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

Abstract

To provide methods of preventing or reducing elevated 4-ethylphenyl sulfate levels in a canine subject.SOLUTION: A method comprises: analyzing a biological sample obtained from a canine subject for the presence of two copies of a minor allele of the single nucleotide polymorphism BICF2P1175095 in the canine subject, thereby identifying the canine subject as having an increased likelihood of elevated levels of 4-ethylphenyl sulfate; and administering tomato pomace to the canine subject; where the presence of two copies of the minor allele of BICF2P1175095 indicates that the canine subject has an increased likelihood of elevated levels of 4-ethylphenyl sulfate within its lifetime. Also provided is a canine food composition that comprises tomato pomace.SELECTED DRAWING: Figure 1

Description

Background Art

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

[0002] Anxiety in dogs (anxiety disorder) is fear, agitation when a dog anticipates a situation where it feels threatened or scared, and the reaction to anxiety. There are dogs that experience disproportionate levels of anxiety. Anxiety can develop into an anxiety disorder and lead to behavioral and other problems. Among them, there are dogs that experience generalized anxiety disorder, presenting a fear response in a wide range of situations where a "normal" pet would be less likely to react. Anxiety disorder can take the form of one of various anxiety disorders, especially generalized anxiety disorder, hyperstimulation anxiety, separation anxiety, confinement, noise phobia, etc.

[0003] Possible causes include genetic factors, prenatal and neonatal stress factors, mother-puppy separation, lack of socialization, unfamiliarity, or having experienced unpleasant results in the past when encountering a stimulus (or a similar stimulus). The most common causes are fear, separation, and aging. Anxiety related to fear can be caused, in particular, by loud noises, unfamiliar people and animals, visual stimuli, new or unfamiliar environments, specific situations, etc. Anxiety related to aging affects older dogs and may be associated with cognitive dysfunction syndrome (CDS). Separation anxiety is a specific anxiety that occurs because a pet cannot find comfort when separated from its family. Approximately 14% of dogs have separation anxiety. Some separation anxiety can be the result of dysfunctional attachment as puppies age and mature. In some cases, separation anxiety can occur when there are changes in the family or daily routine, while in other cases, separation anxiety may be underlying an anxiety state along with other behavioral problems such as phobias.

[0004] Anxiety can lead to destructive behavior (especially towards exits or the owner's possessions), distressed vocalizations, soiling of the house, salivation, pacing, restlessness, inability to settle, loss of appetite, and repetitive or compulsive behaviors. In some cases, anxiety can be involved in aggressive behavior.

[0005] Common symptoms of anxiety in dogs include aggression, urination and defecation indoors, drooling, rapid breathing, destructive behavior, depression, excessive barking, pacing, restlessness, and repetitive or compulsive behaviors. When suffering from anxiety disorders, different dogs exhibit different symptoms and combinations of symptoms.

[0006] Stress in dogs is the dog's reaction to demands for change or adaptation and usually manifests as tension or pressure. A dog experiencing stress can evoke emotions of fear, excitement, hyperactivity, nervousness, hypersensitivity, or irritability. Negative stress, excessive stress, and chronic stress can have an adverse impact on behavior, health, and overall well-being. Stress causes diseases, suppresses the immune system, triggers undesirable behaviors, and increases arousal levels, raising the probability of aggressive behavior.

[0007] Causes of stress in dogs include, in particular, sadness, exposure to conflict, excessive or insufficient stimulation, overcrowded environments, environmental changes (schedule, people, animals, increased noise), punitive training, insufficient social time, scary events, neglect, frustration, and uncertainty.

[0008] Dogs communicate stress in a variety of ways. Indicators that a dog is stressed include dilated pupils, tightness around the eyes, almond / half-moon eyes, yawning, lip / nose licking, rapid breathing, excessive salivation, smiling, teeth chattering, puffed cheeks, bared teeth, a wrinkled muzzle, ears pinned back or erect. Other indicators include a tense body, stretching, excessive hair loss, little or no movement, a low posture, weight shifting to the rear, trembling / shaking, penis exposure, sweaty feet, stiff eyebrows, barking, growling, howling, and snorting. When feeling stressed, a dog's behavior often changes. Behaviors often caused by stress include, among others, restlessness, lack of sleep or excessive sleep, jumping / hyperactivity, irritability, excessive self-grooming, destructive behavior, loss of appetite, compulsive / impulsive behavior, inability to concentrate, hyperactivity, increased urination / defecation, vomiting, and diarrhea.

[0009] A single nucleotide polymorphism (SNP) is a common type of genetic variation. An SNP is a variation of a single base pair at a specific locus. That is, an SNP is a difference in a single base in the DNA sequence that occurs at a specific position within the genome. Typically, for an SNP at a specific position, there are two possible nucleotide variations called alleles at that position. Within a population, the nucleotide variation that most frequently appears at a specific base position within the genome is called the major allele, and the nucleotide variation that is less common at that specific base position is called the minor allele. Like most multicellular organisms, dogs have two sets of chromosomes. Thus, each dog has two copies of each gene or locus, and thus two copies of each SNP. Therefore, for each SNP in a dog's genome, a dog may have two copies of the major allele, or one minor allele and one major allele, or two minor alleles.

[0010] SNPs can act as biological markers. SNPs can be useful for predicting drug response and the risk of developing certain diseases. SNP genotyping refers to the detection of SNPs within the genome. There are numerous methods for detecting SNPs and performing SNP genotyping.

[0011] There is a need to develop improved methods for identifying dogs that have a high likelihood or 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 disorders 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 a 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 single nucleotide polymorphism BICF2P1175095 indicates that the canine subject is likely to have elevated levels of 4-ethylphenyl sulfate, develop stress in the dog, develop anxiety in the dog, and / or inhibit the growth of beneficial microorganisms and promote the growth of harmful microorganisms over its lifetime.

[0014] The method can 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] This method may include analyzing a biological sample obtained from a canine subject by performing at least one nucleic acid analysis technique selected from analysis using a whole-genome SNP chip, single-strand conformational polymorphism analysis (SSCP) assay, restriction fragment length polymorphism (RFLP), automated fluorescence 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 a polypeptide that recognizes nucleotide mismatches, allele-specific PCR, sequence analysis, and SNP genotyping.

[0016] This method may include analyzing a biological sample obtained from a 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] This method may include analyzing a biological sample obtained from a canine subject by performing at least one nucleic acid analysis technique selected from hybridization-based methods consisting of dynamic allele-specific hybridization, molecular beacon method, and SNP microarray; enzyme-based methods consisting of restriction fragment length polymorphism (RFLP), PCR-based methods, flap endonuclease, primer extension method, 5'-nuclease, and oligonucleotide ligation assay; post-amplification methods based on the physical properties of DNA consisting of single-strand conformational 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 sequencing methods.

[0018] A method for preventing or reducing an increase in 4-ethylphenyl sulfate levels in a canine subject, the method comprising detecting the presence of two copies of the 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 providing a nutritional composition comprising an effective amount of tomato pomace to the canine subject.

[0019] A method for treating a canine subject for canine anxiety or canine stress in the canine subject, the method comprising detecting the presence of two copies of the 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 providing a nutritional composition comprising an effective amount of tomato pomace to the canine subject.

[0020] There is provided a food composition for dogs comprising an amount of tomato pomace equal to 0.087 to 0.21% on a dry matter basis.

[0021] There is provided a food composition for dogs comprising an amount of tomato pomace equal to 0.14% on a dry matter basis.

Brief Description of the Drawings

[0022]

Figure 1

Mode for Carrying Out the Invention

[0023] The following description of the preferred embodiments is merely exemplary in nature and is not intended to limit the invention, its application, or uses in any way.

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

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

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

[0027] The term "cat" includes cats that are companion animals known as domestic cats or Felis domesticus. The term "cat" is synonymous with the term "feline".

[0028] A method for treating anxiety and stress in animals, particularly companion animals such as dogs or cats, is provided. The method includes administering to the animal an effective amount of a combination of tomato pomace. The composition includes 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 daily nutrient intake" mean the dry matter intake per day. That is, the water weight is not included in the calculation of daily nutrient consumption. As long as food and food ingredients contain water / moisture, the dry matter represents everything in the sample other than water, including protein, fiber, fat, minerals, etc. The weight of the dry matter is the total weight minus the weight of the moisture. The dry matter intake per day is calculated as the total daily nutrient intake excluding all water. For example, the amount of a component corresponding to a specific percentage of the daily nutrient intake means the amount of that component in dry matter form (also excluding all water) relative to the total amount of dry matter consumed in a day (i.e., excluding all water). A person skilled in the art will readily recognize and understand the amounts and ratios of nutrients expressed as the amount of dry matter, the weight of the dry matter, and the percentage of the dry matter. Since food generally contains a certain amount of water, whether wet, moist, or dry, such water components of the food are excluded when calculating the daily dry matter intake. Water is excluded to calculate the total daily nutrient intake, which is the daily dry matter intake. To calculate the percentage of a component in the total daily intake on a dry matter basis, the moisture is removed from the total intake to obtain the total daily dry matter intake, and the percentage of the component is calculated based on the amount of the component present as dry matter.

[0030] Compositions useful in the method can be pet food compositions such as dog food compositions. Alternatively, tomato pomace may be administered as a supplement, snack, or toy, or otherwise may not be incorporated into the food provided to the animal for daily nutrient intake.

[0031] In some preferred embodiments, the animal is a dog, and the method includes 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 nutrient intake, in some embodiments 0.066 - 0.315% of the daily nutrient intake, in some embodiments 0.087 - 0.21% of the daily nutrient intake, and in some embodiments 0.14% of the daily nutrient intake.

[0032] Compositions and methods for treating anxiety and stress in animals, particularly companion animals such as cats or dogs, are provided. The compositions and methods are useful for treating symptoms of anxiety or stress in an animal in need thereof. The compositions and methods are useful for treating symptoms of anxiety or stress in such animals having elevated 4 - EPS levels. The compositions and methods are useful for reducing the elevation of 4 - EPS levels in companion animals, particularly animals such as dogs, having elevated 4 - EPS levels. 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 removal, reduction in severity, or prevention of one or more symptoms.

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

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

[0036] As used herein, the term "treatment" with respect to anxiety refers to therapeutic and / or prophylactic activity. In a dog with symptoms of anxiety, treatment of the dog's anxiety refers to the removal of symptoms, the halting or suppression of symptom progression, the reduction of symptom severity, and the prevention of symptoms. Treatment that first effects symptom removal, halting, progression suppression, or severity reduction may be continued, and continued treatment may further effect symptom removal, halting, progression suppression, or severity reduction, and / or the prevention of symptom recurrence or onset, or the reduction of the severity of further symptom onset. In some embodiments, prior to treating a dog's anxiety, the dog may be identified as having symptoms of anxiety. In some embodiments, a dog may be treated for anxiety without first identifying symptoms of anxiety. In some embodiments, prior to treatment for anxiety, a dog may be identified as having anxiety or as having a predisposition to developing anxiety. In some embodiments, prior to treatment for anxiety, a dog may be identified as having a high 4-EPS level.

[0037] As used herein, the term "treatment" with respect to stress and stress disorders refers to therapeutic and / or prophylactic activity. In a dog having symptoms of stress or a stress disorder, treatment of the dog's stress refers to the removal of symptoms, the halting or suppression of symptom progression, the reduction of symptom severity, and the prevention of symptoms. Treatment that first effects symptom removal, halting, progression suppression, or severity reduction may be continued, and continued treatment may further effect symptom removal, halting, progression suppression, or severity reduction, and / or the prevention of symptom recurrence or onset, or the reduction of the severity of further symptom onset. In some embodiments, prior to treating a dog's stress, the dog may be identified as having symptoms of stress or a stress disorder. In some embodiments, a dog can be treated for stress or a stress disorder without first identifying symptoms of anxiety. In some embodiments, prior to treatment for stress or a stress disorder, a dog can be identified as having, or being predisposed to develop, stress or a stress disorder. In some embodiments, prior to treatment for stress or a stress disorder, a dog can be identified as having a high 4-EPS level.

[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. Treatment in a dog in which the level of beneficial microbes has decreased and the level of harmful microbes has increased, to halt the levels of beneficial and harmful microbes, or to promote the growth of beneficial microbes and inhibit the growth of harmful microbes. A dog identified as having a factor that inhibits beneficial microbial growth and promotes harmful microbial growth prior to initiation of treatment. Treatment in an animal in which the level of harmful microbes has increased and the level of beneficial microbes has decreased, initially promoting the growth of beneficial microbes, suppressing the growth of harmful microbes, increasing the level of beneficial microbes, and decreasing the level of harmful microbes to a more healthy balance, and then maintaining the levels by continued treatment. In some embodiments, prior to treating a dog for stress, the dog can be identified as having an increased level of harmful microbes and a decreased level of beneficial microbes. In some embodiments, a dog can be treated without the identification of an increase in the level of harmful microbes and a decrease in the level of beneficial microbes in the animal.

[0039] As used herein, the terms "treatment of 4-EPS elevation", "treating 4-EPS elevation", and "treating an elevation in 4-EPS" refer to therapeutic and / or prophylactic activity that reduces 4-EPS levels. In dogs with elevated 4-EPS levels, "treatment of 4-EPS elevation", "treating 4-EPS elevation", and "treating an elevation in 4-EPS" refer to reducing the elevated 4-EPS levels. Treatment can reduce elevated 4-EPS levels to normal, non-elevated levels, or reduced elevated 4-EPS levels. Following reduction of the elevated 4-EPS levels, treatment can prevent an increase in 4-EPS levels or reduce the severity of further progression of an increase in 4-EPS levels. In dogs without an increase in 4-EPS levels, "treatment of 4-EPS elevation", "treating 4-EPS elevation", and "treating an elevation in 4-EPS" refer to halting or reducing 4-EPS levels and preventing the occurrence of an increase in 4-EPS levels, or reducing the severity of the occurrence of an increase in 4-EPS levels. In some embodiments, prior to treatment for 4-EPS, a dog can be identified as having elevated 4-EPS by 4-EPS level measurement. In some embodiments, a dog can be treated for 4-EPS elevation without measuring 4-EPS levels prior to treatment. In some embodiments, prior to treating 4-EPS, a dog can be identified as having a predisposition to 4-EPS elevation. A dog identified as having a predisposition to 4-EPS elevation 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, a dog can be identified as having a predisposition to 4-EPS elevation before starting treatment, with or without measuring 4-EPS levels.

[0040] As used herein, "effective amount", "amount effective", and similar terms refer to the amount of tomato pomace effective to achieve a particular biological result, namely, an increase in 4-EPS levels, treatment of anxiety, stress, and levels of beneficial and harmful microorganisms in the microbiota. 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 sufficient to provide effective treatment and maintenance. The effective amount may be based on several factors including the ideal body weight, age, gender, activity level of the dog, the metabolizable energy of the composition, and the 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 the amount of tomato pomace administered based on total nutrient intake, and the amount of tomato pomace is equal to 0.087 - 0.21% of the total nutrient intake per day. In some embodiments, the effective amount refers to 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 nutrient intake, and the amount of tomato pomace is equal to 0.14% of the total nutrient intake per day. In some embodiments, the effective amount refers to pet food comprising 0.14% tomato pomace.

[0041] In some embodiments, "food", "food composition", or "pet food composition" can be a nutritionally complete diet for an animal such as a dog to which it is provided.

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

[0043] The term "nutrient" refers to a substance that provides nutrition. In some instances, an ingredient may include two or more "nutrients", e.g., a composition may include corn that includes essential nutrients including both protein and carbohydrates.

[0044] The food composition can be provided in the form of pet food to animals including, but not limited to, pets. Pet owners can obtain various types of pet food that are generally known. Options for pet food 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 water content of more than about 65%. Semi-moist pet food generally has a water content of about 20% to about 65% and may contain 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 water content of less than about 15%. Pet treats can typically be semi-moist, chewable treats, any number of forms of dried treats, chewable bones or baked, extruded, or punched-out 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 specific pellet-like components of animal food, such as dog and cat food. In some embodiments, the food kibble has a water 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 puffed to high density. The food kibble can be formed by an extrusion process or a baking process. By way of non-limiting example, 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 uncoated kibble or coated kibble.

[0046] As used herein, the terms "extrude" or "extrusion" refer to the process of sending a pre-treated and / or prepared mixture of ingredients through an extruder. In some embodiments of extrusion, food kibble can be formed by the extrusion process, and a kibble dough containing a mixture of wet and dry ingredients can be extruded under heat and pressure to form 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 also contemplated in their combinations and mixtures and are listed to be within the scope of this specification.

[0047] As contemplated herein, the term "composition" means, but is not limited to, including nutritionally complete and balanced animal food compositions. A "nutritionally complete diet" is a diet that, with respect to diet, can contain sufficient nutrients to maintain the normal health of a healthy dog. Nutritionally complete and balanced pet food compositions, for example 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 providing a diet containing an effective amount of tomato pomace to a dog, a preferred method is contemplated to include providing the dog with pet food containing tomato pomace as an ingredient. In other embodiments, providing a diet containing an effective amount of tomato pomace to a dog is achieved by administering the tomato pomace to the dog as a supplement or treat. Whether delivered in a pet food composition or as a separate supplement or treat, providing tomato pomace to a dog by any means is considered to be providing the dog with a diet containing an effective amount of tomato pomace.

[0049] As used herein, the term "supplemental food" includes, but is not limited to, feed used in another feed to improve the nutritional balance or overall diet performance of an animal. Examples of supplemental foods include, but are not limited to, compositions that are given undiluted as a supplemental food to other feeds, provide a free choice by other portions of the animal's ration that can be used separately, or are diluted and mixed with the animal's normal feed to make a complete feed. For example, the AAFCO guidelines include a discussion of supplemental foods by the Association of American Feed Control Officials (AAFCO), Atlanta, Ga (2012). Supplemental foods can be in various forms, including, for example, powders, liquids, syrups, pills, compositions in capsules, and the like.

[0050] The diet contains an effective amount of tomato pomace and can reduce the elevated levels of 4-EPS in dogs. A diet containing tomato pomace is useful for treating anxiety in dogs. A diet containing tomato pomace is useful for treating stress in dogs. A diet containing tomato pomace is useful for promoting the growth of beneficial microorganisms and inhibiting the growth of harmful microorganisms in the dog-targeted microbiota, particularly in the gastrointestinal tract.

[0051] Genetic predisposition to elevated 4-EPS

[0052] Genetic association studies have identified a genetic marker that can identify dogs with a tendency to have 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 dog genotyping array. Dogs homozygous for the minor allele of SNP BICF2P1175095 tend to have elevated 4-EPS levels. 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 14s of the chromosome 14 pair. Dogs that are homozygous for the minor allele are said to have the homozygous minor allele genotype that is the genotype CC of SNP BICF2P1175095 or the genotype CC as used herein. Dogs with the CC genotype are more likely to develop elevated 4-EPS levels, canine anxiety, and canine stress. In addition to predisposing dogs to develop elevated 4-EPS levels, anxiety, and stress, the CC genotype in dogs is more likely to inhibit the growth of beneficial microorganisms in the dog-targeted microbiota, particularly in the gastrointestinal tract microbiota, and to promote the growth of harmful microorganisms in the dog-targeted microbiota, particularly in the gastrointestinal tract microbiota.

[0053] Dogs identified as having a predisposition 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 in dogs identifies dogs as having a predisposition for increased levels of 4-EPS. Identifying the CC genotype in dogs identifies dogs as having a predisposition for anxiety. Identifying the CC genotype in dogs identifies dogs as having a predisposition for stress. Identifying the CC genotype in dogs identifies dogs as having a predisposition for inhibiting the growth of beneficial microorganisms, and thus, having a lower level of beneficial microorganisms in the microbiota of dogs, particularly in the gastrointestinal tract microbiota, and identifying dogs as having a predisposition for promoting the growth of harmful microorganisms, and thus, having a higher level of harmful microorganisms in the microbiota of dogs, particularly in the gastrointestinal tract microbiota.

[0055] A diet containing an effective amount of tomato pomace to suppress the increase in the 4-EPS level in dogs is particularly beneficial for dogs having the CC genotype and thus being predisposed to an increase in the 4-EPS level. A diet containing an effective amount of tomato pomace is particularly beneficial for dogs having the CC genotype and thus being prone to experiencing anxiety. A diet containing an effective amount of tomato pomace is particularly beneficial for dogs having the CC genotype and thus being prone to experiencing stress. A diet containing an effective amount of tomato pomace is particularly beneficial for dogs having the CC genotype and thus having a predisposition for inhibiting the growth of beneficial microorganisms and promoting the growth of harmful microorganisms in the microbiota of dogs, particularly in the gastrointestinal tract microbiota.

[0056] While not bound by any particular theory, the CC genotype of BICF2P1175095, which is located upstream of the NOD1 gene, can result in a state that favors the growth of harmful microorganisms over the growth of beneficial microorganisms in the gastrointestinal tract of dogs. As a result, beneficial microorganism growth is inhibited in the microbiota of the dog's gastrointestinal tract, and harmful microorganism growth is promoted in the microbiota of the dog's gastrointestinal tract, resulting in a decrease in the level of beneficial microorganisms and an increase in the level of harmful microorganisms. Harmful microorganisms in the microbiota release the metabolic toxin 4-EPS, increasing the level of circulating 4-EPS in the dog. An increase in the level of circulating 4-EPS in the dog can result in the development of anxiety and / or stress in the dog. A diet containing an effective amount of tomato pomace can result in an increase in the level of beneficial microorganisms and a decrease in the level of harmful microorganisms in the dog's microbiota. An increase in the level of beneficial microorganisms and a decrease in the level of harmful microorganisms in the dog's microbiota result in a decrease in 4-EPS released into the circulation, thereby reducing the 4-EPS level.

[0057] A method of identifying a dog subject having a predisposition to an increase in 4-EPS level includes determining that the dog subject has the CC genotype. A method of identifying a dog subject having a predisposition to anxiety includes determining that the dog subject has the CC genotype. A method of identifying a dog subject having a predisposition to stress includes determining that the dog subject has the CC genotype. A method of identifying a dog subject having a predisposition to, in particular, inhibition of beneficial microorganism growth and promotion of harmful microorganism growth in the microbiota of the gastrointestinal tract includes determining that the dog subject has the CC genotype.

[0058] In dogs, a method of reducing, preventing, or reducing the severity of an increase in 4-EPS level includes feeding a diet containing an effective amount of tomato pomace to the dog, which reduces, prevents, or reduces the severity of the increase in 4-EPS level. In some embodiments, a method of reducing, preventing, or reducing the severity of an increase in 4-EPS level in a dog includes identifying the dog as having the CC genotype and feeding a diet containing an effective amount of tomato pomace to the dog, which reduces, prevents, or reduces the severity of the increase in 4-EPS level.

[0059] A method of alleviating, reducing, and / or preventing anxiety in a dog includes feeding a diet containing an effective amount of tomato pomace to the dog to alleviate, reduce, and / or prevent symptoms of dog anxiety and anxiety disorders. In some embodiments, a method of alleviating, reducing, and / or preventing anxiety in a dog includes identifying the dog as having the CC genotype and feeding a diet containing an effective amount of tomato pomace to the dog to alleviate, reduce, and / or prevent the dog's anxiety.

[0060] A method of alleviating, reducing, and / or preventing stress in a dog includes feeding a diet containing an effective amount of tomato pomace to the dog to alleviate, reduce, and / or prevent the dog's stress. In some embodiments, a method of alleviating, reducing, and / or preventing stress in a dog includes identifying the dog as having the CC genotype and feeding a diet containing an effective amount of tomato pomace to the dog to alleviate, reduce, and / or prevent the dog's stress.

[0061] A method for promoting the growth of beneficial microorganisms and inhibiting the growth of harmful microorganisms in dogs involves feeding a diet containing an effective amount of tomato pomace to the dog to promote the growth of beneficial microorganisms and inhibit the growth of harmful microorganisms. In some embodiments, a method for promoting the growth of beneficial microorganisms and inhibiting the growth of harmful microorganisms in dogs involves identifying the dog as having a CC genotype and feeding a diet containing an effective amount of tomato pomace to the dog to promote the growth of beneficial microorganisms and inhibit the growth of harmful microorganisms.

[0062] The method involves analyzing a biological sample obtained from the dog for the presence of two copies of the minor allele C of a single nucleotide polymorphism located at chr14:43309715 as referred to in the CanFam3.1 reference genome. The SNP is named BICF2P1175095 on a commercially available Illumina dog genotyping array. That is, the method involves analyzing a biological sample obtained from the dog 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] Dogs having two copies of the minor allele C of the SNP located at chr14:43309715 (BICF2P1175095), i.e., dogs having the CC genotype, tend to have elevated 4-EPS levels throughout their lives. The CC genotype indicates that the dog has a predisposition to develop anxiety throughout its life, a predisposition to develop stress throughout its life, and a predisposition to inhibit the growth of beneficial microorganisms and promote the growth of harmful microorganisms in its microbiota.

[0064] The likelihood of an increase in the level of 4-ethylphenyl sulfate indicates that the dog is more likely to develop anxiety, more likely to develop stress, and / or more likely to inhibit the growth of beneficial microorganisms and promote the growth of harmful microorganisms. A method of identifying a dog as having an increased likelihood of elevated levels of 4-ethylphenyl sulfate, as a dog having an increased likelihood of developing anxiety, as a dog having an increased likelihood of developing stress, and / or as a dog having an increased likelihood of inhibiting beneficial microbial growth and promoting harmful microbial growth.

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

[0066] A method of identifying a dog as having an increased likelihood of elevated levels of 4-ethylphenyl sulfate indicates that the dog has an increased likelihood of developing anxiety, has an increased likelihood of developing stress, and / or has an increased likelihood of inhibiting beneficial microbial growth and promoting harmful microbial growth, and includes analyzing a biological sample obtained from the dog subject for the presence of two copies of the minor allele C of a single nucleotide polymorphism located at chr14:43309715, as referred to in the CanFam3.1 reference genome, and may be part of a method of treating the dog to prevent or reduce an elevated level of 4-ethylphenyl sulfate, to prevent or alleviate symptoms of dog anxiety, to prevent or alleviate symptoms of an increased likelihood of the dog developing stress, and / or to prevent or reduce the inhibition of beneficial microbial growth and the promotion of harmful microbial growth.

[0067] A method of identifying dogs having a genetic predisposition for an increased level of 4-ethylphenyl sulfate, the development of anxiety in dogs, the development of stress in dogs, and / or the inhibition of beneficial microbial growth and the promotion of harmful microbial growth, as referred to in the CanFam3.1 reference genome, includes analyzing a biological sample obtained from a dog subject presenting symptoms of stress or anxiety in the dog for the presence of two copies of the minor allele C of the single nucleotide polymorphism located at chr14:43309715, and may be part of a method of treating a dog to reduce an increased level of 4-ethylphenyl sulfate, to alleviate symptoms of anxiety in the dog, to alleviate symptoms that increase the likelihood of the dog developing stress, and / or to prevent or reduce the inhibition of beneficial microbial growth and the promotion of harmful microbial growth.

[0068] Single nucleotide polymorphism BICF2P1175095

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

[0070] Nucleotides 1-201 of SEQ ID NO: 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] The genomic sequences containing the disclosed SNPs can be accessed in several ways. One way is to refer to the Illumina Canine HD annotation file found at <ftp: / / webdata2:webdata2@ussdftp.illumina.com / downloads / ProductFiles / CanineHD / CanineHD_B.csv>. Further, the chromosome and position defined by the canine reference genome CanFam3.1 for the SNP is chr14:43309715. Those skilled in the art can identify the target SNP using publicly available interfaces such as the University of California, Santa Cruz Genome Browser and extract the adjacent DNA sequences using the genome browser tools. Further, the canine reference genome is available from many publicly available sources such as <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>. Using these databases, the relevant DNA sequences can be extracted.

[0072] A method for detecting a CC genotype associated with an increased likelihood of elevated 4-EPS levels in dogs is provided. A method for identifying dogs at high risk of developing anxiety, stress, and decreased levels of beneficial microorganisms and increased levels of harmful microorganisms is provided.

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

[0074] In some embodiments, the method includes detecting the CC genotype. That is, the method includes 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) includes examining a DNA sample from a canine 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, in effect, the presence of two copies of the minor allele is detected.

[0075] In some embodiments, the method of detecting the presence of two copies of the minor allele C of SNP BICF2P1175095 (chr14:43309715) includes examining a DNA sample from a canine for the presence of the major allele, and detecting zero copies of the major allele. Detecting zero copies of the major allele effectively detects the presence of two copies of the minor allele. That is, looking for the major allele and finding that there are no copies effectively detects 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 conformational polymorphism analysis (SSCP) assay, restriction fragment length polymorphism (RFLP); automated fluorescence 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 a polypeptide that recognizes 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 physical properties of DNA, and sequencing methods.

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

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

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

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

[0083] In some embodiments, the MassARRAY system is used to detect the presence of the CC genotype. The MassARRAY system is a non-fluorescent detection platform that utilizes mass spectrometry to accurately measure PCR-derived amplicons. By combining mass spectrometry with endpoint PCR, highly multiplexed reactions are possible under universal cycling conditions, providing accurate, rapid, and cost-effective analysis. The MassARRAY system provides a unique solution for target gene testing with limited input material.

[0084] In some embodiments, bead array technology is used for the detection of the CC genotype to be 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 for the detection of the presence of SNP alleles. Illumina BeadArray technology is based on small silica beads that self-organize 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. When the beads are self-organized, a unique decoding process maps the position of each bead and ensures that each bead is individually quality controlled. As a result of this manufacturing process, all BeadChips will undergo stringent testing to ensure the highest possible quality standards. The Infinium assay, unlike many alternative PCR-dependent assays, can be extended to unlimited multiplexing without sacrificing data quality. The simple and 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, greatly reducing labor and sample processing errors. After hybridizing an unlabeled DNA sample to the Beadchip, high call rates and accuracy are obtained by two-step allele detection. Selectivity and specificity are achieved in two steps. Target hybridization to the 50mer oligo bound to the bead provides high selectivity, while enzyme-mediated single-base extension incorporates labeled nucleotides for assay readout. The staining reagent is optimized to provide a higher signal and a more balanced intensity between the red and green channels. These features contribute to high call rates and copy number data with low noise. The Infinium assay generates a two-color (one color for each allele) readout for each SNP in a genotyping test.The intensity values of each of the two-color channels A and B convey information about the allelic ratio at a single genomic locus. Typical studies incorporate values from a large number of samples (hundreds to tens of thousands) to ensure significant statistical representation. When these values are properly normalized and distinct patterns (or clusters) appear when plotted, samples at the assayed locus have the same genotype, exhibit similar signal profiles (A and B values), and aggregate into clusters. In the case of diploid organisms, both allelic loci are expected to exhibit three clusters (AA, AB, and BB). Genotype calling is based on information derived from a standard cluster file that provides statistical data from a representative set of samples. This enables the genotype to be called by referring to the single intensity of the assay against known data for a given locus. Since the calling accuracy is linked 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 discriminates the cluster patterns of genotyping samples and reports an overview.

[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 site. In dynamic allele-specific hybridization, genomic segments are amplified and bound to beads via a PCR reaction with biotinylated primers. The amplified product is then attached to a streptavidin column and washed to remove the non-biotinylated strand. Allele-specific oligonucleotides are then added in the presence of a molecule that fluoresces when bound to double-stranded DNA. Intensity is measured as the temperature increases until the melting temperature (Tm) can be determined. SNPs are detected by a Tm lower than expected. Specifically engineered single-stranded oligonucleotide probes are used in SNP detection using molecular beacons. The oligonucleotide is designed with complementary regions at each end and the probe sequence located between them, such that the probe takes on a hairpin or stem-loop structure in its native isolated state. A fluorophore is attached to one end of the probe and a fluorescence 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 fluoresce. 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 a hairpin configuration and fluoresces. High-density oligonucleotide SNP arrays contain hundreds of thousands of probes arrayed on a small chip, enabling the investigation of many SNPs simultaneously. Several redundant probes designed to have the SNP site at several different locations, as well as containing mismatches to the SNP allele, are used to investigate each SNP. Different amounts of hybridization of the target DNA to each of these redundant probes allow the determination of specific homozygous and heterozygous alleles.

[0086] The CC genotype may be detected using enzyme-based methods. A wide range of enzymes including DNA ligase, DNA polymerase, and nucleases may be used. Examples of enzyme-based methods include methods based on restriction fragment length polymorphism (RFLP), PCR-based methods, methods utilizing flap endonucleases, methods utilizing primer extension, methods utilizing 5'-nucleases, and methods including 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 can use the pattern of fragment lengths 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. The primers are designed such that the two primer pairs overlap at the SNP position, but each is designed to perfectly match only one of the possible SNPs. Alternatively, multiple qPCR reactions can be performed with different primer sets that target each allele separately. Some embodiments utilize flap endonuclease (FEN), an endonuclease that catalyzes structure-specific cleavage. This cleavage is very sensitive to mismatches and can be used to investigate SNPs with high specificity. FEN, called Cleavase, can be combined with two specific oligonucleotide probes to form a triple helix structure recognized by Cleavase with the target DNA. The first probe, called the 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 extends beyond the 3' side of the SNP nucleotide. The allele-specific probe contains a base complementary to the SNP nucleotide.

[0087] Primer extension is a two-step process that first involves hybridization of the probe to the base immediately upstream of the SNP nucleotide, followed by extension of the hybridization primer by DNA polymerase adding a base complementary to the SNP nucleotide. This incorporated base is detected and the SNP allele is determined. The primer extension method is 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. The Sequenom iPLEX SNP genotyping method uses a MassARRAY mass spectrometer. Due to the flexibility and specificity of primer extension, it is suitable for high-throughput analysis. Primer extension probes can be arrayed on a slide and multiple SNPs can 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. In the Infinium assay, over 100,000 SNPs can be genotyped. In the assay, hapten-labeled nucleotides are used in the primer extension reaction. The hapten label is recognized by an antibody, which is then conjugated 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 and uniform 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 amplicons containing the base pair being tested. Methods that utilize 5'-nuclease include those that use the 5'-nuclease activity of Taq DNA polymerase in TaqMan assays for SNP genotyping. TaqMan assays are performed simultaneously with the PCR reaction and the results can be read in real time as the PCR reaction progresses. In methods that include oligonucleotide ligation assays, oligonucleotide DNA ligase catalyzes the ligation of a DNA fragment from the 3' end directly to the 5' end of an adjacent DNA fragment. This mechanism can be used to investigate SNPs by directly hybridizing two probes over the SNP polymorphic site, such 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 are preceded by PCR amplification of the target DNA.

[0089] Some methods for detecting SNP alleles are based on the physical properties of DNA, such as melting temperature and single-stranded higher-order structures. Methods using single-stranded structures are based on single-stranded DNA (ssDNA) that folds into a three-dimensional structure. The three-dimensional structure is sequence-dependent, and most single-base pair mutations change the shape of the structure. When applied to a gel, the three-dimensional shape determines the mobility of the ssDNA and provides a mechanism for distinguishing SNP alleles. This method first involves PCR amplification of the target DNA. The double-stranded PCR product is denatured using heat and formaldehyde to produce ssDNA. The ssDNA is applied to a non-denaturing electrophoresis gel and folds into a three-dimensional structure. Differences in the DNA sequence change the three-dimensional structure and are detected as differences in the mobility of the ssDNA strands. The temperature gradient gel electrophoresis (TGGE) method or the temperature gradient capillary electrophoresis (TGCE) method is based on the principle that partially denatured DNA is more restricted and migrates more slowly in a gel or other porous material. In another method, denaturing high-performance liquid chromatography (DHPLC) uses reverse-phase HPLC to investigate SNPs. In DHPLC, the solid phase has differential affinity for single-stranded DNA 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 to different single-base mismatches with different affinities and can be used in capillary electrophoresis to distinguish all six sets of mismatches. SNPlex is an exclusive genotyping platform sold by Applied Biosystems. The Surveyor nuclease assay uses the Surveyor nuclease, a mismatch endonuclease enzyme that recognizes all base substitutions and small insertions / deletions (indels) and cleaves the 3' side of the mismatch site on both DNA strands. Sequencing technology can also be used for SNP detection. Advancements in sequencing technology have made more practical SNP detection by sequencing possible.

[0090] Genotyping by sequencing using next-generation sequencing technology has become a common practice. Genotyping by sequencing, also known as GBS, is a method for discovering single nucleotide polymorphisms (SNPs) to conduct genotyping studies such as genome-wide association studies (GWAS). GBS reduces the complexity of the genome using restriction enzymes and genotypes multiple DNA samples. After digestion, PCR is performed to increase the fragment pool, and then the GBS library is sequenced using next-generation sequencing technology. With the advancement of next-generation sequencing technologies such as Illumina short-read sequencing by synthesis and PacBio single-molecule real-time sequencing, it has become 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] By applying the methodology outlined above, bioactive dietary components were identified that, in combination, provide a composition, food, and diet that confer a significant benefit to dogs that have been identified as having a predisposition to elevated 4-EPS levels, and thus a high risk of developing anxiety, a high risk of developing stress, and a high risk of inhibiting the growth of beneficial microorganisms and promoting the growth of harmful microorganisms in the canine microbiota, particularly the gut microbiota.

[0093] A method is provided that includes feeding a dog a daily diet that includes tomato pomace in an amount equal to 0.044% to 0.42% of the total daily nutritional intake. In some embodiments, a method is provided that includes feeding a dog a daily diet that includes tomato pomace in an amount equal to 0.066% to 0.315% of the total daily nutritional intake. In some embodiments, a method is provided that includes feeding a dog a daily diet that includes tomato pomace in an amount equal to 0.087% to 0.21% of the total daily nutritional intake. In some embodiments, a method is provided that includes feeding a dog a daily diet that includes 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 composition includes a food composition that may include an effective amount of tomato pomace in combination with from 4% to 75% or more by weight of the total weight of the composition based on dry matter, from 5% to 50% or more by weight of the total weight of the composition based on dry matter, and from 5% to 75% or more by weight of the total weight of the composition based on dry matter of protein, fat, and carbohydrates, respectively, and the food composition is suitable for consumption by dogs.

[0096] The composition administered by the methods provided herein may, in certain embodiments, be formulated as a food composition that is nutritionally balanced and / or nutritionally complete food or diet. In other embodiments, the composition is formulated and prepared as a nutritional supplement, snack, or treat.

[0097] In some embodiments, for example, in addition to an effective amount of tomato pomace, a nutritionally complete and balanced dog food composition may contain 4% - 90%, 5% - 75%, 10% - 60% protein, and 15% - 50% by weight of protein, 0% - 90%, 2% - 80%, 5% - 75%, and 10% - 50% carbohydrates, 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 animals.

[0098] Within the composition, suitable sources of protein, carbohydrates, fat, vitamins, minerals, balancers, etc., particularly for inclusion in the food administered by the methods provided herein, may be selected from conventional materials known to those skilled in the art.

[0099] In some embodiments, proteins useful as components of the food composition include animal - derived proteins such as mammalian - including animal proteins, avian proteins, reptilian, amphibian, fish, invertebrate proteins and combinations thereof; for example, from cows, sheep, pigs, goats, deer, rabbits, horses, kangaroos, their milk, curd, whey or blood, and from 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, reptilian protein sources include crocodiles, lizards, turtles and snakes, fish protein sources include catfish, herring, salmon, tuna, bluefish, cod, flounder, trout, mackerel and their eggs, invertebrate protein sources include lobsters, crabs, clams, mussels or oysters and combinations thereof, meat protein isolates, whey protein isolates, egg proteins, mixtures thereof, etc., and proteins such as soy protein isolates, corn gluten meal, wheat gluten, mixtures thereof, etc.

[0100] In some embodiments, carbohydrates useful as components of the food composition may include, but are not limited to, corn, whole yellow corn, grain sorghum, wheat, barley, rice, millet, polished sake rice, oatmeal, and polysaccharides (e.g., starch and dextrin), as well as one or more of sugars that are metabolized for energy upon hydrolysis (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 components of 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, and mixtures thereof. The fat may be fully incorporated within the food composition, deposited outside 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 canaliculus, 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 including, 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 including, but not limited to, lauric acid, myristic acid, palmitic acid, palmitoleic acid, margaric acid, margaroleic acid, stearic acid, oleic acid, linoleic acid, gamma-linolenic acid, alpha-linolenic acid, stearidonic acid, arachidic acid, gadoleic acid, DHGLA, arachidonic acid, eicosatetraenoic acid, EPA, behenic acid, erucic acid, docosatetraenoic acid, and DPA, among others.

[0105] In some embodiments, the food composition further comprises one or more macronutrients including, but not limited to, water, 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 including, 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 inorganic substances including, 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 including, but not limited to, vitamin A, vitamin C, vitamin D, vitamin E, quinoa, thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, folic acid, vitamin B12, biotin, and choline.

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

[0110] In some embodiments, the food composition further comprises stabilizing substances, such as substances that tend to extend the shelf life of the composition. Potentially suitable examples of such substances 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 ether, starch, starch esters, starch ethers, and modified starches.

[0111] In some embodiments, the food composition can include, for example, colorants, iron oxides, sodium chloride, potassium citrate, potassium chloride, and other edible salts, vitamins, minerals, and flavors as intended additives for coloring, palatability, and nutritional purposes. The amount of such additives in the composition is generally up to 5% (on a dry weight basis 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 have any consistency or moisture content, i.e., the composition may be a wet, semi-moist, or dry food. A "wet" food is generally a food having a moisture content of 60% to over 90%. A "dry" food generally has a moisture content of 3% to 11% and is often manufactured in the form of small pieces or kibbles. A "semi-moist" food generally has a moisture content of 25% to 35%. The food may also include one or more components having a consistency, such as particles or pieces like soft, chewy meat, and kibbles having an outer grain component or coating and an inner "cream" component.

[0114] In some embodiments, foods containing tomato pulp may be prepared in canned or wet form using conventional food preparation processes known to those skilled in the art. Typically, the ground animal protein tissue is mixed with other ingredients such as grains, suitable carbohydrate sources, fats, oils, and preparation ingredients, and contains a vitamin and mineral mixture, inorganic salts, cellulose, beet pulp, etc., and an amount of water sufficient for processing. These ingredients are mixed in a container suitable for heating in the blend of ingredients. For example, heating of the mixture can be carried out using any suitable method, such as by direct steam injection or by using a container equipped with a heat exchanger. After the 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 using other processing aids. When heated to an appropriate temperature, the material is usually in the form of a viscous liquid and is dispensed into cans. The cans are capped and sealed. The sealed cans are then placed in a conventional device designed to sterilize the contents. Sterilization can usually be achieved by heating for an appropriate time to a temperature above about 230°C, depending on the temperature used, the composition, and related factors. The compositions and foods of the present invention may also be added to or combined with the food composition 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 components including dry animal proteins, vegetable proteins, grains, etc. are ground and mixed together. A liquid or wet component including fat, oil, water, animal protein, water, etc. is added and mixed with the dry material. The specific formulations, additional order, combinations, and methods and equipment used to combine the various components can be selected from those known in the art. For example, in certain embodiments, the resulting mixture is processed using an extrusion process where the mixture of dry and wet components is subjected to mechanical work at high pressure and high temperature, passed through small openings or orifices, and cut into kibble, for example, with a rotating knife, and processed into kibble or similar dry pieces. The resulting kibble is dried and optionally coated with one or more topical coating agents including, for example, flavor, fat, oils, powders, etc. The kibble can also be made from dough using a baking method rather than extrusion, in which method, after placing the dough in a mold, a dry heating process is performed.

[0116] When preparing the composition, any component can generally be incorporated into the composition during the compounding process, for example, while and / or after mixing the other components of the composition. The distribution of these components into the composition can be achieved by conventional means. In certain embodiments, the ground animal and / or poultry proteinaceous tissue is mixed with other components including nutritional balancers, inorganic salts, and further mixed with other components including cellulose, beet pulp, fillers, etc. with sufficient water for processing.

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

[0118] A 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, with respect to diet, is a diet that can contain sufficient nutrients to maintain the normal health of a healthy animal. Nutritionally complete and balanced pet food compositions, for example 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, are included in, for example, the Official Publication of The Association of American Feed Control Officials, Inc. (AAFCO), Atlanta, Ga. (2012), which includes discussions on supplementary foods.

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

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

[0121] All publications described herein are incorporated by reference for the purpose of describing and disclosing the materials and methodologies reported in this publication, which may 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 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. The 4-EPS level in plasma can be measured by a commercial laboratory (Metabolon, Durham, NC, USA). The extracted supernatant is split and run on gas chromatography and liquid chromatography mass spectrometry platforms. The peak of 4-EPS is known, and the area under the peak of each sample can be normalized to a known sample. (See also, for example, Evans, A.M., et al. (2009). An integrated untargeted ultra high performance liquid chromatography / electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of small molecule complements of 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 plasma samples. (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] The nucleotide-binding oligomerization domain 1, called NOD1, is an intracellular sensor that detects small peptides derived from the cell wall components of the gut microbiota, which triggers an innate immune response. The innate immune response mediated by NOD1 activation is involved in both host defense against microbial infections and the development of gastrointestinal disorders.

[0127] A genome-wide association study (GWAS) showed a relationship between SNP BICF2P1175095 located upstream of the NOD1 gene and the circulating levels of 4-EPS. Dogs with the CC genotype have a higher risk of impaired intestinal colonization by certain beneficial microorganisms. This may lead to the growth of harmful microorganisms that can potentially affect distal organs such as the brain through specific microbial metabolites that enter the systemic circulation. The known stress-related microbial metabolite 4-EPS is detected up to 9-fold more frequently in dogs with the CC genotype.

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

[0129] Example 3

[0130] A study was completed in which 40 dogs were randomly assigned to a control (20 dogs) or a test group (20 dogs) and fed either a basal diet containing tomato pomace or a food without tomato pomace for 30 days. After a one-month washout period, a crossover was performed to feed the group the food that they had not been given previously. After 30 days, blood samples were collected and 4-ethylphenyl sulfate levels were measured by metabolomics. Therefore, all dogs consumed either a food containing tomato pomace or a food without tomato pomace for 30 days, and at the end of each 30-day feeding period, metabolomics analysis was completed.

[0131] From these results, it was found that dogs that tend to have high 4-EPS may benefit from consuming a diet containing a specific level of tomato pomace. A matched pair analysis comparing the 4-EPS levels of each dog after food intake showed a significant decrease in 4-EPS with the addition of tomato pomace (P = 0.04) (Figure 1).

[0132] Table 1 shows the daily intake of tomato pomace that leads to low levels of 4-EPS. The lowest level of 4-EPS was achieved when an average of 0.14% tomato pomace on a dry matter basis was consumed daily, which corresponds to 0.24 grams of tomato pomace out of a total of about 172 grams per day. Daily consumption of 0.087% - 0.21% tomato pomace on a dry matter basis corresponds to 0.15 grams to 0.35 grams of tomato pomace per 172 grams on a dry matter basis, resulting in a decrease in circulating 4-EPS levels.

Table 1

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

[0134] Example 4

[0135] Saliva samples are obtained from dogs. The samples may be shipped as having been collected at a laboratory in another location, may be partially processed and then shipped to a laboratory in another location, or may be fully processed and analyzed at the laboratory and the collection site. If the samples are shipped as collected to a laboratory in another location or are partially processed and then shipped to a laboratory in another location, some or all of the data collected from the samples by the laboratory may be sent to the collection site and / or the veterinarian and / or the owner or responsible person of the dog. After the saliva samples are obtained, they may be processed for analysis and evaluated for the presence of the CC genotype.

[0136] If the results indicate that the dog has a high likelihood or risk of developing an elevated 4-EPS level, the dog may be administered a composition containing an effective amount of tomato pomace.

[0137] Example 5

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

[0139] At that time, the animal should not eat for 30 minutes or drink for 10 minutes before saliva collection, and the person collecting should not be able to scrape the animal's teeth or cheeks with a sponge, nor should the animal be allowed to chew on the sponge.

[0140] The collection tube provided as part of the PERFORMAgene PG-100 oral collection kit contains a liquid for preserving the DNA sample and is required by the lab for sample analysis. The cap should not be removed before sample collection.

[0141] In the first step of the collection protocol, the sponge is placed in the animal's buccal pouch. Saliva is collected for 30 seconds by removing the sponge and wiping the areas where saliva naturally accumulates (buccal pouch and sublingual area). For animals over 6 months old, moderate restraint may be required.

[0142] Next, hold the tube straight and the cap from the tube is not screwed on. Turn the cap upside down and place the oral swab inside the tube. The cap is tightly screwed on to prevent the liquid sample from leaking during transportation. Invert the tube and shake it vigorously several times, for example 10 times, to completely mix the sample.

[0143] You may clearly write the animal identification number in the blank on the tube label using a permanent marker.

[0144] The step-by-step laboratory protocol for the manual purification of DNA from a 0.5 mL aliquot of Performagene (trademark) samples collected and stored with Performagene chemistry using the PG-100 collection kit is as follows. The reagents required for manual purification are available in the PG-AC1 reagent package or the PG-AC4 reagent package.

[0145] When the DNA sample is collected and mixed with the Performagene solution, the DNA is immediately stabilized. Performagene samples are stable at room temperature for 1 year from the time of collection. Performagene samples can be stored indefinitely at -15°C to -20°C and multiple freeze-thaw cycles can be performed without degrading the DNA.

[0146] In the purification process, the following equipment and reagents are used: a microcentrifuge that can be run at 15,000×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 a similar solution, any glycogen (20 mg / mL) (e.g., Invitrogen Cat. No. 10814-010), ethanol (70%) at room temperature and 5 M NaCl solution.

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

[0148] Incubate the sample in an air incubator at 50°C for at least 2 hours, or in a water incubator at 50°C for at least 1 hour. The DNA in Performagene is stable at room temperature without performing the incubation step. This heat treatment step is essential to ensure that the DNA is properly released and the nuclease is permanently inactivated. This incubation step can be performed at any point after the sample is collected from the animal and before it is purified. Incubation of the entire sample is recommended. The sample can be incubated overnight at 50°C if more convenient. An air incubator requires a longer time because the temperature equilibrium is slower than that of a water incubator.

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

[0150] Next, the mixed 500 μL of 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 the PG-L2P purifier is added to the Eppendorf tube and mixed by vortexing for several seconds. When impurities and inhibitors precipitate, the sample becomes turbid.

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

[0152] Leave the sample at room temperature for 10 minutes to completely precipitate the DNA. Then place the tube in the centrifuge in a known orientation (the DNA pellet may not be visible after centrifugation) and centrifuge at >15,000×g for 2 minutes at room temperature. For example, each tube may be placed in the microcentrifuge such that the hinge part of the cap points 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 visible) at the tip of the tube under the hinge.

[0153] Remove the supernatant with a pipette tip and discard it. The pellet contains DNA. Rotate the tube so that the pellet is on the upper wall, and then safely move the pipette tip along the lower wall to remove all of the supernatant. The supernatant may contain impurities and should be removed as completely as possible. Over-drying of the pellet can make it more difficult to dissolve the DNA. The DNA is first washed by adding 250 μL of 70% ethanol and then left at room temperature for 1 minute. The ethanol is removed with a pipette tip without disturbing the pellet. Washing with 70% ethanol helps to remove residual inhibitors. However, complete removal of the ethanol is essential to prevent inhibition during downstream applications. Therefore, centrifuge the tube for 6 seconds 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. Vortex for at least 5 seconds to assist the dissolution process. Leave at room temperature overnight to ensure complete rehydration of the DNA. The DNA can be quantified and used in downstream applications.

[0155] Assays using fluorescent dyes are more specific than absorbance at 260 nm for quantifying the amount of double-stranded DNA (dsDNA) in a DNA sample. Since they are less interfered with by contaminating RNA, fluorescent dyes such as PicoGreen® or SYBR® Green I may be used to quantify dsDNA for DNA quantification by fluorescence methods. Alternatively, commercially available kits such as Invitrogen's Quant-iT™ PicoGreen dsDNA Assay Kit (catalog number Q-33130) may be used. In either protocol, the purified DNA is preferably diluted 1:50 in TE solution and 5 μL is used in the quantification assay.

[0156] Alternatively, the DNA may be quantified by absorbance, in which case the purified sample is preferably first treated with RNase to digest contaminating RNA and then the RNA fragments are removed by ethanol precipitation of the DNA. DNA from Performagene samples usually contains significantly more RNA than the RNA found in blood samples. Read the absorbance after confirming that the alcohol-precipitated DNA is completely dissolved. 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 an overly large amount of sample, a spectrophotometer cuvette with a capacity of 100 μL or less should be used to read the sample. The absorbance value at 260 nm should be between 0.1 and 1.5. Lower values may be less reliable.

[0157] Dilute a 10 μL aliquot of the purified RNase-treated DNA with 90 μL of TE (1 / 10 dilution) and mix by gently pipetting up and down. Wait for the 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 values are calculated by subtracting the absorbance at 320 nm (A 280 and A 260 values from the A 320 values. DNA concentration in ng / μL = corrected A 260 × 10 (dilution factor) × 50 (conversion factor). A 260 / A 280 ratio: Divide the corrected A 260 by the corrected A 280 . Example 6

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

[0159] Starting with a sample containing purified genomic DNA (200 - 400 ng), the sample is subjected to PCR - free whole - genome amplification to generate fragmented DNA for SNP investigation. The CC genotype, the Illumina Canine HD panel, and an additional 50,000 - 100,000 custom gene markers (SNPs) (The Illumina Canine HD panel, Illumina, Inc. San Diego, CA) may be used. Such a system can be used to investigate samples for the presence of the CC genotype, or the BeadArray technology can be customized to limit the SNPs for screening to fewer.

[0160] The Illumina BeadArray technology is based on small silica beads that self-organize within microwells on a planar silica slide. Each bead is covered with hundreds of thousands of copies of specific oligonucleotides that act as capture arrays in the Infinium assay. When the beads self-organize, a unique decoding process maps the position of each bead, ensuring that each bead is individually quality controlled. As a result of this manufacturing process, all BeadChips are subjected to rigorous testing to guarantee the highest possible quality standards. The Infinium assay, unlike many alternative PCR-dependent assays, can be scaled to unlimited multiplexing without sacrificing data quality. A simple and 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 hybridizing an unlabeled DNA sample to the Beadchip, high call rates and accuracy are obtained through a two-step allele detection process. Selectivity and specificity are achieved in two steps. Target hybridization to the 50mer oligos attached to the beads provides high selectivity, while enzyme-mediated single-base extension incorporates labeled nucleotides for 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 robust, high call rates, and copy number data with low noise. The Infinium assay generates a two-color (one color for each allele) readout for each SNP in genotyping assays. The intensity values of each of the two-color channels A and B convey information about the allelic ratio at a single genomic locus. Typical studies incorporate values from a large number of samples (hundreds to tens of thousands) to ensure significant statistical representation. When these values are properly normalized and distinct patterns (or clusters) appear when plotted, samples at the assayed locus have the same genotype, exhibit similar signal profiles (A and B values), and aggregate into clusters. In the case of diploid organisms, both allelic loci are expected to exhibit three clusters (AA, AB, and BB). Genotype calling is based on information derived from a standard cluster file that provides statistical data from a representative set of samples. This enables the genotype to be called by referring to the single intensity of the assay against known data for a given locus. Since the 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 discriminates the cluster patterns of genotyping samples and reports an overview.

[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 may include providing a daily diet containing an effective amount of tomato pomace to a dog suspected of having elevated 4-EPS. In some embodiments, the method may include providing a daily diet containing an effective amount of tomato pomace to a dog identified as having elevated 4-EPS. In some embodiments, the method may include measuring the dog's 4-EPS to identify dogs having elevated 4-EPS and providing a daily diet containing an effective amount of tomato pomace to the dog.

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

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

[0165] In some embodiments, the dog may initially be identified as presenting, or suspected of presenting, anxiety, an anxiety disorder, or symptoms of anxiety or an anxiety disorder. The dog can then be tested to determine whether its 4-EPS level is elevated by one of the methods described herein. If the result shows that the dog has an elevated 4-EPS value, it is treated by providing it with a daily diet that includes an effective amount of tomato pomace.

[0166] In some embodiments, the dog may initially be identified as presenting, or suspected of presenting, stress, a stress disorder, or symptoms of stress or a stress disorder. Next, a dog is tested to determine whether the 4-EPS level has increased by one of the methods described herein. If the result shows that the dog has an increased 4-EPS value, it is treated by giving it a daily diet containing an effective amount of tomato pomace.

[0167] Example 8

[0168] The following compositions are based on the total amount of nutrients 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 from 0.087% to 0.5%. In some embodiments based on the total weight of the composition on a dry matter basis, the amount of tomato pomace is from 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 from 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 from 0.087% to 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 contain 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 contain 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 contain 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 contain additional plant sources 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 contain additional fruit sources 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% in addition to tomato pomace based on the total weight of the composition on a dry matter basis. In certain embodiments, the composition may contain carbohydrates selected from foxtail millet, glutinous rice, rolled oats, 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 contain the dry weight of a carbohydrate source within a range defined by any two of these values as endpoints.

[0171] Example 9

[0172] Table 2 describes certain embodiments having the proportions of the composition (dry weight % of the component composition).

[0173]

Table 2

[0174] A daily diet containing an effective amount of tomato pomace can provide benefits to dogs identified as being stressed. In some embodiments, the method includes identifying a dog having, suspected of having, or suspected of exhibiting symptoms of stress or a stress disorder, and providing it with a daily diet containing an effective amount of tomato pomace.

[0175] A method is provided that includes feeding a dog a daily diet containing an amount of tomato pomace equal to 0.044% to 0.42% of the daily nutrient intake. In some embodiments, a method is provided that includes feeding a dog a daily diet containing an amount of tomato pomace equal to 0.066% to 0.315% of the daily nutrient intake. In some embodiments, a method is provided that includes feeding a dog a daily diet containing an amount of tomato pomace equal to 0.087% to 0.21% of the daily nutrient intake. In some embodiments, a method is provided that includes feeding a dog a daily diet containing an amount of tomato pomace equal to approximately 0.14% of the daily nutrient intake.

[0176] Example 10

[0177] Table 3 describes the components used in certain embodiments having the composition ratios (dry matter weight % of the component composition). [Table 3]

[0178] Example 11

[0179] Table 4 describes the components used in certain embodiments having the composition ratios (dry matter weight % of the component composition). [Table 4]

[0180] Example 12

[0181] Table 5 describes the components used in certain embodiments having the proportions of the composition (in % of the dry matter weight of the component composition). [Table 5]

[0182] Example 13

[0183] Table 6 describes the components used in certain embodiments having the proportions of the composition (in % of the dry matter weight of the component composition). [Table 6]

[0184] Example 14

[0185] Table 7 describes the components used in certain embodiments having the proportions of the composition (in % of the dry matter weight of the component composition). [Table 7]

[0186] Example 15

[0187] Table 8 describes the components used in certain embodiments having the proportions of the composition (in % of the dry matter weight of the component composition). [Table 8]

[0188] Example 16

[0189] Table 9 describes the components used in certain embodiments having the proportions of the composition (in % of the dry matter weight of the component composition). [Table 9]

Claims

1. A method for preventing or reducing an increase in 4-ethylphenyl sulfate level in a dog, comprising: analyzing a biological sample obtained from the dog to identify the dog as having a high likelihood of an increase in the level of 4-ethylphenyl sulfate, based on the presence of two copies of the minor allele C of a single nucleotide polymorphism (SNP) at position 101 of SEQ ID NO: 1 in the dog; feeding tomato pomace to the dog; wherein the presence of two copies of the minor allele C of the SNP indicates that the dog has a high likelihood of an increase in the level of 4-ethylphenyl sulfate throughout its life.

2. A method for treating a dog for dog anxiety or dog stress, comprising: detecting the presence of two copies of the minor allele C of a single nucleotide polymorphism (SNP) at position 101 of SEQ ID NO: 1 in a biological sample from the dog; feeding tomato pomace to the dog daily; wherein the presence of two copies of the minor allele C of the SNP indicates that the dog has a high likelihood of an increase in the level of 4-ethylphenyl sulfate throughout its life.

3. The method according to claim 1 or 2, wherein the dog is fed tomato pomace corresponding to 0.044% to 0.42% of the daily nutrient intake.

4. The method according to any one of claims 1 to 3, wherein the dog has been previously identified as having dog anxiety disorder.

5. The method according to any one of claims 1 to 3, wherein the dog has been previously identified as having dog stress symptoms.

6. A dog food composition for preventing or reducing an increase in the level of 4-ethylphenyl sulfate in a dog, comprising: tomato pomace in an amount equal to 0.087% to 0.21% of the daily nutrient intake. Preventing or reducing an increase in the level of 4-ethylphenyl sulfate in the dog subject involves, in the dog subject, analyzing a biological sample obtained from the dog subject for the presence of two copies of the minor allele C of the single nucleotide polymorphism (SNP) at position 101 of SEQ ID NO: 1, thereby identifying the dog subject as having a high likelihood of an increase in the level of 4-ethylphenyl sulfate, and the presence of two copies of the minor allele C of the SNP indicates that the dog subject has a high likelihood of an increase in the level of 4-ethylphenyl sulfate throughout its lifetime, a dog food composition. **Claim 7** The dog food composition according to claim 6, comprising tomato pomace present in an amount equal to 0.14% of the daily nutrient intake.

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