Microbial biomarkers of canine idiopathic epilepsy

Abstract

A method of diagnosing canine idiopathic epilepsy is provided, where the method comprises measuring the relative abundance of bacteria in a canine gut microbiome. A decrease or increase in the relative abundance of identified bacteria when compared to a reference sample can indicate the presence of idiopathic epilepsy in a canine. Methods of measuring the gut microbiota as well as methods of treating idiopathic epilepsy are also provided.

Description

[0001] Attorney Docket: 069269.0771

[0002] MICROBIAL BIOMARKERS OF CANINE IDIOPATHIC EPILEPSY

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 718,333, filed November 8, 2024, the contents of which are herein incorporated by reference in its entirety.

[0005] 1. FIELD

[0006] The presently disclosed subject matter relates to methods of diagnosing canine idiopathic epilepsy through microbial biomarkers.

[0007] 2. BACKGROUND

[0008] Epilepsy is a commonly diagnosed neurological condition in canines, with an estimated prevalence of 0.6-0.82% of canines (Heske, L., et al., Vet J, 2014. 202(3): p. 471- 6.; Kearsley-Fleet, L., et al., Vet Rec, 2013. 172(13): p. 338.; Erlen, A., et al., J Vet Intern Med, 2018. 32(5): p. 1665-1676.). This condition can be classified as idiopathic epilepsy (genetic), structural epilepsy (structural issues in the brain), or unknown epilepsy (unknown cause) (www.akcchf.org / canine-health / top-health-concems / epilepsy / AKCCHF-

[0009] Understanding-Canine-Epilepsy-2017.pdf). The treatment for epilepsy will generally include an anti-seizure medication such a phenobarbital or levetiracetam.

[0010] The gastrointestinal tract is a complex ecosystem known as the gut microbiome. In recent years interest it has been found that the gut microbiome has an effect on the nervous system through multiple mechanisms (Fung, T.C., et al., NatNeurosci, 2017. 20(2): p. 145- 155). Thus, the gut microbiome has an important role in the development and function of the central nervous system (Diaz Heijtz, R., et al., Proc Natl Acad Sci U S A, 2011. 108(7): p. 3047-52.; Ogbonnaya, E.S., et al., Biol Psychiatry, 2015. 78(4): p. e7-9.). Often, antiseizure medications are not able to control seizures in epileptic patients. In these cases, the drugs are used in conjunction with diet, such as the ketogenic diet. Studies in recent years have found that the ketogenic diet is effective in humans not because of the diet, but due to the gut microbiome of epilepsy patients (Zhang, Y., et al., Epilepsy Res, 2018. 145: p. 163- 168.; Xie, G., et al., World J Gastroenterol, 2017. 23(33): p. 6164-6171.; Olson, C.A., et al., Cell, 2018. 173(7): p. 1728-1741 ,el3.).

[0011] The gut microbiome of canines with idiopathic epilepsy has yet to be explored. There Attorney Docket: 069269.0771 remains a need in the art for an understanding of the microbial biomarkers of canines with idiopathic epilepsy, as this could lead to diagnosis of the condition and further treatment.

[0012] 3. SUMMARY

[0013] The purpose and advantages of the disclosed subject matter will be set forth in and are apparent from the description that follows, as well as will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the devices particularly pointed out in the written description and claims hereof, as well as from the appended drawings.

[0014] To achieve these and other advantages and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter.

[0015] It is to be understood that both the foregoing general description and the following detailed description and drawings are examples and are provided for purpose of illustration and not intended to limit the scope of the disclosed subject matter in any manner.

[0016] The accompanying drawings, which are incorporated in and constitute part of this specification, are included to illustrate and provide a further understanding of the devices of the disclosed subject matter. Together with the description, the drawings serve to explain the principles of the disclosed subject matter.

[0017] The presently disclosed subject matter provides methods of diagnosing canine idiopathic epilepsy comprising measuring the relative abundance of bacteria, wherein a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a test sample when compared to a reference sample indicates the presence of idiopathic epilepsy. In certain embodiments, an increase in the relative abundance of at least one of Streptococcus tuleliensis. Faecalimonas umbihcala. Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345. Escherichia coH. Clostridium saudiense. Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Phocaeicola sp900546645, Prevotellamassilia sp000437675, Faecalimonas sp900551895, or Fournierella sp002160145 in a test sample when compared to a reference sample further indicates the presence of idiopathic epilepsy. In certain embodiments, the decrease in the relative abundance is at least about 5%. In certain embodiments, the increase in the relative abundance is at least about 0.1%.

[0018] In certain embodiments, the method further comprises measuring the activity of at least one functional metabolic pathway. In certain embodiments, the activity of the at least Attorney Docket: 069269.0771 one functional metabolic pathway in the test sample is enriched. In certain embodiments, the at least one functional metabolic pathway is selected from the group consisting of C5 isoprenoid biosynthesis, tryptophan biosynthesis, lipopolysaccharide biosynthesis, ascorbate biosynthesis, thiamine biosynthesis, EvgS-EvgA 2C-RS, citrate cycle, NAD(P)H: quinone oxidoreductase, SasA-RpaAB 2C-RS, riboflavin biosynthesis, cytochrome d ubiquinol oxidase, FixL-FixJ 2C-RS, PTS system, putative ABC transport system, PrrB-PrrA 2C-RS, and combinations thereof. In certain embodiments, the activity of at least one functional metabolic pathway in the test sample is decreased. In certain embodiments, the at least one functional metabolic pathway selected from the group consisting of sulfonate transport system (TS), osmoprotectant TS, dipeptide TS, glutathione biosynthesis, triacylglycerol biosynthesis, gamma-aminobutyrate (GABA) shunt, putative multiple sugar TS, pyruvate oxidation, PTS system, glutathione TS, d-methionine TS, putative sn-glycerol-phosphate TS, pentose phosphate pathway, maltose / maltodextrin TS, VicK-VicR 2C-RS, putative arabinogalactan oligomer TS, multiple sugar TS, methylgalactoside TS, phosphate TS, putative fructooligosaccharide TS, oligopeptide TS, and combinations thereof.

[0019] In certain embodiments, the test sample and the reference sample are a fecal sample. In certain embodiments, the test sample and the reference sample are sequenced.

[0020] The presently disclosed subject matter further provides methods of treating canine idiopathic epilepsy comprising: a) measuring the relative abundance of bacteria, wherein a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a sample when compared to a reference sample indicates the presence of idiopathic epilepsy; and b) treating idiopathic epilepsy when the presence of idiopathic epilepsy is indicated. In certain embodiments, an increase in the relative abundance of at least one of Streptococcus tuleliensis. Faecalimonas umbihcala. Fusobacterium B sp900554885, Bifidobacterium globosum, BlautiaA sp900541345. Escherichia coH. Clostridium saudiense. Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Phocaeicola sp900546645, Prevotellamassilia sp000437675, Faecalimonas sp900551895, or Fournier ella sp002160145, in a test sample when compared to a reference sample further indicates the presence of idiopathic epilepsy. In certain embodiments, the decrease in the relative abundance is at least about 5%. In certain embodiments, the increase in the relative abundance is at least about 0.1%.

[0021] In certain embodiments, the method further comprises measuring the activity of at least one functional metabolic pathway. In certain embodiments, the activity of at least one Attorney Docket: 069269.0771 functional metabolic pathway in the test sample is enriched. In certain embodiments, the activity of at least one functional metabolic pathway in the test sample is decreased. In certain embodiments, the treatment is selected from the group consisting of an anti-seizure medication, a diet, and combinations thereof. In certain embodiments, the diet is a ketogenic diet. In certain embodiments, the anti-seizure medication is selected from the group consisting of phenobarbital, levetiracetam, potassium bromide, zonisamide, gabapentin, and combinations thereof.

[0022] The presently disclosed subject matter further provides methods of treating idiopathic epilepsy in a canine in need thereof, wherein the method comprises a treatment selected from the group consisting of an anti-seizure medication, a diet, and combinations thereof, wherein the canine has a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a fecal sample when compared to a reference fecal sample, and wherein the canine has an increase in the relative abundance of at least one of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Faecalimonas sp900551895, Phocaeicola sp900546645, Prevotellamassilia sp000437675, or Fournierella sp002160145 in a fecal sample when compared to a reference fecal sample, wherein the method reduces frequency or severity of symptoms associated with idiopathic epilepsy.

[0023] The presently disclosed subject matter further provides methods of diagnosing canine idiopathic epilepsy comprising measuring the relative abundance of bacteria, wherein a decrease in the relative abundance of Prevotella copri o Megamonas funiformis in a test sample when compared to a reference sample and an increase in the relative abundance of bacteria selected from the group consisting of Streptococcus lutetiensis, Escherichia coli, and Clostridium saudiense in a test sample when compared to a reference sample indicates the presence of idiopathic epilepsy.

[0024] In certain embodiments, the decrease in the relative abundance of Prevotella copri ox Megamonas funiformis in a test sample when compared to a reference sample is at least about 5%. In certain embodiments, the decrease in relative abundance of Prevotella copri is from about 5% to about 25%. In certain embodiments, the decrease in relative abundance of Megamonas funiformis is from about 5% to about 25%.

[0025] In certain embodiments, the increase the relative abundance of bacteria selected from the group consisting of Streptococcus lutetiensis, Escherichia coli, and Clostridium Attorney Docket: 069269.0771 saudiense in a test sample when compared to a reference sample is at least about 0.1%.

[0026] In certain embodiments, the method further comprises measuring the activity of at least one functional metabolic pathway. In certain embodiments, the activity of at least one functional metabolic pathway is enriched. In certain embodiments, the at least one functional metabolic pathway is selected from the group consisting of C5 isoprenoid biosynthesis, tryptophan biosynthesis, lipopolysaccharide biosynthesis, ascorbate biosynthesis, thiamine biosynthesis, EvgS-EvgA 2C-RS, citrate cycle, NAD(P)H: quinone oxidoreductase, SasA-RpaAB 2C-RS, riboflavin biosynthesis, cytochrome d ubiquinol oxidase, FixL-FixJ 2C-RS, PTS system, putative ABC transport system, PrrB-PrrA 2C- RS, and combinations thereof.

[0027] In certain embodiments, the activity of at least one functional metabolic pathway is decreased. In certain embodiments, the at least one functional metabolic pathway is selected from the group consisting of sulfonate transport system (TS), osmoprotectant TS, dipeptide TS, glutathione biosynthesis, triacylglycerol biosynthesis, gamma-aminobutyrate (GABA) shunt, putative multiple sugar TS, pyruvate oxidation, PTS system, glutathione TS, d- methionine TS, putative sn-glycerol-phosphate TS, pentose phosphate pathway, maltose / maltodextrin TS, VicK-VicR 2C-RS, putative arabinogalactan oligomer TS, multiple sugar TS, methyl-galactoside TS, phosphate TS, putative fructooligosaccharide TS, oligopeptide TS, and combinations thereof.

[0028] In certain embodiments, the test sample and reference sample are from canines. In certain embodiments, the reference sample is from a healthy canine or a group of healthy canines. In certain embodiments, the test sample and the reference sample is a fecal sample. In certain embodiments, the test sample and the reference sample are sequenced.

[0029] 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The subject matter of the application will be more readily understood from the following detailed description when read in conjunction with the accompanying drawings, in which:

[0031] FIG. 1 illustrates the alpha diversity indices of the gastrointestinal microbiome of control dogs and idiopathic epilepsy dogs;

[0032] FIGS. 2A and 2B provide the analysis of the phylum in the gut microbiota of the control group (FIG. 2A) and the idiopathic epilepsy group (FIG. 2B);

[0033] FIGS. 3A and 3B provide the analysis of the species in the gut microbiota of the Attorney Docket: 069269.0771 control group (FIG. 3 A) and the idiopathic epilepsy group (FIG. 3B);

[0034] FIG. 4 demonstrates the differentially abundant KEGG pathway modules (p<0.01 and LDA score threshold of 2.5) in the control and idiopathic epileptic groups. ‘TS’ - transport system and ‘2C-RS’ - two-component regulatory system; and

[0035] FIG. 5 demonstrates taxonomy differences between healthy and epileptic groups.

[0036] 5. DETAILED DESCRIPTION

[0037] The methods of the present disclosure can be used to determine the presence of canine idiopathic epilepsy. The microbiome of canines with idiopathic epilepsy, methods of measuring the microbiome, and methods of treating idiopathic epilepsy are described herein. These and other aspects of the disclosed subject matter are discussed in more detail below.

[0038] For clarity and not by way of limitation, this detailed description is divided into the following sub-portions:

[0039] 5.1 Definitions;

[0040] 5.2 The Canid;

[0041] 5.3 Microbiome of Canines with Idiopathic Epilepsy;

[0042] 5.4 Methods of Measuring Canine Microbiome; and

[0043] 5.5 Treatment of Idiopathic Epilepsy.

[0044] 5.1. Definitions

[0045] The terms used in this specification generally have their ordinary meanings in the art, within the context of this subject matter and in the specific context where each term is used. Certain terms are defined below to provide additional guidance in describing the compositions and methods of the disclosed subject matter and how to make and use them.

[0046] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of compounds.

[0047] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within three or more than three standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Also, particularly with respect to systems or processes, the term can mean within an order of Attorney Docket: 069269.0771 magnitude, preferably within five-fold, and more preferably within two-fold, of a value.

[0048] As used herein, the terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0049] As used herein, the terms “reduce” or “decrease” refer to a measurable lessening of an end-point (e.g., bacterial taxa, activity of a metabolic pathway) by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the reduction can be from about 0.1% to about 100%.

[0050] As used herein, the term “increase” refers to a measurable augmentation of an endpoint (e.g., bacterial taxa, activity of a metabolic pathway) by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the reduction can be from about 0.1% to about 100%. In certain embodiments, the increase can be at least about 10- fold, about 100-fold, or about 1000-fold or more. In certain embodiments, the increase can be about 100-fold or more, about 1000-fold or more, or about 10,000-fold or more.

[0051] In the detailed description herein, references to “embodiment,” “an embodiment,” “one embodiment,” “in various embodiments,” etc., indicate that the embodiment s) described can include a particular feature, structure, or characteristic, but every embodiment might not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. After reading the description, it will be apparent to one skilled in the relevant art(s) how to implement the disclosure in alternative embodiments.

[0052] 5.2. The Canid

[0053] The methods of the present disclosure can be used to determine the microbiome health of a canid. This genus comprises domestic dogs (Canis lupus familiaris), wolves, coyotes, foxes, jackals, dingoes and the present disclosure can be used for all these animals. In some embodiments, the subject is a domestic dog, herein referred to simply as a dog. Attorney Docket: 069269.0771

[0054] One advantage of the methods of the present disclosure is that they allow a skilled person to determine whether the canid's microbiome is healthy, taking into account the canid's life stage.

[0055] There are numerous different breeds of domestic dogs, which show a diverse habitus. Different breeds also have different life expectancies with smaller dogs generally being expected to live longer than bigger breeds. Accordingly, different breeds are considered to be puppies, adult, senior or geriatric at different time points in their life. A summary of the different life stages is provided in Table 1, below.

[0056] Table 1

[0057] The distinction between toy, small, medium and large breeds is known in the art. In particular, toy breeds comprise distinct breeds including but not limited to Affenpinscher, Australian Silky Terrier, Bichon Frise, Bolognese, Cavalier King Charles Spaniel, Chihuahua, Chinese Crested, Coton De Tulear, English Toy Terrier, Griffon Bruxellois, Havanese, Italian Greyhound, Japanese Chin, King Charles Spaniel, Lowchen (Little Lion Dog), Maltese, Miniature Pinscher, Papillon, Pekingese, Pomeranian, Pug, Russian Toy and Yorkshire Terrier.

[0058] Small breeds are larger on average than toy breeds with an average body weight of up to about 10 kg. Non-limiting exemplary breeds include French Bulldog, Beagle, Dachshund, Pembroke Welsh Corgi, Miniature Schnautzer, Cavalier King Charles Spaniel, Shih Tzu, and Boston Terrier.

[0059] Medium dog breeds have an average weight of about 11 kg to about 26 kg. These dog breeds include, but are not limited to, Bulldog, Cocker Spaniel, Shetland Sheepdog, Border Collie, Basset Hound, Siberian Husky and Dalmatian.

[0060] Large breed are those with an average body weight of at least about 27 kg. Nonlimiting examples include Great Dane, Neapolitan mastiff, Scottish Deerhound, Dogue de Bordeaux, Newfoundland, English mastiff, Saint Bernard, Leonberger and Irish Wolfhound. Attorney Docket: 069269.0771

[0061] Cross-breeds can generally be categorized into toy, small, medium and large dogs depending on their body weight.

[0062] 5.3. Gut Microbiome of Canines with Idiopathic Epilepsy

[0063] The methods of the present disclosure can be used to detect and diagnose the presence of idiopathic epilepsy in canines through analysis of samples from canines. In particular embodiments, the alpha diversity and richness, the relative abundance of microbial bacteria, and the activity of the functional metabolic pathways of canines are used to detect idiopathic epilepsy.

[0064] 5,3.1. Alpha Diversity and Richness

[0065] The term “alpha diversity” is a metric which describes the diversity of the microbiome in the gut in a single sample while the term “beta diversity” compares the diversity of the microbiome in the gut between multiple samples (www. crownl aboratories . com / news / understanding-microbiome-diversity-i s-it-the-secret- to-better-health / ). An increased alpha diversity is typically considered to indicate health (Wilmanski, T., et al., Gut Microbes, 2021. 13(1): p. 1-20.).

[0066] The alpha diversity and richness of canines with idiopathic epilepsy is increased when compared to a reference sample collected from a healthy canine or group of healthy canines. In particular embodiments, the increase in alpha diversity and richness in canines with idiopathic epilepsy is independent of age, breed, diet, home-environment, and / or geographical location of the canine. The alpha diversity and richness in canines with idiopathic epilepsy does not depend on whether the canine has been administered antiseizure medication.

[0067] 5,3.2. Bacteria

[0068] The methods of the present disclosure can be used to determine the presence of idiopathic epilepsy of a canine. This can be achieved by quantitating bacterial species in a sample obtained from the canine to determine their relative abundance. As used herein, “relative abundance” refers to the abundance of the same species in a test sample from a canine when compared to a reference sample. As used herein, a “reference sample” refers to a sample collected from a healthy canine or a group of healthy canines. Healthy bacteria in the gut microbiome, including healthy ranges of the bacteria, is known in the art, as disclosed in U.S. Publication No. 2022 / 0119864, which is incorporated herein by reference.

[0069] In certain embodiments, the relative abundance of the bacteria species corresponds to the abundance or relative abundance of the one or more bacteria species in one or more Attorney Docket: 069269.0771 healthy canine. In certain embodiments, the relative abundance of the one or more bacteria species corresponds to the abundance or relative abundance of the one or more bacteria species in one or more canine with idiopathic epilepsy when compared to healthy canines or a group of healthy canines.

[0070] The increase or decrease in the relative abundance of the particular identified bacteria species indicates a canine has idiopathic epilepsy. These bacteria species include Prevotella copri, Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Faecalimonas sp900551895, Fournierella sp002160145, Megamonas funiformis, Holdemanella sp002299315, Bifidobacterium gallinarum, Bacteroides stercoris, Phocaeicola coprocola, Phocaeicola plebeius, Phocaeicola vulgatus, Ruminococcus B gnavus, Faecalibaterium sp900540455. Phocaeicola sp900546645, Prevotellamassilia sp000437675, and CAG-279. In alternative embodiments, these bacteria species include Prevotella copri, Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Faecalimonas sp900551895, Fournierella sp002160145, Megamonas funiformis, Holdemanella sp002299315, Bifidobacterium gallinarum, and CAG-279.

[0071] In particular embodiments, the test samples is collected from a canine with idiopathic epilepsy and a reference sample is collected a healthy canine or group of healthy canines.

[0072] In certain embodiments, microbial biomarkers in a test sample indicate the presence of idiopathic epilepsy. In particular embodiments, an increase in at least one microbial biomarker in the test sample when compared to a reference sample indicates the present of idiopathic epilepsy. In particular embodiments, a decrease in at least one microbial biomarker in a test sample when compared to a reference sample indicates the present of idiopathic epilepsy.

[0073] In particular embodiments, a decrease in the relative abundance of the Prevotella genus in a test sample when compared to the reference sample indicates the presence of idiopathic epilepsy. In certain embodiments, a decrease in the relative abundance of the bacteria Prevotella copri in a test sample when compared to the reference sample indicates the presence of idiopathic epilepsy. Attorney Docket: 069269.0771

[0074] In particular embodiments, a decrease in the relative abundance of the Megamonas genus in a test sample when compared to the reference sample indicates the presence of idiopathic epilepsy. In certain embodiments, a decrease in the relative abundance of the bacteria Megamonas funiformis in a test sample when compared to the reference sample indicates the presence of idiopathic epilepsy.

[0075] In certain embodiments, the decrease in the relative abundance of Prevotella copri is at least about 5%, at least about 10%, or at least about 12%. In particular embodiments, the decrease in relative abundance of Prevotella copri is from about 5% to about 25%, about 10% to about 20%, or about 12% to about 16%.

[0076] In certain embodiments, the decrease in the relative abundance of Megamonas funiformis is at least about 5%, at least about 10%, or at least about 12%. In particular embodiments, the decrease in relative abundance of Megamonas funiformis is from about 5% to about 25%, about 10% to about 20%, or about 12% to about 16%.

[0077] In particular embodiments, an increase in the relative abundance of at least one genus of Streptococous, Bacleroides. CAG-110, Faecalimonas, Fusobactermium, Bifidobaterium, Blautia, Escherichia, Clostridium, Negativibacillus, Anaerostipes, Phocaeicola, or Fournierella in a test sample when compared to the reference sample of bacteria indicates the presence of idiopathic epilepsy. In certain embodiments, an increase in the relative abundance of at least one species of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Phocaeicola sp900546645, Prevotellamassilia sp000437675, Faecalimonas sp900551895, or Fournierella sp002160145, in a test sample when compared to the reference sample indicates the presence of idiopathic epilepsy. In particular embodiments, an increase in the relative abundance of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 of the bacteria Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Phocaeicola sp900546645, Prevotellamassilia when compared to a reference sample indicates the presence of idiopathic epilepsy. In certain embodiments, an increase in the relative abundance of bacteria selected from the Attorney Docket: 069269.0771 group consisting of Streptococcus lutetiensis, Escherichia coli, Clostridium saudiense, and combinations thereof in a test sample when compared to the reference sample indicates the presence of idiopathic epilepsy.

[0078] In certain embodiments, the increase in the relative abundance of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Phocaeicola sp900546645, Prevotellamassilia sp000437675, Faecalimonas sp900551895, or Fournierella sp002160145 is at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 1%, or at least about 1.5%.

[0079] In particular embodiments, a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a test sample when compared to a reference sample and an increase in the relative abundance of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Phocaeicola sp900546645, Prevotellamassilia sp000437675, Faecalimonas sp900551895, o Fournierella sp002160145 m Mos s xxxp e when compared to a reference sample indicates the presence of idiopathic epilepsy. In particular embodiments, a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a test sample when compared to a reference sample and an increase in the relative abundance of at least one of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Faecalimonas sp900551895, Phocaeicola sp900546645, Prevotellamassilia sp000437675, ox Fournierella sp002160145 in a test sample when compared to a reference sample indicates the presence of idiopathic epilepsy. In certain embodiments, a decrease in the relative abundance of Prevotella copri ox Megamonas funiformis in a test sample when compared to a reference sample and an increase in the relative abundance of bacteria selected from the group consisting of Streptococcus lutetiensis, Escherichia coli, and Clostridium saudiense in a test sample when compared to a reference sample indicates the presence of idiopathic epilepsy. Attorney Docket: 069269.0771

[0080] 5,3.3. Functional Metabolic Pathways

[0081] The gut microbiome of canines with idiopathic epilepsy can exhibit different abundance or activity of functional metabolic pathways when compared to the gut microbiome of a healthy canine, with specific pathways increased or decreased in their relative abundance or activity when compared to healthy canines. These pathways, and the genes therein, can act as biomarkers of idiopathic epilepsy.

[0082] In particular embodiments, the activity of at least one metabolic pathway is increased in canines with idiopathic epilepsy. In alternative embodiments, at activity of at least one metabolic pathway is enriched in canines with idiopathic epilepsy. In particular embodiments, the at least one functional metabolic pathway selected from the group consisting of C5 isoprenoid biosynthesis, tryptophan biosynthesis, lipopolysaccharide biosynthesis, ascorbate biosynthesis, thiamine biosynthesis, EvgS-EvgA two-component regulatory system (2C-RS), citrate cycle, NAD(P)H: quinone oxidoreductase, SasA-RpaAB 2C-RS, riboflavin biosynthesis, cytochrome d ubiquinol oxidase, FixL-FixJ 2C-RS, PTS system, putative ABC transport system, PrrB-PrrA 2C-RS, and combinations thereof.

[0083] In particular embodiments, the activity at least one functional metabolic pathway is decreased in canines with idiopathic epilepsy. Alternatively, the metabolites produced from the at least one functional metabolic pathway are depleted. In certain of these embodiments, the at least one functional metabolic pathway is selected from the group consisting of sulfonate transport system (TS), osmoprotectant TS, dipeptide TS, glutathione biosynthesis, triacylglycerol biosynthesis, gamma-aminobutyrate (GABA) shunt, putative multiple sugar TS, pyruvate oxidation, PTS system, glutathione TS, d-methionine TS, putative sn-glycerol- phosphate TS, pentose phosphate pathway, maltose / maltodextrin TS, VicK-VicR 2C-RS, putative arabinogalactan oligomer TS, multiple sugar TS, methyl-galactoside TS, phosphate TS, putative fructooligosaccharide TS, oligopeptide TS, and combinations thereof. In further embodiments, the GABA shunt pathway activity is decreased in canines with idiopathic epilepsy. In certain embodiments, the activity of the glutathione biosynthesis, glutathione TS, and GABA shunt pathways are decreased in the test sample when compared to a reference sample collected from a healthy canine or a group of healthy canines.

[0084] 5.4. Methods of Measuring Canine Gut Microbiota

[0085] The gut microbiota of canines can be measured using a sample collected from a canine. The sample can be analyzed to measure the relative abundance of bacteria and Attorney Docket: 069269.0771 activity of functional metabolic pathways. These can be used to diagnose idiopathic epilepsy.

[0086] 5,4.1. The Test Sample and Reference Sample

[0087] The test sample is a sample taken from canine that will be tested for the presence of idiopathic epilepsy. The test sample can be a fecal sample. The test sample of the canine is compared to a reference sample to determine the presence of idiopathic epilepsy, as described above in 5.3.

[0088] The reference sample can be a sample from a healthy canine or a group of healthy canines. In particular embodiments, the reference sample is a sample from one healthy canine. In alternative embodiments, the reference sample is a sample from a group of healthy canines. The reference sample can be a fecal sample.

[0089] 5,4.2. Measurement Methods

[0090] The test sample and reference sample can be sequenced using methods known in the art. For example, shotgun metagenomic sequencing is conducted using an Illumina NovaSeq 6000, with a mean read depth of 5 million paired end reads, with read length of 150 base pairs.

[0091] The sequencing reads are then aligned to a canine specific database for taxonomic and functional classification. The relative abundance of each bacterial taxa or relative activity functional metabolic pathway is established and statistical analysis is conducted to establish bacterial groups that are differentially abundant, either increased or decreased, within the gut microbiome of canines with idiopathic epilepsy when compared to reference samples.

[0092] In certain embodiments, the relative abundance of the bacterial taxa or relative activity functional metabolic pathways corresponds to the abundance or relative abundance of the one or more bacterial taxa or relative activity of the one or more functional metabolic pathways in one or more healthy canine. In certain embodiments, the relative abundance of the one or more bacterial taxa or relative activity of the one or more functional metabolic pathways corresponds to the abundance or relative abundance of the one or more bacterial taxa or relative activity of the one or more functional metabolic pathways in one or more canines with idiopathic epilepsy when compared to healthy canines or a group of healthy canines.

[0093] The relative abundance of bacteria, the relative activity of functional metabolic pathways, and combinations thereof described above in section 5.3. can be used individually or together to indicate the presence of idiopathic epilepsy. In particular embodiments, the Attorney Docket: 069269.0771 relative abundance of bacterial taxa in a test sample when compared to a reference sample collected from a healthy canine or group of healthy canines can be used to indicate the presence of idiopathic epilepsy. In certain embodiments, the relative activity of functional metabolic pathways in a test sample when compared to a reference sample collected from a healthy canine or group of healthy canines can be used to indicate the presence of idiopathic epilepsy. In alternative embodiments, the relative abundance of bacterial taxa and relative activity functional metabolic pathways in a test sample when compared to a reference sample collected from a healthy canine or group of healthy canines can be used together to indicate the presence of idiopathic epilepsy.

[0094] The indication of the presence of idiopathic epilepsy can result in a diagnosis of said condition.

[0095] 5.5. Treatment of Idiopathic Epilepsy

[0096] Using the above methods, a canine can be diagnosed with idiopathic epilepsy. After diagnosis, a veterinarian can prescribe treatments or medications known in the art. In particular embodiments, the treatment or medication is an anti-seizure medication, a diet, and combinations thereof. In certain embodiments, the medication is one or more antiseizure medications. In particular embodiments, treatments include a specific diet such as the ketogenic diet. The gut microbiome of a canine with idiopathic epilepsy is not affected by the anti-seizure medications.

[0097] In certain embodiments, the anti-seizure medication is selected from the group consisting of phenobarbital, levetiracetam, potassium bromide, zonisamide, gabapentin, and combinations thereof. In certain embodiments, the canine is provided phenobarbital. In particular embodiments, the canine is provided levetiracetam. In certain embodiments, the canine is provided potassium bromide. In particular embodiments, the canine is provided phenobarbital and levetiracetam. In particular embodiments, the canine is provided phenobarbital and zonisamide. In particular embodiments, the canine is provided levetiracetam and zonisamide. In particular embodiments, the canine is provided levetiracetam and gabapentin. In particular embodiments, the canine is provided potassium bromide and levetiracetam. In particular embodiments, the canine is provided phenobarbital, levetiracetam, and zonisamide. In particular embodiments, the canine is provided phenobarbital, levetiracetam, and potassium bromide. In particular embodiments, the canine is provided phenobarbital, levetiracetam, potassium bromide, and zonisamide. In particular embodiments, the canine is provided phenobarbital, levetiracetam, potassium bromide, Attorney Docket: 069269.0771 zonisamide, and gabapentin.

[0098] The presently disclosed subject matter provides a composition for use in the treatment of canine idiopathic epilepsy. In certain embodiments, the composition comprises an anti-seizure medication, a diet, and combinations thereof.

[0099] The presently disclosed subject matter further provides a composition for the manufacture of a medicament for use in the treatment of canine idiopathic epilepsy. In certain embodiments, the composition comprises an anti-seizure medication, a diet, and combinations thereof.

[0100] The presently disclosed interventions of treatment or adjustment in diet result in reducing frequency or severity of symptoms associated with idiopathic epilepsy in canines, including, but not limited to, seizures (generalized seizure or focal seizure).

[0101] The presently disclosed subject matter further provides methods of treating idiopathic epilepsy in a canine in need thereof, wherein the method comprises a treatment selected from the group consisting of an anti-seizure medication, a diet, and combinations thereof, wherein the canine has a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a fecal sample when compared to a reference fecal sample, and wherein the canine has an increase in the relative abundance of at least one of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Faecalimonas sp900551895, Phocaeicola sp900546645, Prevotellamassilia sp000437675, or Fournierella sp002160145 in a fecal sample when compared to a reference fecal sample, wherein the method reduces frequency or severity of symptoms associated with idiopathic epilepsy.

[0102] The presently disclosed subject matter further provides methods for use in a diagnostic, the methods comprising measuring the relative abundance of Prevotella copri or Megamonas funiformis in a test sample in comparison to a reference sample collected from a healthy canine or group of healthy canines. In certain embodiments, the methods are for use in a diagnostic for canine idiopathic epilepsy. In certain embodiments, a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in the test sample in comparison to the reference sample indicates the presence of idiopathic epilepsy. In certain embodiments, the methods further comprise measuring the relative abundance of bacteria selected from the group consisting of Streptococcus lutetiensis, Escherichia coli, and Clostridium saudiense in a test sample when compared to a reference sample. In certain Attorney Docket: 069269.0771 embodiments, an increase in the relative abundance of the bacteria in comparison to the reference sample indicates the presence of idiopathic epilepsy. In certain embodiments, the diagnostic is an in vitro, in vivo, or ex vivo diagnostic.

[0103] 6. EXAMPLES

[0104] The following examples are merely illustrative of the presently disclosed subject matter and they should not be considered as limiting the scope of the subject matter in any way.

[0105] Example 1.

[0106] Fecal Sample Collection.

[0107] Eighty adult dogs were analyzed in this study. Of these dogs, forty have been diagnosed with either Tier I or Tier II idiopathic epilepsy as per the International Veterinary Epilepsy Task Force Consensus (De Risio, L., et al., BMC Vet Res, 2015. 11: p. 148.), and the other 40 were neurologically-healthy (i.e., non-epileptic). Tier I idiopathic epilepsy (IE) is based on a history of two or more unprovoked epileptic seizures occurring at least 24 hours apart with an age at epileptic onset between 6 months and 6 years, unremarkable interictal physical and neurological examination, and no significant abnormalities on minimum data base blood tests and urinalysis. Tier II IE confidence includes the factors listed in Tier I, as well as unremarkable pre- and post-prandial bile acids, magnetic resonance imaging of the brain and cerebrospinal fluid analysis. In addition, pre- and postprandial bile acids were ideally performed as part of screening, but not a mandatory requirement to be diagnosed as Tier II IE. The epileptic dogs were recruited to a referral neurology specialty hospital (BluePearl North Dallas, Texas, USA), whilst the healthy control dogs were recruited from a blood donation program at a separate referral specialty hospital (BluePearl Tampa, Florida, USA). For enrollment within the blood donor program, the healthy cohort underwent a standard a screening for blood-borne pathogens, as per the enrollment into the blood donor program (Wardrop, K.J., et al., J Vet Intern Med, 2016. 30(1): p. 15-35.). Both groups were confirmed to not have a current or persistent history of gastrointestinal disease. The healthy cohort were also confirmed to not have a history of seizures.

[0108] Owners of all the dogs were required to complete a questionnaire to provide demographic data including age, gender, weight, body condition score Attorney Docket: 069269.0771

[0109] (www.royalcanin.co.uk / wp-content / uploads / 2017 / 02 / BCS-chart-03.12.13.pdf), and breed. The questionnaire also included data on the pet’s diet, veterinary history, co-habiting status, fecal score (www. waitham. com / s3media / 2020-05 / waltham-scoring. pdf.) and for the IE group, details pertaining to frequency and severity of idiopathic epilepsy as well as current anti-seizure medications. Dogs who were fed a raw diet (uncooked ingredients from either livestock or wild animals) were not admitted into the study. Dogs who were fed a predominantly non-commercial (home-cooked) diet were also omitted from the study. Dietary recommendations or prescriptions specific to epilepsy were not given to the IE group prior to sample collection. Dogs were excluded if they had been treated with antimicrobials (including antibiotics and antifungals) or probiotics in the six weeks prior to sample collection (Marshall-Jones, Z.V., et al., Sci Rep, 2024. 14(1): p. 5277.).

[0110] There was no significant difference in age or sex (including neuter status) between the control group and the IE group of dogs analyzed. Weight was categorized into groups (<5kg, 5-10kg, 10-25kg, 25-45kg and >45kg). The two groups were very similar with the majority of dogs falling into the 10-25kg range and of good body condition score (mean 5 / 9). This demographic data is summarized in Tables 2 and 3. The dogs in the IE group were receiving a combination of seizure medication. The most common medication was phenobarbital in combination of levetiracetam. These medications are outlined in Table 4.

[0111] Table 2, Demographic Data of the Control and Idiopathic Epilepsy Groups

[0112] Table 3. Weight Demographic of the Participants Attorney Docket: 069269.0771

[0113] Table 4. Anti-seizure medications that dogs were receiving at time of fecal sampling.

[0114] A ‘pea-sized’ amount of fresh feces was collected from each dog, The fecal samples were either freshly passed, or via digital palpation of a veterinarian or trained veterinary nurse. Feces consistency assessments were conducted immediately upon defecation, according to the 17-point Waltham Feces Scoring System (Moxham, G., Waltham focus, 2001. 11(2): p. 24-5.). Samples were placed into PERFORMAbiome-GUT tubes (DNA Genotek) prior to transportation to a laboratory for aliquoting into Lo-Bind Eppendorf tubes (Eppendorf Ltd.) and storage at -80°C within X days of defecation. Samples were then stored in Lo-Bind Eppendorf tubes.

[0115] Fecal samples were extracted at Diversigen (New Brighton, MN, USA) with the PowerSoil Pro DNA isolation kit (Qiagen) automated for high throughput on the QiaCube HT (Qiagen). Mechanical lysis was completed via bead beating using Powerbead Pro plates (Qiagen) which contain 0.5mm and 0.1mm ceramic beads. Genomic DNA was quantified using the Quant-IT™ PicoGreen™ dsDNA Assay kit and reagents (Invitrogen).

[0116] Bioinformatics and Statistical Methods.

[0117] Libraries were prepared with a proprietary procedure adapted from the Nextera XT kit (Illumina) at Diversigen (New Brighton, MN, USA). Sequence analysis was conducted using the Illumina Novaseq platform. DNA sequences were filtered for low quality (Q-Score < 30) and length (< 50), and adapter sequences were trimmed using Cutadapt (Martin, M., Attorney Docket: 069269.0771

[0118] EMBnet.joumal, 2011. 17(1).). Host (canine) sequences were removed using Bowtie 2 (Langmead, B. and S.L. Salzberg, Nat Methods, 2012. 9(4): p. 357-9.).

[0119] Sequencing reads were mapped using fully gapped alignment with BURST (Al- Ghalith, G. and D. Knights, Preprint at bioRxiv, 2020.) at an identity threshold of 97% to Diversigen’s curated database (DivDB-Canine) containing all bacterial representative genomes in RefSeq with additional manually curated strains. Each sequencing read was assigned to the lowest common ancestor that was consistent across at least 80% of all reference sequences tied for best hit. Kyoto Encyclopaedia of Genes and Genomes Orthology groups (KEGG KOs) (Kanehisa, M., et al., Nucleic Acids Res, 2023. 51(D1): p. D587-D592.) were observed directly via alignment at an identity threshold of 97% using fully gapped alignment with BURST (Al-Ghalith, G. and D. Knights, Preprint at bioRxiv, 2020.). Ambiguously mapped reads were excluded from the resulting functional feature table.

[0120] Based on analysis of mock communities ATCC 1003, low abundant taxa <0.01% were removed from the dataset to prevent potential noise, as were taxa that were only present in one sample.

[0121] Two alpha diversity metrics, namely species richness and the Shannon diversity index, were calculated for each sample. After confirming that all samples were of reasonable count depth (>500,000 counts) the minimum depth from across all samples was found (3,296,842 counts), and 50 random subsets (with replacement) of that size were then taken from each sample. For each sample, the mean Shannon and species richness across the 50 samples was then calculated and used for analysis. Differences in the alpha diversity metrics between cohorts was assessed by fitting linear mixed-effects models with the cohort (IE or control) as the fixed effect, and age category (rounded to the nearest year) as a random effect. Inclusion of size category as a random effect was attempted but was not possible due to singular fit of the model. For cohort (the primary variable of interest), mean estimates and 95% confidence intervals for the cohorts are provided, alongside a 95% confidence interval for the contrast between cohorts with associated p-value. Other variables are graphed, but not tested for significance.

[0122] To assess beta diversity (differences between samples), non-metric multidimensional scaling (nMDS) was carried out, using the Bray-Curtis dissimilarity between each pair of samples. Samples were then plotted by their scores for the first 2 dimensions, colored by cohort, with point shapes possibly used to visualize a second grouping, and then overlaid with 95% data ellipses where possible. Attorney Docket: 069269.0771

[0123] Linear discriminant analysis Effect Size (LEfSe) was also conducted on the count data. Firstly, Kruskal-Wallis tests were applied to filter the data to those exhibiting significant differences between the two cohorts. Then, a Linear Discriminant Analysis (LDA) was conducted to find the linear combination of features best separating the two groups. All features achieving at least -1+2 importance in the LDA are plotted, colored by the group in which each feature exhibits greater abundance.

[0124] A differential abundance analysis was performed to highlight significant taxa and pathway abundances between disease and control group using 'Lefser ’ package in R. LefSE was performed with a LDA threshold of 3 for taxa (Wilcoxon p-value < 0.05) for taxa and LDA of 2.5 for KEGG modules (p-value < 0.01). The differences of the abundant taxa were plotted at all taxonomic ranks and for pathways at module level.

[0125] Analysis of Gut Microbiota.

[0126] The alpha diversity, gut microbiota relative abundance, beta diversity, and functional metabolic pathways of the idiopathic epilepsy and the control dogs was analyzed. These results are discussed below.

[0127] Alpha Diversity

[0128] The dogs in the IE group had increased alpha diversity richness (measured by Shannon, p value < 0.01 and Richness, p value <0.05) when compared to the control group. The mean Shannon Index was 2.82 and 2.51 with a mean richness of 127.33 and 117.36 for the IE and control groups respectively. This increased richness and alpha diversity in IE group could possibly be explained by several species (FIG. 1) which were significantly enriched in the diseased group (Thomas, W.B. and C.W. Dewey, C.W. Dewey and R.C. da Costa, Editors. 2015, Wiley-Blackwell, 2016. p. 249-267.).

[0129] When single characteristics were evaluated across groups including age, size category of dog, sex, and neuter status, there was no difference in alpha diversity. Neither fecal score nor meal format (wet, dry, and combinations of wet and dry) resulted in a difference in alpha diversity. For the IE group, no alpha diversity difference was identified for any single anti-seizure medication and combinations of anti-seizure medications.

[0130] Gut Microbiota Relative Abundance

[0131] The most abundant phyla in the control and IE groups were analyzed (FIGS. 2 A and 2B). The following phyla were predominant in the study in both groups: Bacteroidetes (healthy: 68.66%; IE: 59.21%), Firmicutes (healthy: 21.64%; IE: 27.73%), Proteobacteria (healthy: 2.96%; IE: 5.68%), Actinobacteriota (healthy: 1.96%; IE: 1.84%), and Attorney Docket: 069269.0771

[0132] Fusobacteriota (healthy: 1.62%; IE: 2.91%). The most common bacterial class in both groups was Bacteroidia (healthy: 68.66%; IE: 59.21%) and Clostridia (healthy: 13.75%; IE: 19.42%).

[0133] The most abundant identifiable species were then identified (FIGS. 3A, 3B, and 4). Overall, Prevotella coprr \\!zs the most abundant (healthy: 38.19%; IE: 23.79%), followed by Phocaeicola sp900546645 (healthy: 5.61%; IE: 6.99%),

[0134] Prevotellamassilia sp000437675 (healthy: 3.63%; IE: 3.89%) and Megamonas funiformis (healthy: 4.21%; IE: 2.24%).

[0135] Several significantly (p<0.05 and LDA threshold 3) differentially abundant taxa at various taxonomy levels were identified in control and epileptic groups. Overall, 13 species were enriched in the IE group and 6 species in the control group (FIG. 5). Significant depletion of health associated Prevotella copri in the IE group was observed, as well as a marked increase in some microbes such as Streptococcus lutetiensis, Escherichia coli, and Clostridium saudiense in IE group known to produce pathogenic conditions.

[0136] Beta Diversity

[0137] The results of the nMDS analysis found that there were no differences in beta diversity between the control and IE groups in regard to sex, neuter status, age, body condition score, diet format (dry, wet, or mixture of dry and wet), or fecal score.

[0138] Functional Metabolic Pathways

[0139] Analysis of the activity of functional metabolic pathway modules revealed a number of pathways that were differentially abundant between the idiopathic epileptic group and the control group (Fig. 4). Two pathway modules of particular interest were glutathione biosynthesis, conversion of glutamate to glutathione, and glutathione transport system pathways which were found to be differentially abundant in the control group. The third pathway that was of particular interest was the GABA (gamma-aminobutyrate) shunt pathway, which was found to be depleted in the epileptic group.

[0140] * * *

[0141] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. As such, the particular features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features Attorney Docket: 069269.0771 disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0142] It will be apparent to those skilled in the art that various modifications and variations can be made in the systems and methods of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter include modifications and variations that are within the scope of the appended claims and their equivalents.

[0143] Various patents and patent applications are cited herein, the contents of which are hereby incorporated by reference herein in their entireties.

Claims

Attorney Docket: 069269.0771WHAT IS CLAIMED IS:

1. A method of diagnosing canine idiopathic epilepsy comprising measuring the relative abundance of bacteria, wherein a decrease in the relative abundance oiPrevotella copri or Megamonas funiformis in a test sample when compared to a reference sample indicates the presence of idiopathic epilepsy.

2. The method of claim 1, wherein an increase in the relative abundance of at least one of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Faecalimonas sp900551895, Phocaeicola sp900546645, Prevotellamassilia sp000437675, or Fournierella sp002160145 in a test sample when compared to a reference sample further indicates the presence of idiopathic epilepsy.

3. The method of claim 1, wherein the decrease in the relative abundance is at least about 5%.

4. The method of claim 2, wherein the increase in the relative abundance is at least about 0.1%.

5. The method of claim 1, further comprising measuring the activity of at least one functional metabolic pathway.

6. The method of claim 5, wherein the activity of the at least one functional metabolic pathway in a test sample is enriched.

7. The method of claim 6, wherein the at least one functional metabolic pathway is selected from the group consisting of C5 isoprenoid biosynthesis, tryptophan biosynthesis, lipopolysaccharide biosynthesis, ascorbate biosynthesis, thiamine biosynthesis, EvgS- EvgA 2C-RS, citrate cycle, NAD(P)H: quinone oxidoreductase, SasA-RpaAB 2C-RS, riboflavin biosynthesis, cytochrome d ubiquinol oxidase, FixL-FixJ 2C-RS, PTS system, putative ABC transport system, PrrB-PrrA 2C-RS, and combinations thereof.

8. The method of claim 5, wherein the activity of at least one functional metabolic pathway in a test sample is decreased.Attorney Docket: 069269.07719. The method of claim 8, wherein at least one functional metabolic pathway selected from the group consisting of sulfonate transport system (TS), osmoprotectant TS, dipeptide TS, glutathione biosynthesis, triacylglycerol biosynthesis, gamma-aminobutyrate (GABA) shunt, putative multiple sugar TS, pyruvate oxidation, PTS system, glutathione TS, d-methionine TS, putative sn-glycerol-phosphate TS, pentose phosphate pathway, maltose / maltodextrin TS, VicK-VicR 2C-RS, putative arabinogalactan oligomer TS, multiple sugar TS, methyl -galactoside TS, phosphate TS, putative fructooligosaccharide TS, oligopeptide TS, and combinations thereof.

10. The method of claim 1, wherein the test sample and the reference sample are a fecal sample.

11. The method of claim 1, wherein the test sample and the reference sample are sequenced.

12. A method of treating canine idiopathic epilepsy comprising: a) measuring the relative abundance of bacteria, wherein a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a sample when compared to a reference sample indicates the presence of idiopathic epilepsy; and b) treating idiopathic epilepsy when the presence of idiopathic epilepsy is indicated.

13. The method of claim 12, wherein an increase in the relative abundance of at least one of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Phocaeicola sp900546645, Prevotellamassilia sp000437675, Faecalimonas sp900551895, or Fournier ella sp002160145 in a test sample when compared to a reference sample further indicates the presence of idiopathic epilepsy.

14. The method of claim 12, wherein the decrease in the relative abundance is at least about 5%.

15. The method of claim 14, wherein the increase in the relative abundance is at least about 0.1%.Attorney Docket: 069269.077116. The method of claim 13, further comprising measuring at least one functional metabolic pathway.

17. The method of claim 17, wherein the at least one functional metabolic pathway in the test sample is enriched.

18. The method of claim 17, wherein the at least one functional metabolic pathway in the test sample is decreased.

19. The method of claim 12, wherein the treatment is selected from the group consisting of an anti-seizure medication, a diet, and combinations thereof.

20. The method of claim 18, wherein the diet is a ketogenic diet.

21. The method of claim 18, wherein the anti-seizure medication is selected from the group consisting of phenobarbital, levetiracetam, potassium bromide, zonisamide, gabapentin, and combinations thereof.

22. A method of treating idiopathic epilepsy in a canine in need thereof, wherein the method comprises a treatment selected from the group consisting of an anti-seizure medication, a diet, and combinations thereof, wherein the canine has a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a fecal sample when compared to a reference fecal sample, and wherein the canine has an increase in the relative abundance of at least one of Streptococcus lutetiensis, Faecalimonas umbilicata, Fusobacterium B sp900554885, Bifidobacterium globosum, Blautia A sp900541345, Escherichia coli, Clostridium saudiense, Fusobacterium A sp900555845, Negativibacillus sp000435195, Anaerostipes caccae, Phocaeicola sp900544075, Faecalimonas sp900551895, Phocaeicola sp900546645, Prevotellamassilia sp000437675, or Fournierella sp002160145 in a fecal sample when compared to a reference fecal sample, wherein the method reduces frequency or severity of symptoms associated with idiopathic epilepsy.

23. A method of diagnosing canine idiopathic epilepsy comprising measuring the relativeAttorney Docket: 069269.0771 abundance of bacteria, wherein a decrease in the relative abundance of Prevotella copri or Megamonas funiformis in a test sample when compared to a reference sample and an increase in the relative abundance of bacteria selected from the group consisting of Streptococcus lutetiensis, Escherichia coli, and Clostridium saudiense in a test sample when compared to a reference sample indicates the presence of idiopathic epilepsy.

24. The method of claim 23, wherein the decrease in the relative abundance of Prevotella copri o Megamonas funiformis in a test sample when compared to a reference sample is at least about 5%.

25. The method of claim 24, wherein the decrease in relative abundance of Prevotella copri is from about 5% to about 25%.

26. The method of claim 24, wherein the decrease in relative abundance of Megamonas funiformis is from about 5% to about 25%.

27. The method of claim 23, wherein the increase the relative abundance of bacteria selected from the group consisting of Streptococcus lutetiensis, Escherichia coli, and Clostridium saudiense in a test sample when compared to a reference sample is at least about 0.1%.

28. The method of claims 23-27, further comprising measuring the activity of at least one functional metabolic pathway.

29. The method of claim 28, wherein the activity of at least one functional metabolic pathway in a test sample is enriched.

30. The method of claims 28-29, wherein the at least one functional metabolic pathway is selected from the group consisting of C5 isoprenoid biosynthesis, tryptophan biosynthesis, lipopolysaccharide biosynthesis, ascorbate biosynthesis, thiamine biosynthesis, EvgS-EvgA 2C-RS, citrate cycle, NAD(P)H: quinone oxidoreductase, SasA-RpaAB 2C-RS, riboflavin biosynthesis, cytochrome d ubiquinol oxidase, FixL-FixJ 2C-RS, PTS system, putative ABC transport system, PrrB-PrrA 2C-RS, and combinations thereof.

31. The method of claims 29, wherein the activity of at least one functional metabolicAttorney Docket: 069269.0771 pathway in a test sample is decreased.

32. The method of claims 29 and 32, wherein the at least one functional metabolic pathway selected from the group consisting of sulfonate transport system (TS), osmoprotectant TS, dipeptide TS, glutathione biosynthesis, triacylglycerol biosynthesis, gamma- aminobutyrate (GABA) shunt, putative multiple sugar TS, pyruvate oxidation, PTS system, glutathione TS, d-methionine TS, putative sn-glycerol-phosphate TS, pentose phosphate pathway, maltose / maltodextrin TS, VicK-VicR 2C-RS, putative arabinogalactan oligomer TS, multiple sugar TS, methyl-galactoside TS, phosphate TS, putative fructooligosaccharide TS, oligopeptide TS, and combinations thereof.

33. The method of claims 23-32, wherein the test sample and reference sample are from canines.

34. The method of claim 33, wherein the reference sample is from a healthy canine or a group of healthy canines.

35. The method of claims 23-34, wherein the test sample and the reference sample is a fecal sample.

36. The method of claims 23-35, wherein the test sample and the reference sample are sequenced.

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