Diagnosis and treatment of chronic kidney disease in felines

Abstract

The present invention relates to a method for diagnosing chronic kidney disease in felines, including early stage chronic kidney disease, by using a microbiome containing specific genera and species. In one embodiment, the method may include measuring the absolute abundance of fecal bacteria, including Faecalibacterium, Turicibacter, Streptococcus, Bifidobacterium, Bacteroides, Escherichia coli, and C. hyranonis, calculating a dysbiosis index based on the fecal bacteria, and determining that the feline has chronic kidney disease if the dysbiosis index is 0.5 or more.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 335,973, filed April 28, 2022, the disclosure of which is incorporated by reference in its entirety into this specification. [Background technology]

[0002]

[0002] The kidneys have five main functions: they filter waste products (e.g., urea and creatinine) from the body, regulate electrolytes (e.g., potassium, calcium, phosphorus, and sodium), produce erythropoietin (which stimulates the bone marrow to produce red blood cells), produce renin (which controls blood pressure), and produce and concentrate urine.

[0003]

[0003] Chronic kidney disease ("CKD") is a progressive kidney disease with four stages: loss of renal reserve, renal dysfunction, azotemia, and uremic. The kidney has a large inherent reserve because normal kidney function requires only about 30% of the kidney's capacity. Renal capacity decreases over time for various reasons, such as aging, kidney-damaging diseases, and medicines. Renal dysfunction is characterized by a decline in kidney function and is generally observed when about 70% of kidney function is lost (i.e., when only about 30% of the kidney's capacity is available). Typically, clinical signs during the stages of loss of renal reserve and renal dysfunction are not obvious, making CKD difficult to detect. Stage 1 CKD does not have azotemia and generally cannot be diagnosed because there are no obvious clinical symptoms. Stage 2 CKD is mild azotemia with no or mild clinical signs. Stage 3 is moderate azotemia with clinical signs. Stage 4 is severe azotemia with clinical signs.

[0004]

[0004] CKD is a terminal disease and one of the leading causes of death in felines. Therefore, there is a need for compositions and methods for diagnosing and preventing CKD in felines, particularly for diagnosing early CKD. There is also a need for compositions and methods for treating CKD that result in partial or complete relief.

[0005] [Summary of the Invention]

[0005] The present disclosure generally relates to diagnosing chronic kidney disease in felines, and in one aspect, diagnosing early CKD. In one embodiment, a method for diagnosing CKD in a feline may include measuring the absolute abundance of fecal bacteria including Faecalibacterium, Turicibacter, Streptococcus, Bifidobacterium, Bacteroides, E. coli, and C. hiranonis, calculating a dysbiosis index based on the fecal bacteria, and determining that the feline has CKD if the dysbiosis index is 0.5 or greater.

[0006] In another embodiment, the method for diagnosing early stage CKD in a feline comprises using Lactobacillus animalis, Subdoligranulum variabile, Catenibacterium mitsuokai, Collinsella aerofaciens, Ruminococcus obeum, Eubacterium biforme, Lactobacillus reuteri, Bifidobacterium pseudocatenulatum, Coprococcus comes, Megasphaera elsdenii, Lactobacillus subtilis ... elsdenii, Lachnospiraceae Bacterium_1_1_57FAA, Faecalibacterium prausnitzii, Eubacterium hallii, Ruminococcaceae Bacterium_D16, Dorea longicatena, Clostridium hiranonis, Acidaminococcus fermentans, Saccharomyces cerevisiae, Streptococcus parauberis, Acidaminococcus instinii intestini, Helicobacter canis, Bacteroides coprocolacoprocola), and combinations thereof; and determining that the feline has early CKD if the absolute abundance of the biomarker is within the ranges set forth in Table 5.

[0007]

[0007] In yet another embodiment, a method for diagnosing early-stage CKD in a feline may include measuring the absolute abundance of bacteria in a genera selected from the group consisting of Catenibacterium, Lactobacillus, Coprococcus, Megasphaera, Helicobacter, Eubacterium, Faecalibacterium, Acidaminococcus, Bifidobacterium, Subdoligranulum, Allobaculum, Escherichia, Enterococcus, and combinations thereof, and determining that the feline has early-stage CKD if the absolute abundance of the biomarkers is within the ranges set forth in Table 3.

[0008]

[0008] Furthermore, a method for enabling treatment or slowing the progression of CKD in a feline can include the steps of diagnosing CKD in a feline as disclosed herein and recommending a composition for the feline, wherein the composition treats or slows the progression of CKD in the feline.

[0009]

[0009] Furthermore, a method for enabling treatment or delaying the progression of early CKD in a feline can include the steps of diagnosing early CKD in a feline as disclosed herein and recommending a composition for the feline, wherein the composition treats or delays the progression of early CKD in the feline.

[0010]

[0010] Additional features and advantages are described herein, and will be apparent from the detailed description that follows. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] definition

[0011] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to "a fecal bacteria or bacterium" or "the fecal bacteria or bacterium" includes two or more such bacteria or bacteria. The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y." As used herein, the terms "example" and "for example," particularly when followed by a list of terms, are used merely exemplary and illustratively and are not exclusive or inclusive.

[0012]

[0012] As used herein, "about" is understood to refer to a number within a numerical range, for example, within a range of -10% to +10%, within a range of -5% to +5%, or in one embodiment within a range of -1% to +1% of the referenced number, and in a specific embodiment within a range of -0.1% to +0.1% of the referenced number. Furthermore, all numerical ranges herein should be understood to include all integers, whole or fractional, within the range. Furthermore, these numerical ranges should be interpreted as supporting claims directed to any number or subset of numbers within the range. For example, a disclosure of 1 to 10 should be interpreted as corresponding to ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0013]

[0013] As used herein, "between" includes the endpoints. For example, a dysbiosis index between 0.5 and 1.2 (0.5-1.2) includes cases where dysbiosis is 0.5 or 1.2.

[0014]

[0014] All percentages expressed herein are by weight of the composition on a dry matter basis unless otherwise stated. Those skilled in the art will understand that the term "dry matter basis" means that the concentration or percentage of an ingredient in a composition is measured or specified after all free moisture in the composition has been removed. When reference is made to pH, the value corresponds to the pH measured at 25°C with standard equipment. "Amount" may be the total amount of the referenced ingredient per serving of the composition or the total amount per discrete unit of the composition, and / or may be the weight percent of the referenced ingredient by dry weight. Additionally, "amount" includes zero; for example, a recitation of an amount of a compound does not necessarily mean that the compound is present unless accompanied by a range excluding zero.

[0015]

[0015] As used herein, "absolute abundance" refers to the amount of each microbial organism calculated by the base 10 logarithm of bacterial DNA abundance measured by a quantitative PCR (qPCR) assay.

[0016]

[0016] As used herein, "chronic kidney disease" or "CKD" refers to a sustained decline in renal function over an extended period of time in felines.

[0017] As used herein, "early stage chronic kidney disease" refers to stage 1 or stage 2 chronic kidney disease (CKD) or CKD1 / 2 according to the IRIS (International Renal Research Association) guidelines (http: / / www.iris-kidney.com / guidelines / ).

[0018]

[0018] As used herein, "late stage chronic kidney disease" refers to stage 3 or stage 4 chronic kidney disease (CKD) or CKD3 / 4 according to the IRIS (International Renal Research Association) guidelines (http: / / www.iris-kidney.com / guidelines / ).

[0019]

[0019] As used herein, the "dysbiosis index" or "DI" is calculated using the qPCR cycle threshold (Ct value) obtained for each bacterial group. To eliminate sample-to-sample variability, the Ct value of each individual bacterial group was normalized by dividing it by the Ct value of the total bacteria. The DI is set by the nearest centroid classifier algorithm, where the DI is defined as the difference between the Euclidean distance between the centroid of the test sample and the centroid of the healthy class and the Euclidean distance between the centroid of the test class and the centroid of the diseased class. The DI is mathematically calculated as follows: The DI of a text sample z is defined as:

number

number

[0020]

[0020] The methods disclosed herein may be absent any step not specifically disclosed herein. Thus, the disclosure of an embodiment using the term "comprising" includes the disclosure of an embodiment "essentially comprising" the specified step, and an embodiment "consisting of" the specified step. Unless otherwise indicated and directly stated, any embodiment disclosed herein can be combined with any other embodiment disclosed herein.

[0021]

[0021] Unless otherwise specified, all technical and scientific terms and any abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any compositions, methods, products, or other techniques or materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred compositions, methods, products, or other techniques or materials are described herein.

[0022]

[0022] All patents, patent applications, publications, and other references cited or referred to in this specification are hereby incorporated by reference to the extent permitted by law. The discussion of these references is intended only to summarize the assertions made in the references. No admission is made that such patents, patent applications, publications, or references, or any portion thereof, are relevant prior art to the present invention, and the right to challenge the accuracy and pertinence of such patents, patent applications, publications, and other references is expressly reserved.

[0023] Embodiment

[0023] The inventors have discovered that kidney disease (CKD) can be diagnosed based on a dysbiosis index and a specific microbiome, including specific genera and species. Furthermore, the inventors have discovered that the microbiome can be used to diagnose early stage CKD in felines. Such a method allows for inexpensive and efficient diagnosis of a variety of conditions that can be difficult and expensive to diagnose.

[0024] In one embodiment, a method for diagnosing CKD in a feline may include measuring the absolute abundance of fecal bacteria including Faecalibacterium, Turicibacter, Streptococcus, Bifidobacterium, Bacteroides, Escherichia coli, and C. hyranonis, calculating a dysbiosis index based on the fecal bacteria, and determining that the feline has CKD if the dysbiosis index is 0.5 or greater. Additionally, the method may also include measuring other bacteria. In one aspect, the fecal bacteria may include Blautia. In another aspect, the fecal bacteria may include Fusobacterium.

[0025]

[0025] Generally, a feline may be diagnosed as having CKD when the dysbiosis index is 0.5 or greater. However, in one embodiment, the determining step may also include determining that the feline has early CKD when the dysbiosis index is between 0.5 and 1.2. Additionally, in another embodiment, the method may further determine that the feline has late CKD when the dysbiosis index is greater than 1.2.

[0026] In another embodiment, the method for diagnosing early stage CKD in a feline comprises using Lactobacillus animalis, Subdoligranulum variabile, Catenibacterium mitsuokai, Collinsella aerofaciens, Ruminococcus obeum, Eubacterium biforme, Lactobacillus reuteri, Bifidobacterium pseudocatenulatum, Coprococcus comethus, Megasphaera elsdenii, Lachnospiraceae Bacterium_1_1_57FAA, Faecalibacterium prausnitzii, Eubacterium hallii, The method may include measuring the absolute abundance of biomarkers selected from the group consisting of Ruminococcus bacterium_D16, Dorea longicathena, Clostridium hyranonis, Acidaminococcus fermentans, Saccharomyces cerevisiae, Streptococcus parauberis, Acidaminococcus instinii, Helicobacter canis, Bacteroides coprolita, and combinations thereof, and determining that the feline has early stage CKD if the absolute abundance of the biomarkers is within the ranges set forth in Table 5.

[0027]

[0027] Generally, felines can be diagnosed as having early stage CKD by various biomarkers, i.e. bacteria, discussed herein, having a particular absolute abundance. However, in one embodiment, the diagnosis can be based on at least two biomarkers. In another embodiment, the diagnosis can be based on at least three biomarkers. In yet another embodiment, the diagnosis can be based on at least four biomarkers. In yet other embodiments, the diagnosis can be based on five biomarkers, six biomarkers, seven biomarkers, eight biomarkers, nine biomarkers, ten biomarkers, fifteen biomarkers, or more biomarkers.

[0028]

[0028] In yet another embodiment, a method for diagnosing early-stage CKD in a feline may include measuring the absolute abundance of bacteria in a genera selected from the group consisting of Catenibacterium, Lactobacillus, Coprococcus, Megasphaera, Helicobacter, Eubacterium, Faecalibacterium, Acidaminococcus, Bifidobacterium, Subdoligranulum, Allobaculum, Escherichia, Enterococcus, and combinations thereof, and determining that the feline has early-stage CKD if the absolute abundance of the biomarkers is within the ranges set forth in Table 3.

[0029]

[0029] Generally, felines may be diagnosed as having early stage CKD by the presence of bacteria of the various genera discussed herein having a particular absolute abundance. However, in one embodiment, the diagnosis may be based on at least two genera. In another embodiment, the diagnosis may be based on at least three genera. In yet another embodiment, the diagnosis may be based on at least four genera. In another embodiment, the diagnosis may be based on at least five genera. In yet other embodiments, the diagnosis may be based on six genera, seven genera, eight genera, nine genera, ten genera, or more genera.

[0030]

[0030] Furthermore, a method for enabling treatment or slowing the progression of CKD in a feline can include the steps of diagnosing CKD in a feline as disclosed herein and recommending a composition for the feline, wherein the composition treats or slows the progression of CKD in the feline.

[0031]

[0031] Furthermore, a method for enabling treatment or delaying the progression of early CKD in a feline can include the steps of diagnosing early CKD in a feline as disclosed herein and recommending a composition for the feline, wherein the composition treats or delays the progression of early CKD in the feline.

[0032]

[0032] Compositions useful for treating CKD, including early CKD, include pet foods and dietary supplements. Such compositions may include medium chain triglycerides, omega-3 fatty acids, vitamin E, vitamin C, B vitamins (including thiamine, riboflavin, pantothenic acid, niacin, pyridoxine, folic acid, biotin, and cobalamin), L-arginine, or sulfur-containing amino acids such as taurine. The compositions may be wet pet foods, semi-moist pet foods, or dry pet foods, such as kibbles.

[0033]

[0033] Generally, the medium chain triglycerides can be from about 0.5% to about 60% by weight of the composition. In one embodiment, the medium chain triglycerides can be from about 1% to about 20% by weight of the composition. In other embodiments, the medium chain triglycerides can be from about 1% to about 15%, from about 1% to about 10%, or from about 2% to about 10% by weight of the composition. The medium chain triglycerides may be prepared by any known method, such as direct esterification, rearrangement, fractionation, and / or transesterification. For example, the medium chain triglycerides may be prepared from a vegetable oil feedstock, such as coconut oil, via a rearrangement process. The chain length and distribution of the medium chain triglycerides may vary depending on the feedstock oil. For example, MCTs containing 1-10% C6, 30-60% C8, 30-60% C10, and 1-10% C12 can be derived from palm oil and / or coconut oil. In some embodiments, at least a portion of the MCTs are obtained from coconut oil, while in other embodiments, the compositions contain no coconut oil. Semi-synthetic esterification of octanoic acid to glycerin can produce MCTs containing at least about 95% C8, of which in some embodiments the remaining fatty acids are C6 and C10. Also useful herein are mixtures containing MCTs having about 50% total C8 and / or about 50% total C10.

[0034]

[0034] Non-limiting examples of suitable omega-3 fatty acids include eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), alpha linolenic acid (ALA), and mixtures thereof. In one embodiment, the omega-3 fatty acids may range from about 0.2% to about 3% by weight of the composition. In some embodiments, the omega-3 fatty acids may be at least about 0.2%, at least about 1.0%, or at least about 2.0% by weight.

[0035]

[0035] Non-limiting examples of suitable sulfur-containing amino acids include methionine, cysteine, homocysteine, taurine, and mixtures thereof.

[0036] In one embodiment, the composition further comprises (i) carnitine, (ii) lysine and methionine, (iii) an antioxidant such as glutathione, and (iv) a mixture thereof. The composition may be high in protein, for example, at least about 20%, at least about 25%, or even at least about 30% by weight of the composition is protein. In addition, the composition may have balanced amounts of magnesium, sodium, and potassium, for example, a ratio of potassium to sodium of about 5:1 to about 1:1, and in one embodiment, about 5:1 to about 2:1, and an amount of magnesium of about 0.08% to about 0.25% by weight, and in one embodiment, about 0.10% to about 0.15% by weight. At least a portion of the magnesium, sodium, and potassium may be provided as isolated compounds (e.g., salts). Alternatively, or in addition, at least a portion of the magnesium, sodium, and potassium may be provided by one or more food products. For example, magnesium can be obtained from wheat bran, whole grains, green leafy vegetables, meat, beans, and bananas, and potassium and sodium can be obtained from meat, fish, whole grains, yogurt, bananas, sweet potatoes, pumpkin, beans, and tomatoes.

[0037] In one embodiment, the complete nutrition as defined by the Association of American Feed Control Officials (AAFCO) and for the intended animal species (e.g., cat) is provided by the pet food composition. In another embodiment, the composition may be a dietary supplement. Such dietary supplements may be added to the food composition, or may be administered together with the food composition, or may be administered separately.

[0038]

[0038] The pet food composition may include meat, such as emulsified meat. Examples of suitable meat include poultry, beef, pork, lamb, and fish, including those types of meat that are particularly suitable for pets. The meat may include any additional parts of an animal, including offal. Some or all of the meat may be provided as one or more meat meals, i.e., meat that has been dried and ground to form particles of substantially uniform size, as defined by AAFCO. Additionally or alternatively, vegetable proteins such as pea protein, corn protein (e.g., ground corn or corn gluten), wheat protein (e.g., ground wheat or wheat gluten), soy protein (e.g., soybean meal, soy concentrate, or soy isolate), and rice protein (e.g., ground rice or rice gluten) may be used.

[0039]

[0039] The pet food composition disclosed herein may include one or more of vegetable oil, flavoring, coloring, or water. Non-limiting examples of suitable vegetable oils include soybean oil, corn oil, cottonseed oil, sunflower oil, canola oil, peanut oil, and safflower oil. In some embodiments, the lipids in the composition may include MCT and one or more of any vegetable oil, any fish oil, any meat-derived lipid, and any omega-3 fatty acid.

[0040]

[0040] Non-limiting examples of suitable flavoring agents include yeast, tallow, rendered animal meals (e.g., poultry, beef, lamb, and pork), flavor extracts or blends (e.g., grilled beef), and animal digestives. Non-limiting examples of suitable coloring agents include FD&C colors such as Blue No. 1, Blue No. 2, Green No. 3, Red No. 3, Red No. 40, Yellow No. 5, and Yellow No. 6; natural colors such as caramel color, annatto, chlorophyllin, cochineal, betanin, turmeric, saffron, paprika, lycopene, elderberry juice, pandan, and butterfly pea; titanium dioxide; and any suitable food coloring agent known to those skilled in the art.

[0041]

[0041] The pet food compositions disclosed herein may optionally contain further ingredients such as starches, humectants, oral care ingredients, preservatives, amino acids, fibre, prebiotics, sugars, animal oils, aromas, other oils in addition to or as an alternative to the vegetable oils, salts, vitamins, minerals, probiotic microorganisms, bioactive molecules or combinations thereof.

[0042]

[0042] Non-limiting examples of suitable starches include cereals such as corn, rice, wheat, barley, oats, potatoes, peas, beans, cassava, and mixtures of these cereals, and suitable starches may be at least partially contained in any flour. Non-limiting examples of suitable humectants include salt, sugars, propylene glycol, and polyhydric glycols such as glycerin and sorbitol. Non-limiting examples of suitable oral care ingredients include alfalfa nutrient concentrate containing chlorophyll, baking soda, phosphates (e.g., tricalcium phosphate, acid pyrophosphate, tetrasodium pyrophosphate, metaphosphate, and orthophosphate), peppermint, cloves, parsley, and ginger. Non-limiting examples of suitable preservatives include potassium sorbate, sorbic acid, sodium methyl para-hydroxybenzoate, calcium propionate, propionic acid, and combinations thereof.

[0043]

[0043] The specific amount of each additional ingredient in the pet food compositions disclosed herein will depend on a variety of factors, such as the ingredients contained in the first food ingredient and any second food ingredient, the species of the animal, the age, weight, health, sex, and diet of the animal, the rate of consumption by the animal, and the purpose for which the food product is administered to the animal. Thus, the ingredients and their amounts can vary widely.

[0044]

[0044] Additionally, the composition may have, in whole or in part, a specific protein to phosphorus ratio of about 5:1 to about 15:1. This ratio may provide a palatability enhancing effect on a renal diet, and this ratio may be present in the base material or in a coating (e.g., kibble) applied to the base material. Other compositions may include protein, fat, fiber, and carbohydrates, and may optionally include functional ingredients that reduce renal damage or enhance renal function, including functional ingredients that regulate or reduce the levels of blood catabolites, regulate or reduce the levels of phosphorus, reduce blood urea nitrogen levels (BUN) or BUN / creatine ratio levels. Functional ingredients include, but are not limited to, conjugated linoleic acid. Generally, with respect to pet food products, functional ingredients act to mitigate the adverse effects of a high protein diet. Natural or synthetic functional ingredients are contemplated. Synthetic and semi-synthetic preparations of conjugated linoleic acid (i.e., isomerization of vegetable oils, for example, by the ruminal bacterium Butyrivibrio fibrisolvens) have been described and are believed to be suitable for the present invention (see, e.g., U.S. Pat. Nos. 6,410,761, 6,380,409, and 5,554,646, each of which is incorporated by reference herein in its entirety). In embodiments where the functional ingredient is conjugated linoleic acid, the amount can be calculated as part of either the functional ingredient content or the fat content. EXAMPLES

[0045]

[0045] The following non-limiting examples are illustrative of embodiments of the present disclosure.

[0046] Example 1 Feline CKD Study

[0046] Twenty-eight cats without renal disease (healthy controls), 21 cats with IRIS stage 1 or stage 2 chronic kidney disease (CKD), and 9 cats with CKD stage 3 or stage 4 were enrolled in the study. The cats were diagnosed and staged according to the IRIS guidelines (http: / / www.iris-kidney.com / guidelines / )A).

[0047] Dysbiosis Index (DI)

[0047] Fecal DNA samples from three groups of cats with and without CKD were assessed for the abundance of 10 bacterial groups, including total bacteria, Faecalibacterium, Turicibacter, Escherichia coli, Streptococcus, Blautia, Fusobacterium, Clostridium hyranonis, Bifidobacterium, and Bacteroides. qPCR primer sets, protocols, and dysbiosis index (DI) were as previously reported by Sung et al. Journal of Feline Medicine and Surgery, March 2022. A DI < 0.5 indicates normobiosis, whereas a DI ≥ 0.5 indicates dysbiosis.

[0048] Metagenomics sequencing

[0048] The Illumina DNA library prep kit utilizes a bead-based transposome complex to tag genomic DNA. Tagmentation is a one-step process that fragments DNA and then tags it with adapter sequences. After saturation with input DNA, the bead-based transposome complex fragments a set number of DNA molecules. This fragmentation provides the flexibility to use a wide range of DNA inputs to generate normalized libraries with consistent fragment sizes. After tagmentation, limited-cycle PCR adds Illumina DNA Prep-specific index adapter sequences to the ends of the DNA fragments. A sample purification bead (SPB) cleanup step then purifies the library for use on an Illumina sequencer. The double-stranded DNA library is denatured prior to hybridization of a biotin probe oligonucleotide pool. A total of 100ng of DNA was used per sample, following the user guide without any modifications: Illumina DNA Prep Reference Guide (Illumina DNA Prep Reference Guide (1000000025416)). Quality control and quantification for pooling was based on quantification with Qubit Hs dsDNA kit and quality check of small samples on TapeStation using HSD5000 kit.

[0049] Two PE-150 sequencing runs were performed on the NextSeq2000 using P3-300 cycles chemistry. The first run was loaded at 750 pM and the second run at 650 pM with 2% Phix added. The sequencing runs were demultiplexed on the NextSeq2000 with DRAGEN.

[0050] Bioinformatics and statistical analysis

[0050] The sequencing data from the two runs were combined and the paired-end sequences were merged using the software PEAR (https: / / cme.hits.org / exelixis / web / software / pear / ) Paired-End read merger. The software Trimmomatic (http: / / www.usadellab.org / cms / ?page=trimmomatic), a flexible read trimming tool for Illumina sequencing data, was used to trim low-quality sequences. The software KneadData (https: / / huttenhower.sph.harvard.edu / kneaddata / ) was used to separate and remove host genome sequences from microbial genome DNA sequences. Microbial profiling was performed using the metagenomic phylogenetic analysis MetaPhlAn2 (https: / / huttenhower.sph.harvard.edu / metaphlan2 / #:~:text=metaphLaN%202.0,from%20metagenomic%20shotgun%20sequencing%20data.) The nonparametric Kruskal–Wallis test was used to compare microbial composition data among the three groups of cats: non-CKD, CKD1 / 2, and CKD3 / 4, and the mean for each group was calculated.

[0051] result

[0051] Table 1 shows the total bacteria and bacterial genera / species that differed in abundance between the non-CKD group, the CKD stage 1 or stage 2 group, and the CKD stage 3 or stage 4 group, along with the dysbiosis index for each category.

[0052] [Table 1]

[0053]

[0052] Table 2 shows the bacterial genera that differed in abundance between the non-CKD group, the CKD stage 1 or stage 2 group, and the CKD stage 3 or stage 4 group.

[0054] [Table 2]

[0055]

[0053] Table 3 shows the diagnostic ranges for distinguishing between normal, early CKD, and late CKD disease states in cats based on the genera in Table 2.

[0056] [Table 3]

[0057]

[0054] Table 4 shows the bacterial genus species that differ in abundance between the non-CKD group, the CKD stage 1 or stage 2 group, and the CKD stage 3 or stage 4 group.

[0058] [Table 4]

[0059]

[0055] Table 5 shows the diagnostic ranges for distinguishing between normal, early CKD, and late CKD disease states in cats based on the genus and species in Table 4.

[0060] [Table 5]

[0061]

[0056] It should be understood that various changes and modifications to the present preferred embodiment described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

Claims

1. 1. A method for diagnosing chronic kidney disease in a feline, comprising: measuring the absolute abundance of fecal bacteria including Faecalibacterium, Turicibacter, Streptococcus, Bifidobacterium, Bacteroides, E. coli, and C. hiranonis; calculating a dysbiosis index based on said fecal bacteria; determining that the feline has chronic kidney disease if the dysbiosis index is 0.5 or greater; A method comprising:

2. 2. The method of claim 1, wherein the fecal bacteria comprises Blautia.

3. 2. The method of claim 1, wherein the fecal bacteria comprises Fusobacterium.

4. 2. The method of claim 1, further comprising determining that the feline has early stage chronic renal disease when the dysbiosis index is between 0.5 and 1.

2.

5. 2. The method of claim 1, further comprising determining that the feline has late stage chronic renal disease when the dysbiosis index is greater than 1.

2.

6. 1. A method for diagnosing early stage chronic kidney disease in a feline comprising: Lactobacillus animalis, Subdoligranulum variabile, Catenibacterium mitsuokai, Collinsella aerofaciens, Ruminococcus obeum, Eubacterium biforme, Lactobacillus reuteri, Bifidobacterium pseudocatenulatum, pseudocatenulatum, Coprococcus comes, Megasphaera elsdenii, Lachnospiraceae bacterium_1_1_57FAA, Faecalibacterium prausnitzii, Eubacterium hallii, Ruminococceae bacterium_D16, Dorea longicatena, Clostridium hiranonis measuring the absolute abundance of biomarkers selected from the group consisting of: Saccharomyces hiranonis, Acidaminococcus fermentans, Saccharomyces cerevisiae, Streptococcus parauberis, Acidaminococcus intestini, Helicobacter canis, Bacteroides coprocola, and combinations thereof; if the absolute abundance of the biomarker is within 【Table 1】 determining that the feline has early stage chronic kidney disease; A method comprising:

7. The method of claim 6, wherein the determining step is based on at least two biomarkers.

8. The method of claim 6, wherein the determining step is based on at least three biomarkers.

9. 7. The method of claim 6, wherein the determining step is based on at least four biomarkers.

10. 1. A method for diagnosing early stage chronic kidney disease in a feline comprising: determining the absolute abundance of bacteria in genera selected from the group consisting of Catenibacterium, Lactobacillus, Coprococcus, Megasphaera, Helicobacter, Eubacterium, Faecalibacterium, Acidaminococcus, Bifidobacterium, Subdoligranulum, Allobaculum, Escherichia, Enterococcus, and combinations thereof; if the absolute abundance of the biomarker is within 【Table 2】 determining that the feline has early stage chronic kidney disease; A method comprising:

11. The method of claim 10 , wherein the determining step is based on at least two genera.

12. The method of claim 10 , wherein the determining step is based on at least three genera.

13. The method of claim 10 , wherein the determining step is based on at least four genera.

14. 11. A method for treating or slowing the progression of chronic kidney disease (CKD) in a feline, comprising the steps of diagnosing CKD in the feline according to claim 1 and recommending a composition for the feline, wherein the composition treats or slows the progression of CKD in the feline.

15. 13. A method for treating or delaying the progression of early chronic kidney disease (CKD) in a feline, comprising the steps of diagnosing early CKD in the feline according to claim 6 or claim 10 and recommending a composition for the feline, wherein the composition treats or delays the progression of early CKD in the feline.

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