Methods, kits, and compositions for dogs at risk of kidney / bladder calcium oxalate stone formation

Abstract

The disclosure relates in part to a method of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject, kits related to the same, and composition related to the same.

Description

METHODS, KITS, AND COMPOSITIONS FOR DOGS AT RISK OF KIDNEY / BLADDER CALCIUM OXALATE STONE FORMATIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63 / 665,632, filed June 28, 2024, which is incorporated herein by reference in its entirety.FIELD

[0002] The disclosure relates in part to a method of determining susceptibility to developing calcium oxalate stones in kidney and / or bladder in a canine subject, kits related to the same, and composition related to the same.BACKGROUND

[0003] Previously, microbiome assessment in a canine, specifically focusing on the measurement of taxonomic abundance and diversity was used to determine stone formation propensity. Further, evaluating the response to dietary interventions such as inulin, as well as proposing the use of antibiotics, probiotics, adjusted dietary patterns, and fecal microbial transplantation as methods to prevent occurrence and recurrence of kidney stones were paired with such microbiome assessment. One commonly studied microorganism, Oxalobacter formigenes, is an oxalate degrading bacterium identified in humans and its abundance levels show an inverse relationship in calcium oxalate stone formers compared with the healthy controls.

[0004] Previous methods also included measuring the urinary microbiome or stone microbiome. These samples are more challenging to collect. Further, methods such as the analysis of the stones microbiome, require extraction and analysis of the kidney and / or bladder stone itself.

[0005] Previous methods addressed microbial abundance and / or diversity, focusing on the presence / absence or abundance of specific bacterial groups or the overall microbiome composition, without addressing the functional capacity of the microbiome.SUMMARY

[0006] In some embodiments, the disclosure relates to a method of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject. The methodcomprises analyzing a fecal sample of the canine subject to determine an abundance of gastrointestinal microbiomc metabolic pathways. The pathways involve at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. The abundance is a measure of at least one of respective microbial gene sequence abundance, respective microbial mRNA sequence abundance, or respective metabolite level abundance for the pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. Relative to control canine values, the canine subject has an increased susceptibility to developing calcium oxalate kidney and / or bladder stones when the analyzing shows at least one of (i) an increase in pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, or (g) microbial oxalate degradation, or (ii) a decrease in pathways for (f) microbial branched amino acid biosynthesis in the canine subject.

[0007] In some aspects, the disclosure relates to a method of treating or preventing calcium oxalate kidney stones and / or bladder stones in a canine subject. The method comprises: determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject. The method also comprises feeding the canine subject a stone forming preventative diet at least when the step of determining results in a determination that the canine subject is more susceptible to developing calcium oxalate kidney and / or bladder stones than a control canine subject or control canine population. The determining step comprises analyzing a fecal sample of the canine subject to determine an abundance of gastrointestinal microbiome metabolic pathways. The pathways involve at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. The abundance is a measure of at least one of respective microbial gene sequence abundance, respective microbial mRNA sequence abundance, or respective metabolite level abundance for the pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinolbiosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. Relative to control canine values, the canine subject has an increased susceptibility to developing calcium oxalate kidney and / or bladder stones when the analyzing shows at least one of (i) an increase in pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, or (g) microbial oxalate degradation, or (ii) a decrease in pathways for (f) microbial branched amino acid biosynthesis in the canine subject.

[0008] In some embodiments, the disclosure relates to a kit for identifying a canine subject having an increased risk for developing calcium oxalate kidney and / or bladder stones from a biological sample from the canine. The kit comprises at least one detection reagent selected from: at least one nucleic acid primer, at least one fluorescence tag, or at least one stain specific to a combination of two or more metabolites. The kit also comprises instructions for use.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A and IB illustrate that metabolic functional capacity captures more variability than microbial species abundance. FIG. 1A illustrates principal component analysis based on microbes. FIG. IB illustrates principal component analysis based on functional pathways.

[0010] FIG. 2 illustrates abundance levels of microbial phylloquinol (Vitamin KI) biosynthesis pathway in the healthy dogs (n = 447), dogs with a history of calcium oxalate kidney and / or bladder stone formation (n = 19), and dogs with a history of calcium oxalate kidney and / or bladder stone formation fed Hill’s diet specific to the condition (n = 7). Different letters on the bars indicate the significance at P value < 0.05 by mixed model analyses. Each bar represents Mean(superpathway of phylloquinol biosynthesis).

[0011] FIG. 3 illustrates abundance levels of microbial menaquinol (Vitamin K2) biosynthesis pathways in the healthy dogs (n = 447), dogs with a history of calcium oxalate kidney and / or bladder stone formation (n = 19) and dogs with a history of calcium oxalate kidney and / or bladder stone formation fed Hill’s diet specific to the condition (n = 7). Different letters on the bars indicate the significance at P value < 0.05 by mixed model analyses. In each of the Healthy, Stone formers (no feeding specific to condition), and Stone formers (feeding specific to condition) panels, the bars represent, from left to right, (1) Mean(superpathway of menaquinaol-6-biosynthesis I), (2) Mean(superpathway of menaquinaol-7-biosynthesis), (3) Mcan(supcrpathway of mcnaquinaol-8-biosynthcsis I), (4) Mcan(supcrpathway of mcnaquinaol-8-biosynthesis II), (5) Mean(superpathway of menaquinaol-10-biosynthesis), (6) Mean(superpathway of menaquinaol-9-biosynthesis), (7) Mean(superpathway of menaquinaol- 11-biosynthesis), (8) Mean(superpathway of menaquinaol-12-biosynthesis), (9) Mean(superpathway of menaquinaol-13-biosynthesis), (10) Mean(superpathway of demthylmenaquinaol-6-biosynthesis I), (11) Mean(superpathway of demthylmenaquinaol-6- biosynthesis II), (12) Mean(superpathway of demthylmenaquinaol-8-biosynthesis), and (13) Mean(superpathway of demthylmenaquinaol-9-biosynthesis).

[0012] FIG. 4 illustrates abundance levels of various microbial arginine biosynthesis pathways in the healthy dogs (n =447), dogs with a history of calcium oxalate kidney and / or bladder stone formation (n =19) and dogs with a history of calcium oxalate kidney and / or bladder stone formation fed Hill’s diet specific to the condition (n =7). Different letters on the bars indicate the significance at P value < 0.05 by mixed model analyses. In each of the Healthy, Stone formers (no feeding specific to condition), and Stone formers (feeding specific to condition) panels, the bars represent, from left to right, (1) Mean(L-arginine biosynthesis I (via L-ornithine), (2) Mean(L- arginine biosynthesis II (acetyl cycle), (3) Mean(L-arginine biosynthesis III (via N-acetyl-L- citrullline), and (4) Mean(L-arginine biosynthesis IV (archaebacteria).

[0013] FIG. 5 illustrates abundance levels of microbial ubiquinol (Coenzyme Q10) biosynthesis pathway in the healthy dogs (n =447), dogs with a history of calcium oxalate kidney and / or bladder stone formation (n =19) and dogs with a history of calcium oxalate kidney and / or bladder stone formation fed Hill’s diet specific to the condition (n =7). Different letters on the bars indicate the significance at P value < 0.05 by mixed model analyses. In each of the Healthy, Stone formers (no feeding specific to condition), and Stone formers (feeding specific to condition) panels, the bars represent, from left to right, (1) Mean(uiquinol-7-biosynthesis (prokaryotic), (2) Mean(uiquinol-9-biosynthesis (prokaryotic), (3) Mean(uiquinol-10-biosynthesis (prokaryotic), (4) Mean(uiquinol-8-biosynthesis (prokaryotic), and (4) Mean(superpathway of uiquinol-8- biosynthesis (prokaryotic).

[0014] FIG. 6 illustrates abundance levels of microbial allantoin degradation pathway in the healthy dogs (n =447), dogs with a history of calcium oxalate kidney and / or bladder stone formation (n =19) and dogs with a history of calcium oxalate kidney and / or bladder stoneformation fed Hill’s diet specific to the condition (n =7). Different letters on the bars indicate the significance at P value < 0.05 by mixed model analyses. In each of the Healthy, Stone formers (no feeding specific to condition), and Stone formers (feeding specific to condition) panels, the bars represent, from left to right, (1) Mean(allantoin degradation IV (anaerobic), (2) Mean(allantoin degradation to glyoxylate III, and (3) Mean(superpathway of allantoin degradation in yeast.

[0015] FIG. 7 illustrates abundance levels of microbial branched amino acids biosynthesis pathway in the healthy dogs (n =447), dogs with a history of calcium oxalate kidney and / or bladder stone formation (n =19) and dogs with a history of calcium oxalate kidney and / or bladder stone formation fed Hill’s diet specific to the condition (n =7).). Different letters on the bars indicate the significance at P value < 0.05 by mixed model analyses.

[0016] FIG. 8 illustrates abundance levels of microbial enzymes involved in oxalate degradation pathway in the healthy dogs (n =447), dogs with a history of calcium oxalate kidney and / or bladder stone formation (n =19) and dogs with a history of calcium oxalate kidney and / or bladder stone formation fed Hill’s diet specific to the condition (n =7). Different letters on the bars indicate the significance at P value < 0.05 by mixed model analyses. In each of the Healthy, Stone formers (no feeding specific to condition), and Stone formers (feeding specific to condition) panels, the bars represent, from left to right, (1) Mean(OXALYL-COA-DECARBOXYLASE-RXN-4.1.1.8- Oxalyl-CoA decarboxylase) and (2) Mean(RXN0- 1382-2.8.3.16-Formyl-CoA transferase).

[0017] FIG. 9A illustrates that selected six microbial functional pathways and their variations captures 79.7 % variability to differentiate healthy dogs vs. dogs with a history of calcium oxalate kidney and / or bladder stone formation by principal component analyses.

[0018] FIG. 9B illustrates a PCA loading matrix showing the contribution of the selected pathway variables in FIG. 9A by the first three components. From top to bottom, each variable listed on the right is represented by three elements: Prinl, Prin2, and Prin3 in order from top to bottom.

[0019] FIGS. 10A and 10B illustrate abundance levels of serum BUN levels (FIG. 10A) and BUN / creatinine ratio (FIG. 10B) in the healthy dogs (n =447), dogs with a history of calcium oxalate kidney and / or bladder stone formation (n =19) and dogs with a history of calcium oxalate kidney and / or bladder stone formation fed Hill’s diet specific to the condition (n =7). Different letters on the bars indicate the significance at P value < 0.05 by mixed model analyses.DETAILED DESCRIPTION

[0020] The following description of the preferred cmbodimcnt(s) is merely exemplary in nature and is in no way intended to limit the invention. The description of illustrative embodiments is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description. The following description of the preferred embodiment(s) is merely exemplary in nature and is in no way intended to limit the invention. The description of illustrative embodiments is intended to be read in connection with the accompanying drawings, which are to be considered pail of the entire written description.

[0021] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context dictates otherwise. The singular form of any class of the ingredients refers not only to one chemical species within that class, but also to a mixture of those chemical species; for example, the term “protein” in the singular form, may refer to a mixture of compounds each of which is also considered a protein. The terms “a” (or “an”), “one or more” and “at least one” may be used interchangeably herein. The terms “comprising”, “including”, and “having” may be used interchangeably. The term “include” should be interpreted as “include, but are not limited to”. The term “including” should be interpreted as “including, but are not limited to”.

[0022] As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range. In addition, all references cited herein are hereby incorporated by reference in their entireties. In the event of a conflict in a definition in the present disclosure and that of a cited reference, the present disclosure controls.

[0023] Unless otherwise defined, all technical and scientific terms and associated acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Further, it should be understood that the present invention is not limited to any particular method, protocol, and reagent described herein. The described methods, protocols, and reagents are merely examples.

[0024] As used herein, the term “biological sample” may be used interchangeably with the terms “sample,” “specimen,” “biomaterial” and “biological material.” A biological sample refers to any organic material obtained from a pet including bodily fluids such as blood, saliva, and urine; tissue samples such as from a biopsy or fur; and other clinical specimens such as exhaled breathcondensate. A biological sample could be obtained in a noninvasive and / or invasive manner. For example, a biological sample may be provided in such noninvasive ways as via a swabbing of a mouth, a collection of fur, or a urination. In other examples, a biological sample may be provided in such invasive ways as via a taking of blood via a needle or a removal of tissue via a biopsy.

[0025] In certain embodiments, the biological sample may further comprise one or more excipients. Excipients may be added to the biological sample at any time. For example, an excipient may be added to the biological sample during collection, transportation, preparation and / or analysis of the sample.

[0026] The addition of excipients is well known in the art. Such excipients should be present in amounts that do not impair the purpose and effect provided by the invention. An excipient may be included as a stabilizer, preservative, processing aid, pH buffer, bulking agent, diluent, color reagent and dye. For example, ethylenediaminetetraacetic acid (EDTA) may be added to a biological sample during collection to preserve the biological sample.

[0027] Examples of excipients may include boric acid and derivatives thereof, dimethyl sulfoxide (DMSO), ethanol, polyethylene glycol, ethylenediaminetetraacetic acid (EDTA), formic acid and derivatives thereof, protease inhibitors, sodium salts such as sodium citrate and sodium metabisulfate, and protease inhibitors.

[0028] The detection of a disease, disorder, or other condition via a biological sample may enable point-of-care diagnosis for diseases, disorders, or other conditions. Methods to quantify one or more biomarkers within a biological sample are well known in the art (e.g., colorimetric reporting, nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy, mass spectrometry, etc.). Although the disclosure may provide many examples relating to fecal samples, it should be understood that such examples are for illustration purposes only. The examples may extend to any biological sample, including a bodily fluid or biological tissue.

[0029] As used herein, the term “biomarker” may be used interchangeably with “biological marker” and is used to refer to any measurable substance that could be used to examine organ function or any other biological state or condition. In certain embodiments, a biomarker may include a protein such as an immunoglobulin, a polynucleotide such as DNA and RNA, and a metabolite. In a preferred embodiment, the biomarker is a metabolic biomarker. In some embodiments, the biomarker is a nucleic acid sequence specific for a gene or mRNA encoding anenzyme involved in a metabolic pathway. The metabolic pathway may be prokaryotic, archacbactcrial, or eukaryotic. The metabolic pathway may be a microbial pathway.

[0030] Detection and quantitation of biomarkers in a sample (such as a fecal sample) may be performed at any time after collection. For example, quantification of a biomarker within a biological sample may be performed within a short time period (e.g., within 2-minutes, within 5- mintes, etc.) or after a longer storage, transportation, preservation, or incubation phase (e.g., within 6 hours, within, 72 hours, etc.). In certain embodiments, the biological sample is stored, transported, or preserved at temperatures ranging from about -200°C to about 30°C. For example, a urine sample is preferably stored, transported, and preserved at about 22°C or 4°C. For another example, a tissue biopsy is preferably stored at -20°C for short term storage and -80°C for long term storage. It should be understood that these examples are for illustration purposes only and that the proper temperatures depend on the type of biological sample used in the present invention. The biological sample should be stored at the proper temperature for the specific type of biological sample as commonly understood by one of ordinary skill in the art.

[0031] One or more biomarkers within a sample may be used as a diagnostic tool for any type of disease, disorder, or other condition. For example, one or more biomarkers may indicate a propensity (e.g., likelihood of developing) a pet may have for a disease, disorder, and / or condition. In a preferred embodiment, one or more biomarkers are used to diagnose or identify the propensity of urolithiasis, kidney disease, or bladder disease in pets. In an even more preferred embodiments, 30-60 biomarkers are used to diagnose or identify the propensity of urolithiasis, kidney disease, or bladder disease in pets.

[0032] Quantified metabolites may include but are not limited to, metabolites found in the pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0033] In some embodiments, microbial pathways that are the object of one or more biomarkers herein are part of at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. In some embodiments, the microbial pathways comprise pathways from allof a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (c) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0034] In some embodiments, microbial pathways that are the object of one or more biomarkers herein are part of (a) microbial phylloquinol biosynthesis.

[0035] In some embodiments, microbial pathways that are the object of one or more biomarkers herein are part of (b) microbial menaquinol biosynthesis. In some embodiments, the microbial menaquinol biosynthesis pathways targeted by biomarkers herein comprise pathways in at least one of or all of the superpathway of menaquinol-6 biosynthesis I, the superpathway of menaquinol- 7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, the superpathway of menaquinol-8 biosynthesis II, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol- 10 biosynthesis, the superpathway of menaquinol- 11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol- 13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-6 biosynthesis II, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis. In some embodiments, the pathways comprise those in at least one of or all of the superpathway of menaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol- 10 biosynthesis, the superpathway of menaquinol- 11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol- 13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis.

[0036] In some embodiments, microbial pathways that are the object of one or more biomarkers herein are part of (c) microbial arginine biosynthesis. In some embodiments, the microbial arginine biosynthesis pathways targeted by biomarkers herein comprise pathways in at least one of or all of L-arginine biosynthesis I via L-ornithine, L-arginine biosynthesis II (acetyl cycle), L-arginine biosynthesis III via N-acetyl-L-citruline, and L-arginine biosynthesis IV (archaebacterial).

[0037] In some embodiments, microbial pathways that are the object of one or more biomarkers herein are part of (d) microbial ubiquinol biosynthesis. In some embodiments, the microbial ubiquinol biosynthesis pathways targeted by biomarkers herein comprise pathways in at least oneof or all of ubiquinol-7-biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol-10-biosynthcsis (prokaryotic), ubiquinol-8-biosynthcsis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic).

[0038] In some embodiments, microbial pathways that are the object of one or more biomarkers herein are part of (e) microbial allantoin degradation. In some embodiments, the microbial allantoin degradation pathways targeted by biomarkers herein comprise pathways in at least one of or all of allantoin degradation IV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast.

[0039] In some embodiments, microbial pathways that are the object of one or more biomarkers herein are part of (f) microbial branched amino acid biosynthesis.

[0040] In some embodiments, microbial pathways that are the object of one or more biomarkers herein are part of (g) microbial oxalate degradation. In some embodiments, the microbial oxalate degradation pathways targeted by biomarkers herein comprise at least one of or both of OXALYL- COA-DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO- 1382-2.8.3.16- Formyl-CoA transferase.

[0041] In some embodiments, the biomarkers comprise nucleic acid sequences for enzymes within one or more of the above listed pathways. In some embodiments, the nucleic acid sequences are objects of one or more of polymerase chain reaction, reverse transcription, and sequencing.

[0042] In some embodiments, analyzing an abundance of a superpathway or pathway comprises analyzing an abundance of less than the total number of enzymes related to the superpathway or pathway. In some embodiments, analyzing an abundance of a superpathway or pathway comprises analyzing an abundance of five, four, three, two, or one enzyme related to the superpathway or pathway. In some embodiments, analyzing the abundance of an enzyme comprises analyzing a nucleic acid encoding the enzyme. In some embodiments, the nucleic acid is a gene encoding the enzyme. In some embodiments, the nucleic acid is an mRNA encoding the enzyme. In some embodiments, the analyzing comprises at least one of a polymerase chain reaction, a reverse transcription, or sequencing of the nucleic acid.

[0043] In some embodiments, the disclosure relates to identifying gastrointestinal microbiome metabolic pathways as potential biomarkers for a gastrointestinal microbiome metabolic test, to identify the dogs at risk of developing stone formation. In some embodiments, the identifying is paired with a nutritional solution designed to optimize the microbiome metabolic characteristics(including metabolic capacity and metabolite production) to reduce the risk of kidney and / or bladder calcium oxalate stone formation and enhance overall kidney and bladder function of dogs.

[0044] The functional capacity of the microbiome is a measure that puts less emphasis on which bacterial taxa are present and more emphasis on the metabolic capabilities of the bacteria that are present. This measure is a more robust measure (less sensitive to small changes in microbial composition) and allows that a given metabolic capacity may be present in one or more bacterial groups.

[0045] In some embodiments, the disclosure relates to a method of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject. In some embodiments, the method comprises analyzing any one of the above listed biomarkers in a biologic sample from the canine subject in order to determine an abundance of one or more metabolic pathway in the canine subject. In some embodiments, the biologic sample may be a fecal sample.

[0046] In some embodiments, the disclosure relates to a method of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject. The method comprises analyzing a fecal sample of the canine subject to determine an abundance of gastrointestinal microbiome metabolic pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. The abundance is a measure of at least one of respective microbial gene sequence abundance, respective microbial mRNA sequence abundance, or respective metabolite level abundance for the pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. Relative to control canine values, the canine subject has an increased susceptibility to developing calcium oxalate kidney and / or bladder stones when the analyzing shows at least one of (i) an increase in pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, or (g) microbial oxalate degradation, or (ii) a decrease in pathways for (f) microbial branched amino acid biosynthesis in the canine subject.

[0047] In some embodiments, the step of analyzing a fecal sample of the canine subject to determine an abundance of pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation comprises analyzing at least two of, at least three of, at least four of, at least five of, or at least six of the abundance of pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation

[0048] In some embodiments, the step of analyzing a fecal sample of the canine subject to determine an abundance of pathways for at least one of (a) microbial phylloquinol biosynthesis,(b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation comprises analyzing the abundance of pathways for all of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis,(c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0049] In some embodiments, the analyzing the abundance of pathways for (a) microbial phylloquinol biosynthesis comprises analyzing an abundance of the superpathway of phylloquinol biosynthesis.

[0050] In some embodiments, the step of determining analyzing the abundance of pathways for (a) comprises determining susceptibility when the abundance (RPKM) of at least one enzyme or all enzymes in the superpathway of phylloquinol biosynthesis is at least about 1.5 fold, about 2 fold, about 3 fold, or about 4 fold more than a control canine subject not susceptible. In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of at least one or all enzymes listed in reference to FIG. 2 for phylloquinol biosynthesis is about as or as shown in FIG. 2 for stone formers in comparison to a healthy canine subject.

[0051] In some embodiments, the analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of at least one of superpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway ofmenaquinol-8 biosynthesis T, superpathway of menaquinol-8 biosynthesis II, superpathway of mcnaquinol-9 biosynthesis, superpathway of mcnaquinol-10 biosynthesis, superpathway of menaquinol-11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-6 biosynthesis II, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

[0052] In some embodiments, the analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of at least one of or all of superpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol- 10 biosynthesis, superpathway of menaquinol-11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

[0053] In some embodiments, the analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of all of superpathway of menaquinol- 6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol- 10 biosynthesis, superpathway of menaquinol-11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-6 biosynthesis II, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

[0054] In some embodiments, the step of determining after analyzing the abundance of pathways for (b) comprises determining susceptibility when the RPKM abundance of at least one enzyme or all enzymes in menaquinol biosynthesis are at least about 1.5 fold, about 2 fold, about 3 fold, or about 4 fold more than a control canine subject not susceptible. In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of at least one or all of the enzymes in menaquinol biosynthesis listed in reference to FIG. 3 is about as or as shown in FIG. 3 for stone formers in comparison to a healthy canine subject for at least one of the above superpathways for abundance of pathways for (b). In some embodiments, the step of determiningcomprises determining susceptibility when the abundance (RPKM) of enzymes in menaquinol biosynthesis listed in reference to FIG. 3 is about as or as shown in FIG. 3 for stone formers in comparison to a healthy canine subject for all of the above superpathways.

[0055] In some embodiments, the analyzing the abundance of pathways for (c) microbial arginine biosynthesis comprises analyzing an abundance of at least one of L-arginine biosynthesis I via L- omithine, L-arginine biosynthesis II (acetyl cycle), L-arginine biosynthesis III via N-acetyl-L- citruline, and L-arginine biosynthesis IV (archaebacterial). In some embodiments, the analyzing the abundance of pathways for (c) microbial arginine biosynthesis comprises analyzing an abundance of all of L-arginine biosynthesis I via L-omithine, L-arginine biosynthesis II (acetyl cycle), L-arginine biosynthesis III via N-acetyLL-citruline, and L-arginine biosynthesis IV ( arch acb acterial) .

[0056] In some embodiments, the step of determining after analyzing the abundance of pathways for (c) comprises determining susceptibility when the RPKM abundance of at least one enzyme or all enzymes in arginine biosynthesis are at least about 1.5 fold, about 2 fold, about 3 fold, or about 4 fold more than a control canine subject not susceptible. In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of at least one or all of the enzymes in arginine biosynthesis listed in reference to FIG. 4 is about as or as shown in FIG. 4 for stone formers in comparison to a healthy canine subject for at least one of the above pathways for abundance of pathways for (c). In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of enzymes in arginine biosynthesis as listed in reference to FIG. 4 is about as or as shown in FIG. 4 for stone formers in comparison to a healthy canine subject for all of the above pathways.

[0057] In some embodiments, the analyzing the abundance of pathways for (d) microbial ubiquinol biosynthesis comprises analyzing an abundance of at least one of ubiquinol-7- biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol- 10-biosynthesis (prokaryotic), ubiquinol-8-biosynthesis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic). In some embodiments, the abundance of pathways for (d) microbial ubiquinol biosynthesis comprises analyzing an abundance of all of ubiquinol-7-biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol- 10-biosynthesis (prokaryotic), ubiquinol-8-biosynthesis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic).

[0058] In some embodiments, the step of determining after analyzing the abundance of pathways for (d) comprises determining susceptibility when the RPKM abundance of at least one enzyme or all enzymes in ubiquinol biosynthesis are at least about 1.5 fold, about 2 fold, about 3 fold, or about 4 fold more than a control canine subject not susceptible. In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of at least one or all of the enzymes in ubiquinol biosynthesis as listed in reference to FIG. 5 is as shown in FIG. 5 for stone formers in comparison to a healthy canine subject for at least one of the above pathways for abundance of pathways for (c). In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of enzymes in arginine biosynthesis listed in reference to FIG. 5 is about as or as shown in FIG. 5 for stone formers in comparison to a healthy canine subject for all of the above pathways.

[0059] In some embodiments, the analyzing the abundance of pathways for (e) microbial allantoin degradation comprises analyzing an abundance of at least one of allantoin degradation IV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast. In some embodiments, the analyzing the abundance of pathways for (e) microbial allantoin degradation comprises analyzing an abundance of all of allantoin degradation IV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast.

[0060] In some embodiments, the step of determining after analyzing the abundance of pathways for (e) comprises determining susceptibility when the RPKM abundance of at least one enzyme or all enzymes in microbial allantoin degradation are at least about 1.5 fold, about 2 fold, about 3 fold, or about 4 fold more than a control canine subject not susceptible. In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of at least one or all of the enzymes in microbial allantoin degradation as listed in reference to FIG. 6 is about as or as shown in FIG. 6 for stone formers in comparison to a healthy canine subject for at least one of the above pathways, for abundance of pathways for (c). In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of enzymes in allantoin degradation listed in reference to FIG. 6 is about as or as shown in FIG. 6 for stone formers in comparison to a healthy canine subject for all of the above pathways.

[0061] In some embodiments, the analyzing the abundance of pathways for (f) branched amino acid biosynthesis comprises analyzing an abundance of the enzymes of branched amino acids biosynthesis.

[0062] In some embodiments, the step of determining analyzing the abundance of pathways for(f) comprises determining susceptibility when the abundance (RPKM) of at least one enzyme or all enzymes in the pathway of branched amino acid biosynthesis is at least about 1.5 fold, about 2 fold, about 3 fold, or about 4 fold more than a control canine subject not susceptible. In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of at least one or all enzymes for branched chain amino acid biosynthesis is about as or as shown in FIG. 7 for stone formers in comparison to a healthy canine subject.

[0063] In some embodiments, the analyzing the abundance of pathways for (g) microbial oxalate degradation comprises analyzing an abundance of at least one of OXALYL-COA- DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO- 1382-2.8.3.16- Formyl-CoA transferase. In some embodiments, the analyzing the abundance of pathways for (g) microbial oxalate degradation comprises analyzing an abundance of both of OXALYL-COA- DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO- 1382-2.8.3.16- Formyl-CoA transferase.

[0064] In some embodiments, the step of determining analyzing the abundance of pathways for(g) comprises determining susceptibility when the abundance (RPKM) of at least one enzyme or all enzymes in the microbial oxalate degradation is at least about 1.5 fold, about 2 fold, about 3 fold, or about 4 fold more than a control canine subject not susceptible. In some embodiments, the step of determining comprises determining susceptibility when the abundance (RPKM) of at least one or all enzymes for microbial oxalate degradation is about as or as shown in FIG. 7 for stone formers in comparison to a healthy canine subject. The at least one or all may be selected from OXALYL-COA-DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO- 1382-2.8.3.16-Formyl-Co A transferase.

[0065] In some embodiments, the susceptibility to developing is a susceptibility to recurrence of calcium oxalate kidney and / or bladder stones in the canine subject.

[0066] In some embodiments, the susceptibility to developing is a susceptibility to developing a first case of calcium oxalate kidney and / or bladder stones in the canine subject.

[0067] In some embodiments, the canine subject is a senior canine. In some embodiments, the canine subject is a male canine. In some embodiments, the canine subject is a small breed of dog. In some embodiments, the canine subject is a mini breed of dog.

[0068] In some embodiments, the analyzing comprises sequencing nucleic acid in the fecal sample and identifying sequences encoding enzymes within the gastrointestinal microbiomc metabolic pathways for the at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0069] In some embodiments, determining the abundance of the gastrointestinal microbiome metabolic pathways comprises quantifying identified sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for the at least one of the (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0070] In some embodiments, quantifying identified sequences comprises quantifying sequences for the a) microbial phylloquinol biosynthesis. In some embodiments, quantifying identified sequences comprises quantifying sequences for a subset or all of the superpathway for phylloquinol biosynthesis.

[0071] In some embodiments, quantifying identified sequences comprises quantifying sequences for the (b) microbial menaquinol biosynthesis. In some embodiments, quantifying identified sequences comprises quantifying sequences for at least one of, a combination of a subset of, or all of the superpathway of menaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, the superpathway of menaquinol-8 biosynthesis II, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol-10 biosynthesis, the superpathway of menaquinol- 11 biosynthesis, the superpathway of menaquinol-12 biosynthesis, the superpathway of menaquinol-13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-6 biosynthesis II, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis. In some embodiments, the combination of a subset of is a combination of two of, three of, four of, five of, six of, seven of, eight of, nine of, ten of, eleven of, or twelve of the aforementioned.

[0072] In some embodiments, quantifying identified sequences comprises quantifying sequences for the (c) microbial arginine biosynthesis. In some embodiments, quantifying identified sequencescomprises quantifying sequences for at least one of, a combination of a subset of, or all of the L- argininc biosynthesis I via L-ornithinc, the L-argininc biosynthesis II (acetyl cycle), the L-argininc biosynthesis III via N-acetyl-L-citruline, and the L-arginine biosynthesis IV (archaebacterial). In some embodiments, the combination of a subset of is a combination of two of, or three of the aforementioned.

[0073] In some embodiments, quantifying identified sequences comprises quantifying sequences for the (d) microbial ubiquinol biosynthesis. In some embodiments quantifying identified sequences comprises quantifying sequences for at least one of, a subset of, or all of the ubiquinol- 7-biosynthesis (prokaryotic), the ubiquinol-9-biosynthesis (prokaryotic), the ubiquinol- 10- biosynthesis (prokaryotic), the ubiquinol-8-biosynthesis (prokaryotic), and the superpathway of ubiquinol-8 biosynthesis (prokaryotic). In some embodiments, the combination of a subset of is a combination of two of, three, or four of the aforementioned.

[0074] In some embodiments, quantifying identified sequences comprises quantifying sequences for the (e) microbial allantoin degradation. In some embodiments, quantifying identified sequences comprises quantifying sequences for at least one of, a combination of a subset of, or all of the allantoin degradation IV (anaerobic), the allantoin degradation to glyoxylate III, and the superpathway of allantoin degradation in yeast.

[0075] In some embodiments, quantifying identified sequences comprises quantifying sequences for the (f) microbial branched amino acid biosynthesis.

[0076] In some embodiments, quantifying identified sequences comprises quantifying sequences for the (g) microbial oxalate degradation. In some embodiments, quantifying identified sequences comprises quantifying sequences for at least one of or both of OXALYL-COA- DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO-1382-2.8.3.16- Formyl-CoA transferase.

[0077] In some embodiments, determining the abundance of the gastrointestinal microbiome metabolic pathways comprises quantifying identified sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for all of the (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0078] In some embodiments, the step of sequencing a nucleic acid comprises sequencing a DNA. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the sequencing comprises converting the mRNA to cDNA and sequencing the cDNA.

[0079] In some embodiments, the method further comprises comparing the abundance of the gastrointestinal microbiome metabolic pathways in the subject canine to control abundance values of the gastrointestinal microbiome metabolic pathways in one or more control canine.

[0080] In some embodiments, the disclosure relates to a method of treating or preventing calcium oxalate kidney and / or bladder stones in a canine subject. The method comprises conducting a method of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in the canine subject. The method of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in the canine subject, in some embodiments, is as described herein. In some embodiments, the method further comprises feeding the canine subject a stone forming preventative diet. In some embodiments, the method further comprises feeding the canine subject a stone forming preventative diet upon a determination that the canine subject is susceptible to developing calcium oxalate kidney and / or bladder stones.

[0081] The step of determining susceptibility in a method of treating or preventing calcium oxalate kidney and / or bladder stones in a canine subject, in some embodiments, comprises analyzing any one of the above listed biomarkers in a biologic sample from the canine subject in order to determine an abundance of one or more metabolic pathway in the canine subject. In some embodiments, the biologic sample may be a fecal sample.

[0082] The step of determining susceptibility in a method of treating or preventing calcium oxalate kidney and / or bladder stones in a canine subject, in some embodiments, comprises analyzing a fecal sample of the canine subject to determine an abundance of gastrointestinal microbiome metabolic pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. The abundance is a measure of at least one of respective microbial gene sequence abundance, respective microbial mRNA sequence abundance, or respective metabolite level abundance for the pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial argininebiosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. Relative to control canine values, the canine subject has an increased susceptibility to developing calcium oxalate kidney and / or bladder stones when the analyzing shows at least one of (i) an increase in pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, or (g) microbial oxalate degradation, or (ii) a decrease in pathways for (f) microbial branched amino acid biosynthesis in the canine subject.

[0083] In some embodiments of a method of treating or preventing, the step of determining susceptibility in a method of treating or preventing calcium oxalate kidney and / or bladder stones in a canine subject, the step of analyzing a fecal sample of the canine subject to determine an abundance of pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation comprises analyzing at least two of, at least three of, at least four of, at least five of, or at least six of the abundance of pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation

[0084] In some embodiments of a method of treating or preventing, the step of determining susceptibility in a method of treating or preventing calcium oxalate kidney and / or bladder stones in a canine subject, the step of analyzing a fecal sample of the canine subject to determine an abundance of pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation comprises analyzing the abundance of pathways for all of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. In some embodiments, the analyzing the abundance of pathways for (a) microbial phylloquinolbiosynthesis comprises analyzing an abundance of the superpathway of phylloquinol biosynthesis.

[0085] In some embodiments of a method of treating or preventing, the analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of at least one of superpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol-10 biosynthesis, superpathway of menaquinol- 11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-6 biosynthesis II, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

[0086] In some embodiments of a method of treating or preventing, the analyzing the abundance of pathways for (a) microbial phylloquinol biosynthesis comprises analyzing an abundance of the superpathway of phylloquinol biosynthesis.

[0087] In some embodiments of a method of treating or preventing, the analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of at least one of or all of superpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol- 7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol-10 biosynthesis, superpathway of menaquinol- 11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

[0088] In some embodiments, the analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of all of superpathway of menaquinol- 6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol-10 biosynthesis, superpathway of menaquinol- 11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway ofdernethylmenaquinol-6 biosynthesis TI, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of dcmcthylmcnaquinol-9 biosynthesis.

[0089] In some embodiments of a method of treating or preventing, the analyzing the abundance of pathways for (c) microbial arginine biosynthesis comprises analyzing an abundance of at least one of L-arginine biosynthesis I via L-omithine, L-arginine biosynthesis II (acetyl cycle), L- arginine biosynthesis III via N-acetyl-L-citruline, and L-arginine biosynthesis IV (archaebacterial). In some embodiments, the analyzing the abundance of pathways for (c) microbial arginine biosynthesis comprises analyzing an abundance of all of L-arginine biosynthesis I via L-omithine, L-arginine biosynthesis II (acetyl cycle), L-arginine biosynthesis III via N-acetyl-L-citruline, and L-arginine biosynthesis IV (archaebacterial).

[0090] In some embodiments of a method of treating or preventing, the analyzing the abundance of pathways for (d) microbial ubiquinol biosynthesis comprises analyzing an abundance of at least one of ubiquinol-7-biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol- 10-biosynthesis (prokaryotic), ubiquinol-8-biosynthesis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic). In some embodiments, the abundance of pathways for (d) microbial ubiquinol biosynthesis comprises analyzing an abundance of all of ubiquinol-7- biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol- 10-biosynthesis (prokaryotic), ubiquinol-8-biosynthesis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic).

[0091] In some embodiments of a method of treating or preventing, the analyzing the abundance of pathways for (e) microbial allantoin degradation comprises analyzing an abundance of at least one of allantoin degradation IV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast. In some embodiments, the analyzing the abundance of pathways for (e) microbial allantoin degradation comprises analyzing an abundance of all of allantoin degradation IV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast.

[0092] In some embodiments of a method of treating or preventing, the analyzing the abundance of pathways for (f) branched amino acid biosynthesis comprises analyzing an abundance of the enzymes of branched amino acids biosynthesis.

[0093] In some embodiments of a method of treating or preventing, the analyzing the abundance of pathways for (g) microbial oxalate degradation comprises analyzing an abundance of at leastone of OXALYL-COA-DECARBOXYLASE-RXN-4.1 .1 ,8-Oxalyl-CoA decarboxylase and RXNO-1382-2.8.3.16-Formyl-CoA transferase. In some embodiments, the analyzing the abundance of pathways for (g) microbial oxalate degradation comprises analyzing an abundance of both of OXALYL-COA-DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO-1382-2.8.3.16-Formyl-CoA transferase.

[0094] The step of determining susceptibility after any one or more of (a), (b), (c), (d), (e), (f), and (g) in some embodiments, is as set for the above regarding method of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject.

[0095] In some embodiments of a method of treating or preventing, the susceptibility to developing is a susceptibility to recurrence of calcium oxalate kidney and / or bladder stones in the canine subject.

[0096] In some embodiments of a method of treating or preventing, the susceptibility to developing is a susceptibility to developing a first case of calcium oxalate kidney and / or bladder stones in the canine subject.

[0097] In some embodiments of a method of treating or preventing, the canine subject is a senior canine. In some embodiments, the canine subject is a male canine. In some embodiments, the canine subject is a small breed of dog. In some embodiments, the canine subject is a mini breed of dog.

[0098] In some embodiments of a method of treating or preventing, the analyzing comprises sequencing nucleic acid in the fecal sample and identifying sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for the at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0099] In some embodiments of a method of treating or preventing, determining the abundance of the gastrointestinal microbiome metabolic pathways comprises quantifying identified sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for the at least one of the (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0100] In some embodiments of a method of treating or preventing, quantifying identified sequences comprises quantifying sequences for the a) microbial phylloquinol biosynthesis. In some embodiments, quantifying identified sequences comprises quantifying sequences for a subset or all of the superpathway for phylloquinol biosynthesis.

[0101] In some embodiments of a method of treating or preventing, quantifying identified sequences comprises quantifying sequences for the (b) microbial menaquinol biosynthesis. In some embodiments, quantifying identified sequences comprises quantifying sequences for at least one of, a combination of a subset of, or all of the superpathway of menaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, the superpathway of menaquinol-8 biosynthesis II, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol-10 biosynthesis, the superpathway of menaquinol- 11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol-13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-6 biosynthesis II, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis. In some embodiments, the combination of a subset of is a combination of two of, three of, four of, five of, six of, seven of, eight of, nine of, ten of, eleven of, or twelve of the aforementioned.

[0102] In some embodiments of a method of treating or preventing, quantifying identified sequences comprises quantifying sequences for the (c) microbial arginine biosynthesis. In some embodiments, quantifying identified sequences comprises quantifying sequences for at least one of, a combination of a subset of, or all of the L-arginine biosynthesis I via L-ornithine, the L- arginine biosynthesis II (acetyl cycle), the L-arginine biosynthesis III via N-acetyl-L-citruline, and the L-arginine biosynthesis IV (archaebacterial). In some embodiments, the combination of a subset of is a combination of two of, or three of the aforementioned.

[0103] In some embodiments of a method of treating or preventing, quantifying identified sequences comprises quantifying sequences for the (d) microbial ubiquinol biosynthesis. In some embodiments quantifying identified sequences comprises quantifying sequences for at least one of, a subset of, or all of the ubiquinol-7-biosynthesis (prokaryotic), the ubiquinol-9-biosynthesis (prokaryotic), the ubiquinol- 10-biosynthesis (prokaryotic), the ubiquinol-8-biosynthesis (prokaryotic), and the superpathway of ubiquinol-8 biosynthesis (prokaryotic). In someembodiments, the combination of a subset of is a combination of two of, three, or four of the aforementioned.

[0104] In some embodiments of a method of treating or preventing, quantifying identified sequences comprises quantifying sequences for the (e) microbial allantoin degradation. In some embodiments, quantifying identified sequences comprises quantifying sequences for at least one of, a combination of a subset of, or all of the allantoin degradation IV (anaerobic), the allantoin degradation to glyoxylate III, and the superpathway of allantoin degradation in yeast.

[0105] In some embodiments of a method of treating or preventing, quantifying identified sequences comprises quantifying sequences for the (f) microbial branched amino acid biosynthesis.

[0106] In some embodiments of a method of treating or preventing, quantifying identified sequences comprises quantifying sequences for the (g) microbial oxalate degradation. In some embodiments, quantifying identified sequences comprises quantifying sequences for at least one of or both of OXALYL-COA-DECARBOXYLASE-RXN-4.1.1 ,8-Oxalyl-CoA decarboxylase and RXNO-1382-2.8.3.16-Formyl-CoA transferase.

[0107] In some embodiments of a method of treating or preventing, determining the abundance of the gastrointestinal microbiome metabolic pathways comprises quantifying identified sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for all of the (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

[0108] In some embodiments of a method of treating or preventing, the step of sequencing a nucleic acid comprises sequencing a DNA. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the sequencing comprises converting the mRNA to cDNA and sequencing the cDNA.

[0109] In some embodiments of a method of treating or preventing, the method further comprises comparing the abundance of the gastrointestinal microbiome metabolic pathways in the subject canine to control abundance values of the gastrointestinal microbiome metabolic pathways in one or more control canine.

[0110] In some embodiments, “treating or preventing” relates to preventing. In some embodiments, “treating or preventing” relates to treating. In some embodiments, “treating orpreventing” relates to preventing and the preventing comprises reducing the risk of developing calcium oxalate kidney and / or bladder stones. In some embodiments, “treating or preventing” relates to preventing and the preventing comprises optimizing the subject’s microbiome. In some embodiments, “treating or preventing” relates to treating and the treating comprises reducing conditions leading to development of calcium oxalate kidney and / or bladder stones. In some embodiments, “treating or preventing” relates to treating and the treating comprises optimizing the subject’s microbiome.

[0111] In some embodiments, the stone forming preventative diet comprises a composition disclosed herein. In some embodiments, the stone forming preventative diet comprises at least one of protein content from about 14 to about 32 wt. %, calcium content from about 0.6 to about 1.6 wt. %, phosphorous content from about 0.2 to about 0.9 wt. %, magnesium content from about 0.04 to about 0.2 wt. %, sodium content from about 0.2 to about 0.5 wt. %, potassium content from about 0.5 to about 1.1 wt. %, and chloride dry content from about 0.4 to about 1.3 wt. %. The wt. % is based on dry weight of the stone forming preventative diet. In some embodiments, the chloride wet content is about 1.5 wt. % based on hydrated weight of the stone forming preventative diet.

[0112] In some embodiments, the stone forming preventative diet comprise all of the protein content from about 14 to about 32 wt. %, the calcium content from about 0.6 to about 1.6 wt. %, the phosphorous content from about 0.2 to about 0.9 wt. %, the magnesium content from about 0.04 to about 0.2 wt. %, the sodium content from about 0.2 to about 0.5 wt. %, the potassium content from about 0.5 to about 1.1 wt. %, and the chloride dry content from about 0.4 to about 1.3 wt. %.

[0113] In some embodiments, the disclosure relates to a kit for identifying a canine subject having an increased risk for developing calcium oxalate kidney and / or bladder stones from a biological sample from the canine. The kit comprises at least one detection reagent selected from one or more of: at least one nucleic acid primer, at least one fluorescence tag, or at least one stain specific to a combination of two or more metabolites. In some embodiment, the kit further comprises instructions for use. In some embodiment, the kit further comprises a vessel for collecting the biological sample. In some embodiment, the biological sample is a fecal sample. In some embodiment, the at least one detection reagent is for analyzing the fecal sample to determine an abundance of gastrointestinal microbiome metabolic pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial argininebiosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. In some embodiment, the at least one detection reagent comprises at least one of a polymerase chain reaction primer set or a sequencing primer specific for an enzyme in at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation. In some embodiments, the enzyme(s) are in any one or more pathway described herein. In some embodiments, the instructions for use instruct a user thereof to compare the abundance of the gastrointestinal microbiome metabolic pathways in the canine subject to an abundance of the gastrointestinal microbiome metabolic pathways in a control canine. In some embodiments, the control canine is a healthy canine. In some embodiments, the instructions instruct that relative to the control canine values, the canine subject has an increased susceptibility to developing calcium oxalate kidney and / or bladder stones when the analyzing shows at least one of (i) an increase in pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, or (g) microbial oxalate degradation, or (ii) a decrease in pathways for (f) microbial branched amino acid biosynthesis in the canine subject.

[0114] In some embodiments, the disclosure relates to a composition. The composition comprises a pet for treating any of the disease states or conditions described herein. In some embodiments, the composition comprises high levels of arginine, low levels of tryptophan, and optimal amounts of protein.

[0114] In some embodiments, the pet food composition is in the form of a kibble. In some embodiments, the composition is in the form of multi-layer kibble and / or a multi-layer kibble comprising a coating. In some embodiments, the coating comprises a palatant. The term "palatability", as used herein, encompasses all the various properties of food sensed by animals such as texture, taste, and aroma. In certain embodiments, the composition has a palatability equal to that of a control composition.

[0115] In some embodiments, the kibble is formed by extrusion. In some embodiments, the composition is in a form selected from: a loaf, a stew, a “meat and gravy” form, a gruel, shredswith a moisture content greater than 50%”, and a product that could be pushed through a syringe. In some embodiments, the composition comprises 6% wt. to about 12% wt. moisture.

[0116] In some embodiments, the kibble comprises a binder. In some embodiments the binder comprises one of the following, a combinations of two or more of, or all of the following: monosaccharides, glucose, fructose, mannose, arabinose; disaccharides, trisaccharides, sucrose, lactose, maltose, trehalose, lactulose, com syrup solids, rice syrup solids, dextrins (for example, com, wheat, rice and tapioca dextrin), one or more maltodextrin, starch (for example, one or more of rice, wheat, corn, potato, tapioca starches or chemical modified starches), alginates, chitosans; gums (for example, carrageen and gum Arabic), polyols (for example, glycerol, sorbitol, mannitol, xylitol, erythritol, esters of polyols (for example, sucrose esters, polyglycol esters, glycerol esters, polyglycerol esters, and sorbitan esters), sorbitol), molasses, honey, gelatin; peptides, proteins, and modified proteins (for example, whey liquid, whey powder, whey concentrate, whey isolate, whey protein isolate, high lactose whey by-product, meat broth solids (for example, chicken broth, chicken broth solids, soy protein, and egg white)).

[0117] In some embodiments, the binder includes but is not limited to a lipid and / or lipid derivative. In some embodiments, the lipids are in combination with water and / or other binder components. In some embodiments, the lipids include plant fats (for example, soybean oil, com oil, rapeseed oil, olive oil, safflower oil, palm oil, coconut oil, palm kernel oil, and partially and fully hydrogenated derivatives thereof), animal fats and partially and fully hydrogenated derivatives thereof, and waxes.

[0118] In some embodiments, the composition comprises additional ingredients including but not limited to, additives, minerals, vitamins, sources of carbohydrates, fat, protein, additional fiber, amino acids, carotenoids, antioxidants, fatty acids, glucose mimetics, probiotics, prebiotics, and others.

[0119] In some embodiments, the composition comprises additives known in the art. In some embodiments, the additives are present in amounts that do not impair the purpose and effect provided by the composition. Examples of additives include substances with a stabilizing effect, organoleptic substances, processing aids, and substances that provide nutritional benefits.

[0120] Substances with a stabilizing effect may increase the shelf life of the composition. Examples comprise preservatives, antioxidants, synergists and sequestrants, packaging gases, stabilizers, emulsifiers, thickeners, gelling agents, and humectants. Examples of emulsifiers and / orthickening agents include gelatin, cellulose ethers, starch, starch esters, starch ethers, and modified starches.

[0121] In some embodiments, the composition comprises additives for coloring, palatability, and nutritional purposes. In some embodiments, these comprise colorants, salts (including but not limited to sodium chloride, potassium citrate, potassium chloride, and other edible salts), vitamins, minerals, and flavoring. The amount of an additives in the composition typically is up to about 5% by weight (on a dry matter basis of the composition). In some embodiments, the composition comprises other additives. In some embodiments, the other additives comprise antioxidants, omega-3 fatty acids, omega-6 fatty acids, glucosamine, chondroitin sulfate, vegetable extracts, and herbal extracts.

[0122] In some embodiments, the composition comprises vitamins and minerals in amounts required to avoid deficiency and maintain health. These amounts are readily available in the art. The Association of American Feed Control Officials (AAFCO) provides recommended amounts of such ingredients for dogs and cats (see Association of American Feed Control Officials. Official Publication, pp. 126-140 (2003)). Minerals may specifically be maintained at optimum levels known by those skilled in the art to reduce the incidence of stone formation.

[0123] In some embodiment, the composition comprises vitamins. In some embodiments, the vitamins comprise at least one, a combination of two or more of, or all of vitamin A, vitamin B 1 (thiamine or related sources such as thiamine mononitrate), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid or related sources such as calcium pantothenate), vitamin B6 (pyridoxine or related sources such as pyridoxine hydrochloride), vitamin B8 (folic acid), vitamin B 12, vitamin C (ascorbic acid), vitamin D (such as a vitamin D3 supplements), vitamin E, vitamin H (biotin), vitamin K, acetate, choline and choline related sources such as choline chloride, and inositol.

[0124] In some embodiment, the composition comprises minerals and trace elements. In some embodiments, the minerals and trace elements comprise calcium, phosphorus, sodium, potassium, magnesium, copper, zinc, choline, and iron salts. In some embodiments, mineral sources comprise sodium selenite, monosodium phosphate, calcium carbonate, potassium chloride, ferrous sulfate, zinc oxide, manganese sulfate, copper sulfate, manganous oxide, potassium iodide, and / or cobalt carbonate.

[0125] The term “carbohydrate” as used herein includes polysaccharides (for example, starches and dextrins) and sugars (for example, sucrose, lactose, maltose, glucose, and fructose) that arc metabolized for energy when hydrolyzed. Examples of high carbohydrate ingredients suitable for inclusion in the compositions disclosed herein include but are not limited to, corn, grain sorghum, wheat, barley, and rice.

[0126] In certain embodiments, the carbohydrate component comprises a mixture of one or more carbohydrate sources. Examples of carbohydrate or carbohydrate ingredients may comprise cereals, grains, com, wheat, rice, oats, corn grits, sorghum, grain sorghum / milo, wheat bran, oat bran, amaranth, Durum, and / or semolina.

[0127] The skilled artisan could manipulate the texture of the final product by properly balancing carbohydrate sources. For example, short chain polysaccharides lend to be sticky and gluey, and longer chain polysaccharides are less sticky and gluey than the shorter chain; the desired texture of this hybrid food is achieved by longer chain polysaccharide and modified starches such as native or modified starches, cellulose and the like.

[0128] The carbohydrate mixture may additionally comprise optional components such as added salt, spices, seasonings, vitamins, minerals, flavorants, colorants, and the like. The amount of the optional additives is at least partially dependent on the nutritional requirements for different life stages of animals.

[0129] In some embodiments, the composition comprises about 5% wt. to about 25% wt. of fat. In some embodiments, sources of fats or fat ingredients comprise poultry fat, chicken fat, turkey fat, pork fat, lard, tallow, beef fat, vegetable oils, com oil, soy oil, cottonseed oil, palm oil, palm kernel oil, linseed oil, canola oil, rapeseed oil, fish oil, menhaden oil, anchovy oil, and / or olestra.

[0130] In some embodiments, the composition comprises about 5% wt. to about 30% wt. of protein. The term “protein” means a polypeptide, or a peptide, or a polymer of amino acids. The term encompasses naturally occurring and non-naturally occurring (synthetic) polymers and polymers in which artificial chemical mimetics are substituted for one or more amino acids. The term also encompasses fragments, variants, and homologs that have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. The term encompasses polymers of any length, including polymers containing from about 2 to 1000, from 4 to 800, from 6 to 600, and from 8 to 400 amino acids. The term includes amino acidpolymers that are synthesized and that are isolated and purified from natural sources. Under some embodiments, the terms “polypeptide,” “peptide,” or “protein” arc used interchangeably.

[0131] Protein may be supplied by any of a variety of sources known to the skilled artisan, including plant sources, animal sources, microbial sources, or a combination of these. For example, animal sources may include meat, meat-by products, seafood, dairy, eggs, etc. Meats, for example, may include animal flesh (for example, poultry fish, and mammals (for example, cattle, pigs, sheep, and goats)). Meat by-products may include, for example, lungs, kidneys, brain, livers, stomachs, and intestines. Plant protein includes, for example, soybean, cottonseed, and peanuts. Microbial sources may be used to synthesize amino acids (for example, lysine, threonine, tryptophan, methionine) or intact protein such as protein from sources listed below.

[0132] Examples of protein or protein ingredients may comprise chicken meals, chicken, chicken by-product meals, lamb, lamb meals, turkey, turkey meals, beef, beef by-products, viscera, fish meal, enterals, kangaroo, white fish, venison, soybean meal, soy protein isolate, soy protein concentrate, com gluten meal, corn protein concentrate, distillers dried grains, and / or distillers dried grain solubles and single-cell proteins, for example yeast, algae, and / or bacteria cultures.

[0133] The protein can be intact, completely hydrolyzed, or partially hydrolyzed. The protein content of foods may be determined by any number of methods known by those of skill in the art, for example, as published by the Association of Official Analytical Chemists in Official Methods of Analysis (“OMA”), method 988.05. The amount of protein in a composition disclosed herein may be determined based on the amount of nitrogen in the composition according to methods familiar to one of skill in the art.

[0134] Examples of amino acids may comprise 1 -Tryptophan, Taurine, Histidine, Carnosine, Alanine, Cysteine, Arginine, Methionine, Tryptophan, Lysine, Asparagine, Aspartic acid, Phenylalanine, Valine, Threonine, Isoleucine, Histidine, Leucine, Glycine, Glutamine, Taurine, Tyrosine, Homocysteine, Ornithine, Citruline, Glutamic acid, Proline, and / or Serine. Sources of carotenoids may include lutein, astaxanthin, zeaxanthin, bixin, lycopene, and / or beta-carotene. Sources of antioxidant ingredients may comprise tocopherols (vitamin E), vitamin C, vitamin A, plant-derived materials, carotenoids (described above), selenium, and / or CoQlO (Co-enzyme Q10). In a preferred embodiment, the pet food composition contains high levels of arginine and derivatives thereof and / or low levels of tryptophan and derivatives thereof. In another preferredembodiment, the pet food composition contains high levels of polyunsaturated fatty acids (e.g., alpha linolenic, arachidonic, EPA and DHA)

[0135] Examples of fatty acid ingredients may comprise arachidonic acid, alpha-linolenic acid, gamma linolenic acid, linoleic acid, eicosapentanoic acid (EPA), docosahexanoic acid (DHA), and / or fish oils as a source of EPA and / or DHA. Sources of glucose mimetics may comprise glucose anti-metabolites including 2-deoxy D-glucose, 5-thio-D-glucose, 3-O-methylglucose, anhydrosugars including 1,5-anhydro-D-glucitol, 2,5-anhydro-D-glucitol, and 2,5-anhydro-D- mannitol, mannoheptulose, and / or avocado extract comprising mannoheptulose.

[0136] Still other ingredients may include beef broth, brewers dried yeast, egg, egg product, flax meal, DL methionine, amino acids, leucine, lysine, arginine, cysteine, cystine, aspartic acid, polyphosphates, sodium pyrophosphate, sodium tripolyphosphate; zinc chloride, copper gluconate, stannous chloride, stannous fluoride, sodium fluoride, triclosan, glucosamine hydrochloride, chondroitin sulfate, green lipped mussel, blue lipped mussel, methyl sulfonyl methane (MSM), boron, boric acid, phytoestrogens, phytoandrogens, genistein, diadzein, L- camitine, chromium picolinate, chromium tripicolinate, chromium nicotinate, acid / base modifiers, potassium citrate, potassium chloride, calcium carbonate, calcium chloride, sodium bisulfate; eucalyptus, lavender, peppermint, plasticizers, colorants, flavorants, sweeteners, buffering agents, slip aids, carriers, pH adjusting agents, natural ingredients, stabilizers, biological additives such as enzymes (including proteases and lipases), chemical additives, coolants, chelants, denaturants, drug astringents, emulsifiers, external analgesics, fragrance compounds, humectants, opacifying agents (such as zinc oxide and titanium dioxide), antifoaming agents (such as silicone), preservatives (such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), propyl gallate, benzalkonium chloride, EDTA, benzyl alcohol, potassium sorbate, parabens and mixtures thereof), reducing agents, solvents, hydrotropes, solubilizing agents, suspending agents (non-surfactant), solvents, viscosity increasing agents (aqueous and non-aqueous), sequestrants, and / or keratolytics.

[0137] The probiotic component may comprise any suitable bacteria, yeast, microorganisms, and / or mixtures of any thereof. Various probiotic microorganisms are known in the art. In certain embodiments, the probiotic component may comprise bacteria of the order Lactobacillales; bacteria of the genus Bacillus, Bacteroides, and / or Bifidobacterium; yeast of the orderSaccharomycetales including the genus Saccharomyces and Candida; and / or mixtures of any thereof. The probiotic may or may not form a spore.

[0138] In some embodiments, the composition comprises polyphenols. In some embodiments, the polyphenol source comprises a phenolic compound selected from ellagic acid; gallic acid; protocatechuic acid; p-hydroxybenzoic acid; catechin; and a combination of two or more thereof. In some embodiments, the polyphenol source comprises pecan shells, or any other component of the pecan nut. Examples of further sources of polyphenols may comprise tea extract, rosemary extract, rosemarinic acid, coffee extract, pecan shells, caffeic acid, turmeric extract, blueberry extract, grape extract, grapeseed extract, and / or soy extract.

[0139] The component of the composition may be determined by any of the variety of methods for feed analysis known by the skilled artisan. Feed analysis may be done to measure any of the nutritional content listed herein including moisture, protein, fiber, carbohydrate, energy, vitamin, mineral, energy, fat, and ash content.

[0140] Protein content may be measured and reported in any of the variety of methods known to one skilled in the art. Protein may be reported as crude protein (CP) to measure both true protein content and non-protein nitrogen. Crude protein content may be further differentiated between degradable intake protein (DIP), undegradable intake protein (UIP) and metabolizable protein (MP). In certain embodiments, protein content may be differentiated to include heat damaged protein or insoluble crude protein (ICP), adjusted crude protein (ACP), and digestible protein (DP).

[0141] Fiber content may be measured and reported in any of the variety of methods known to one skilled in the art. Fiber content may be reported as total dietary fiber (TDF, a combination of soluble and insoluble fiber) crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF) and / or acid detergent lignin (ADL). Crude fiber is generally known to estimate the indigestible portion of plant material found in pet food compositions. ADF measures cellulose and lignin, components of plant cell walls. NDF measures the total material found in plant cell walls and includes hemicellulose in addition to the fiber content measured as ADF. ADL measures only the lignin portion of a plant cell wall.

[0142] Energy content may be measured and reported in any of the variety of methods known to one skilled in the art. Energy content may be reported as digestible energy (DE), metabolizable energy (ME), net energy (NE), total digestible nutrient (TDN), ether extract (EE), relative feed value (RFV), and relative forage quality (RFQ).

[0143] Embodiments will now be further described by way of the following, non-limiting, examples.Examples

[0144] The following examples describe a non-invasive and convenient method for identifying dogs at risk of calcium oxylate stone formation and then treating with a nutritional intervention.

[0145] The exemplary method for identifying dogs at risk of calcium oxylate stone formation is a non-invasive test administered using an easily collected sample (feces) to obtain predicted gastrointestinal microbiome metabolic function. This test can be used to determine if the features of a dog's microbiome are consistent with the features of dogs known to have developed calcium oxalate kidney and / or bladder stones. The method of nutritional intervention is a nutrition solution designed to optimize the microbiome metabolic characteristics (including metabolic capacity and metabolite production) so that they are less similar to dogs known to have developed calcium oxalate kidney and / or bladder stones; additionally, the promotion of certain features consistent with reduced likelihood of stone development may also be sought. The method of treating may be paired with the method of identifying.

[0146] Since calcium oxalate kidney and / or bladder stones are common, they often occur in canine subjects with other common comorbidities, including dental disease, overweight / obesity, urinary tract infection, otitis, and atopic dermatitis. Calcium oxalate kidney and / or bladder stones are common in senior dogs, especially male, small, and mini breed dogs. Therefore, nutrition solutions can be tailored to the canine subject’s personalized risk profile, addressing not only the risk of calcium oxalate kidney and / or bladder stone formation but also addressing the nutrition needs associated with one or more comorbidities, or age, sex, breed, or life stage concerns. Such solutions are more individualized and address a wider range of the canine subject’s nutrition needs, simplifying nutrition and medical management for the canine subject, the owner, and the veterinarian. Microbiome modulating technologies could include nutrition technologies aimed at optimizing oxalate degradation, butyrate production, vitamin K biosynthesis, arginine biosynthesis, or optimizing the calcium / oxalate ratio. For example, among small breed, overweight male dogs with a history of calcium oxalate kidney and / or bladder stones, nutrition solutions that have reduced energy and controlled protein levels with fiber blends aimed at optimizing short chain fatty acid production, postbiotics and / or vitamin levels to promote the growth of oxalate metabolizing bacteria and promoting microbial community health, controlled minerals (especiallycalcium), and offered in an appropriately sized format (for example, small bite format for small and mini breeds) may reduce the risk of calcium oxalate stone recurrence while addressing overall health including overall kidney and bladder health. A wide range of potentially appropriate foods as described above with additional ingredients to support the microbiome are possible, enabling even greater personalization of the food to reduce the risk of calcium oxalate kidney and / or bladder stones while addressing other comorbidities, underlying health concerns, or age, sex, breed, or life stage concerns.

[0147] These examples focus on calcium oxalate kidney and / or bladder stones in dogs and includes a simple, non-invasive test, aimed at detecting and addressing alterations in gastrointestinal microbiome function with a goal of improving metabolic capacity of the gastrointestinal microbiome as well as overall kidney and bladder function. They also address both the stone condition as well as the dog’s personalized risk profile, including addressing comorbidities as well as concerns associated with age, sex, breed and / or lifestage.

[0148] The results provided herein were derived from a cross-sectional observational study where fecal samples collected from healthy dogs and dogs with a history of calcium oxalate stone formers. Overall, the data was derived from 447 healthy dogs and 36 stone formers. Among these stone forming dogs, 19 dogs were fed a food that was not specific to the condition and 7 dogs were fed a food specific to the condition (Hill’s Prescription Diet c / d Multicare; Table 1). Food intake was assessed using a 24 hr recall completed by pet owners for the pet, >98% of pet owners verified that the reported food intake was typical for their pet. Fecal samples were collected from these dogs using a fecal loop or a scoop from the whole stool and shotgun metagenomics sequencing was performed using the Illumina platform by CosmosID (Germantown, MD, USA). Briefly, fecal DNA was extracted using the QIAGEN DNeasy PowerSoil Prokit (Qiagen Inc.,) following the manufacturer’s protocol and quantified using a Qubit 4 fluorometer with Qubit™ dsDNA HS Assay Kit (Thermofisher Scientific). DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina) and IDT Unique Dual Indexes with total DNA input of Ing. Per manufacturer’ s protocol, genomic DNA was fragmented using a proportional amount of Illumina Nextera XT fragmentation enzyme. Unique dual indexes were added to each sample followed by 12 cycles of PCR to construct libraries. DNA libraries were purified using AMpure magnetic Beads (Beckman Coulter) and eluted in QIAGEN EB buffer. DNA libraries were again quantified using Qubit 4 fluorometer and Qubit™ dsDNA HS Assay Kit. Libraries were then sequenced onIllumina HiSeq X platform with paired reads (2 X 15Obp) to a target depth of 6M reads. All raw sequences were processed using their proprietary bioinformatics workflow to deliver microbiomc abundance data, functional pathways, and their enzymatic functions. Microbiome functional pathways and enzymatic functions were normalized by reads per kilo million (RPKM) before performing the statistical analyses. Besides the fecal microbiome analyses, we also collected the blood samples to measure serum BUN and creatinine levels. To identify the microbiome functional pathways, we used linear mixed model regression analysis followed by Tukey post-hoc significance test with FDR corrected P-values using JMP Pro v!7.Table 1

[0149] Referring to FIGS. 1 A and IB, these data show that metabolic functional capacity better explains the variability in the microbiomc than microbial species abundance.

[0150] Based on the analyses herein, six gut microbiome functional pathways were identified as significantly different between healthy dogs vs. dogs with a history of calcium oxalate kidney and / or bladder stone formation, and those pathways further impacted in the dogs with a history of calcium oxalate kidney and / or bladder stone formation fed a Hill’s food specific to the condition.

[0151] Vitamin K metabolism composed of phylloquinone (Vitamin KI), and menaquinones (vitamin K2) represents a family of structurally and functionally related fat-soluble vitamers plays a vital role in cardiovascular health, inflammation, calcium transport, chronic kidney disease, cognition, glucose homeostasis and cancer (McCabe et al 2013; Pilkey et al 2007). Vitamin K2 production mostly relied on the gut bacteria either converting dietary Vitamin KI to Vitamin K2 or dietary Vitamin K3 (menadione) to Vitamin K2. Several studies reported impairment of Vitamin K2 levels and Vitamin K2 dependent proteins, specifically matrix Gia protein associated with stone formers in rodent and human studies (see review, Chimel et al 2023). FIGS. 2 and 3 show that gut microbial Vitamin KI (phylloquinone) and Vitamin K2 (menaquinone) functional pathways are significantly different between healthy dogs vs. dogs with a history of calcium oxalate kidney and / or bladder stone formation showing significant increase and further enhanced when fed with Hill’s food specific to the condition. The gut microbiome of the dogs with a history of stone formation may be sensitive to the presence of a subclinical deficiency of Vitamin K levels and may be combating the insufficiency by upregulating vitamin K metabolic pathways, which are further increased when a food specific to the condition (Hill’s Prescription Diet c / d Multicare) is fed.

[0152] Similarly, referring to FIG. 4, it was also observed that microbial arginine biosynthesis pathway was also significantly increased in the dogs with a history of stone formation vs. healthy dogs. Arginine biosynthesis pathway was further increased when dogs with a history of calcium oxalate kidney and / or bladder stone formation were fed a food specific to the condition (Hill’s Prescription Diet c / d Multicare as a preventive measure to reduce the risk of stone recurrence. Recently a study (Liu et al 2021) demonstrated that arginine could ameliorate calcium oxalate crystals to renal cell adhesion in a concentration dependent way. Another study demonstrated that feeding of L-arginine prevents the retention of calcium oxalate crystals in hyperoxaluric rats by protecting the renal cells from oxidative stress. (Pragasam et al 2021).

[0153] Referring to FIG. 5, it was also identified that gut microbial ubiquinol biosynthesis pathway was significantly increased in the dogs with a history of calcium oxalate kidney and / or bladder stone formation vs. healthy dogs and further increased when dogs with a history of calcium oxalate kidney and / or bladder stone formation fed a food specific to the condition (Hill’s Prescription Diet c / d Multicare).

[0154] Referring to FIG. 6, allantoin degradation pathways were significantly increased in the dogs with a history of calcium oxalate kidney and / or bladder stone formation vs. healthy dogs and further enhanced the allantoin degradation pathway capability when the dogs with a history of calcium oxalate kidney and / or bladder stone formation fed a food specific to the condition (Hill’s Prescription Diet c / d Multicare) to reduce allantoin levels. Similarly, referring to FIG. 7, branched amino acids biosynthesis pathways are also significantly decreased in the dogs with a history of calcium oxalate kidney and / or bladder stone formation compared with the healthy dogs and further decreased when the dogs with a history of calcium oxalate kidney and / or bladder stone formation fed a food specific to the condition (Hill’s Prescription Diet c / d Multicare).

[0155] Oxalobacterformigen.es was the first bacterial species identified to degrade oxalate in the humans and later several other bacterial species such as Lactobacillus sp., Bifidobacterium sp., and others can degrade oxalate (Liu et al 2021). The two genes involved in the microbial oxalate degradation pathway are: Oxalyl-CoA decarboxylase and formyl-CoA transferase. Referring to FIG. 8, abundance levels of oxalyl-CoA decarboxylase and formyl-CoA transferase are increased in the dogs with a history of calcium oxalate kidney and / or bladder stone formation vs. healthy dogs and further increased significantly when the dogs with a history of calcium oxalate kidney and / or bladder stone formation fed Hill’s food specific to the condition (FIG. 8). These data suggest that a food specific to the condition (Hill’s Prescription Diet c / d Multicare) increases the gut microbial functional capacity to degrade oxalate to prevent the recurrence of calcium oxalate stone formation and improves renal function by lessening the burden.

[0156] Referring to FIGS. 9A and 9B, a principal component analysis was performed with the selected functional pathways which showed that PCI explains 74.1 % variability in the data (PCI 74.1% & PC25.6 %) highlighting the critical role of these pathways in differentiating healthy dogs vs. dogs with a history of calcium oxalate kidney and / or bladder stone formation (FIG. 9A). In addition, the principal component loading matrix shows how these functional pathways variables contribute based on their loading coefficients in the first three principal components (FIG. 9B).This analysis clearly suggests that these functional pathways are the key contributors to differentiate healthy dogs vs. dogs with a history of calcium oxalate kidney and / or bladder stone formation.

[0157] Overall, this example identified key gut microbial functional pathways as potential biomarkers to identify dogs that have a propensity to be calcium oxalate stone formers and provides a method to prevent the recurrence of calcium oxalate stone formation. This example also provides a means to improve overall renal function with the provided nutritional solution. Further, it is evident that dogs with a history of stone formation that were fed Hill’s food specific to the condition decreased in serum BUN and BUN / creatinine ratio, which are considered markers for improving kidney function (FIG. 10).

[0158] While the present invention has been described with reference to several embodiments, which embodiments have been set forth in considerable detail for the purposes of making a complete disclosure of the invention, such embodiments are merely exemplary and are not intended to be limiting or represent an exhaustive enumeration of all aspects of the invention. The scope of the invention is to be determined from the claims appended hereto. Further, it will be apparent to those of skill in the art that numerous changes may be made in such details without departing from the spirit and the principles of the invention.

Claims

CLAIMSWhat Is Claimed Is:

1. A method of treating or preventing calcium oxalate kidney and / or bladder stones in a canine subject, the method comprising: determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject, and feeding the canine subject a stone forming preventative diet.

2. The method of claim 1, wherein the step of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject comprises: analyzing a fecal sample of the canine subject to determine an abundance of gastrointestinal microbiome metabolic pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation, wherein the abundance is a measure of at least one of respective microbial gene sequence abundance, respective microbial mRNA sequence abundance, or respective metabolite level abundance for the pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation, and relative to control canine values, the canine subject has an increased susceptibility to developing calcium oxalate kidney and / or bladder stones when the analyzing shows at least one of (i) an increase in pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, or (g) microbial oxalate degradation, or (ii) a decrease in pathways for (f) microbial branched amino acid biosynthesis in the canine subject.

3. The method of claim 1, wherein the step of analyzing a fecal sample of the canine subject to determine an abundance of pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbialubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation comprises analyzing at least two of, at least three of, at least four of, at least five of, or at least six of the abundance of pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

4. The method of claim 1, wherein the step of analyzing a fecal sample of the canine subject to determine an abundance of pathways for at least one of (a) microbial phylloquinol biosynthesis,(b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation comprises analyzing the abundance of pathways for all of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis,(c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

5. The method of any one of claims 1-4, wherein analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of at least one of superpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol- 10 biosynthesis, superpathway of menaquinol- 11 biosynthesis, superpathway of menaquinol- 12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-6 biosynthesis II, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

6. The method of claim 5, wherein analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of superpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol- 10biosynthesis, superpathway of menaquinol-11 biosynthesis, superpathway of menaquinol- 12 biosynthesis, superpathway of mcnaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

7. The method of claim 5, wherein analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of all of superpathway of menaquinol- 6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol- 10 biosynthesis, superpathway of menaquinol- 11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-6 biosynthesis II, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

8. The method of any one of claims 1-7, wherein analyzing the abundance of pathways for (c) microbial arginine biosynthesis comprises analyzing an abundance of at least one of L-arginine biosynthesis I via L-omithine, L-arginine biosynthesis II (acetyl cycle), L-arginine biosynthesis III via N-acetyl-L-citruline, and L-arginine biosynthesis IV (archaebacterial).

9. The method of claim 8, wherein analyzing the abundance of pathways for (c) microbial arginine biosynthesis comprises analyzing an abundance of all of L-arginine biosynthesis I via L- omithine, L-arginine biosynthesis II (acetyl cycle), L-arginine biosynthesis III via N-acetyl-L- citruline, and L-arginine biosynthesis IV (archaebacterial).

10. The method of any one of claims 1-9, wherein analyzing the abundance of pathways for (d) microbial ubiquinol biosynthesis comprises analyzing an abundance of at least one of ubiquinol-7-biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol- 10- biosynthesis (prokaryotic), ubiquinol-8-biosynthesis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic).

11. The method of claim 10, wherein analyzing the abundance of pathways for (d) microbial ubiquinol biosynthesis comprise analyzing an abundance of all of ubiquinol-7- biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol- 10-biosynthesis (prokaryotic), ubiquinol-8-biosynthesis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic).

12. The method of any one of claims 1-11, wherein analyzing the abundance of pathways for (e) microbial allantoin degradation comprise analyzing an abundance of at least one of allantoin degradation IV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast.

13. The method of claim 12, wherein analyzing the abundance of pathways for (e) microbial allantoin degradation comprise analyzing an abundance of all of allantoin degradation IV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast.

14. The method of any one of claims 1-13, wherein analyzing the abundance of pathways for (g) microbial oxalate degradation comprise analyzing an abundance of at least one of OXALYL-COA-DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO- 1382-2.8.3.16-Formyl-Co A transferase.

15. The method of claim 14, wherein analyzing the abundance of pathways for (g) microbial oxalate degradation comprise analyzing an abundance of both of OXALYL-COA- DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO- 1382-2.8.3.16- Formyl-CoA transferase.

16. The method of any one of claims 1-15, wherein the susceptibility to developing is a susceptibility to recurrence of calcium oxalate kidney and / or bladder stones in the canine subject.

17. The method of any one of claims 1-15, wherein the susceptibility to developing is a susceptibility to developing a first case of calcium oxalate kidney and / or bladder stones in the canine subject.

18. The method of any one of claims 1-17, wherein the canine subject is a senior canine.

19. The method of any one of claims 1-18, wherein the canine subject is a male canine.

20. The method of any one of claims 1-19, wherein the canine subject is a small breed of dog.

21. The method of any one of claims 1-19, wherein the canine subject a mini breed of dog.

22. The method of any one of claims 1-21, wherein the analyzing comprises sequencing nucleic acid in the fecal sample and identifying sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for the at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

23. The method of claim 22, wherein determining the abundance of the gastrointestinal microbiome metabolic pathways comprises quantifying identified sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for the at least one of the (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

24. The method of claim 23, wherein quantifying identified sequences comprises quantifying sequences for the (b) microbial menaquinol biosynthesis.

25. The method of claim 24, wherein quantifying identified sequences comprises quantifying sequences for at least one of the superpathway of mcnaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol-10 biosynthesis, the superpathway of menaquinol- 11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol- 13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-6 biosynthesis II, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis.

26. The method of claim 24, wherein quantifying identified sequences comprises quantifying sequences for the superpathway of menaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol-10 biosynthesis, the superpathway of menaquinol-11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol- 13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis.

27. The method of claim 24, wherein quantifying identified sequences comprises quantifying sequences for all of the superpathway of menaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol-10 biosynthesis, the superpathway of menaquinol-11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol- 13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-6 biosynthesis II, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis.

28. The method of claim 23, wherein quantifying identified sequences comprises quantifying sequences for the (c) microbial arginine biosynthesis.

29. The method of claim 28, wherein quantifying identified sequences comprises quantifying sequences for at least one of the L-arginine biosynthesis I via L-omithine, the L- arginine biosynthesis II (acetyl cycle), the L-arginine biosynthesis III via N-acetyl-L-citruline, and the L-arginine biosynthesis IV (archaebacterial).

30. The method of claim 28, wherein quantifying identified sequences comprises quantifying sequences for all of the L-arginine biosynthesis I via L-ornithine, the L-arginine biosynthesis II (acetyl cycle), the L-arginine biosynthesis III via N-acetyl-L-citruline, and the L- arginine biosynthesis IV (archaebacterial).

31. The method of claim 23, wherein quantifying identified sequences comprises quantifying sequences for the (d) microbial ubiquinol biosynthesis.

32. The method of claim 31, wherein quantifying identified sequences comprises quantifying sequences for at least one of the ubiquinol-7-biosynthesis (prokaryotic), the ubiquinol- 9-biosynthesis (prokaryotic), the ubiquinol- 10-biosynthesis (prokaryotic), the ubiquinol-8- biosynthesis (prokaryotic), and the superpathway of ubiquinoL8 biosynthesis (prokaryotic).

33. The method of claim 31, wherein quantifying identified sequences comprises quantifying sequences for all of the ubiquinol-7-biosynthesis (prokaryotic), the ubiquinol-9- biosynthesis (prokaryotic), the ubiquinol- 10-biosynthesis (prokaryotic), the ubiquinol-8- biosynthesis (prokaryotic), and the superpathway of ubiquinol-8 biosynthesis (prokaryotic).

34. The method of claim 23, wherein quantifying identified sequences comprises quantifying sequences for the (e) microbial allantoin degradation.

35. The method of claim 34, wherein quantifying identified sequences comprises quantifying sequences for at least one of the allantoin degradation IV (anaerobic), the allantoin degradation to glyoxylate III, and the superpathway of allantoin degradation in yeast.

36. The method of claim 34, wherein quantifying identified sequences comprises quantifying sequences for all of the allantoin degradation IV (anaerobic), the allantoin degradation to glyoxylate III, and the superpathway of allantoin degradation in yeast.

37. The method of claim 23, wherein quantifying identified sequences comprises quantifying sequences for the (g) microbial oxalate degradation.

38. The method of claim 37, wherein quantifying identified sequences comprises quantifying sequences for at least one of the 0XALYL-C0A-DECARB0XYLASE-RXN-4.1.1.8- Oxalyl-CoA decarboxylase and the RXNO-1382-2.8.3.16-Formyl-CoA transferase.

39. The method of claim 37, wherein quantifying identified sequences comprises quantifying sequences for both of the 0XALYL-C0A-DECARB0XYLASE-RXN-4.1.1.8- Oxalyl-CoA decarboxylase and the RXNO-1382-2.8.3.16-Formyl-CoA transferase.

40. The method of any one of claims 1-39, wherein determining the abundance of the gastrointestinal microbiome metabolic pathways comprises quantifying identified sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for all of the (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

41. The method of any one of claims 22-40, wherein the nucleic acid is DNA.

42. The method of any one of claims 22-40, wherein the nucleic acid is mRNA.

43. The method of claim 42, wherein the sequencing comprises converting the mRNA to cDNA and sequencing the cDNA.

44. The method of any one of claims 1-43 further comprising comparing the abundance of the gastrointestinal microbiomc metabolic pathways in the subject canine to control abundance values of the gastrointestinal microbiome metabolic pathways in one or more control canine.

45. The method of any one of claims 1-44, wherein the stone forming preventative diet comprises at least one or all of: protein content from about 14 to about 32 wt. %, calcium content from about 0.6 to about 1.6 wt. %, phosphorous content from about 0.2 to about 0.9 wt. %, magnesium content from about 0.04 to about 0.2 wt. %, sodium content from about 0.2 to about 0.5 wt. %, potassium content from about 0.5 to about 1.1 wt. %, and chloride dry content from about 0.4 to about 1.3 wt. %, wherein the wt. % is based on dry weight of the stone forming preventative diet.

46. The method of claim 45, wherein the chloride wet content is about 1.5 wt. % based on hydrated weight of the stone forming preventative diet.

47. The method of claim any one of claims 1-46, wherein the step of feeding the canine subject a stone forming preventative diet occurs upon a determination that the canine subject is susceptible to developing calcium oxalate kidney and / or bladder stones.

48. A kit for identifying a canine subject having an increased risk for developing calcium oxalate kidney and / or bladder stones from a biological sample from the canine, the kit comprising: at least one detection reagent selected from: at least one nucleic acid primer, at least one fluorescence tag, or at least one stain specific to a combination of two or more metabolites; and instructions for use.

49. The kit of claim 48 further comprising a vessel for collecting the biological sample.

50. The kit of claim 49, wherein the biological sample is a fecal sample.

51. The kit according to claim 50, wherein the at least one detection reagent is for analyzing the fecal sample to determine an abundance of gastrointestinal microbiome metabolic pathwaysfor at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (c) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

52. The kit according to any one of claims 48-51, wherein the instructions for use instruct a user thereof to compare the abundance of the gastrointestinal microbiome metabolic pathways in the canine subject to an abundance of the gastrointestinal microbiome metabolic pathways in a control canine.

53. The kit according to claim 52, wherein the control canine is a healthy canine.

54. The kit according to any one of claims 51 to 53, wherein the instructions instruct that relative to the control canine values, the canine subject has an increased susceptibility to developing calcium oxalate kidney and / or bladder stones when the analyzing shows at least one of (i) an increase in pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, or (g) microbial oxalate degradation, or (ii) a decrease in pathways for (f) microbial branched amino acid biosynthesis in the canine subject.

55. A method of determining susceptibility to developing calcium oxalate kidney and / or bladder stones in a canine subject comprising: analyzing a fecal sample of the canine subject to determine an abundance of gastrointestinal microbiome metabolic pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation, wherein the abundance is a measure of at least one of respective microbial gene sequence abundance, respective microbial mRNA sequence abundance, or respective metabolite level abundance for the pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinolbiosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation, and relative to control canine values, the canine subject has an increased susceptibility to developing calcium oxalate kidney and / or bladder stones when the analyzing shows at least one of (i) an increase in pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, or (g) microbial oxalate degradation, or (ii) a decrease in pathways for (f) microbial branched amino acid biosynthesis in the canine subject.

56. The method of claim 55, wherein the step of analyzing a fecal sample of the canine subject to determine an abundance of pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation comprises analyzing at least two of, at least three of, at least four of, at least five of, or at least six of the abundance of pathways for (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

57. The method of claim 55, wherein the step of analyzing a fecal sample of the canine subject to determine an abundance of pathways for at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation comprises analyzing the abundance of pathways for all of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

58. The method of any one of claims 55-57, wherein analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of at least one ofsuperpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of mcnaquinol-8 biosynthesis I, superpathway of mcnaquinol-8 biosynthesis II, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol- 10 biosynthesis, superpathway of menaquinol- 11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-6 biosynthesis II, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

59. The method of claim 58, wherein analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of superpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol- 10 biosynthesis, superpathway of menaquinol- 11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

60. The method of claim 58, wherein analyzing the abundance of pathways for (b) microbial menaquinol biosynthesis comprises analyzing an abundance of all of superpathway of menaquinol-6 biosynthesis I, superpathway of menaquinol-7 biosynthesis, superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, superpathway of menaquinol-9 biosynthesis, superpathway of menaquinol- 10 biosynthesis, superpathway of menaquinol- 11 biosynthesis, superpathway of menaquinol-12 biosynthesis, superpathway of menaquinol-13 biosynthesis, superpathway of demethylmenaquinol-6 biosynthesis I, superpathway of demethylmenaquinol-6 biosynthesis II, superpathway of demethylmenaquinol-8 biosynthesis I, and superpathway of demethylmenaquinol-9 biosynthesis.

61. The method of any one of claims 55-60, wherein analyzing the abundance of pathways for (c) microbial arginine biosynthesis comprises analyzing an abundance of at least one of L- arginine biosynthesis I via L-omithine, L-arginine biosynthesis II (acetyl cycle), L-arginine biosynthesis III via N-acetyl-L-citruline, and L-arginine biosynthesis IV (archaebacterial).

62. The method of claim 61, wherein analyzing the abundance of pathways for (c) microbial arginine biosynthesis comprises analyzing an abundance of all of L-arginine biosynthesis I via L-omithine, L-arginine biosynthesis II (acetyl cycle), L-arginine biosynthesisIII via N-acetyl-L-citruline, and L-arginine biosynthesis IV (archaebacterial).

63. The method of any one of claims 55-62, wherein analyzing the abundance of pathways for (d) microbial ubiquinol biosynthesis comprises analyzing an abundance of at least one of ubiquinol-7-biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol- 10- biosynthesis (prokaryotic), ubiquinol-8-biosynthesis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic).

64. The method of claim 63, wherein analyzing the abundance of pathways for (d) microbial ubiquinol biosynthesis comprise analyzing an abundance of all of ubiquinol-7- biosynthesis (prokaryotic), ubiquinol-9-biosynthesis (prokaryotic), ubiquinol- 10-biosynthesis (prokaryotic), ubiquinol-8-biosynthesis (prokaryotic), and superpathway of ubiquinol-8 biosynthesis (prokaryotic).

65. The method of any one of claims 55-64, wherein analyzing the abundance of pathways for (e) microbial allantoin degradation comprise analyzing an abundance of at least one of allantoin degradation IV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast.

66. The method of claim 65, wherein analyzing the abundance of pathways for (e) microbial allantoin degradation comprise analyzing an abundance of all of allantoin degradationIV (anaerobic), allantoin degradation to glyoxylate III, and superpathway of allantoin degradation in yeast.

67. The method of any one of claims 55-66, wherein analyzing the abundance of pathways for (g) microbial oxalate degradation comprise analyzing an abundance of at least one ofOXALYL-COA-DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO- 1382-2.8.3.16-Formyl-Co A transferase.

68. The method of claim 67, wherein analyzing the abundance of pathways for (g) microbial oxalate degradation comprise analyzing an abundance of both of OXALYL-COA- DECARBOXYLASE-RXN-4.1.1.8-Oxalyl-CoA decarboxylase and RXNO- 1382-2.8.3.16- Formyl-CoA transferase.

69. The method of any one of claims 55-68, wherein the susceptibility to developing is a susceptibility to recurrence of calcium oxalate kidney and / or bladder stones in the canine subject.

70. The method of any one of claims 55-68, wherein the susceptibility to developing is a susceptibility to developing a first case of calcium oxalate kidney and / or bladder stones in the canine subject.

71. The method of any one of claims 55-70, wherein the canine subject is a senior canine.

72. The method of any one of claims 55-71 wherein the canine subject is a male canine.

73. The method of any one of claims 55-72, wherein the canine subject is a small breed of dog.

74. The method of any one of claims 55-72, wherein the canine subject a mini breed of dog.

75. The method of any one of claims 55-74, wherein the analyzing comprises sequencing nucleic acid in the fecal sample and identifying sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for the at least one of (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

76. The method of claim 75, wherein determining the abundance of the gastrointestinal microbiome metabolic pathways comprises quantifying identified sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for the at least one of the (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial arginine biosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

77. The method of claim 76, wherein quantifying identified sequences comprises quantifying sequences for the (b) microbial menaquinol biosynthesis.

78. The method of claim 77, wherein quantifying identified sequences comprises quantifying sequences for at least one of the superpathway of menaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol- 10 biosynthesis, the superpathway of menaquinol- 11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol- 13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-6 biosynthesis II, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis.

79. The method of claim 77, wherein quantifying identified sequences comprises quantifying sequences for the superpathway of menaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol- 10 biosynthesis, the superpathway of menaquinol-11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol- 13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis.

80. The method of claim 77, wherein quantifying identified sequences comprises quantifying sequences for all of the superpathway of mcnaquinol-6 biosynthesis I, the superpathway of menaquinol-7 biosynthesis, the superpathway of menaquinol-8 biosynthesis I, superpathway of menaquinol-8 biosynthesis II, the superpathway of menaquinol-9 biosynthesis, the superpathway of menaquinol-10 biosynthesis, the superpathway of menaquinol- 11 biosynthesis, the superpathway of menaquinol- 12 biosynthesis, the superpathway of menaquinol- 13 biosynthesis, the superpathway of demethylmenaquinol-6 biosynthesis I, the superpathway of demethylmenaquinol-6 biosynthesis II, the superpathway of demethylmenaquinol-8 biosynthesis I, and the superpathway of demethylmenaquinol-9 biosynthesis.

81. The method of claim 76, wherein quantifying identified sequences comprises quantifying sequences for the (c) microbial arginine biosynthesis.

82. The method of claim 81, wherein quantifying identified sequences comprises quantifying sequences for at least one of the L-arginine biosynthesis I via L-omithine, the L- arginine biosynthesis II (acetyl cycle), the L-arginine biosynthesis III via N-acetyl-L-citruline, and the L-arginine biosynthesis IV (archaebacterial).

83. The method of claim 81, wherein quantifying identified sequences comprises quantifying sequences for all of the L-arginine biosynthesis I via L-ornithine, the L-arginine biosynthesis II (acetyl cycle), the L-arginine biosynthesis III via N-acetyl-L-citruline, and the L- arginine biosynthesis IV (archaebacterial).

84. The method of claim 76, wherein quantifying identified sequences comprises quantifying sequences for the (d) microbial ubiquinol biosynthesis.

85. The method of claim 84, wherein quantifying identified sequences comprises quantifying sequences for at least one of the ubiquinol-7-biosynthesis (prokaryotic), the ubiquinoL 9-biosynthesis (prokaryotic), the ubiquinol- 10-biosynthesis (prokaryotic), the ubiquinoL8- biosynthesis (prokaryotic), and the superpathway of ubiquinol-8 biosynthesis (prokaryotic).

86. The method of claim 84, wherein quantifying identified sequences comprises quantifying sequences for all of the ubiquinol-7-biosynthcsis (prokaryotic), the ubiquinol-9- biosynthesis (prokaryotic), the ubiquinol-10-biosynthesis (prokaryotic), the ubiquinol-8- biosynthesis (prokaryotic), and the superpathway of ubiquinol-8 biosynthesis (prokaryotic).

87. The method of claim 76, wherein quantifying identified sequences comprises quantifying sequences for the (e) microbial allantoin degradation.

88. The method of claim 87, wherein quantifying identified sequences comprises quantifying sequences for at least one of the allantoin degradation IV (anaerobic), the allantoin degradation to glyoxylate III, and the superpathway of allantoin degradation in yeast.

89. The method of claim 87, wherein quantifying identified sequences comprises quantifying sequences for all of the allantoin degradation IV (anaerobic), the allantoin degradation to glyoxylate III, and the superpathway of allantoin degradation in yeast.

90. The method of claim 76, wherein quantifying identified sequences comprises quantifying sequences for the (g) microbial oxalate degradation.

91. The method of claim 90, wherein quantifying identified sequences comprises quantifying sequences for at least one of the OXAL YL-COA-DECARBOXYLASE-RXN-4.1.1.8- Oxalyl-CoA decarboxylase and the RXNO-1382-2.8.3.16-Formyl-CoA transferase.

92. The method of claim 90, wherein quantifying identified sequences comprises quantifying sequences for both of the OXAL YL-COA-DECARBOXYLASE-RXN-4.1.1.8- OxalyLCoA decarboxylase and the RXNO-1382-2.8.3.16-Formyl-CoA transferase.

93. The method of any one of claims 55-92, wherein determining the abundance of the gastrointestinal microbiome metabolic pathways comprises quantifying identified sequences encoding enzymes within the gastrointestinal microbiome metabolic pathways for all of the (a) microbial phylloquinol biosynthesis, (b) microbial menaquinol biosynthesis, (c) microbial argininebiosynthesis, (d) microbial ubiquinol biosynthesis, (e) microbial allantoin degradation, (f) microbial branched amino acid biosynthesis, and (g) microbial oxalate degradation.

94. The method of any one of claims 75-93, wherein the nucleic acid is DNA.

95. The method of any one of claims 75-93, wherein the nucleic acid is mRNA.

96. The method of claim 95, wherein the sequencing comprises converting the mRNA to cDNA and sequencing the cDNA.

97. The method of any one of claims 55-96 further comprising comparing the abundance of the gastrointestinal microbiome metabolic pathways in the subject canine to control abundance values of the gastrointestinal microbiome metabolic pathways in one or more control canine.

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