Methods and compositions for treating intestinal dysbiosis

Abstract

Methods for assessing microbiome health in a companion animal, such as a dog, as well as compositions and food products for improving intestinal health, treating intestinal dysbiosis, and / or treating intestinal disease in a companion animal, such as a dog, are provided.

Description

[0001] This application is a divisional application of the application filed on January 17, 2020, with international application number PCT / US2020 / 014201, national application number 202080021648.0, and entitled "Method and Composition for Treating Intestinal Ecology Disorders". Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 794,542, filed January 18, 2019, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The currently disclosed topics relate to methods, compositions, and foods for assessing gut health in companion animals such as dogs, improving gut health, treating gut dysbiosis, and / or treating intestinal diseases in companion animals such as dogs. Background Technology

[0004] For pet owners, fecal consistency, especially the extremes of loose or dry stools, is a key indicator of pet health. Therefore, the impact of diet and composition on canine fecal quality is important for optimizing pet health and nutrition. Dietary intake, including dry matter volume and nutrient composition, affects fecal consistency. Changes in fecal quality and the composition of gut health-related microbiota, as well as bacterial fermentation products such as butyrate, are associated with fiber intake (Wakshlag et al., 2011). Extensive meta-analyses of human studies have revealed a link between high fiber intake (particularly whole grains and cereal-derived fiber) and a reduced incidence of colorectal cancer, suggesting a potential link between the gut microbiome and long-term clinical health in human subjects. However, research on the gut microbiome and its association with animal health is limited. Therefore, there is a need to understand the relationship between the gut microbiome and animal health. Furthermore, new methods and compositions are needed for treating microbiome-related gut diseases. Summary of the Invention

[0005] The currently disclosed subject provides a pet food containing sugar beet pulp at a concentration between about 0.5% w / w and about 6% w / w, or at a daily dose between 0.5 g and 90 g, depending on the size and food consumption of the companion animal (such as a dog). In some embodiments, the concentration of sugar beet pulp is between about 0.5% w / w and about 1% w / w. In some embodiments, the concentration of sugar beet pulp is about 0.8% w / w. The pet food may be a topper, and in some embodiments, the topper is fed to the companion animal such that the companion animal receives a dose of sugar beet pulp between about 0.5 g / day and about 90 g / day.

[0006] In some embodiments, the pet food further contains additional prebiotics. In some embodiments, the pet food also contains additional fiber. In some embodiments, the pet food also contains probiotics.

[0007] The currently disclosed subject provides a pet food containing a quantity of bacteria selected from species of the family Trichophytonceae to effectively improve the gut health and / or fecal quality of companion animals. Lachnospiraceae sp. ), Prevotella coccidioides ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ), species of the genus Dornos ( Dorea sp. ), species of the genus *Ruminococcus* ( Ruminococcaceae sp. ), species of the genus Bacteroides ( Bacteroides sp. ), species of the genus Broutella ( Blautia sp. ), species of Erysipelothrix family ( Erysipelotrichaceae sp. ), species of the family Spirulinaceae ( Lachnospiraceae sp. The group consisting of ) and any combination thereof. In some embodiments, the bacteria are selected from *Proteus vulgaris* (…). Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme The bacteria are selected from the group consisting of any combination thereof. In some embodiments, the bacteria are selected from the group consisting of any bacteria containing 16S rRNA, wherein the 16S rRNA contains a nucleotide sequence having at least about 95% sequence identity with any one of SEQ ID NO: 1-14 or identical to any one of SEQ ID NO: 1-14. In some embodiments, the bacteria are selected from the group consisting of: denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, denovo943 and any combination thereof. In some embodiments, the amount of bacteria is between about 1 x 10^6 bacteria. 4 CFU to approximately 1x10 14 Between CFUs.

[0008] In some embodiments, the pet food also contains beet pulp. The pet food may include an amount of beet pulp that effectively improves the gut health and / or stool quality of the companion animal. In some embodiments, beet pulp is present in the pet food at a concentration between about 0.5% w / w and about 6% w / w, or at a dose between about 0.5 g and 90 g per day. In some embodiments, beet pulp is present in the pet food at a concentration between about 0.5% w / w and about 1% w / w. In some embodiments, beet pulp is present in the pet food at a concentration of about 0.8% w / w.

[0009] In some embodiments, the pet food contains probiotics in addition to bacteria and / or prebiotics. In some embodiments, the pet food improves the companion animal's gut health and / or stool quality within approximately 14 days after administration.

[0010] In some embodiments, the pet food is a dietary supplement. In some embodiments, the dietary supplement is added on top of the pet food as a topper. In some embodiments, the dietary supplement is then mixed into the entire product. In some embodiments, the pet food is dog food. In some embodiments of any of the foregoing methods, the companion animal is a dog.

[0011] Furthermore, the currently disclosed subject matter also provides a method for treating gut dysbiosis and / or improving gut health in companion animals in need. In some embodiments, the method includes administering an effective amount of any of the pet foods disclosed herein to the companion animal to treat gut dysbiosis and / or improve gut health.

[0012] The currently disclosed subject matter provides a method for determining the intestinal health status of companion animals. In some embodiments, the currently disclosed subject matter provides a method for determining the intestinal health status of companion animals in need, such as animals with poor fecal quality or diarrhea or intestinal dysbiosis. In some embodiments, the method includes: measuring a first amount of gut microbiota in a companion animal and detecting bacteria denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, and denovo943; and further bacteria denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, and denovo4881, and using the relative amounts of these bacteria to determine the health of the companion animal.

[0013] In some implementations, the method includes: a) Measure a first amount of first gut microbiota and / or a second amount of second gut microbiota in the companion animal; b) Comparing a first quantity of the gut microbiota to a first reference quantity of the first gut microbiota, and / or comparing a second quantity of the gut microbiota to a second reference quantity of the second gut microbiota, wherein the reference quantity of the gut microbiota is determined based on the number of gut microbiota in multiple healthy companion animals; and c) Determine the gut health status of the companion animal when the first amount of the first gut microbiota is higher than the first reference amount of the first gut microbiota, and / or when the second amount of the second gut microbiota is lower than the second reference amount of the second gut microbiota.

[0014] In some embodiments, a method for determining the gut health status of a companion animal includes: a) measuring a first amount of a first gut microbiota from a first sample collected from the companion animal; b) comparing the first amount of the first gut microbiota to a first reference amount of the first gut microbiota; and / or c) measuring a second amount of a second gut microbiota from a second sample collected from the companion animal; d) comparing the second amount of the second gut microbiota to a second reference amount of the second gut microbiota; wherein the gut health status of the companion animal is determined to be healthy when the first amount of the gut microbiota is higher than the first reference amount of the first gut microbiota and / or when the second amount of the second gut microbiota is lower than the second reference amount of the second gut microbiota; and wherein the first reference amount of the first gut microbiota and the second reference amount of the second gut microbiota are determined based on the number of gut microbiota from multiple healthy companion animals.

[0015] In some embodiments, the first gut microbiota is one or more bacteria containing 16S rRNA, said 16S rRNA containing a nucleotide sequence having at least about 95% sequence identity with any one of SEQ ID NO:1-14 or the same as any one of SEQ ID NO:1-14.

[0016] In some embodiments, the first gut microbiota is selected from the group consisting of denovo 1184, denovo 1244, denovo 1696, denovo 2407, denovo 2451, denovo 283, denovo 3487, denovo 4154, denovo 4328, denovo 4681, denovo 498, denovo 5338, denovo 6995, denovo 943, and any combination thereof. In some embodiments, the first gut microbiota is selected from *Bacillus prednisii* (…). Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ) and any combination thereof.

[0017] In some embodiments, the second gut microbiota is one or more bacteria containing 16S rRNA, said 16S rRNA containing a nucleotide sequence having at least about 95% sequence identity with any one of SEQ ID NO:15-26 or the same as any one of SEQ ID NO:15-26.

[0018] In some implementations, the second gut microbiota is selected from the group consisting of denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, denovo4881, and any combination thereof.

[0019] In some embodiments, the method further includes providing a customized recommendation for a treatment regimen and / or further monitoring of the gut microbiota when the first amount of the first gut microbiota is lower than the first reference amount of the first gut microbiota, and / or when the second amount of the second gut microbiota is higher than the second reference amount of the second gut microbiota. In some embodiments, the treatment regimen is pet food as disclosed herein.

[0020] In some implementations, the amount of first and / or second intestinal bacteria is measured from a fecal sample of the companion animal.

[0021] In some embodiments, the method measures the amount of at least three microorganisms. In some embodiments, the method measures the amount of at least ten microorganisms. In some embodiments, the method measures the amount of about five to twenty-six microorganisms. In some embodiments, the method measures the amount of about ten to twenty-six microorganisms.

[0022] The currently disclosed subject matter provides a method for treating gut dysbiosis and / or improving gut health in companion animals in need. In some embodiments, the method includes: a) Measure the first quantity of one or more gut or fecal microorganisms in a companion animal; b) Treatment regimens administered to companion animals for the treatment of intestinal diseases and / or to improve intestinal health; c) Measure a second amount of the subject's gut microbiota after step b); and d) Determine the animal's gut health status or response to treatment.

[0023] In some embodiments, the method further includes continuing the treatment regimen when a second amount of gut microbiota changes compared to a first amount.

[0024] On the other hand, this disclosure is characterized by a method for treating intestinal dysbiosis and / or improving intestinal health in companion animals in need, the method comprising: a) Measure the first amount of gut microbiota from samples collected from the companion animal; and b) Administer a treatment regimen to the companion animal to treat the gut dysbiosis and / or improve gut health; The effectiveness of the treatment regimen is determined by measuring a second amount of gut microbiota in the subject after performing step b) and determining the animal’s gut health status or response to the treatment regimen based on the difference between the first amount and the second amount of gut microbiota.

[0025] In some embodiments, the gut microbiota is one or more bacteria containing 16S rRNA, said 16S rRNA comprising a nucleotide sequence having at least about 95% sequence identity with any one of SEQ ID NO: 1-14 or identical to any one of SEQ ID NO: 1-14. In some embodiments, the gut microbiota is selected from the group consisting of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, denovo943, and any combination thereof. In some embodiments, the method further comprises: c) if, after step b), the second amount of the gut microbiota increases compared to the first amount of the gut microbiota, then continuing the treatment regimen. In some embodiments, the gut microbiota is selected from *Bacillus prednisii* (…). Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme Groups consisting of (b) and any combination thereof. In some embodiments, a second quantity of gut bacteria is measured between approximately 3 days or approximately 7 days and approximately 14 days after step b).

[0026] In other embodiments, the gut microbiota is one or more bacteria comprising 16S rRNA, the 16S rRNA comprising a nucleotide sequence having at least about 95% sequence identity with any one of SEQ ID NO: 15-26 or identical to any one of SEQ ID NO: 15-26. In some embodiments, the gut microbiota is selected from the group consisting of denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, denovo4881, and any combination thereof. In some embodiments, the method further includes c) continuing the treatment regimen if the second amount of the gut microbiota is reduced compared to the first amount of the gut microbiota after step b).

[0027] In some embodiments, the gut microbiota are one or more bacteria containing 16S rRNA, said 16S rRNA containing at least about 95% identity with any one of SEQ ID NO: 1-26 or the same nucleotide sequence as any one of SEQ ID NO: 1-26, and said method further comprises: continuing to administer the treatment regimen when a second amount of gut microbiota increases for bacteria containing 16S rRNA (which contains at least about 95% identity with any one of SEQ ID NO: 1-14 or the same nucleotide sequence as any one of SEQ ID NO: 1-26), and / or when a decrease in the amount of gut microbiota occurs for bacteria containing 16S rRNA (which contains at least about 95% identity with any one of SEQ ID NO: 15-26 or the same nucleotide sequence as any one of SEQ ID NO: 15-26) compared to the first amount of gut microbiota.

[0028] In some embodiments, a second amount of gut bacteria is measured between approximately 3 days or approximately 7 days and approximately 14 days after step b). In some embodiments, the treatment regimen includes a dietary regimen. In some embodiments, the dietary regimen includes administering an effective amount of any pet food disclosed herein. In a particular embodiment, the companion animal is a dog.

[0029] In some embodiments of any of the aforementioned methods, the sample is a fecal sample collected from a companion animal.

[0030] In some implementations, microarrays are used to determine the amount of gut microbes.

[0031] In some embodiments, the method measures the amount of at least three microorganisms. In some embodiments, the method measures the amount of at least ten microorganisms. In some embodiments, the method measures the amount of about five to twenty-six microorganisms. In some embodiments, the method measures the amount of about ten to twenty-six microorganisms.

[0032] On the other hand, this article discloses a pet food for treating intestinal ecological disorders in companion animals, the pet food comprising beet pulp, wherein the concentration of beet pulp in the pet food is from about 0.1% w / w to about 10% w / w.

[0033] In some embodiments, the pet food is a dietary supplement or functional food. In some embodiments, the pet food is a cover food. In some embodiments, the cover food is fed to the companion animal at a dose of about 0.5 g / day to about 90 g / day of beet pulp. In some embodiments, the concentration of beet pulp in the pet food is between about 0.5% w / w and about 6% w / w.

[0034] On the other hand, a feature of this disclosure is that any pet food disclosed herein is used to treat gut dysbiosis or improve gut health in companion animals.

[0035] In some implementations of any pet food, the pet food is dog food.

[0036] On the other hand, this disclosure is characterized by the use of beet pulp in dietary supplements or pet food for the treatment or prevention of ecological disorders in companion animals. In some embodiments, the companion animal experiences dietary changes. In further embodiments, the concentration of beet pulp in the dietary supplement or pet food is between about 0.5% w / w and about 6.0% w / w. In some embodiments, the companion animal is a dog.

[0037] In some implementations, dietary supplements or pet food are given to the companion animal for at least approximately 3 days. In other implementations, dietary supplements or pet food are given to the companion animal for at least approximately 7 days.

[0038] On the other hand, this disclosure is characterized by the use of beet pulp in dietary supplements or pet food for the treatment or prevention of ecological disorders in companion animals, wherein the companion animals are undergoing dietary changes. In some embodiments, the concentration of beet pulp in the dietary supplement or pet food is between about 0.1% w / w and about 10% w / w.

[0039] In another aspect, this disclosure is characterized by the use of any of the pet foods disclosed herein for the treatment or prevention of intestinal ecological disorders in companion animals, or for the improvement of intestinal health in companion animals.

[0040] In some embodiments for any public use, pet food is dog food. In some embodiments, companion animal is a dog.

[0041] In another aspect, this disclosure is characterized by a health assessment tool for monitoring the intestinal health status or ecological imbalance of companion animals, comprising one or more probes for detecting amounts of one or more microorganisms, said microorganisms comprising 16S rRNA, said 16S rRNA containing a nucleotide sequence having at least about 95% sequence identity with or identical to the nucleotide sequence of any one of SEQ ID NO: 1-26.

[0042] In some embodiments, the health assessment tool includes a microarray of one or more probes. In some embodiments, the probes detect the 16S rRNA sequence of one or more microorganisms. In a further embodiment, the health assessment tool includes probes for detecting at least about three of one or more microorganisms. In some embodiments, the health assessment tool includes probes for detecting between about five and about 26 of one or more microorganisms.

[0043] In a further embodiment of the health assessment tool, the one or more microorganisms are selected from the group consisting of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, denovo943, denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, denovo4881, and any combination thereof. In some embodiments, the amount of microorganisms is measured from fecal samples of companion animals. In some embodiments, the health assessment tool monitors gut health or dysbiosis by comparing the amount of the one or more microorganisms to a reference amount of the one or more microorganisms. In a further embodiment, the companion animal is a dog. Attached Figure Description

[0044] Figure 1 A study design was described to compare the effects of three digestive health components on fecal quality and fecal microbiome.

[0045] Figure 2A and Figure 2B The fecal scoring system used in Example 1 is described.

[0046] Figure 3A and Figure 3B The average fecal mass fraction overnight at the enclosure was depicted. Figure 3A It is all 21 days of the feeding phase for each diet and Figure 3B It is during the last 7 days of the 21-day feeding period. SBP may have been adjusted within the past 7 days.

[0047] Figure 4A and Figure 4B The percentage of total feces from overnight enclosures with an unacceptable fecal fraction (all +3.75) is depicted. Figure 4A It is all 21 days of the feeding phase for each diet and Figure 4B It is during the last 7 days of the 21-day feeding period.

[0048] Figure 5 PLS correlation plots depicting the abundance data of 26 OTUs, where the projected importance (VIP) score of the variables is >1. Sample and OTU descriptors have been removed for easier visualization and replaced with a color guide for localization purposes (samples represented by diets on the y-axis: A, reference diet; B, beet pulp; C, cellulose; and D, pea protein). Fecal samples are represented by separate horizontal rows and clustered according to similarity, while bacterial OTUs are represented by individual columns in the heat plot. Color coding in the plot indicates the degree and direction of correlation. Detailed Implementation

[0049] To date, there remains a need for novel methods and compositions for treating gut dysbiosis and other gut diseases that target the gut microbiome. This application relates to methods, compositions, and foods for improving gut health in companion animals, treating gut dysbiosis, and / or treating gut diseases, based at least in part on the findings that animal foods containing beet pulp can promote gut health and that changes in gut microbiota are associated with gut health status.

[0050] For clarity rather than limitation, the detailed description of the currently disclosed subject matter is divided into the following sections: 1. Definition; 2. Gut bacteria and related health assessment tools; 3. Food; and 4. Health assessment and treatment methods.

[0051] 1. Definition

[0052] The terms used in this specification generally have their ordinary meanings in the art, in the context of this invention, and in the specific context in which each term is used. Some terms are discussed below or elsewhere in the specification to provide additional guidance to practitioners in describing the methods and compositions of the invention and how they are prepared and used.

[0053] As used herein, the use of the terms "a" or "an" in conjunction with the term "comprising" in the claims and / or specification can mean "a," but is also consistent with the meanings of "one or more," "at least one," and "one or more." Furthermore, the terms "having," "comprising," "containing," and "comprising" are interchangeable, and those skilled in the art will recognize that these terms are open-ended.

[0054] The terms “about” or “approximately” refer to a range of acceptable error for a particular value as determined by those skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” can mean within three or more standard deviations. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, preferably within a value, up to five times, and more preferably up to two times.

[0055] The term "effective treatment" or "effective amount" of a substance refers to an amount of treatment or substance sufficient to produce a beneficial or desired outcome, including clinical outcomes, and therefore, "effective treatment" or "effective amount" depends on the context of its application. In the case of administering a composition (such as pet food) to improve immunity, digestive function, and / or reduce inflammation, the effective amount of the composition described herein is an amount sufficient to improve stool quality, digestive health, immunity, digestive function, and / or reduce inflammation, as well as alleviate symptoms and / or reduce the likelihood of digestive disorders and / or inflammation. The effective treatment described herein is a treatment sufficient to improve the microbiome, stool quality, digestive health, immunity, digestive function, and / or reduce inflammation, as well as alleviate symptoms and / or reduce the likelihood of digestive disorders and / or inflammation. The reduction can be approximately 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% in the severity of symptoms of digestive disorders or inflammation, or a reduction in the likelihood of digestive disorders or inflammation. An effective dose can be administered once or multiple times. The likelihood of effective treatment described herein refers to the probability of treatment sufficient to alter the microbiome, or to treat or improve digestive disorders and / or inflammation, and to alleviate symptoms.

[0056] As used herein and as is well known in the art, “treatment” is a method for obtaining a beneficial or desired outcome, including clinical outcomes. For the purposes of this subject matter, a beneficial or desired clinical outcome includes, but is not limited to, the reduction or improvement of one or more symptoms, a reduction in the severity of the condition, a stable (i.e., non-worsening) state of the condition, prevention of disease, delay or slowing of disease progression, and / or improvement or remission of the disease state. A reduction may be a decrease in the severity of complications or symptoms of about 0.01%, about 0.1%, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 99%. “Treatment” can also mean an extension of survival compared to the expected survival without treatment.

[0057] As used herein and as is well known in the art, "probiotics" are preparations or compositions containing microorganisms that provide health benefits when consumed. Microorganisms include, but are not limited to, bacteria, fungi, yeasts, and archaea. In some embodiments, probiotics may modify the microbiome in a GI system to enhance the balance of the microbiome within the GI system, for example, by acting as an inoculum for an increase in beneficial microbiota, and / or by antagonizing the growth of harmful microorganisms. In some embodiments, the probiotics are animal probiotics, such as feline or canine probiotics.

[0058] As used herein and as is well known in the art, a "prebiotic" is a substance or composition that can induce the growth or activity of one or more beneficial microorganisms, such as one or more probiotics, including bacteria, fungi, yeasts, and archaea. In some embodiments, a prebiotic can modify the microbiome in a GI system to enhance the balance of the microbiome in the GI system. In some embodiments, a prebiotic is non-digestible for animals. In some embodiments, a prebiotic can induce the growth or activity of one or more animal probiotics, such as feline or canine probiotics.

[0059] The terms "pet food," "pet food composition," "pet food," or "final pet food" refer to products or compositions intended for consumption by companion animals such as cats, dogs, guinea pigs, rabbits, birds, or horses. For example, but not as a limitation, a companion animal can be a "domestic" dog, such as a domestic dog. Canis lupus familiaris In some implementations, the companion animal can be a "domestic" cat, such as a house cat ( Felis domesticus "Pet food" or "pet food composition" or "pet food" or "final pet food" includes any food, feed, treats, food supplements, liquids, beverages, snacks, toys (chewable and / or consumable toys) and meal replacements or meal substitutes.

[0060] In this article, "individual" or "subject" refers to a vertebrate, such as a human, or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, livestock animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0061] 2. Gut microbiota and related health assessment tools

[0062] The currently published subject provides information on gut microbiota and their combinations, based at least in part on the finding that variations in the gut microbiota population within the microbiome are associated with gut health and stool quality in subjects. Subjects may be, for example, companion animals, such as dogs.

[0063] In some implementations, gut microbiota can be used to indicate a subject's gut health. In some implementations, gut microbiota are associated with a subject's health status or gut dysbiosis. For example, the subject may be a companion animal, such as a dog.

[0064] In some embodiments, gut microbiota indicates the healthy gut status of a subject. In some embodiments, the subject is a companion animal, such as a dog. For example, the health status of the companion animal can be determined based on reference values ​​from multiple healthy companion animals of the same type. In some embodiments, the gut microbiota are bacteria comprising 16S ribosomal RNA (rRNA) comprising a nucleotide sequence having at least about 95% sequence identity with any of the nucleotide sequences in SEQ ID NO:1-26, and any combination thereof.

[0065] In some implementations, the gut microbiota is selected from the *Trichophyton* species (…). Lachnospiraceae sp. ), Prevotella coccidioides ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ), species of the genus Dornos ( Dorea sp. ), species of the genus *Ruminococcus* ( Ruminococcaceae sp. ), species of the genus Bacteroides ( Bacteroides sp. Species of the genus *Broutella* Blautia sp. ), species of Erysipelothrix family ( Erysipelotrichaceae sp. ), species of the family Spirulinaceae ( Lachnospiraceae sp. The group consisting of ) and any combination thereof. In some embodiments, the bacteria are selected from *Proteus vulgaris* ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme The group consisting of ( ) and any combination thereof. In a further embodiment, gut microbiota is an organism comprising a nucleotide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity with any of the nucleotide sequences in SEQ ID NO:1-26.

[0066] In some implementations, the gut microbiota are bacteria selected from the group consisting of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, denovo943, and any combination thereof.

[0067] In some embodiments, each of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, and denovo943 comprises a 16S rRNA sequence containing a nucleotide sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) sequence identity with the nucleotide sequence of SEQ ID NO:1-14.

[0068] In some implementations, each of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, and denovo943 comprises a 16S rRNA sequence containing the nucleotide sequence of SEQ ID NO:1-14.

[0069] Gut microbiota can indicate the health of the microbiome or the state of gut dysbiosis in the microbiome of companion animals. In some embodiments, gut microbiota include species selected from the order Clostridium (… Clostridiales sp. ), species of Clostridium ( Clostridia sp. ), species of the Bacillus family ( Mogibacteriaceae sp .), species of the family Spirulinaceae ( Lachnospiraceae sp. ), species of the Clostridium family ( Clostridiaceae sp. ), Peptostreptococcus species ( Peptostreptococcaceae sp. Bacteria and groups of bacteria formed by any combination of them.

[0070] In some implementations, the bacteria are selected from the group consisting of denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, denovo4881, and any combination thereof.

[0071] In some embodiments, each of denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, and denovo4881 comprises a 16S rRNA containing a nucleotide sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) sequence identity with the nucleotide sequence of SEQ ID NO:15-26.

[0072] In some embodiments, each of denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, and denovo4881 comprises a 16S rRNA containing the nucleotide sequence described in SEQ ID NO:15-26.

[0073] denovo1184 [SEQ ID NO: 1]:

[0074] CCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTCTCTGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCACTTACCTCTCCAGTACTCTAGCTCAACAGTTTCCAAAGCAGTCCCGTGGTTAAGCCTCGGGCTTTCACTTCAGACTTGCCGAGCCGT CTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTCGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTCATTCTCTTCCCTGCTGATAGAGCTTTACGTACCGAAGTACTTCTTCACTCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATCCCCCA

[0075] denovo1244 [SEQ ID NO: 2]:

[0076] CCTGTTTGCTACCCACACTTTCGAGCCTCAGCGTCAGTTGGTGCCCAGTAGGCCGCCTTCGCCACTGGTGTTCCTCCCGATATCTACGCATTCCACCGCTACACCGGGAATTCCGCCTACCTCTGCACTACTCAAGAAAAACAGTTTTGAAAGCAGTTTATGGGTTGAGCCCATAGATTTCACTTCCAACTTGTCTTCCCGCCTGCGCTCCCTTTACACCCAGTAATTCCGGACAACGCTTGTGACCTACGTTTTACCGCGGCTGCTGGCACGTAGTTAGCCGTCACTTCCTTGTTGGGTACCGTCATTATCTTCCCCAACAACAGGAGTTTACAATCCGAAGACCTTCTTCCTCCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCA

[0077] denovo1696 [SEQ ID NO: 3]:

[0078] CCTGTTTGATACCCACACTTTCGAGCATGAACGTCAGTTACGGCTTAGTGTGCTGCCTTCGCAATCGGAGTTCTTCGTGATATCTAAGCATTTCACCGCTACACCACGAATTCCGCACACCTCAACCGCACTCAAGGACGCCAGTATCAACTGCAATTTTAAGGTTGAGCCCCAAACTTTCACAGCTGACTTAACGACCCGTCTGCGCTCCCTTTAAACCCAATAAATCCGGATAACGCTCGCATCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGATGCTTATTCATAAGGTACATACAAGCTCCCACACGTGGGAGGTTTTATTCCCTTATAAAAGAAGTTTACAATCCGTAGGACCTTCATCCTTCACGCTACTTGGCTGGTTCAGACTCTCGTCCATTGACCAATATTCCTCA

[0079] denovo2407 [SEQ ID NO: 4]:

[0080] CCTATTTGCTCCCCACGCTTTCGTGCTTCAGTGTCAGAATCCAGACCAGACGGCCGCCTTCGCCACCGGTGTTCTTCCATATATCTACGCATTTTACCGCTACACATGGAGTTCCGCCGTCCTCTTCTGTTCTCTAGCTGATCAGTTTCCAGAGCAAGTACGGGTTGAGCCCATACCTTTTACTCCAGACTTGATCTGCCACCTACGCACCCTTTACGCCCAATCATTCCGGATAACGCTCGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTAAGATACCATCACTCACTCATCATTCCCTATGAGTGCCGTTTTTCTCTTACAACAGAGCTTTACGATCCGAAGACCTTCCTCACTCACGCGGCATTGCTCGTTCAGGGTTCCCCCCATTGACGAAAATTCCCTA

[0081] denovo2451 [SEQ ID NO: 5]:

[0082] CCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTTATCGTCCAGTAAGCCGCCTTCGCCACTGATGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTACCTCTCCGACACTCTAGAAGCACAGTTTCCAAAGCAGTCACGGGGTTGAGCCCCGGGCTTTCACTTCAGACTTGCACTTCCGTCTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTCATTTTCTTCCCTGCTGATAGAAGTTTACATACCGAAATACTTCTTCCTTCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCA

[0083] denovo283 [SEQ ID NO: 6]:

[0084] CCTGTTTGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTAGTGCCCAGCAGGCCGCCTTCGCCACTGGTGTTCCTCCCGATATCTACGCATTCCACCGCTACACCGGGAATTCCGCCTGCCTCTGCACCACTCAAGATTTGCAGTTTTGAATGCGAGAAGGGGTTGAGCCCCTCCATTAAACATCCAACTTGCAAACCCGCCTGCGCTCCCTTTACACCCAGTAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTCCTCCTCGGGTACCGTCATACTTCGTCCCCGAAGACAGAGGTTTACAATCCGAAAATCTTCTTCCCTCACGCGGCGTCGCTGCATCAGAGTTTCCTCCATTGTGCAATATTCCCCA

[0085] denovo3487 [SEQ ID NO: 7]:

[0086] CCTGTTTGATACCCGCACTTTCGAGCATCAGCGTCAGTTACGGTCCAGTAAGCTGCCTTCGCAATCGGAGTTCTTCGTGATATCTAAGCATTTCACCGCTACACCACGAATTCCGCCTACCTATACCGCACTCAAGAAATCCAGTATCAACTGCAATTTTACGGTTGAGCCGCAAACTTTCACAACTGACTTAAACTTCCGCCTACGCTCCCTTTAAACCCAATAAATCCGGATAACGCTCGGATCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGATCCTTATTCATACGGTACATACAAAAAAGCACACGTGCTTCACTTTATTCCCGTATAAAAGAAGTTTACAACCCATAGGGCAGTCATCCTTCACGCTACTTGGCTGGTTCAGACTCTCGTCCATTGACCAATATTCCTCA

[0087] denovo4154 [SEQ ID NO: 8]:

[0088] CCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTTACCGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTACCCCTCCGGTACTCAAGATCAACAGTTTCCAATGCAGTCCAGGGGTTGAGCCCCTGCCTTTCACATCAGACTTGCTGCTCCGTCTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTCATTATCTTCCCTGCTGATAGAAGTTTACATACCGAGATACTTCTTCCTTCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCA

[0089] denovo4328 [SEQ ID NO: 9]:

[0090] CCTGTTTGATACCCACACTTTCGAGCATGAACGTCAGTTACAGTTTAGCAAGCTGCCTTCGCAATCGGGGTTCTTCGTGATATCTAAGCATTTCACCGCTACACCACGAATTCCGCCTGCCTCAACTGCACTCAAGGAAACCAGTATCAACTGCAATTTTACGGTTGAGCCGCAAACTTTCACAACTGACTTAATCTCCCGTCTGCGCTCCCTTTAAACCCAATAAATCCGGATAACGCTCGCATCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGATGCTTATTCATACGGTACATACAAAATCCTACACGTAGGAAACTTTATTCCCGTATAAAAGAAGTTTACAATCCGTAGGACCTTCATCCTTCACGCTACTTGGCTGGTTCAGGCTCTCGCCCATTGACCAATATTCCTCA

[0091] denovo4681 [SEQ ID NO: 10]:

[0092] CCTATTTGCTCCCCACGCTTTCGTGCCTGAGCGTCAGTTACAGACTAGCAAGCCGCCTTCGCCACCGGTGTTCCTCCATATATCTATGCATTTTACCGCTACACATGGAATTCCACTTGCCCCTTCTGCACTCTAGTTCACCAGTTTCTAAGCCTGGATGGGGTTGAGCCCCACAATTTAAGCTTAAACTTAATAAACCGCCTGCGCACCCTTTACGCCCAATAATTCCGGATAACGCTCGTCACCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGTGACTTTCTGGTAAAGTACCGTCACTTACGTAGTTGATACGTAACATTCTTCCTTTACAACAGAGCTTTACAAGCCGAAGACCTTCATCACTCACGCGGCATTGCTCGGTCAGGCTTGCGCCCATTGCCGAAAATTCCCTA

[0093] denovo498 [SEQ ID NO: 11]:

[0094] CCTGTTTGATACCCACACTTTCGAGCCTCAATGTCAGTTGCAGCTTAGCAGGCTGCCTTCGCAATCGGAGTTCTTCGTGATATCTAAGCATTTCACCGCTACACCACGAATTCCGCCTGCCTCAACTGCACTCAAGATATCCAGTATCAACTGCAATTTTACGGTTGAGCCGCAAACTTTCACAACTGACTTAAACATCCATCTACGCTCCCTTTAAACCCAATAAATCCGGATAACGCTCGGATCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGATCCTTATTCATAAAGTACATGCAAACGGGTATGCATACCCGACTTTATTCCTTTATAAAAGAAGTTTACAACCCATAGGGCAGTCATCCTTCACGCTACTTGGCTGGTTCAGGCTCTCGCCCATTGACCAATATTCCTCA

[0095] denovo5338 [SEQ ID NO: 12]:

[0096] CCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTTACTGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCACTTACCTCTCCAGCACTCTAGCAGAACAGTTTCCAAAGCAGTCCCGGGGTTGAGCCCCGGGCTTTCACTTCAGACTTGCTCCGCCGTCTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTCATTTTCTTCCCTGCTGATAGAGCTTTACATACCGAGATACTTCTTCACTCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCA

[0097] denovo6995 [SEQ ID NO: 13]:

[0098] CCTGTTCGCTCCCCACGCTTTCGAGTCTCAGCGTCAGTTATAGTCCAGCAAGCCGCCTTCGCCACCGGTGTTCTTCCTGATATCTACGCATTTCACCGCTACACCAGGAATTCCGCTTGCCCTTCCTACACTCTAGCTGTACAGTTTCAAAAGCAGTCTTGGGGTTGAGCCCCAAGTTTTCACTCCTGACTTGTACTGCCGCCTACACTCCCTTTACACCCAGTAAATCCGGATAACGCTTGCCCCATACGTATTACCGCGGCTGCTGGCACGTATTTAGCCGGGGCTTCTTCTGTAGGTACCGTCATTATCTTCCCTACTGATAGAGCTTTACATACCGAAATACTTCTTCACTCACGCGGCGTCGCTGCATCAGAGTTGCCTCCATTGTGCAATATTCCCCA

[0099] denovo943 [SEQ ID NO: 14]:

[0100] CCTGTTTGATACCCACACTTTCGAGCCTCAATGTCAGTTGCAGCTTGGCGGACTGCTTTCGCAATCGGGGTTCTTCGTGATATCTAAGCATTTCACCGCTACACCACGAATTCCATCCGCCTCAAATGCACTCAAGAAAACCAGTATCAACTGCAATTTTACGGTTGAGCCGCAAACTTTCACAGCTGACTTAATCTCCCATCTACGCTCCCTTTAAACCCAATAAATCCGGATAACGCTCGCATCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGATGCTTATTCATAAGGTACATACAAACAGGTACACGTACCTGACTTTATTCCCTTATAAAAGAAGTTTACAACCCATAGGGCAGTCTTCCTTCACGCTACTTGGCTGGTTCAGACTCTCGTCCATTGACCAATATTCCTCA

[0101] denovo1214 [SEQ ID NO: 15]:

[0102] CCTGTTTGCTCCCCACGCTTTCGTACCTCAGTGTCAGTTACAGTCCAGAAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTTCCTCTCCTGCACTCAAGTTTGCCAGTTCGCAGGGCGAACAATGGTTGAGCCATTGCCTTAAACCCTGCGCTTGGTAAACCACCTACGTACCCTTTACGCCCAATAATTCCGGATAACGCTTGCCCCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGGGCTTCCTCCAAGGGTACCGTCATTTGTTTCTTCCCCAAGGACAGAGCTTTACGACCCAAAGGCCTTCATCGCTCACGCGGCGTTGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCA

[0103] denovo1400 [SEQ ID NO: 16]:

[0104] CCTGTTCGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATAAGCCTGGCAGACCGCCTTCGCCTCCGGTATTCCTCCTGATCTCTGCGCATTTCACCGCTACACCAGGAATTCTGCCTGCCTCGACAATACTCCAGCTGCCCGGTTTGCGGTGACCTCCCAGGGTTGAGCCCTGGACTTTTACACCGCACCTAAACAACCACCTGCGTGCCCTTTACGCCCAATAATTCCGGATAACGCTCGCCCCCTACGTATTACCGCAGCTGCTGGCACGTAGTTGGCTGGGGCTTGCTTACCGGGTACCGTCATCGTCTTCCCCGGTAACAGAGCTTTACAGAACGAATCCCTTCTTCACTCACGCGGCATCGCTGCGTCAGAGTTGCCTCCATTGCGCAATATTCCCCA

[0105] denovo1762 [SEQ ID NO: 17]:

[0106] CCTGTTTGCTCCCCACGCTTTCGAGCCTCAACGTCAGTTACCGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCGCTTACCTCTCCGGCACTCAAGTCCCACAGTTTCCAATGCAATCCAGAAGTTGAGCCTCTGCCTTTCACATCAGACTTGCAGAACCGTCTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCTTAGTCAGGTACCGTCATTTTCTTCCCTGCTGATAGAGCTTTACATCACGAATGACTTCTTCACTCACGCGGCGTCGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCA

[0107] denovo2014 [SEQ ID NO: 18]:

[0108] CCTGTTCGCTCCCCGCGCTCTCGCGCCTCAGCGTCAGTGTCCGTCCGGCGGGCCGCCTTCGCCTCCGGTGTTCCTCCTGGCCTCTGCGCATTTCACCGCTACGCCAGGAGTTCCGCCCGCCCCTCCGGCACTCCAGCCGCCCGGTCCGGGGCGCTTGCCCGGGGTTGGGCCCCGGCTTTTCACACCCCGCCTAAGCGGCCGCCTGCGCGCGCTTTACGCCCAGTGATTCCGGACAACGCTCGCCCCCCACGTATTGCCGCGGCTGCTGGCACGTGGTTGGCCGGGGCTTTCGCGCCGGGTTCAGTCATTTCTTCCTCCCCGGCTACGGGGCTTTACGGGCCGAGGCCCTTCGTCGCCCACGCGGCGTCGCTGCGTCAGAGTTCCCTCCATTGCGCAATATTCCCCA

[0109] denovo2197 [SEQ ID NO: 19]:

[0110] CCTGTTTGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGTCCAGAGAATCGCCTTCGCCACTGGTGTTCTTCCTAATCTCTACGCATTTCACCGCTACACTAGGAATTCCATTCTCCTCTCCTGCACTCTAGACTTCCAGTTTGAAATGCAGCACCCAAGTTGAGCCCGGGTATTTCACATCTCACTTAAAAGTCCGCCTACGCTCCCTTTACGCCCAGTAAATCCGGACAACGCTCGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTCCTCCTCAGGTACCGTCATTATCGTCCCTGAAGACAGAGCTTTACAACCCGAAGGCCGTCATCACTCACGCGGCGTTGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCA

[0111] denovo2368 [SEQ ID NO: 20]:

[0112] CCTGTTCGCTCCCCGCGCTCTCGCGCCTCAGCGTCAGTGTCCGTCCGGCGGGCCGCCTTCGCCTCCGGTGTTCCTCCCGGCCTCTGCGCATTTCACCGCTACGCCGGGAGTTCCGCCCGCCCCTCCGGCGCTCCAGCCGCCCGGTCCGGGGCGCTTGCCCGGGGTTGGGCCCCGGCTTTTCACACCCCGCCTAAGCGGCCGCCTGCGCGCGCTTTACGCCCAGTGATTCCGGACAACGCTCGCCCCCCACGTATTGCCGCGGCTGCTGGCACGTGGTTGGCCGGGGCTTTCGTGCCGGGTTCAGTCATTTCCTTCCTCCCCGGCGGCGGGGCTTTACGGGCCTGGGCCCTTCATCGCCCACGCGGCGTCGCTGCGTCAGAGTTGCCTCCATTGCGCAATATTCCCCA

[0113] denovo3663 [SEQ ID NO: 21]:

[0114] CCTGTTCGCTCCCCGCGCTCTCGCGCCTTAGCGTCAGTGTGCGTCCGGCGGGCCGCCTTCGCCTCCGGTGTTCCCCCTGGCCTCTGCGCATTTCACCGCTACGCCAGGGGTTCCGCCCGCCTCTCCGCCACTCCAGCCGCCCGGTCCGGGGCGCTTGCCCGGGGTTGGGCCCCGGCTTTTAACGCCCCGCCTAAGCGGCCGCCTGCGCGCGCTTTACGCCCAGTGATTCCGGACAACGCTCGCCCCCCACGTATTGCCGCGGCTGCTGGCACGTGGTTGGCCGGGGCTTTCTTGCCGGGTTGCGTCATTTTTTTCCTCCCCGGCGGCGGGGCTTTACGGGCCTGAGCCCTTCATCGCCCACGCGGCATCGCTGCGTCAGAGTTCCCTCCATTGCGCAATATTCCCCA

[0115] denovo4206 [SEQ ID NO: 22]:

[0116] CCTGTTTGCTCCCCACGCTTTCGTGCCTCAGTGTCAGTTACAGTCCAGAAAGCCGCCTTCGCTACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCCACTTTCCTCTCCTGCACTCAAGTTTCCCAGTTTCAAGAGCTTACTACGGTTAAGCCGTAGCCTTTCACTCCTGACTTAAGAAACCACCTACGCACCCTTTACGCCCAGTAAATCCGGATAACGCTAGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCCTCCTCAAGTACCGTCATTATCTTCCTTGAGGACAGAGTTTTACGACCCGAAGGCCTTCATCACTCACGCGGCGTTGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCA

[0117] denovo4485 [SEQ ID NO: 23]:

[0118] CCTGTTTGCTCCCCACGCTCTCGCGCCTTAGCGTCAGTCTCTTCCCGGCAGGCCGCCTTCGCCTCCGGTATTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCTGCCTGCCTCTAAAGGACTCCAGCCTCTCGGTTCCTGTCGCCTCCCCGGGTTGGGCCCGGGACTTTTACAACAGGCCTTCTAGGCCGCCTACGCGCGCTTTACGCCCAGTAATTCCGGACAACGCTCGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTTAAAGACGGGTGACATCTCTCTTCTCCCCGTCGACTGAGCTCTGCGGGACGAATCCCTTCCTCACTCACGCGGCATCGCTGCGTCAGAGTTTCCTCCATTGCGCAATATTCCCCA

[0119] denovo6368 [SEQ ID NO: 24]:

[0120] CCTGTTTGCTCCCCACGCTCTCGCGCCTGAGCGTCAGTCTCTCTCCAGCAGGCCGCCTTCGCCTCCGGTATTCCTCCTGATCTCTGCGTATTTCACCACTACACCAGGAATTCTGCCTGCCTCTAGAGCACTCTAGTCAGCTGGTTCTTCCTGCTTACCCGGGGTGGGCCCGGGCCTTTTACAAAAAGCCTCTCTGACCGCCTGCGCGCGCTTTACGCCCAATGATTCCGGACAACGCTCGCCCCCTACGTATTGCCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTAAAGTCAGGTAGTCTCTCTCTTGCCTGACGACTGAGCTCTGCGGGACGAATCCCTTCCTCACTCACGCGGCATCGCTGCGTCAGAGTTGCCTCCATTGCGCAATATTCCCCA

[0121] denovo7117 [SEQ ID NO: 25]:

[0122] CCTGTTTGCTCCCCACGCTTTCGTGCCTCAGTGTCAGTTACAGTCCAGAGAGCCGCCTTCGCAACTGGTATTCCTCCTAATATCTACGCATTTCACCGCTACACTAGGAATTCTACTCTCCTCTCCTGCACTCAAGTTTCTCAGTTTCAAAGGCTTACTACGGTTGAGCCGTAGCCTTTCACCTCTGACTTAAGAAACCACCTACGCACCCTTTACGCCCAGTAATTCCGGATAACGCTAGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTCCTCCTCAAGTACCGTCATTATCTTCCTTGAGGACAGAGCTTTACGACCCGAAGGCCTTCATCGCTCACGCGGCGTTGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCA

[0123] denovo4881 [SEQ ID NO: 26]:

[0124] CCTGTTTGCTCCCCACGCTCTCGCGCCTGAGCGTCAGTGTCTGTCCGGCAGGCCGCCTTCGCCTCTGGTATTCCTCCTGATCTCTGCGCATTTCACCGCTACACCAGGAATTCTGCCTGCCTCTCCAGCACTCGAGCCGCGCGGTTCGGGATGCCCGAACAGGGTTGGGCCCTGATCTTTCACATCCCGCCTTCGCGGCCGC CTGCGCGCCCTTTACGCCCAGTCATTCCGGACAACGCTCGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTGCTTGTCAGGTACCGTCTCTCTCTTCCCTGACAACAGAGCTTTACGGGACGAATCCCTTCTTCGCTCACGCGGCATTGCTGCGTCAGAGTTGCCTCCATTGCGCAATATTCCCCA

[0125] In the context of this invention, the "percentage of identity" or "sequence identity" between two sequences (e.g., nucleic acid or amino acid sequences) is intended to represent the percentage of identical nucleotides or amino acid residues between two sequences to be compared after optimal alignment (optimal alignment). This percentage is purely statistical, and the differences between the two sequences are randomly distributed and lie across their entire length. Sequence comparisons between two nucleic acid or amino acid sequences are typically performed by comparing these sequences after optimal alignment, which can be done via fragments or "comparison windows." In addition to manual methods, similarity can be obtained through Smith and Waterman’s local homology algorithm (1981) [Advances in Applied Mathematics (Ad. App. Math). 2:482], Neddleman and Wunsch’s local homology algorithm (1970) [J. Mol. Biol. 48:443], Pearson and Lipman’s (1988) similarity study method [Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 85:2444], computer software using these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or through BLAST N or BLAST P comparison software.

[0126] The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing the two sequences in an optimal alignment, wherein the nucleic acid or amino acid sequences to be compared may contain additions or deletions relative to a reference sequence for optimal alignment between the two sequences. The percentage of identity is calculated as follows: determine the number of identical positions of nucleotide or amino acid residues between the two sequences, divide that number of identical positions by the total number of positions in the comparison window, and then multiply the result by 100 to obtain the percentage of identity between the two sequences.

[0127] For example, the BLAST program can be used. The “BLAST 2 Sequence” (Tatusova et al., “Blast 2 Sequence – A New Tool for Comparing Protein and Nucleotide Sequences,” FEMS Microbiol Lett. 174:247-250) is available at www.ncbi.nlm.nih.gov. The parameters used are the default ones (specifically, for the parameters “open gap penalty”: 5 and “expanded gap penalty”: 2; the selected matrix is ​​the “BLOSUM62” matrix as proposed by the program). The percentage of identity between the two sequences being compared is calculated directly by the program. Other programs, such as “ALIGN” or “Megalign” (DNASTAR) software, can also be used.

[0128] Amino acid sequences having at least about 80%, preferably at least about 85%, at least about 90%, at least about 95%, and at least about 98% identity with a reference amino acid sequence, particularly those with certain modifications relative to the comparative reference sequence, especially the deletion, addition, or substitution of at least one amino acid, truncation, or extension, are preferred. In cases where one or more continuous or discontinuous amino acids are substituted, the preferred substitution is where the substituted amino acid is replaced by an "equivalent" amino acid. The expression "equivalent amino acid" is intended herein to mean any amino acid that can be substituted by one of the basic structural amino acids without substantially altering the biological activity of the corresponding antibody, and is, for example, defined later, particularly in the examples. These equivalent amino acids can be determined by their structural homology with the amino acids they replace, or by the results of comparative experiments on the biological activities of different antibodies that can be performed.

[0129] Table 1 illustrates, through non-limiting examples, the possibility of substitutions being made without causing profound changes in the biological activity of the corresponding modified amino acid sequence, with reverse substitutions naturally conceivable under the same conditions.

[0130] Table 1

[0131] The currently disclosed subject matter provides health assessment tools related to the microorganisms disclosed herein. In some embodiments, the health assessment tools are used to monitor the gut health status or gut dysbiosis of a subject. In some non-limiting embodiments, the subject may be a companion animal (e.g., a dog). In some embodiments, the health assessment tool includes one or more probes for detecting amounts of one or more microorganisms disclosed herein. In some embodiments, the health assessment tool includes a microarray of one or more probes for detecting amounts of one or more microorganisms disclosed herein. In some embodiments, the probes comprise nucleic acid probes for detecting a signature gene of the microorganisms disclosed herein. In some embodiments, the probes detect the 16S rRNA sequence of the microorganisms disclosed herein, such as a 16S rRNA sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) or 100% sequence identity with any of the nucleotide sequences in SEQ ID NO:1-26. In some embodiments, the probes comprise antibodies, such as monoclonal antibodies. Antibodies can bind to the surface proteins / antigens of microorganisms disclosed herein, and can be, for example, naturally occurring or synthetic antibodies.

[0132] In some implementations, the amount of microorganisms is measured from a fecal sample of a subject (e.g., a companion animal, such as a dog). In some implementations, health assessment tools monitor gut health or dysbiosis by comparing the amount of one or more microorganisms to a reference amount of one or more microorganisms.

[0133] In some embodiments, the health assessment tool includes a probe for detecting at least about 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 26, or more of the microorganisms disclosed herein. In some embodiments, the health assessment tool includes a probe for detecting about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, or about 26 of the microorganisms disclosed herein. In some implementations, the health assessment tool includes the microorganisms disclosed herein for detecting between about 1 and about 500, between about 1 and about 100, between about 1 and about 26, between about 5 and about 100, between about 5 and about 26, between about 10 and about 26, between about 15 and about 50, or between about 50 and about 100.

[0134] In some embodiments, the one or more microorganisms include 16S rRNA containing at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) homology or identity with any of the sequences in Table 5 (such as any of SEQ ID NO: 27-293).

[0135] In some embodiments, the one or more microorganisms include bacteria containing 16S rRNA, said 16S rRNA comprising a nucleotide sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) or 100% sequence identity with any of the nucleotide sequences in SEQ ID Nos: 1-26.

[0136] In a particular embodiment, one or more microorganisms include bacteria containing 16S rRNA, said 16S rRNA comprising a nucleotide sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) or 100% sequence identity with any of the nucleotide sequences in SEQ ID Nos: 1-14.

[0137] In other embodiments, the one or more microorganisms include bacteria containing 16S rRNA, the 16S rRNA comprising a nucleotide sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) or 100% sequence identity with any of the nucleotide sequences in SEQ ID Nos: 15-26.

[0138] In some embodiments, the bacteria are selected from the group consisting of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, denovo943, denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, denovo4881, and any combination thereof.

[0139] 3. Pet food

[0140] The currently disclosed subject provides a pet food for improving the gut health of subjects. Subjects can be companion animals, such as dogs or cats.

[0141] In some embodiments, the pet food contains an effective amount of beet pulp. In some embodiments, the beet pulp is untreated beet pulp. In some embodiments, the beet pulp is cooked or sterilized or included in extruded or processed products.

[0142] In some implementations, beet pulp is present in pet food at concentrations between about 0.01% w / w and about 10% w / w, between about 0.1% w / w and about 5% w / w, between about 0.5% w / w and about 4% w / w, between about 0.5% w / w and about 3% w / w, between about 0.5% w / w and about 2% w / w, between about 0.5% w / w and about 1.5% w / w, between about 0.5% w / w and about 1.2% w / w, between about 0.5% w / w and about 1% w / w, between about 0.5% w / w and about 0.9% w / w, or between about 0.5% w / w and about 0.8% w / w. In some embodiments, beet pulp is present in pet food at concentrations between about 0.1% w / w and about 10% w / w, about 0.1% w / w and about 5% w / w, about 0.5% w / w and about 4% w / w, about 0.8% w / w and about 3% w / w, about 0.8% w / w and about 2% w / w, about 0.8% w / w and about 1.5% w / w, about 0.8% w / w and about 1% w / w, about 1% w / w and about 10% w / w, about 1% w / w and about 5% w / w, about 2% w / w and about 5% w / w, or about 1% w / w and about 2% w / w. In some embodiments, beet pulp is present in pet food at a concentration of about 0.8% w / w.

[0143] Pet food can be fed to companion animals, such as, but not limited to, dogs or cats. In some implementations, the intake of beet pulp for companion animals ranges from about 0.5g / day to about 90g / day.

[0144] This document also discloses a pet food containing an effective amount of gut microbiota, such as any of the bacteria disclosed herein, which are associated with a healthy gut condition in companion animals. As used herein, the term "healthy" means a companion animal that has not been diagnosed with a disease known to affect the microbiome. Examples of such diseases include, but are not limited to, irritable bowel syndrome, ulcerative colitis, Crohn's disease, and inflammatory bowel disease. Preferably, a healthy companion animal does not suffer from dysbiosis. Dysbiosis refers to an imbalance in the gut microbiome due to key bacteria (such as Bifidobacteria) bifidobacteria ), such as Bifidobacterium infantis subsp. ( B. longum subsp. infantis This is caused by insufficient levels of certain substances or an overabundance of harmful bacteria in the gut. Methods for detecting dysbiosis are well-known in the field.

[0145] The effective amount of gut microbiota disclosed herein in pet food refers to the amount required to improve immunity, digestive function, and / or reduce inflammation in companion animals (e.g., dogs) when ingested or consumed; the amount required to improve fecal quality, digestive health, immunity, digestive function, and / or reduce inflammation; the amount required to alleviate symptoms and / or reduce the likelihood of digestive disorders and / or inflammation; the amount required to improve the microbiome, fecal quality, digestive health, immunity, digestive function, and / or reduce inflammation; and / or the amount required to alleviate symptoms and / or reduce the likelihood of digestive disorders and / or inflammation. In some embodiments, the gut microbiota is selected from the group consisting of any bacteria comprising 16S rRNA, said 16S rRNA comprising a nucleotide sequence having at least about 95% sequence identity with a nucleotide sequence of any one of SEQ ID NO: 1-14 and any combination thereof. In some embodiments, the bacteria are selected from species of the family Trichophytoncidae (…). Lachnospiraceae sp. ), Prevotella coccidioides ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius Holdemania [Eubacterium] biforme ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ), species of the genus Dornos ( Dorea sp. ), species of the genus *Ruminococcus* ( Ruminococcaceae sp. ), species of the genus Bacteroides ( Bacteroides sp. ), species of the genus Broutella ( Blautia sp. ), species of the genus Erysipelothrix ( Erysipelotrichaceae sp. ), species of the genus *Helicobacter* ( Lachnospiraceae sp. The group consisting of ) and any combination thereof. In some embodiments, the bacteria are selected from *Proteus vulgaris* ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme The group consisting of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, denovo943, and any combination thereof. In some embodiments, the bacteria are selected from the group consisting of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, denovo943, and any combination thereof.

[0146] In some implementations, the amount of bacteria contained in pet food is between approximately 1,000 CFU and approximately 100 trillion CFU. In some implementations, the amount of bacteria is between approximately 1,000 CFU and approximately 10 trillion CFU, between approximately 1 million CFU and approximately 1 trillion CFU, between approximately 1 billion CFU and approximately 1 trillion CFU, between approximately 1 billion CFU and approximately 100 billion CFU, between approximately 100 million CFU and approximately 100 billion CFU, between approximately 1 billion CFU and approximately 50 billion CFU, between approximately 100 million CFU and approximately 50 billion CFU, or between approximately 1 billion CFU and approximately 10 billion CFU. In some implementations, the bacteria contained in pet food are at least about 1,000 CFU, at least about 1 million CFU, at least about 10 million CFU, at least about 100 million CFU, at least about 1 billion CFU, at least about 10 billion CFU, at least about 100 billion CFU, or more.

[0147] In some embodiments, the pet food also contains an effective amount of beet pulp. An effective amount of beet pulp refers to the amount required, when ingested or consumed by a subject (e.g., a companion animal, such as a dog), in the companion animal to: improve immunity, digestive function, and / or reduce inflammation; improve stool quality, digestive health, immunity, digestive function, and / or reduce inflammation; alleviate symptoms and / or reduce the likelihood of digestive disorders and / or inflammation; improve the microbiome, stool quality, digestive health, immunity, digestive function, and / or reduce inflammation; and / or alleviate symptoms and / or reduce the likelihood of digestive disorders and / or inflammation.

[0148] In some embodiments, pet food is a dietary supplement, for example, applied on top of pet food as a topper or subsequently mixed into the whole product. In some embodiments, pet food is a treat product or chewable, or a coarse-grind-based treat or supplement product. In some embodiments, pet food is cat food or dog food. In some embodiments, the food is a dog food product. In some embodiments, pet food is dry pet food. In some embodiments, pet food is wet pet food.

[0149] In some implementations, the concentration of beet pulp in the wet pet food is between about 0.01% w / w and about 10% w / w, about 0.1% w / w and about 5% w / w, about 0.5% w / w and about 4% w / w, about 0.5% w / w and about 3% w / w, about 0.5% w / w and about 2% w / w, about 0.5% w / w and about 1.5% w / w, about 0.5% w / w and about 1.2% w / w, about 0.5% w / w and about 1% w / w, about 0.5% w / w and about 0.9% w / w, or about 0.5% w / w and about 0.8% w / w. In some embodiments, the concentration of beet meal is between about 0.1% w / w and about 10% w / w, about 0.1% w / w and about 5% w / w, about 0.5% w / w and about 4% w / w, about 0.8% w / w and about 3% w / w, about 0.8% w / w and about 2% w / w, about 0.8% w / w and about 1.5% w / w, about 0.8% w / w and about 1% w / w, about 1% w / w and about 10% w / w, about 1% w / w and about 5% w / w, about 2% w / w and about 5% w / w, or about 1% w / w and about 2% w / w. In some embodiments, the concentration of beet meal is about 0.8% w / w.

[0150] In some embodiments, any pet food disclosed herein may also contain additional active agents. Non-limiting examples of additional active agents that may be present in formulations of the subject matter of this disclosure include nutrients (e.g., amino acids, peptides, proteins, fatty acids, carbohydrates, sugars, nucleic acids, nucleotides, vitamins, minerals, etc.), prebiotics, probiotics, antioxidants and / or agents that enhance the microbiome, improve gastrointestinal health, and improve animal health.

[0151] In some embodiments, the pet food contains one or more probiotics. In some embodiments, the probiotics are animal probiotics. In some embodiments, the animal probiotics are feline probiotics. In other embodiments, the animal probiotics are canine probiotics. In some embodiments, the probiotics are Bifidobacteria (…). Bifidobacterium ), Lactobacillus ( Lactobacillus ), lactic acid bacteria ( lactic acid bacterium ) and / or Enterococci ( Enterococcus In some implementations, probiotics are selected from the group consisting of any organisms derived from lactic acid bacteria, and more specifically from the genera *Lactococcus*. Lactococcus spp. ), Pediococcus ( Pediococcus spp. Bifidobacterium spp. Bifidobacterium spp. (such as Bifidobacterium longum) B. longumBifidobacterium bifidum ( B. bifidum Bifidobacterium pseudolongum ( B. pseudolongum Bifidobacterium animalis ( B. animalis ), Bifidobacterium infantis ( B infantis Lactobacillus ()), Lactobacillus genus ( Lactobacillus spp. (e.g., Lactobacillus bulgaricus) L. bulgaricus ), Lactobacillus acidophilus ( L. acidophilus ), Lactobacillus brevis ( L. brevis Lactobacillus casei ( L casei Lactobacillus rhamnosus ( L. rhamnosus Lactobacillus plantarum ( L. plantarum Lactobacillus reuteri ( L. reuteri ), Lactobacillus fermentum ( L. fermentum ), Enterococcus ( Enterococcus spp. (such as Enterococcus faecalis) E. faecium ), Prevotella spp. ( Prevotella spp. ), Fusobacterium genus ( Fusobacterium spp ), Proteobacterium spp. Alloprevotella spp (and any combination thereof). In some embodiments, probiotics are included in pet food administered to companion animals, wherein the amount of probiotics administered to companion animals is from about 1 colony-forming unit (CFU) to about 100 billion CFU per day to maintain GI microbiota or gut health. In some embodiments, probiotics are administered to companion animals from about 1 colony-forming unit (CFU) to about 20 billion CFU per day to maintain gut microbiota or gut health. In some embodiments, probiotics are administered to companion animals in amounts from about 1 billion CFU to about 20 billion CFU per day to maintain GI microbiota. In some embodiments, probiotics are administered to companion animals in amounts from about 100 million to about 100 billion live bacteria per day. In some embodiments, probiotics are administered to companion animals in amounts from about 100 million to about 10 billion live bacteria per day. In some embodiments, probiotics are administered to companion animals in amounts from about 100 million to about 10 billion live bacteria per day. 4 CFU to 1×10 14 CFU is administered to companion animals.

[0152] In further embodiments, additional prebiotics may be included, such as fructooligosaccharides (FOS), xylooligosaccharides (XOS), galactooligosaccharides (GOS), dextran, galactan, arabinogalactan, inulin, and / or mannose oligosaccharides. The additional prebiotics may be administered through pet food in amounts sufficient to actively stimulate the microbiome or gut microbiota and / or cause the proliferation of one or more probiotics in the companion animal.

[0153] In some embodiments, pet food may also contain additional additives present in the pet food in amounts that do not impair the purposes and effects provided by the subject matter currently disclosed. Examples of contemplated additives include, but are not limited to, substances that functionally improve health, substances with stabilizing effects, sensory substances, processing aids, substances that enhance palatability, coloring substances, and substances that provide nutritional benefits. In some embodiments, stabilizing substances include, but are not limited to, substances that can increase the shelf life of the product. Such substances include, but are not limited to, preservatives, synergists and chelating agents, packaging gases, stabilizers, emulsifiers, thickeners, gelling agents, and humectants. Examples of emulsifiers and / or thickeners include, but are not limited to, gelatin, cellulose ethers, starch, starch esters, starch ethers, and modified starches.

[0154] Other exemplary additives include additives for coloring, palatability, and nutritional purposes, such as colorants; iron oxide, sodium chloride, potassium citrate, potassium chloride, and other edible salts; vitamins; minerals; and flavorings. The amount of such additives in a product is typically up to about 5% (based on dry product).

[0155] The pet foods disclosed herein can be formulated as dietary supplements for companion animals. For example, a dietary supplement can be a feed used in conjunction with another feed to improve nutritional balance or overall performance. A dietary supplement can also be a composition that is fed undiluted as a supplement to other feeds, freely chosen with other portions of an animal's ration available separately, or diluted and mixed with the animal's regular feed to produce a complete feed. For example, AAFCO provides a discussion of supplements in American Feed Control Officials, Incorp. (Official Publication, p220 (2003)). Supplements can be in various forms, including, for example, powders, liquids, syrups, pills, tablets, or capsule compositions. Other forms of supplements are known to those skilled in the art.

[0156] The pet food disclosed herein can also be formulated as a treat. In some embodiments, the treat includes, for example, a composition administered to the animal to induce it to eat outside of mealtimes. In some embodiments, the pet food is a canine treat, such as a dog bone. Treats can be nutritious, wherein the product contains one or more nutrients and may, for example, have a composition for food as described above. Non-nutritional treats include any other non-toxic treats.

[0157] In some embodiments, the gut microbiota and / or beet pulp of the subject matter of this disclosure may be incorporated into the composition during formulation processing, such as during and / or after mixing with other components of the product. These components may be dispensed into the product in a conventional manner known to those skilled in the art.

[0158] Furthermore, in some embodiments, the pet food of this disclosure can be prepared in canned or wet form using conventional companion animal food processes. In such embodiments, ground animal (e.g., mammals, poultry, and / or fish) protein tissue can be mixed with other ingredients, such as milk fish oil, grains, other nutritionally balanced ingredients, special-purpose additives (e.g., vitamin and mineral blends, inorganic salts, cellulose and beet pulp, fillers, etc.), and sufficient water for processing is added. These ingredients are mixed in a container suitable for heating while mixing the ingredients. The mixture can be heated using any suitable method, such as by direct steam injection or by using a container equipped with a heat exchanger. After the last ingredient is added, the mixture is heated to a temperature range of approximately 50°F to approximately 212°F. Temperatures outside this range are acceptable but commercially impractical without the use of other processing aids. When heated to the appropriate temperature, the material is typically in the form of a viscous liquid. The viscous liquid is filled into cans. The lids are placed on the cans, and the containers are sealed airtight. The sealed cans are then placed in conventional equipment designed for sterilizing the contents. This is typically achieved by heating to a temperature above approximately 230°F for an appropriate time, depending on factors such as the temperature used and the composition.

[0159] In some embodiments, the pet food of this disclosure can be prepared in a dry form using conventional methods. The dry ingredients used in the dried pet food include, for example, animal protein sources, plant protein sources, grains, etc., which are ground and mixed together. Wet or liquid ingredients, including fats, oils, animal protein sources, water, etc., can then be added to and mixed with the dry mixture. The mixture can then be processed into coarsely ground or similar dry flakes. In some embodiments, the pet food is a coarsely ground material. The coarsely ground material can be formed using an extrusion process, in which the mixture of dry and wet ingredients is mechanically processed under high pressure and high temperature and forced through a small opening and cut into coarsely ground material by a rotating blade. The wet coarsely ground material can then be dried and optionally coated with one or more localized coatings, such as flavorings, fats, oils, powders, etc. In some embodiments, the coarsely ground material can also be made from raw dough using a baking process instead of an extrusion process, wherein the raw dough is placed in a mold prior to dry heat processing.

[0160] In some implementations, the snacks of the currently disclosed subject matter can be prepared by, for example, extrusion or baking processes similar to those described above for dry foods.

[0161] 4. Treatment methods and health assessment

[0162] This disclosure also provides methods for enhancing or improving the microbiome, improving gut health, and / or treating gut dysbiosis in subjects in need. In some embodiments, the subjects are companion animals, such as dogs or cats. In some embodiments, the method can improve the companion animal's immunity, digestive function, and / or alleviate dysbiosis.

[0163] Such methods include administering an effective amount of any pet food disclosed herein to a subject. The method may further include monitoring the gut microbiota in a companion animal, such as any or more of the gut microbiota disclosed herein. In some embodiments, the gut microbiota are measured in a fecal sample from the subject. In some other embodiments, the gut microbiota are measured in a sample from the subject's intestines. In some embodiments, the subject is a companion animal, such as a dog.

[0164] In some implementations, pet food may be administered to subjects approximately 20 times to once daily, approximately 10 times to once daily, or approximately 5 times to once daily. In some implementations, pet food may be administered to subjects once daily, twice daily, three times daily, four times daily, five times daily, six times daily, seven times daily, eight times daily, nine times daily, ten times daily, or more. In some implementations, pet food may be administered to subjects every two days, every three days, every four days, every five days, every six days, once a week, once every two weeks, once every three weeks, or once a month. In some implementations, pet food may be administered to animals in a constant manner, for example, where the animal is graze on a continuously available supply of the main food.

[0165] In some embodiments, the dosage of pet food is between about 1 mg / kg body weight per day and about 5000 mg / kg body weight per day. In some embodiments, the dosage of pet food is between about 5 mg / kg body weight per day and about 1000 mg / kg body weight per day, between about 10 mg / kg body weight per day and about 500 mg / kg body weight per day, between about 10 mg / kg body weight per day and about 250 mg / kg body weight per day, between about 10 mg / kg body weight per day and about 200 mg / kg body weight per day, between about 20 mg / kg body weight per day and about 100 mg / kg body weight per day, between about 20 mg / kg body weight per day and about 50 mg / kg body weight per day, or any intermediate range thereof. In some embodiments, the dosage of pet food is at least about 1 mg / kg body weight per day, at least about 5 mg / kg body weight per day, at least about 10 mg / kg body weight per day, at least about 20 mg / kg body weight per day, at least about 50 mg / kg body weight per day, at least about 100 mg / kg body weight per day, at least about 200 mg / kg body weight per day, or more. In some implementations, the dosage of pet food is not more than about 5 mg / kg body weight per day, not more than about 10 mg / kg body weight per day, not more than about 20 mg / kg body weight per day, not more than about 50 mg / kg body weight per day, not more than about 100 mg / kg body weight per day, not more than about 200 mg / kg body weight per day, not more than about 500 mg / kg body weight per day or more.

[0166] In some implementations, the amount of pet food is reduced during the feeding of the companion animal. In some implementations, the concentration of pet food is increased during the feeding of the companion animal. In some implementations, the concentration of pet food is modified according to the age of the companion animal.

[0167] In some non-limiting embodiments, the currently disclosed subject matter provides a method for determining the intestinal health status of a companion animal in need. In some embodiments, the method includes: a) Measure the first amount of the first gut microbiota and / or the second amount of the second gut microbiota in companion animals; b) Comparing a first quantity of first gut microbiota to a first reference quantity of first gut microbiota, and / or comparing a second quantity of second gut microbiota to a second reference quantity of second gut microbiota, wherein the reference quantity of gut microbiota is determined based on the quantity of gut microbiota in multiple healthy companion animals; and c) Determine the gut health status of the companion animal when the first amount of gut microbiota is higher than the first reference amount of the first gut microbiota, and / or when the second amount of the second gut microbiota is lower than the second reference amount of the second gut microbiota.

[0168] In some embodiments, the first gut microbiota is one or more bacteria containing 16S rRNA, said 16S rRNA comprising a nucleotide sequence having at least about 95% sequence identity with a nucleotide sequence of any one of SEQ ID NO: 1-14.

[0169] In some embodiments, the first gut microbiota is selected from the group consisting of denovo 1184, denovo 1244, denovo 1696, denovo 2407, denovo 2451, denovo 283, denovo 3487, denovo 4154, denovo 4328, denovo 4681, denovo 498, denovo 5338, denovo 6995, denovo 943, and any combination thereof. In some embodiments, the first gut microbiota is selected from *Bacillus prednisii* (…). Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ) and any combination thereof.

[0170] In some embodiments, the second gut microbiota is one or more bacteria containing 16S rRNA, said 16S rRNA comprising a nucleotide sequence having at least about 95% sequence identity with the nucleotide sequence of any one of SEQ ID NO: 15-26.

[0171] In some implementations, the second gut microbiota is selected from the group consisting of denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, denovo4881, and any combination thereof.

[0172] In some non-limiting embodiments, the currently disclosed subject matter provides a method for treating gut dysbiosis and / or improving gut health in companion animals in need. In some embodiments, the method includes: a) Measure the first quantity of one or more gut microbes in a companion animal; b) Treatment regimens administered to companion animals to treat intestinal diseases and / or improve intestinal health; c) Measure a second amount of the subject's gut microbiota after step b); and d) Determine the animal's gut health status or response to treatment.

[0173] In some implementations, the method further includes continuing the treatment regimen when a second amount of gut microbiota changes compared to a first amount.

[0174] In some embodiments, the first gut microbiota is one or more bacteria containing 16S rRNA, said 16S rRNA comprising a nucleotide sequence having at least about 95% sequence identity with a nucleotide sequence of any one of SEQ ID NO: 1-14, and wherein step d) comprises continuing to administer the treatment regimen when the second amount of gut microbiota is increased compared to the first amount of gut microbiota.

[0175] In some embodiments, the first gut microbiota is selected from the group consisting of denovo 1184, denovo 1244, denovo 1696, denovo 2407, denovo 2451, denovo 283, denovo 3487, denovo 4154, denovo 4328, denovo 4681, denovo 498, denovo 5338, denovo 6995, denovo 493, and any combination thereof. In some embodiments, the first gut microbiota is selected from *Bacillus prednisii* (…). Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ) and groups consisting of any combination thereof.

[0176] In some embodiments, a second quantity of gut microbiota is measured between approximately 7 and approximately 14 days after step b). In some embodiments, the quantity of gut microbiota increases within approximately 21 days, approximately 14 days, approximately 12 days, approximately 10 days, approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, or approximately 1 day after step b). In some embodiments, the quantity of gut bacteria increases within approximately 1 day to approximately 21 days, approximately 1 day to approximately 14 days, approximately 3 days to approximately 14 days, approximately 5 days to approximately 14 days, approximately 7 days to approximately 14 days, approximately 10 days to approximately 14 days, or approximately 7 days to approximately 21 days after step b).

[0177] In some embodiments, the second gut microbiota is one or more bacteria containing 16S rRNA, said 16S rRNA containing at least about 95% identity with any one of SEQ ID NO: 15-26 or the same nucleotide sequence as any one of SEQ ID NO: 15-26, wherein step d) includes continuing the treatment regimen when the second amount of gut microbiota is reduced compared to the first amount of gut microbiota.

[0178] In some implementations, the second gut microbiota is selected from the group consisting of denovo1214, denovo1400, denovo1762, denovo2014, denovo2197, denovo2368, denovo3663, denovo4206, denovo4485, denovo6368, denovo7117, denovo4881, and any combination thereof.

[0179] In some embodiments, a second quantity of gut microbiota is measured between approximately 7 and approximately 14 days after step b). In some embodiments, the quantity of gut microbiota decreases within approximately 21 days, approximately 14 days, approximately 12 days, approximately 10 days, approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, or approximately 1 day after step b). In some embodiments, the quantity of gut bacteria decreases within approximately 1 day to approximately 21 days, approximately 1 day to approximately 14 days, approximately 3 days to approximately 14 days, approximately 5 days to approximately 14 days, approximately 7 days to approximately 14 days, approximately 10 days to approximately 14 days, or approximately 7 days to approximately 21 days after step b).

[0180] In some embodiments, the reference level for gut microbiota is the average level of gut microbiota across multiple healthy companion animals. In some embodiments, the reference level for gut microbiota is within approximately three standard deviations of the average level of gut microbiota across multiple healthy companion animals. In some embodiments, the reference level for gut microbiota is within approximately two standard deviations of the average level of gut microbiota across multiple healthy companion animals. In some embodiments, the reference level for gut microbiota is within approximately one standard deviation of the average level of gut microbiota across multiple healthy companion animals.

[0181] In some embodiments, the quantity of gut microbiota can be determined by any method known in the art. In some embodiments, the method includes, but is not limited to, antibody-based detection methods for detecting microbial-associated proteins / antigens, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blotting; and methods for detecting microbial-associated 16S rRNA, such as real-time polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), DNA sequencing, and microarray analysis. In some embodiments, the microarray comprises probes for detecting any gut microbiota disclosed herein.

[0182] In some embodiments, the treatment may be any treatment for ecological disorders known in the art. In some embodiments, the treatment includes the treatments disclosed herein.

[0183] In some implementations, the amount of gut bacteria is measured from a fecal sample taken from the subject.

[0184] Example

[0185] The subject matter disclosed herein will be better understood by referring to the following embodiments, which are provided as examples of the invention and not as limiting.

[0186] Example 1

[0187] introduce

[0188] Revealing whether there are associations between the canine microbiome, fecal quality, and dietary factors, and if so, revealing the nature of these associations with the advancement of deep sequencing methods, could be feasible. This knowledge would be beneficial in supporting an understanding of the health and characteristics of a healthy microbiome and in achieving dietary control of the gut microbiome for a healthy gastrointestinal tract.

[0189] Three ingredients were incorporated into wet pet food blocks in previously identified gravy products for improving fecal quality. The digestive health ingredients included beet pulp (0.8% w / w), cellulose (0.5% w / w), and pea protein (0.25% w / w). In a feeding study of 24 dogs, the three diets were compared to a commercial control diet containing 0.5% beet pulp. The primary measures of the study included assessment of fecal quality and fecal microbiota. Fecal quality studies require accurate determination of fecal form, and therefore fecal consistency grading scales are used to assess fecal quality. One such grading system is the Waltham fecal scoring system, which classifies fecal form according to a 17-point scale, from grade 1 (represented by hard and dry feces) to grade 5 (watery diarrhea) (Moxham, 2001).

[0190] method

[0191] Learning Plan

[0192] A cohort of 24 captive dogs was recruited. Three diets were produced for the study: a wet pet food block based on a commercially formulated gravy pouch diet and diets containing the same basic formula but with increased levels of beet pulp, cellulose, or pea protein. Dogs received each diet for 21 days to obtain sufficient fecal quality data and to stabilize the gut microbiota after dietary changes. Dogs were fed each of the three test diets in the commercially formulated diet and a balanced Latin square study design to allow each dog to experience the sequential effect of each diet (…). Figure 1 ).

[0193] Record the fecal mass fraction, number of defecations and wet weight of overnight defecation every day, and collect two fresh fecal samples from each dog at the beginning (days 2 and 4[+1]) and the end (days 18 and 20[+1]).

[0194] animal

[0195] The group consisted of 12 Beagles and 12 Labrador Retrievers. At the start of the study, the animals were between 2.0 and 6.8 years old (mean 4.16 years). All but one female dog were spayed / neutered. The study group was divided into 17 females and 7 males.

[0196] diet

[0197] Animals were fed three equal daily portions at standard times. All four diets were based on the same wet pet food block in the gravy pouch product and included a reference diet and three test diets with altered levels of beet pulp, cellulose, or pea protein. Each pouch contains approximately 100g of product.

[0198] Dogs were transitioned from their dry food diet to a standard commercial bag-sized block diet of chicken gravy and vegetables (100g bag). The transition was conducted over a 14-day period. A Latin square study design was used to test the diets based on the dietary rotation to produce a balanced diet.

[0199] Dogs were assigned to one of four feeding groups 1, 2, 3, or 4, forming a diet group of six dogs in triplicate of enclosures (Table 2). Dogs in these groups received food in the same rotation order, thus also forming dedicated social and exercise groups throughout the study period.

[0200] Table 2. Feeding groups and dietary rotations across the study cohorts.

[0201] Data collection

[0202] Throughout the study, the quality of all overnight stools was scored daily using a 17-point stool quality rating scale, and the incidence of poor stools (outside the acceptable range of 1.5–3.75) was recorded. Figure 2A and Figure 2B Fecal samples were collected on days 2, 4, 18, and 20 for assessing the microbiome (or +1 if no samples were generated by the target date).

[0203] During the study, data for the following covariates were collected for inclusion analysis to determine whether differences in the microbiome were associated with adult life stage, older life stage, and late life stage.

[0204] Daily and overnight stool scores for each pair

[0205] Daily food intake Weight and physical condition score All collected stool samples were scored using a 17-point stool quality rating scale, and the incidence of poor stool samples (outside the acceptable range of 1.5–3.75) was recorded.

[0206] Fecal sample collection and processing

[0207] Collect fresh fecal samples, with the most frequent sample representing the first bowel movement of the day to ensure sample safety. Most samples are freshly generated in grass paddocks or during walks. Collect samples immediately, but no more than 15 minutes after defecation. After collection, divide the feces into three 100mg and two 400mg aliquots in sterile 2ml Lo-Bind Eppendorf tubes. Store samples at -80°C.

[0208] Fecal processing: 100 mg portions of feces were treated with the QIAamp Power Faecal DNA Kit (Qiagen) to extract DNA from the feces, following the manufacturer's instructions. Following DNA extraction, the DNA concentration achieved for each sample was determined using a standard nanodroplet DNA quantification method. The fecal DNA was then diluted 1:10 before preparing an Illumina high-throughput DNA sequencing library via PCR amplification of the 16S rDNA locus (V4-6 region; Fadrosh et al., 2014). DNA sequencing was performed at a depth of 160 samples / run using the Miseq Illumina system (Chemistry v.3; 2 x 300 bp paired-end sequencing).

[0209] A quality threshold of at least 1,000 sequence reads per sample was defined; sequence data failing to meet this level were removed from the analysis. Sequence data were denoised to remove chimeras and clustered into hypothetical taxa based on 98% sequence identity. The resulting operational taxa (OTU) data were reduced to the non-rare fraction by removing taxa representing <0.01% of sequences in <2 animals from any given group. After reduction to the non-rare fraction of the population, OTU identification based on a single taxa reference sequence was reanalyzed, selected as the most representative sequence in the cluster. These sequences were used to query select Greengenes (McDonald et al., 2012) and Silva (ed. 132; Yilmaz et al., 2014) databases to confirm sequences in these databases with a similarity criterion of within 98% identity compared to the non-rare taxa reference sequence. Then, classification and assignment are performed based on sequence identity with top-level database hits, which are first evaluated based on the top 10 hits generated by a database search for each reference sequence. In cases where discrepancies exist between searches, Greengenes assignment is used.

[0210] Group comparison identification is based on the relative abundance of individual taxa detected compared to the total sequence within the sample.

[0211] Statistical methods

[0212] Prior to analysis, rare OTUs in the data were grouped into individual pseudo-OTUs. In at least two samples from any diet, non-rare (abundance sequences) were classified as OTUs, with a proportion greater than 0.01% of the total sequences. Multivariate methods such as multi-group principal component analysis (mgPCA), multivariate analysis (MFA), and partial least squares discriminant analysis (PLSDA) were used to convert the data into proportions and applied to the OTU data, using +2 and +4 as the numerator and denominator, followed by a log10 transformation.

[0213] Using animal ID as the grouping variable, mgPCA with a variance-free scale was applied to prevent particularly variable animal-dominated outcomes.

[0214] The MFA used to create the spider diagram was applied to the reformulated data, where rows correspond to diet and time, and columns correspond to the proportion of OTUs in each animal's block, with the mean of each block centered but not scaled by variance.

[0215] PLSDA was applied to OTU data, with responses to combined dietary and time variables per animal and multilevel adjustments. The number of components was adjusted using 3-fold cross-validation, and influential OTUs were selected by identifying those with a variable importance (VIP) score greater than 1 in the projection. The results of PLSDA were visualized using clustering image plots.

[0216] Univariate analyses were performed using generalized linear mixed-effects models with a binomial error distribution and a logit link function applied to individual OTUs. The response for each model was OTU count + 2 and total sample count - OTU count + 4 (i.e., success and failure). Fixed effects included diet, time, and their interactions, and random effects were included for animals to explain repeated measurements. Observational-level random effects were also included to explain over-dispersion.

[0217] The univariate generalized linear mixture model with the same fixed and random structure described above is also applicable to each phylum, family, and genus level, replacing the OTU counts with the sum of OTU counts within that level.

[0218] Shannon diversity and total sample readings were modeled using a linear mixed-effects model, where diversity / total readings were used as a response, a fixed effect of diet, time and their interaction, and a random effect of the animal to account for repeated measurements.

[0219] The average values ​​for all diet and time combinations were estimated from all models, with 95% confidence intervals. The following comparisons were also performed. Dietary intake at different times Between different times of each diet Dietary patterns between different time points, i.e., dietary slope For univariate analysis, multiple comparison correction was performed using the Benjamini-Hochberg procedure to maintain a false discovery rate of 5%.

[0220] All analyses were performed using R version 3.5.1 and the libraries lme4, multcomp, optimx, FactoryMineR (MFA), and mixOmics (mgPCA and PLSDA).

[0221] result

[0222] Analysis of the consistency between fecal samples and diet

[0223] Fecal quality analysis included assessment of fecal consistency (quality) scores; the proportion of unacceptable feces; defecation frequency; and overnight fecal wet weight. Fecal scores generated from overnight enclosures throughout the 21-day feeding period were analyzed to obtain fecal scores for the entire period. Additionally, comparative data between groups were evaluated during the last 7 days of the period. For all diets supplemented with digestive health components (0.8% w / w beet pulp, 0.5% w / w cellulose, and 0.25% w / w pea protein), the mean fecal score of this group was significantly higher than the optimal score of 2.5 compared to the commercial diet containing 0.5% beet pulp (Figure 3A). After adjustment for a period of time, when considering data from the last 7 days of the feeding period, the mean fecal quality scores of the reference control diet and SBP were numerically lower, and no significant difference was detected when dogs received a diet containing beet pulp compared to the reference diet. When dogs received a diet containing cellulose and pea protein, even in the last 7 days, the mean fecal score of the group remained significantly higher than the optimal fecal score of the group (…). Figure 3B ).

[0224] Throughout the study, all unacceptable feces scored above 3.75 (i.e., diarrhea), and no feces in this group produced a consistency score of 1.5 or lower (i.e., dry feces) (unacceptable dry feces). Consistent with the data describing the average fecal score, dogs receiving the reference diet produced significantly fewer bowel movements with a consistency score of 3.75 or higher compared to the diet containing fiber and pea protein. A significant difference was not detected in the proportion of unacceptable feces when animals received a diet containing beet pulp compared to the reference diet. Figure 4A and Figure 4B However, compared to the standard diet alone, the proportion of unacceptable feces exceeding a consistency score of 3.75 (consistent with diarrhea) significantly increased when cellulose or pea protein was supplemented instead of beet pulp. When animals received beet pulp, the level of diarrheal feces was significantly lower compared to cellulose and pea protein. These same patterns were detected throughout the full 21 days and the last 7 days of feeding when animals received the reference diet, compared to diets containing beet pulp, and the levels of unacceptable feces were very similar.

[0225] Correlation analysis between microbiome and diet and dietary changes

[0226] A total of 340 bacterial operational taxonomic units (OTUs) at the species level were identified. Partial least squares discriminant analysis (PLSDA) was used to analyze the abundance data of the 340 individual OTUs and the “rare” group, generating correlation plots. When the bacterial taxa with the least influence on driving sample clustering (variable importance in projection; VIP score <1) were removed, and the PLS-DA analysis was repeated, a subset of 26 taxa was retained. Figure 5 These correlation plots show that the composition of the fecal microbiota was more similar when animals were fed diets containing beet pulp (0.5% and 0.8% w / w), while other clusters contained samples produced when animals were fed cellulose and pea protein diets. A subset of the 26 bacterial taxa identified in the correlation plots contributed most to the compositional differences when animals were fed diets containing cellulose and pea protein compared to diets containing beet pulp. Of these 26 organisms that had a greater impact on sample clustering in the correlation plots, a subset of 14 OTUs appeared to produce a stronger signal (indicated by higher abundance, thus highlighted as red signals in the correlation plots), while the abundance of clusters enriched at the beginning of samples across all diets was lower (blue signals) when animals were fed a reference or beet pulp diet compared to diets containing cellulose and pea protein (p = <0.01; lower clusters, Figure 3). These include species identified as belonging to the family Trichophytonceae (…). Lachnospiraceae sp. ), Prevotella coccidioides ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ), species of the genus Dornos ( Dorea sp. ), species of the genus *Ruminococcus* ( Ruminococcaceae sp. ), species of the genus Bacteroides ( Bacteroides sp. ), species of the genus Broutella ( Blautia sp. ), 2 species of Bacteroides Bacteroides sp.2 ), species of the genus Erysipelothrix ( Erysipelotrichaceae sp. ), 3 species of Bacteroides Bacteroides sp.3 ), species of the genus *Helicobacter* ( Lachnospiraceae sp. ), 4 species of Bacteroides Bacteroides sp.4 ) and 5 species of Bacteroides Bacteroides sp.5 The literature reports some links between these species and human and cat health, as well as the production of short-chain fatty acids, including butyrate. Health associations in humans, cats, and other mammals are described in the family Trichophyceae (…). Lachnospiraceae ), Erysipelothrix family ( Erysipelotrichaceae ) and Rumenococci ( Ruminococcaceae ) and Dornos spp. ( Golden ) and Broutella spp. Blueberry ) species, and Proteus vulgaris ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ) and Dimorphic Halldmann's Eubacterium ( Holdemania [Eubacterium] biforme ).

[0227] The second group of 12 organisms appeared to be more abundant in the clusters that began to enrich at this stage, and less abundant when animals were fed a diet containing beet pulp (Table 2). Most of these species appear to be novel organisms from the order Clostridium, and some are so novel that they could not be identified outside the order Clostridium by sequence similarity searches of public databases containing previously detected organisms. Since all 12 organisms were not confirmed outside the family level by comparison with known bacterial species, they appear to be novel species from the intestines of canines.

[0228] discuss

[0229] At the dosage used, when added to wet pet food blocks in a gravy diet prior to processing, all previously reported components that improve fecal quality to achieve optimal fractions actually resulted in a significant change in optimal consistency compared to animals receiving a commercial diet containing 0.5% beet pulp. Of the components used, at the levels of inclusion in wet pet food blocks in a gravy pet food context, beet pulp appeared to have a smaller effect on fecal fraction than cellulose and pea protein, and this effect was less pronounced after a period of adjustment. Figure 4B When animals were fed diets containing beet pulp, there was no significant difference in the mean fecal fraction. No significant difference was observed in the proportion of loose stools classified as diarrhea (>3.75) between diets containing different levels of beet pulp; however, significantly higher levels of diarrheal feces were observed when animals were fed diets without beet pulp (which contained feed-grade levels of cellulose and pea protein).

[0230] Analysis of the fecal microbiota revealed that the composition was more similar when animals were fed diets containing varying levels of beet pulp than when fed diets containing cellulose or pea protein. The relative abundance of 26 microbial subsets identified in feces changed when animals were fed diets containing both cellulose and pea protein compared to diets containing beet pulp. Several of these 26 bacteria have previously been identified as being associated with the health of humans, cats, and other mammals. The abundance of these 26 organisms also changed more significantly in the first 2–4 days after the dietary change, suggesting that even with relatively small changes in nutrient intake, there is an underlying microbial imbalance (dysbiosis) associated with dietary transitions. The observed signs of microbial dysbiosis reflected the effects of the diet on fecal output consistency (fecal coarseness) and the rate of unacceptable fecal output; a diet containing 0.5% beet pulp supported more solid feces compared to the looser feces produced when animals were fed diets containing pea protein and cellulose. After a period of adjustment to a diet containing higher levels of beet pulp (0.8%), the average fecal fraction (looser stool) of the dogs did not increase significantly.

[0231] Because dietary changes are often associated with temporary loose stools, poor stool quality, or even diarrhea, there is considerable interest in managing these changes. Research suggests that dietary changes lead to alterations in the composition of the gut microbiota, which may be related to the observed poor stool quality.

[0232] Furthermore, the data suggest that the bacterial species detailed in Table 2 may be associated with more solid feces and less frequent diarrheal feces in dogs, while those in Table 2 (new species currently represented by DNA sequences) may represent species associated with poor fecal quality and diarrheal events in other healthy dogs. Management of these and related bacterial species in the gut microbiome could serve as a biological lever to control or reduce diet-related diarrheal events associated with dietary changes or poor fecal quality / diarrhea.

[0233] Bacterial species also represent organisms associated with gut health or healthy stool quality in dogs (Table 2) and reduced stool quality or diarrhea in other healthy dogs (Table 3). Therefore, bacterial species represent presumptive markers of gut health (Table 2) or poor stool quality (Table 3) and can be used to assess gut health in healthy dogs.

[0234] Table 2. Compared to diets containing cellulose and pea protein, bacterial taxa (OTUs) presented in the PLSDA cluster were more abundant in diets containing beet pulp, and were presented in a low abundance form at the beginning of the post-dietary change phase (i.e., in dietary disorders).

[0235] Table 3. Compared with diets containing cellulose and pea protein, bacterial taxa (OTUs) presented in the PLSDA cluster were less abundant in diets containing beet pulp, but were more abundant at the beginning of the phase following dietary changes.

[0236]

[0237] Table 4.

[0238]

[0239]

[0240] Table 5. 16S rRNA sequences of gut bacteria

[0241] References: 1. WAKSHLAG JJ, SIMPSON KW, STRUBLE AM, DOWD SE. Negative fecal characteristics are associated with pH and fecal floral alterations during dietary change in dogs. International Journal of Applied Research in Veterinary Medicine. 2011;9(3):278. 2. The Waltham Faeces Scoring System - a tool for veterinarians and petowners: how does your pet rate? Waltham Focus. 2001;11(2):24-5. 3. ANTHARAM, VC, LI, EC, ISHMAEL, A., SHARMA, A., MAI, V., RAND, KH & WANG, GP 2013. Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial diarrhea. J Clin Microbiol, 51, 2884-92. 4. ANTHARAM, VC, MCEWEN, DC, GARRETT, TJ, DOSSEY, AT, LI, EC, KOZLOV, AN, MESBAH, Z. & WANG, GP 2016. An Integrated Metabolomic and Microbiome Analysis Identified Specific Gut Microbiota Associated with Fecal Cholesterol and Coprostanol in Clostridium difficile Infection. PLoS One, 11, e0148824. 5. BARRY KA, HERNOT DC, VAN LOO J, FAHEY GC JR, DE GODOY MR. Fructan supplementation of senior Beagle dogs affects stool metabolite concentrations and fecal microbiota concentrations, but not nitrogen partitioning in excreta. J Anim Sci. 2014 Nov;92(11):4964-71. 6. BELL ET, SUCHODOLSKI JS, ISAIAH A, FLEEMAN LM, COOK AK, STEINER JM, MANSFIELD CS. Faecal microbiota of Beagle dogs with insulin-treated diabetes mellitus. PLoS One. 2014 Oct 3;9(10):e108729. 7. BELOSHAPKA, AN, DOWD, SE, SUCHODOLSKI, JS, STEINER, JM, DUCLOS, L. & SWANSON, KS 2013. Fecal microbial communities of healthy adult dogs fed raw meat-based diets with or without inulin or yeast cell wall extracts as assessed by 454 pyrosequencing. FEMS Microbiol Ecology, 84, 532-41. 8. BERMINGHAM, EN, YOUNG, W., KITTELMANN, S., KERR, KR, SWANSON, KS, ROY, NC & THOMAS, DG 2013. Dietary format alters fecal bacterial populations in the domesticcat (Felis catus). Microbiologyopen, 2, 173-81. 9. DAVID, LA, MAURICE, CF, CARMODY, RN, GOOTENBERG, DB, BUTTON, JE, WOLFE, BE, LING, AV, DEVLIN, AS, VARMA, Y., FISCHBACH, MA AND BIDDINGER, SB, 2014. Diet rapidly and reproducibly alters the human gut microbiome. Nature, 505(7484), p.559. 10. DE GODOY, MR, KERR, KR & FAHEY, GC, JR. 2013. Alternative dietary fiber sources in companion animal nutrition. Nutrients, 5, 3099-117. 11. DEUSCH, O., O'FLYNN, C., COLYER, A., MORRIS, P., ALLAWAY, D., JONES, PG & SWANSON, KS 2014. Deep Illumina-based shotgun sequencing reveals dietary effects on the structure and function of the fecal microbiome of growing kittens. PLoS One, 9, e101021. 12. FADROSH, DW, MA, B., GAJER, P., SENGAMALAY, N., OTT, S., BROTMAN, RM, & RAVEL, J. (2014). An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome, 2(1), 6. 13. FERRARIO C, STATELLO R, CARNEVALI L, et al. How to Feed the Mammalian Gut Microbiota: Bacterial and Metabolic Modulation by Dietary Fibers. Front Microbiol. 2017; 8:1749. Published 2017 Sep 12. doi:10.3389 / fmicb.2017.01749 14. HOODA, S., VESTER BOLER, BM, KERR, KR, DOWD, SE & SWANSON, KS 2013. The gut microbiome of kittens is affected by dietary protein:carbohydrate ratio and associated with blood metabolite and hormone concentrations. British Journal of Nutrition, 109, 1637-46. 15. MARCHESI, JR, ADAMS, DH, FAVA, F., HERMES, GD, HIRSCHFIELD, GM, HOLD, G., QURAISHI, MN, KINROSS, J., SMIDT, H., TUOHY, KM, THOMAS, LV, ZOETENDAL, EG & HART, A. 2016. The gut microbiota and host health: a new clinicalfrontier. Gut, 65, 330-9. 16.MCDONALD D, PRICE MN, GOODRICH J, NAWROCKI EP, DESANTIS TZ, PROBSTA, ANDERSEN GL, KNIGHT R, HUGENHOLTZ P. An improved greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 2012; 6(3):610–8. 17. MIDDELBOS, IS, VESTER BOLER, BM, QU, A., WHITE, BA, SWANSON, KS & FAHEY, GC, JR. 2010. Phylogenetic characterization of fecal microbial communities of dogs fed diets with or without supplemental dietary fiber using 454 pyrosequencing. PLoS One, 5, e9768. 18. MINAMOTO, Y., DHANANI, N., MARKEL, ME, STEINER, JM & SUCHODOLSKI, JS 2014. Prevalence of Clostridium perfringens, Clostridium perfringens enterotoxin and dysbiosis in fecal samples of dogs with diarrhea. Veterinary Microbiol, 174, 463-73. 19. MINAMOTO, Y., OTONI, CC, STEELMAN, SM, BUYUKLEBLEBICI, O., STEINER, JM, JERGENS, AE & SUCHODOLSKI, JS 2015. Alteration of the fecal microbiota and serum metabolite profiles in dogs with idiopathic inflammatory bowel disease. Gut Microbes, 6, 33-47. 20. MOXHAM, G. WALTHAM Faeces Scoring System - a tool for veterinarians and pet owners: how does your pet rate? Waltham Focus. 2001;11(2):24-5. 21. PANASEVICH, MR, KERR, KR, DILGER, RN, FAHEY, GC, JR., GUERIN-DEREMAUX, L., LYNCH, GL, WILS, D., SUCHODOLSKI, JS, STEER, JM, DOWD, SE & SWANSON, KS 2015. Modulation of the fecal microbiome of healthy adult dogs by inclusion of potato fiber in the diet. British Journal of Nutrition, 113, 125-33. 22. SIMPSON JM, MARTINEAU B, JONES WE, BALLAM JM, MACKIE RI. Characterization of fecalbacterial populations in canines: effects of age, breed and dietary fiber. Microb Ecology. 2002;44(2):186-97. 23. SUCHODOLSKI JS, CAMACHO J, STEINER JM. Analysis of bacterial diversity in the canine duodenum, jejunum, ileum, and colon by comparative 16S rRNA gene analysis. FEMS Microbiol Ecology. 2008 Dec;66(3):567-78. 24. SUCHODOLSKI JS, MARKEL ME, GARCIA-MAZCORRO JF, UNTERER S, HEILMANNRM, DOWD SE, KACHROO P, IVANOV I, MINAMOTO Y, DILLMAN EM, et al. The fecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease. PLoS One. 2012;7:e51907. 25. SUCHODOLSKI, JS, DOWD, SE, WILKE, V., STEINER, JM & JERGENS, AE 2012a. 16S rRNA gene pyrosequencing reveals bacterial dysbiosis in the duodenum of dogs with idiopathic inflammatory bowel disease. PLoS One, 7, e39333. 26. SUCHODOLSKI, JS, MARKEL, ME, GARCIA-MAZCORRO, JF, UNTERER, S., HEILMANN, RM, DOWD, SE, KACHROO, P., IVANOV, I., MINAMOTO, Y., DILLMAN, EM, STEINER, JM, COOK, AK & TORESSON, L. 2012b. The fecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease. PLoS One, 7, e51907. 27. SUCHODOLSKI, JS, 2016. Diagnosis and interpretation of intestinal dysbiosis in dogs and cats. The Veterinary Journal, 215, pp.30-37. 28. SUNVOLD GD, FAHEY GC, MERCHEN NR, TITGEMEYER EC, BOURQUIN LD, BAUER LL et al. Dietary fiber for dogs: IV. In vitro fermentation of selected fiber sources by dog ​​fecal inoculum and in vivo digestion and metabolism of fiber-supplemented diets. Journal of Animal Science. 1995; 73(4):1099. 29. VANDEPUTTE D, FALONY G, VIEIRA-SILVA S, et al. Stool consistency is strongly associated with gut microbiota richness and composition, enterotypes and bacterial growth rates. Gastrointestinal Tract (Gut) 2016;65:57-62. 30. VAZQUEZ-BAEZA, Y., HYDE, ER, SUCHODOLSKI, JS & KNIGHT, R. 2016. Dog and human inflammatory bowel disease rely on overlapping yet distinct dysbiosis networks. Nat Microbiol, 1, 16177. 31. VÁZQUEZ-BAEZA, Y., HYDE, ER, SUCHODOLSKI, JS AND KNIGHT, R., 2016. Dog and human inflammatory bowel disease rely on overlapping yet distinct dysbiosis networks. Nature Microbiology, 1(12), p.16177. 32. VICKERS, RJ, SUNVOLD, GD, KELLEY, RL & REINHART, GA2001. Comparison offermentation of selected fructooligosaccharides and other fiber substrates by canine colonic microflora. American Journal of Veterinary Research, 62, 609-615. 33. WAKSHLAG, JJ, SIMPSON, KW, STRUBLE, AM & DOWD, SE2011. Negative fecal characteristics are associated with pH and fecal flora alterations during dietary changes in dogs. International Journal of Applied Research in Veterinary Medicine, 9, 278. 34. YILMAZ P, PARFREY LW, YARZA P, GERKEN J, PRUESSE E, QUAST C, SCHWEER T, PEPLIES J, LUDWIG W, GLÖCKNER FO. The SILVA and “All-species Living Tree Project (LTP)” taxonomic frameworks. Nucleic Acids Res. 2014;42(Databaseissue):643–8.

[0242] Although the subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications may be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described in the specification. As will be readily understood by those skilled in the art from the subject matter of this disclosure, existing or future processes, machines, manufactures, compositions of matter, means, methods, or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized based on the subject matter of this disclosure. Therefore, the appended claims are intended to include such processes, machines, manufactures, compositions of matter, means, methods, or steps within their scope.

[0243] Patents, patent applications, publications, product descriptions and agreements are cited in their entirety in this application, and their disclosures are incorporated herein by reference in their entirety for all purposes.

Claims

1. A pet food comprising beet pulp at a concentration between about 0.5% w / w and about 6% w / w, wherein the pet food contains a quantity of bacteria effective in improving the gut health and / or fecal quality of companion animals, said bacteria being selected from species of the family Trichophytonceae (…). Lachnospiraceae sp. ), Prevotella coccidioides ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ), species of the genus Dornos ( Dorea sp. ), species of the genus *Ruminococcus* ( Ruminococcaceae sp .), species of the genus Bacteroides ( Bacteroides sp. ), species of the genus Broutella ( Blautia sp. ), species of Erysipelothrix family ( Erysipelotrichaceae sp. ), species of the family Spirulinaceae ( Lachnospiraceae sp. ) and any combination thereof.

2. The pet food of claim 1, wherein the concentration of the beet pulp is between about 0.6% w / w and about 1% w / w.

3. The pet food of claim 2, wherein the concentration of beet pulp is about 0.8% w / w.

4. The pet food as claimed in any one of claims 1-3, wherein the pet food is a cover.

5. The pet food of claim 4, wherein the cover is fed to the companion animal at a dose of about 0.5 g / day to about 90 g / day of beet pulp.

6. The pet food as claimed in any one of claims 1-5, wherein the pet food further comprises additional fiber, probiotics and / or prebiotics.

7. The pet food as claimed in claim 1, wherein the bacteria are selected from *Pleurotus erythrorhizium anisopliae* (…). Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ) and any combination thereof.

8. The pet food of claim 7, wherein the bacteria comprises 16S ribosomal RNA (rRNA), the 16S ribosomal RNA (rRNA) comprising a nucleotide sequence having at least about 95% sequence identity with any of the nucleotide sequences in SEQ ID NO: 1-14 or identical to the nucleotide sequences in any of SEQ ID NO: 1-14.

9. The pet food of claim 8, wherein the bacteria are selected from the group consisting of denovo1184, denovo1244, denovo1696, denovo2407, denovo2451, denovo283, denovo3487, denovo4154, denovo4328, denovo4681, denovo498, denovo5338, denovo6995, denovo943, and any combination thereof.

10. The pet food according to any one of claims 1-3, wherein the effective amount of said bacteria is between about 10,000 CFU and about 100 trillion CFU.

11. The pet food according to any one of claims 1-3, wherein, in addition to the bacteria, the pet food also contains probiotics.

12. The pet food of any one of claims 1-3, wherein when applied to the companion animal, the pet food improves the companion animal's gut health and / or fecal quality.

13. The pet food of any one of claims 1-3, wherein the pet food improves the intestinal health and / or fecal quality of the companion animal within about 14 days after being administered to the companion animal.

14. The pet food as claimed in any one of claims 1-3, wherein the pet food is a dietary supplement.

15. The pet food as claimed in any one of claims 1-3, wherein the pet food is dog food.

16. The pet food as claimed in any one of claims 1-3, wherein the companion animal is a dog.

17. Use of the pet food according to any one of claims 1-16 in the preparation of a medicament for treating intestinal dysbiosis and / or improving intestinal health in companion animals in need.

18. A pet food for treating intestinal ecological disorders in companion animals, said pet food comprising beet pulp and selected species from the family Trichophytonceae (…). Lachnospiraceae sp. ), Prevotella coccidioides ( Faecalibacterium prausnitzii ), Bacteroides commonis ( Bacteroides plebeius ), Dimorphic Halldman's Eubacterium ( Holdemania [Eubacterium] biforme ), species of the genus Dornos ( Dorea sp. ), species of the genus *Ruminococcus* ( Ruminococcaceae sp .), species of the genus Bacteroides ( Bacteroides sp. ), species of the genus Broutella ( Blautia sp. ), species of Erysipelothrix family ( Erysipelotrichaceae sp. ), species of the family Spirulinaceae ( Lachnospiraceae sp. The group consisting of bacteria of the group consisting of beet pulp and any combination thereof, wherein the concentration of beet pulp in the pet food is from about 0.1% w / w to about 10% w / w.

19. The pet food of claim 18, wherein the pet food is a dietary supplement or a functional food.

20. The pet food as claimed in claim 18 or 19, wherein the pet food is a cover.

21. The pet food of any one of claims 18-19, wherein the cover is fed to the companion animal at a dose of about 0.5 g / day to about 90 g / day of beet pulp.

22. The pet food of any one of claims 18-19, wherein the concentration of beet pulp in the pet food is between about 0.5% w / w and about 6% w / w.

23. The pet food as described in any one of claims 1-16, for treating intestinal ecological disorders or improving the intestinal health of companion animals.

24. The pet food as claimed in any one of claims 18-19, wherein the pet food is dog food.

25. The pet food of claim 23, wherein the pet food is dog food.

26. Use of beet pulp in the preparation of dietary supplements or pet foods for the treatment or prevention of ecological disorders in companion animals.

27. The use as claimed in claim 26, wherein the companion animal undergoes dietary changes.

28. The use as described in claim 26 or 27, wherein the concentration of beet pulp in the dietary supplement or the pet food is between about 0.5% w / w and about 6.0% w / w.

29. The use as claimed in any one of claims 26-27, wherein the companion animal is a dog.

30. The use as described in any one of claims 26-27, wherein the companion animal is fed the dietary supplement or the pet food for at least about 3 days.

31. The use as described in claim 30, wherein the companion animal is fed the dietary supplement or the pet food for at least about 7 days.

32. Use of beet pulp in dietary supplements or pet food for the treatment or prevention of ecological disorders in companion animals, wherein the companion animals are subjected to dietary changes.

33. The use as described in claim 32, wherein the concentration of the beet pulp in the dietary supplement or pet food is between about 0.1% w / w and about 10% w / w.

34. The use of the pet food as described in any one of claims 1-16 for the treatment or prevention of intestinal ecological disorders in companion animals, or for the improvement of the intestinal health of said companion animals.

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