mycoplasma hominis, Neisseria weaveri, Abiotrophia defectiva, Streptococcus unclassified, Haemophilus unclassified, Moraxella osloensis, Bergeyalla unclassified, and Mycoplasma lipophilum', the late colonizers are Treponema HMT 249, Filif actor alocis, Porphyromonas HMT 285, Treponema parvum, Treponema HMT 490, Prevotella intermedia, Treponema HMT 951, Treponema HMT 257, Treponema HMT 227, Treponema HMT 234, Treponema HMT 238, Treponema HMT 237, Fusobacterium nucleatum vincentii, Tannerella forsythia, Treponema denticola, Porphyromonas gingivalis, Treponema HMT 235, Treponema HMT 239, Treponema HMT 258, Campylobacter rectus, and Treponema unclassified', and the positive effect phyla are Actinobacteria and Proteobacteria.

[0021] According to certain embodiments of the kits disclosed herein, the components for detecting a relative abundance are chosen from components for next generation sequencing technology, preferably by 16s rRNA amplicon sequencing and metagenomics, targeted multiplex qPCR technology, or customized microarray technology.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The features and advantages of aspects of the disclosure will be apparent from the following more detailed description of certain embodiments of the disclosure and as illustrated in the accompanying drawings in which:

[0023] FIG. 1 is a plot showing tooth gingival scores for healthy (T2, best) and unhealthy (Tl, worst) teeth, as described in Example 1.

[0024] FIG. 2 is a plot showing tooth gingival scores plotted against whole mouth plaque grading for light, medium, and heavy grades, as described in Example 1.

[0025] FIG. 3A is a bar graph showing whole mouth plaque scores for dogs fed a control food or a test food for 28 days, as described in Example 3, wherein an * indicates statistical significance.

[0026] FIG. 3B is a bar graph showing whole mouth calculus scores for dogs fed a control food or a test food for 28 days, as described in Example 3, wherein an * indicates statistical significance.

[0027] FIG. 3C is a bar graph showing whole mouth gingivitis scores for dogs fed a control food or a test food for 28 days, as described in Example 3.

[0028] FIG. 4 is a graph showing the canine TMGI scores for dogs fed a control food or a test food for 28 days, as described in Example 3.

[0029] It should be understood that the various aspects of the disclosure are not limited to the compositions, arrangements, and instrumentality shown in the figures.DETAILED DESCRIPTION

[0030] For illustrative purposes, the principles of the present disclosure are described by referencing various exemplary embodiments thereof. Although certain embodiments of the disclosure are specifically described herein, one of ordinary skill in the art will readily recognize that the same principles are equally applicable to and can be employed in other compositions and methods. Before explaining the disclosed embodiments in detail, it is to be understood that the disclosure is not necessarily limited in its application to the details of any particular embodiment disclosed. The terminology used herein is for the purpose of description and not of limitation.

[0031] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context dictates otherwise. The singular form of any class of the ingredients refers not only to one ingredient within that class, but also to a mixture of those ingredients. The terms “a” (or “an”), “one or more” and “at least one” may be used interchangeablyherein. The terms “comprising”, “including”, and “having” may be used interchangeably. The term “include” should be interpreted as “include, but arc not limited to”. The term “including” should be interpreted as “including, but are not limited to”.

[0032] As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range. Thus, a range from 1-5, includes specifically 1, 2, 3, 4 and 5, as well as subranges such as 2-5, 3-5, 2-3, 2-4, 1-4, etc. The term “about” when referring to a number means any number within a range of 10% of the number. For example, the phrase “about 2 wt%” refers to a number between and including 1.8 wt% and 2.2 wt%.

[0033] All references cited herein are hereby incorporated by reference in their entireties. In the event of a conflict in a definition in the present disclosure and that of a cited reference, the present disclosure controls.

[0034] The abbreviations and symbols as used herein, unless indicated otherwise, take their ordinary meaning. The abbreviation “wt.%” or “wt%” means percent by weight with respect to the pet food composition. The symbol “°” refers to a degree, such as a temperature degree or a degree of an angle. The symbols “h”, “min”, “mL”, “nm”, “ m” means hour, minute, milliliter, nanometer, and micrometer, respectively. The abbreviation “UV-VIS” referring to a spectrometer or spectroscopy, means Ultraviolet-Visible. The abbreviation “rpm” means revolutions per minute.

[0035] Any member in a list of species that are used to exemplify or define a genus, may be mutually different from, or overlapping with, or a subset of, or equivalent to, or nearly the same as, or identical to, any other member of the list of species. Further, unless explicitly stated, such as when reciting a Markush group, the list of species that define or exemplify the genus is open, and it is given that other species may exist that define or exemplify the genus just as well as, or better than, any other species listed.

[0036] All components and elements positively set forth in this disclosure can be negatively excluded from the claims. In other words, the pet food compositions of the instant disclosure can be free or essentially free of all components and elements positively recited throughout the instant disclosure. In some instances, the pet food compositions of the present disclosure may be substantially free of non-incidental amounts of the ingredient(s) or compound(s) described herein. A non-incidental amount of an ingredient or compound is the amount of that ingredient or compound that is added into the pet food composition by itself. For example, a pet foodcomposition may be substantially free of a non-incidental amount of an ingredient or compound, although such ingrcdicnt(s) or compound(s) may be present as part of a raw material that is included as a blend of two or more compounds. Substantially free, unless other defined or described herein, typically refers to an ingredient or compound in an amount of about 2 wt% or less, about 1.5 wt% or less, about 1 wt% or less, about 0.5 wt% or less, about 0.1 wt% or less, or about 0.05 wt% or less, or about 0.01 wt% or less, based on the total weight of the pet food composition on a dry matter basis.

[0037] Some of the various categories of components identified may overlap. In such cases where overlap may exist and the pet food composition includes both components (or the composition includes more than two components that overlap), an overlapping compound does not represent more than one component. For example, certain components or ingredients may be characterized as both an ancient grain and an amaranth. If a particular pet food care composition recites both an ancient grain and an amaranth, a compound that may be characterized as both an ancient grain and an amaranth will serve only as either an ancient or an amaranth — not both.

[0038] As used herein, the term “pet” could be used interchangeably with “companion animal” and refers to an animal of any species kept by a caregiver as a pet or any animal of a variety of species that have been widely domesticated as pets, including canines (Cards familiaris) and felines (Fells domeslicus). Thus, a pet may include but is not limited to, working dogs, pet dogs, cats kept for rodent control (i.e., farm cats), pet cats, ferrets, birds, reptiles, rabbits, and fish.

[0039] To the extent that food and food ingredients contain water / moisture, the dry matter represents everything in the sample other than water including, for example, protein, fiber, fat, carbohydrates, minerals, etc. Dry matter weight is the total weight minus the weight of any water. The skilled artisan would readily recognize and understand nutritional amounts and percentages expressed as dry matter amounts, dry matter weights and dry matter percentages.

[0040] Dry matter intake per day is calculated as the total nutritional intake per day excluding all water. For example, an amount of an ingredient equal to a specific percent of daily nutritional intake refers to the amount of that ingredient in dry matter form (i.e., excluding all water) relative to the total amount of dry matter consumed (also excluding all water) in a day.

[0041] “Daily nutritional intake” and “total nutritional intake per day” refer to dry matter intake per day. That is, water weight is not included in calculating the amount of nutrition consumed per day. To calculate percent of an ingredient of total daily intake on a dry matter basis, water isremoved from the total intake to give total daily dry matter intake and the percentage of the ingredient is based on amount of ingredient present as dry matter.

[0042] As used herein, an “ingredient” refers to any component of a pet food composition. The term “nutrient” refers to a substance that provides nourishment and thus has a nutrient value. In some cases, an ingredient may comprise more than one “nutrient,” for example, a composition may comprise com comprising important nutrients including both protein and carbohydrate.

[0043] Certain embodiments disclosed herein include a method of identifying a companion animal, e.g., a canine, as being at risk of having an oral disease or condition or at risk of developing an oral disease or condition. Aspects of the disclosure are directed to a Tooth Microbiome Gingival Index (TMGI) to classify a companion animal’s tooth as having healthy or unhealthy conditions, as may be determined through its subgingival plaque microbiome composition. Also disclosed herein are nutritional solutions to mitigate the risk of developing an oral disease or condition, e.g. gingivitis, or to treat or prevent such an oral disease or condition in a companion animal.

[0044] Also disclosed herein are kits for identifying a companion animal as being at risk of having an oral disease or condition or at risk of developing an oral disease or condition. In certain embodiments, the kit comprises a device for collecting at least one plaque sample from the companion animal. Optionally, the kit may further comprise instructions for analyzing results of a microbiome analysis performed from the plaque sample that has been obtained. In certain embodiments, the kit further comprises a device for determining a relative abundance in the plaque sample of one or more of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, positive effect phyla, and optionally negative effect phyla. In certain embodiments, the device for determining relative abundance comprises PCR primers for amplifying 16s rDNA from the plaque sample.

[0045] In accordance with a further aspect of the disclosure, a method is provided for treating, preventing, or mitigating an oral disease or condition in a companion animal, the method comprising administering an oral disease-mitigating diet, such as a pet food composition as disclosed herein, to the companion animal. In some instances, the method may include providing and / or feeding the animal the oral disease-mitigating diet for 1 or more days, preferably 5 or more days, preferably 7 or more days, 10, 14, 21, 28, 30, or 42 or more days. The method may include feeding the animal one time a day, two times a day, three times a day, or in some embodiments four or more times a day.Methods of Identifying a Companion Animal at Risk of Oral Disease or Condition

[0046] Disclosed herein a method of identifying whether a companion animal is at risk of an oral disease or condition. As used herein, the term “at risk of oral disease or condition” indicates that the companion animal has an oral disease or condition or is at risk of developing an oral disease or condition. An oral disease or condition may include any oral disease or condition recognized in the art, such as, for example, periodontitis, dental plaque, calculus, or gingivitis. In certain embodiments, the companion animal has gingivitis or is at risk of developing gingivitis. In certain embodiments, the companion animal is a canine, and in certain embodiments, the companion animal is a feline.

[0047] The methods disclosed herein comprise a step of obtaining from the companion animal a plaque sample. The plaque sample may be obtained by any means known in the art, such as by contacting the surface of the companion animal’s tooth, including for example from the gingival region or the subgingival region, with a device (e.g., a swab, a paper point, a filter paper strip) designed to collect the plaque sample. The plaque sample may then remain on the device for further processing or may be transferred from the device to a collection device for further processing.

[0048] After the plaque sample has been obtained from the tooth of the companion animal, the methods disclosed herein further comprise analyzing the plaque sample to determine the relative abundance of microbiotic species in the plaque sample. In certain embodiments, the relative abundance of at least one of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, positive effect phyla, and / or negative effect phyla may be determined.

[0049] Microbiotic species disclosed herein are referred to by the taxonomic identification as set forth in the Human Oral Microbiome Database (HOMD), available at www.homd.org. The HOMD database includes 774 species in multiple phyla, including, for example, Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Fusobacteria, Proteobacteria, Saccharibacteria, Spirochaetes, Synergistetes, and Tenericutes, found in teeth, gingival sulcus, gingiva, tongue, cheek, lip, and palate. In various embodiments, the HOMD (or expanded HOMD, known as “eHOMD”) may facilitate the comparison of gene sequences (e.g., 16S rRNA gene fragments) by providing a systematic naming scheme for taxa, including unnamed taxa, identified through sequencing techniques. For example, taxa in HOMD and eHOMD may be defined by their sequence similarity (e.g., at least about 95%, about 98%, about 98.5%, about 99%,about 99.5% or about 100% sequence identity) across 16s rRNA gene sequences and assigned a Human Microbial Taxon (HMT) number. The HMT number may then be used to search and retrieve the sequence-based taxon from the database.

[0050] Relative abundance of microbiotic species may be determined by any means known in the art. In certain embodiments, relative abundance is determined by next generation sequencing technology, targeted multiplex qPCR technology, or customized microarray technology. In certain embodiments, relative abundance of a microbiotic species may be determined by 16s rRNA amplicon sequencing.

[0051] As used herein, “positive effect microbiotic species” indicates bacterial species that are known to have a positive effect on the oral health of a companion animal. In certain embodiments, a positive effect microbiotic species may be identified in the art, and in certain embodiments, a positive effect microbiotic species may be identified by the presence of the microbiotic species obtained from a plaque sample of a companion animal from a healthy tooth.

[0052] In certain exemplary embodiments, positive effect microbiotic species may be selected from one or more of the following microbiotic species: Moraxella osloensis, Acinetobacter johnsonii, Moraxellaceae spp., Haemophilus spp., Bergeyella HMT 22, Streptococcus unclassified, and Solobacterium moorei.

[0053] As used herein, “negative effect microbiotic species” indicates bacterial species that are known to have a negative effect on the oral health of a companion animal. In certain embodiments, a negative effect microbiotic species may be identified in the art, and in certain embodiments, a negative effect microbiotic species may be identified by the presence of the microbiotic species obtained from a plaque sample of a companion animal from an unhealthy tooth.

[0054] In certain exemplary embodiments, negative effect microbiotic species may be selected from one or more of the following microbiotic species: Treponema denticola, Fusobacterium nucleatum_subsp_vincentii, Treponema HMT 490, Ruminococcaceae spp., Mollicutres_[G-2] bacterium HMT 906, Peptostreptococcaceae_[XI][G-8] bacterium HMT 382, Treponema HMT 239, Escherichia coli, Treponema HMT 238, Fastidiosipila sanguinis, and Mycoplasma lipophilum.

[0055] Microbiotic species involved in the formation and growth of dental plaque may be divided into “early colonizers” and “late colonizers.” As used herein, “early colonizers” indicate microbiotic species that are present in plaque, including plaque biofilm, and may appear de novoon a clean tooth surface that is otherwise substantially free of bacteria within a brief period of time, rising in abundance early on during the plaque formation process. Early colonizers of plaque formation may include facultative anaerobes and / or saccharolytic species that grow on salivary mucus and other glycoproteins.

[0056] As the plaque biofilm continues to grow and early colonizers develop, oxygen may be progressively depleted, such that anaerobic microbiotic species rise in abundance in the plaque biofilm. These species that increase in abundance later in time during the development of the plaque biofilm are termed late colonizers. As used herein, “late colonizers” indicate microbiotic species that are present in plaque, including plaque biofilm, and may appear after the colonization of early colonizer species. Late colonizers may include proteolytic obligate anaerobes and may, in certain embodiments, be capable of co-existing with other microbiotic species already established in the plaque colonies.

[0057] In certain embodiments, early colonizer microbiotic species may include at least one of Moraxella nonliquifaciens, Bergeyella HMT 422, Capnocytophaga spp., Escherichia coli, Mycoplasma hominis, Neisseria weaveri, Abiotrophia defectiva, Streptococcus spp., Haemophilus spp., Moraxella osloensis, Bergeyalla spp., and Mycoplasma lipophilum.

[0058] In certain embodiments, late colonizer microbiotic species may include at least one of Treponema HMT 249, Filifactor alocis, Porphyromonas HMT 285, Treponema parvum, Treponema HMT 490, Prevotella intermedia, Treponema HMT 951, Treponema HMT 257, Treponema HMT 227, Treponema HMT 234, Treponema HMT 238, Treponema HMT 237, Fusobacterium nucleatum vincentii, Tannerella forsythia, Treponema denticola, Porphyromonas gingivalis, Treponema HMT 235, Treponema HMT 239, Treponema HMT 258, Campylobacter rectus, and Treponema spp.

[0059] As used herein, the term “positive effect phyla” refers to microbial species that are taxonomically assigned to phyla, wherein the presence of the phyla in plaque is associated with a positive effect on the oral health of a companion animal. Likewise, the term “negative effect phyla” as used herein refers to microbial species that are taxonomically assigned to phyla, wherein the presence of the phyla in plaque is associated with a negative effect on the oral health of a companion animal.

[0060] In certain embodiments, positive effect phyla may include at least one of Actinobacteria and Proteobacteria.

[0061] In certain embodiments, negative effect phyla may include at least one of Spirochaetes, Bacteriodetes, Firmicutes, and Fusobacteria. In certain embodiments, the negative effect phylum is Spirochaetes .

[0062] In the methods disclosed herein for identifying a companion animal as being at risk of an oral disease or condition, an average of the relative abundance of each of the positive effect microbiotic species, the negative effect microbiotic species, the early colonizers, the late colonizers, and the positive effect phyla may be determined from the plaque sample obtained from the companion animal. The relative abundance averages may then be used to calculate a TMGI score according to the following formula:

[0063] In certain embodiments, a TMGI value that falls at or below a certain threshold indicates that the companion animal is at risk of an oral disease or condition, and in certain embodiments, a TMGI value that falls above a certain threshold indicates that the companion animal is not at risk of an oral disease or condition or is at a lowered risk of an oral disease or condition. In certain embodiments, the threshold value is from about -0.9 to about -0.15, such as about -0.10 to about - 0.14, or about -0.11 to about -0.13. In certain embodiments, the threshold value is about -0.12, and in certain embodiments, the threshold value is -0.12.

[0064] In certain embodiments, the TGMI score may be calculated prior to or immediately prior to performing a dental prophylaxis on the companion animal, and in certain embodiments, the TGMI score may be calculated after or immediately after performing a dental prophlaxis or cleaning on the companion animal.Method of Determining Whether a Diet Improves Oral Health of Companion Animal

[0065] Also disclosed herein are methods for determining whether a diet, such as a pet food composition, improves the oral health of a companion animal as well as method of preventing or treating an oral disease or condition in a companion animal by identifying a companion animal at risk for an oral disease or condition according to the methods disclosed herein and administering to the companion animal an oral-disease mitigating diet, such as an oral disease-mitigating pet food composition.

[0066] In certain embodiments, the method of determine whether a diet improves the oral health of a companion animal comprises obtaining a first plaque sample from a tooth of a companion animal and determining from the first plaque sample an initial relative abundance of each ofpositive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, and positive effect phyla in the first plaque sample. The methods disclosed herein further comprise calculating a first TMGI score, wherein the first TMGI score is equal to the log of the average initial relative abundances of positive effect species, early colonizers, and positive effect species over the average initial relative abundances of negative effect species and late colonizers, according to the formula:

[0067] The methods disclosed herein for determining whether a diet, such as a pet food composition, improves the oral health of a companion animal further comprise administering the diet to the companion animal after obtaining the first plaque sample. In certain embodiments, the diet may be administered to the animal for any period of time, including for at least about 7 days, such as at least about 14 days, at least about 21 days, at least about 28 days, at least about 1 month, at least about 2 months, or at least about 3 months, and in certain embodiments, the diet may be administered after calculation of a first TMGI score and / or after administering a dental prophylaxis cleaning to the companion animal.

[0068] In the methods disclosed herein, after administering the diet to the companion animal for a period of time, the method further comprises obtaining a second plaque sample from the companion animal. In certain embodiments, the second plaque sample may be obtained from the same tooth as the first plaque sample, and in certain embodiments, the second plaque sample may be obtained from a different tooth as the first plaque sample. After obtaining the second plaque sample, the methods disclosed herein further comprise using the second plaque sample to determine a second relative abundance of each of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, and positive effect phyla in the second plaque sample.

[0069] After determining the second relative abundances, the methods disclosed herein further comprise calculating a second TMGI score, wherein the second TMGI score is equal to the log of the average second relative abundances of positive effect species, early colonizers, and positive effect species over the average second relative abundances of negative effect species and late colonizers, according to the formula:TMGI =

[0070] In certain embodiments of the methods disclosed herein, the oral disease-mitigating diet, such as the pet food composition, improves the oral health of the companion animal if the second TMGI score is higher than the first TMGI score. In certain embodiments, the second TGMI score is greater than about -0.9 to about -0.15, such as greater than about -0.10 to about -0.14, or greater than about -0.11 to about -0.13. In certain embodiments, the initial TMGI score is less than or equal to about -0.12, and in certain embodiments, the second TMGI score is greater than or equal to about -0.12. In certain embodiments, the initial TMGI score is less than or equal to -0.12, and the second TMGI score is greater than or equal to -0.12.

[0071] In any of the methods disclosed herein, in certain embodiments the companion animal is a canine, such as a small breed canine. The definition of the term “small breed canine” as used herein indicates a dog that, as a full-grown adult, has a small stature and does not refer to any particular breed or genetic origin of the dog. In certain instances, a small breed canine may weigh, as an adult, less than about 40 pounds, such as less than about 35 pounds, less than about 30 pounds, less than about 25 pounds, less than about 20 pounds, less than about 15 pounds, less than about 10 pounds, or less than about 5 pounds. In certain embodiments, the small breed canine weighs about 3 pounds to about 35 pounds, such as from about 3 pounds to about 30 pounds, about 3 pounds to about 25 pounds, about 3 pounds to about 20 pounds, about 3 pounds to about 15 pounds, about 3 pounds to about 10 pounds, about 3 to about 5 pounds; from about 5 pounds to about 35 pounds, about 5 pounds to about 30 pounds, about 5 pounds to about 25 pounds, about 5 pounds to about 20 pounds, about 5 pounds to about 15 pounds, about 5 pounds to about 10 pounds; or from about 10 pounds to about 35 pounds, about 10 pounds to about 30 pounds, about 10 pounds to about 25 pounds, about 10 pounds to about 20 pounds, or about 10 pounds to about 15 pounds. Solely by way of example, exemplary small-breed canines may include or have characteristics of at least one of the following dog breeds: Yorkshire terrier, chihuahua, Pomeranian, bichon fries, Boston terrier, border terrier, maltese, Cavalier King Charles spaniel, shih tzu, dachshund, pug, Havanese, beagle, Jack Russell terrier, Brussels Griffon, schnauzer, papillon, llasa apso, corgi, Scottish terrier, West Highland terrier, silky terrier, cocker spaniel, sheltie, toy poodle, French bulldog, and / or Norwich terrier. A small breed canine may be a mixture of more than one breed,some of which may he small breed canines and some of which may be considered medium or large breed canines.

[0072] In any of the embodiments disclosed herein, the oral disease or condition may be any oral disease or condition known to afflict companion animals. In certain embodiments, the oral disease or condition is selected from at least one of periodontitis, dental plaque, dental tartar, or gingivitis, and in certain embodiments, the oral disease is gingivitis.Oral Disease-Mitigating Diets and Pet Food Compositions

[0073] In accordance with one aspect of the disclosure, the oral disease-mitigating diet may be a pet food composition, and in certain embodiments, the pet food composition may comprise at least one ingredient believed to treat an oral disease or condition in the companion animal and / or other ingredients known for use in a pet food composition. In certain embodiments of the methods disclosed herein, the pet food composition may be administered together with an additional treatment method, e.g., topical and / or oral medication.

[0074] In certain embodiments, the pet food composition may mitigate an oral disease or condition through either mechanical means, chemical means, or a combination of mechanical and chemical means.

[0075] In certain embodiments, the pet food composition disclosed herein may be a nutritionally complete diet. A “nutritionally complete diet” may be a diet that may include sufficient nutrients for maintenance of normal health of a healthy animal on the diet. In certain aspects, the pet food composition(s) disclosed herein may be a nutritionally complete diet, and in certain embodiments, the pet food composition may not be a nutritionally complete diet, but may be used to supplement an animal’s otherwise nutritionally complete diet, e.g., as a treat or a supplement. In certain embodiments, the pet food composition may be blended with a nutritionally complete diet and / or balanced food diet.

[0076] According to another aspect of the disclosure, provided is an oral disease-mitigating pet food composition that includes a fat source; a protein; fiber; ash; and carbohydrate. For example, a nutritionally complete and balanced pet food composition may comprise: about 0 to about 90%, preferably about 5% to 60%, by weight of carbohydrates; about 5% to about 70%, preferably about 10% to about 60%, more preferably about 20% to about 50%, by weight of protein; about 1% to about 50%, preferably about 2% to about 40%, more preferably about 3% to about 15%, by weight of fat; about 0.1% to about 40%, preferably about 1% to about 30%, more preferably about 15%to about 50%, by weight of total dietary fiber; about 0 to about 15%, preferably about 2% to about 8%, by weight of vitamins and minerals, antioxidants, and other nutrients which support the nutritional needs of the animal.

[0077] Suitable components, such as those listed herein, may be included or excluded from the formulations for the pet food compositions depending on the specific combination of other ingredients and the form of the pet food compositions. In some embodiments, the pet food compositions disclosed herein may be in the form of a standalone pet food, as a supplement to pet food, as a pet food treat, or the like.

[0078] The pet food compositions are formulated to include fat in an amount that may vary, but typically is in the range of about 8 to about 50 wt%, endpoints included, based on the total weight of the pet food composition on a dry matter basis. For example, the pet food composition may include fat in an amount ranging from about 10 to about 50 wt%, about 12 to about 50 wt%, about 14 to about 50 wt%, about 16 to about 50 wt%, about 18 to about 50 wt%, about 20 to about 50 wt%, about 22 to about 50 wt%, about 24 to about 50 wt%; from about 8 to about 40 wt%, about 10 to about 40 wt%, about 12 to about 40 wt%, about 14 to about 40 wt%, about 16 to about 40 wt%, about 18 to about 40 wt%, about 20 to about 40 wt%, about 22 to about 40 wt%, about 24 to about 40 wt%; from about 8 to about 35 wt%, about 10 to about 35 wt%, about 12 to about 35 wt%, about 14 to about 35 wt%, about 16 to about 35 wt%, about 18 to about 35 wt%, about 20 to about 35 wt%, about 22 to about 35 wt%, about 24 to about 35 wt%; about 8 to about 30 wt%, about 10 to about 30 wt%, about 12 to about 30 wt%, about 14 to about 30 wt%, about 16 to about 30 wt%, about 18 to about 30 wt%, about 20 to about 30 wt%, about 22 to about 30 wt%, about 24 to about 30 wt%; from about 8 to about 27 wt%, about 10 to about 27 wt%, about 12 to about 27 wt%, about 14 to about 27 wt%, about 16 to about 27 wt%, about 18 to about 27 wt%, about 20 to about 27 wt%, about 22 to about 27 wt%; from about 8 to about 24 wt%, about 10 to about 24 wt%, about 12 to about 24 wt%, about 14 to about 24 wt%, about 16 to about 24 wt%, about 18 to about 24 wt%, about 20 to about 24 wt%; from about 8 to about 22 wt%, about 10 to about 22 wt%, about 12 to about 22 wt%, about 14 to about 22 wt%, about 16 to about 22 wt%, about 18 to about 22 wt%; from about 8 to about 22 wt%, about 10 to about 22 wt%, about 12 to about 22 wt%, about 14 to about 22 wt%, about 16 to about 22 wt%, about 18 to about 22 wt%; from about 8 to about 20 wt%, about 10 to about 20 wt%, about 12 to about 20 wt%, about 14 to about20 wt%, about 16 to about 20 wt%, including ranges and subranges thereof, based on the total weight of the pct food composition on a dry matter basis.

[0079] The term “fat” generally refers to a lipid or mixture of lipids that may generally be a solid or a liquid at ordinary room temperatures (e.g., 25 °C) and pressures (e.g., 1 atm). In some instances, the fat may be a viscous liquid or an amorphous solid at standard room temperature and pressure. The fat may be incorporated completely within the food composition, deposited on the outside of the pet food composition, or a mixture of the two methods.

[0080] The fat may comprise dietary fats, such as triglycerides. In some embodiments, the triglyceride may comprise about 20 to about 100%, about 40 to about 100%, about 50 to about 100%, about 60 to about 100%, about 70 to about 100%, about 80 to about 100%, about 90 to about 100%, of the total amount of fat in the pet food composition. In further embodiments, the triglyceride comprises about 20 to about 95%, about 40 to about 95%, about 50 to about 95%, about 60 to about 95%, about 70 to about 95%, about 80 to about 95%, about 90 to about 95%, of the total amount of fat in the pet food composition. In additional embodiments, the triglyceride comprises about 20 to about 90%, about 40 to about 90%, about 50 to about 90%, about 60 to about 90%, about 70 to about 90%, about 80 to about 90%, of the total amount of fat in the pet food composition. In yet further embodiment, the triglyceride comprises about 20 to about 80%, about 40 to about 80%, about 50 to about 80%, about 60 to about 80%, about 70 to about 80%, of the total amount of fat in the pet food composition.

[0081] Fat can be supplied by any of a variety of sources known by those skilled in the art, including meat, meat by-products, canola oil, fish oil such as anchovy oil and menhaden oil, and plants. Meat fat sources include poultry fat, turkey fat, pork fat, lard, tallow, and beef fat. Plant fat sources include wheat, flaxseed, rye, barley, rice, sorghum, com, oats, millet, wheat germ, com germ, soybeans, peanuts, and cottonseed, as well as oils derived from these and other plant fat sources such as com oil, soybean oil, cottonseed oil, palm oil, palm kernel oil, linseed oil, canola oil, rapeseed oil, and / or olestra.

[0082] In some cases, the fat in the compositions is crude fat. Crude fat may be included into the compositions in the amounts disclosed above with respect of the total fat, such as from about 8 to about 50 wt%, based on the total weight of the pet food composition on a dry matter basis. In some embodiments, the pet food composition comprises emde fat in an amount of about 10 to about 40 wt%, about 12 to about 35 wt%, about 14 to about 30 wt%, about 16 to about 24 wt%, based onthe total weight of the pet food composition on a dry matter basis. In some cases, it may be preferable that about 50 wt% or more, about 60 wt% or more, about 70 wt% or more, about 80 wt% or more, or about 90 wt% or more of the total fat is obtained from an animal source. Alternatively, about 50 wt% or more, about 60 wt% or more, about 70 wt% or more, about 80 wt% or more, or about 90 wt% or more of the total fat may be obtained from a plant source.

[0083] The pet food compositions typically include protein in an amount ranging from about 15 to about 55 wt%, based on the total weight of the pet food composition on a dry matter basis. In some instances, the total amount of protein in the pet food composition is in a range from about 15 to about 50 wt%, about 15 to about 48 wt%, about 15 to about 46 wt%, about 15 to about 44 wt%, about 15 to about 42 wt%, about 15 to about 40 wt%, about 15 to about 38 wt%, about 15 to about 36 wt%, about 15 to about 34 wt%; from about 20 to about 55 wt%, about 20 to about 50 wt%, about 20 to about 48 wt%, about 20 to about 46 wt%, about 20 to about 44 wt%, about 20 to about 42 wt%, about 20 to about 40 wt%, about 20 to about 38 wt%, about 20 to about 36 wt%, about 20 to about 34 wt%; from about 25 to about 55 wt%, about 25 to about 50 wt%, about 25 to about 48 wt%, about 25 to about 46 wt%, about 25 to about 44 wt%, about 25 to about 42 wt%, about 25 to about 40 wt%, about 25 to about 38 wt%, about 25 to about 36 wt%, about 25 to about 34 wt%; from about 30 to about 55 wt%, about 30 to about 50 wt%, about 30 to about 48 wt%, about 30 to about 46 wt%, about 30 to about 44 wt%, about 30 to about 42 wt%, about 30 to about 40 wt%, about 30 to about 38 wt%, or about 30 to about 36 wt%, including ranges and subranges therebetween, based on the total weight of the pet food composition on a dry matter basis.

[0084] The protein of the pet food composition comprises one or more amino acids selected from tryptophan, taurine, histidine, carnitine, carnosine, alanine, cysteine, arginine, methionine (including DL-methionine, D-methionine, and L-methionine), tryptophan, lysine, asparagine, aspartate (aspartic acid), phenylalanine, valine, threonine, isoleucine, histidine, leucine, glycine, glutamine, tyrosine, homocysteine, ornithine, citruline, glutamate (glutamic acid), proline, and / or serine, and a combination of two or more thereof. The pet food composition may comprise two or more amino acids. For instance, the pet food composition may include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more amino acids. In some embodiments, the pet food composition includes glycine and proline, and optionally one or more additional amino acids.

[0085] In some cases, the one or more amino acid(s) may comprise essential amino acids. Essential amino acids arc amino acids that cannot be synthesized de novo, or in sufficient quantities by an organism and thus must be supplied in the diet. Essential amino acids vary from species to species, depending upon the organism's metabolism. For example, it is generally understood that the essential amino acids for dogs and cats (and humans) are phenylalanine, leucine, methionine, lysine, isoleucine, valine, threonine, tryptophan, histidine and arginine. In addition, taurine, while technically not an amino acid but a derivative of cysteine, is an essential nutrient for cats.

[0086] Protein may be supplied by any of a variety of sources known by those of ordinary skill in the art including plant sources, animal sources, microbial sources or a combination of these. For example, animal sources may include meat, meat-by products, seafood, dairy, eggs, etc. Meats, for example, may include animal flesh such as poultry, fish, and mammals including cattle, pigs, sheep, goats, and the like. Meat by-products may include, for example, lungs, kidneys, brain, livers, stomachs and intestines. Plant protein includes, for example, soybean, cottonseed, and peanuts. Microbial sources may be used to synthesize amino acids (e.g., lysine, threonine, tryptophan, methionine) or intact protein such as protein from sources listed below.

[0087] Examples of protein or protein ingredients may comprise chicken meals, chicken, chicken by-product meals, lamb, lamb meals, turkey, turkey meals, beef, beef by-products, viscera, fish meal, enterals, white fish, venison, soybean meal, soy protein isolate, soy protein concentrate, com gluten meal, corn protein concentrate, distillers dried grains, and / or distillers dried grain solubles and single-cell proteins, for example yeast, algae, and / or bacteria cultures.

[0088] The protein can be intact, completely hydrolyzed, or partially hydrolyzed. The protein content of foods may be determined by any number of methods known by those of skill in the art, for example, as published by the Association of Official Analytical Chemists in Official Methods of Analysis (“OMA”), method 988.05. The amount of protein in a composition disclosed herein may be determined based on the amount of nitrogen in the composition according to methods familiar to one of skill in the art.

[0089] The pet food compositions are typically formulated to include fiber in an amount from about 10 to about 60 wt%, based on the total weight of the pet food composition on a dry matter basis. For instance, the amount of fiber present in the pet food composition may be from about 10 to about 55 wt%, about 10 to about 50 wt%, about 10 to about 45 wt%, about 10 to about 40 wt%, about 10 to about 35 wt%, about 10 to about 30 wt%; from about 15 to about 60 wt%, about 15 toabout 55 wt%, about 15 to about 50 wt%, about 15 to about 45 wt%, about 15 to about 40 wt%, about 15 to about 35 wt%, about 15 to about 30 wt%; from about 20 to about 60 wt%, about 20 to about 55 wt%, about 20 to about 50 wt%, about 20 to about 45 wt%, about 20 to about 40 wt%, about 20 to about 35 wt%, about 20 to about 30 wt%; from about 25 to about 60 wt%, about 25 to about 55 wt%, about 25 to about 50 wt%, about 25 to about 45 wt%, about 25 to about 40 wt%, about 25 to about 35 wt%; from about 30 to about 60 wt%, about 30 to about 55 wt%, about 30 to about 50 wt%, about 30 to about 45 wt%, about 30 to about 40 wt%; from about 35 to about 60 wt%, about 35 to about 55 wt%, about 35 to about 50 wt%, about 35 to about 45 wt%; from about 40 to about 60 wt%, about 40 to about 55 wt%, about 40 to about 50 wt%; from about 45 to about 60 wt%, about 45 to about 55 wt%; from about 50 to about 60 wt%, including ranges and subranges thereof, based on the total weight of the pet food composition on a dry matter basis.

[0090] The total amount of fiber present in the pet food composition generally comprises an amount of crude fiber and dietary fiber. The amount of crude fiber and / or dietary fiber may be present in the pet food compositions in any of the above amounts disclosed for the total amount of fiber. Crude fiber includes indigestible components contained in cell walls and cell contents of plants such as grains, e.g., hulls of grains such as rice, com, and beans.

[0091] Dietary fiber refers to components of a plant that are resistant to digestion by an animal's digestive enzymes. Dietary fiber includes soluble fiber and insoluble fiber. Soluble fibers are resistant to digestion and absorption in the small intestine and undergo complete or partial fermentation in the large intestine, e.g., beet pulp, guar gum, chicory root, psyllium, pectin, blueberry, cranberry, squash, apples, oats, beans, citrus, barley, or peas. Insoluble fibers can be supplied by any of a variety of sources, including, for example, cellulose, whole-wheat products, wheat oat, com bran, flax seed, grapes, celery, green beans, cauliflower, potato skins, fruit skins (e.g., pear skin), vegetable skins, peanut hulls, almond shell, walnut shell, pecan shell, citrus pulp, beet pulp, and soy fiber. In some embodiments, the dietary fiber may be chosen from pecan shell, citms pulp, beet pulp, pear skin, and a combination of two or more thereof. Cmde fiber includes indigestible components contained in cell walls and cell contents of plants such as grains, for example, hulls of grains such as rice, com, and beans.

[0092] The pet food composition may further comprise ash. As described herein, ash consists of compounds that are not organic or water, generally produced by combustion of biological materials. The ash may be present in the pet food composition in an amount ranging from about 1to about 10 wt%, based on the total weight of the food composition on a dry weight basis, including all amounts and sub-ranges there -between. In some embodiment, the ash may be present in the food composition in an amount ranging from about 1 to about 8 wt%, about 1 to about 6 wt%, about 1 to about 5 wt%, about 1 to about 4 wt%, about 1 to about 3 wt%, about 1 to about 2 wt%; from about 2 to about 10 wt%, about 2 to about 8 wt%, about 2 to about 6 wt%, about 2 to about 5 wt%, about 2 to about 4 wt%; from about 3 to about 10 wt%, about 3 to about 8 wt%, about 3 to about 6 wt%, about 3 to about 5 wt%; from about 4 to about 10 wt%, about 4 to about 8 wt%, about 4 to about 6 wt%; from about 5 to about 10 wt%, about 5 to about 8 wt%, or any range or subrange thereof, based on the total weight of the food composition on a dry weight basis.

[0093] The pet food composition may include carbohydrates, e.g., in an amount up to about 65 wt%, based on the total weight of the pet food composition on a dry matter basis. The term “carbohydrate” as used herein includes polysaccharides (e.g., starches and dextrins) and sugars (e.g., sucrose, lactose, maltose, glucose, and fructose) that are metabolized for energy when hydrolyzed. One skilled in the ail could manipulate the texture of the final product by properly balancing carbohydrate sources. For example, short chain polysaccharides lend to be sticky and gluey, and longer chain polysaccharides are less sticky and gluey than the shorter chain; the desired texture of this hybrid food is achieved by longer chain polysaccharide and modified starches such as native or modified starches, cellulose and the like. The carbohydrate mixture may additionally comprise optional components such as added salt, spices, seasonings, vitamins, minerals, flavorants, colorants, and the like. The amount of the optional components is at least partially dependent on the nutritional requirements for different life stages of animals.

[0094] Carbohydrates can be supplied by any of a variety of sources known by those skilled in the art, including, but not limited to, oat fiber, cellulose, peanut hulls, beet pulp, parboiled rice, cornstarch, corn gluten meal, cereal, and sorghum. Grains supplying carbohydrates can include, but are not limited to, wheat, durum, semolina, corn, barley, and rice. In certain embodiments, the carbohydrate component comprises a mixture of one or more carbohydrate sources. Carbohydrates content of foods can be determined by any number of methods known by those of skill in the art.

[0095] Generally, carbohydrate percentage can be calculated as nitrogen free extract (“NFE”), which can be calculated as follows: NFE %=100%-(moisture %)-(protein %)-(fat %)-(ash %)- (crude fiber %) The amount of carbohydrate, e.g., calculated as NFE, present in the composition may be from an amount up to about 65 wt%, an amount up to about 60 wt%, an amount up toabout 55 wt%, an amount up to about 50 wt%, an amount up to about 45 wt%, an amount up to about 40 wt%, an amount up to about 35 wt%, an amount up to about 30 wt%, an amount up to about 25 wt%, an amount up to about 20 wt%, an amount up to about 15 wt%, an amount up to about 10 wt%, an amount up to about 5 wt%; about 1 to about 65 wt%, about 1 to about 55 wt%, about 1 to about 50 wt%, about 1 to about 45 wt%, about 1 to about 40 wt%, about 1 to about 35 wt%; about 1 to about 30 wt%, about 1 to about 25 wt%, about 1 to about 20 wt%, about 1 to about 15 wt%, about 1 to about 10 wt%, about 1 to about 5 wt%; about 5 to about 65 wt%, about 5 to about 55 wt%, about 5 to about 50 wt%, about 5 to about 45 wt%, about 5 to about 40 wt%, about 5 to about 35 wt%; about 5 to about 30 wt%, about 5 to about 25 wt%, about 5 to about 20 wt%, about 5 to about 15 wt%; about 10 to about 65 wt%, about 10 to about 55 wt%, about 10 to about 50 wt%, about 10 to about 45 wt%, about 10 to about 40 wt%, about 10 to about 35 wt%; about 10 to about 30 wt%, about 10 to about 25 wt%; about 15 to about 65 wt%, about 15 to about 55 wt%, about 15 to about 50 wt%, about 15 to about 45 wt%, about 15 to about 40 wt%, about 15 to about 35 wt%; about 15 to about 30 wt%; about 20 to about 65 wt%, about 20 to about 55 wt%, about 20 to about 50 wt%, about 20 to about 45 wt%, about 20 to about 40 wt%, about 20 to about 35 wt%; about 25 to about 65 wt%, about 25 to about 55 wt%, about 25 to about 50 wt%, about 25 to about 45 wt%, about 25 to about 40 wt%, about 25 to about 35 wt%; about 30 to about 65 wt%, about 30 to about 55 wt%, about 30 to about 50 wt%, about 30 to about 45 wt%; about 35 to about 65 wt%, about 35 to about 55 wt%, about 35 to about 50 wt%; about 40 to about 65 wt%, about 40 to about 55 wt%, about 45 to about 65 wt%, about 45 to about 55 wt%; or about 50 to about 65 wt%, including ranges and subranges thereof, based on the total weight of the pet composition on a dry matter basis.

[0096] In certain embodiments, the pet food composition comprises moisture. The moisture may be present at various amounts or concentrations. In one embodiment, moisture may be present in an amount of from about 3 to about 20 wt%, based on the total weight of the pet food composition. For example, moisture may be present in an amount of about 3 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, about 10 wt%, about 10.5 wt%, about 11 wt%, about 11.5 wt%, about 12 wt%, about 12.5 wt%, about 13 wt%, about 13.5 wt%, about 14 wt%, about 14.5 wt%, or about 15 wt%, based on the total weight of the pet food composition. In another example, moisture may be present in an amount of from about 6% to about 12%, about 9% to about 13%, about 9% to about11 %, or about 9% to about 13%, based on the total weight of the pet food composition. In certain embodiments, moisture is present in an amount of about 5% to about 12%, about 6% to about 11%, or about 7% to about 10%, based on the total weight of the pet food composition. In further embodiments, moisture is present in an amount of about 65% to about 85%, about 60% to about 80%, or about 60% to about 75%, based on the total weight of the pet food composition.

[0097] The pet food compositions disclosed herein may be wet or dry compositions, and the ingredients can be either incorporated into the food composition and / or on the surface of any composition component, such as, for example, by spraying, agglomerating, dusting, or precipitating on the surface. Additionally, the pet food compositions may be formulated and produced to be in various forms and / or consistencies. For instance, the pet food compositions may, for example, be a dry, moist or semi-moist animal food composition. “Semi-moist” refers to a food composition containing from about 25 to about 35% moisture. “Moist” food refers to a food composition that has a moisture content of about 60 to 90% or greater. “Dry” food refers to a food composition with about 3 to about 12% moisture content and is often manufactured in the form of small bits or kibbles.

[0098] The food products may also include components of more than one consistency, for example, soft, chewy meat-like particles or pieces as well as kibble having an outer coating and an inner “core” component. In some embodiments, the pet food compositions may be in the form of a kibble or food kibble. As used herein, the term “kibble” or “food kibble” refers to a particulate pellet, e.g., like a component of feline or canine feeds. In some embodiments, a food kibble has a moisture, or water, content of less than 15% by weight. Food kibbles may range in texture from hard to soft. Food kibbles may range in internal structure from expanded to dense. Food kibbles may be formed by an extrusion process or a baking process. In non-limiting examples, a food kibble may have a uniform internal structure or a varied internal structure. For example, a food kibble may include a core and a coating to form a coated kibble. It should be understood that when the term “kibble” or “food kibble” is used, it can refer to an uncoated kibble or a coated kibble.

[0099] The composition of the present disclosure can additionally comprise other additives in amounts and combinations familiar to one of skill in the art. Such additives should be present in amounts that do not impair the purpose and effect provided by the disclosure. Examples of additives include substances with a stabilizing effect, organoleptic substances, processing aids, and substances that provide nutritional benefits.

[0100] Stabilizing substances may include, by way of example, substances that tend to increase the shelf life of the pct food composition. Other examples of other such additives potentially suitable for inclusion in the compositions of the disclosure include, for example, preservatives, antioxidants, synergists and sequestrants, packaging gases, stabilizers, emulsifiers, thickeners, gelling agents, and humectants. Examples of emulsifiers and / or thickening agents include gelatin, cellulose ethers, starch, starch esters, starch ethers, and modified starches. Additives for coloring, palatability, and nutritional purposes can include colorants, salts (including, but not limited to, sodium chloride, potassium citrate, potassium chloride, and other edible salts), vitamins, minerals, and flavoring. Other additives can include glucosamine, chondroitin sulfate, vegetable extracts, herbal extracts, etc.

[0101] The concentration of such additives in the pet food composition typically can be up to about 5 wt%, based on the total weight of the pet food composition on a dry matter basis. For example, the additives may be present in an amount from about 0.01 to about 5 wt%, about 0.01 to about 4 wt%, about 0.01 to about 4 wt%, about 0.01 to about 3 wt%, about 0.01 to about 2 wt%, about 0.01 to about 1 wt%; about 0.1 to about 5 wt%, about 0.1 to about 4 wt%, about 0.1 to about 4 wt%, about 0.1 to about 3 wt%, about 0.1 to about 2 wt%, about 0.1 to about 1 wt%; about 1 to about 5 wt%, about 1 to about 4 wt%, about 1 to about 4 wt%, about 1 to about 3 wt%, about 1 to about 2 wt%; about 2 to about 5 wt%, about 2 to about 4 wt%, about 2 to about 4 wt%, about 2 to about 3 wt%; about 3 to about 5 wt%, about 3 to about 4 wt%; or about 4 to about 5 wt%, based on the total weight of the pet food composition on a dry matter basis. In some embodiments, the concentration of such additives (particularly where such additives are primarily nutritional balancing agents, such as vitamins and minerals) is from about 0 to about 2.0% by weight, based on the total weight of the pet food composition on a dry matter basis. The amount of additives comprising vitamins may be in addition to the amount of vitamin B discussed above. In some embodiments, the concentration of such additives (again, particularly where such additives are primarily nutritional balancing agents) is from about 0 to about 1.0% by weight, based on the total weight of the pet food composition on a dry matter basis. Although the list of foregoing additives may be potentially suitable in some embodiments, one or more of the foregoing additives may be excluded from other embodiments of the pet food composition.

[0102] In certain embodiments, the pet food composition is an extruded food product having a solid, uniform, expanded composition having fibrous striations extending transversely through amatrix microstructure. The food product, when chewed by the animal, unlike baked on other extruded products, docs not crumble but instead fractures along the matrix striations and hence offers the animal the intended teeth cleansing benefits stemming from the mechanical cleansing and other abrasive contacts with the separated matrix layers in the chewed striated product. In addition, as the striated fibrous product does not crumble as the animal chews on the product, the product clings in adhered contact with the teeth for an extended time, prolonging the mechanical dental cleansing action. In certain embodiments, the expanded, striated pet food composition may have a density of about 10 to about 35 lbs / ft3, and a nutritional content as follows;Ingredient % by WeightCarbohydrate about 35 to about 70Protein about 10 to about 35Fat about 10 to about 20Fiber about 10 to about 25Nutritional additives about 0.01 to about 0.40

[0103] In certain embodiments of the pet food composition, the moisture content of the expanded extrudate is adjusted to the range of about 5 to about 11%. At moisture levels below 5%, the composition may become too hard to be easily chewed by the animal, and at moisture levels above 11%, the hardness decreases and the mechanical cleaning efficacy of a striated product may become compromised. As disclosed in U.S. 5,431,927, incorporated by reference herein, maximum mechanical cleaning efficacy of the striated product may be achieved at a density of about 20 to about 30 pounds per cubic foot and a fiber level of about 15 to about 20% by weight. At these fiber levels, the composition has the desired degree of striation to achieve the desired degree of self-adhesion and tooth clinging characteristics.

[0104] To further improve palatability and energy (caloric) levels, the dried, extruded striated product may be coated with about 1 to about 13% additional fat.

[0105] While the striated product disclosed herein can be of several shapes, the shapes that may be the most desirable for mechanical cleaning efficacy include a cylindrical or disc shape. In certain embodiments, the pet food composition comprises disc-shaped pellets having a thickness of about 0.32 to about 0.70 inches and a diameter of about 0.7 to about 1.2 inches, as disclosed in U.S. 5,431,927, incorporated by reference herein.

[0106] In specific embodiments, the pet food compositions and food products are formulated to address specific nutritional differences between species and breeds of animals, as well as one of more of the attributes of the animal. For example, cat foods arc typically formulated based upon the life stage, age, size, weight, body composition, and breed.

[0107] Sources of proteins, carbohydrates, fats, vitamins, minerals, balancing agents, and the like, suitable for inclusion in the pet food compositions, and particularly in the food products to be administered in methods provided herein, may be selected from among those conventional materials known to those of ordinary skill in the art.

[0108] The pet food composition may be produced by various methods to achieve the desired pet food composition or desired form for the pet food composition. For example, dry food may be baked or extruded, then cut into individual shaped portions, such as kibbles. In some embodiments, the pet food composition may be prepared in a canned or wet form using conventional food preparation processes known to those of ordinary skill in the art. Typically, ground animal proteinaceous tissues are mixed with the other ingredients, such as cereal grains, suitable carbohydrate sources, fats, oils, and balancing ingredients, including special purpose additives such as vitamin and mineral mixtures, inorganic salts, cellulose, beet pulp and the like, and water in an amount sufficient for processing. The ingredients are mixed in a vessel suitable for heating while blending the components. Heating the mixture is carried out using any suitable manner, for example, direct steam injection or using a vessel fitted with a heat exchanger. Following addition of all of the ingredients of the formulation, the mixture may be heated to a temperature of from 50 °F to 212 °F. Although temperatures outside this range can be used, they may be commercially- impractical without the use of other processing aids. When heated to the appropriate temperature, the material will typically be in the form of thick liquid, which is dispensed into cans. A lid is applied and the container is hermetically sealed. The sealed can is then placed in convention equipment designed for sterilization of the contents. Sterilization is usually accomplished by heating to temperatures of greater than 230 °C for an appropriate time depending on the temperature used, the nature of the composition, and related factors. The pet food compositions and food products of the present disclosure can also be added to or combined with food compositions before, during, or after their preparation.

[0109] In some embodiments, the food products may be prepared in a dry form using convention processes known to those of ordinary skill in the art. Typically, dry ingredients, including driedanimal protein, plant protein, grains and the like are ground and mixed together. Liquid or moist ingredients, including fats, oils water, animal protein, water, and the like arc added combined with the dry materials. The specific formulation, order of addition, combination, and methods and equipment used to combine the various ingredients can be selected from those known in the art. For example, in certain embodiments, the resulting mixture is process into kibbles or similar dry pieces, which are formed using an extrusion process in which the mixture of dry and wet ingredients is subjected to mechanical work at high pressure and temperature, forced through small openings or apertures, and cut off into the kibbles, e.g., with a rotating knife. The resulting kibble can be dried and optionally coated with one or more topical coatings comprising, e.g., flavors, fats, oils, powdered ingredients, and the like. Kibbles may also be prepared from dough by baking, rather than extrusion, in which the dough is placed into a mold before dry-heat processing.

[0110] In preparing a composition, any ingredient generally may be incorporated into the composition during the processing of the formulation, e.g., during and / or after mixing of the other components of the composition. Distribution of these components into the composition can be accomplished by conventional means. In certain embodiments, ground animal and / or poultry proteinaceous tissues are mixed with other ingredients, including nutritional balancing agents, inorganic salts, and may further include cellulose, beet pulp, bulking agents and the like, along with sufficient water for processing.Kits

[0111] Further provided herein are kits for use in the methods disclosed according to any of the aforementioned embodiments. In certain embodiments, the kits disclosed herein may be used to identify a companion animal that is at risk of an oral disease or condition. In certain embodiments, the kit comprises a device for collection a plaque sample, e.g., a subgingival plaque sample, from a tooth of a companion animal. The kit may further comprise or may alternatively components for detecting a relative abundance of each of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, positive effect phyla, and negative effect phyla in the plaque sample.

[0112] In certain embodiments disclosed herein, the components for detecting a relative abundance are chosen from components for next generation sequencing technology, preferably by 16s rRNA amplicon sequencing and metagenomics, targeted multiplex qPCR technology, or customized microarray technology.

[0113] In certain embodiments, the kits disclosed herein comprise at least one component for detecting a relative abundance of positive effect microbiotic species, and in certain embodiments, the positive effect microbiotic species are selected from at least one of Moraxella osloensis, Acinetobacter johnsonii. Moraxellaceae unclassified, Haemophilus unclassified, Bergeyella HMT 22, Streptococcus unclassified, and Solobacterium moorei. In certain embodiments, the kits disclosed herein comprise at least one component for detecting a relative abundance of negative effect microbiotic species, and in certain embodiments, the negative effect microbiotic species are selected from at least one of Treponema denticola, Fusobacterium nucleatum_subsp_vincentii, Treponema HMT 490, Ruminococcaceae unclassified, Mollicutres_[G-2] bacterium HMT 906, Peptostreptococcaceae_[Xl][G-8] bacterium HMT 382, Treponema HMT 239, Escherichia coli, Treponema HMT 238, Fastidiosipila sanguinis, and Mycoplasma lipophilum.

[0114] In certain embodiments of the disclosure, the kits comprise at least one component for detecting a relative abundance of early colonizers, and in certain embodiments, the early colonizers are selected from at least one of Moraxella nonliquifaciens, Bergeyella HMT 422, Capnocytophaga unclassified, Escherichia coli, Mycoplasma hominis, Neisseria weaveri, Abiotrophia defectiva, Streptococcus unclassified, Haemophilus unclassified, Moraxella osloensis, Bergeyalla unclassified, and Mycoplasma lipophilum. In certain embodiments of the disclosure, the kits comprise at least one component for detecting a relative abundance of late colonizers, and in certain embodiments, the late colonizers are selected from at least one of Treponema HMT 249, Filifactor alocis, Porphyromonas HMT 285, Treponema parvum, Treponema HMT 490, Prevotella intermedia, Treponema HMT 951, Treponema HMT 257, Treponema HMT 227, Treponema HMT 234, Treponema HMT 238, Treponema HMT 237, Fusobacterium nucleatum vincentii, Tannerella forsythia, Treponema denticola, Porphyromonas gingivalis, Treponema HMT 235, Treponema HMT 239, Treponema HMT 258, Campylobacter rectus, and Treponema unclassified.

[0115] In certain embodiments, the kit comprises at least one component for detecting a relative abundance of positive effect phyla, and in certain embodiments, the positive effect phyla are selected from at least one of Actinobacteria and Proteobacteria.EXAMPLES

[0116] The following Examples are provided to further describe certain embodiments of the disclosure and are not to be construed to limit the scope of the disclosure.Example 1 - Canine Survey Data

[0117] Scoring and sample collection: For the caninc colony survey data analysis, subgingival plaque samples were collected from dogs in their annual dental examination. For a given dog, two subgingival plaque samples were collected, with one from a healthy (best) tooth and a second from an unhealthy (worst) tooth. In some instances, for a dog that did not have an unhealthy tooth, subgingival plaque samples were collected from two different healthy teeth. Healthy and unhealthy teeth were confirmed based on individual gingivitis scores. The dental grading and charting for all animals used in this survey data sample collection was performed by a single dental assistant.

[0118] Tooth gingivitis scores were measured using a modified scoring method, as disclosed in Loe, H. & Silness, J, Periodontal Disease in Pregnancy. I. Prevalence and Severity, Acta Odontol Scand. 1963, 21:533-51. Briefly, the scoring method was performed on the mesial, buccal, and distal portions of a given tooth by following the metrics: 0 = normal gingiva; 0.5 = mild inflammation with slight redness; 1 = moderate inflammation and redness, but no bleeding on probing; 2 = moderate inflammation with severe redness and bleeding on probing; and 3 = severe inflammation and redness, edema, ulceration, and spontaneous bleeding on probing. The scores were summed to represent the individual tooth gingivitis score on a scale of 0-9. A summed individual tooth gingivitis score of less than 3 was considered healthy, and 3 or more was considered unhealthy.

[0119] Besides individual tooth gingivitis scores, whole mouth gingivitis scores and whole mouth plaque scores were also measured for each dog. Whole mouth gingivitis scores were graded as light, medium, and heavy using the following criteria: light = little to no inflammation, no bleeding on probing on majority to all gingival surfaces; medium = moderate inflammation with moderate redness, bleeding on probing on majority to all gingival surfaces; and heavy = severe inflammation with marked redness, hypertrophy, and / or ulceration, immediate bleeding on probing on majority to all gingival surfaces.

[0120] Similarly, whole mouth plaque scores were graded as light, medium, and heavy using the following criteria: light = light to no coverage and light intensity (stained with eosin) on all teeth; medium = moderate to full coverage and moderate intensity (stained with eosin) on all teeth; and heavy = moderate to full coverage and heavy intensity (stained with eosin) on all teeth.

[0121] In addition, scores for each tooth recession (i.e., measurement of tooth root exposure caused by loss of gum tissue and / or retraction of the gingival margin from the crown of the tooth),pockets (measurement of pathologically deepened gingival sulcus occurring secondary to coronal movement of the gingival margin), and attachment loss (measurement of pathological detachment of collagen fibers from the cemental surface with the concomitant apical migration of the junctional or pocket epithelium onto the root surface) scores were measured from the tooth subgingival plaque samples collected.

[0122] In total, 692 subgingival plaque samples were collected from 347 dogs, including mixed genders (168 male and 179 female), intact, neutered, or spayed dogs. The age of the dogs ranged from 1.9 years to 14.8 years. The time period ranged from 0.3 years to 1.9 years between the last dental prophylaxis exam to the current dental prophylaxis exam for grading and subgingival plaque sample collection.

[0123] Distribution of subgingival plaque analysis along with individual tooth scores and whole mouth plaque scores - metadata analysis: Tooth gingivitis scores ranges from 0 to 6 in the canine dental survey study discussed above in Example 1. However, as shown in Figure 1, there was a clear distinction showing that gingivitis scores less than 3 were considered as the healthy (T2, best) tooth and gingivitis scores at 3 or more were considered unhealthy (Tl, worst) tooth.

[0124] In addition, tooth gingivitis scores were plotted against whole mouth plaque scores, but, as shown in Figure 2, there was no clear trend of the tooth gingivitis score associating with whole mouth plaque score grading. Both higher (unhealthy, greater than or equal to 3) and lower (healthy, less than 3) tooth gingivitis scores were represented equally regardless of the whole mouth plaque score grading of light, medium, and heavy. See Figure 2. These data suggest that overall plaque building was not a causal factor for gingivitis, which is contrary to well-accepted understanding in the art. However, the presence of specific bacterial species or the imbalance of subgingival microbiome composition has the potential to trigger gum inflammation and gingivitis. Further, the tooth gingivitis scores were plotted against the scores of recession, pocket, and attachment loss. Interestingly, the higher recession, pocket, and attachment loss scores were associated with tooth gingivitis scores, and the presence of specific bacterial species or an imbalance of subgingival microbiome composition has the potential to lead to inflammation and gingivitis. Further, the categorization of healthy versus unhealthy teeth based on the gingivitis scores was corroborated by other measures such as recession, pocket, and attachment loss scores.Example 2 - Data Analysis, Results, and Calculation of TMGI Scores

[0125] Canine Dental Survey Subgingival Microbiome Data Filtering and Analysis: Tooth subgingival plaque microbiomc data were analyzed at the species level. Only the operational taxonomic unit (OTU) species present in at least 70% of all the samples were considered in the statistical analysis. The individual operational taxonomic unit (OTU) counts were analyzed by a negative binomial mixed model to study how age, time between the last current dental prophylaxis, number of diets fed ten days prior to the dental survey, and tooth gingivitis score affect the relative abundance of each OTU. All p values were adjusted for false discovery rate (FDR) using the Benjamini-Hochberg procedure. Principal coordinate analysis plots based on the Manhattan distance were made to visualize the proximity of the subgingival microbial compositions. All the statistical microbiome analyses were carried out in R-4.2.3.

[0126] Tooth Gingivitis Condition Classification Modeling: Considering the OTU results from the negative binomial mixed models, relevant literature appearance, and prevalence in the internal oral microbiome studies, 54 species were selected to go into the classification modeling. Five groups of bacteria, including as Early Colonizers, Late Colonizers, and three phyla (Actinobacteria, Proteobacteria, and Spirochaeles) were included in the modeling, as well as eleven blood chemistry variables, thirteen disease indicators, and age.

[0127] The modeling was done in Dataiku to predict the binary tooth gingivitis condition (“healthy” if the tooth gingivitis score was less than 3 and “unhealthy” if it was 3 or greater). The canine dental survey study data were randomly split 80 / 20 to the training and test sets. The training set was used for model training, and a five-fold cross-validation was used for hyperparameter tuning. The test set was used to evaluate the model’s performance. Random forest (RF), logistic regression, LightGBM, and XGBoost algorithms were used to find the best performing model based on the ROC AUC metric.

[0128] Subgingival microbiome analyses: Principal coordinate analysis plots showed no obvious trend in subgingival microbiome compositions for whole mouth plaque scores and whole mouth gingivitis scores. However, a trend was observed for the individual tooth gingivitis scores. Higher tooth gingivitis scores trended with the positive side of the first axis, and lower tooth gingivitis scores trended with the negative side (variances explained: 23.1%). This data suggests that subgingival microbiome composition potentially plays a causal role in inducing gingivitis. It’s reasonable that subgingival sulcus creates an anaerobic environment compared to supragingival, which helps to proliferate anaerobic bacteria, including pathobionts (also considered latecolonizers). The persistence of pathobionts can trigger inflammation in gingival tissue (gingivitis), impact the host immune response, which further disturbs the plaque microbiomc balance and eventually leads to gingivitis, to chronic gingivitis, and progression into irreversible periodontitis. In addition, based on the literature, the bacterial species were listed as early colonizers and late colonizers to calculate the early-to-late colonizer ratio in the dental survey data, which provided an indicator of microbial composition balance. Overall, 12 bacterial species were listed as early colonizers, and 21 bacterial species were listed as late colonizers. Early colonizers included the species Moraxella, Bergeyella, Capnocytophaga, Neisseria, Escherichia, and Haemophilus. Similarly, late colonizers included the species Treponema, Filifactor, Porphyromonas, Prevotella, Fusobacterium, Tannerella, and Campylobacter. It has been demonstrated that these late colonizers serve as a predictive cluster with an increased probability of having a periodontal site in a human patient with chronic periodontitis.

[0129] From the negative binomial mixed model results, significant tooth gingivitis score effects were found in 90 bacterial species. The effect coefficients were grouped by phylum, and their densities were plotted. Those 90 bacterial species belonged to nine different phyla, where the tooth gingivitis score was positively associated with the species in the Spirochaetes phylum, and negatively associated with the species in the Proteobacteria and Actinobacteria phyla.

[0130] Modeling for subgingival microbiome biomarkers: For the prediction outcome, tooth gingivitis scores were dichotomized into two tooth gingivitis conditions: healthy, if tooth gingivitis scores were less than 3, and unhealthy, if tooth gingivitis scores were 3 or greater than 3. This cutoff was also based on other measurements, such as recession, pocket, and attachment loss scores. Recession, pocket, and attachment loss scores were measurable when the given tooth gingivitis score was 3 or above. Among the four models, the best performing model was random forest, and the model had a ROC AUC of 74.5% (0.745), accuracy of 74% (0.743), precision of 73% (0.731), and recall of 74% (0.742).

[0131] The top nineteen features (excluding the early-to-late colonizers ratio) from the best performing model (random forest) and the top features from the second best performing model (logistic regression) were identified and combined for a total of 22 features, after excluding overlap. These 22 features were used for further canine tooth microbiome gingivitis index development. The majority of those features were highly abundant in healthy teeth (low toothgingivitis scores less than 3), except three were highly abundant in unhealthy teeth (higher tooth gingivitis scores of 3 and greater than 3).

[0132] The features that were highly abundant in unhealthy teeth were as follows:Bacteria; Firmicutes; Clostridia; Clostridiales; Peptostreptococcaceae_[XI]; Peptostreptococcaceae_[XI][G-8]; bacterium_HMT_382 (OTU 058),Bacteria; Spirochetes; Spirochaetia; Spirochaetales; Spirochaetaceae; Treponema; sp._HMT_239 (OTU 060); andBacteria; Spirochaetes; Spirochaetia; Spirochaetales; Spirochaetaceae; Treponema; denticola (OTU 006).

[0133] The rest of the features abundant in the healthy teeth included species belonging to various genera considered early colonizers. However, the top four among 20 included the following:Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Moraxellaceae; Moraxellaceae_unclassified; Moraxellaceae_unclassified (OTU 014);Bacteria; Proteobacteria; Gammaproteobacteria; Pasteurellales; Pasteurellaceae; Haemophilus; Haemophilus_unclassified (OTU 017);Bacteria; Firmicutes; Bacilli; Lactobacillales; Streptococcaceae; Streptococcus; Streptococcus_unclassified (OTU 054); and early colonizers and early-to-late colonizers ratio (E2L).

[0134] Development of Canine Tooth Microbiome Gingival Index ( CTMG1): CTMGI was created by using the most important 22 features from the best performing (random forest) and the second best performing (logistic regression) models. These features were determined to be either positive or negative based on their effects on the tooth gingivitis condition outcome, and arc set forth below in Table 1.

[0135] Table 1 - Top 22 Features Identified by Random Forest and Logistic Regression Models

[0136] The canine TMGI was calculated using the formula TMGI = log[average relative abundance (positive features) / average relative abundance (negative features)].

[0137] The classification cutoff for the TMGI was set at -0.12 to maximize the sum of sensitivity and specificity. It provided a ROC AUC of 0.761, sensitivity of 0.701, and specificity of 0.752. TMGI offers an approach to classifying the tooth gingivitis condition that is easier than the random forest model, with a similar performance. As disclosed herein, if the TMGI is greater than about -0.12, the tooth gingivitis condition is healthy. If the TMGI is less than or equal to about -0.12, the tooth gingivitis condition is unhealthy.Example 3 - Ad Hoc Feeding Study Shows an Increasing Canine TMGI Score

[0138] Foods used in the study: Two foods were used in this study, a control food (Purina Dog Chow Complete Adult with real chicken, dry) and a test food (Hill’s Pet Nutrition PD Canine Adult t / d Small Bites, dry).

[0139] Experimental Feeding Design: The study was performed on a total of 40 clinically healthy small breed adult dogs, as determined based on their blood CBC / serum chemistry and urinalysis. All dogs’ ages ranged from 1.5 to 7.4 years. All animals were of mixed gender and reproductive status (intact, neutered, or spayed). The feeding study was performed using a two-armed parallel design. Briefly, all 40 dogs were grouped into two groups of 20 dogs each, based on gender, age, and history of whole mouth plaque scores measured before the feeding study started, which was considered as a baseline.

[0140] Before starting the feeding trial, full mouth dental prophylaxis (supra- and subgingival on the buccal and lingual sides) and a dental examination were performed. Biofilm removal at supragingival and subgingival levels was confirmed with the use of eosin dye or a dental flashlight during prophylaxis. After dental prophylaxis, one group was fed the control food for 28 days, and the other group was fed the test diet for 28 days. On Day 28, dogs were graded under anesthesia using the Logan-Boyce method (Logan & Boyce, 1994) for dental plaque and calculus. Whole mouth plaque and calculus accumulation was quantified using the Veterinary Oral Health Council (VOHC) metrics and those outlined below, applied to the “whole tooth” (no splitting for plaque and calculus). Each tooth’s dental plaque score was based on the coverage and thickness (eosin stain intensity) by using the numerical score as follows for coverage: 0 = no plaque detected; 1 = 1-24%; 2 = 25-49%; 50-74%; and 4 = 75-100%. The numerical score for thickness was as follows: 1 = light, pink to light red; 2 = medium, red. Each tooth calculus was scored (Warrick & Gorrel, 1997) based on the coverage using the following score for coverage: 0 = no calculus detected; 1 = 1-24%; 2 = 25-49%; 50-74%; and 4 = 75-100%. Each tooth gingivitis score was measured based on a modified method (Loe & Silness, 1963) using the following metrics: 0 = normal gingiva; 1 = moderate inflammation and redness, but no bleeding on probing; 2 = moderate inflammation with severe redness and bleeding on probing; and 3 = severe inflammation and redness, edema, ulceration, and spontaneous bleeding. The whole mouth plaque, calculus, and gingivitis mean1021326-00-WD-01-HL score was calculated by averaging the total tooth scores for each animal. All dogs were allowed to do their routine activities, except they were not allowed to have chew toys or treats.

[0141] Subgingival plaque sample collection: Sub-gingival plaque samples were also collected for microbiome analyses. For every subject, subgingival plaque samples were collected from healthy and unhealthy teeth. Healthy and unhealthy teeth were confirmed based on individual teeth gingivitis scores. A tooth gingivitis score of 0 = normal gingiva and was considered healthy, while tooth gingivitis scores of 1 or more than 1 were considered unhealthy. If the subject did not have an unhealthy tooth, then subgingival plaque was collected from two different healthy teeth.

[0142] Microbiome Sequencing: Subgingival plaque samples were analyzed for microbiome sequencing by extracting total DNA from frozen samples using the Qiagen DNeasy Power Biofilm extraction kit. Following total DNA extraction, 16s rDNA amplicon was developed from the samples by employing PCR using primer sets spanning the VI and V3 hypervariable regions, and the amplicons were subsequently analyzed qualitatively by an Agilent 2100 Bioanalyzer. Further, index PCR was performed, followed by library quantification, normalization, and pooling the samples by following the manufacturer’s instructions and modifications. The final pooled sample library was loaded into a MiSeq v3 sample loading cartridge kit, and the cartridge was placed in a MiSeq (Illumina) sequencer for sequencing the amplicons. The sample sequences were demultiplexed by using MiSeq’s in-built metagenomics workflow to obtain FASTQ files. FASTQ files were processed by employing Mothur software to classify the sequence reads using the Human Oral Microbiome Database (HOMD v 15.11), followed by custom modifications.

[0143] The test diet (Hills PD Canine Adult t / d Small Bites (dry) used in this study was previously shown to be effective by significantly reducing whole mouth plaque, calculus, and gingivitis scores compared to the control diet due to the mechanical effect delivered by proprietary fibrous striated structural matrix technology, as described, for example, in U.S. 5,500,239 and U.S. 5,431,927, incorporated by reference herein. In this study, the efficacy of the test diet versus the control diet was demonstrated by reducing whole mouth plaque, calculus, and gingivitis scores in small breed dogs via collected subgingival plaque samples for microbiome analyses as described in the methods section above.

[0144] The dogs fed the test diet significantly reduced whole mouth plaque and calculus scores compared to the control diet, as shown in Figure 3 A and Figure 3B, respectively. A non- significant decrease was observed in the whole mouth gingivitis scores, as shown in Figure 3C. The non-significant decrease in whole mouth gingivitis scores is due to the study period, where each food fed for 28 days might not be enough to develop gingivitis, and it is reasonable that the mean gingivitis scores range from 0 to 3.

[0145] The TMGI was also calculated for the microbiome samples collected from this feeding study, to show the efficacy of the test diet to improve oral microbiome health in addition to the physical reduction of plaque buildup. Based on the analyses, the dogs fed the test diet showed a significantly (p value = 0.021) higher mean TMGI score (1.36) compared to the dogs fed the control diet (0.66). See Figure 4. This data suggest that the test diet shifted or maintained the subgingival microbiome towards a healthier tooth gingivitis condition by 28 days.

Claims

CLAIMSWhat Is Claimed Is:

1. A method of identifying a companion animal as being at risk of an oral disease or condition comprising: obtaining a first plaque sample from a tooth of the companion animal; determining a relative abundance of each of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, and positive effect phyla in the first plaque sample; and calculating a first Tooth Microbiome Gingival Index (TMGI) score, wherein the first TMGI score equalswherein the companion animal is identified as being at risk of the oral disease or condition if the first TMGI score is less than about -0.12.

2. A method of determining whether a pet food composition improves the oral health of a companion animal, the method comprising: identifying a companion animal as being at risk of an oral disease or condition according to the method of claim 1 ; administering the pet food composition to the companion animal for at least about 7 days; obtaining a second plaque sample from a tooth of the companion animal after administering the pet food composition; determining a second relative abundance of each of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, and positive effect phyla in the second plaque sample; calculating a second TMGI score, wherein the second TMGI score equalswherein the pet food composition improves the oral health of the companion animal if the second TMGI score is higher than the first TMGI score.

3. The method of claim 2, wherein the second TMGI score is greater than about -0.12.

4. The method of claim 2 or 3, wherein the pet food composition is administered for at least about 14 days, such as at least about 21 days or at least about 28 days.

5. The method of any one of claims 2-4, wherein prior to administering the pet food composition, the companion animal is administered a dental prophylaxis cleaning.

6. The method according to any one of the preceding claims, wherein the companion animal is a canine.

7. The method according to claim 6, wherein the canine is a small breed canine.

8. The method according to any one of the preceding claims, wherein the oral disease or condition is periodontitis, dental plaque, calculus, or gingivitis, preferably gingivitis.

9. The method according to any one of the preceding claims, wherein the first plaque sample is a subgingival plaque sample.

10. The method according to any of claims 2-9, wherein the second plaque sample is a subgingival plaque sample.

11. The method according any one of the preceding claims, wherein the positive effect microbiotic species and negative effect microbiotic species are determined by next generation sequencing technology, preferably by 16s rRNA amplicon sequencing and metagenomics, targeted multiplex qPCR technology, or customized microarray technology.

12. The method according to any one of the preceding claims, wherein the positive effect microbiotic species are selected from at least one of Moraxella osloensis, Acinetobacter johnsonii, Moraxellaceae spp., Haemophilus spp., Bergeyella HMT 22, Streptococcus spp., and Solobacterium moorei.

13. The method according to any one of the preceding claims, wherein the negative effect microbiotic species are selected from at least one of Treponema denticola, Fusobacterium nucleatum_subsp_vincentii, Treponema HMT 490, Ruminococcaceae spp., Mollicutres_[G-2] bacterium HMT 906, Peptostreptococcaceae_[XI][G-8] bacterium HMT 382, Treponema HMT 239, Escherichia coli, Treponema HMT 238, Fastidiosipila sanguinis, and Mycoplasma lipophilum.

14. The method according to any one of the preceding claims, wherein the early colonizers are selected from at least one of Moraxella nonliquifaciens, Bergeyella HMT 422, Capnocytophaga spp., Escherichia coli, Mycoplasma hominis, Neisseria weaveri, Abiotrophia defectiva, Streptococcus spp., Haemophilus spp., Moraxella osloensis, Bergeyalla spp., and Mycoplasma lipophilum.

15. The method according to any one of the preceding claims, wherein the late colonizers are selected from at least one of Treponema HMT 249, Filifactor alocis, Porphyromonas HMT 285, Treponema parvum, Treponema HMT 490, Prevotella intermedia, Treponema HMT 951, Treponema HMT 257, Treponema HMT 227, Treponema HMT 234, Treponema HMT 238, Treponema HMT 237, Fusobacterium nucleatum vincentii, Tannerella forsythia, Treponema denticola, Porphyromonas gingivalis, Treponema HMT 235, Treponema HMT 239, Treponema HMT 258, Campylobacter rectus, and Treponema spp.

16. The method according to any one of the preceding claims, wherein the positive effect phyla are selected from at least one of Actinobacteria and Proteobacteria.

17. The method of claim 16, wherein the positive effect phyla are Actinobacteria and Proteobacteria.

18. The method according to any one of the preceding claims, further comprising determining a relative abundance of negative effect phyla.

19. The method according to claim 18, wherein the negative effect phylum are chosen from at least one of Spirochaetes and Firmicute.

20. A method for preventing or treating an oral disease or condition in a companion animal comprising identifying a companion animal at risk for the oral disease or condition according to the method of claim 1, and administering to the companion animal an oral-disease mitigating pet food composition.

21. A kit for identifying a companion animal as being at risk of an oral disease or condition comprising: a device for collecting a plaque sample from a tooth of the companion animal; and components for detecting a relative abundance of each of positive effect microbiotic species, negative effect microbiotic species, early colonizers, late colonizers, positive effect phyla, and negative effect phyla in the plaque sample.

22. The kit according to claim 21, wherein: the positive effect microbiotic species are Moraxella osloensis, Acinetobacter johnsonii, Moraxellaceae unclassified, Haemophilus unclassified, Bergeyella HMT 22, Streptococcus unclassified, and Solobacterium moorei', the negative effect microbiotic species are Treponema denticola, Fusobacterium nucleatum_subsp_vincentii, Treponema HMT 490, Ruminococcaceae unclassified, Mollicutres_[G-2] bacterium HMT 906, Peptostreptococcaceae_[XI][G-8] bacterium HMT 382, Treponema HMT 239, Escherichia coli, Treponema HMT 238, Fastidiosipila sanguinis, and Mycoplasma lipophilum', the early colonizers are Moraxella nonliquifaciens, Bergeyella HMT 422, Capnocytophaga unclassified, Escherichia coli, Mycoplasma hominis, Neisseria weaveri, Abiotrophia defectiva, Streptococcus unclassified, Haemophilus unclassified, Moraxella osloensis, Bergeyalla unclassified, and Mycoplasma lipophilum', the late colonizers are Treponema HMT 249, Filifactor alocis, Porphyromonas HMT 285, Treponema parvum, Treponema HMT 490, Prevotella intermedia, Treponema HMT 951, Treponema HMT 257, Treponema HMT 227, Treponema HMT 234, Treponema HMT 238,Treponema HMT 237, Fusobacterium nucleatum vincentii, Tannerella forsythia, Treponema denticola, Porphyromonas gingivalis, Treponema HMT 235, Treponema HMT 239, Treponema HMT 258, Campylobacter rectus, and Treponema unclassified', and the positive effect phyla are Actinobacteria and Proteobacteria.

23. The kit according to claim 21 or 22, wherein the components for detecting a relative abundance are chosen from components for next generation sequencing technology, preferably by 16s rRNA amplicon sequencing and metagenomics, targeted multiplex qPCR technology, or customized microarray technology.

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