Compositions and methods for treating and reducing the risk of conditions associated with elevated 4-ethylphenylsulfate in dogs using tomato pomace and methods for identifying dogs at risk for such conditions

Abstract

Methods for identifying canine subjects at a high risk of developing elevated levels of 4-ethylphenyl sulfate, canine stress, canine anxiety, and / or inhibition of beneficial microbial growth and promotion of harmful microbial growth are disclosed. The methods include analyzing a biological sample obtained from the canine subject for the presence of two copies of the minor allele of the single nucleotide polymorphism BICF2P1175095 in the canine subject. Methods for treating identified canine subjects by administering an effective amount of tomato pomace are also disclosed. Methods for treating canine subjects for elevated 4-EPS levels, canine anxiety, or canine stress are disclosed. Canine food compositions containing tomato pomace are disclosed.

Description

[Background technology]

[0001] The microbial toxin 4-ethylphenylsulfate (4-EPS) is a metabolite produced by gut bacteria. Among other microbial metabolites, 4-EPS enters the systemic circulation. In dogs, elevated levels of 4-EPS in the blood have been associated with stress, anxiety, brain damage, and other behavioral problems. Reductions in 4-EPS levels have been shown to reduce symptoms of stress and anxiety.

[0002] Canine anxiety is a fearful, agitated, or anxious response to anticipating a threatening or frightening situation. Some dogs experience disproportionate levels of anxiety. Anxiety can develop into an anxiety disorder, leading to behavioral and other problems. Some dogs experience generalized anxiety, where they have fearful reactions to a wide range of situations that "normal" pets would not react to. Anxiety can take the form of a number of anxiety disorders, including generalized anxiety disorder, overstimulation anxiety, separation anxiety, confinement, and noise phobia, among others.

[0003] Causes may include genetic factors, prenatal and neonatal stressors, separation from the mother and puppy, lack of socialization, unfamiliarity, or previous unpleasant outcomes when encountering the stimulus (or similar stimuli). The most common causes are fear, separation, and aging. Fear-related anxiety can be triggered by loud noises, strange people or animals, visual stimuli, new or unfamiliar environments, and certain situations, among others. Age-related anxiety affects older dogs and may be associated with cognitive dysfunction syndrome (CDS). Separation anxiety is a specific anxiety that arises from a pet's inability to feel at ease when separated from its family. Approximately 14% of dogs have separation anxiety. Some separation anxiety may be the result of dysfunctional attachments as puppies age and mature. In some cases, separation anxiety may occur with changes in the home or daily routine, while in others separation anxiety may be due to an underlying anxiety condition along with other behavioral issues such as phobias.

[0004] Anxiety can lead to destructive behavior (especially towards exits or the owner's belongings), distress vocalizations, house-soiling, salivation, pacing, fidgeting, restlessness, loss of appetite, and repetitive or obsessive behaviors. In some cases, anxiety can contribute to aggressive behavior.

[0005] Common symptoms of anxiety in dogs include aggression, urinating or defecating inside the house, drooling, rapid breathing, destructive behavior, depression, excessive barking, pacing, fidgeting, and repetitive or obsessive behaviors. Different dogs will exhibit different symptoms and combinations of symptoms when suffering from anxiety.

[0006] Stress in dogs is a dog's response to changes and demands to adapt, and is usually manifested as tension or pressure. Dogs experiencing stress may develop feelings of fear, excitement, hyperactivity, nervousness, irritability, or tantrums. Negative, excessive, and chronic stress can have adverse effects on behavior, health, and overall well-being. Stress can cause disease, suppress the immune system, induce unwanted behaviors, and increase arousal and the likelihood of aggressive behavior.

[0007] Causes of stress in dogs include sadness, exposure to conflict, excessive or insufficient stimulation, overcrowding, environmental changes (increased schedules, people, animals, noise), punitive training, insufficient social time, frightening events, neglect, frustration, and uncertainty, among others.

[0008] Dogs communicate that they are stressed in a variety of ways. Indicators that a dog is stressed include dilated pupils, tightness around the eyes, cetacean / half-moon eyes, yawning, licking lips / nose, rapid breathing, excessive salivation, smiling, grinding teeth, puffing out cheeks, baring teeth, furrowed muzzle, and pulled-back or erect ears. Other indicators include tense body, stretching, excessive hair loss, little or no movement, low body position, shifting weight backwards, trembling / shaking, exposed penis, sweaty paws, stiff brows, barking, growling, howling, and snorting. Dogs' behavior often changes when they are stressed. Behaviors that are often brought on by stress include fidgeting, lack of sleep or excessive sleep, jumping / irritability, tantrums, excessive self-grooming, destructive behavior, loss of appetite, obsessive / impulsive behavior, difficulty concentrating, hyperactivity, increased urination / defecation, vomiting and diarrhea, among others.

[0009] Single nucleotide polymorphisms (SNPs) are a common type of genetic variation. A SNP is a single base pair variation at a particular locus. That is, a SNP is a difference in a single base in a DNA sequence that occurs at a particular location in the genome. Typically, for a SNP at a particular location, there are two possible nucleotide variations called alleles at that location. Within a population, the nucleotide variation that occurs most commonly at a particular base location in the genome is called the major allele, and the nucleotide variation that is less common at that particular base location is called the minor allele. Dogs, like most multicellular organisms, have two sets of chromosomes. Thus, each dog has two copies of each gene or locus, and therefore two copies of each SNP. Thus, for each SNP in the dog's genome, the dog may have two copies of the major allele, or one minor allele and one minor allele, or two minor alleles.

[0010] SNPs can act as biological markers. SNPs can be useful in predicting drug response and risk of developing certain diseases. SNP genotyping refers to the detection of SNPs in the genome. There are many methods to detect SNPs and perform SNP genotyping.

[0011] There is a need to develop improved methods for identifying dogs that are likely or at high risk of developing anxiety and stress, methods for reducing the risk of anxiety and stress in dogs, and methods for treating anxiety and stress in dogs.There is a need for methods and compositions for reducing elevated levels of 4-EPS in dogs.There is a need for methods and compositions for treating or reducing elevated levels of the severity of anxiety in dogs.There is a need for methods and compositions for treating or reducing elevated levels of the severity of stress in dogs. Summary of the Invention

[0012] A method is provided that includes analyzing a biological sample obtained from the canine subject for the presence of two copies of the minor allele of single nucleotide polymorphism BICF2P1175095 in the canine subject.

[0013] The presence of two copies of the minor allele of the single nucleotide polymorphism BICF2P1175095 indicates that the canine subject will be more likely to have elevated levels of 4-ethylphenylsulfate, more likely to develop canine stress, more likely to develop canine anxiety, and / or more likely to inhibit the growth of beneficial microorganisms and promote the growth of harmful microorganisms over the course of their lifetime.

[0014] The method may include analyzing a biological sample obtained from the canine subject by performing DNA sequencing, restriction enzyme digestion, polymerase chain reaction (PCR), hybridization, real-time PCR, reverse transcriptase PCR, or ligase chain reaction.

[0015] The method may include analyzing a biological sample obtained from the canine subject by performing at least one nucleic acid analysis technique selected from analysis using a whole genome SNP chip, single-stranded conformational polymorphism analysis (SSCP) assay, restriction fragment length polymorphism (RFLP), automated fluorescent sequencing; clamped denaturing gel electrophoresis (CDGE), denaturing gradient gel electrophoresis (DGGE), mobility shift analysis, restriction enzyme analysis, heteroduplex analysis, chemical mismatch cleavage (CMC), RNase protection assay, use of polypeptides that recognize nucleotide mismatches, allele-specific PCR, sequence analysis, and SNP genotyping.

[0016] The method may include analyzing a biological sample obtained from the canine subject by performing at least one nucleic acid analysis technique selected from hybridization-based methods, enzyme-based methods, post-amplification methods based on the physical properties of DNA, and sequencing methods.

[0017] The method may include analyzing a biological sample obtained from the canine subject by performing at least one nucleic acid analysis technique selected from the group consisting of a hybridization-based method selected from the group consisting of dynamic allele-specific hybridization, molecular beacon methods, and SNP microarrays; an enzyme-based method selected from the group consisting of restriction fragment length polymorphism (RFLP), PCR-based methods, flap endonuclease, primer extension methods, 5'-nuclease, and oligonucleotide ligation assays; a post-amplification method based on the physical properties of DNA selected from the group consisting of single-stranded conformation polymorphism analysis, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high-resolution amplicon melting, DNA mismatch binding proteins, SNPlex, and surveyor nuclease assays; and a sequencing method.

[0018] A method for preventing or reducing elevated levels of 4-ethylphenyl sulfate in a canine subject, comprising detecting the presence of two copies of a minor allele of BICF2P1175095 in a biological sample from the canine subject, and administering to the canine subject a composition comprising an effective amount of tomato pomace, such as by feeding the canine subject a nutritional composition comprising an effective amount of tomato pomace.

[0019] A method of treating a canine subject for canine anxiety or canine stress in the canine subject, comprising detecting the presence of two copies of a minor allele of BICF2P1175095 in a biological sample from the canine subject, and administering to the canine subject a composition comprising an effective amount of tomato pomace, such as by feeding the canine subject a nutritional composition comprising an effective amount of tomato pomace.

[0020] A dog food composition is provided that includes tomato pomace in an amount equal to 0.087-0.21% on a dry matter basis.

[0021] A dog food composition is provided which includes tomato pomace in an amount equal to 0.14% on a dry matter basis. [Brief description of the drawings]

[0022] [Figure 1] Figure 1 shows a matched pair analysis using data generated in Example 2. Figure 1 shows a comparison of the levels of 4-EPS in each dog fed a diet containing tomato pomace (CTRL) versus a diet not containing tomato pomace (TEST). The results show that dogs fed the test food diet without tomato pomace had higher 4-EPS levels compared to the 4-EPS levels observed when fed the control food diet containing tomato pomace (P=0.04). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The following description of the preferred embodiments is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.

[0024] As used in this invention and in the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0025] As used herein, the term "companion animal" includes any non-human animal suitable for being kept as a pet by humans, including, but not limited to, dogs, cats, rabbits and rodents. Particular embodiments are therapeutic formulations and methods for dogs and / or cats. In one particular aspect, the present invention is directed to therapeutic formulations and methods for dogs.

[0026] The term "canine" includes canines that are companion animals, such as Canis familiaris, working dogs, etc. The term "dog" is a synonym of the term "canine."

[0027] The term "cat" includes the companion animal cat known as the domestic cat or house cat or Felis domesticus. The term "cat" is a synonym of the term "feline."

[0028] A method is provided for treating anxiety and stress in an animal, particularly a companion animal such as a dog or cat. The method comprises administering to the animal an effective amount of a tomato pomace combination. The composition comprises an effective amount of tomato pomace. The effective amount of tomato pomace is 0.044-0.42% of the daily nutritional intake, in some embodiments 0.066-0.315% of the daily nutritional intake, in some embodiments 0.087-0.21% of the daily nutritional intake, and in some embodiments 0.14% of the daily nutritional intake.

[0029] "Daily nutrient intake" and "total nutrient intake per day" refer to the amount of dry matter intake per day. That is, the weight of water is not included in the calculation of the amount of nutrient consumed per day. To the extent that foods and food ingredients contain water / moisture, dry matter represents everything in the sample other than water, including protein, fiber, fat, minerals, etc. The weight of dry matter is the total weight minus the weight of water. Daily dry matter intake is calculated as the total nutrient intake per day excluding all water. For example, the amount of an ingredient that represents a particular percentage of daily nutrient intake refers to the amount of that ingredient in dry matter form (also excluding all water) relative to the total amount of dry matter consumed per day (i.e., excluding all water). Those skilled in the art will readily recognize and understand the amount of dry matter, the weight of dry matter, and the amount and percentage of nutrition expressed as a percentage of dry matter. Because foods, whether moist, moist, or dry, generally contain a certain amount of water, the water content of such foods is excluded when calculating the daily dry matter intake. To calculate total daily nutrient intake, which is the daily dry matter intake, water is excluded. To calculate the percentage of an element in total daily intake on a dry matter basis, water is removed from the total intake to give the total daily dry matter intake, and the percentage of the element is calculated based on the amount of the element present as dry matter.

[0030] The composition useful in the method can be a pet food composition, such as a dog food composition. Alternatively, the tomato pomace can be administered as a supplement, treat, or toy, or otherwise not incorporated into the food provided to the animal for its daily nutritional intake.

[0031] In some preferred embodiments, the animal is a dog and the method comprises administering to the dog an effective amount of tomato pomace daily, the effective amount of tomato pomace administered to the dog per day is 0.044-0.42% of the daily nutritional intake, in some embodiments 0.066-0.315% of the daily nutritional intake, in some embodiments 0.087-0.21% of the daily nutritional intake, and in some embodiments 0.14% of the daily nutritional intake.

[0032] Compositions and methods are provided for treating anxiety and stress in animals, particularly companion animals such as cats or dogs.The compositions and methods are useful for treating symptoms of anxiety or stress in animals in need thereof.The compositions and methods are useful for treating symptoms of anxiety or stress in such animals with elevated 4-EPS levels.The compositions and methods are useful for reducing elevated 4-EPS levels in companion animals, particularly animals with elevated 4-EPS levels, such as dogs.In some embodiments, the compositions and methods are for treating canine anxiety or canine stress in dogs.

[0033] As used herein, the term "treatment" refers to the elimination, reduction in severity, or prevention of one or more symptoms.

[0034] As used herein, the term "anxiety" refers to anxiety, anxiety disorders, and symptoms of anxiety and anxiety disorders.

[0035] As used herein, the term "stress" refers to stress, stress disorders, and symptoms of stress and stress disorders.

[0036] As used herein, the term "treatment" with respect to anxiety refers to therapeutic and / or preventive activity. In dogs with symptoms of anxiety, treatment of anxiety in dogs refers to the elimination of symptoms, the halting or suppression of the progression of symptoms, the reduction of the severity of symptoms, and the prevention of symptoms. Treatment that initially eliminates, stops, suppresses the progression, or reduces the severity of symptoms may be continued, and continued treatment may further eliminate, stop, suppress the progression, or reduce the severity of symptoms, and / or prevent recurrence or onset of symptoms, or reduce the severity of further onset of symptoms. In some embodiments, before treating anxiety in dogs, the dog may be identified as having symptoms of anxiety. In some embodiments, the dog may be treated for anxiety without identifying symptoms of anxiety before treatment. In some embodiments, before treating anxiety, the dog may be identified as having anxiety or being predisposed to developing anxiety. In some embodiments, before treating anxiety, the dog may be identified as having high 4-EPS levels.

[0037] As used herein, the term "treatment" with respect to stress and stress disorder refers to therapeutic and / or preventive activity. In dogs with symptoms of stress or stress disorder, treating the dog with stress refers to removing symptoms, stopping or inhibiting the progression of symptoms, reducing the severity of symptoms, and preventing symptoms. Treatment that initially removes, stops, inhibits the progression, or reduces the severity of symptoms may be continued, and continued treatment may further remove, stop, inhibit the progression, or reduce the severity of symptoms, and / or prevent recurrence or onset of symptoms, or reduce the severity of further onset of symptoms. In some embodiments, before treating the dog with stress, the dog may be identified as having symptoms of stress or stress disorder. In some embodiments, the dog may be treated for stress or stress disorder without identifying symptoms of anxiety before treatment. In some embodiments, before treating for stress or stress disorder, the dog may be identified as having stress or stress disorder or as being predisposed to developing stress or stress disorder. In some embodiments, before treating for stress or stress disorder, the dog may be identified as having high 4-EPS levels.

[0038] As used herein, the term "treatment" with respect to promoting beneficial microbial growth and inhibiting harmful microbial growth refers to therapeutic and / or prophylactic activity. A treatment that stops the levels of beneficial and harmful microorganisms, or promotes the growth of beneficial microorganisms and inhibits the growth of harmful microorganisms in dogs with reduced levels of beneficial microorganisms and elevated levels of harmful microorganisms. A dog that is identified as having a predisposition to inhibit beneficial microbial growth and promote harmful microbial growth before the start of treatment. A treatment that initially promotes the growth of beneficial microorganisms and inhibits the growth of harmful microorganisms, increases the level of beneficial microorganisms and decreases the level of harmful microorganisms to a more healthy balance in animals with elevated levels of harmful microorganisms and decreased levels of beneficial microorganisms, and then maintains the levels by continuing treatment. In some embodiments, before treating the dog for stress, the dog may be identified as having elevated levels of harmful microorganisms and decreased levels of beneficial microorganisms. In some embodiments, the dog may be treated without identifying elevated levels of harmful microorganisms and decreased levels of beneficial microorganisms in the animal.

[0039] As used herein, the terms "treatment of elevated 4-EPS", "treating against elevated 4-EPS", and "treating elevated 4-EPS" refer to therapeutic and / or preventative activity in which 4-EPS levels are reduced. In dogs with elevated 4-EPS levels, "treatment of elevated 4-EPS", "treating against elevated 4-EPS", and "treating elevated 4-EPS" refer to the reduction of elevated 4-EPS levels. Treatment may reduce elevated 4-EPS levels to normal, non-elevated levels, or reduced elevated 4-EPS levels. Following the reduction of elevated 4-EPS levels, treatment may prevent elevated 4-EPS levels or reduce the severity of further progression of elevated 4-EPS levels. In dogs that do not have elevated 4-EPS levels, "treating elevated 4-EPS", "treating for elevated 4-EPS" and "treating elevated 4-EPS" refer to stopping or decreasing 4-EPS levels and preventing the occurrence of elevated 4-EPS levels or reducing the severity of the occurrence of elevated 4-EPS levels. In some embodiments, prior to treatment for 4-EPS, the dog may be identified as having elevated 4-EPS by measuring 4-EPS levels. In some embodiments, the dog may be treated for elevated 4-EPS without measuring 4-EPS levels prior to treatment. In some embodiments, prior to treatment for 4-EPS, the dog may be identified as having a predisposition to elevated 4-EPS. A dog identified as having a predisposition to elevated 4-EPS may have elevated 4-EPS at the time of treatment, in which case the treatment is therapeutic, or may not have elevated 4-EPS, in which case the treatment is prophylactic, or treatment may be performed without determining 4-EPS levels. In some embodiments, dogs can be identified as predisposed to elevated 4-EPS prior to initiating treatment, with or without measuring 4-EPS levels.

[0040] As used herein, "effective amount", "effective dose", and similar terms refer to an amount of tomato pomace effective to achieve a particular biological outcome, i.e., increasing 4-EPS levels, treating anxiety, stress, and levels of beneficial and harmful microbes in the microbiome. In certain embodiments, an effective amount of the composition is administered for a time sufficient to affect treatment. In certain embodiments, the method includes administration and consumption of a composition comprising tomato pomace for a period of time sufficient to provide effective treatment and maintenance. The effective amount may be based on several factors, including the dog's ideal body weight, age, sex, activity level, metabolizable energy of the composition, and frequency of feeding the composition, e.g., once, twice, or three times a day, as well as other compositions fed to the dog. In some embodiments, the effective amount refers to an amount of tomato pomace administered based on total nutritional intake, where the amount of tomato pomace is equal to 0.087-0.21% of the total nutritional intake per day. In some embodiments, the effective amount refers to a pet food comprising 0.087-0.21% tomato pomace. In some embodiments, the effective amount refers to the amount of tomato pomace administered based on total nutritional intake, the amount of tomato pomace being equal to 0.14% of the total nutritional intake per day. In some embodiments, the effective amount refers to a pet food that includes 0.14% tomato pomace.

[0041] A "food," "food composition," or "pet food composition" may, in some embodiments, be a nutritionally complete diet for the animal, such as a dog, to which it is fed.

[0042] As used herein, a "component" refers to any element of a composition.

[0043] The term "nutrient" refers to a substance that provides nutrition. In some cases, an ingredient may contain more than one "nutrient", for example, a composition may contain corn, which contains important nutrients including both protein and carbohydrates.

[0044] The food composition can be provided to animals, including but not limited to pets, in the form of pet food. Various types of pet food are available to pet owners as are commonly known. Pet food choices include, but are not limited to, wet pet food, semi-moist pet food, dry pet food, and pet treats. Wet pet food generally has a moisture content greater than about 65%. Semi-moist pet food generally has a moisture content of about 20% to about 65% and may include humectants, potassium sorbate, and other ingredients to prevent microbial growth (bacteria and mold). Dry pet food, including but not limited to food kibble, generally has a moisture content less than about 15%. Pet treats can typically be semi-moist, chewable treats, dry treats in any number of forms, chewable bones or baked, extruded, or punched treats, confectionery treats, or other types of treats known to those skilled in the art.

[0045] As used herein, the term "kibble" or "food kibble" refers to a particular pellet-like component of an animal's diet, such as dog and cat diet. In some embodiments, the food kibble has a moisture content or moisture of less than 15% by weight. The food kibble can range in texture from hard to soft. The food kibble can range in internal structure from puffy to dense. The food kibble can be formed by an extrusion process or a baking process. In non-limiting examples, the food kibble can have a uniform internal structure or a varying internal structure. For example, the food kibble may include a core and a coating to form a coated kibble. It should be understood that when the term "kibble" or "food kibble" is used, it can refer to an uncoated kibble or a coated kibble.

[0046] As used herein, the term "extrude" or "extrusion" refers to the process of passing a pre-treated and / or prepared ingredient mixture through an extruder. In some embodiments of extrusion, the food kibble is formed by an extrusion process, in which a kibble dough containing a mixture of wet and dry ingredients can be extruded under heat and pressure to form a food kibble. Any type of extruder can be used, examples of which include, but are not limited to, single screw extruders and twin screw extruders. The sources, ingredients, and lists of ingredients described below are listed such that combinations and mixtures thereof are also contemplated and are within the scope of this specification.

[0047] As intended herein, composition is meant to include, but is not limited to, nutritionally complete and balanced animal food compositions. A "nutritional complete diet" is a diet that can contain sufficient nutrients to maintain the normal health of a healthy dog ​​in terms of diet. Nutritionally complete and balanced pet food compositions, such as for dogs, are well known to those skilled in the art. For example, substances such as nutrients and ingredients suitable for nutritionally complete and balanced animal feed compositions, and their recommended amounts, can be found, for example, in the Official Publication of the Association of American Feed Control Officials, Inc. (AAFCO), Atlanta, Ga., (2012).

[0048] When feeding dog with a diet containing an effective amount of tomato pomace, it is contemplated that a preferred method comprises feeding dog with a pet food that contains tomato pomace as an ingredient.In another embodiment, feeding dog with a diet containing an effective amount of tomato pomace is achieved by administering tomato pomace to dog as a supplement or treat.Providing tomato pomace to dog by any means, whether delivered in pet food composition or as a separate supplement or treat, is considered to feed dog with a diet containing an effective amount of tomato pomace.

[0049] As used herein, the term "supplement" includes, but is not limited to, a feed that is used in another feed to improve the nutritional balance or performance of the total diet of an animal. Supplements include, but are not limited to, compositions that are fed undiluted as a supplement to another feed, provide free choice with other portions of the animal's ration that can be used separately, or are diluted with the animal's regular feed and mixed to create a complete diet. For example, AAFCO guidelines include a discussion of supplements in the Association of American Feed Control Officials (AAFCO), Atlanta, Ga (2012). Supplements can be in a variety of forms, including, for example, powders, liquids, syrups, pills, encapsulated compositions, and the like.

[0050] The diet comprises an effective amount of tomato pomace, and can reduce the elevated level of 4-EPS in dogs.The diet comprising tomato pomace is useful for treating anxiety in dogs.The diet comprising tomato pomace is useful for treating stress in dogs.The diet comprising tomato pomace is useful for promoting beneficial microbial growth and inhibiting harmful microbial growth in the microbiota of dog subjects, particularly in the gastrointestinal track.

[0051] Genetic predisposition to elevated 4-EPS

[0052] Genetic association studies have identified a genetic marker that can identify dogs predisposed to elevated levels of 4-EPS. The genetic marker is a SNP located upstream of the NOD1 gene at chr14:43309715 using the CanFam3.1 reference genome, and is designated BICF2P1175095 on the commercially available Illumina canine genotyping array. Dogs homozygous for the minor allele of SNP BICF2P1175095 are predisposed to elevated levels of 4-EPS. The minor allele of SNP BICF2P1175095 is C. Dogs have 39 pairs of chromosomes. Dogs homozygous for the minor allele of SNP BICF2P1175095 have the minor allele present on each of the two chromosome 14 pairs. Dogs that are homozygous for minor alleles are said to have homozygous minor allele genotype, which is SNP BICF2P1175095 genotype CC or genotype CC as used herein.Dogs with CC genotype are more likely to develop elevated 4-EPS levels, dog anxiety and dog stress.In addition to dog's predisposition to develop elevated 4-EPS levels, anxiety and stress, dog CC genotype is more likely to inhibit beneficial microbial growth in canine subject's microbiota, especially in the microbiota of gastrointestinal track, and promote harmful microbial growth in canine subject's microbiota, especially in the microbiota of gastrointestinal track.

[0053] Dogs identified as being predisposed to elevated levels of 4-EPS, such as identifying dogs as having the CC genotype, can be treated through a diet containing an effective amount of tomato pomace.

[0054] Identifying the CC genotype of the canine subject identifies the canine subject as being predisposed to elevated levels of 4-EPS. Identifying the CC genotype of the canine subject identifies the canine subject as being predisposed to anxiety. Identifying the CC genotype of the canine subject identifies the canine subject as being predisposed to stress. Identifying the CC genotype of the canine subject identifies the canine subject as being predisposed to inhibition of beneficial microbial growth, and therefore to lower levels of beneficial microorganisms in the canine subject's microbiota, particularly the microbiota of the gastrointestinal track, and also identifies the canine subject as being predisposed to promotion of harmful microbial growth, and therefore to higher levels of harmful microorganisms in the canine subject's microbiota, particularly the microbiota of the gastrointestinal track.

[0055] The diet containing tomato pomace in an effective amount to suppress the increase of 4-EPS level in dogs is particularly beneficial for dog subjects who have CC genotype and therefore tend to have increased 4-EPS level.The diet containing tomato pomace in an effective amount is particularly beneficial for dog subjects who have CC genotype and therefore tend to experience anxiety.The diet containing tomato pomace in an effective amount is particularly beneficial for dog subjects who have CC genotype and therefore tend to experience stress.The diet containing tomato pomace in an effective amount is particularly beneficial for dog subjects who have CC genotype and therefore tend to inhibit beneficial microbial growth and promote harmful microbial growth in the microflora of dog subjects, particularly the microflora of the gastrointestinal track.

[0056] Without being bound by any particular theory, the CC genotype of BICF2P1175095, located upstream of the NOD1 gene, may result in a condition that favors harmful microbial growth over beneficial microbial growth in the gastrointestinal track of dogs. As a result, beneficial microbial growth is inhibited in the microbiota of the gastrointestinal track of dogs, and harmful microbial growth is promoted in the microbiota of the gastrointestinal track of dogs, resulting in a decrease in the level of beneficial microorganisms and an increase in the level of harmful microorganisms. The harmful microorganisms in the microbiota release the metabolic toxin 4-EPS, increasing the level of circulating 4-EPS in dogs. The increase in the level of circulating 4-EPS in dogs may result in the development of anxiety and / or stress in dogs. A diet containing an effective amount of tomato pomace may result in an increase in the level of beneficial microorganisms and a decrease in the level of harmful microorganisms in the microbiota of dogs. The increase in the level of beneficial microorganisms and the decrease in the level of harmful microorganisms in the microbiota of dogs results in a decrease in 4-EPS released into the circulation, thereby decreasing the 4-EPS level.

[0057] The method for identifying a canine subject that is predisposed to elevated 4-EPS levels comprises determining that the canine subject has the CC genotype. The method for identifying a canine subject that is predisposed to anxiety comprises determining that the canine subject has the CC genotype. The method for identifying a canine subject that is predisposed to stress comprises determining that the canine subject has the CC genotype. The method for identifying a canine subject that is predisposed to inhibiting beneficial microbial growth and promoting harmful microbial growth, particularly in the microflora of the gastrointestinal track, comprises determining that the canine subject has the CC genotype.

[0058] The method of reducing elevated 4-EPS levels, preventing elevated 4-EPS levels, or reducing the severity of elevated 4-EPS levels in a canine subject comprises feeding the canine subject a diet comprising tomato pomace in an effective amount, thereby reducing elevated 4-EPS levels, preventing elevated 4-EPS levels, or reducing the severity of elevated 4-EPS levels.In some embodiments, the method of reducing elevated 4-EPS levels, preventing elevated 4-EPS levels, or reducing the severity of elevated 4-EPS levels in a canine subject comprises identifying the canine subject as having a CC genotype, and feeding the canine subject a diet comprising tomato pomace in an effective amount, thereby reducing elevated 4-EPS levels, preventing elevated 4-EPS levels, or reducing the severity of elevated 4-EPS levels.

[0059] A method for reducing, reducing, and / or preventing anxiety in a canine subject comprises feeding the canine subject a diet comprising an effective amount of tomato pomace to reduce, reduce, and / or prevent anxiety and symptoms of anxiety disorder in the canine. In some embodiments, a method for reducing, reducing, and / or preventing anxiety in a canine subject comprises identifying the canine subject as having a CC genotype and feeding the canine subject a diet comprising an effective amount of tomato pomace to reduce, reduce, and / or prevent anxiety in the canine.

[0060] A method of reducing, reducing, and / or preventing stress in a canine subject comprises feeding the canine subject a diet comprising an effective amount of tomato pomace to reduce, reduce, and / or prevent stress in the canine. In some embodiments, a method of reducing, reducing, and / or preventing stress in a canine subject comprises identifying the canine subject as having a CC genotype and feeding the canine subject a diet comprising an effective amount of tomato pomace to reduce, reduce, and / or prevent stress in the canine.

[0061] A method for promoting beneficial microbial growth and inhibiting harmful microbial growth in a canine subject comprises feeding the canine subject a diet comprising an effective amount of tomato pomace to promote beneficial microbial growth and inhibit harmful microbial growth. In some embodiments, a method for promoting beneficial microbial growth and inhibiting harmful microbial growth in a canine subject comprises identifying the canine subject as having a CC genotype and feeding the canine subject a diet comprising an effective amount of tomato pomace to promote beneficial microbial growth and inhibit harmful microbial growth.

[0062] The method includes analyzing a biological sample obtained from a canine subject for the presence of two copies of the single nucleotide polymorphism minor allele C, which is located at chr14:43309715 as referred to in the CanFam3.1 reference genome. The SNP is named BICF2P1175095 on the commercially available Illumina canine genotyping array. That is, the method includes analyzing a biological sample obtained from a canine subject for the presence of the CC genotype. In some embodiments, the sample is analyzed by performing DNA sequencing, restriction enzyme digestion, polymerase chain reaction (PCR), hybridization, real-time PCR, reverse transcriptase PCR, or ligase chain reaction.

[0063] Canine subjects with two copies of the minor allele C of the SNP located at chr14:43309715 (BICF2P1175095), i.e., with a CC genotype, are prone to elevated 4-EPS levels in their lifetime. The CC genotype indicates that the canine subject is predisposed to developing anxiety in their lifetime, that the canine subject is predisposed to developing stress in their lifetime, and that the canine subject is predisposed to inhibiting beneficial microbial growth and promoting harmful microbial growth in their microbiota.

[0064] An increased likelihood of having elevated levels of 4-ethylphenylsulfate indicates an increased likelihood of the dog developing anxiety, an increased likelihood of the dog developing stress, and / or an increased likelihood of inhibiting beneficial microbial growth and promoting harmful microbial growth. A method of identifying a dog as having an increased likelihood of having elevated levels of 4-ethylphenylsulfate is a method of identifying a dog as having an increased likelihood of developing anxiety, as having an increased likelihood of developing stress, and / or as having an increased likelihood of inhibiting beneficial microbial growth and promoting harmful microbial growth.

[0065] In dogs exhibiting symptoms of canine stress or canine anxiety, the methods provided herein include analyzing a biological sample obtained from the canine subject for the presence of two copies of the minor allele C of a single nucleotide polymorphism located at chr14:43309715, as referenced in the CanFam3.1 reference genome, as part of a method for diagnosing a dog as having a genetic predisposition to having canine stress or canine anxiety.

[0066] The method of identifying a dog as being more likely to have elevated levels of 4-ethylphenylsulfate, indicating that the dog is more likely to develop anxiety, that the dog is more likely to develop stress, and / or that the inhibition of beneficial microbial growth and promotion of harmful microbial growth is more likely, comprises analyzing a biological sample obtained from the canine subject for the presence of two copies of the minor allele C of a single nucleotide polymorphism located at chr14:43309715, as referenced in the CanFam3.1 reference genome, and may be part of a method of treating a dog to prevent or reduce elevated levels of 4-ethylphenylsulfate, to prevent or reduce symptoms of anxiety in the dog, to prevent or reduce symptoms that make the dog more likely to develop stress, and / or to prevent or reduce the inhibition of beneficial microbial growth and promotion of harmful microbial growth.

[0067] A method for identifying a dog having a genetic predisposition to increased likelihood of elevated levels of 4-ethylphenylsulfate, developing canine anxiety, developing canine stress, and / or inhibiting beneficial microbial growth and promoting harmful microbial growth, comprises analyzing a biological sample obtained from a canine subject exhibiting symptoms of canine stress or canine anxiety for the presence of two copies of the minor allele C of a single nucleotide polymorphism located at chr14:43309715, as referenced in the CanFam3.1 reference genome, and may be part of a method for treating a dog to reduce elevated levels of 4-ethylphenylsulfate, to alleviate symptoms of canine anxiety, to alleviate symptoms that increase the likelihood of the dog developing stress, and / or to prevent or reduce inhibiting beneficial microbial growth and promoting harmful microbial growth.

[0068] Single nucleotide polymorphism BICF2P1175095

[0069] As mentioned above, SNP BICF2P1175095 is located on chromosome 14, 43309715 (chr14:43309715) of the CanFam3.1 reference genome. SEQ ID NO:1 is 201 nucleotides long and shows a sequence including SNP BICF2P1175095 (chr14:43309715) and 100 adjacent nucleotides upstream and downstream of the SNP. Position 101 of SEQ ID NO:1 is the position of the SNP, with the minor allele being C and the major allele being A, indicated as [C / A].

[0070] Nucleotides of SEQ ID NO: 1-201: 5'TATTTGTCTT GAAATTTCAT TATAAGCTTA ATTTTTCCTT GTTGTTGGTA TCAGACTACC GTGTATGCTT GTTTTCTGTT TCCCTCCACG GCAATCTACC[C / A]AAATAAAAT GAGGTGTGGT TCCTTTGTCC TTTCTGTAAC TCTCAGTCCT CCCCCCCACC CCATATCCTT TACTTGAGGA GGGAGACTAC ATCTAATTTG G-3'

[0071] Genomic sequences containing the disclosed SNPs can be accessed in several ways.<ftp: / / webdata2:webdata2@ussdftp.illumina.com / downloads / ProductFiles / CanineHD / CanineHD_B.csv> The purpose of the present invention is to refer to the Illumina dog HD annotation file found at: http: / / www.illumina.com / canine_hd / annotation_files / . Additionally, the chromosome and location defined by the canine reference genome CanFam3.1 for the SNP is chr14:43309715. One of skill in the art can use publicly available interfaces, such as the University of California, Santa Cruz Genome Browser, to identify SNPs of interest and extract flanking DNA sequences using genome browser tools. Additionally, the canine reference genome can be found at:<ftp: / / ftp.ensembl.org / pub / release-94 / fasta / canis_familiaris / dna / > or<http: / / hgdownload.cse.ucsc.edu / goldenPath / canFam3 / bigZips / > or<ftp: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCA / 000 / 002 / 285 / GCA_000002285.2_CanFam3.1> These databases are available from many public sources, including genomic DNA sequences. These databases can be used to extract relevant DNA sequences.

[0072] Methods are provided for detecting CC genotypes associated with an increased likelihood of elevated 4-EPS levels in a canine subject. Methods are provided for identifying dogs at high risk for developing anxiety, stress, and reduced levels of beneficial microorganisms and elevated levels of harmful microorganisms.

[0073] In some embodiments, the sample is a genomic DNA sample.In some embodiments, the sample is obtained from the blood, saliva, hair root, nasal swab, or oral swab of canine subject.In some embodiments, the biological sample is a genomic DNA sample collected from canine subject using a commercially available kit such as PERFORMAgene PG-100 Oral sample collection it (DNA Genotek, OraSure Technologies, Inc., Bethlehem, PA).

[0074] In some embodiments, the method comprises detecting CC genotype. That is, the method comprises detecting the presence of two copies of the minor allele C of SNP BICF2P1175095 (chr14:43309715). In some embodiments, detecting the presence of two copies of the minor allele C of SNP BICF2P1175095 (chr14:43309715) comprises investigating a DNA sample from the dog for the presence of the minor allele C and the major allele A, and detecting the presence of the minor allele and the absence of the major allele A. If the minor allele is detected and the major allele is not detected, it actually detects the presence of two copies of the minor allele.

[0075] In some embodiments, a method for detecting the presence of two copies of the minor allele C of SNP BICF2P1175095 (chr14:43309715) comprises examining a DNA sample from a dog for the presence of the major allele and detecting zero copies of the major allele. Detecting zero copies of the major allele is actually detecting the presence of two copies of the minor allele. That is, looking for the major allele and finding no copies is actually detecting the presence of two copies of the minor allele.

[0076] In some embodiments, the CC genotype is detected using a method comprising at least one nucleic acid analysis technique selected from the following: DNA sequencing, restriction enzyme digestion, polymerase chain reaction (PCR), hybridization, real-time PCR, reverse transcriptase PCR, or ligase chain reaction.

[0077] In some embodiments, the CC genotype is detected by performing at least one nucleic acid analysis technique selected from the group consisting of: analysis using a whole genome SNP chip, single strand conformation polymorphism analysis (SSCP) assay, restriction fragment length polymorphism (RFLP); automated fluorescent sequencing, clamped denaturing gel electrophoresis (CDGE), denaturing gradient gel electrophoresis (DGGE), mobility shift analysis, restriction enzyme analysis, heteroduplex analysis, chemical mismatch cleavage (CMC), RNase protection assay, use of polypeptides that recognize nucleotide mismatches, allele-specific PCR, sequence analysis, and SNP genotyping.

[0078] In some embodiments, the CC genotype is detected using a method selected from the group consisting of hybridization-based methods, enzyme-based methods, post-amplification methods based on the physical properties of DNA, and sequencing methods.

[0079] In some embodiments, the CC genotype is detected using a method selected from the type of methods consisting of: a hybridization-based method selected from the group consisting of dynamic allele-specific hybridization, molecular beacon methods and SNP microarrays; an enzyme-based method selected from the group consisting of restriction fragment length polymorphism (RFLP), PCR-based methods, flap endonuclease, primer extension methods, 5'-nuclease and oligonucleotide ligation assays; a post-amplification method based on the physical properties of DNA selected from the group consisting of single-stranded conformation polymorphism analysis, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high-resolution amplicon melting curve analysis, DNA mismatch binding proteins, SNPlex, and surveyor nuclease assays; and a sequencing method.

[0080] In some embodiments, the CC genotype is detected using a high density array containing genetic markers, including genetic markers for interrogating SNPs.

[0081] In some embodiments, the CC genotype is detected using a low density array containing genetic markers for interrogating the SNPs.

[0082] In some embodiments, CC genotype is detected using high density array containing genetic markers. Examples of arrays include commercially available microarrays such as GeneChip® Canine Genome 2.0 Array (Affymetrix, Thermo Fisher Scientific, Waltham, MA), Dog Genome Microarray (Core Life Sciences, Irvine CA), Illumina Canine HD Panel and additional 50,000-100,000 custom genetic markers (SNPs) (Illumina Canine HD Panel and additional 50,000-100,000 custom genetic markers (SNPs), such as Infinium® iSelect®, Custom Genotyping Assays (Illumina, Inc. San Diego, CA).

[0083] In some embodiments, the MassARRAY system is used to detect the presence of CC genotypes. The MassARRAY system is a non-fluorescent detection platform that utilizes mass spectrometry to accurately measure PCR-derived amplicons. Combining mass spectrometry with end-point PCR allows for highly multiplexed reactions under universal cycling conditions, providing accurate, rapid, and cost-effective analysis. The MassARRAY system offers a unique solution for targeted genetic testing with limited input materials.

[0084] In some embodiments, bead array technology is used to detect the CC genotypes being detected. For example, Illumina BeadArray technology and Infinium HD assay (Illumina, Inc. San Diego, CA) may be used. In some embodiments, bead array technology is used to detect the presence of SNP alleles. Illumina BeadArray technology is based on small silica beads that self-assemble within microwells on a planar silica slide. Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that acts as a capture sequence in the Infinium assay. Once the beads self-assemble, a proprietary decoding process maps the location of each bead, ensuring that each bead is individually quality controlled. As a result of this manufacturing process, every BeadChip undergoes rigorous testing to ensure the highest possible quality standards. Unlike many alternative PCR-dependent assays, the Infinium assay can be scaled to unlimited multiplexing without compromising data quality. A simple, streamlined workflow is common to all products, regardless of the number of SNPs being investigated. Similarly, the data acquisition process and analysis are the same. The Infinium assay protocol features single-tube sample preparation and whole genome amplification without PCR or ligation steps, significantly reducing labor and sample processing errors. After hybridization of unlabeled DNA samples to the Beadchip, two-step allele detection results in high call rates and accuracy. Selectivity and specificity are achieved in two steps. Target hybridization to bead-bound 50mer oligos provides high selectivity, while enzymatic single-base extension also incorporates labeled nucleotides for the assay readout. The staining reagents are optimized to provide higher signals and more balanced intensities between the red and green channels. These features contribute to precision, high call rates, and copy number data with low noise. The Infinium assay generates a dual-color (one color for each allele) readout for each SNP in a genotyping test.The intensity values ​​of each dual-color channel A and B convey information about the allelic ratio at a single genomic locus. A typical study incorporates values ​​from a large number of samples (hundreds to tens of thousands) to ensure meaningful statistical representation. When these values ​​are properly normalized and plotted distinct patterns (or clusters) emerge, it is expected that samples have identical genotypes at the assayed loci, exhibit similar signal profiles (A and B values), and aggregate into clusters. For diploid organisms, biallelic loci are expected to exhibit three clusters (AA, AB, and BB). Genotype calling is based on information derived from a standard cluster file, which provides statistical data from a representative set of samples. This allows genotypes to be called by referencing a single intensity of an assay against known data for a given locus. Since calling accuracy is tied to the quality of the cluster data, an efficient and robust clustering algorithm is essential for accurate genotyping. The Illumina Gebtrain2 algorithm accurately and efficiently identifies and summarizes cluster patterns of genotyped samples.

[0085] SNP alleles may be detected using hybridization-based methods. Examples of hybridization-based methods include dynamic allele-specific hybridization, methods using molecular beacons, and methods using SNP microarrays, including high-density oligonucleotide SNP arrays or low-density oligonucleotide SNP arrays. SNPs can be investigated by hybridizing complementary DNA probes to the SNP sites. In dynamic allele-specific hybridization, genome segments are amplified and bound to beads via a PCR reaction with biotinylated primers. The amplified products are then attached to a streptavidin column and washed to remove the non-biotinylated strand. An allele-specific oligonucleotide is then added in the presence of a molecule that fluoresces when bound to double-stranded DNA. The intensity is measured as the temperature is increased until the melting temperature (Tm) can be determined. SNPs are detected by a lower Tm than expected. Specifically engineered single-stranded oligonucleotide probes are used in SNP detection using molecular beacons. An oligonucleotide is designed with a complementary region at each end and a probe sequence located between them, so that the probe assumes a hairpin or stem-loop structure in its native isolated state. A fluorophore is attached to one end of the probe and a fluorescent quencher is attached to the other end. The fluorophore is in close proximity to the quencher when the oligo is in the hairpin configuration and the molecule does not emit fluorescence. The probe sequence is complementary to the genomic DNA used in the assay. When the probe sequence of the molecular beacon encounters its target genomic DNA during the assay, it anneals and hybridizes. The oligo no longer assumes the hairpin configuration and emits fluorescence. High-density oligonucleotide SNP arrays contain hundreds of thousands of probes arrayed on a small chip, allowing many SNPs to be interrogated simultaneously. Several redundant probes designed to have the SNP site in several different locations, as well as containing mismatches with the SNP alleles, are used to interrogate each SNP.Differential amounts of hybridization of the target DNA to each of these redundant probes allows the determination of specific homozygous and heterozygous alleles.

[0086] CC genotypes may be detected using enzyme-based methods. A wide range of enzymes may be used, including DNA ligases, DNA polymerases, and nucleases. Examples of enzyme-based methods include restriction fragment length polymorphism (RFLP)-based methods, PCR-based methods, methods that utilize flap endonucleases, methods that utilize primer extension, methods that utilize 5'-nucleases, and methods that include oligonucleotide ligation assays. RFLP methods for detecting SNPs use many different restriction endonucleases to digest genomic samples. Whether the enzyme cuts the expected restriction site can be confirmed by determining the fragment length via a gel assay. RFLP assays are designed to include enzymes that cut in the presence or absence of the SNP, and the pattern of fragment lengths can be used to determine the presence or absence of the SNP. PCR-based methods include tetra-primer amplification refractory mutation system PCR, or ARMS-PCR, and multiple qPCR reactions. Tetra-primer amplification refractory mutation system PCR, or ARMS-PCR, uses two pairs of primers to amplify two alleles in one PCR reaction. Primers are designed such that two primer pairs overlap at the SNP position, but each perfectly matches only one of the possible SNPs. Alternatively, multiple qPCR reactions can be performed with different primer sets targeting each allele separately. Some embodiments utilize Flap Endonuclease (FEN), an endonuclease that catalyzes structure-specific cleavage. This cleavage is highly sensitive to mismatches and can be used to interrogate SNPs with high specificity. FEN, called Cleavase, can combine with two specific oligonucleotide probes and form a triple altite structure with the target DNA that is recognized by Cleavase. The first probe, called Invader oligonucleotide, is complementary to the 3' end of the target DNA. The last base of the Invader oligonucleotide is a non-matching base that overlaps with the SNP nucleotide in the target DNA.The second probe is an allele-specific probe that is complementary to the 5' end of the target DNA but also extends beyond the 3' side of the SNP nucleotide. The allele-specific probe contains a base that is complementary to the SNP nucleotide.

[0087] Primer extension is a two-step process that first involves hybridization of a probe to the base immediately upstream of the SNP nucleotide, followed by DNA polymerase extending the hybridization primer by adding a base complementary to the SNP nucleotide. This incorporated base is detected and the SNP allele is determined. Primer extension methods are used in several assay formats. These formats use a wide range of detection techniques, including methods such as MALDI-TOF mass spectrometry (see Sequenom) and ELISA. Sequenom's iPLEX SNP genotyping method uses a MassARRAY mass spectrometer. The flexibility and specificity of primer extension makes it suitable for high-throughput analysis. Primer extension probes can be arrayed on a slide, allowing many SNPs to be genotyped at once. This technique, called arrayed primer extension (APEX), has several advantages over methods based on differential hybridization of probes.

[0088] Illumina Incorporated's Infinium assay is an example of a whole genome genotyping pipeline based on primer extension. The Infinium assay can genotype over 100,000 SNPs. The assay uses hapten-labeled nucleotides in a primer extension reaction. The hapten label is recognized by an antibody, which is then coupled to a detectable signal. APEX-2 is an array primer extension genotyping method that can identify hundreds of SNPs or mutations in parallel using efficient homogeneous multiplex PCR (up to 640-plex) and four-color single-base extension on a microarray. Multiplex PCR requires two oligonucleotides per SNP / mutation and generates an amplicon containing the tested base pair. Methods that utilize 5'-nuclease include using the 5' nuclease activity of Taq DNA polymerase in the TaqMan assay for SNP genotyping. The TaqMan assay is performed simultaneously with the PCR reaction, allowing the results to be read in real time as the PCR reaction progresses. In the method involving oligonucleotide ligation assay, oligonucleotide DNA ligase catalyzes the ligation from the 3' end of a DNA fragment to the 5' end of the directly adjacent DNA fragment. This mechanism can be used to investigate SNPs by hybridizing two probes directly on the SNP polymorphic site, so that ligation can occur if the probes are identical to the target DNA. Examples of other post-amplification methods for detecting SNPs include methods based on the physical properties of DNA. Such methods first involve PCR amplification of the target DNA.

[0089] Some methods to detect SNP alleles are based on physical properties of DNA, such as melting temperature and single-stranded conformation. Methods using single-stranded conformation are based on single-stranded DNA (ssDNA) folding into a tertiary structure. The conformation is sequence-dependent, and most single base pair mutations change the shape of the structure. When applied to a gel, the tertiary shape determines the mobility of the ssDNA, providing a mechanism to distinguish SNP alleles. The method first involves PCR amplification of the target DNA. Double-stranded PCR products are denatured using heat and formaldehyde, producing ssDNA. The ssDNA is applied to a non-denaturing electrophoretic gel and allowed to fold into a tertiary structure. Differences in DNA sequence change the tertiary structure and are detected as differences in the mobility of the ssDNA strands. Temperature gradient gel electrophoresis (TGGE) or temperature gradient capillary electrophoresis (TGCE) methods are based on the principle that partially denatured DNA is more confined and migrates slower in a gel or other porous material. In another method, denaturing high performance liquid chromatography (DHPLC) uses reversed-phase HPLC to survey SNPs. In DHPLC, the solid phase has differential affinity for single-stranded and double-stranded DNA. Another method used is high-resolution melting of the entire amplicon. DNA mismatch-binding proteins may be used to detect SNPs. The MutS protein from Thermus aquaticus binds different single-base mismatches with different affinities and can be used in capillary electrophoresis to distinguish all six pairs of mismatches. SNPlex is a proprietary genotyping platform sold by Applied Biosystems. The Surveyor Nuclease assay uses Surveyor Nuclease, a mismatch endonuclease enzyme that recognizes all base substitutions and small insertions / deletions (indels) and cleaves 3' to the mismatch site on both DNA strands. Sequencing techniques can also be used for SNP detection. Advances in sequencing technology make SNP detection by sequencing more practical.

[0090] Genotyping by sequencing using next-generation sequencing technologies has become a common practice. Genotyping by sequencing, also known as GBS, is a method to discover single nucleotide polymorphisms (SNPs) to conduct genotyping studies such as genome-wide association studies (GWAS). GBS uses restriction enzymes to reduce the complexity of the genome and genotype multiple DNA samples. After digestion, PCR is performed to increase the fragment pool, and then the GBS library is sequenced using next-generation sequencing technologies. With the advancement of next-generation sequencing technologies such as Illumina short-read sequencing by synthesis and PacBio single-molecule real-time sequencing, it is becoming more feasible to perform GBS. In the future, the development of new technologies such as nanopore single-molecule sequencing may enable whole genome sequencing / genotyping.

[0091] Compositions and Formulations

[0092] Application of the methodology outlined above has identified bioactive dietary components that are combined to provide compositions, foods, and diets that provide significant benefit to dogs that have been identified as predisposed to having elevated 4-EPS levels and therefore at high risk for developing anxiety disorders, at high risk for developing anxiety stress, and at high risk for inhibiting the growth of beneficial microorganisms and promoting the growth of harmful microorganisms in the canine microbiota, particularly the gut microbiota.

[0093] Methods are provided that include feeding the dog a daily diet comprising tomato pomace in an amount equal to 0.044%-0.42% of the total daily nutritional intake. In some embodiments, methods are provided that include feeding the dog a daily diet comprising tomato pomace in an amount equal to 0.066%-0.315% of the total daily nutritional intake. In some embodiments, methods are provided that include feeding the dog a daily diet comprising tomato pomace in an amount equal to 0.087%-0.21% of the total daily nutritional intake. In some embodiments, methods are provided that include feeding the dog a daily diet comprising tomato pomace in an amount equal to 0.14% of the total daily nutritional intake.

[0094] In some embodiments, the food is a nutritionally complete diet for adult dogs. In certain aspects, the food is a nutritionally complete diet formulated for mature companion dogs.

[0095] In some embodiments, the compositions include food compositions that may include an effective amount of tomato pomace in combination with protein in an amount of from 4% to 75% or more by weight of the total composition on a dry matter basis, fat in an amount of from 5% to 50% or more by weight of the total composition on a dry matter basis, and carbohydrates in an amount of from 5% to 75% or more by weight of the total composition on a dry matter basis, wherein the food composition is suitable for consumption by a dog.

[0096] The compositions administered in the methods provided herein may, in certain embodiments, be formulated as food compositions that are nutritionally balanced and / or nutritionally complete foods or meals, hi other embodiments, the compositions are formulated and prepared as dietary supplements, treats, or toys.

[0097] In some embodiments, for example, in addition to an effective amount of tomato pomace, a nutritionally complete and balanced dog food composition may include 4%-90%, 5%-75%, 10%-60%, and 15%-50% protein, 0%-90%, 2%-80%, 5%-75%, and 10%-50% carbohydrate, 2%-60%, 5%-50%, and 10%-35% fat. The composition may further contain 0-15% or 2%-8% vitamins and minerals, antioxidants, and other nutrients that support the nutritional needs of the animal.

[0098] Sources of protein, carbohydrates, fats, vitamins, minerals, balancing agents, and the like suitable for inclusion in the compositions, particularly in foods administered in the methods provided herein, may be selected from conventional materials known to those of skill in the art.

[0099] In some embodiments, proteins useful as ingredients in food compositions are proteins from animals, such as animal proteins including mammalian proteins, avian proteins, reptile, amphibian, fish, invertebrate proteins, and combinations thereof; for example, from cows, sheep, pigs, goats, deer, rabbits, horses, kangaroos, their milk, curds, whey, or blood, and organs such as smooth muscle, striated muscle, liver, kidney, intestine, or heart; additional avian protein sources include turkeys, geese, ducks, ostriches, quails, pigeons, their eggs, and organs such as smooth muscle, striated muscle, liver, kidney, intestine, or heart. amphibian sources include frogs or salamanders, reptile protein sources include alligators, lizards, turtles and snakes, fish protein sources include catfish, herring, salmon, tuna, bluefish, cod, halibut, trout, swordfish and their eggs, invertebrate protein sources may include proteins such as lobster, crab, clams, mussels or oysters and combinations thereof, meat protein isolate, whey protein isolate, egg protein, mixtures thereof and the like, as well as soy protein isolate, corn gluten meal, wheat gluten, mixtures thereof and the like.

[0100] In some embodiments, carbohydrates useful as ingredients of the food composition may include, but are not limited to, one or more of corn, whole yellow corn, grain sorghum, wheat, barley, rice, millet, sake rice extract, oatmeal, and polysaccharides (e.g., starch and dextrin), and sugars that are metabolized to energy when hydrolyzed (e.g., sucrose, lactose, maltose, glucose, and fructose). Examples of additional carbohydrate sources suitable for inclusion in the compositions disclosed herein include fruits and non-tomato pomace vegetables.

[0101] Fats useful as ingredients in the food composition may be from any source, including but not limited to poultry fat, beef tallow, lard, choice white grease, soybean oil, corn oil, canola oil, sunflower oil, mixtures thereof, etc. The fat may be fully incorporated within the food composition, deposited on the outside of the food composition, or a mixture of the two methods.

[0102] In some embodiments, the composition further comprises an effective amount of one or more substances selected from the group consisting of glucosamine, chondroitin, chondroitin sulfate, methylsulfonylmethane (MSM), creatine, antioxidants, Perna canaliculata, omega-3 fatty acids, omega-6 fatty acids, and mixtures thereof.

[0103] In some embodiments, the food composition further comprises one or more amino acids, such as, but not limited to, arginine, histidine, isoleucine, leucine, lysine, methionine (including DL-methionine and L-methionine), phenylalanine, threonine, tryptophan, valine, taurine, carnitine, alanine, aspartate, cystine, glutamate, glutamine, glycine, proline, serine, tyrosine, and hydroxyproline.

[0104] In some embodiments, the food composition further comprises one or more fatty acids, such as, but not limited to, lauric acid, myristic acid, palmitic acid, palmitoleic acid, margaric acid, margaroleic acid, stearic acid, oleic acid, linoleic acid, g-linoleic acid, a-linoleic acid, stearidonic acid, arachidic acid, gadoleic acid, DHGLA, arachidonic acid, eicosatetraacetic acid, EPA, behenic acid, erucic acid, docosatetraacetic acid, and DPA.

[0105] In some embodiments, the food composition further comprises one or more macronutrients, such as, but not limited to, moisture, protein, fat, crude fiber, ash, dietary fiber, soluble fiber, insoluble fiber, raffinose, and stachyose.

[0106] In some embodiments, the food composition further comprises one or more micronutrients, such as, but not limited to, beta-carotene, alpha-lipoic acid, glucosamine, chondroitin sulfate, lycopene, lutein, and quercetin.

[0107] In some embodiments, the food composition further comprises one or more minerals, such as, but not limited to, calcium, phosphorus, potassium, sodium, chlorine, iron, copper, manganese, zinc, iodine, selenium, cobalt, sulfur, fluorine, chromium, boron, and oxalate.

[0108] In some embodiments, the food composition further comprises one or more other vitamins, such as, but not limited to, vitamin A, vitamin C, vitamin D, vitamin E, quinoa grain, thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, folic acid, vitamin B12, biotin, and choline.

[0109] In some embodiments, the food composition further comprises fiber, which may be provided from a variety of sources, including, for example, cellulose, beet pulp, peanut shells, and soy fiber.

[0110] In some embodiments, food composition further comprises stabilizing material, for example, material that tends to extend the shelf life of composition.Potentially suitable examples of such material include, for example, preservatives, antioxidants, synergists and scavengers, packaging gases, stabilizers, emulsifiers, thickeners, gelling agents, and humectants.Examples of emulsifiers and / or thickeners include, for example, gelatin, cellulose ethers, starches, starch esters, starch ethers, and modified starches.

[0111] In some embodiments, the food composition may contain additives intended for color, palatability, and nutritional purposes, such as colorants, iron oxides, sodium chloride, potassium citrate, potassium chloride, and other edible salts, vitamins, minerals, and flavors. The amount of such additives in the composition is generally up to 5% (based on the dry weight of the composition).

[0112] Preparation of the Composition

[0113] Compositions containing tomato pomace may be prepared as foods suitable for consumption by dogs. These foods may be of any consistency or moisture content, i.e., the compositions may be moist, semi-moist, or dry foods. "Moist" foods are generally foods with a moisture content of 60% to 90% or more. "Dry" foods are generally foods with a moisture content of 3% to 11% and are often produced in the form of pieces or kibbles. "Semi-moist" foods generally have a moisture content of 25% to 35%. Foods may also include components with one or more consistencies, such as, for example, soft, chewy meat-like particles or pieces, and kibbles with an outer grain component or coating and an inner "creamy" component.

[0114] In some embodiments, foods containing tomato pomace may be prepared in canned or wet form using conventional food preparation processes known to those skilled in the art. Typically, ground animal protein tissue is mixed with other ingredients such as grains, suitable carbohydrate sources, fats, oils, and preparation ingredients, including vitamin and mineral mixtures, inorganic salts, cellulose, beet pulp, etc., and a sufficient amount of water for processing. These ingredients are mixed in a vessel suitable for providing heating during blending of the ingredients. Heating of the mixture can be achieved using any suitable method, such as, for example, by direct steam injection or by using a vessel equipped with a heat exchanger. After addition of all the ingredients of the formulation, the mixture is heated to a temperature between 50°F and 212°F. Temperatures outside this range can be used, but may not be commercially practical without other processing aids. Once heated to the appropriate temperature, the material is usually in the form of a viscous liquid and is dispensed into cans. A lid is attached and the container is sealed. The sealed can is then placed in conventional equipment designed to sterilize the contents. Pasteurization can be achieved by heating, usually to temperatures above about 230° C., for an appropriate time, depending on the temperature and composition used and related factors. The compositions and foods of the present invention may also be added to or combined with food compositions before, during, or after their preparation.

[0115] In some embodiments, the food may be prepared in dry form using conventional processes known to those skilled in the art. Typically, dry ingredients, including dry animal protein, vegetable protein, grains, etc., are ground and mixed together. Liquid or wet ingredients, including fats, oils, water, animal protein, water, etc., are added and mixed with the dry materials. The particular formulations, order of addition, combinations, and methods and equipment used to combine the various ingredients can be selected from those known in the art. For example, in certain embodiments, the resulting mixture is processed into kibbles or similar dry pieces, formed using an extrusion process, in which the mixture of dry and wet ingredients is subjected to mechanical work at high pressure and high temperature, forced through small openings or apertures, and cut into kibbles, for example, with a rotating knife. The resulting kibbles are dried and optionally coated with one or more topical coatings, including, for example, flavors, fats, oils, powders, etc. Kibbles can also be made from dough using a baking process rather than extrusion, in which the dough is placed in a mold followed by a dry heating process.

[0116] When preparing the composition, optional ingredients can generally be incorporated into the composition during the compounding process, e.g., during and / or after mixing with other ingredients of the composition. Distribution of these ingredients into the composition can be accomplished by conventional means. In certain embodiments, comminuted animal and / or poultry proteinaceous tissues are mixed with other ingredients including nutritional balancing agents, inorganic salts, and further mixed with other ingredients including cellulose, beet pulp, fillers, etc. with sufficient water for processing.

[0117] In some embodiments, the compositions are formulated to be more chewable. In certain embodiments, the compositions and foods are formulated to address one or more of the specific nutritional differences between animal species and breeds, as well as animal attributes. For example, dog foods, for example, are typically formulated based on life stage, age, size, weight, body composition, and breed.

[0118] The composition comprising an effective amount of tomato pomace is formulated as a nutritionally complete diet to meet the needs of mature adult dogs.A nutritionally complete diet is a diet that can contain sufficient nutrients to maintain the normal health of a healthy animal with respect to diet.Nutritionally complete and balanced pet food compositions, such as for companion dogs, are well known to those skilled in the art.For example, substances such as nutrients and ingredients suitable for nutritionally complete and balanced animal feed compositions, and their recommended amounts, include the discussion of supplements in, for example, Official Publication of The Association of American Feed Control Officials, Inc. (AAFCO), Atlanta, Ga. (2012).

[0119] In another embodiment, the treats comprising an effective amount of tomato pomace can be prepared, for example, by extrusion or baking processes similar to those described below for dry food, to provide an edible product. Treats include, for example, compositions that are given to animals during non-meal times to entice them to eat. Treats can be nutritious, and the compositions can include one or more nutrients and have, for example, the compositions described above for food. Non-nutritional treats include any other treats that are non-toxic. The compositions can be coated on the therapeutic agent, incorporated into the therapeutic agent, or both.

[0120] In another embodiment, an animal toy is provided that is a chewable or consumable toy. Such toys are typically prepared by coating any existing toy with an effective amount of tomato pomace. Thus, toys include, for example, chewable toys. Toys intended for dogs include, for example, artificial bones. In certain embodiments, the composition of the present invention can form a coating on the surface of the toy or on the surface of a toy part, can be incorporated into a part of the toy, or the entire toy, or both. There is a wide range of suitable toys currently available on the market. See, for example, U.S. Pat. No. 5,339,771 (and references disclosed in U.S. Pat. No. 5,339,771). Also, see, for example, U.S. Pat. No. 5,419,283 (and references disclosed in U.S. Pat. No. 5,419,283). It should be recognized that the present invention contemplates both partially consumable toys (e.g., toys that include plastic components) and fully consumable toys (e.g., rawhide and various artificial bones). It should further be appreciated that the present invention contemplates a toy for use by companion animals, particularly dogs or cats.

[0121] All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing materials and methodology reported in the publications that might be used in connection with the present invention.

[0122] Further areas of applicability of the present invention will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. EXAMPLES

[0123] Example 1

[0124] Blood is drawn to determine the plasma metabolic profile. 4-EPS levels in plasma can be measured by a commercial testing laboratory (Metabolon, Durham, NC, USA). The extracted supernatant is split and run on gas chromatography and liquid chromatography mass spectrometry platforms. The peak for 4-EPS is known, and the area under the peak for each sample can be normalized to the known sample. (See also, e.g., Evans, A.M., et al. (2009). An integrated non-targeted ultra-high performance liquid chromatography / electrospray ionization tandem mass spectrometry platform for identification and relative quantification of small molecule complements in biological systems. Anal. Chem. 81, 6656-6667.) Gas chromatography (for hydrophobic molecules) and liquid chromatography (for hydrophilic molecules) are used to identify and provide relative quantification of metabolites such as 4-EPS present in the plasma sample. (See also, for example, Ballet, C. et al. (2018) New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites, Chem. Sci., 9, 6233-6239; Akiyama, Y et al. (2012) A Metabolomic Approach to Clarifying the Effect of AST-120 on 5 / 6 Nephrectomized Rats by Capillary Electrophoresis with Mass Spectrometry (CE-MS) Toxins 4(11), 1309-1322; and Kikuchi K, et al. (2010) Metabolomic search for uremic toxins as indicators of the effect of an oral sorbent AST-120 by liquid chromatography / tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 878:2997-3002.)

[0125] Example 2

[0126] Nucleotide-binding oligomerization domain 1, called NOD1, is an intracellular sensor that detects small peptides derived from cell wall components of the gut microbiota, which triggers innate immune responses. NOD1 activation-mediated innate immune responses are involved in both host defense against microbial infections and the development of gastrointestinal disorders.

[0127] Genome-wide association study (GWAS) showing an association between SNP BICF2P1175095 located upstream of the NOD1 gene and circulating levels of 4-EPS. Dogs with the CC genotype are at increased risk for impaired gut colonization by certain beneficial microorganisms. This may lead to the proliferation of harmful microorganisms that can affect distal organs such as the brain via certain microbial metabolites that enter the systemic circulation. 4-EPS, a known stress-related microbial metabolite, is detected nine times more frequently in dogs with the CC genotype.

[0128] An effective amount of tomato pomace can reduce 4-EPS levels in dogs, and therefore tomato pomace is particularly beneficial for dogs with the CC genotype.

[0129] Example 3

[0130] Forty dogs were randomly assigned to a control (20 dogs) or experimental group (20 dogs) and completed a 30-day study on either a basal diet containing tomato pomace or a diet without tomato pomace. After a one-month washout period, a crossover was performed to feed the groups with the previously unfed diet. After 30 days, blood samples were taken to measure 4-ethylphenyl sulfate levels by metabolomics. Thus, all dogs received either a diet with or without tomato pomace for 30 days and completed metabolomic analysis at the end of each 30-day feeding period.

[0131] These results suggest that dogs that tend to have high 4-EPS may benefit from consuming a food containing certain levels of tomato pomace.Matched-pair analysis comparing the levels of 4-EPS in each dog after food intake showed a significant decrease in 4-EPS with the addition of tomato pomace (P = 0.04) (Figure 1).

[0132] Daily intakes of tomato pomace leading to low levels of 4-EPS are shown in Table 1. The lowest levels of 4-EPS were achieved with an average daily intake of 0.14% tomato pomace on a dry matter basis, which corresponds to 0.24 g tomato pomace per 172 g total per day. Daily consumption of 0.087% to 0.21% tomato pomace on a dry matter basis, which corresponds to 0.15 to 0.35 g tomato pomace per 172 g total per day, resulted in lower circulating 4-EPS levels. [Table 1]

[0133] When provided in an effective amount, tomato pomace reduces circulating levels of 4-EPS. Anti-stress foods for pets can be formulated by including an effective amount of tomato pomace, thereby reducing blood levels of 4-EPS, a microbial toxin, the increases of which are associated with stress, anxiety, and brain damage. Such pet foods thereby address stress-related issues associated with elevated circulating levels of 4-EPS in pets.

[0134] Example 4

[0135] A saliva sample is obtained from the dog. The sample may be shipped as collected to a laboratory at another location, partially processed and then shipped to a laboratory at another location, or may be fully processed and analyzed at the laboratory and collection location. If the sample is shipped as collected to a laboratory at another location, partially processed and then shipped to a laboratory at another location, some or all of the data collected from the sample by the laboratory may be sent to the collection location and / or to the veterinarian and / or the dog's owner or responsible person. After the saliva sample is obtained, it may be processed for analysis and evaluated for the presence of the CC genotype.

[0136] If the results indicate that the dog is likely or at high risk of developing elevated 4-EPS levels, the dog may be administered a composition comprising an effective amount of tomato pomace.

[0137] Example 5

[0138] Samples are collected from dogs using a PERFORMAgene PG-100 oral collection kit.

[0139] In this regard, the animal should not eat for 30 minutes or drink for 10 minutes prior to saliva collection, and the individual performing the collection cannot scrape the animal's teeth or cheeks with a sponge or allow the animal to chew on the sponge.

[0140] The collection tube provided as part of the PERFORMAgene PG-100 Oral Collection Kit contains a liquid that preserves the DNA sample and is required by the lab to analyze the sample. The cap should not be removed prior to sample collection.

[0141] In the first step of the collection protocol, a sponge is placed in the cheek pouch of the animal's mouth. Saliva is collected for 30 seconds by removing the sponge and swabbing saliva in areas where it naturally accumulates (cheek pouch and under the tongue). For animals older than 6 months, moderate restraint may be required.

[0142] Next, hold the tube upright and the cap from the tube is not screwed on. Turn the cap upside down and place the buccal swab into the tube. The cap is screwed on tightly to prevent leakage of the liquid sample during transport. Invert the tube and shake vigorously multiple times, for example 10 times, to thoroughly mix the sample.

[0143] A permanent marker may be used to clearly write the animal identification number in the blank space on the tube label.

[0144] A step-by-step laboratory protocol for manual purification of DNA from 0.5 mL aliquots of Performagene™ samples collected and stored with Performagene chemistry using the PG-100 collection kit is as follows: Reagents required for manual purification are available in the PG-AC1 or PG-AC4 reagent packages.

[0145] When a DNA sample is collected and mixed with the Performagene solution, the DNA is immediately stabilized. Performagene samples are stable at room temperature for one year from the time of collection. Performagene samples can be stored indefinitely at -15°C to -20°C and can undergo multiple freeze-thaw cycles without degradation of the DNA.

[0146] For the purification process, use the following equipment and reagents: a microcentrifuge capable of running at 15,000 x g, an air or water incubator at 50 °C, ethanol (95%-100%) at room temperature, DNA buffer: TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or similar solution, glycogen of choice (20 mg / mL) (e.g., Invitrogen Cat. No. 10814-010), ethanol (70%) at room temperature and 5 M NaCl solution.

[0147] In the first step, the sample is mixed by shaking vigorously for 5 seconds to ensure that the viscous sample mixes properly with the Performagene solution.

[0148] The samples are incubated for a minimum of 2 hours in a 50°C air incubator or 1 hour in a 50°C water incubator. The DNA in Performagene is stable at room temperature without an incubation step. This heat treatment step is essential to ensure that the DNA is properly released and that the nucleases are permanently inactivated. This incubation step can be performed at any time after the sample is taken from the animal and before it is purified. Incubation of the entire sample is recommended. Samples can be incubated overnight at 50°C if more convenient. Longer times are required in air incubators because temperature equilibration is slower than in water incubators.

[0149] Optionally, the collection sponge may be removed. The cap is removed and the collection sponge is pressed against the inside of the tube to extract as much sample as possible. The sponge and cap are discarded. Removal of the sponge is determined by workflow preference.

[0150] Next, 500 μL of the mixed Performagene sample is transferred to a 1.5 mL microcentrifuge tube. The remainder of the Performagene sample can be stored at room temperature or frozen (-15°C to -20°C). Then, 20 μL (1 / 25 volume) of PG-L2P purifier is added to the Eppendorf tube and mixed by vortexing for a few seconds. The sample will become cloudy as impurities and inhibitors precipitate.

[0151] Incubate the sample on ice for 10 min (room temperature incubation is substituted but slightly less effective in removing impurities) and then centrifuge at 15,000 × g for 5 min at room temperature. Longer centrifugation (up to 15 min) may be beneficial to reduce the turbidity (high A320) of the final DNA solution. Transfer the clear supernatant with a pipette tip to a fresh microcentrifuge tube and discard the pellet containing the turbid impurities. To 500 μL of the supernatant, add 25 μL (1 / 20 volume) of 5 M NaCl, followed by mixing. The addition of NaCl is necessary to ensure efficient recovery of DNA. To 500 μL of the supernatant, add 600 μL of room temperature 95%-100% ethanol, followed by gentle mixing by inversion 10 times. During mixing with ethanol, the DNA precipitates. The DNA may appear as clumps of DNA fibers or as a fine precipitate, depending on the amount of DNA in the sample. Even if no clumps are seen, recover the DNA by carefully following the next steps.

[0152] The samples are left at room temperature for 10 minutes to allow the DNA to fully precipitate. The tubes are then placed in the centrifuge in a known orientation (the DNA pellet may not be visible after centrifugation) and centrifuged at >15,000 x g for 2 minutes at room temperature. For example, each tube may be placed in a microcentrifuge with the hinged portion of the cap pointing away from the center of the rotor. After centrifugation, the position of the pellet can be located (even if it is too small to be easily seen) and is at the tip of the tube below the hinge.

[0153] The supernatant is removed with a pipette tip and discarded. The pellet contains the DNA. By rotating the tube so that the pellet is on the upper wall, the pipette tip can be safely moved along the lower wall and all of the supernatant can be removed. The supernatant may contain impurities and should be removed as completely as possible. Over-drying of the pellet can make dissolving the DNA more difficult. The DNA is washed by first adding 250 μL of 70% ethanol, then left at room temperature for 1 min. The ethanol is removed with a pipette tip without disturbing the pellet. The 70% ethanol wash helps to remove residual inhibitors. However, complete removal of the ethanol is essential to prevent inhibition during downstream applications. Therefore, the tube is centrifuged for 6 s to pool any remaining ethanol and remove it with a pipette tip.

[0154] Add 100 μL of DNA buffer (e.g., TE buffer) to the tube to dissolve the DNA pellet. Vortexing for at least 5 seconds aids in the dissolution process. Leave at room temperature overnight to ensure complete rehydration of the DNA. DNA can be quantified and used in downstream applications.

[0155] Assays using fluorescent dyes are more specific than absorbance at 260 nm to quantify the amount of double-stranded DNA (dsDNA) in a DNA sample. To quantify DNA by fluorescence methods, fluorescent dyes such as PicoGreen® or SYBR® Green I may be used to quantify dsDNA, as there is less interference from contaminating RNA. Alternatively, commercially available kits such as Invitrogen's Quant-iT™ PicoGreen dsDNA Assay Kit (Cat. No. Q-33130) may be used. In both protocols, purified DNA is preferably diluted 1:50 in TE solution, and 5 μL is used in the quantitative assay.

[0156] Alternatively, DNA may be quantified by absorbance, in which case the purified sample is preferably first treated with RNase to digest contaminating RNA, followed by removal of RNA fragments by ethanol precipitation of the DNA. DNA from Performagene samples contains clearly more RNA than that usually found in blood samples. Ensure that the alcohol-precipitated DNA is completely dissolved, then read the absorbance. An absorbance of 1.0 at 260 nm corresponds to a concentration of 50 ng / μL (50 μg / mL) of pure dsDNA. To avoid using excessively large volumes of sample, a spectrophotometer cuvette capable of reading samples with a volume of 100 μL or less should be used. Absorbance values ​​at 260 nm should be between 0.1 and 1.5. Lower values ​​may be less reliable.

[0157] Dilute a 10 μL aliquot of purified RNase-treated DNA with 90 μL of TE (1 / 10 dilution) and mix by gently pipetting up and down. Wait for air bubbles to disappear. TE is used in the reference (blank) cell. Absorbance is measured at 320 nm, 280 nm, and 260 nm. The corrected A 280 and A 260 The value is A 280 and A 260 value to 320 nm (A 320 ) and subtract the absorbance at 0.5 μg / μL. DNA concentration in ng / μL = Corrected A 260 × 10 (dilution factor) × 50 (conversion factor). A 260 / A 280 Ratio: Corrected A 260 Corrected A 280 Divide by. Example 6

[0158] Genome-wide DNA analysis using Illumina BeadChip technology and the Infinium HD assay can be used to detect CC genotypes. Preferably, the test is part of a panel that includes interrogation of one or more other clinically significant SNPs.

[0159] Starting with a sample containing purified genomic DNA (200-400 ng), the sample undergoes PCR-free whole genome amplification to generate fragmented DNA interrogated for SNPs. CC genotypes, the Illumina Canine HD Panel, and an additional 50,000-100,000 custom genetic markers (SNPs) (The Illumina Canine HD Panel, Illumina, Inc. San Diego, CA) may be used. Such a system can be used to interrogate samples for the presence of CC genotypes, or BeadArray technology can be customized to limit the number of SNPs for screening.

[0160] Illumina BeadArray technology is based on tiny silica beads that self-assemble within microwells on a planar silica slide. Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that acts as a capture sequence in the Infinium assay. Once the beads self-assemble, a proprietary decoding process maps the location of each bead, ensuring that each bead is individually quality-controlled. As a result of this manufacturing process, every BeadChip undergoes rigorous testing to ensure the highest possible quality standards. Unlike many alternative PCR-dependent assays, the Infinium assay can be scaled to unlimited multiplexing without compromising data quality. A simple, streamlined workflow is common across all products, regardless of the number of SNPs being investigated. Similarly, the data acquisition process and analysis are the same. The Infinium assay protocol features single-tube sample preparation and whole genome amplification without PCR or ligation steps, significantly reducing labor and sample handling errors. After hybridization of unlabeled DNA samples to the Beadchip, two-step allele detection results in high call rates and accuracy. Selectivity and specificity are achieved in two steps. Target hybridization to bead-bound 50mer oligos provides high selectivity, while enzymatic single-base extension also incorporates labeled nucleotides for assay readout. Staining reagents are optimized to provide higher signal and more balanced intensity between red and green channels. These features contribute to precision, high call rates, and copy number data with low noise. Infinium assays generate a dual-color readout for each SNP (one color for each allele) in genotyping studies. Intensity values ​​of each dual-color channel A and B convey information about the allele ratio at a single genomic locus. A typical study incorporates values ​​from a large number of samples (hundreds to tens of thousands) to ensure meaningful statistical representation.When these values ​​are properly normalized and plotted distinct patterns (or clusters) emerge, samples have identical genotypes at the assayed loci, exhibit similar signal profiles (A and B values), and aggregate into clusters. For diploid organisms, biallelic loci are expected to exhibit three clusters (AA, AB, and BB). Genotype calling is based on information derived from standard cluster files, which provide statistical data from a representative set of samples. This allows genotypes to be called by referencing a single intensity of an assay against known data for a given locus. Since calling accuracy is tied to the quality of the cluster data, efficient and robust clustering algorithms are essential for accurate genotyping. The Illumina Gebtrain2 algorithm accurately and efficiently identifies and summarizes cluster patterns for genotyped samples.

[0161] Example 7

[0162] A daily diet containing tomato pomace provides significant benefits to dogs identified as having high levels of 4-EPS. In some embodiments, the method can include feeding a daily diet containing an effective amount of tomato pomace to a dog suspected of having elevated 4-EPS. In some embodiments, the method can include feeding a daily diet containing an effective amount of tomato pomace to a dog identified as having elevated 4-EPS. In some embodiments, the method can include measuring 4-EPS in a dog to identify the dog as having elevated 4-EPS, and feeding the dog a daily diet containing an effective amount of tomato pomace.

[0163] In some embodiments, the method may include measuring 4-EPS in a dog to identify the dog as having elevated 4-EPS, comparing the measured 4-EPS level with a positive reference standard, and feeding the dog a daily diet containing an effective amount of tomato pomace. The positive reference standard corresponds to a 4-EPS level that is considered to be elevated, particularly for dogs of comparable size, weight, age, and breed. If the measured value is equal to or greater than the positive reference standard, the dog is identified as having elevated 4-EPS and is treated by feeding the dog a daily diet containing an effective amount of tomato pomace.

[0164] In some embodiments, the method may include measuring 4-EPS in a dog to identify the dog as having elevated 4-EPS, comparing the measured 4-EPS level of the dog subject with the measured 4-EPS level of a positive control sample, and feeding the dog a daily diet containing an effective amount of tomato pomace.The positive control sample is a representative sample that has a concentration of 4-EPS that is considered to be high for dogs of a similar size, weight, age, and breed.If the measured 4-EPS level of the dog subject is equal to or greater than the measured 4-EPS level of the positive control sample, the dog is identified as having elevated 4-EPS and is treated by feeding the dog a daily diet containing an effective amount of tomato pomace.

[0165] In some embodiments, the dog may be initially identified as exhibiting or suspected of anxiety, anxiety disorder, or symptoms of anxiety or anxiety disorder. The dog may then be tested to determine whether it has elevated 4-EPS levels by one of the methods described herein. If the results show that the dog exhibits elevated 4-EPS levels, it is treated by feeding it a daily diet containing an effective amount of tomato pomace.

[0166] In some embodiments, the dog may be initially identified as exhibiting or suspected of stress, stress disorder, or symptoms of stress or stress disorder. The dog may then be tested to determine whether it has elevated 4-EPS levels by one of the methods described herein. If the results show that the dog exhibits elevated 4-EPS levels, it is treated by feeding it a daily diet containing an effective amount of tomato pomace.

[0167] Example 8

[0168] The compositions below are based on total nutritional amounts provided per day.

[0169] In some embodiments based on the total weight of the composition on a dry matter basis, the amount of tomato pomace is 0.087-0.5%. In some embodiments based on the total weight of the composition on a dry matter basis, the amount of tomato pomace is 0.044% to about 0.42%. In some embodiments based on the total weight of the composition on a dry matter basis, the amount of tomato pomace is 0.066% to about 0.315%. In some embodiments based on the total weight of the composition on a dry matter basis, the amount of tomato pomace is 0.087-0.21%. In some embodiments based on the total weight of the composition on a dry matter basis, the amount of tomato pomace is about 0.14%.

[0170] In certain embodiments, the composition may include chicken in an amount of 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, or 25% based on the total weight of the composition on a dry matter basis. In certain embodiments, the composition may include egg protein in an amount of 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% based on the total weight of the composition on a dry matter basis. In certain embodiments, the composition may include corn gluten meal in an amount of 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% based on the total weight of the composition on a dry matter basis. In certain embodiments, the composition may comprise an additional plant source in an amount of 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, or 1.9%, or 2.0%, based on the total weight of the composition on a dry matter basis. In certain embodiments, the composition may comprise an additional fruit source in an amount of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%, based on the total weight of the composition on a dry matter basis, in addition to tomato pomace. In certain embodiments, the composition may comprise a carbohydrate selected from millet, sake rice, oat groats, and combinations thereof in an amount of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% based on the total weight of the composition on a dry matter basis. In certain aspects of these embodiments, the compositions of the invention may comprise a dry weight of a carbohydrate source within a range defined by any two of these values ​​as endpoints.

[0171] Example 9

[0172] Table 2 lists certain embodiments with compositional percentages (% of dry weight of component compositions).

[0173] [Table 2]

[0174] A daily diet comprising an effective amount of tomato pomace may provide benefit to dogs identified as stressed. In some embodiments, the method comprises identifying a dog having or suspected of having stress, a stress disorder, or suspected of exhibiting symptoms of stress or a stress disorder, and feeding it a daily diet comprising an effective amount of tomato pomace.

[0175] Methods are provided that include feeding the dog a daily diet that includes tomato pomace in an amount equal to 0.044%-0.42% of the daily nutritional intake. In some embodiments, methods are provided that include feeding the dog a daily diet that includes tomato pomace in an amount equal to 0.066%-0.315% of the daily nutritional intake. In some embodiments, methods are provided that include feeding the dog a daily diet that includes tomato pomace in an amount equal to 0.087%-0.21% of the daily nutritional intake. In some embodiments, methods are provided that include feeding the dog a daily diet that includes tomato pomace in an amount equal to about 0.14% of the daily nutritional intake.

[0176] Example 10

[0177] Table 3 lists the ingredients used in certain embodiments with the composition percentages (% by weight of dry matter of the ingredient composition). [Table 3]

[0178] Example 11

[0179] Table 4 lists the ingredients used in certain embodiments with the composition percentages (% by weight of dry matter of the ingredient composition). [Table 4]

[0180] Example 12

[0181] Table 5 lists the ingredients used in certain embodiments with the composition percentages (% by weight of dry matter of the ingredient composition). [Table 5]

[0182] Example 13

[0183] Table 6 lists the ingredients used in certain embodiments with the percentages of the composition (% by weight of dry matter of the ingredient composition). [Table 6]

[0184] Example 14

[0185] Table 7 lists the ingredients used in certain embodiments with the composition percentages (% by weight of dry matter of the ingredient composition). [Table 7]

[0186] Example 15

[0187] Table 8 lists the ingredients used in certain embodiments with the composition percentages (% by weight of dry matter of the ingredient composition). [Table 8]

[0188] Example 16

[0189] Table 9 lists the ingredients used in certain embodiments with the composition percentages (% by weight of dry matter of the ingredient composition). [Table 9]

Claims

1. 1. A method for identifying a canine subject as being likely to have elevated levels of 4-ethylphenyl sulfate, comprising: analyzing a biological sample obtained from a canine subject for the presence of two copies of a minor allele C of a single nucleotide polymorphism (SNP) at position 101 of SEQ ID NO:1 in said canine subject; The presence of two copies of the minor allele C of the SNP indicates that the canine subject will be more likely to have elevated levels of 4-ethylphenylsulfate during his or her lifetime.

2. 10. The method of claim 1, wherein the sample is analyzed by performing DNA sequencing, restriction enzyme digestion, polymerase chain reaction (PCR), hybridization, real-time PCR, reverse transcriptase PCR, or ligase chain reaction.

3. The method of claim 1 or 2, wherein the sample is a genomic DNA sample.

4. The method of any one of claims 1 to 3, wherein the sample is obtained from blood, saliva, follicle root, nasal swab, or oral swab of the canine subject.

5. 5. The method of any one of claims 1 to 4, wherein the sample is analyzed by performing at least one nucleic acid analysis technique selected from analysis using a whole genome SNP chip, single strand conformation polymorphism analysis (SSCP) assay, restriction fragment length polymorphism (RFLP), automated fluorescent sequencing; clamped denaturing gel electrophoresis (CDGE), denaturing gradient gel electrophoresis (DGGE), mobility shift analysis, restriction enzyme analysis, heteroduplex analysis, chemical mismatch cleavage (CMC), RNase protection assay, use of polypeptides that recognize nucleotide mismatches, allele-specific PCR, sequence analysis, and SNP genotyping.

6. 5. The method according to any one of claims 1 to 4, wherein the sample is analysed by carrying out at least one nucleic acid analysis technique selected from hybridisation-based methods, enzyme-based methods, post-amplification methods based on the physical properties of DNA and sequencing methods.

7. 5. The method of any one of claims 1 to 4, wherein the sample is analyzed by performing at least one nucleic acid analysis technique selected from the group consisting of: a hybridization-based method selected from the group consisting of dynamic allele-specific hybridization, molecular beacon method and SNP microarray; an enzyme-based method selected from the group consisting of restriction fragment length polymorphism (RFLP), PCR-based method, flap endonuclease, primer extension method, 5'-nuclease and oligonucleotide ligation assay; a post-amplification method based on the physical properties of DNA selected from the group consisting of single-stranded conformation polymorphism analysis, temperature gradient gel electrophoresis, denaturing high performance liquid chromatography, high resolution amplicon melting, DNA mismatch binding protein, SNPlex, and surveyor nuclease assay; and a sequencing method.

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