Microbial compositions for canine intestinal disease and dysbiosis and methods of use

Abstract

To provide a composition and a method for treating dogs in response to intestinal diseases or in terms of prevention.SOLUTION: The present disclosure relates to isolated microbial-microbial ensembles comprising novel strains of microbes and compositions comprising them. Further, the present disclosure teaches methods of utilizing the described microbes, microbial compositions, and compositions comprising them, in methods of treating or preventing gastrointestinal disease or dysbiosis in dogs. In certain aspects, the present disclosure provides methods of treating or preventing morbidity and mortality caused by GI etiology or autoimmunity.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 972,337, filed February 10, 2020, and U.S. Provisional Patent Application No. 63 / 083,178, filed September 25, 2020, each of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to isolated, biologically pure microorganisms that have particular utility in the prevention and treatment of intestinal disease or dysbiosis in dogs. The disclosed microorganisms may be utilized in their isolated, biologically pure state or formulated into compositions. The present disclosure provides microbial ensembles containing at least two members of the disclosed microorganisms, as well as methods for utilizing the microbial ensembles. Additionally, the present disclosure provides methods for modulating the gastrointestinal microbiome of dogs.

[0003] Sequence Listing Description The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the Sequence Listing is named ASBI_018_03WO_SeqList_ST25.txt. The text file is 112kb, was created on February 9, 2021, and has been submitted electronically via EFS-Web. [Background technology]

[0004] Dogs are colonized by microorganisms in the gastrointestinal (GI) tract, skin, and other epithelial and tissue niches, such as the oral cavity, ocular surface, and vagina. The GI tract harbors a rich and diverse microbial community. It is a complex system, providing an environment or niche for many different species or organisms, including diverse strains of bacteria. Hundreds of different species may form commensal communities within the GI tract of healthy animals, and this assortment of organisms evolves from birth to ultimately form a functionally mature microbial population. Within these populations, interactions between microbial strains and between microorganisms and the host, such as the host immune system, shape the community structure, with resource availability and competition affecting microbial distribution. Such resources can be food, habitat, availability of growing space, or physical structures to which microorganisms can attach. For example, the host's diet contributes to shaping the GI tract microbiota.

[0005] A healthy microbiota provides multiple benefits to the host, including resistance to colonization against a wide range of pathogens, biosynthesis and absorption of essential nutrients, and immune stimulation that maintains a healthy intestinal epithelium and well-regulated systemic immunity. In dysbiosis or common intestinal diseases, the composition and function of the microbiota can be lost or significantly altered, resulting in increased susceptibility to pathogens, altered metabolic profiles, or the induction of proinflammatory signals that can lead to local or systemic inflammation or autoimmunity. Therefore, the gut microbiota plays a critical role in the pathogenesis of many diseases and disorders, including various pathogenic infections of the intestine. For example, when the normal gut microbiota is disrupted by the use of broad-spectrum antibiotics, dogs become more susceptible to pathogenic infections. Many of these diseases and disorders are chronic conditions that significantly reduce the animal's quality of life and can be fatal.

[0006] To date, gut microbiome replacement or supplementation in dogs has been limited primarily to oral supplementation with freeze-dried microorganisms typically found in yogurt and fecal transplants. Both have the potential to treat varying degrees of intestinal dysbiosis. Oral supplements are easier to administer, but selecting the appropriate microorganisms for the most effective outcome is challenging. Fecal transplants are considered a procedure of last resort because of the potential for transmission of infectious or allergenic substances between hosts, the potential for transmission of hundreds of unknown strains from donor to patient, and the difficulty of performing them on a large scale. Furthermore, fecal transplants are inherently non-standardized and inconsistent, and various desirable and / or undesirable substances may be transmitted in any given donation. Therefore, there is a need for defined compositions that can be utilized to treat a spectrum of diseases and conditions associated with intestinal disease by reducing an animal's susceptibility to infection and / or promoting the restoration of a healthy gut microbiome.

[0007] Therefore, there is a need for effective, well-defined, and reproducible treatments for enteric and dysbiotic disorders in dogs. To prepare a therapeutic agent with commercial potential, we designed a bacterial composition of isolated bacterial strains with multiple beneficial properties based on our understanding of those bacterial strains and our analysis of properties that enhance the utility and commercialization of the bacterial composition.

[0008] Therefore, in response to the need for durable, efficient, and effective compositions and methods for the treatment of intestinal diseases and GI dysbiosis, particularly serious pathogenic infections, as well as for the maintenance of general GI health, we provide compositions and methods for treating dogs in response to or as a preventative measure against intestinal disease. Summary of the Invention [Means for solving the problem]

[0009] In some embodiments, the disclosure provides a microbial composition comprising: (a) a purified microbial population comprising one or more bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1-333; and (b) one or more carriers suitable for administration to a canine. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 19. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 172. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 172. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 237. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that includes SEQ ID NO: 237. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 326. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence comprising SEQ ID NO: 326. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 327. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 328. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 328. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 329. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that comprises SEQ ID NO: 329.In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 330. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence comprising SEQ ID NO: 330. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NO: 331. In some embodiments, the purified microbial population comprises bacteria having a 16S nucleic acid sequence that comprises SEQ ID NO: 331. In some embodiments, one or more bacteria comprise a 16S nucleic acid sequence that shares at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 19, 172, 237, and 326-331. In some embodiments, one or more bacteria comprise a 16S nucleic acid sequence selected from SEQ ID NOs: 19, 172, 237, and 326-331. In some embodiments, one or more bacteria comprise a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NOs: 19, 172, and 326. In some embodiments, the one or more bacteria comprise the 16S nucleic acid sequences of SEQ ID NOs: 19, 172, and 326. In some embodiments, the one or more bacteria comprise a 16S nucleic acid sequence that shares at least 97% sequence identity with SEQ ID NOs: 19, 172, 237, 326, 328, 330, and 331. In some embodiments, the one or more bacteria comprise the 16S nucleic acid sequences of SEQ ID NOs: 19, 172, 237, 326, 328, 330, and 331.

[0010] In some embodiments, the microbial composition is comprised of spores. In some embodiments, the microbial composition is embedded in wax. In some embodiments, the microbial composition is encapsulated. In some embodiments, the encapsulated microbial composition comprises a polymer selected from the group consisting of a sugar polymer, an agar polymer, an agarose polymer, a protein polymer, and a lipid polymer. In some embodiments, the microbial composition is preserved in a glass matrix.

[0011] In some embodiments, the one or more carriers are selected from the group consisting of edible feed-grade materials, aluminosilicate-containing minerals, zeolites, calcium carbonate, prebiotics, and flavoring agents. In some embodiments, the prebiotics are inulin, oligosaccharides, and / or vitamins. In some embodiments, the one or more carriers are inulin, calcium carbonate, activated carbon, and / or yucca. In some embodiments, the flavoring agent is dry yeast, cheese flavoring, beef flavoring, fish flavoring, poultry flavoring, and / or pork flavoring.

[0012] In some embodiments, the one or more bacteria are present in an amount of about 10 per gram of the composition. 2 ~about 10 15 In some embodiments, the one or more bacteria are present in the composition at a concentration of at least 10 cells per gram of the composition. 2 The cells are present in the composition at a concentration of 1000 cells.

[0013] In some embodiments, the microbial composition is mixed with or sprinkled on animal feed. In some embodiments, the microbial composition is formulated as a tablet, pill, capsule, powder, solution, suspension, or emulsion. In some embodiments, the microbial composition is formulated as a pill. In some embodiments, the microbial composition is formulated as food. In some embodiments, the microbial composition is formulated as dry food, wet food, kibble, or raw food.

[0014] In some embodiments, the disclosure provides a method for the preparation of a nucleic acid sequence comprising: (a) (i) Askusk9_546A deposited as NRRL Accession No. B-67972; (ii) Askusk9_672A deposited as NRRL Accession No. B-67973; (iii) Askusk9_210B deposited as NRRL Accession No. B-67974; (iv) Askusk9_51G deposited as NRRL Accession No. B-67975; (v) Askusk9_33E deposited as NRRL Accession No. B-67976; (vi) Provided is a microbial composition comprising at least one isolated microbial strain selected from the group consisting of (i) Ascusk9_0G deposited as NRRL Accession No. B-67977, (ii) Ascusk9_38A deposited as NRRL Accession No. B-67987, (iii) Ascusk9_17A deposited as NRRL Accession No. B-67986, and (ix) Ascusk9_2A deposited as NRRL Accession No. B-67985; and (b) one or more carriers suitable for administration to a dog.

[0015] In some embodiments, the present disclosure provides a method of imparting at least one desirable trait in a dog, the method comprising administering to the dog a microbial composition described herein. In some embodiments, the at least one desirable trait is selected from the group consisting of improved stool consistency, increased regularity of bowel movements, reduced incidence of diarrhea, reduced incidence of constipation, reduced straining during bowel movements, reduced antibiotic side effects, improved oral hygiene, livelier eyes, increased energy, increased appetite, improved coat and coat quality, reduced incidence of infectious or non-infectious diseases, increased lifespan, and / or improved dog performance.

[0016] In some embodiments, the present disclosure provides a method of maintaining or improving gastrointestinal health in a dog, the method comprising administering to the dog a microbial composition described herein.

[0017] In some embodiments, the disclosure provides methods of treating or preventing gastrointestinal dysbiosis or gastrointestinal disease in a dog, the methods comprising administering to the dog a microbial composition described herein. In some embodiments, the microbial composition reduces the incidence of diarrhea, improves stool consistency, reduces straining during defecation, reduces constipation, increases bowel regularity, reduces dysbiosis, and / or reduces bowel disease in the dog.

[0018] In some embodiments, the present disclosure provides a method for modulating the microbiome of a dog, the method comprising administering a composition described herein to the dog. In some embodiments, the modulation of the microbiome is an increase in the proportion of one or more bacteria in the microbiome, the increase being measured compared to a dog to which the one or more bacteria were not administered. In some embodiments, the modulation of the microbiome is a decrease in the proportion of one or more bacteria present in the microbiome before administration of the composition, the decrease being measured compared to the dog's microbiome before administration of the composition.

[0019] In some embodiments, the present disclosure provides a method for increasing a dog's resistance to colonization by a pathogenic microorganism, the method comprising administering a composition described herein, wherein the ability of the pathogen to colonize the gastrointestinal tract of the dog is reduced.

[0020] In some embodiments, the present disclosure provides a method of treating a dog for the presence of at least one pathogenic microorganism, the method comprising administering a composition described herein. In some embodiments, after administration of the composition, the relative abundance of the at least one pathogenic microorganism is reduced to less than 5% relative abundance in the gastrointestinal tract. In some embodiments, the relative abundance of the at least one pathogenic microorganism is reduced to less than 1% relative abundance in the gastrointestinal tract. In some embodiments, the at least one pathogenic microorganism is undetectable in the gastrointestinal tract.

[0021] In some embodiments, the microbial composition is administered once daily for the life of the dog. In some embodiments, the microbial composition is administered twice daily for the life of the dog. In some embodiments, the microbial composition is administered to the dog once daily, twice daily, three times daily, once weekly, twice weekly, three times weekly, once every two weeks, or once monthly for a period of one month, two months, three months, six months, or twelve months.

[0022] In some embodiments, the present disclosure provides a dog feed supplement capable of increasing desirable phenotypic traits in dogs, the feed supplement comprising (a) a microbial composition described herein present at a concentration that does not naturally occur in dogs, and (b) an acceptable carrier.

[0023] In some embodiments, the present disclosure provides a dog feed supplement capable of increasing desirable phenotypic traits in dogs, the feed supplement comprising: (a) one or more bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity with SEQ ID NOs: 19, 172, and 326; and (b) an acceptable carrier, wherein the one or more bacteria are present in an amount of at least 10 per gram of the composition. 2 It is present in the dog feed supplement at a concentration of 1000 cells.

[0024] In some embodiments, the disclosure provides a dog feed supplement for treating or preventing gastrointestinal dysbiosis or gastrointestinal disease in dogs, the feed supplement comprising: (a) one or more bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity with SEQ ID NOs: 19, 172, 237, 326, 328, 330, and 331; and (b) an acceptable carrier, wherein the one or more bacteria are present in an amount of at least 10 per gram of the composition. 2 It is present in the dog feed supplement at a concentration of 1000 cells.

[0025] In some embodiments, the present disclosure provides a dog feed supplement capable of increasing desirable phenotypic traits in dogs, the feed supplement comprising Megamonas sp. and an acceptable carrier, wherein the Megamonas sp. is present in an amount of at least 10 per gram of the composition. 2 It is present in dog food supplements at a concentration of 1000 cells / ml.

[0026] In some embodiments, the disclosure provides a dog feed supplement for treating or preventing gastrointestinal dysbiosis or gastrointestinal disease in dogs, the feed supplement comprising Megamonas sp. and an acceptable carrier, wherein the Megamonas sp. is present in an amount of at least 10 per gram of the composition. 2 It is present in the dog feed supplement at a concentration of 1000 cells.

[0027] In some embodiments, the present disclosure provides an isolated microbial strain selected from any one of the microbial strains in Table 1.

[0028] In some embodiments, the present disclosure provides an isolated microbial strain selected from the group consisting of: (a) Askusk9_546A deposited as NRRL Accession No. B-67972; (b) Askusk9_672A deposited as NRRL Accession No. B-67973; (c) Askusk9_210B deposited as NRRL Accession No. B-67974; (d) Askusk9_210B deposited as NRRL Accession No. B-67975; _51G, (e) Askusk9_33E deposited as NRRL Accession No. B-67976, (f) Askusk9_0G deposited as NRRL Accession No. B-67977, (g) Askusk9_38A deposited as NRRL Accession No. B-67987, (h) Askusk9_17A deposited as NRRL Accession No. B-67986, and (i) Askusk9_2A deposited as NRRL Accession No. B-67985.

[0029] In some embodiments, the present disclosure provides an isolated microbial strain comprising a polynucleotide sequence that shares at least 90% sequence identity with any one of SEQ ID NOs: 1-333.

[0030] In some embodiments, the present disclosure provides a substantially pure culture of the isolated microbial strains described herein.

[0031] Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure Some microorganisms described in this application have been deposited at the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) Culture Collection (NRRL®), located at 1815 N. University St., Peoria, IL 61604, USA. Some microorganisms described in this application have been deposited at the Bigelow National Center for Marine Algae and Microbiota, located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA. Some microorganisms described in this application have been deposited at the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20108, USA.

[0032] The deposit was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. ATCC, NRRL, and Bigelow National Center for Marine Algae and Microbiota accession numbers and corresponding deposit dates are provided in Table 1.

[0033] In Table 1, the closest taxa predicted using the microbial BLAST algorithm are listed in column 1. The strains specified in the table below have been deposited in the Native Microbials, Inc. laboratory since at least April 2019. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17] [Table 1-18] [Table 1-19] [Brief explanation of the drawings]

[0034] [Figure 1] 1 illustrates the general workflow of one embodiment of a method for determining the absolute abundance of one or more active microbial strains. [Figure 2]

[0023] A general workflow of one embodiment is shown for a method to determine the co-occurrence of one or more, or two or more active microbial strains in a sample with one or more metadata (environmental) parameters, and then utilize cluster analysis and community detection methods on the network of relationships determined. [Figure 3A] A list of MIC score cutoffs for target microorganisms for use in this disclosure is provided, with many microorganisms contributing to anti-IBD / anti-diarrheal conditions occurring above about 0.5 MIC and many microorganisms contributing to IBD / diarrheal conditions occurring below about 0.5 MIC. [Figure 3B] Furthermore, we demonstrate the emergence of three socially distinct groups that are expected to be effective in treating and preventing IBD / diarrhea. [Figure 4A] The impact on the IBD / diarrhea state (indicated by the MIC score on the Y-axis) is shown as a function of the number of network connections shared by the microorganisms (X-axis). Two populations emerge: microorganisms contributing to the IBD / diarrhea state and microorganisms contributing to the anti-IBD / diarrhea state. [Figure 4B] IBD / diarrhea states (pro- and anti-) associated with highly linked and unlinked microorganisms are shown, with highly linked microorganisms showing a tendency to contribute to anti-IBD / diarrhea states (high MIC scores). [Figure 5A] 1 shows the alpha diversity of the fecal microbiome of dog #1 before and after daily administration of a microbial supplement over a 30-day period. [Figure 5B] 1 shows the taxonomic diversity of the fecal microbiome of dog #1 before and after daily administration of a microbial supplement for 30 days. [Figure 5C] Figure 5 shows the taxa at the genus level in the fecal microbiome of dog #1 before and after daily administration of a microbial supplement over a 30-day period. The microbial supplement included Askusk9_51G, Askusk9_546A, and Askusk9_0G (Figures 5A-5C). [Figure 6] Principal coordinate analysis (PCoA) of four individual case studies in dogs is shown. Dogs #1 and #4 were healthy dogs supplemented with Ascusk9_51G, Ascusk9_546A, and Ascusk9_0G for 30 days. Dogs #2 and #3 were dogs diagnosed with chronic intestinal disease and supplemented with Ascusk9_51G, Ascusk9_546A, Ascusk9_0G, Ascusk9_210B, Ascusk9_17A, Ascusk9_2A, and Ascusk9_33E for 30 days. [Figure 7A] 1 shows the alpha diversity of the fecal microbiome of dog #2 before and after daily administration of a microbial supplement over a 30-day period. [Figure 7B] 1 shows the taxonomic diversity of the fecal microbiome of dog #2 before and after daily administration of a microbial supplement over a 30-day period. [Figure 7C] The taxa at the genus level in the fecal microbiome of dog #2 are shown before and after daily administration of a microbial supplement over a 30-day period. The microbial supplement included Askusk9_51G, Askusk9_546A, Askusk9_0G, Askusk9_210B, Askusk9_17A, Askusk9_2A, and Askusk9_33E (Figures 7A-7C). [Figure 8A]1 shows the alpha diversity of the fecal microbiome of dog #3 before and after daily administration of a microbial supplement over a 30-day period. [Figure 8B] 1 shows the taxonomic diversity of the fecal microbiome of dog #3 before and after daily administration of a microbial supplement for 30 days. [Figure 8C] The taxa at the genus level in the fecal microbiome of dog #3 are shown before and after 30 days of daily administration of a microbial supplement containing Askusk9_51G, Askusk9_546A, Askusk9_0G, Askusk9_210B, Askusk9_17A, Askusk9_2A, and Askusk9_33E (Figures 8A-8C). [Figure 9A] 1 shows the alpha diversity of the fecal microbiome of dog #4 before and after daily administration of a microbial supplement over a 30-day period. [Figure 9B] 1 shows the taxonomic diversity of the fecal microbiome of dog #4 before and after daily administration of a microbial supplement for 30 days. [Figure 9C] Figure 9 shows the taxa at the genus level in the fecal microbiome of dog #4 before and after 30 days of daily administration of a microbial supplement containing Askusk9_51G, Askusk9_546A, and Askusk9_0G (Figures 9A-9C). [Figure 10] Individual microbial isolates (Ascusk9_546A and Ascusk9_17A) demonstrate the ability to compete with E. coli in vitro. DETAILED DESCRIPTION OF THE INVENTION

[0035] definition Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the subject matter of the present disclosure.

[0036] The term "a" or "an" may refer to one or more of that entity, i.e., to multiple referents. As such, the terms "a" or "an," "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to an "element" by the open-ended article "a" or "an" does not exclude the possibility that multiple elements are present, unless the context clearly requires that only one of the element be present.

[0037] Throughout this specification, the terms "one embodiment," "embodiment," "one aspect," or "aspect" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0038] As used herein, in certain embodiments, the term "about" or "approximately" when preceding a numerical value indicates a range of plus or minus 10% of the value.

[0039] As used herein, the terms "microorganism" or "microbe" should be interpreted broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains, bacteria and archaea, the eukaryotic fungi and protozoa, and viruses. In some embodiments, the present disclosure refers to the "microorganisms" in Table 1, or "microorganisms" incorporated by reference. This characterization can refer not only to the predicted taxonomic microbial identifiers in the table, but also to the identified strains of the microorganisms listed in the table.

[0040] The term "microbial community" refers to a group of microorganisms comprising two or more species or strains. Unlike a microbial ensemble, a microbial community need not perform a common function or be involved in, linked to, or correlated with a recognizable parameter, such as a phenotypic trait of interest (e.g., reduced GI enteropathy or dysbiosis).

[0041] As used herein, "isolated," "isolated," "isolated microorganism," and similar terms are intended to mean that one or more microorganisms have been separated from at least one of the materials with which they are associated in a particular environment (e.g., soil, water, animal tissue).

[0042] The microorganisms of the present disclosure may include spores and / or plant cells. In some embodiments, the microorganisms of the present disclosure include viable but non-culturable (VBNC) or quiescent microorganisms. See Liao and Zhao (U.S. Patent Application Publication No. 2015 / 267163A1). In some embodiments, the microorganisms of the present disclosure include microorganisms in biofilms. See Merritt et al. (U.S. Patent No. 7,427,408).

[0043] Thus, an "isolated microorganism" does not exist in its naturally occurring environment; rather, through various techniques described herein, the microorganism has been removed from its natural environment and placed in a non-naturally occurring state of existence. Thus, an isolated strain or isolated microorganism may exist, for example, as a biologically pure culture or as a spore (or other form of the strain) associated with an acceptable carrier.

[0044] As used herein, "spore" or "spores" refers to structures produced by bacteria and fungi that are adapted for survival and dispersal. Although spores are generally characterized as dormant structures, spores can differentiate through the process of germination. Germination is the differentiation of a spore into a vegetative cell capable of metabolic activity, growth, and reproduction. Germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are the unit of asexual reproduction and, in some cases, are a necessary structure in the fungal life cycle. Bacterial spores are usually viable structures that may be non-conductive for the survival or growth of vegetative cells.

[0045] As used herein, "microbial composition" refers to a composition comprising one or more microorganisms of the present disclosure, and in some embodiments, the microbial composition is administered to an animal of the present disclosure.

[0046] As used herein, "carrier," "acceptable carrier," or "pharmaceutical carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions, and aqueous dextrose and glycerol solutions are preferably used as carriers, and in some embodiments, are used for injectable solutions. In some embodiments, gelling agents are used as carriers. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavoring agent, and a coloring agent. The choice of carrier can be determined with regard to the intended route of administration and standard pharmaceutical practice. Hardee and Baggo (1998. Development and Formulation of Veterinary Dosage Forms. 2 ndEd.CRC Press.504 pg.), EW Martin(1970.Remington's Pharmaceutical Sciences.17 th Ed. Mack Pub. Co.), and Blaser et al. (U.S. Patent Application Publication No. 2011 / 0280840A1).

[0047] In some embodiments, the carrier may be granular in structure, such as sand or sand particles. In further embodiments, the carrier may be dry, as opposed to moist or wet. In some embodiments, the carrier may be a nutritional and / or prebiotic substance selected from fructooligosaccharides, inulin, isomalto-oligosaccharides, lactitol, lactosucrose, lactulose, pyrodextrin, soybean oligosaccharides, transgalactooligosaccharides, xylooligosaccharides, and vitamins. In some embodiments, the carrier may be in solid or liquid form. In some embodiments, the carrier may be zeolite, calcium carbonate, aluminosilicate-containing minerals, magnesium carbonate, trehalose, chitosan, shellac, albumin, starch, nonfat dry milk, sweet whey powder, maltodextrin, lactose, and inulin. In some embodiments, the carrier is water or saline.

[0048] In some embodiments, one or more supports may be a zeolite. In some embodiments, the zeolite is a natural or synthetic zeolite. In some embodiments, the zeolite is selected from heulandite, analcite, chabazite, clinoptilolite, natrium, stilbite, and phillipsite.

[0049] The terms "bioensemble," "microbial ensemble," or "synthetic ensemble" refer to a composition identified by the disclosed methods, systems, and / or devices and containing one or more active microorganisms that do not naturally occur in ratios or amounts that do not occur in a naturally occurring environment and / or in nature. A bioensemble is a subset of a microbial community of individual microbial species, or strains of species, that can be described as performing a common function or as being involved in, leading to, or correlating with a recognizable parameter, such as a phenotypic trait of interest (e.g., reduced incidence of GI intestinal disease or dysbiosis). A bioensemble may include two or more species or strains of microorganisms. In some cases, microorganisms coexist symbiotically within the community.

[0050] In certain embodiments of the present disclosure, the isolated microorganism exists as an isolated, biologically pure culture. One of skill in the art will understand that an isolated, biologically pure culture of a particular microorganism indicates that the culture is substantially free (within scientific reason) of other organisms and contains only the individual microorganism in question. The culture may contain various concentrations of the microorganism. The present disclosure notes that isolated, biologically pure microorganisms are often "necessarily distinct from impure or impure material." See, e.g., In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins); In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microorganisms); and Parke-Davis & Co. v HK Mulford & Co., 189 F.95 (SDNY 1911) (discussing Learned Hand purified adrenaline), aff'd in part, rev'd in part, 196 F.496 (2d Cir. 1912), which are incorporated herein by reference. Furthermore, in some aspects, the present disclosure provides specific quantitative measures of concentration or purity limits that must be found in isolated, biologically pure microbial cultures. In certain embodiments, the presence of these purity values ​​is an additional attribute that distinguishes the microorganisms of the present disclosure from those present in their natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limits for microbially produced vitamin B12).

[0051] As used herein, "individual isolate" should be interpreted to mean a composition or culture containing a predominance of a single genus, species, or strain of a microorganism after separation from one or more other microorganisms. The phrase should not be interpreted as indicating the extent to which the microorganism has been isolated or purified. However, an "individual isolate" can contain substantially only one genus, species, or strain of a microorganism.

[0052] As used herein, "microbiome" refers to the collection of microorganisms and the physical environment of the microorganisms that inhabit the digestive or gastrointestinal tract of an animal (i.e., the microbiome has both biological and physical components). The microbiome is dynamic and can be modulated by many naturally occurring and artificial conditions (e.g., dietary changes, disease, antimicrobial agents, the influx of additional microorganisms, etc.). Modulation of the GI microbiome can be achieved through administration of one or more compositions of the present disclosure and can take the form of: (a) increasing or decreasing specific families, genera, species, or functional classes of microorganisms (i.e., altering the biological components of the GI microbiome) and / or (b) increasing or decreasing volatile fatty acids in the GI tract, increasing or decreasing pH, or increasing or decreasing any other physical parameter important to gastrointestinal health (i.e., altering the abiotic components of the GI microbiome).

[0053] As used herein, "probiotic" refers to a substantially pure microorganism (i.e., a single isolate) or a mixture of desired microorganisms, and may also include any additional components that can be administered to a dog to restore the microbiota. The probiotic or microbial inoculant compositions of the present disclosure may be administered with agents to enable the microorganisms to survive the environment of the gastrointestinal tract, i.e., to withstand low pH and grow in the gastrointestinal environment. In some embodiments, the composition (e.g., microbial composition) is a probiotic in some aspects.

[0054] As used herein, "prebiotics" refers to agents that increase the number and / or activity of one or more desired microorganisms.Non-limiting examples of prebiotics that can be useful in the method of the present disclosure include fructooligosaccharides (e.g., oligofructose, inulin, inulin-type fructans), galactooligosaccharides, amino acids, alcohols, and mixtures thereof.See Ramirez-Farias et al. (2008.Br.J.Nutr.4:1-10) and Pool-Zobel and Sauer (2007.J.Nutr.137:2580-2584, and supplements).

[0055] As used herein, the term "growth medium" refers to any medium suitable for supporting the growth of microorganisms. By way of example, the medium may be natural or artificial, including gastrin-supplemented agar, LB medium, serum, and tissue culture gel. Of course, the medium may be used alone or in combination with one or more other media. It may also be used with or without the addition of exogenous nutrients.

[0056] As used herein, the term "relative abundance" refers to the number or percentage of microorganisms present in the gastrointestinal tract or other organ system relative to the number or percentage of total microorganisms present in that organ system. Relative abundance may also be determined for a particular type of microorganism, such as a bacterium, fungus, virus, and / or protozoan, relative to the total number or percentage of bacteria, fungi, viruses, and / or protozoans present. In one embodiment, relative abundance is determined by PCR. In another embodiment, relative abundance is determined by colony-forming unit (cfu) or plaque-forming unit (pfu) assays performed on samples from the gastrointestinal tract or other organ system of interest.

[0057] The medium may be amended or enriched with additional compounds or components, e.g., components that may aid in the interaction with and / or selection of specific microbial groups. For example, antibiotics (such as penicillin) or sterilizing agents (e.g., quaternary ammonium salts and oxidizing agents) can be present, and / or physical conditions (e.g., salinity, nutrients (e.g., organic and inorganic minerals (e.g., phosphorus, nitrogen salts, ammonia, potassium, and micronutrients such as cobalt and magnesium), pH, and / or temperature), methionine, prebiotics, ionophores, and beta-glucans) can be amended.

[0058] As used herein, the term "dog" includes mammals of the genera Canis, Atelocynus, Cerdocyon, Chrysocyon, Cuon, Dusicyon, Lycalopex, Lycaon, Nyctereute, Otocyon, Speothos, Urocyon, and Vulpes.

[0059] As used herein, the terms "canine" and "canid" are used interchangeably to refer to mammals such as: domestic dog, short-eared dog, coyote, jackal, black-backed jackal, side-striped jackal, wolf, Abyssinian wolf, African golden wolf, gray wolf, dingo, crab-eating dog, maned wolf, dhole, Clupeo's fox, Darwin's fox, Japanese dog, Pampas fox, Sechura fox, African wild dog, raccoon dog, bat-eared dog dog), bush dog, gray fox, island gray fox, arctic fox, Bengal fox, Blanford's fox, Cape fox, Corsac fox, fennec fox, kit fox, black-tailed fox, red fox, silver fox, white-tailed fox, swift fox, and Tibetan fox, or any hybrids and / or crossbreeds thereof.

[0060] As used herein, "improved" should be interpreted broadly to encompass an improvement in a property of interest compared to a control group or compared to a known mean amount associated with that property. In this disclosure, "improved" does not necessarily require the data to be statistically significant (i.e., p<0.05); rather, any quantifiable difference demonstrating that one value (e.g., mean treatment value) is different from another value (e.g., mean control value) may be elevated to the level of "improved."

[0061] As used herein, "inhibition and suppression" and similar terms should not be construed as requiring complete inhibition or suppression, although in some embodiments this may be desirable.

[0062] As used herein, the term "marker" or "unique marker" refers to an indicator of a unique microbial type, strain, or activity of a microbial strain. Markers can be measured in biological samples and include, but are not limited to, nucleic acid-based markers such as ribosomal RNA genes, peptide- or protein-based markers, and / or metabolite or other small molecule markers.

[0063] As used herein, the term "metabolite" refers to an intermediate or product of metabolism. In one embodiment, a metabolite is a small molecule. Metabolites have a variety of functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, as cofactors for enzymes, in defense, and in interactions with other organisms (e.g., pigments, odorants, and pheromones). Primary metabolites are directly involved in normal growth, development, and reproduction. Secondary metabolites are not directly involved in these processes but usually have important ecological functions. Examples of metabolites include, but are not limited to, antibiotics and pigments such as resins and terpenes. Some antibiotics use primary metabolites as precursors, such as actinomycin, which is produced from the primary metabolite tryptophan. As used herein, metabolites include small hydrophilic carbohydrates, large hydrophobic lipids, and complex natural compounds.

[0064] As used herein, the term "genotype" refers to the genetic makeup of an individual cell, cell culture, tissue, organism, or group of organisms.

[0065] As used herein, the term "allele" refers to any of one or more alternative forms of a gene, all of which are associated with at least one trait or characteristic. In diploid cells, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. Because the present disclosure, in embodiments, relates to QTLs, i.e., genomic regions that may contain one or more genes or regulatory sequences, in some cases, the term "allele" will more accurately refer to "haplotypes" (i.e., alleles of a chromosomal segment) rather than alleles; however, in these instances, the term "allele" should be understood to include the term "haplotype." Alleles are considered identical if they express a similar phenotype. Sequence differences are possible but inconsequential as long as they do not affect the phenotype.

[0066] As used herein, the term "locus" (loci) means a specific location or place or site on a chromosome where, for example, a gene or genetic marker is found.

[0067] As used herein, the term "genetically linked" refers to two or more traits that are highly co-inherited during breeding, such that they are difficult to separate via breeding.

[0068] As used herein, "recombination" or "recombination event" refers to the crossing over or independent assortment of chromosomes. The term "recombinant" refers to an organism that has a new genetic makeup that results from a recombination event.

[0069] As used herein, the term "molecular marker" or "genetic marker" refers to an indicator used in a method for visualizing differences in nucleic acid sequence characteristics. Examples of such indicators include restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequences (CAPS) markers, or isozyme markers, or any combination of markers described herein that define specific genetic and chromosomal locations. Markers also include polynucleotide sequences encoding 16S or 18S rRNA, and internal transcribed spacer (ITS) sequences, which are sequences found between the small subunit and large subunit rRNA genes and have proven particularly useful for revealing the relationship or distinction between them when compared with each other. Mapping molecular markers near alleles is a procedure that can be performed by an average person skilled in molecular biology techniques.

[0070] The primary structure of the major rRNA subunit, the 16S, contains a specific combination of conserved, variable, and hypervariable regions that evolve at different rates, enabling the elucidation of both very ancient lineages, such as domains, and more recent lineages, such as genera. The secondary structure of the 16S subunit contains approximately 50 helices, with approximately 67% of the residues base-paired. These highly conserved secondary structural features are of crucial functional importance and can be used to ensure positional homology in multiple sequence alignments and phylogenetic analyses. Over the past several decades, the 16S rRNA gene has become the most sequenced taxonomic marker and is the cornerstone of the current phylogenetic classification of bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro. 12:635-45).

[0071] Sequence identity of 94.5% or less for two 16S rRNA genes is strong evidence for a separate genus, 86.5% or less for a separate family, 82% or less for a separate order, 78.5% or less for a separate class, and 75% or less for a separate phylum. Comparative analysis of 16S rRNA gene sequences allows for the establishment of taxonomic thresholds useful not only for the classification of cultured microorganisms but also for the classification of many environmental sequences. (Yarza et al. 2014. Nature Rev. Micro. 12:635-45)

[0072] As used herein, the term "trait" refers to a characteristic or phenotype. For example, in relation to some embodiments of the present disclosure, a reduced incidence of gastrointestinal dysbiosis, a reduced severity of gastrointestinal dysbiosis, a reduced incidence of diarrhea, a reduced severity of diarrhea, a reduced incidence of irritable bowel disease (IBD), a reduced severity of irritable bowel disease, a reduced incidence of gastrointestinal pathogen colonization, a reduced incidence of gastrointestinal pathogen-induced illness, a reduced frequency of gastrointestinal pathogen carriage, a reduced amount of primary bile acids present in the feces, an increased amount of secondary bile acids present in the feces, an increased production of fatty acids in the GI tract, an increased polysaccharide and lignin degradation, an increased fat, starch, and / or protein digestion, an increased pH balance, an increased vitamin availability, a reduced likelihood or incidence of mortality, a reduced likelihood or incidence of morbidity, an increased production of antimicrobial agents, an increased mammalian and / or microbial synthesis of vitamins, a reduced alpha diversity of the gastrointestinal microbiome, and / or a combination thereof, wherein the increase or decrease is determined by comparing the increase or decrease to an animal not administered the composition.

[0073] The term "trait" may also refer to the predominance of short-, medium-, and long-chain fatty acids produced or available systemically or in the gastrointestinal tract, such as improved digestibility, increased breakdown of cellulose, lignin, and hemicellulose, increased concentrations of GI or systemic fatty acids such as acetate, propionate, and butyrate, etc.

[0074] Traits can be inherited in a dominant or recessive manner, or in a partial or incomplete dominance manner. Traits may be monogenic (i.e., determined by a single locus) or polygenic (i.e., determined by multiple loci), or may result from the interaction of one or more genes with the environment.

[0075] In the context of the present disclosure, a trait may also result from the interaction of one or more canine genes and one or more microbial genes.

[0076] As used herein, the term "homozygosity" refers to the genetic condition that exists when two identical alleles are present at a particular locus, but are individually located on corresponding pairs of homologous chromosomes within a cell of a diploid organism. Conversely, as used herein, the term "heterozygosity" refers to the genetic condition that exists when two different alleles are present at a particular locus, but are individually located on corresponding pairs of homologous chromosomes within a cell of a diploid organism.

[0077] As used herein, the term "phenotype" refers to the observable characteristics of an individual cell, cell culture, organism (e.g., a dog), or group of organisms that result from the interaction between the genetic makeup (i.e., genotype) of an individual and the environment.

[0078] As used herein, the terms "chimeric" or "recombinant" when describing a nucleic acid or protein sequence refers to a nucleic acid or protein sequence that links at least two heterologous polynucleotides or two heterologous polypeptides into a single macromolecule, or that reconstitutes one or more elements of at least one naturally occurring nucleic acid or protein sequence. For example, the term "recombinant" can refer to the artificial combination of two otherwise separate segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acid by genetic engineering techniques.

[0079] As used herein, a "synthetic nucleotide sequence" or "synthetic polynucleotide sequence" is a nucleotide sequence that is not known to occur in nature or is not naturally occurring. Generally, such a synthetic nucleotide sequence will contain at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.

[0080] As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or their analogs. The term refers to the primary structure of the molecule and thus includes double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. It also includes modified nucleic acids, such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, etc. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably.

[0081] As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and / or regulatory sequences necessary for their expression. Genes may also include non-expressed DNA segments, such as those that form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0082] As used herein, the terms "homologous," "homolog," or "ortholog" are known in the art and refer to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms "homology," "homologous," "substantially similar," and "substantially corresponding" are used interchangeably herein. They refer to nucleic acid fragments in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a particular phenotype. These terms also refer to modifications of the nucleic acid fragments of the present disclosure, such as the deletion or insertion of one or more nucleotides, that do not substantially alter the functional properties of the resulting nucleic acid fragment compared to the original, unmodified fragment. Thus, as one of skill in the art will understand, the present disclosure encompasses more than specific exemplary sequences. These terms describe the relationship between a gene found in one species, subspecies, variety, strain, or culture and the corresponding or equivalent gene in another species, subspecies, variety, strain, or culture. For purposes of this disclosure, homologous sequences are compared. "Homologous sequences" or "homologs" or "orthologs" are thought, believed, or known to be functionally related. Functional relationships can be demonstrated in any one of several ways, including, but not limited to, (a) degree of sequence identity and / or (b) identical or similar biological function. Preferably, both (a) and (b) are demonstrated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M.A. Usubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK), ALIGN Plus (Scientific and Educational Software, Pennsylvania), and AlignX (Vector NTI, Invitrogen, Carlsbad, CA).Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters.

[0083] As used herein, the term "nucleotide change" refers to, for example, a nucleotide substitution, deletion, and / or insertion, as is well understood in the art. For example, mutations contain changes that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or the way the protein is made.

[0084] As used herein, the term "protein modification" refers to, for example, amino acid substitutions, modifications, deletions, and / or insertions that are well understood in the art.

[0085] As used herein, the term "at least a portion" or "fragment" of a nucleic acid or polypeptide refers to a portion having the minimum size characteristic of such a sequence, or any larger fragment of a full-length molecule, up to and including the full-length molecule. A fragment of a polynucleotide of the present disclosure can encode a biologically active portion of a gene regulatory element. A biologically active portion of a gene regulatory element can be prepared by isolating a portion of one of the polynucleotides of the present disclosure that contains the gene regulatory element and assessing activity as described herein. Similarly, a portion of a polypeptide can be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, etc., up to the full-length polypeptide. The length of the portion used will depend on the particular application. A portion of a nucleic acid useful as a hybridization probe can be as short as 12 nucleotides, and in some embodiments, 20 nucleotides. A portion of a polypeptide useful as an epitope can be as short as 4 amino acids. A portion of a polypeptide that performs the function of the full-length polypeptide is generally longer than 4 amino acids.

[0086] Variant polynucleotide also encompasses the sequence derived from mutagenic and recombinogenic procedures such as DNA shuffling.Such DNA shuffling strategies are known in the art.For example, see Stemmer (1994) PNAS 91:10747-10751, Stemmer (1994) Nature 370:389-391, Crameri et al. (1997) Nature Biotech.15:436-438, Moore et al. (1997) J.Mol.Biol.272:336-347; Zhang et al. (1997) PNAS 94:4504-4509, Crameri et al. (1998) Nature 391:288-291, and U.S. Patent No. 5,605,793 and U.S. Patent No. 5,837,458. For PCR amplification of the polynucleotides disclosed herein, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York). Also see Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and See also "PCR Applications" (Academic Press, New York), Innis and Gelfand, eds. (1995) "PCR Strategies" (Academic Press, New York), and Innis and Gelfand, eds. (1999) "PCR Methods Manual" (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.

[0087] As used herein, the term "primer" refers to an oligonucleotide that can anneal to an amplification target and attract DNA polymerase, thereby serving as a starting point for DNA synthesis when placed under conditions that induce the synthesis of primer extension products, i.e., in the presence of nucleotides and a polymerization agent, such as a DNA polymerase, at an appropriate temperature and pH. (Amplification) primers are preferably single-stranded for maximum efficiency in amplification. Primers are preferably oligodeoxyribonucleotides. Primers must be long enough to prime the synthesis of extension products in the presence of a polymerization agent. The exact length of a primer depends on many factors, including the temperature and composition (A / T vs. G / C content) of the primer. A pair of bidirectional primers, consisting of one forward and one reverse primer, is commonly used in DNA amplification techniques, such as PCR amplification.

[0088] The term "stringency" or "stringent hybridization conditions" refers to hybridization conditions that affect hybrid stability, e.g., temperature, salt concentration, pH, formamide concentration, etc. These conditions are empirically optimized to maximize specific binding of primers or probes to their target nucleic acid sequences and minimize nonspecific binding. The term used includes reference to conditions under which a probe or primer hybridizes to its target sequence to a detectably greater extent than other sequences (e.g., at least twice the background level). Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences will specifically hybridize at higher temperatures. Generally, stringent conditions are selected to be approximately 5°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Typically, stringent conditions are those in which the salt concentration is less than about 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion (or other salt), at a pH of 7.0 to 8.3, and the temperature is at least about 30°C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes or primers (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. Exemplary low stringency or "reduced stringency" conditions include hybridization in a buffer of 30% formamide, 1 M NaCl, and 1% SDS at 37°C, followed by a wash in 2×SSC at 40°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, and 1% SDS at 37°C, followed by a wash in 0.1×SSC at 60°C. Hybridization procedures are well known in the art and are described, for example, in Ausubel et al., 1998 and Sambrook et al., 2001.In some embodiments, stringent conditions include hybridization in 0.25 M NaHPO buffer (pH 7.2) containing 1 mM NaEDTA and 0.5-20% (e.g., 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) sodium dodecyl sulfate at 45°C, followed by 5x washes with SSC containing 0.1% (w / v) sodium dodecyl sulfate at 55°C to 65°C.

[0089] As used herein, "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. A promoter sequence consists of proximal and more distal upstream elements, the latter often referred to as enhancers. Thus, an "enhancer" is a DNA sequence capable of stimulating promoter activity, and may be a promoter's native element or a heterologous element inserted to enhance the level or tissue specificity of the promoter. A promoter may be derived entirely from a native gene, or may be composed of different elements derived from different naturally occurring promoters, or may even contain synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, at different developmental stages, or in response to different environmental conditions. It is further recognized that, in most cases, the exact boundaries of regulatory sequences have not been fully defined, and therefore DNA fragments with some variation may have identical promoter activity.

[0090] As used herein, a "constitutive promoter" is a promoter that is active under most conditions and / or during most developmental stages. The use of constitutive promoters in expression vectors used in biotechnology has several advantages, including, for example, high-level production of proteins used to select transgenic cells or organisms, high-level expression of reporter proteins or scorable markers that allow for easy detection and quantification, high-level production of transcription factors that are part of a regulated transcription system, production of compounds that require ubiquitous activity in an organism, and production of compounds required at all stages of development. Non-limiting exemplary constitutive promoters include the CaMV 35S promoter, opine promoter, ubiquitin promoter, alcohol dehydrogenase promoter, and the like.

[0091] As used herein, a "non-constitutive promoter" refers to a promoter that is active under certain conditions, in certain types of cells, and / or during certain developmental stages. For example, tissue-specific, tissue-preferential, cell-type-specific, cell-type-preferential, inducible promoters, and promoters under developmental control are non-constitutive promoters. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues.

[0092] As used herein, an "inducible" or "repressible" promoter is a promoter that is under the control of chemical or environmental factors. Examples of environmental conditions that can affect transcription by an inducible promoter include anaerobic conditions, certain chemicals, the presence of light, acidic or basic conditions, etc.

[0093] As used herein, "tissue-specific" promoter refers to a promoter that only initiates transcription in specific tissues.Unlike the constitutive expression of genes, tissue-specific expression is the result of several levels of gene regulation interaction.Therefore, in the art, it is sometimes desirable to use promoters from homologous or closely related species to achieve efficient and reliable expression of transgenes in specific tissues.This is one of the main reasons why a large number of tissue-specific promoters isolated from specific tissues can be found in both scientific and patent literature.

[0094] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is controlled by the other. For example, a promoter is operably linked to a coding sequence if it can regulate the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. In another example, the complementary RNA region of the present disclosure can be operably linked directly or indirectly to a target mRNA 5', or 3', or within the target mRNA, or the first complementary region is 5' to the target mRNA and its complement is 3'.

[0095] As used herein, the phrases "recombinant construct," "expression construct," "chimeric construct," "construct," and "recombinant DNA construct" are used interchangeably herein. A recombinant construct includes an artificial combination of nucleic acid fragments, such as regulatory and coding sequences, that are not found together in nature. For example, a chimeric construct may contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from that found in nature. Such constructs can be used alone or in combination with a vector. When a vector is used, the choice of vector depends on the method used to transform host cells, which is well known to those skilled in the art. For example, a plasmid vector can be used. Those skilled in the art are well aware of the genetic elements that must be present on a vector to successfully transform, select, and grow host cells containing any of the isolated nucleic acid fragments of the present disclosure. Those skilled in the art also recognize that different, independent transformation events result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86). Therefore, multiple events must be screened to obtain a line exhibiting the desired expression level and pattern. Such screening can be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblot analysis of protein expression, or phenotypic analysis, among others. Vectors can be plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, etc., which can replicate autonomously or integrate into the host cell chromosome. Vectors can also be naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, polylysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, etc., which do not replicate autonomously.As used herein, the term "expression" refers to the production of a functional end-product, such as mRNA or a protein (precursor or mature).

[0096] In some embodiments, the cell or organism has at least one heterologous trait. As used herein, the term "heterologous trait" refers to a phenotype imparted to a host cell or transgenic organism transformed with an exogenous DNA segment, heterologous polynucleotide, or heterologous nucleic acid. Various phenotypic changes are of interest to the present disclosure, including, but not limited to, reduced severity of GI intestinal disorders and dysbiosis (e.g., reduced diarrhea, bloody diarrhea, etc.). These results can be achieved by providing increased expression of heterologous products or endogenous products in an organism using the methods and compositions of the present disclosure.

[0097] As used herein, the term "MIC" refers to maximum information coefficient. MIC is a type of non-parametric network analysis that identifies a score (MIC score) between the active microbial strains of the present disclosure and at least one measured metadata (e.g., ability to treat / prevent GI dysbiosis or enteric disease). Additionally, U.S. Patent Application No. 15 / 217,575, filed July 22, 2016 (issued January 10, 2017 as U.S. Patent No. 9,540,676), is incorporated herein by reference in its entirety.

[0098] Next, as shown in Figure 2, maximum information coefficients (MIC) are calculated between strains and metadata 3021a and between strains 3021b. The results are pooled to create a list of all relationships and their corresponding MIC scores 3022. If the relationship score is below a given threshold 3023, the relationship is considered / qualified as unrelated 3023b. If the relationship is above a given threshold 3023, the relationship is considered / qualified as related 2023a and is subject to further network analysis 3024. The following code fragment illustrates an exemplary methodology for such analysis, according to one embodiment. Read total list of relationships file as links threshold=0.8 for i in range(len(links)): if links>=threshold multiplier[i]=1 else multiplier[i]=0 end if links_temp=multiplier*links final_links=links_temp[links_temp!=0] savetxt(output_file, final_links) output_file.close()

[0099] In some embodiments, compositions of the present disclosure comprise one or more bacteria and / or one or more fungi having an MIC score of at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.4, at least about 0.45, at least about 0.5, at least about 0.55, at least about 0.6, at least about 0.65, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.9, or at least about 0.95.

[0100] In some embodiments, compositions of the present disclosure comprise one or more bacteria and / or one or more fungi with an MIC score of at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.80, at least 0.85, at least 0.9, or at least 0.95.

[0101] Based on the output of the network analysis, active strains are selected 3025 for preparing products (e.g., ensembles, aggregates, and / or other synthetic groups) containing the selected strains. The output of the network analysis may also be used to inform the selection of strains for further product composition testing.

[0102] The use of thresholds for analysis and determination is discussed above. Depending on the implementation and application, thresholds can be set as follows: (1) empirically determined (e.g., setting a cutoff number to remove a specific or significant portion of low-level reads based on distribution levels), (2) any non-zero value, (3) percentile-based, (4) only lines with a normalized second marker (i.e., activity) read greater than the normalized first marker (cell count) read, (5) the log2 fold change between activity and abundance or cell count, (6) a normalized second marker (activity) read greater than the average second marker (activity) read for the entire sample (and / or sample set), and / or any of the above thresholds in addition to a statistical threshold (i.e., significance test).

[0103] As used herein, "shelf-stable" refers to the functional properties and novel utility acquired by a microorganism formulated according to the present disclosure, which allows the microorganism to exist in a useful / active state outside of its natural environment (i.e., with significantly different properties) in the gastrointestinal tract. Thus, shelf-stability is a functional attribute created by the formulation / composition of the present disclosure, and indicates that the microorganism formulated in a shelf-stable composition can exist outside the gastrointestinal tract and under ambient conditions for a period of time that can be determined depending on the specific formulation utilized, but generally means that the microorganism can be formulated to exist in a composition that is stable under ambient conditions for at least several days, generally at least one week. Thus, a "shelf-stable dog supplement" refers to a composition comprising one or more microorganisms of the present disclosure, which are formulated in a composition that is stable under ambient conditions for at least one week, and in which the microorganisms (e.g., whole cells, spores, or lysed cells) contained in the composition, when administered, can confer one or more beneficial phenotypic properties to a dog (e.g., improved GI health and / or modulation of the gastrointestinal microbiome).

[0104] Prior application The subject matter of this case is separate from the subject matter of previous Ascus Biosciences, Inc. applications. The microbial 16S and / or ITS sequences of the present disclosure are considered separate from those of any previous Native Microbials, Inc. applications.

[0105] Isolated microorganisms In some aspects, the present disclosure provides isolated microorganisms, including the novel microbial strains presented in Table 1.

[0106] In other aspects, the present disclosure provides isolated whole microbial cultures of the microorganisms identified in Table 1. These cultures may contain various concentrations of the microorganisms.

[0107] In some aspects, the present disclosure provides for utilizing one or more microorganisms selected from Table 1 to increase a phenotypic trait of interest in a dog.

[0108] In some embodiments, the present disclosure provides isolated microbial species belonging to the following taxonomic families: Prevotellaceae, Veillonellaceae, Lachnospiraceae, Clostridiaceae, Succinivibrionaceae, Erysipelotrichaceae, Eubacteriaceae, Peptostreptococcaceae, Enterobacteriaceae, Acetobacteraceae, Sutterellaceae, Coriobacteriaceae, Bacteroidaceae, Bacillaceae, Caulobacteraceae, Actinomycetaceae, Ruminococcaceae, Porphyromonadaceae, Fusobacteriaceae, Moraxellaceae, Carnobacteriaceae, Clostridiales, Aerococcaceae, Streptosporangiaceae, Bifidobacteriaceae, Fusobacteriaceae, and Lactobacillaceae.

[0109] In further embodiments, the isolated microbial species is selected from the group consisting of Prevotella, Megamonas, Ruminococcus, Clostridium, and Clostridium. sensu stricto, Lachnospiraceae, Anaerobiospirillum, Catenibacterium, Eubacterium, Holdemanella, Clostridium XI, Allobaculum, Morganella, Acidicaldus, Parasutterella, Collinsella, Blautia, Bacteroides, Bacillus, Lactonifactor, Brevundimonas, Dialister, Actinomyces, Coprococcus, Cellulsilyticum, Acetanaerobacterium, Faecalibacterium, Murimonas, Clostridium XIVa, Parabacteroides, Cetobacterium, Clostridium XVIII, Odoribacter, Terrisporobacter, Turicibacter, Fusinateibacter, Kandleria, Butyricicoccus, Veillonella, Acinetobacter, Enterococcus, P araprevotella, Thermaerobacter, Bulleidia, Aerococcus, Robinoniella, Erysipelotrichaceae, Streptosporangium, Bifidobacterium, Clostridium III, Pediococcus, Fusobacterium, Glautia, Sarcina, Jeotgalibaca, and Megasphaera.

[0110] Additionally, the present disclosure relates to microorganisms having characteristics substantially similar to the characteristics of the microorganisms identified in Table 1.

[0111] The isolated microbial species, and novel strains of such species, identified in this disclosure can confer beneficial properties or traits to dogs.

[0112] For example, an isolated microorganism listed in Table 1, or a microbial population of such microorganisms, can promote a reduced incidence of gastrointestinal dysbiosis, a reduced severity of gastrointestinal dysbiosis, a reduced incidence of diarrhea, a reduced severity of diarrhea, a reduced incidence of irritable bowel disease (IBD), a reduced severity of irritable or inflammatory bowel disease, a reduced incidence of chronic bowel disease, a reduced severity of chronic bowel disease, a reduced incidence of gastrointestinal pathogen colonization, a reduced incidence of gastrointestinal pathogen-induced illness, a reduced frequency of gastrointestinal pathogen carriage, a reduced amount of primary bile acids present in the feces, an increased amount of secondary bile acids present in the feces, an increased production of fatty acids in the GI tract, an increased fat, starch, and / or protein digestion, an increased pH balance, an increased vitamin availability, a reduced likelihood or incidence of mortality, a reduced likelihood or incidence of morbidity, an increased production of antimicrobial agents, an increased mammalian and / or microbial synthesis of vitamins, a reduced alpha diversity of the gastrointestinal microbiome, and / or a combination thereof, wherein the increase or decrease is determined by comparing the increase or decrease to an animal not administered the composition.

[0113] Heinken et al. (2019. Microbiome. 7:75; 18pgs) identified a link between secondary bile acid metabolism in gut microbes and the distinct metabolic capabilities of these gut microbes in inflammatory bowel disease. The gut microbiome appears to have the ability to regulate overall bile acid production and secondary bile acid production.

[0114] In some embodiments, the isolated microbial strain is a genetically modified microorganism of the present disclosure. In some embodiments, the genetically modified or recombinant microorganism comprises a polynucleotide sequence that does not naturally occur in the microorganism. In some embodiments, the microorganism may comprise a heterologous polynucleotide. In further embodiments, the heterologous polynucleotide may be operably linked to one or more polynucleotides native to the microorganism.

[0115] In some embodiments, the heterologous polynucleotide may be a reporter gene or a selectable marker. In some embodiments, the reporter gene may be selected from any of the fluorescent protein family (e.g., GFP, RFP, YFP, etc.), β-galactosidase, or luciferase. In some embodiments, the selectable marker may be selected from neomycin phosphotransferase, hygromycin phosphotransferase, aminoglycoside adenyltransferase, dihydrofolate reductase, acetolactase synthase, bromoxynil nitrilase, β-glucuronidase, dihydrogolate reductase, and chloramphenicol acetyltransferase. In some embodiments, the heterologous polynucleotide may be operably linked to one or more promoters.

[0116] In some embodiments, the isolated microbial strain expresses a transgenic or native polypeptide selected from a cellulase (e.g., endocellulase, exocellulase, and glucosidase), a pectinase, an amylase, an amylopectinase, a ligninase, and a phytase.

[0117] Microbial composition In some embodiments, the present disclosure provides microbial compositions comprising a combination of at least any two microorganisms selected from among the microorganisms identified in Table 1. In some embodiments, the present disclosure provides microbial compositions comprising at least one microorganism selected from among the microorganisms identified in Table 1.

[0118] In certain embodiments, the compositions of the present disclosure include two microorganisms, or three microorganisms, or four microorganisms, or five microorganisms, or six microorganisms, or seven microorganisms, or eight microorganisms, or nine microorganisms, or ten or more microorganisms, wherein the microorganisms of the composition are different microbial species or different microbial species strains.

[0119] In some embodiments, the present disclosure provides a microbial composition comprising at least one or at least two isolated microbial species belonging to the following genera: Prevotella, Megamonas, Ruminococcus, Clostridium, Clostridium sensu stricto, Lachnospiraceae, Anaerobiospirillum, Catenibacterium, Eubacterium, Holdemanella, Clostridium XI, Allobaculum, Morganella, Acidicaldus, Parasutterella, Collinsella, Blautia, Bacteroides, Bacillus, Lactonifactor, Brevundimonas, Dialister, Actinomyces, Coprococcus, Cellulsilyticum, Acetanaerobacterium, Faecalibacterium, Murimonas, Clostridium XIVa, Parabacteroides, Cetobacterium, Clostridium XVIII, Odoribacter, Terrisporobacter, Turicibacter, Fusicatenibacter, Kandleria, Butyricicoccus, Veillonella, Acinetobacter, Enterococcus, Paraprevotella, Thermaerobacter, Bulleidia, Aerococcus, Robinsoniella, Erysipelotrichaceae, Streptosporangium, Bifidobacterium, Clostridium III, Pediococcus, Fusobacterium, Glautia, Sarcina, Jeotgalibaca, and Megasphaera. Specific novel strains of species of these aforementioned genera can be found in Table 1.

[0120] In some embodiments, the present disclosure provides a microbial composition comprising at least one or at least two isolated microbial species belonging to the following families: Prevotellaceae, Veillonellaceae, Lachnospiraceae, Clostridiaceae, Succinivibrionaceae, Erysipelotrichaceae, Eubacteriaceae, Peptostreptococcaceae, Enterobacteriaceae, Acetobacteraceae, Sutterellaceae, Coriobacteriaceae, Bacteroidaceae, Bacillaceae, Caulobacteraceae, Actinomycetaceae, Ruminococcaceae, Porphyromonadaceae, Fusobacteriaceae, Moraxellaceae, Carnobacteriaceae, Clostridiales, Aerococcaceae, Streptosporangiaceae, Bifidobacteriaceae, Fusobacteriaceae, and Lactobacillaceae.

[0121] Specific novel strains of species in these aforementioned genera can be found in Table 1.

[0122] In certain embodiments, the present disclosure provides microbial compositions comprising the species grouped in Tables 2 through 8. With respect to Tables 2 through 8, letters A through I represent a non-limiting selection of the microorganisms of the present disclosure, as defined below. A = strain designation Askusk9_546A identified in Table 1 ; B = strain designation Askusk9_672A identified in Table 1 ; C = strain designation Askusk9_210B identified in Table 1; D = strain designation Askusk9_2A identified in Table 1; E = strain designation Askusk9_33E identified in Table 1 ; F = strain designation Askusk9_51G identified in Table 1 G = strain designation Askusk9_0G identified in Table 1 ; H = strain designation Askusk9_38A identified in Table 1 , and I = strain designation Askusk9_17A identified in Table 1 . [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0123] In some embodiments, the microbial composition can be selected from any member group of Tables 2-8.

[0124] Isolated Microorganisms - Raw Materials In certain embodiments, the microorganisms of the composition are not naturally found in association with the same animal. In some aspects, the microbial species or strains are not found in dogs of the same breed. In some aspects, the microbial species or strains are not found in dogs of the same age or the same approximate age. In some aspects, the microbial species or strains are not found in dogs fed the same diet. In some aspects, the microbial species or strains are not found in dogs with the same approximate activity level. In some aspects, all of the microbial species forming the microbial community are found in association with animals from the same geographic location. In other aspects, each microbial species forming the composition is from a different geographic location. Geographic locations can be defined based on the predominant soil type in the area, the predominant climate in the area, the predominant plant communities present in the area, the predominant plant communities present in the area, the distance between areas, the average rainfall in the area, etc.

[0125] In some embodiments, the microorganisms of the composition are not naturally found in association with the same animal species, hi some embodiments, the microorganisms of the composition are found in the same animal species but separated by geographic region.

[0126] In certain embodiments, at least one microbial species that is a member of the microbial community induced by the methods of the present disclosure is native to or obtained from a geographic region that is at least about 1 m, 10 m, 100 m, 1 km, 10 km, 100 km, 1,000 km, 10,000 km, 20,000 km, 30,000 km, or 40,000 km from the location of the animals whose phenotypic traits are to be increased based on the taught methods.

[0127] The microorganisms of the present disclosure were obtained from the gastrointestinal tract of dogs from various locations in the United States, among other locations.

[0128] Isolated Microorganisms - Microbial Culture Techniques The microorganisms in Table 1 were matched to their closest taxa using the Ribosomal Database Project (RDP) classification tools for 16s rRNA sequences and the User-friendly Nordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences. Examples of microorganisms that fit the closest taxonomy can be found in Lan et al. (2012, PLOS One. 7(3):e32491), Schloss and Westcott (2011, Appl. Environ. Microbiol. 77(10):3219-3226), and Koljalg et al. (2005, New Phytologist. 166(3):1063-1068).

[0129] Isolation, identification, and cultivation of the microorganisms of this disclosure can be carried out using standard microbiological techniques. Examples of such techniques are described in Gerhardt, P. (ed.) Methods for General and Molecular Microbiology. American Society for Microbiology, Washington, DC (1994) and Lennette, E. H. (ed.). Manual of Clinical Microbiology, Third Edition. American Society for Microbiology, Washington, DC (1980), each of which is incorporated by reference.

[0130] Isolation can be achieved by streaking the specimen onto a solid medium (e.g., a nutrient agar plate) to obtain a single colony characterized by the phenotypic traits described herein (e.g., Gram positive / negative, able to sporulate aerobically / anaerobically, cell morphology, carbon source metabolism, acid / base production, enzyme secretion, metabolic secretion, etc.) and reducing the chance of working with a contaminated culture.

[0131] For example, for the microorganisms of the present disclosure, biologically pure isolates can be obtained through repeated subculture of a biological sample, with streaking onto solid media after each subculture to obtain individual colonies or colony-forming units. Methods for preparing, thawing, and growing freeze-dried bacteria are generally known, see, for example, Gherna, R.L. and C. Reddy. 2007. Culture Preservation, pp. 1019-1033. In C. Reddy, T.J. Beveridge, J.A. Breznak, G.A. Marzluf, T.M. Schmidt, and L.R. Snyder, eds. American Society for Microbiology, Washington, DC, p. 1033, incorporated herein by reference. Thus, freeze-dried liquid preparations and cultures stored long-term at -70°C in a solution containing glycerol are contemplated for use in providing the formulations of the present disclosure.

[0132] The microorganisms of the present disclosure can be grown in liquid media under aerobic or anaerobic conditions. The medium for growing the bacterial strains of the present disclosure includes a carbon source, a nitrogen source, and inorganic salts, as well as specially required substances such as vitamins, amino acids, and nucleic acids. Examples of suitable carbon sources that can be used for microbial growth include, but are not limited to, starch, peptone, yeast extract, amino acids, sugars such as glucose, arabinose, mannose, glucosamine, and maltose, salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, and malonic acid, alcohols such as ethanol and glycerol, and fats and oils such as soybean oil, rice bran oil, olive oil, corn oil, and sesame oil. The amount of carbon source added varies depending on the type of carbon source, typically between 1 and 100 grams per liter of medium. Preferably, glucose, starch, and / or peptone are included in the medium at a concentration of 0.1-5% (w / v) as the primary carbon source. Examples of suitable nitrogen sources that can be used to grow the bacterial strains of the present disclosure include, but are not limited to, amino acids, yeast extract, tryptone, beef extract, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, or combinations thereof. The amount of nitrogen source varies depending on the type of nitrogen source and is typically 0.1-30 grams per liter of medium. Inorganic salts such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferrous chloride, ferrous chloride, manganous sulfate, manganous chloride, zinc sulfate, zinc chloride, copper sulfate, calcium chloride, sodium chloride, calcium carbonate, and sodium carbonate can be used alone or in combination. The amount of inorganic acid varies depending on the type of inorganic salt and is typically 0.001-10 grams per liter of medium. Examples of specially required substances include, but are not limited to, vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, dry yeast, and combinations thereof. Cultivation can be carried out at a temperature that allows growth of the microbial strain, essentially between 20°C and 46°C.In some aspects, the temperature range is 30°C to 39°C. For optimal growth, in some embodiments, the medium may be adjusted to a pH of 6.0 to 7.4. It will be appreciated that commercially available media, such as Nutrient Broth or Nutrient Agar available from Difco, Detroit, MI, may also be used to culture the microbial strain. It will be appreciated that the incubation time may vary depending on the type of culture medium used and the concentration of sugar as the primary carbon source.

[0133] In some embodiments, the culture is continued for 24 to 96 hours. The microbial cells thus obtained are isolated using methods well known in the art. Examples include, but are not limited to, membrane filtration and centrifugation. The pH may be adjusted using sodium hydroxide, and the culture may be dried using a freeze dryer until the moisture content is 4% or less. Microbial co-cultures can be obtained by growing each strain as described above. In some embodiments, polycultures of microorganisms can be obtained by growing two or more strains described above. It will be understood that microbial strains can be cultured together if compatible culture conditions are available.

[0134] Isolated Microorganisms - Microbial Strains Microorganisms can be differentiated into genera based on polyphasic taxonomy, which incorporates all available phenotypic and genotypic data into a consensus classification (Vandamme et al. al.1996.Polyphasic taxonomy, a consensus One accepted genotypic method for defining species is based on overall genomic relatedness, and under standard conditions, a ΔT of 5°C or less is used. mUsing DNA-DNA hybridization (the difference in melting temperature between homologous and heterologous hybrids), strains that share approximately 70% or more relatedness are considered members of the same species. Therefore, populations that share more than the aforementioned 70% threshold can be considered variants of the same species. Another acceptable genotypic method for defining species is to isolate the marker genes disclosed herein, sequence these genes, and align these sequenced genes from multiple isolates or variants. If one or more of the sequenced genes share at least 97% sequence identity, the microorganisms are considered to belong to the same species.

[0135] 16S or 18S rRNA sequences or ITS sequences are often used to distinguish species and strains, and two organisms from which the sequences were obtained are said to be of different species or strains if one of the aforementioned sequences shares less than a certain % sequence identity from a reference sequence.

[0136] Thus, microorganisms can be considered to be of the same species if they share at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across 16S or 18S rRNA sequences, or ITS1 or ITS2 sequences.

[0137] Furthermore, certain microbial strains can be defined as sharing at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across 16S or 18S rRNA sequences, or ITS1 or ITS2 sequences.

[0138] In one embodiment, the microbial strain of the present disclosure includes a microbial strain having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, or , 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0139] In further embodiments, the microbial strains of the present disclosure include a microbial strain having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 96.10%, 96.11%, 96.12%, 96.13%, 96.14%, 96.15%, 96.16%, 96.17%, 96.18%, 96.19%, 96.20%, 96.21%, 96.22%, 96.23%, 96.24%, 96.25%, 96.26%, 96.27%, 96.28%, 96.29 ... 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity.

[0140] Comparison of 23S rRNA sequences to a reference sequence is also possible.

[0141] Uncultivable microorganisms often cannot be assigned to a specific species in the absence of phenotyping, and a microorganism may be given the candidatus designation within a genus if its 16S or 18S rRNA sequence, or ITS sequence, conforms to the principle of identity with known species.

[0142] One approach is to observe the distribution of a large number of lineages of closely related species in sequence space and identify clusters of lineages that are well resolved from other clusters. This approach, developed by assessing clustering patterns using concatenated sequences of multiple core (housekeeping) genes, is called multilocus sequence analysis (MLSA) or multilocus sequence phylogenetic analysis. MLSA has been successfully used to explore clustering patterns of large numbers of lineages that are assigned to very closely related species by current taxonomic methods, to examine relationships among a small number of lineages within a genus or broader taxonomic group, and to address specific taxonomic questions. More generally, this method can be used to determine whether bacterial species exist, i.e., whether large populations of similar lineages always fall into well-resolved clusters, or whether a genetic continuum exists where, in some cases, no clear classification into clusters is observed.

[0143] To more accurately determine the genus, phenotypic traits, such as morphological, biochemical, and physiological characteristics, are determined and compared with the prototype of the reference genus. Colony morphology may include color, shape, pigmentation, mucus production, etc. Cellular characteristics are described as shape, size, Gram reaction, extracellular material, presence of endospores, presence and position of flagella, motility, and inclusion bodies. Biochemical and physiological characteristics describe the growth of the organism in various temperature ranges, pH, salinity, and atmospheric conditions, and growth in the presence of various sole carbon and nitrogen sources. One skilled in the art would be reasonably informed of the phenotypic traits that define the genus of the present disclosure.

[0144] In one embodiment, the microorganisms taught herein were identified using 16S rRNA gene sequences and ITS sequences. It is known in the art that 16S rRNA contains hypervariable regions that can provide species / strain-specific signature sequences useful for bacterial identification, and that ITS sequences can also provide species / strain-specific signature sequences useful for fungal identification.

[0145] Phylogenetic analysis using rRNA gene and / or ITS sequences can also be used to define "substantially similar" species within a common genus and to define "substantially similar" strains of a given taxonomic species. Additionally, physiological and / or biochemical characteristics of isolates can be used to highlight both subtle and significant differences between strains that may result in advantageous behavior or outcomes in dogs.

[0146] The compositions of the present disclosure may include combinations of fungal spores and bacterial spores, fungal spores and bacterial vegetative cells, fungal vegetative cells and bacterial spores, or fungal vegetative cells and bacterial vegetative cells. In some embodiments, the compositions of the present disclosure include bacteria only in the form of spores. In some embodiments, the compositions of the present disclosure include bacteria only in the form of vegetative cells. In some embodiments, the compositions of the present disclosure include bacteria in the absence of fungi. In some embodiments, the compositions of the present disclosure include fungi in the absence of bacteria. In some embodiments, the compositions of the present disclosure include VBNC bacteria and / or fungi. In some embodiments, the compositions of the present disclosure include quiescent bacteria and / or fungi. In some embodiments, the compositions of the present disclosure include dormant bacteria and / or fungi.

[0147] Bacterial spores may include endospores and akinetes. Fungal spores can include non-ballistospores, ballistospores, autospores, immobile spores, zoospores, vegetative spores, megaspores, microspores, meiospores, chlamydospores, uredospores, teliospores, oospores, carpospores, tetraspores, sporangiospores, zygospores, ascospores, basidiospores, ascospores, and asciospores.

[0148] In some embodiments, spores of the compositions of the present disclosure germinate upon administration to an animal. In some embodiments, spores of the compositions of the present disclosure germinate only upon administration to an animal.

[0149] Microbial composition In some embodiments, the microorganisms of the present disclosure are combined in a microbial composition.

[0150] In some embodiments, the microbial composition comprises animal feed, such as grains and grain by-products (such as barley, corn, oats, sorghum, wheat, distillers' grains, sweet bran, etc.), starch (such as tapioca), protein (such as oilseed cake, vegetable waste, corn by-products, wheat by-products, etc.), lean animal protein (such as chicken, turkey, duck, beef, buffalo, wild boar, pork, fish, lamb, rabbit, etc.), animal meal (such as chicken meal, beef meal, wild boar meal, pork meal, buffalo meat meal, bone meal, fish meal, rabbit meal, lamb meal, turkey meal, duck meal, etc.), animal fat (such as beef fat, chicken fat, tallow, etc.), and / or non-nitrogenous protein. The animal feed for the microbial composition may further comprise commercially available dry or wet animal feed. In some embodiments, the microbial composition comprises vitamins and / or their metabolites, minerals, urea, trace elements, emulsifiers, aromatizing products, binders, colorants, odorants, thickeners, antibiotics, etc. In some embodiments, the microbial composition comprises one or more of an ionophore, a vaccine, an antibiotic, an antiparasitic, a virucide, a nematicide; an amino acid such as methionine, glutamine, valine, glycine, cysteine, homocysteine, aspartic acid, and arginine; a bioactive molecule such as fish oil, oregano, carnitine, pantoate, pantothenate, and aspartate; and an enzyme.

[0151] In some embodiments, the vitamins include vitamins B5, B1, B2, B3, B6, B9, B12, H, C, A, D, E, or K, and combinations thereof. In some embodiments, the microbial composition includes a microorganism that synthesizes vitamins B5, B1, B2, B3, B6, B9, B12, H, C, A, D, E, and / or K. In some embodiments, the microbial composition includes a microorganism that synthesizes vitamin B5. In some embodiments, metabolites of vitamins B5, B1, B2, B3, B6, B9, B12, H, C, A, D, E, or K are contemplated as one or more components of the microbial compositions of the present disclosure. In one embodiment, pantothenate is a component of the microbial composition of the present disclosure. In one embodiment, a component of the microbial composition of the present disclosure includes one or more precursors utilized by mammalian or microbial biosynthesis.

[0152] In some embodiments, the microbial composition of the present disclosure is solid. When a solid composition is used, it may be desirable to include one or more carrier materials, including, but not limited to, mineral earth carriers, food-grade carriers, and / or carriers of plant origin. Examples of such carriers include, but are not limited to, silica, talc, kaolin, limestone, chalk, clay, dolomite, diatomaceous earth, activated carbon, calcium sulfate, magnesium sulfate, magnesium oxide, zeolite, calcium carbonate, magnesium carbonate, trehalose, chitosan, shellac, albumin, starch, yucca root, skim milk powder, sweet whey powder, maltodextrin, lactose, inulin, dextrose, whey protein, wheat flour (e.g., chickpea flour, sweet potato flour, or soy flour), yucca, sugar, soybean meal, maltodextrin, spices, herbs, cereal meal, bark meal, wood flour, and nutshell meal.

[0153] In some embodiments, the microbial compositions of the present disclosure are liquid. In further embodiments, the liquid comprises a solvent, which may include water, alcohol, saline, or a carbohydrate solution, and other animal-safe solvents. In some embodiments, the microbial compositions of the present disclosure comprise a binder, such as an animal-safe polymer, carboxymethyl cellulose, starch, or polyvinyl alcohol.

[0154] In some embodiments, the microbial compositions of the present disclosure include thickening agents such as silica, clay, natural extracts of seeds or seaweed, synthetic derivatives of cellulose, guar gum, locust bean gum, alginates, and methylcellulose. In some embodiments, the microbial compositions include anti-settling agents such as modified starch, polyvinyl alcohol, and xanthan gum.

[0155] In some embodiments, the microbial compositions of the present disclosure comprise flavorings, such as dry yeast, cheese flavorings, fish flavorings, pork flavorings, chicken flavorings, and / or beef flavorings.

[0156] In some embodiments, the microbial compositions of the present disclosure include a colorant comprising an organic chromophore classified as azo, acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, or xanthene, including nitroso, nitro, monoazo, bisazo, and polyazo. In some embodiments, the microbial compositions of the present disclosure include micronutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc. In some embodiments, the microbial compositions include both natural and artificial dyes. In some embodiments, the dye is green.

[0157] In some embodiments, the microbial compositions of the present disclosure comprise an animal-safe virucide, parasiticide, bactericide, fungicide, or nematicide.

[0158] In some embodiments, the microbial compositions of the present disclosure comprise saccharides (e.g., monosaccharides, disaccharides, trisaccharides, polysaccharides, oligosaccharides, etc.), polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides, proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acid polymers, silica, inorganic salts, and combinations thereof. In further embodiments, the microbial compositions comprise polymers such as agar, agarose, Gelrite, gellan gum, and the like. In some embodiments, the microbial compositions comprise plastic capsules, emulsions (e.g., water and oil), membranes, and artificial membranes. In some embodiments, emulsions or linked polymer solutions can comprise the microbial compositions of the present disclosure. See Harel and Bennett (U.S. Patent No. 8,460,726 B2).

[0159] In some embodiments, the microbial composition of the present disclosure comprises one or more oxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators, and combinations thereof. In one embodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators are not chemically active when the microbial composition is mixed with food and / or water administered to an animal. In one embodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators are not chemically active when administered to an animal.

[0160] In some embodiments, the microbial compositions of the present disclosure are present in a solid form (e.g., dispersed freeze-dried spores) or in a liquid form (microorganisms dispersed in a storage medium). In some embodiments, the microbial compositions of the present disclosure are added in dry form to a liquid immediately prior to administration to form a suspension.

[0161] In some embodiments, the microbial compositions of the present disclosure include one or more preservatives. The preservatives may be in liquid or gaseous form. Preservatives include monosaccharides, disaccharides, trisaccharides, polysaccharides, acetic acid, ascorbic acid, calcium ascorbate, erythorbic acid, isoascorbic acid, erythrobic acid, potassium nitrate, sodium ascorbate, sodium erythrobate, sodium isoascorbate, sodium nitrate, sodium nitrite, nitrogen, benzoic acid, calcium sorbate, ethyl lauroyl alginate, methyl p-hydroxybenzoate, methylparaben, potassium acetate, potassium benzoate, potassium bisulfite, potassium diacetate, potassium lactate, potassium metabisulfite, potassium sorbate, propyl p-hydroxybenzoate, propylparaben, sodium acetate, sodium benzoate, sodium bisulfite, sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulfite, sodium salt of methyl p-hydroxybenzoate, p-hydroxybenzoic acid ... salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of methyl p-hydroxybenzoate, sodium salt of Sodium salt of propylhydroxybenzoate, sodium sulfate, sodium sulfite, sodium dithionite, sulfite, calcium propionate, dimethyl bicarbonate, natamycin, potassium sorbate, potassium bisulfite, potassium metabisulfite, propionic acid, sodium diacetate, sodium propionate, sodium sorbate, sorbic acid, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, butylated hydroxyanisole, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), citric acid, citric acid esters of monoglycerides and / or diglycerides, L-cysteine, L-cysteine ​​hydrochloride, guaiac oil (gum guaiacum, guaiac gum, lecithin, lecithin citrate, monoglyceride citrate, monoisopropyl citrate, propyl gallate, sodium metabisulfite, tartaric acid, tertiary butylhydroquinone, stannous chloride, thiodipropionic acid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin, sulfur dioxide, formic acid, or tocopherol(s).

[0162] In some embodiments, the microbial compositions of the present disclosure include a carrier that reduces fecal odor. In some embodiments, the microbial compositions include activated carbon to reduce fecal odor. In some embodiments, the microbial compositions include yucca root to reduce fecal odor.

[0163] In some embodiments, the microbial compositions of the present disclosure comprise bacterial and / or fungal cells in spore, vegetative, and / or lysed cell forms. In one embodiment, the lysed cell forms act as mycotoxin-binding agents, e.g., mycotoxins that bind to dead cells.

[0164] In some embodiments, the microbial composition is storage-stable in a refrigerator (35-40°F) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days. In some embodiments, the microbial composition is shelf-stable in a refrigerator (35-40°F) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks. In some embodiments, the microbial composition is shelf-stable in a refrigerator (35-40°F) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 years.

[0165] In some embodiments, the microbial composition is storage-stable at room temperature (68-72°F) or at 50-77°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial composition is storage stable at room temperature (68-72°F) or at 50-77°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks. In some embodiments, the microbial composition is storage-stable at room temperature (68-72°F) or at 50-77°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 years.

[0166] In some embodiments, the microbial composition is storage stable at -23 to 35°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial composition is storage stable at -23 to 35°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks. In some embodiments, the microbial composition is storage stable at -23 to 35°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 years.

[0167] In some embodiments, the microbial composition is storage-stable at 77-100°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial composition is storage stable at 77-100°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks. In some embodiments, the microbial composition is storage stable at 77-100°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 years.

[0168] In some embodiments, the microbial composition is storage-stable at 101-213°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial composition is storage stable at 101-213°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks. In some embodiments, the microbial composition is storage stable at 101-213°F for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 years.

[0169] In some embodiments, the microbial compositions of the present disclosure exhibit a microbial activity of about 1-100, about 1-95, about 1-90, about 1-85, about 1-80, about 1-75, about 1-70, about 1-65, about 1-60, about 1-55, about 1-50, about 1-45, about 1-40, about 1-35, about 1-30, about 1-25, about 1-20, about 1-15, about 1-10, about 1-5, about 5-100, about 5-95, about 5-90, about 5-85, about 5-80, about 5-60, about 5-75, about 1-70, about 1-65, about 1-60, about 1-55, about 1-50, about 1-45, about 1-40, about 1-35, about 1-30, about 1-25, about 1-20, about 1-15, about 1-10, about 1-5, about 5-100, about 5-95, about 5-90, about 5-85, about 5-80, about 5-85, about 5-95, about 5-90, about 5-85, about 5-80, about 5-95, about 5-100, about 5-100, about 5-105 ... ~75, about 5~70, about 5~65, about 5~60, about 5~55, about 5~50, about 5~45, about 5~40, about 5~35, about 5~30, about 5~25, about 5~20, about 5~15, about 5~10, about 10~100, about 10~95, about 10~90, about 10~85, about 10~80, about 10~75 , about 10 to 70, about 10 to 65, about 10 to 60, about 10 to 55, about 10 to 50, about 10 to 45, about 10 to 40, about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10 to 15, about 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85, about 15 to 80, about 15 to 75, approximately 15-70, approximately 15-65, approximately 15-60, approximately 15-55, approximately 15-50, approximately 15-45, approximately 15-40, approximately 15-35, approximately 15-30, approximately 15-25, approximately 15-20, approximately 20-100, approximately 20-95, approximately 20-90, approximately 20-85, approximately 20-80, approximately 20-75, approximately 20-70, approximately 20-65, approximately 20-60, approximately 20-55, approximately 20-50, approximately 20-45, approximately 20-40, approximately 20-35, approximately 20-30, approximately 20-25, approximately 25-100, approximately 25-95, approximately 25-90, approximately 25-85, approximately 25-80, approximately 25-75, approximately 25-70, approximately 25-65 , about 25 to 60, about 25 to 55, about 25 to 50, about 25 to 45, about 25 to 40, about 25 to 35, about 25 to 30, about 30 to 100, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 80, about 30 to 75, about 30 to 70, about 30 to 65, about 30 to 60, about 30 to 55, about 30 ~50, about 30~45, about 30~40, about 30~35, about 35~100, about 35~95, about 35~90, about 35~85, about 35~80, about 35~75, about 35~70, about 35~65, about 35~60, about 35~55, about 35~50, about 35~45, about 35~40, about 40~100,Approximately 40-95, approximately 40-90, approximately 40-85, approximately 40-80, approximately 40-75, approximately 40-70, approximately 40-65, approximately 40-60, approximately 40-55, approximately 40-50, approximately 40-45, approximately 45-100, approximately 45-95, approximately 45-90, approximately 45-85, approximately 45-80, approximately 45-75, approximately 45-70, approximately 45-65, approximately 45-6 0, about 45-55, about 45-50, about 50-100, about 50-95, about 50-90, about 50-85, about 50-80, about 50-75, about 50-70, about 50-65, about 50-60, about 50-55, about 55-100, about 55-95, about 55-90, about 55-85, about 55-80, about 55-75, about 55-70, about 55 ~65, about 55~60, about 60~100, about 60~95, about 60~90, about 60~85, about 60~80, about 60~75, about 60~70, about 60~65, about 65~100, about 65~95, about 65~90, about 65~85, about 65~80, about 65~75, about 65~70, about 70~100, about 70~95, about 70~90, It has storage stability of about 70 to 85, about 70 to 80, about 70 to 75, about 75 to 100, about 75 to 95, about 75 to 90, about 75 to 85, about 75 to 80, about 80 to 100, about 80 to 95, about 80 to 90, about 80 to 85, about 85 to 100, about 85 to 95, about 85 to 90, about 90 to 100, about 90 to 95, and 95 to 100 weeks.

[0170] In some embodiments, the microbial compositions of the present disclosure exhibit a microbial activity of 1-100, 1-95, 1-90, 1-85, 1-80, 1-75, 1-70, 1-65, 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-100, 5-95, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-60, 5-75, 5-85, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-65, 5-75, 5-85, 5-9 ...100, 5-100, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5-105, 5- 50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-100, 10-95, 10-90, 10-85, 10-80, 10-75, 10-70, 10-65, 10-60, 10-55, 10-50, 10-45, 10-40, 10 ~35, 10~30, 10~25, 10~20, 10~15, 15~100, 15~95, 15~90, 15~85, 15~80, 15~75, 15~70, 15~65, 15~60, 15~55, 15~50, 15~45, 15~40, 15~35, 15~30, 15~25, 15-20, 20-100, 20-95, 20-90, 20-85, 20-80, 20-75, 20-70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-100, 25-95, 25-90, 25 ~85, 25~80, 25~75, 25~70, 25~65, 25~60, 25~55, 25~50, 25~45, 25~40, 25~35, 25~30, 30~100, 30~95, 30~90, 30~85, 30~80, 30~75, 30~70, 30~65, 30~60, 30~55, 30~50, 30~45, 30~40, 30~35, 35~100, 35~95, 35~90, 35~85, 35~80, 35~75, 35~70, 35~65, 35~60, 35~55, 35~50, 35~45, 35~40, 40~100, 40~95, 40 ~90, 40~85, 40~80, 40~75, 40~70, 40~65, 40~60, 40~55, 40~50, 40~45, 45~100, 45~95, 45~90, 45~85, 45~80, 45~75, 45~70, 45~65, 45~60, 45~55, 45~50,50~100, 50~95, 50~90, 50~85, 50~80, 50~75, 50~70, 50~65, 50~60, 50~55, 55~100, 55~95, 55~90, 55~85, 55~80, 55~75, 55~70, 55~65, 55~60, 60~100, 60~95, 60~90, 60~85, 60~80, 60~75, 60~70, 60~65, 65~100, 65~ Storage stability is 95, 65-90, 65-85, 65-80, 65-75, 65-70, 70-100, 70-95, 70-90, 70-85, 70-80, 70-75, 75-100, 75-95, 75-90, 75-85, 75-80, 80-100, 80-95, 80-90, 80-85, 85-100, 85-95, 85-90, 90-100, 90-95, and 95-100 weeks.

[0171] In some embodiments, the microbial compositions of the present disclosure exhibit a microbial activity of about 1-36, about 1-34, about 1-32, about 1-30, about 1-28, about 1-26, about 1-24, about 1-22, about 1-20, about 1-18, about 1-16, about 1-14, about 1-12, about 1-10, about 1-8, about 1-6, about 1-4, about 1-2, about 4-36, about 4-34, about 4-32, about 4-30, about 4-28, about 4-26, about 4-24, about 4-22, about 4-50, about 50-57 ... ~20, about 4~18, about 4~16, about 4~14, about 4~12, about 4~10, about 4~8, about 4~6, about 6~36, about 6~34, about 6~32, about 6~30, about 6~28, about 6~26, about 6~24, about 6~22, about 6~20, about 6~18, about 6~16, about 6~14, about 6~12, about 6~ 10, about 6-8, about 8-36, about 8-34, about 8-32, about 8-30, about 8-28, about 8-26, about 8-24, about 8-22, about 8-20, about 8-18, about 8-16, about 8-14, about 8-12, about 8-10, about 10-36, about 10-34, about 10-32, about 10-30, about 10-2 8, about 10-26, about 10-24, about 10-22, about 10-20, about 10-18, about 10-16, about 10-14, about 10-12, about 12-36, about 12-34, about 12-32, about 12-30, about 12-28, about 12-26, about 12-24, about 12-22, about 12-20, about 12 ~18, about 12-16, about 12-14, about 14-36, about 14-34, about 14-32, about 14-30, about 14-28, about 14-26, about 14-24, about 14-22, about 14-20, about 14-18, about 14-16, about 16-36, about 16-34, about 16-32, about 16-30, about 1 6-28, approximately 16-26, approximately 16-24, approximately 16-22, approximately 16-20, approximately 16-18, approximately 18-36, approximately 18-34, approximately 18-32, approximately 18-30, approximately 18-28, approximately 18-26, approximately 18-24, approximately 18-22, approximately 18-20, approximately 20-36, approximately 20-34, approximately 20-32, approximately 20-30, about 20-28, about 20-26, about 20-24, about 20-22, about 22-36, about 22-34, about 22-32, about 22-30, about 22-28, about 22-26, about 22-24, about 24-36, about 24-34, about 24-32, about 24-30, about 24-28, about 24-26,Storage stability is maintained for about 26 to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28, about 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30 to 36, about 30 to 34, about 30 to 32, about 32 to 36, about 32 to 34, and about 34 to 36 months.

[0172] In some embodiments, the microbial compositions of the present disclosure exhibit a cell cycle survival rate of 1-36, 1-34, 1-32, 1-30, 1-28, 1-26, 1-24, 1-22, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 4-36, 4-34, 4-32, 4-30, 4-28, 4-26, 4-24, 4-22, 4-20, 4-18, 4-16, 4-14 at refrigerated temperatures (35-40°F), room temperature (68-72°F), 50-77°F, -23-35°F, 70-100°F, or 101-213°F. , 4-12, 4-10, 4-8, 4-6, 6-36, 6-34, 6-32, 6-30, 6-28, 6-26, 6-24, 6-22, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-36, 8-34, 8-32, 8-30, 8-28, 8-26, 8-24, 8-22, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-36, 10-34, 10-32, 10-30, 10-28, 10-26, 10-24, 10-22, 10-20, 10-18, 10-16, 10-14, 10-12 , 12-36, 12-34, 12-32, 12-30, 12-28, 12-26, 12-24, 12-22, 12-20, 12-18, 12-16, 12-14, 14-36, 14-34, 14-32, 14-30, 14-28, 14-26, 14-24, 14-22, 14-20, 14-18, 14-16, 16-36, 16-34, 16-32, 16-30, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 18-36, 18-34, 18-32, 18-30, 18-28, 18-26, 18-24 , 18-22, 18-20, 20-36, 20-34, 20-32, 20-30, 20-28, 20-26, 20-24, 20-22, 22-36, 22-34, 22-32, 22-30, 22-28, 22-26, 22-24, 24-36, 24-34, 24-32, 24-30, 24-28, 24-26, 26-36, 26-34, 26-32, 26-30, 26-28, 28-36, 28-34, 28-32, 28-30, 30-36, 30-34, 30-32, 32-36, 32-34, 34-36 months.

[0173] In some embodiments, the microbial compositions of the present disclosure exhibit a microbial activity of about 1-36, about 1-34, about 1-32, about 1-30, about 1-28, about 1-26, about 1-24, about 1-22, about 1-20, about 1-18, about 1-16, about 1-14, about 1-12, about 1-10, about 1-8, about 1-6, about 1-4, about 1-2, about 4-36, about 4-34, about 4-32, about 4-30, about 4-28, about 4-26, about 4-24, about 4-22, about 4-50, about 50-57 ... ~20, about 4~18, about 4~16, about 4~14, about 4~12, about 4~10, about 4~8, about 4~6, about 6~36, about 6~34, about 6~32, about 6~30, about 6~28, about 6~26, about 6~24, about 6~22, about 6~20, about 6~18, about 6~16, about 6~14, about 6~12, about 6~ 10, about 6-8, about 8-36, about 8-34, about 8-32, about 8-30, about 8-28, about 8-26, about 8-24, about 8-22, about 8-20, about 8-18, about 8-16, about 8-14, about 8-12, about 8-10, about 10-36, about 10-34, about 10-32, about 10-30, about 10-2 8, about 10-26, about 10-24, about 10-22, about 10-20, about 10-18, about 10-16, about 10-14, about 10-12, about 12-36, about 12-34, about 12-32, about 12-30, about 12-28, about 12-26, about 12-24, about 12-22, about 12-20, about 12 ~18, about 12-16, about 12-14, about 14-36, about 14-34, about 14-32, about 14-30, about 14-28, about 14-26, about 14-24, about 14-22, about 14-20, about 14-18, about 14-16, about 16-36, about 16-34, about 16-32, about 16-30, about 1 6-28, approximately 16-26, approximately 16-24, approximately 16-22, approximately 16-20, approximately 16-18, approximately 18-36, approximately 18-34, approximately 18-32, approximately 18-30, approximately 18-28, approximately 18-26, approximately 18-24, approximately 18-22, approximately 18-20, approximately 20-36, approximately 20-34, approximately 20-32, approximately 20-30, about 20-28, about 20-26, about 20-24, about 20-22, about 22-36, about 22-34, about 22-32, about 22-30, about 22-28, about 22-26, about 22-24, about 24-36, about 24-34, about 24-32, about 24-30, about 24-28, about 24-26,It has storage stability for about 26-36, about 26-34, about 26-32, about 26-30, about 26-28, about 28-36, about 28-34, about 28-32, about 28-30, about 30-36, about 30-34, about 30-32, about 32-36, about 32-34, and about 34-36 years.

[0174] In some embodiments, the microbial compositions of the present disclosure exhibit a cell cycle survival rate of 1-36, 1-34, 1-32, 1-30, 1-28, 1-26, 1-24, 1-22, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 4-36, 4-34, 4-32, 4-30, 4-28, 4-26, 4-24, 4-22, 4-20, 4-18, 4-16, 4-1 4, 4-12, 4-10, 4-8, 4-6, 6-36, 6-34, 6-32, 6-30, 6-28, 6-26, 6-24, 6-22, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-36, 8-34, 8-32, 8-30, 8-28, 8-26, 8-24, 8-22, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-36, 10-34, 10-32, 10-30, 10-28, 10-26, 10-24, 10-22, 10-20, 10-18, 10-16, 10-14, 10-1 2, 12-36, 12-34, 12-32, 12-30, 12-28, 12-26, 12-24, 12-22, 12-20, 12-18, 12-16, 12-14, 14-36, 14-34, 14-32, 14-30, 14-28, 14-26, 14-24, 14-22, 14-20, 14-18, 14-16, 16-36, 16-34, 16-32, 16-30, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 18-36, 18-34, 18-32, 18-30, 18-28, 18-26, 18-2 Storage stability for 4, 18-22, 18-20, 20-36, 20-34, 20-32, 20-30, 20-28, 20-26, 20-24, 20-22, 22-36, 22-34, 22-32, 22-30, 22-28, 22-26, 22-24, 24-36, 24-34, 24-32, 24-30, 24-28, 24-26, 26-36, 26-34, 26-32, 26-30, 26-28, 28-36, 28-34, 28-32, 28-30, 30-36, 30-34, 30-32, 32-36, 32-34, 34-36.

[0175] In some embodiments, the microbial compositions of the present disclosure exhibit a microbial activity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98% relative humidity.

[0176] In some embodiments, the microbial compositions of the present disclosure contain microbial compounds at 0.750, 0.700, 0.650, 0.600, 0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350, 0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0. Water activity (a) of less than 150, 0.140, 0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080, 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, 0.010, or 0.005 w )

[0177] In some embodiments, the microbial compositions of the present disclosure comprise a microbial composition of about 0.750, about 0.700, about 0.650, about 0.600, about 0.550, about 0.500, about 0.475, about 0.450, about 0.425, about 0.400, about 0.375, about 0.350, about 0.325, about 0.300, about 0.275, about 0.250, about 0.225, about 0.200, about 0.190, about 0.180, about 0.170, about 0.160, about 0.280, about 0.300, about 0.350, about 0.325, about 0.3 ... a water activity (a) of less than about 50, about 0.140, about 0.130, about 0.120, about 0.110, about 0.100, about 0.095, about 0.090, about 0.085, about 0.080, about 0.075, about 0.070, about 0.065, about 0.060, about 0.055, about 0.050, about 0.045, about 0.040, about 0.035, about 0.030, about 0.025, about 0.020, about 0.015, about 0.010, or about 0.005 w )

[0178] The water activity value is determined by the method of saturated aqueous solutions (Multon, "Techniques d'Analyse E De Controle Dans Les Industries Agroalimentaires" APRIA (1981)) or by direct measurement using a viable Robotronic BT hygrometer or other hygrometer or hygrometer.

[0179] In some embodiments, the microbial composition comprises at least two different microorganisms, and the at least two microorganisms are present in the composition in a ratio of 1:2, 1:3, 1:3, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:40, 1:50, 1:60, 1:100, 1:125, 1:150, 1:175, or 1:200 or vice versa. In some embodiments, the microbial composition comprises at least three different microorganisms, and the three microorganisms are present in the composition in a ratio of 1:2:1, 1:1:2, 2:2:1, 1:3:1, 1:1:3, 3:1:1, 3:3:1, 1:5:1, 1:1:5, 5:1:1, 5:5:1, or 1:5:5.

[0180] In some aspects, the microbial composition comprises a probiotic microorganism. In some aspects, the microbial composition comprises a prebiotic substance. In some aspects, the probiotic microorganism is known to competitively eliminate enteric pathogens.

[0181] Encapsulated Composition In some embodiments, the microorganism or microbial composition of the present disclosure is encapsulated in an encapsulation composition. The encapsulation composition protects the microorganism from external stressors before entering the dog's gastrointestinal tract. In some embodiments, the external stressors include thermal and physical stressors associated with pelleting and extrusion. In some embodiments, the external stressors include chemicals present in the composition. The encapsulation composition further creates an environment that may be beneficial to the microorganism, such as minimizing the oxidative stress of an aerobic environment for anaerobic microorganisms. For encapsulation compositions for microorganisms and methods of encapsulating microorganisms, see Kalsta et al. (US Pat. No. 5,104,662 A), Ford (US Pat. No. 5,733,568 A), and Mosbach and Nilsson (US Pat. No. 4,647,536 A).

[0182] In one embodiment, the compositions of the present disclosure exhibit heat tolerance, which is used interchangeably with heat resistance and heat resistance. In one embodiment, the heat-resistant compositions of the present disclosure are resistant to high temperatures associated with feed manufacturing, mixing of the disclosed feeds and compositions, storage in high heat environments, etc. In one embodiment, the heat-resistant compositions of the present disclosure are resistant to heat killing and denaturation of cell wall components and the intracellular environment.

[0183] In one embodiment, the compositions of the present disclosure exhibit pH tolerance, which is used interchangeably with acid tolerance and base tolerance. In one embodiment, the pH-tolerant compositions of the present disclosure are tolerant to low pH associated with one or more feed preparation processes. In one embodiment, the pH-tolerant compositions of the present disclosure are tolerant to low pH associated with one or more environments in an animal's gastrointestinal tract, such as the stomach, duodenum, jejunum, ileum, cecum, proximal colon, distal colon, and rectum. In one embodiment, the pH-tolerant compositions of the present disclosure are tolerant to high pH associated with one or more feed preparation processes. In one embodiment, the pH-tolerant compositions of the present disclosure are tolerant to rapid pH fluctuations (high to low, low to high, high to neutral, low to neutral, neutral to high, and neutral to low) associated with one or more feed preparation processes. In one embodiment, the pH-tolerant compositions of the present disclosure are tolerant to low pH associated with one or more environments in an animal's gastrointestinal tract, such as the stomach.

[0184] In one embodiment, the encapsulation is a reservoir-type encapsulation. In one embodiment, the encapsulation is a matrix-type encapsulation. In one embodiment, the encapsulation is a coated matrix-type encapsulation. Burgain et al. (2011. J. Food Eng. 104:467-483) discloses numerous encapsulation embodiments and techniques, all of which are incorporated by reference.

[0185] In some embodiments, the compositions of the present disclosure are encapsulated in one or more of the following: gellan gum, xanthan gum, potassium carrageenan, cellulose acetate phthalate, chitosan, starch, milk fat, whey protein, calcium alginate, raftilose, raftiline, pectin, sugars, glucose, maltodextrin, gum arabic, guar, seed flour, alginate, dextrin, dextran, cellulose, gelatin, albumin, casein, gluten, acacia gum, tragacanth, wax, paraffin, stearic acid, monodiglycerides, and diglycerides. In some embodiments, the compositions of the present disclosure are encapsulated in one or more of a polymer, carbohydrate, sugar, plastic, glass, polysaccharide, lipid, wax, oil, fatty acid, or glyceride. In one embodiment, the microbial composition is encapsulated in glucose. In one embodiment, the microbial composition is encapsulated in a glucose-containing composition. In one embodiment, the formulation of the microbial composition comprises a glucose sequestrant. In one embodiment, the formulation of the microbial composition comprises a glucose encapsulating composition.

[0186] In some embodiments, encapsulation of the compositions of the present disclosure is carried out by extrusion, emulsification, coating, agglomeration, freeze-drying, vitrification, foam drying, evaporative preservation, vacuum drying, or spray drying.

[0187] In some embodiments, the encapsulated compositions of the present disclosure are vitrified. In some embodiments, encapsulation involves drying the compositions of the present disclosure in the presence of a substance that forms a glassy, ​​amorphous solid state, a process known as vitrification, thereby encapsulating the composition. In some embodiments, the vitrified composition is protected from degradative conditions that typically destroy or degrade microorganisms. Many common substances possess vitrifying properties, i.e., they form a glassy solid state under certain conditions. These substances include some sugars, including sucrose and maltose, as well as other more complex compounds, such as polyvinylpyrrolidone (PVP). When any solution dries, the molecules in the solution can crystallize, or vitrify. Solutes with extensive dissymmetry can be good vitrifying agents because they prevent crystal nucleation during drying. A substance that inhibits the crystallization of another substance, such as raffinose in the presence of sucrose, can result in the combined substance forming a good vitrified solution. See U.S. Patent Nos. 5,290,765 and 9,469,835.

[0188] In some embodiments, a microbial composition encapsulated in a vitrification material is produced. Vitrification compositions can be made by selecting a mixture containing cells, combining the mixture with a sufficient amount of one or more vitrifying solutes to protect the mixture and inhibit destructive reactions during drying, and drying the combination by exposing the combination to a desiccant or drying conditions at a temperature above the temperature at which the combination freezes but below the temperature at which the vitrifying solute achieves a vitrified state, at about standard atmospheric pressure, until the combination is substantially dry.

[0189] In one embodiment, the encapsulation composition comprises microcapsules having a multiplicity of liquid cores encapsulated in a solid shell material. For purposes of this disclosure, a "multiplicity" of cores is defined as two or more.

[0190] The first category of useful fusible shell materials is normally solid fats, including fats that already have a suitable hardness, as well as animal or vegetable oils that are hydrogenated until their melting points are high enough for the purposes of this disclosure. Depending on the desired process and storage temperature and the specific material selected, the specific fat may be either a normally solid or a normally liquid material. As used herein, the terms "normally solid" and "normally liquid" refer to the state of the material at the desired temperature for storing the resulting microcapsules. Because fats and hydrogenated oils do not strictly have melting points, the term "melting point" is used herein to describe the minimum temperature at which the fusible material softens or becomes liquid enough to successfully emulsify and spray-cool, and therefore corresponds roughly to the maximum temperature at which the shell material has sufficient integrity to prevent the release of the choline core. "Melting point" is similarly defined herein for other materials that do not have a distinct melting point.

[0191] Specific examples of fats and oils useful herein (some of which require hydrogenation) include: animal fats such as beef tallow, mutton tallow, lamb fat, lard or pork fat, fish oil, and whale oil, vegetable oils such as canola oil, cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, linseed oil, tung oil, and castor oil, fatty acid mono- and diglycerides, free fatty acids such as stearic acid, palmitic acid, and oleic acid, and mixtures thereof. The above list of fats and oils is not exhaustive but merely illustrative.

[0192] Specific examples of fatty acids include linoleic acid, gamma-linoleic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosatetraenoic acid, vaccenic acid, nervonic acid, mead acid, erucic acid, gondoic acid, elaidic acid, oleic acid, palitoleic acid, stearidonic acid, eicosapentaenoic acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid These include melissic acid, henatriacontylic acid, lacceroic acid, psyllic acid, gedic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontanoic acid, and octatriacontanoic acid.

[0193] Another category of fusible materials useful for encapsulating shell materials are waxes. Representative waxes contemplated for use herein include animal waxes such as beeswax, lanolin, shell wax, and Chinese insect wax; vegetable waxes such as carnauba, candelilla, bayberry, and sugarcane; mineral waxes such as paraffin, microcrystalline petroleum, ozokerite, ceresin, and montan; synthetic waxes such as low molecular weight polyolefins (e.g., CARBOWAX) and polyol ether-esters (e.g., sorbitol); Fischer-Tropsch synthetic waxes, and mixtures thereof. If the core is aqueous, water-soluble waxes such as CARBOWAX and sorbitol are not contemplated herein.

[0194] Still other fusible compounds useful herein are fusible natural resins such as rosin, balsam, shellac, and mixtures thereof.

[0195] In some embodiments, the microorganism or microbial composition is embedded in a wax, such as the waxes described in this disclosure.

[0196] In some embodiments, the microorganisms or microbial compositions are embedded in wax balls. In some embodiments, the microorganisms or microbial compositions are already encapsulated before being embedded in the wax balls. In some embodiments, the wax balls are 10 microorganisms, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns, 400 microns, 450 microns, 500 microns, 550 microns, 600 microns, 650 microns, 700 microns, 750 microns, 800 microns, 850 microns, 900 microns, 950 microns, or 1,000 microns.

[0197] In some embodiments, the wax balls are about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, about 500 microns, about 550 microns, about 600 microns, about 650 microns, about 700 microns, about 750 microns, about 800 microns, about 850 microns, about 900 microns, about 950 microns, or about 1,000 microns.

[0198] In some embodiments, the wax balls are 10 to 20 microns, 10 to 30 microns, 10 to 40 microns, 10 to 50 microns, 10 to 60 microns, 10 to 70 microns, 10 to 80 microns, 10 to 90 microns, 10 to 100 microns, 10 to 250 microns, 10 to 500 microns, 10 to 750 microns, 10 to 1,000 microns, 20 to 30 microns, 20 to 40 microns, 20 to 50 microns, 20 to 60 microns, 20 to 70 microns, 20 to 80 microns, 20 to 90 microns, 20 to 100 microns, 20 to 250 microns, 20 to 500 microns, 20 to 750 microns, 20 to 1,000 microns, 30 to 40 microns, 30 to 50 microns, 30 to 60 microns, 30 to 70 microns, 30 to 80 microns, 30 to 90 microns, 30 to 100 microns, 30-250 microns, 30-500 microns, 30-750 microns, 30-1,000 microns, 40-50 microns, 40-60 microns, 40-70 microns, 40-80 microns, 40-90 microns, 40-100 microns, 40-250 microns, 40-500 microns, 40-750 microns, 40-1,000 microns, 50-60 microns, 50-70 microns Ron, 50-80 microns, 50-90 microns, 50-100 microns, 50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70 microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns, 60-500 microns, 60-750 microns, 60-1,000 microns, 70-80 microns 70-90 microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns, 70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250 microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000 microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns, 90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns, 100-1,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns, 500-750 microns, 500-1,000 microns, or 750-1,000 microns.

[0199] In some embodiments, the wax balls are about 10 to 20 microns, about 10 to 30 microns, 10 to 40 microns, about 10 to 50 microns, about 10 to 60 microns, about 10 to 70 microns, about 10 to 80 microns, about 10 to 90 microns, about 10 to 100 microns, about 10 to 250 microns, about 10 to 500 microns, about 10 to 750 microns, about 10 to 1,000 microns, about 20 to 30 microns, about 20 to 40 microns, Approximately 20 to 50 microns, approximately 20 to 60 microns, approximately 20 to 70 microns, approximately 20 to 80 microns, approximately 20 to 90 microns, approximately 20 to 100 microns, approximately 20 to 250 microns, approximately 20 to 500 microns, approximately 20 to 750 microns, approximately 20 to 1,000 microns, approximately 30 to 40 microns, approximately 30 to 50 microns, approximately 30 to 60 microns, approximately 30 to 70 microns, approximately 30 to 80 microns, approximately 30 to 90 microns, approximately 30 to 100 microns Clonal, approximately 30 to 250 microns, approximately 30 to 500 microns, approximately 30 to 750 microns, approximately 30 to 1,000 microns, approximately 40 to 50 microns, approximately 40 to 60 microns, approximately 40 to 70 microns, approximately 40 to 80 microns, approximately 40 to 90 microns, approximately 40 to 100 microns, approximately 40 to 250 microns, approximately 40 to 500 microns, approximately 40 to 750 microns, approximately 40 to 1,000 microns, approximately 50 to 60 microns, approximately 50 to 70 microns about 50 to 80 microns, about 50 to 90 microns, about 50 to 100 microns, about 50 to 250 microns, about 50 to 500 microns, about 50 to 750 microns, about 50 to 1,000 microns, about 60 to 70 microns, about 60 to 80 microns, about 60 to 90 microns, about 60 to 100 microns, about 60 to 250 microns, about 60 to 500 microns, about 60 to 750 microns, about 60 to 1,000 microns, about 70 to 80 microns 70 to 90 microns, about 70 to 90 microns, about 70 to 100 microns, about 70 to 250 microns, about 70 to 500 microns, about 70 to 750 microns, about 70 to 1,000 microns, about 80 to 90 microns, about 80 to 100 microns, about 80 to 250 microns, about 80 to 500 microns, about 80 to 500 microns, about 80 to 750 microns, about 80 to 1,000 microns, about 90 to 100 microns, about 90 to 250 microns, about 90 to 500 microns, about 90 to 750 microns, about 90 to 1,000 microns, about 100 to 250 microns, about 100 to 500 microns, about 100 to 750 microns, about 100 to 1,000 microns, about 250 to 500 microns, about 250 to 750 microns, about 250 to 1,000 microns, about 500 to 750 microns, about 500 to 1,000 microns, or about 750 to 1,000 microns.

[0200] In accordance with the present disclosure, various adjunct materials are contemplated for incorporation into the fusible material. For example, antioxidants, light stabilizers, dyes and lakes, flavorings, essential oils, anti-caking agents, fillers, pH stabilizers, sugars (mono-, di-, tri-, and polysaccharides), etc. may be incorporated into the fusible material in amounts that do not detract from their usefulness for the present disclosure.

[0201] Core materials contemplated herein comprise from about 0.1% to about 50%, from about 1% to about 35%, or from about 5% to about 30% by weight of the microcapsule. In some embodiments, core materials contemplated herein comprise up to about 30% by weight of the microcapsule. In some embodiments, core materials contemplated herein comprise about 5% by weight of the microcapsule. Core materials are contemplated as either liquids or solids at the intended storage temperatures of the microcapsules.

[0202] The core may contain other additives well known in the pharmaceutical arts, including edible sugars such as sucrose, glucose, maltose, fructose, lactose, cellobiose, monosaccharides, disaccharides, trisaccharides, and polysaccharides, and mixtures thereof; artificial sweeteners such as aspartame, saccharin, cyclamate, and mixtures thereof; edible acids such as acetic acid (vinegar), citric acid, ascorbic acid, tartaric acid, and mixtures thereof; edible starches such as corn starch, vegetable protein hydrolysates, water-soluble vitamins such as vitamin C, water-soluble pharmaceuticals, water-soluble nutritional materials such as ferrous sulfate, flavorings, salt, monosodium glutamate, antibacterial agents such as sorbic acid, antifungal agents such as potassium sorbate, sorbic acid, sodium benzoate, and benzoic acid, food-grade pigments and dyes, and mixtures thereof. Other potentially useful supplemental core materials will be apparent to those skilled in the art.

[0203] Emulsifiers can be used to aid in the formation of a stable emulsion. Exemplary emulsifiers include glyceryl monostearate, polysorbate esters, ethoxylated mono- and diglycerides, and mixtures thereof.

[0204] For ease of processing, and particularly for successful formation of a reasonably stable emulsion, the viscosities of the core and shell materials should be similar at the temperature at which the emulsion is formed. Specifically, the ratio of shell viscosity to core viscosity, expressed in centipoise or equivalent units and both measured at the temperature of the emulsion, should be from about 22:1 to about 1:1, desirably from about 8:1 to about 1:1, and preferably from about 3:1 to about 1:1. While a 1:1 ratio is ideal, viscosity ratios within the recited ranges are useful.

[0205] The encapsulation composition is not limited to the microcapsule compositions disclosed above. In some embodiments, the encapsulation composition encapsulates the microbial composition in an adhesive polymer, which can be natural or synthetic, without toxic effects. In some embodiments, the encapsulation composition may be a matrix selected from a sugar matrix, a gelatin matrix, a polymer matrix, a silica matrix, a starch matrix, a foam matrix, a glass / glass matrix, and the like. See Pirzio et al. (U.S. Patent No. 7,488,503). In some embodiments, the encapsulating composition may be selected from polyvinyl acetate, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, celluloses including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose, polyvinylpyrrolidone, starch, modified starch, dextrin, maltodextrin, polysaccharides including alginates and chitosan, monosaccharides, fats, fatty acids including oils, proteins including gelatin and zein, gum arabic, shellac, vinylidene chloride and vinylidene chloride copolymers, calcium lignosulfonate, acrylic copolymers, polyvinyl acrylate, polyethylene oxide, acrylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomer, and polychloroprene.

[0206] In some embodiments, the encapsulating composition comprises at least one layer of encapsulation, hi some embodiments, the encapsulating composition comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 layers of encapsulation / encapsulant.

[0207] In some embodiments, the encapsulation composition comprises at least two layers of encapsulation. In some embodiments, each layer of encapsulation imparts a different property to the composition. In some embodiments, no two consecutive layers impart the same property. In some embodiments, at least one layer of the at least two layers of encapsulation imparts thermal stability, storage stability, UV resistance, moisture resistance, hydrophobicity, hydrophilicity, lipophobicity, lipophilicity, pH stability, acid resistance, and base resistance.

[0208] In some embodiments, the encapsulation composition comprises two layers of encapsulation, a first layer providing thermal and / or shelf stability and a second layer providing pH resistance.

[0209] In some embodiments, the encapsulation layers provide for sustained release of the microbial composition held in the center of the encapsulation layer, and in some embodiments, the more layers there are, the longer the time after administration before the microbial composition is exposed.

[0210] In some embodiments, the encapsulating shells of the present disclosure have a diameter of at most 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, m, 400μm, 410μm, 420μm, 430μm, 440μm, 450μm, 460μm, 470μm, 480μm, 490μm, 500μm, 510μm, 520μm, 530μm, 540μm, 550μm, 560μm, 570μm, 580μm, 590μm, 60 0μm, 610μm, 620μm, 630μm, 640μm, 650μm, 660μm, 670μm, 680μm, 690μm, 700μm m, 710μm, 720μm, 730μm, 740μm, 750μm, 760μm, 770μm, 780μm, 790μm, 800μm, 8 10μm, 820μm, 830μm, 840μm, 850μm, 860μm, 870μm, 880μm, 890μm, 900μm, 910 μm, 920μm, 930μm, 940μm, 950μm, 960μm, 970μm, 980μm, 990μm, 1000μm, 1010 μm, 1020μm, 1030μm, 1040μm, 1050μm, 1060μm, 1070μm, 1080μm, 1090μm, 110 0μm, 1110μm, 1120μm, 1130μm, 1140μm, 1150μm, 1160μm, 1170μm, 1180μm, 119 0μm, 1200μm, 1210μm, 1220μm, 1230μm, 1240μm, 1250μm, 1260μm, 1270μm, 12 80μm, 1290μm, 1300μm, 1310μm, 1320μm, 1330μm, 1340μm, 1350μm, 1360μm, 1 370μm, 1380μm, 1390μm, 1400μm, 1410μm, 1420μm, 1430μm, 1440μm, 1450μm, 1460μm, 1470μm, 1480μm, 1490μm, 1500μm, 1510μm, 1520μm, 1530μm, 1540μm,1550μm, 1560μm, 1570μm, 1580μm, 1590μm, 1600μm, 1610μm, 1620μm, 1630μm, 1640μm, 1650μm, 1660μm, 1670μm, 1680μm, 1690μm, 1700μm, 1710μm, 1720μm ,1730μm,1740μm,1750μm,1760μm,1770μm,1780μm,1790μm,1800μm,1810μm,1820μm,1830μm,1840μm,1850μm,1860μm,1870μm,1880μm,1890μm,1900μm m, 1910μm, 1920μm, 1930μm, 1940μm, 1950μm, 1960μm, 1970μm, 1980μm, 1990μm, 2000μm, 2010μm, 2020μm, 2030μm, 2040μm, 2050μm, 2060μm, 2070μm, 2080 μm, 2090μm, 2100μm, 2110μm, 2120μm, 2130μm, 2140μm, 2150μm, 2160μm, 2170μm, 2180μm, 2190μm, 2200μm, 2210μm, 2220μm, 2230μm, 2240μm, 2250μm, 226 0μm, 2270μm, 2280μm, 2290μm, 2300μm, 2310μm, 2320μm, 2330μm, 2340μm, 2350μm, 2360μm, 2370μm, 2380μm, 2390μm, 2400μm, 2410μm, 2420μm, 2430μm, 24 40μm, 2450μm, 2460μm, 2470μm, 2480μm, 2490μm, 2500μm, 2510μm, 2520μm, 2530μm, 2540μm, 2550μm, 2560μm, 2570μm, 2580μm, 2590μm, 2600μm, 2610μm, 2 620μm, 2630μm, 2640μm, 2650μm, 2660μm, 2670μm, 2680μm, 2690μm, 2700μm, 2710μm, 2720μm, 2730μm, 2740μm, 2750μm, 2760μm, 2770μm, 2780μm, 2790μm, 2800μm, 2810μm, 2820μm, 2830μm, 2840μm, 2850μm, 2860μm, 2870μm, 2880μm, 2890μm, 2900μm, 2910μm, 2920μm, 2930μm, 2940μm, 2950μm, 2960μm, 2970μm,It can be 2980 μm, 2990 μm, or 3000 μm thick.

[0211] In some embodiments, the encapsulating composition of the present disclosure has a viscosity of 0.750, 0.700, 0.650, 0.600, 0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350, 0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0.175, 0.180, 0.195, 0.200, 0.250, 0.225, 0.200, 0.250, 0.300, 0.375, 0.350, 0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0.175, 0.180, 0.195, 0.200, 0.250, 0.2 ...50, 0.225, 0.200, 0.250, 0.250, 0.250, 0.250, 0.250, 0.250, 0.250, 0.2 Water activity (a) of less than 50, 0.140, 0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080, 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, 0.010, or 0.005 w )

[0212] In some embodiments, the encapsulating composition of the present disclosure has a viscosity of about 0.750, about 0.700, about 0.650, about 0.600, about 0.550, about 0.500, about 0.475, about 0.450, about 0.425, about 0.400, about 0.375, about 0.350, about 0.325, about 0.300, about 0.275, about 0.250, about 0.225, about 0.200, about 0.190, about 0.180, about 0.170, about 0.160, about 0.180, about 0.190, about 0.200, about 0.250, about 0.225, about 0.200, about 0.375, about 0.350, about 0.325, about 0.300, about 0.275, about 0.250, about 0.225, about 0.200, about 0.190, about 0.180, about 0.170, about 0.160, about 0.180, about 0.200, about 0.250, about 0.225 ...50, about 0.200, about 0.250, about 0.250, about 0.200, about 0.250, about 0.25 a water activity (a) of less than about 50, about 0.140, about 0.130, about 0.120, about 0.110, about 0.100, about 0.095, about 0.090, about 0.085, about 0.080, about 0.075, about 0.070, about 0.065, about 0.060, about 0.055, about 0.050, about 0.045, about 0.040, about 0.035, about 0.030, about 0.025, about 0.020, about 0.015, about 0.010, or about 0.005 w )

[0213] In one embodiment, the microorganism is first dried by spray drying, freeze drying, or foam drying together with an excipient, which may include one or more sugars, sugar alcohols, disaccharides, trisaccharides, polysaccharides, salts, amino acids, amino acid salts, or polymers.

[0214] In some embodiments, the microorganism or composition comprising the microorganism is ground to a size of 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns, 400 microns, 450 microns, 500 microns, 550 microns, 600 microns, 650 microns, 700 microns, 750 microns, 800 microns, 850 microns, 900 microns, 950 microns, or 1,000 microns.

[0215] In some embodiments, the microorganism or composition comprising the microorganism is ground to a size of about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, about 500 microns, about 550 microns, about 600 microns, about 650 microns, about 700 microns, about 750 microns, about 800 microns, about 850 microns, about 900 microns, about 950 microns, or about 1,000 microns.

[0216] In some embodiments, the microorganism or composition comprising the microorganism is sized to a depth of 10 to 20 microns, 10 to 30 microns, 10 to 40 microns, 10 to 50 microns, 10 to 60 microns, 10 to 70 microns, 10 to 80 microns, 10 to 90 microns, 10 to 100 microns, 10 to 250 microns, 10 to 500 microns, 10 to 750 microns, 10 to 1,000 microns, 20 to 30 microns, 20 to 40 microns, 20 to 50 microns, 20 to 60 microns, 20 to 70 microns, 20 to 80 microns, 20 to 90 microns, 20 to 100 microns, 20 to 250 microns, 20 to 500 microns, 20 to 750 microns, 20 to 1,000 microns, 30 to 40 microns, 30 to 50 microns, 30 to 60 microns, 30 to 70 microns, 30 to 80 microns, 30 to 90 microns, 30 to 100 microns, 3 ...30 to 50 microns, 30 to 60 microns, 30 to 7 ~100 microns, 30~250 microns, 30~500 microns, 30~750 microns, 30~1,000 microns, 40~50 microns, 40~60 microns, 40~70 microns, 40~80 microns, 40~90 microns, 40~100 microns, 40~250 microns, 40~500 microns, 40~750 microns, 40~1,000 microns, 50~60 microns, 50~70 Microns, 50-80 microns, 50-90 microns, 50-100 microns, 50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70 microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns, 60-500 microns, 60-750 microns, 60-1,000 microns, 70-80 microns 70-90 microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns, 70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250 microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000 microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns, 90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns, 100-1,The powder is ground to a size of 2,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns, 500-750 microns, 500-1,000 microns, or 750-1,000 microns.

[0217] In some embodiments, the microorganism or composition comprising the microorganism has a diameter of about 10 to 20 microns, about 10 to 30 microns, 10 to 40 microns, about 10 to 50 microns, about 10 to 60 microns, about 10 to 70 microns, about 10 to 80 microns, about 10 to 90 microns, about 10 to 100 microns, about 10 to 250 microns, about 10 to 500 microns, about 10 to 750 microns, about 10 to 1,000 microns, about 20 to 30 microns, about 20 to 300 microns, about 20 to 400 microns, about 10 to 500 microns, about 10 to 600 microns, about 10 to 700 microns, about 10 to 800 microns, about 10 to 90 microns, about 10 to 100 microns, about 10 to 250 microns, about 10 to 500 microns, about 10 to 750 microns, about 10 to 1,000 microns, about 20 to 3 ... 40 microns, about 20 to 50 microns, about 20 to 60 microns, about 20 to 70 microns, about 20 to 80 microns, about 20 to 90 microns, about 20 to 100 microns, about 20 to 250 microns, about 20 to 500 microns, about 20 to 750 microns, about 20 to 1,000 microns, about 30 to 40 microns, about 30 to 50 microns, about 30 to 60 microns, about 30 to 70 microns, about 30 to 80 microns, about 30 to 90 microns, about 30 ~100 microns, about 30 to 250 microns, about 30 to 500 microns, about 30 to 750 microns, about 30 to 1,000 microns, about 40 to 50 microns, about 40 to 60 microns, about 40 to 70 microns, about 40 to 80 microns, about 40 to 90 microns, about 40 to 100 microns, about 40 to 250 microns, about 40 to 500 microns, about 40 to 750 microns, about 40 to 1,000 microns, about 50 to 60 microns, about 50 to 70 microns, about 50 to 80 microns, about 50 to 90 microns, about 50 to 100 microns, about 50 to 250 microns, about 50 to 500 microns, about 50 to 750 microns, about 50 to 1,000 microns, about 60 to 70 microns, about 60 to 80 microns, about 60 to 90 microns, about 60 to 100 microns, about 60 to 250 microns, about 60 to 500 microns, about 60 to 750 microns, about 60 to 1,000 microns, about 70 to 80 microns 70 to 90 microns, about 70 to 90 microns, about 70 to 100 microns, about 70 to 250 microns, about 70 to 500 microns, about 70 to 750 microns, about 70 to 1,000 microns, about 80 to 90 microns, about 80 to 100 microns, about 80 to 250 microns, about 80 to 500 microns, about 80 to 500 microns, about 80 to 750 microns, about 80 to 1,000 microns, about 90 to 100 microns, about 90 to 250 microns, about 90 to 500 microns, about 90 to 750 microns, about 90 to 1,The powder is ground to a size of about 1,000 microns, about 100 to 250 microns, about 100 to 500 microns, about 100 to 750 microns, about 100 to 1,000 microns, about 250 to 500 microns, about 250 to 750 microns, about 250 to 1,000 microns, about 500 to 750 microns, about 500 to 1,000 microns, or about 750 to 1,000 microns.

[0218] In some embodiments, the microorganisms or compositions comprising microorganisms are combined with a wax, fat, oil, fatty acid, or fatty alcohol and spray-congealed into beads of about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, about 500 microns, about 550 microns, about 600 microns, about 650 microns, about 700 microns, about 750 microns, about 800 microns, about 850 microns, about 900 microns, about 950 microns, or about 1,000 microns.

[0219] In some embodiments, the microorganism or composition comprising the microorganism is combined with a wax, fat, oil, fatty acid, or fatty alcohol to form a microsphere having a particle size of 10-20 microns, 10-30 microns, 10-40 microns, 10-50 microns, 10-60 microns, 10-70 microns, 10-80 microns, 10-90 microns, 10-100 microns, 10-250 microns, 10-500 microns, 10-750 microns. , 10-1,000 microns, 20-30 microns, 20-40 microns, 20-50 microns, 20-60 microns, 20-70 microns, 20-80 microns, 20-90 microns, 20-100 microns, 20-250 microns, 20-500 microns, 20-750 microns, 20-1,000 microns, 30-40 microns, 30-50 microns, 30-60 microns, 30-70 microns, 30-80 microns, 30-90 microns, 30-100 microns, 30-250 microns, 30-500 microns, 30-750 microns, 30-1,000 microns, 40-50 microns, 40-60 microns, 40-70 microns, 40-80 microns, 40-90 microns, 40-100 microns, 40-250 microns, 40-500 microns, 40-750 microns, 40-1,000 microns, 50-60 microns Ron, 50-70 microns, 50-80 microns, 50-90 microns, 50-100 microns, 50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70 microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns, 60-500 microns, 60-750 microns, 60-1,000 microns, 70-80 microns 70-90 microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns, 70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250 microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000 microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns, 90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns, 100-1,It is spray-congealed into beads of 2,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns, 500-750 microns, 500-1,000 microns, or 750-1,000 microns.

[0220] In some embodiments, the microorganism or composition comprising the microorganism is combined with a wax, fat, oil, fatty acid, or fatty alcohol to form a microsphere of about 10-20 microns, about 10-30 microns, 10-40 microns, about 10-50 microns, about 10-60 microns, about 10-70 microns, about 10-80 microns, about 10-90 microns, about 10-100 microns, about 10-250 microns, about 10-500 microns, about 10-750 microns, about 10- 1,000 microns, approximately 20 to 30 microns, approximately 20 to 40 microns, approximately 20 to 50 microns, approximately 20 to 60 microns, approximately 20 to 70 microns, approximately 20 to 80 microns, approximately 20 to 90 microns, approximately 20 to 100 microns, approximately 20 to 250 microns, approximately 20 to 500 microns, approximately 20 to 750 microns, approximately 20 to 1,000 microns, approximately 30 to 40 microns, approximately 30 to 50 microns, approximately 30 to 60 microns, approximately 30 to 70 microns, approximately 30 to 80 microns about 30 to 90 microns, about 30 to 100 microns, about 30 to 250 microns, about 30 to 500 microns, about 30 to 750 microns, about 30 to 1,000 microns, about 40 to 50 microns, about 40 to 60 microns, about 40 to 70 microns, about 40 to 80 microns, about 40 to 90 microns, about 40 to 100 microns, about 40 to 250 microns, about 40 to 500 microns, about 40 to 750 microns, about 40 to 1,000 microns, about 50 to 60 microns about 50 to 70 microns, about 50 to 80 microns, about 50 to 90 microns, about 50 to 100 microns, about 50 to 250 microns, about 50 to 500 microns, about 50 to 750 microns, about 50 to 1,000 microns, about 60 to 70 microns, about 60 to 80 microns, about 60 to 90 microns, about 60 to 100 microns, about 60 to 250 microns, about 60 to 500 microns, about 60 to 750 microns, about 60 to 1,000 microns, about 70 to 80 microns 70-90 microns, approximately 70-90 microns, approximately 70-100 microns, approximately 70-250 microns, approximately 70-500 microns, approximately 70-750 microns, approximately 70-1,000 microns, approximately 80-90 microns, approximately 80-100 microns, approximately 80-250 microns, approximately 80-500 microns, approximately 80-500 microns, approximately 80-750 microns, approximately 80-1,The mixture is spray-congealed into beads having a diameter of about 1,000 microns, about 90 to 100 microns, about 90 to 250 microns, about 90 to 500 microns, about 90 to 750 microns, about 90 to 1,000 microns, about 100 to 250 microns, about 100 to 500 microns, about 100 to 750 microns, about 100 to 1,000 microns, about 250 to 500 microns, about 250 to 750 microns, about 250 to 1,000 microns, about 500 to 750 microns, about 500 to 1,000 microns, or about 750 to 1,000 microns.

[0221] In some embodiments, the microorganism or a composition comprising the microorganism is combined with a wax, fat, oil, fatty acid, or fatty alcohol and a water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol, and spray-solidified into beads whose size is described herein. In some embodiments, the water-soluble polymer, salt, polysaccharide, sugar, or sugar alcohol functions as a disintegrant. In some embodiments, the disintegrant forms pores when the beads are dispersed in the GI tract of an animal.

[0222] In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified so that the disintegrant dissolves within 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes after administration. In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified so that the disintegrant dissolves within about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes after administration.

[0223] In some embodiments, the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol composition is modified so that the disintegrant dissolves within 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 hours after administration. In some embodiments, the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol composition is modified so that the disintegrant dissolves within about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, or about 12 hours after administration.

[0224] In some embodiments, the composition of the water soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves at a temperature of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50°C. In some embodiments, the composition of the water soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves at a temperature of at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50°C.

[0225] In some embodiments, the composition of the water soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is such that the disintegrant has a viscosity of at least 3.8, 3.9, 4.4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29, 6.30, 6.31, 6.32, 6.33, 6.34, 6.35, 6.36, 6.37, 6.38, 6.39, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.46, 6.47, 6.48, 6.49, 6.50, 6.51, 6.52, 6.53, 6.54, 6.55, 6.56, 6.57, 6.58, 6.59, 6.60, 6.61, 6.62, and modified to dissolve at a pH of 4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0.In some embodiments, the composition of the water soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is such that the disintegrant has a viscosity of at least about 3.8, at least about 3.9, at least about 4. at least about 4.1, at least about 4.2, at least about 4.3, at least about 4.4, at least about 4.5, at least about 4.6, at least about 4.7, at least about 4.8, at least about 4.9, at least about 5.0, at least about 5.1, at least about 5.2, at least about 5.3, at least about 5.4, at least about 5.5, at least about 5.6, at least about 5.7, at least about 5.8, at least about 5.9, at least about 6.0, at least about 6.2, at least about 6.3, at least about 6.4, at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8, at least about 6.9, at least about 7.0, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9 at least about 6.8, at least about 6.9, at least about 7.0, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, at least about 8.0, at least about 8.1, at least about 8.2, at least about 8.3, at least about 8.4, at least about 8.5, at least about 8.6, at least about 8.7, at least about 8.8, at least about 8.9, at least about 9.0, at least about 9.1, at least about 9.2, at least about 9.3, at least about 9.4, at least about 9.5, at least about 9.6, at least about 9.7, at least about 9.8, at least about 9.9, or at least about 10.0.

[0226] In some embodiments, the microorganism or composition comprising the microorganism is coated with a polymer, polysaccharide, sugar, sugar alcohol, gel, wax, fat, fatty alcohol, or fatty acid.

[0227] In some embodiments, the microorganism or a composition comprising the microorganism is coated with a polymer, polysaccharide, sugar, sugar alcohol, gel, wax, fat, fatty alcohol, or fatty acid.

[0228] In some embodiments, the coating of the microorganism or composition comprising the microorganism is modified to dissolve within 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes after the coating is administered, hi some embodiments, the coating of the microorganism or composition comprising the microorganism is modified to dissolve within about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes after the coating is administered.

[0229] In some embodiments, the coating of the microorganism or composition comprising the microorganism is modified to dissolve within 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 hours after the coating is administered. In some embodiments, the coating of the microorganism or composition comprising the microorganism is modified to dissolve within about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, or about 12 hours after the coating is administered.

[0230] In some embodiments, the coating of the microorganism or composition comprising the microorganism is modified such that the coating dissolves at a temperature of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50°C. In some embodiments, the coating of the microorganism or composition comprising the microorganism is modified such that the coating dissolves at a temperature of at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50°C.

[0231] In some embodiments, the coating of the microorganism or composition comprising the microorganism is at least 3.8, 3.9, 4.4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29, 6.30, 6.31, 6.32, 6.33, 6.34, 6.35, 6.36, 6.37, 6.38, 6.39, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.46, 6.47, 6.48, 6.49, 6.50, 6.51, 6.52, 6.53, 6.54, 6.55, 6.56, 6.57, 6.58, 6.59, 6.60, 6.61, 6.62, 6.63, 6.64, 6.65, 6 Modified to dissolve at pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0. In some embodiments, the coating of the microorganism or composition comprising the microorganism is at least about 3.8, at least about 3.9, at least about 4. at least about 4.1, at least about 4.2, at least about 4.3, at least about 4.4, at least about 4.5, at least about 4.6, at least about 4.7, at least about 4.8, at least about 4.9, at least about 5.0, at least about 5.1, at least about 5.2, at least about 5.3, at least about 5.4, at least about 5.5, at least about 5.6, at least about 5.7, at least about 5.8, at least about 5.9, at least about 6.0, at least about 6.2, at least about 6.3, at least about 6.4, at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8 , at least about 6.9, at least about 7.0, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, at least about 8.0, at least about 8.1, at least about 8.2, at least about 8.3, at least about 8.4, at least about 8.5, at least about 8.6, at least about 8.7, at least about 8.8, at least about 8.9, at least about 9.0, at least about 9.1, at least about 9.2, at least about 9.3, at least about 9.4, at least about 9.5, at least about 9.6, at least about 9.7, at least about 9.8, at least about 9.9, or at least about 10.0.

[0232] animal feed In some embodiments, the animal feed comprises dry food, kibble, wet food, treats, cookies, biscuits, frozen food, fresh food, preserved food, dehydrated food, pasteurized food, raw food, freeze-dried food, and home-cooked food. In some embodiments, the animal feed is pet food.

[0233] In some embodiments, the composition of the present disclosure is mixed with animal feed. In some embodiments, the composition of the present disclosure is sprinkled on top of the animal feed. In some embodiments, the animal feed can be in various forms, such as kibble, wet food, dry food, raw food, etc.

[0234] In some embodiments, the composition of the present disclosure is mixed into the feed itself. In one embodiment, the composition of the present disclosure is mixed into the feed in a feed grinder. In one embodiment, the composition of the present disclosure is mixed into or on the feed just before feeding.

[0235] In some embodiments, the feed of the present disclosure may be supplemented with water, premix(s), commercially available formulated feed, and mixtures thereof.

[0236] In some embodiments, the microbial composition of the present disclosure may be mixed with animal feed. Individual components of the animal feed may be mixed with the microbial composition before feeding the animal. The microbial composition of the present disclosure may be mixed with the feed and pelleted into kibble.

[0237] In some embodiments, the microbial compositions of the present disclosure may be mixed with animal feed at various stages of the animal's development.

[0238] Administration of Microbial Compositions In some embodiments, the microbial compositions of the present disclosure are administered to animals via the oral route. In some embodiments, the microbial compositions are administered via direct injection into the gastrointestinal tract. In further embodiments, direct injection administration delivers the microbial composition directly to the stomach. In some embodiments, the microbial compositions are administered to the buccal cavity, oral cavity, stomach, duodenum, jejunum, ileum, cecum, proximal colon, distal colon, and rectum. In some embodiments, the microbial compositions of the present disclosure are administered to animals via the anus. In some embodiments, the microbial compositions are directed to the buccal cavity, oral cavity, stomach, duodenum, jejunum, ileum, cecum, proximal colon, distal colon, and rectum of the animal. In further embodiments, anal administration is in the form of an inserted suppository.

[0239] In some embodiments, the microbial composition comprises a total of at least 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32 mL , administered in dosage volumes including 33mL, 34mL, 35mL, 36mL, 37mL, 38mL, 39mL, 40mL, 41mL, 42mL, 43mL, 44mL, 45mL, 46mL, 47mL, 48mL, 49mL, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 200mL, 300mL, 400mL, 500mL, 600mL, 700mL, 800mL, 900mL, or 1,000mL.

[0240] In some embodiments, the microbial composition comprises a total of, or at least, 10 18 , 10 17 , 10 16 , 10 15 , 10 14 , 10 13 , 10 12 , 10 11 , 10 10 , 10 9 , 10 8 , 10 7, 10 6 , 10 5 , 10 4 , 10 3 , or 10 2 The dose is administered in a volume containing microbial cells.

[0241] In some embodiments, the microbial composition is mixed with feed and administration is by ingestion of the microbial composition with the feed. In some embodiments, the dose of the microbial composition is 10 or more per gram or per milliliter of composition. 2 ~10 12 , 10 3 ~10 12 , 10 4 ~10 12 , 10 5 ~10 12 , 10 6 ~10 12 , 10 7 ~10 12 , 10 8 ~10 12 , 10 9 ~10 12 , 10 10 ~10 12 , 10 11 ~10 12 , 10 2 ~10 11 , 10 3 ~10 11 , 10 4 ~10 11 , 10 5 ~10 11 , 10 6 ~10 11 , 10 7 ~10 11 , 10 8 ~10 11 , 10 9 ~10 11 , 10 10 ~10 11 , 10 2 ~10 10 , 10 3 ~10 10 , 10 4 ~10 10 , 10 5 ~10 10 , 10 6 ~1010 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9 、10 5 ~10 9 、10 6 ~10 9 、10 7 ~10 9 、10 8 ~10 9 、10 2 ~10 8 、10 3 ~10 8 、10 4 ~10 8 、10 5 ~10 8 、10 6 ~10 8 、10 7 ~10 8 、10 2 ~10 7 、10 3 ~10 7 、10 4 ~10 7 、10 5 ~10 7 、10 6 ~10 7 、10 2 ~10 6 、10 3 ~10 6 、10 4 ~10 6 、10 5 ~10 6 、10 2 ~10 5 、10 3 ~10 5 、10 4 ~10 5 、10 2 ~10 4 、10 3 ~10 4 、10 2 ~10 3, 10 12 , 10 11 , 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , or 10 2 are administered so that there are a total of microbial cells.

[0242] In some embodiments, the administered dose of the microbial composition is 10 2 ~10 18 , 10 3 ~10 18 , 10 4 ~10 18 , 10 5 ~10 18 , 10 6 ~10 18 , 10 7 ~10 18 , 10 8 ~10 18 , 10 9 ~10 18 , 10 10 ~10 18 , 10 11 ~10 18 , 10 12 ~10 18 , 10 13 ~10 18 , 10 14 ~10 18 , 10 15 ~10 18 , 10 16 ~10 18 , 10 17 ~10 18 , 10 2 ~10 12 , 10 3 ~10 12 , 10 4 ~10 12 , 10 5 ~10 12 , 10 6 ~10 12 , 10 7 ~10 12 , 10 8 ~10 12、10 9 ~10 12 、10 10 ~10 12 、10 11 ~10 12 、10 2 ~10 11 、10 3 ~10 11 、10 4 ~10 11 、10 5 ~10 11 、10 6 ~10 11 、10 7 ~10 11 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9 、10 5 ~10 9 、10 6 ~10 9 、10 7 ~10 9 、10 8 ~10 9 、10 2 ~10 8 、10 3 ~10 8 、10 4 ~10 8 、10 5 ~10 8 、106 ~10 8 , 10 7 ~10 8 , 10 2 ~10 7 , 10 3 ~10 7 , 10 4 ~10 7 , 10 5 ~10 7 , 10 6 ~10 7 , 10 2 ~10 6 , 10 3 ~10 6 , 10 4 ~10 6 , 10 5 ~10 6 , 10 2 ~10 5 , 10 3 ~10 5 , 10 4 ~10 5 , 10 2 ~10 4 , 10 3 ~10 4 , 10 2 ~10 3 , 10 18 , 10 17 , 10 16 , 10 15 , 10 14 , 10 13 , 10 12 , 10 11 , 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , or 10 2 Contains a total of microbial cells.

[0243] In some embodiments, the microbial composition comprises one or more bacteria comprising a 16S nucleic acid sequence selected from SEQ ID NOs: 1-333. In some embodiments, the one or more bacteria are present in an amount of 10 or more per gram or milliliter of the composition. 2 ~10 12、10 3 ~10 12 、10 4 ~10 12 、10 5 ~10 12 、10 6 ~10 12 、10 7 ~10 12 、10 8 ~10 12 、10 9 ~10 12 、10 10 ~10 12 、10 11 ~10 12 、10 2 ~10 11 、10 3 ~10 11 、10 4 ~10 11 、10 5 ~10 11 、10 6 ~10 11 、10 7 ~10 11 、10 8 ~10 11 、10 9 ~10 11 、10 10 ~10 11 、10 2 ~10 10 、10 3 ~10 10 、10 4 ~10 10 、10 5 ~10 10 、10 6 ~10 10 、10 7 ~10 10 、10 8 ~10 10 、10 9 ~10 10 、10 2 ~10 9 、10 3 ~10 9 、10 4 ~10 9 、10 5 ~10 9 、10 6 ~10 9 、107 ~10 9 , 10 8 ~10 9 , 10 2 ~10 8 , 10 3 ~10 8 , 10 4 ~10 8 , 10 5 ~10 8 , 10 6 ~10 8 , 10 7 ~10 8 , 10 2 ~10 7 , 10 3 ~10 7 , 10 4 ~10 7 , 10 5 ~10 7 , 10 6 ~10 7 , 10 2 ~10 6 , 10 3 ~10 6 , 10 4 ~10 6 , 10 5 ~10 6 , 10 2 ~10 5 , 10 3 ~10 5 , 10 4 ~10 5 , 10 2 ~10 4 , 10 3 ~10 4 , 10 2 ~10 3 , 10 12 , 10 11 , 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 In some embodiments, the one or more bacteria are present in the microbial composition at a concentration of at least 10 cells per gram or per milliliter of the composition.2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 12 , 10 11 , 10 13 , 10 14 , 10 15 It exists at a concentration of cells.

[0244] In some embodiments, the composition is administered one or more times daily, hi some aspects, the composition is administered with food each time the animal is fed. In some embodiments, the composition is administered 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, 9-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day. In some embodiments, the composition is administered once per day. In some embodiments, the composition is administered twice per day.

[0245] In some embodiments, the microbial composition is administered 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, 9-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week.

[0246] In some embodiments, the microbial composition is administered 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, 9-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

[0247] In some embodiments, the microbial composition is administered 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, 9-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per year.

[0248] In some embodiments, the microbial composition is administered to the animal throughout its lifetime. In some embodiments, the microbial composition is administered only to young animals. In some embodiments, the microbial composition is administered only to adult animals. In some embodiments, the microbial composition is administered only to older animals. In some aspects, the microbial composition is administered to the animal during the first 5%, 10%, 15%, 20%, or 25% of the animal's expected lifespan. In some aspects, the microbial composition is administered to the animal during the last 5%, 10%, 15%, 20%, or 25% of the animal's expected lifespan.

[0249] In some embodiments, the microbial compositions are administered chronically to animals to improve their overall health. In some embodiments, the microbial compositions are administered once daily, twice daily, three times daily, once weekly, twice weekly, three times weekly, once every two weeks, or once monthly throughout the lifespan of the animal to improve its overall health. In some embodiments, long-term administration using the microbial compositions described herein improves one or more traits in dogs. In some embodiments, long-term administration using the microbial compositions described herein reduces diarrhea, improves stool consistency, reduces the incidence of infectious or non-infectious diseases, extends lifespan, and / or improves animal performance. In some embodiments, long-term administration using the microbial compositions described herein improves gastrointestinal health, reduces inflammation, and / or stabilizes the gastrointestinal microbiome.

[0250] In some embodiments, the microbial composition is acutely administered to an animal to improve its overall health. In some embodiments, the microbial composition is administered once daily, twice daily, three times daily, once weekly, twice weekly, three times weekly, once every two weeks, or once monthly for a period of one month, two months, three months, six months, or twelve months to improve the animal's overall health. In some embodiments, the microbial composition is administered once daily for a period of one month. In some embodiments, acute administration using the microbial compositions described herein improves one or more traits in dogs. In some embodiments, acute administration using the microbial compositions described herein reduces diarrhea, reduces dysbiosis, reduces enteric disease, reduces the incidence of infectious or non-infectious diseases, extends lifespan, and / or improves animal performance. In some embodiments, acute administration using the microbial compositions described herein improves gastrointestinal health, reduces inflammation, and / or stabilizes the gastrointestinal microbiome.

[0251] In some embodiments, the type of diet fed to an animal corresponds to the type of microbial composition administered to the animal. In some embodiments, an animal exhibiting GI dysbiosis or GI enteropathy receives a first microbial composition. In some embodiments, the animal receiving the first microbial composition is administered a second microbial composition (different from the first) to reduce the severity of GI dysbiosis or GI enteropathy. In some embodiments, the animal administered the first microbial composition is administered a third microbial composition (different from the first and second) to reduce the severity of GI dysbiosis or GI enteropathy. In some embodiments, the animal administered at least a first, at least a second, or at least a third microbial composition is administered a subsequent microbial composition (different from the previously administered microbial composition) to maintain a healthy microbiota state in the gastrointestinal tract.

[0252] In some embodiments, an animal exhibiting GI dysbiosis or GI enteropathy receives a first microbial composition. In further embodiments, the same animal, fed a different diet, receives a second microbial composition, where the first microbial composition is different from the second microbial composition. In some embodiments, the same animal, further fed a different diet, receives a third microbial composition, where the first microbial composition is different from the second and third microbial compositions. In some embodiments, the same animal, further fed a different diet, receives a fourth microbial composition, where the first microbial composition is different from the second, third, and fourth microbial compositions. In some embodiments, the same animal, further fed a different diet, receives a fifth microbial composition, where the first microbial composition is different from the second, third, fourth, and fifth microbial compositions.

[0253] In some embodiments, feed can be uniformly coated with one or more layers of the microorganisms and / or microbial compositions disclosed herein using conventional methods of mixing, spraying, or combinations thereof through the use of treatment application equipment specifically designed and manufactured to apply the coating accurately, safely, and efficiently. Such equipment uses various types of coating techniques, such as rotary coaters, drum coaters, fluidized bed technology, spouted beds, rotary mist, or combinations thereof. Liquid treatments, such as those disclosed herein, can be applied either through a rotating "atomizer" disk or spray nozzle, which evenly distributes the microbial composition on the feed as it moves through the spray pattern. In some aspects, the feed is then further mixed or tumbled for a period of time to achieve additional treatment distribution and drying.

[0254] In some embodiments, the coating of the feed of the present disclosure has a thickness of up to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 90μm, 400μm, 410μm, 420μm, 430μm, 440μm, 450μm, 460μm, 470μm, 480μm, 490 μm, 500μm, 510μm, 520μm, 530μm, 540μm, 550μm, 560μm, 570μm, 580μm, 590μm, 600μm, 610μm, 620μm, 630μm, 640μm, 650μm, 660μm, 670μm, 680μm, 690μm, 70 0μm, 710μm, 720μm, 730μm, 740μm, 750μm, 760μm, 770μm, 780μm, 790μm, 800μm , 810μm, 820μm, 830μm, 840μm, 850μm, 860μm, 870μm, 880μm, 890μm, 900μm, 9 10μm, 920μm, 930μm, 940μm, 950μm, 960μm, 970μm, 980μm, 990μm, 1000μm, 101 0μm, 1020μm, 1030μm, 1040μm, 1050μm, 1060μm, 1070μm, 1080μm, 1090μm, 11 00μm, 1110μm, 1120μm, 1130μm, 1140μm, 1150μm, 1160μm, 1170μm, 1180μm, 11 90μm, 1200μm, 1210μm, 1220μm, 1230μm, 1240μm, 1250μm, 1260μm, 1270μm, 1 280μm, 1290μm, 1300μm, 1310μm, 1320μm, 1330μm, 1340μm, 1350μm, 1360μm, 1 370μm, 1380μm, 1390μm, 1400μm, 1410μm, 1420μm, 1430μm, 1440μm, 1450μm, 1460μm, 1470μm, 1480μm, 1490μm, 1500μm, 1510μm, 1520μm, 1530μm, 1540μm,1550μm, 1560μm, 1570μm, 1580μm, 1590μm, 1600μm, 1610μm, 1620μm, 1630μm, 1640μm, 1650μm, 1660μm, 1670μm, 1680μm, 1690μm, 1700μm, 1710μm, 1720μm ,1730μm,1740μm,1750μm,1760μm,1770μm,1780μm,1790μm,1800μm,1810μm,1820μm,1830μm,1840μm,1850μm,1860μm,1870μm,1880μm,1890μm,1900μm m, 1910μm, 1920μm, 1930μm, 1940μm, 1950μm, 1960μm, 1970μm, 1980μm, 1990μm, 2000μm, 2010μm, 2020μm, 2030μm, 2040μm, 2050μm, 2060μm, 2070μm, 2080 μm, 2090μm, 2100μm, 2110μm, 2120μm, 2130μm, 2140μm, 2150μm, 2160μm, 2170μm, 2180μm, 2190μm, 2200μm, 2210μm, 2220μm, 2230μm, 2240μm, 2250μm, 226 0μm, 2270μm, 2280μm, 2290μm, 2300μm, 2310μm, 2320μm, 2330μm, 2340μm, 2350μm, 2360μm, 2370μm, 2380μm, 2390μm, 2400μm, 2410μm, 2420μm, 2430μm, 24 40μm, 2450μm, 2460μm, 2470μm, 2480μm, 2490μm, 2500μm, 2510μm, 2520μm, 2530μm, 2540μm, 2550μm, 2560μm, 2570μm, 2580μm, 2590μm, 2600μm, 2610μm, 2 620μm, 2630μm, 2640μm, 2650μm, 2660μm, 2670μm, 2680μm, 2690μm, 2700μm, 2710μm, 2720μm, 2730μm, 2740μm, 2750μm, 2760μm, 2770μm, 2780μm, 2790μm, 2800μm, 2810μm, 2820μm, 2830μm, 2840μm, 2850μm, 2860μm, 2870μm, 2880μm, 2890μm, 2900μm, 2910μm, 2920μm, 2930μm, 2940μm, 2950μm, 2960μm, 2970μm,The thickness is 2980 μm, 2990 μm, or 3000 μm.

[0255] In some embodiments, the microbial cells can be freely coated onto any number of compositions, or they can be formulated into a liquid or solid composition before being coated onto the composition. For example, a solid composition containing a microorganism can be prepared by mixing a solid carrier with a spore suspension until the solid carrier is impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

[0256] In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents such as, for example, activated carbon, minerals, vitamins, prebiotics, oligosaccharides, fiber, and other agents that can improve product quality or combinations thereof.

[0257] The coating method and composition of the above-mentioned method known in the art can be particularly useful when they are modified by adding one of the embodiments of the present disclosure.Such coating method and apparatus for their application are disclosed in, for example, U.S. Patent Nos. 8,097,245 and 7,998,502, and PCT Patent Publication Nos. WO2008 / 076975, WO2010 / 138522, WO2011 / 094469, WO2010 / 111347 and WO2010 / 111565, each of which is incorporated herein by reference.

[0258] In some embodiments, the microorganisms or microbial compositions of the present disclosure exhibit a synergistic effect on one or more of the traits described herein when in contact with one or more of the microorganisms or microbial compositions. The synergistic effect obtained by the methods taught can be quantified, for example, according to Colby's formula (i.e., (E) = X + Y - (X * Y / 100)). See Colby, RS, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations," 1967. Weeds. Vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, "synergistic" is intended to reflect an increased result / parameter / effect beyond additive amounts.

[0259] In some embodiments, the microorganisms or microbial compositions of the present disclosure may be administered via a drench. In one embodiment, the drench is an oral drench. Drench administration involves utilizing a drench kit / applicator / syringe that injects / releases a liquid containing the microorganism or microbial composition into the buccal cavity and / or esophagus of the animal.

[0260] In some embodiments, the microorganisms or microbial compositions of the present disclosure may be administered in a time-release manner. The composition may be coated with a chemical composition or contained in a mechanical device or capsule that instead releases the microorganism or microbial composition once over a period of time. In one embodiment, the microorganism or microbial composition is administered to the animal in a time-release capsule. In one embodiment, the composition may be coated with a chemical composition or contained in a mechanical device or capsule that releases the microorganism or microbial composition once over a period of several hours after ingestion.

[0261] In some embodiments, the microorganism or microbial composition is administered in a sustained release manner over 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, or 1 to 100 hours.

[0262] In some embodiments, the microorganism or microbial composition is administered in a sustained release manner for 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, or 1 to 30 days.

[0263] In some embodiments, compositions of the present disclosure may use a variety of formulations for administration to dogs, including formulations such as tablets, compressed tablets, pills, powders, granules, solutions, suspensions, emulsions, elixirs, lotions, creams, gels, ointments, tinctures, pastes, foams, aerosols, washes, sprays, suppositories, or dressings. The form of the resulting formulation will depend on many factors, including the intended method of administration (e.g., oral administration, enteral administration, parenteral administration, and topical administration to the skin, nasal cavity, or buccal cavity).

[0264] In some embodiments, oral administration of the microorganism or microbial composition may be in the form of a solid or liquid composition. Solid dosage forms may be tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets, which may be enteric coated, sugar coated, or film coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be combined with other ingredients known to those skilled in the art and provided in non-effervescent or effervescent form. In other embodiments, oral dosage forms include osmotic-controlled release oral delivery systems (OROS). In other embodiments, the oral dosage form may comprise a matrix-implanted dosage form or related device. In some embodiments, the oral dosage form of the present invention may comprise an orally disintegrating tablet. Pharmaceutically acceptable carriers utilized in tablets include binders, lubricants, diluents, disintegrants, colorants, flavoring agents, and wetting agents.

[0265] Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions, and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars, such as sucrose, and may contain preservatives. Emulsions are two-phase systems (typically oil-in-water or water-in-oil) in which one liquid is dispersed in the form of small globules throughout another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers, and preservatives. Suspensions can use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules to be reconstituted into liquid oral dosage forms include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form, may include organic acids and a source of carbon dioxide. Coloring and flavoring agents can be used in all of the above dosage forms.

[0266] microorganisms As used herein, the term "microorganism" should be interpreted broadly and includes, but is not limited to, the two prokaryotic domains, bacteria and archaea, as well as the eukaryotic fungi, protozoa, and viruses.

[0267] By way of example, microorganisms can include species of the following genera: Prevotella, Megamonas, Ruminococcus, Clostridium, Clostridium sensu stricto, Lachnospiraceae, Anaerobiospirillum, Catenibacterium, Eubacterium, Holdemanella, Clostridium. XI, Allobaculum, Morganella, Acidicaldus, Parasutterella, Collinsella, Blautia, Bacteroides, Bacillus, Lactonifactor, Brevundimonas, Dialister, Actinomyces, Coprococcus, Cellulsilyticum, Acetanaerobacterium, Faecalibacterium, Murimonas, Clostridium XIVa, Parabacteroides, Cetobacterium, Clostridium XVIII, Odoribacter, Terrisporobacter, Turicibacter, Fusinateibacter, Kandleria, Butyricicoccus, Veillonella, Acinetobacter, Enterococcus, P araprevotella, Thermaerobacter, Bulleidia, Aerococcus, Robinoniella, Erysipelotrichaceae, Streptosporangium, Bifidobacterium, Clostridium III, Pediococcus, Fusobacterium, Glautia, Sarcina, Jeotgalibaca, and Megasphaera.

[0268] In certain embodiments, the microorganism is non-culturable, which should be taken to mean that the microorganism is not known to be culturable or is difficult to cultivate using methods known to those of skill in the art.

[0269] In one embodiment, the microorganisms are obtained from animals (e.g., mammals, reptiles, birds, etc.), soil (e.g., rhizosphere), air, water (e.g., marine, freshwater, wastewater sludge), sediment, oil, plants (e.g., roots, leaves, stems), agricultural products, and extreme environments (e.g., acid mine drainage or hydrothermal systems). In a further embodiment, the microorganisms are obtained from a marine or freshwater environment, such as an ocean, river, or lake. In a further embodiment, the microorganisms can be from the surface of a body of water, or from any depth within a body of water (e.g., a deep-sea sample).

[0270] The microorganisms of the present disclosure can be isolated in substantially pure cultures or mixed cultures. They can be concentrated, diluted, or provided at the natural concentrations found in the source material. For example, microorganisms from saline sediments can be isolated for use in the present disclosure by suspending the sediments in fresh water and allowing the sediments to settle to the bottom. The water containing most of the microorganisms can be removed by decantation after a suitable settling period and administered to the GI tract of a dog, or it can be concentrated by filtration or centrifugation, diluted to a suitable concentration, and administered to the GI tract of a dog with most of the salt removed. As a further example, microorganisms from mineralized or toxic sources can be similarly processed to recover microorganisms for application to dogs to minimize potential injury to the animal.

[0271] In another embodiment, the microorganism is used in crude form, not isolated from the source material in which the microorganism naturally occurs.For example, the microorganism is provided in combination with the source material in which it exists, such as fecal matter, other compositions found in the gastrointestinal tract.In this embodiment, the source material can contain one or more microbial species.

[0272] In some embodiments, a mixed population of microorganisms is used in the methods of the present disclosure.

[0273] In embodiments of the present disclosure in which microorganisms are isolated from source materials (e.g., materials in which they occur naturally), any one or combination of several standard techniques that would be readily known to one of skill in the art can be used. However, by way of example, these generally employ processes by which solid or liquid cultures of a single microorganism can be obtained in substantially pure form, usually by physical separation on the surface of a solid microbial growth medium or by volumetric dilution isolation into a liquid microbial medium. These processes may include isolation from dry material, liquid suspensions, slurries, or homogenates, in which the material is spread in a thin layer on an appropriate solid gel growth medium, or serial dilutions of the material made into a sterile medium and inoculated into a liquid or solid culture medium.

[0274] Although not required, in one embodiment, the material containing the microorganisms may be pretreated prior to the isolation process to grow all microorganisms in the material, remove specific microorganisms in the material, and / or shift the distribution of microorganisms in the material. The microorganisms can then be isolated from the enriched material as disclosed above.

[0275] In certain embodiments, as described herein above, the microorganisms can be used in crude form and do not need to be isolated from the animal or culture medium. For example, feces or growth medium containing microorganisms identified as beneficial for reducing GI dysbiosis can be obtained and used as a crude source of microorganisms for the next round of the method or at the end of the method. For example, fresh feces can be obtained and optionally processed.

[0276] Microbiome shifts and microbial abundance In some embodiments, the canine microbiome, including the gastrointestinal microbiome, comprises a diverse array of microorganisms with a wide range of metabolic capabilities. The microbiome is influenced by various factors, such as diet, changes in the animal's metabolism, and breeding. The end products of primary decomposition ultimately support a range of microorganisms that produce various organic acids along with hydrogen and carbon dioxide. Due to the complex and interconnected nature of the microbiome, changing the diet and thus the substrates of primary decomposition can alter both organic acid profiles, potentially having a cascading effect on the metabolism of intestinal microorganisms.

[0277] In some aspects, the present disclosure is directed to administering the microbial compositions described herein to modulate or shift the microbiome of a dog. Additional methods of administering microbial compositions to modulate the microbiome are described in International PCT Application Publication No. WO2018 / 218211, the entire contents of which are incorporated herein by reference.

[0278] In some embodiments, the microbiome is shifted through administration of one or more microorganisms to one or more sections of the gastrointestinal tract. In some embodiments, the microbiome is shifted through administration of one or more microorganisms to the gastrointestinal tract. In further embodiments, the one or more microorganisms are microorganisms selected from Table 1. In some embodiments, the microbiome shift or modulation involves a reduction or loss of specific microorganisms that were present prior to administration of one or more microorganisms of the present disclosure. In some embodiments, the microbiome shift or modulation involves an increase in microorganisms that were present prior to administration of one or more microorganisms of the present disclosure. In some embodiments, the microbiome shift or modulation involves the gain of one or more microorganisms that were not present prior to administration of one or more microorganisms of the present disclosure. In further embodiments, the gain of one or more microorganisms is a microorganism that was not specifically included in the administered microbial composition.

[0279] In some embodiments, administration of a microorganism of the present disclosure results in sustained modulation of the microbiome such that the administered microorganism is present in the microbiome for at least 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, 9-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0280] In some embodiments, administration of a microorganism of the present disclosure results in sustained modulation of the microbiome such that the administered microorganism is present in the microbiome for at least 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, 9-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

[0281] In some embodiments, administration of a microorganism of the present disclosure provides that the administered microorganism is at least 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8 , 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, 9-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months of exposure to the microbiome, resulting in sustained modulation of the microbiome.

[0282] In some embodiments, the presence of the administered microorganism is detected by sampling the gastrointestinal tract and using primers to amplify the 16S or 18S rDNA sequence, or the ITS rDNA sequence, of the administered microorganism. In some embodiments, the administered microorganism is one or more of the microorganisms selected from Table 1. In some embodiments, the administered microorganism is one or more of the microorganisms comprising an rDNA sequence selected from SEQ ID NOs: 1-331.

[0283] In some embodiments, a dog's microbiome is measured by amplifying polynucleotides collected from gastrointestinal samples, where the polynucleotides can be 16S or 18S rDNA fragments or ITS rDNA fragments of microbial rDNA. In one embodiment, the microbiome is fingerprinted by denaturing gradient gel electrophoresis (DGGE), where amplified rDNA fragments are sorted by where they denature, forming unique banding patterns on the gel that can be used to compare the microbiomes of the same dog over time or multiple microbiomes. In another embodiment, the microbiome is fingerprinted by terminal restriction fragment length polymorphism (T-RFLP), where labeled PCR fragments are digested using restriction enzymes and then sorted by size. In a further embodiment, data collected from the T-RFLP method is evaluated by non-metric multidimensional scaling (nMDS) ordinations and PERMANOVA statistics to identify differences in the microbiome and allow for the identification and measurement of shifts in the microbiome. See Coelho et al. (2018. BMC Microbiome, 6:12).

[0284] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome, increasing the number and / or type of microorganisms belonging to one or more of the following taxa: Bacteroidales, Selenomonadales, Clostridiales, Aeromonadales, Erysipelotrichales, Enterobacteriales, Rhodospirillales, Burkholderiales, Coriobacteriales, Bacillales, Caulobacterales, Actinomycetales, Fusobacteriales, Pseudomonadales, Lactobacillales, Bifidobacteriales, Prevotellaceae ae, Veillonellaceae, Lachnospiraceae, Clostridiaceae, Succinivibrionaceae, Erysipelotrichaceae, Eubacteriaceae, Peptostreptococcaceae, Enterobacteriaceae, Acetobacteraceae, Sutterellacea e, Coriobacteriaceae, Bacteroidaceae, Bacillaceae, Caulobacteraceae, Actinomycetacea, Ruminococcaceae, Porphyromonadaceae, Fusobacteriaceae, Moraxellaceae, Carnobacteriaceae, Clostridiales XVII, Aerococcaceae, Streptosporangiaceae, Bifidobacteriaceae, Lactobacillaceae, Prevotella, Megamonas, Ruminococcus, Clostridium, Clostridium sensu stricto, Lacnospiracea, Anaerobiospirillum, Catenibacterium, Eubacterium, Holdemanella, ClostridiumXI, Allobaculum, Morganella, Acidicaldus, Parasutterella, Collinsella, Blautia, Bacteroides, Bacillus, Lactonifactor, Brevundimonas, Dialister, Actinomyces, Coprococcus, Ruminococcus, Cellulosilyticum, Acetanaerobacterium, Faecalibacterium, Murimonas, Clostridium XIVa, Parabacteroides, Cetobacterium, Clostridium XVIII, Odoribacter, Terrisporobacter, Turicibacter, Fusicatenibacter, Kandleria, Butyricicoccus, Beillonella, Acinetobacter, Enterococcus, Paraprevotella, Thermaerobacter, Bulleidia, Aerococcus, Robinsoniella, Erysipelotrichaceae, Streptosporangium, Bifidobacterium, Clostridium III, Pediococcus, and Megasphaera.

[0285] In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of microorganisms belonging to one or more taxa disclosed herein by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of microorganisms belonging to one or more taxa disclosed herein by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0286] In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the number and / or type of microorganisms belonging to one or more taxa disclosed herein. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the number and / or type of microorganisms belonging to one or more taxa disclosed herein by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the number and / or type of microorganisms belonging to one or more taxa disclosed herein by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0287] In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the number and / or type of microorganisms belonging to one or more taxa disclosed herein. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the number and / or type of microorganisms belonging to one or more taxa disclosed herein by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the number and / or type of microorganisms belonging to one or more taxa disclosed herein by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0288] In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of microorganisms belonging to one or more taxa disclosed herein. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of microorganisms belonging to one or more taxa disclosed herein by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of microorganisms belonging to one or more taxa disclosed herein by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0289] In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of volatile fatty acid (VFA)-producing microorganisms. In some embodiments, VFAs include acetic acid, butyric acid, propionic acid, isobutyric acid, isovaleric acid, and valeric acid. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of VFA-producing microorganisms by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that increases the number and / or type of VFA-producing microorganisms by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0290] In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of microorganisms utilized as a protein source for the animal. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of microorganisms utilized as a protein source for the animal by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of microorganisms utilized as a protein source in an animal by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0291] In some embodiments, administration of one or more microbial compositions to a dog results in a shift in the microbiome in which the number, type, and / or relative abundance of microorganisms in the GI tract increases, but the increased number, type, and / or relative abundance of microorganisms is not present in the dog's hindgut.

[0292] In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of vitamin-synthesizing microorganisms. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of vitamin-synthesizing microorganisms by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that increases the number and / or type of vitamin-synthesizing microorganisms by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0293] In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the overall alpha diversity of the microbial community. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the overall alpha diversity of the microbial community by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial compositions results in a microbiome shift that reduces the overall alpha diversity of the microbial community by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0294] In some embodiments, administration of a microorganism of the present disclosure results in a modulation or shift in the microbiome, which in turn results in a desired phenotype or improved trait.

[0295] Diversity of canine microbial composition Dogs have been found to exhibit high inter-animal variability in GI tract microbial diversity. Increased variability in GI tract microbial composition may lead to a reduced ability to achieve a stable microbial composition. Low variability, in turn, leads to significant differences in health, weight, and other characteristics that affect animal survival. See Shabat SKB et al. (ISME J 10:2958-2972).

[0296] In some embodiments, administration of one or more microorganisms and / or microbial compositions of the present disclosure reduces the variability of the gut microbiome in dogs and further establishes a stable canine microbiome.

[0297] In some embodiments, gut microbiome variability is measured as the total number of species present in the gastrointestinal tract at one or more locations.

[0298] In some embodiments, administration of one or more microorganisms and / or microbial compositions of the present disclosure reduces the time required for the gastrointestinal microbiome to reach a steady state.

[0299] In some embodiments, administration of one or more microorganisms and / or microbial compositions of the present disclosure results in the dog of the present disclosure reaching a steady state of the gastrointestinal microbiome and reducing variability in the GI microbiome.

[0300] In some embodiments, the GI microbiome of a dog is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1000, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2500, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 90 ... A steady state of the GI microbiome is reached when the microbiome contains about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, or about 10,000 different species.

[0301] In some embodiments, a stable state of the canine intestinal microbiome is reached when the canine GI microbiome contains about 10 to about 50, about 10 to about 100, about 50 to about 100, about 50 to about 200, about 100 to about 150, about 100 to about 200, about 100 to about 400, about 200 to about 500, about 200 to about 700, about 400 to about 800, about 500 to about 1,000, about 500 to about 2,000, about 1,000 to about 2,000, about 1,000 to about 5,000, about 5,000 to about 7,000, about 5,000 to about 10,000, or about 8,000 to about 10,000 different species.

[0302] In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of feed-transitioning dogs reach a steady state after administration of one or more microorganisms and / or organism ensembles of the present disclosure.

[0303] MIC scoring According to the methods provided herein, a sample is processed to detect the presence of one or more microbial types in the sample (FIG. 1, 1001; FIG. 2, 2001). The absolute number of one or more microbial types in the sample is determined (FIG. 1, 1002; FIG. 2, 2002). The determination of the presence of one or more organism types and the absolute number of at least one organism type can be performed in parallel or sequentially. For example, in the case of a sample containing a microbial community including bacteria (i.e., one microbial type) and fungi (i.e., a second microbial type), the user in one embodiment detects the presence of one or both organism types in the sample (FIG. 1, 1001; FIG. 2, 2001). In a further embodiment, the user determines the absolute number of at least one organism type in the sample. In this example, the number of bacteria, fungi, or a combination thereof in the sample (FIG. 1, 1002; FIG. 2, 2002).

[0304] In one embodiment, the sample, or a portion thereof, is subjected to flow cytometry (FC) analysis to detect the presence and / or number of one or more microbial types (FIG. 1, 1001, 1002; FIG. 2, 2001, 2002). In one flow cytometer embodiment, individual microbial cells are detected in at least about 300 s -1 , or at least about 500*s -1 , or at least about 1000*s -1 The cells pass through the illumination zone at a velocity of 0.05 .mu.m. However, one skilled in the art will recognize that this velocity may vary depending on the type of instrument used. An electronically gated detector measures the magnitude of the pulses, which represent the degree of scattered light. These pulse magnitudes are electronically sorted into "bins" or "channels," allowing for the display of a histogram of the number of cells having a particular quantitative property (e.g., cell staining property, diameter, cell membrane) versus channel number. Such analysis allows for the determination of the number of cells in each "bin," which in the embodiments described herein are "microorganism-type" bins, such as bacteria, fungi, nematodes, protozoa, archaea, algae, dinoflagellates, viruses, viroids, etc.

[0305] In one embodiment, a sample is stained with one or more fluorescent dyes, which are specific to a particular microbial type and can be detected via a flow cytometer or some other detection and quantification method that utilizes fluorescence, such as fluorescence microscopy. The method can provide quantification of cell number and / or cell volume of a given organism type in a sample. In a further embodiment, as described herein, flow cytometry is utilized to determine the presence and amount of a specific first marker and / or a specific second marker of the organism type, such as enzyme expression, cell surface protein expression, etc. For example, two- or three-variable histograms or contour plots of light scatter versus fluorescence from a cell membrane stain (versus fluorescence from a protein or DNA stain) can also be generated, thus providing an impression of the distribution of various properties of interest among cells throughout a population. Many such displays of multiparameter flow cytometry data are commonly used and suitable for use with the methods described herein.

[0306] In one embodiment of processing a sample to detect the presence and number of one or more microbial types, a microscopic assay is employed (FIG. 1, 1001, 1002). In one embodiment, the microscope is an optical microscope, which uses visible light and a lens system to magnify an image of a small sample. Digital images can be captured by a charge-coupled device (CCD) camera. Other microscopic techniques include, but are not limited to, scanning electron microscopy and transmission electron microscopy. Microbial types are visualized and quantified according to the aspects provided herein.

[0307] In another embodiment of the present disclosure, each sample, or a portion thereof, is subjected to fluorescence microscopy to detect the presence and number of one or more microbial types. Various fluorescent dyes can be used to directly stain cells in the sample, and total cell numbers can be quantified using epifluorescence microscopy, as well as flow cytometry as described above. Useful dyes for quantifying microorganisms include, but are not limited to, acridine orange (AO), 4,6-diamino-2-phenylindole (DAPI), and 5-cyano-2,3-ditolyltetrazolium chloride (CTC). Viability can be estimated by viability staining methods such as the LIVE / DEAD® Bacterial Viability Kit (Bac-Light™), which contains two nucleic acid stains: the green-fluorescent SYTO 9™ dye, which permeates all membranes, and the red-fluorescent propidium iodide (PI) dye, which permeates cells with damaged membranes. Thus, cells with damaged membranes stain red, while cells with intact membranes stain green. Fluorescence in situ hybridization (FISH) extends epifluorescence microscopy, allowing for the rapid detection and enumeration of specific organisms. FISH uses fluorescently labeled oligonucleotide probes (typically 15–25 base pairs) that specifically bind to biological DNA in a sample, allowing visualization of cells using epifluorescence or confocal laser scanning microscopy (CLSM). Catalytic reporter deposition fluorescence in situ hybridization (CARD-FISH) improves on FISH by using oligonucleotide probes labeled with horseradish peroxidase (HRP) to amplify the signal intensity obtained from the microorganisms under study. FISH can be combined with other techniques to characterize microbial communities. One combination technique is high-affinity peptide nucleic acid (PNA)-FISH, in which probes have an enhanced ability to penetrate the extracellular polymeric substance (EPS) matrix. Another example is LIVE / DEAD-FISH, which combines a cell viability kit with FISH and has been used to evaluate the efficiency of disinfection in drinking water distribution systems.

[0308] In another embodiment, each sample, or a portion thereof, is subjected to Raman microspectroscopy to determine the presence and absolute number of at least one microbial type (Figure 1, 1001-1002; Figure 2, 2001-2002). Raman microspectroscopy is a non-destructive, label-free technique that can detect and measure single-cell Raman spectra (SCRS). A typical SCRS provides a unique biochemical "fingerprint" of a single cell. SCRS contains rich information about the biomolecules present within it, including nucleic acids, proteins, carbohydrates, and lipids, enabling characterization of different cell types, physiological changes, and cellular phenotypes. Raman microscopy examines the scattering of laser light by the chemical bonds of different cellular biomarkers. SCRS is the summation of the spectra of all biomolecules in a single cell and represents the phenotypic profile of the cell. Cell phenotype, as a result of gene expression, usually reflects the genotype. Therefore, under identical growth conditions, different microbial types give distinct SCRSs corresponding to their genotypic differences and can therefore be distinguished by their Raman spectra.

[0309] In yet another embodiment, a sample, or a portion thereof, is centrifuged to determine the presence and number of at least one microbial type (Figure 1, 1001-1002; Figure 2, 2001-2002). This process uses centrifugal force generated by a centrifuge to sediment a heterogeneous mixture. Denser components of the mixture move away from the axis of the centrifuge, while less dense components of the mixture move toward the axis. Centrifugation can fractionate a sample into cytoplasmic, membrane, and extracellular portions. It can also be used to determine the localization information of biological molecules of interest. Furthermore, centrifugation can be used to fractionate total microbial community DNA. Because different prokaryotic communities have different guanine + cytosine (G+C) content in their DNA, density gradient centrifugation based on G+C content is a method for distinguishing between types of organisms and the number of cells associated with each type. This technique generates a fractionated profile of the entire community DNA, showing DNA abundance as a function of G+C content. Total community DNA can be physically separated into highly purified fractions, each representing a different G+C content, which can be analyzed by additional molecular techniques, such as denaturing gradient gel electrophoresis (DGGE) / amplified ribosomal DNA restriction fragment analysis (ARDRA) (see discussion herein), to assess the diversity of the total microbial community and the presence / abundance of one or more microbial types.

[0310] In another embodiment, the sample, or a portion thereof, is stained to determine the presence and number of at least one microbial type (Figure 1, 1001-1002; Figure 2, 2001-2002). Stains and dyes can be used to visualize biological tissues, cells, or organelles within cells. Stains can be used in conjunction with microscopy, flow cytometry, or gel electrophoresis to visualize or mark cells or biological molecules specific to different microbial types. In vivo staining is the process of staining biological tissue, while in vitro staining refers to staining cells or structures removed from their biological environment. Examples of specific staining techniques for use with the methods described herein include, but are not limited to, Gram staining to determine the Gram state of bacteria, endospore staining to identify the presence of endospores, Ziehl-Neelsen staining, hematoxylin and eosin staining for examining thin sections of tissue, Papanicolaou staining for examining cell samples from various body secretions, Periodic Acid-Schiff staining for carbohydrates, Masson's trichrome staining using a three-color staining protocol to distinguish cells from surrounding connective tissue, Romanowsky staining (or common variations including Wright's stain, Jenner's stain, May-Grinwald stain, Leschmann's stain, and Giemsa stain) for examining blood or bone marrow samples, silver staining to reveal protein and DNA, Sudan staining for lipids, and Conklin's staining to detect true endospores.Common biological stains include acridine orange for cell cycle determination, Bismarck brown for acid mucins, carmine for glycogen, carmine alum for nuclei, Coomassie blue for proteins, cresyl violet for acidic components of neuronal cytoplasm, crystal violet for cell walls, DAPI for nuclei, eosin for cytoplasmic material, cell membranes, some extracellular structures and red blood cells, ethidium bromide for DNA, acid fuchsin for collagen, smooth muscle or mitochondria, hematoxylin for nuclei, Hoechst stain for DNA, iodine for starch, malachite green for bacteria and for spores in the Gimenez staining technique, methyl green for chromatin, methylene blue for animal cells, neutral red for Nissl substance, Nile blue for nuclei, Nile red for lipophilic entities, osmium tetroxide for lipids, rhodamine for fluorescence microscopy, and safranin for nuclei. Stains are also used to enhance contrast in transmission electron microscopy and include phosphotungstic acid, osmium tetroxide, ruthenium tetroxide, ammonium molybdate, cadmium iodide, carbohydrazide, ferric chloride, hexamine, indium trichloride, lanthanum nitrate, lead acetate, lead citrate, lead(II) nitrate, periodic acid, phosphomolybdic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate, sodium chloroaurate, thallium nitrate, thiosemicarbazide, uranyl acetate, uranyl nitrate, and vanadyl sulfate.

[0311] In another embodiment, a sample, or a portion thereof, is subjected to mass spectrometry (MS) to determine the presence and number of at least one microbial type (Figure 1, 1001-1002; Figure 2, 2001-2002). As discussed below, MS can also be used to detect the presence and expression of one or more unique markers in a sample (Figure 1, 1003-1004; Figure 2, 2003-2004). MS is used, for example, to detect the presence and quantity of protein and / or peptide markers specific to a microbial type, thus providing an estimate of the number of each microbial type in a sample. Quantification can be performed either with stable isotope labeling or without labeling. De novo sequencing of peptides can also be performed directly from MS / MS spectra or sequence tagging (creating short tags that can be matched against databases). MS can also reveal post-translational modifications of proteins and identify metabolites. MS can be used in conjunction with chromatography and other separation techniques (e.g., gas chromatography, liquid chromatography, capillary electrophoresis, ion mobility, etc.) to improve mass resolution and determination.

[0312] In another embodiment, a sample, or a portion thereof, is subjected to lipid analysis to determine the presence and number of at least one microbial type (Figure 1, 1001-1002; Figure 2, 2001-2002). Fatty acids are present in a relatively constant proportion of cellular biomass, and characteristic fatty acids exist in microbial cells that can distinguish microbial types within a community. In one embodiment, fatty acids are extracted by saponification followed by derivatization to obtain their respective fatty acid methyl esters (FAMEs), which are then analyzed by gas chromatography. The FAME profile of one embodiment is then compared to a reference FAME database, and the fatty acids and corresponding microbial signatures are identified by multivariate statistical analysis.

[0313] In embodiments of the methods provided herein, the number of unique first markers and the abundance of each unique first marker in a sample or portion thereof (e.g., a sample aliquot) are measured (Figure 1, 1003; Figure 2, 2003). The unique markers are markers of the microbial strain. Those skilled in the art will understand that depending on the unique markers probed and measured, the entire sample need not be analyzed. For example, if the unique marker is specific to a bacterial strain, the fungal portion of the sample need not be analyzed. As described above, in some embodiments, measuring the absolute abundance of one or more organism types in a sample includes separating the sample by organism type, for example, via flow cytometry.

[0314] Any marker specific to the organism strain can be employed herein. For example, the marker can include, but is not limited to, the small subunit ribosomal RNA gene (16S / 18S rDNA), the large subunit ribosomal RNA gene (23S / 25S / 28S rDNA), the intercalary 5.8S gene, cytochrome c oxidase, beta tubulin, elongation factor, RNA polymerase, and internal transcribed spacer (ITS).

[0315] Ribosomal RNA genes (rDNA), particularly the small subunit ribosomal RNA gene (18S rDNA) in eukaryotes and 16S rRNA in prokaryotes, are the primary targets for assessing organism types and strains in microbial communities. However, the large subunit ribosomal RNA gene, 28S rDNA, has also been targeted. rDNAs are suitable for taxonomic identification because (i) they are ubiquitous in all known organisms, (ii) they contain both conserved and variable regions, and (iii) there is an exponentially expanding database of their sequences available for comparison. In sample community analysis, the conserved regions serve as annealing sites for corresponding universal PCR and / or sequencing primers, while the variable regions can be used for phylogenetic differentiation. Furthermore, the high copy number of rDNA in cells facilitates its detection in environmental samples.

[0316] The internal transcribed spacer (ITS), located between the 18S and 28S rDNA, is also targeted. The ITS is transcribed but is spliced ​​away before ribosome assembly. The ITS region consists of two highly variable spacers, ITS1 and ITS2, and the intervening 5.8S gene. This rDNA operon occurs in multiple copies in the genome. The ITS region is highly variable because it does not encode ribosomal components.

[0317] In one embodiment, the unique RNA marker may be an mRNA marker, an siRNA marker, or a ribosomal RNA marker.

[0318] Functional genes encoding proteins can be used as unique first markers herein, including, but not limited to, the recombinase A gene family (bacterial RecA, archaeal RadA and RadB, eukaryotic Rad51 and Rad57, and phage UvsX), RNA polymerase subunit (RpoB) genes involved in transcription initiation and elongation, and chaperonins. Candidate marker genes have also been identified for bacteria and archaea: ribosomal protein S2 (rpsB), ribosomal protein S10 (rpsJ), ribosomal protein L1 (rplA), translation elongation factor EF-2, translation initiation factor IF-2, metalloendopeptidase, ribosomal protein L22, ffh signal recognition particle protein, ribosomal protein L4 / L1e (rplD), ribosomal protein L2 (rplB), ribosomal protein S9 (rpsI), ribosomal protein L3 (rplC), phenylalanyl-tRNA synthetase beta subunit, ribosomal protein L14b / L23e (rplN), ribosomal protein S5, ribosomal protein S19 (rpsS), ribosomal protein S7, ribosomal protein L16 / L10E (rplP), ribosomal protein S13 (rpsM), and phenylalanyl-tRNA synthetase beta subunit. Niralanyl-tRNA synthetase α subunit, ribosomal protein L15, ribosomal protein L25 / L23, ribosomal protein L6 (rplF), ribosomal protein L11 (rplK), ribosomal protein L5 (rplE), ribosomal protein S12 / S23, ribosomal protein L29, ribosomal protein S3 (rpsC), ribosomal protein S11 (rpsK), ribosomal protein L10, ribosomal protein S8, tRNA pseudouridine synthase B, ribosomal protein L18P / L5E, ribosomal protein S15P / S13e, porphobilinogen deaminase, ribosomal protein S17, ribosomal protein L13 (rplM), phosphoribosylformylglycine amidine cycloligase (rpsE), ribonuclease HII, and ribosomal protein L24. Other bacterial marker gene candidates include: transcription elongation protein NusA ( nusA );rpoB DNA-dependent RNA polymerase subunit beta (rpoB), GTP-binding protein EngA, rpoC DNA-dependent RNA polymerase subunit beta', priA primosome assembly protein, transcription-repair coupling factor, CTP synthase (pyrG), secY preprotein translocase subunit SecY, GTP-binding protein Obg / CgtA, DNA polymerase I, rpsF 30S ribosomal protein S6, poA DNA-dependent RNA polymerase subunit alpha, peptide chain release factor 1, rplI 50S ribosomal protein L9, polyribonucleotide nucleotidyltransferase, tsf elongation factor Ts(tsf), rplQ 50S ribosomal protein L17, tRNA(guanine-N(1)-)-methyltransferase (rplS), rplY Putative 50S ribosomal protein L25, DNA repair protein RadA, glucose-inhibited fission protein A, ribosome binding factor A, DNA mismatch repair protein MutL, smpB SsrA-binding protein (smpB), N-acetylglucosaminyltransferase, S-adenosyl-methyltransferase MraW, UDP-N-acetylmuramoylalanine-D-glutamate ligase, rplS 50S ribosomal protein L19, rplT 50S ribosomal protein L20 (rplT), ruvA Holliday junction DNA helicase, ruvB Holliday junction DNA helicase B, serS seryl-tRNA synthetase, rplU 50S ribosomal protein L21, rpsR 30S ribosomal protein S18, DNA mismatch repair protein MutS, rpsT 30S ribosomal protein S20, DNA repair protein RecN, frr ribosome recycling factor (frr), recombination protein RecR, protein of unknown function UPF0054, miaA tRNA isopentenyltransferase, GTP-binding protein YchF, chromosome replication initiator protein DnaA, dephospho-CoA kinase, 16S rRNA processing protein RimM, ATP-cone domain protein, 1-deoxy-D-xylulose 5-phosphate reductoisomerase,2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, fatty acid / phospholipid biosynthesis protein PlsX, tRNA(Ile)-lysine synthetase, dnaG DNA primase (dnaG), ruvC Holliday junction resolvase, rpsP 30S ribosomal protein S16, recombinase A recA, riboflavin biosynthesis protein RibF, glycyl-tRNA synthetase beta subunit, trmU tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase, rpmI 50S ribosomal protein L35, hemE uroporphyrinogen decarboxylase, rod shape-determining protein, rpmA 50S ribosomal protein L27 (rpmA), peptidyl-tRNA hydrolase, translation initiation factor IF-3 (infC), UDP-N-acetylmuramyl-tripeptide synthetase, rpmF 50S ribosomal protein L32, rpIL 50S ribosomal protein L7 / L12 (rpIL), leuS leucyl-tRNA synthetase, ligA NAD-dependent DNA ligase, cell division protein FtsA, GTP-binding protein TypA, ATP-dependent Clp protease, ATP-binding subunit ClpX, DNA replication and repair protein RecF, and UDP-N-acetylenolpyruvoylglucosamine reductase.

[0319] Phospholipid fatty acids (PLFAs) can also be used as unique first markers according to the methods described herein. PLFAs are rapidly synthesized during microbial growth, are not found in storage molecules, and degrade rapidly during cell death, providing an accurate census of the current, living community. All cells contain fatty acids (FAs), which can be extracted and esterified to form fatty acid methyl esters (FAMEs). When FAMEs are analyzed using gas chromatography-mass spectrometry, the resulting profile constitutes a "fingerprint" of the microorganisms in the sample. The chemical composition of membranes of organisms in the domains Bacteria and Eukaryotes is composed of fatty acids linked to glycerol by ester bonds (phospholipid fatty acids (PLFAs)). In contrast, the membrane lipids of archaea are composed of long, branched hydrocarbons linked to glycerol by ether bonds (phospholipid ether lipids (PLELs)). This is one of the most widely used non-genetic criteria for distinguishing between the three domains. In this context, phospholipids derived from microbial cell membranes, characterized by a variety of acyl chains, are excellent signature molecules, as such lipid structural diversity can be associated with specific microbial taxa.

[0320] As provided herein, to determine whether an organism strain is active, the expression level of one or more specific second markers, which may be the same as or different from the first marker, is measured (Figure 1, 1004; Figure 2, 2004).Specific first markers are described above.Specific second markers are markers of microbial activity.For example, in one embodiment, the mRNA or protein expression of any of the above-mentioned first markers is considered a specific second marker for the purposes of this disclosure.

[0321] In one embodiment, a microorganism is considered active if the expression level of the second marker exceeds or is at a threshold level (e.g., a control level) (Figure 1, 1005; Figure 2, 2005). In one embodiment, activity is determined by whether the expression level of the second marker changes by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% compared to the threshold level, which in some embodiments is the control level.

[0322] In one embodiment, the second unique marker is measured at the protein, RNA, or metabolite level. The second unique marker is the same as or different from the first unique marker.

[0323] As described above, several unique first markers and unique second markers can be detected according to the methods described herein. Furthermore, detection and quantification of the unique first markers are performed according to methods known to those skilled in the art (Figures 1, 1003-1004; Figures 2, 2003-2004).

[0324] In one embodiment, nucleic acid sequencing (e.g., gDNA, cDNA, rRNA, mRNA) is used to determine the absolute cell number of the specific first marker and / or the specific second marker.Sequencing platforms include, but are not limited to, Sanger sequencing and high-throughput sequencing available from Roche / 454 Life Sciences, Illumina / Solexa, Pacific Biosciences, Ion Torrent and Nanopore.Sequencing methods can be amplicon sequencing of specific DNA or RNA sequences, or whole metagenomic / transcriptome shotgun sequencing.

[0325] Traditional Sanger sequencing (Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl. Acad. Sci. USA, 74, pp. 5463-5467, incorporated herein by reference in its entirety) relies on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerases during in vitro DNA replication and is suitable for use in the methods described herein.

[0326] In another embodiment, the sample, or a part thereof, is subjected to nucleic acid extraction, the amplification of the DNA of interest (such as rRNA gene) with suitable primers, and the construction of a clone library using a sequencing vector.The selected clones are then sequenced by Sanger sequencing to recover the nucleotide sequence of the DNA of interest, allowing the number of unique microbial strains in the sample to be calculated.

[0327] 454 pyrosequencing from Roche / 454 Life Sciences produces long reads and can be used in the methods described herein (Margulies et al. (2005) Nature, 437, pp. 376-380; U.S. Patent Nos. 6,274,320, 6,258,568, and 6,210,891, each of which is incorporated herein in its entirety for all purposes). The nucleic acid to be sequenced (e.g., amplicon or nebulized genomic / metagenomic DNA) has a specific adapter attached to either end by PCR or ligation. The DNA with the adapter is immobilized on small beads suspended in a water-in-oil emulsion (ideally, one bead carries one DNA fragment). An emulsion PCR step is then performed to generate multiple copies of each DNA fragment, resulting in a set of beads, each containing multiple cloned copies of the same DNA fragment. Each bead is then placed into a well of a fiber-optic chip that also contains the enzymes necessary for the sequencing-by-synthesis reaction. The addition of a base (such as A, C, G, or T) triggers the release of pyrophosphate, generating a flash of light that is recorded to deduce the sequence of the DNA fragment in each well. Approximately one million reads can be achieved per run, with reads up to 1,000 bases long. Paired-end sequencing can be performed, which generates pairs of reads, each beginning at one end of a given DNA fragment. Molecular barcodes can be created and placed between the adapter sequence and the sequence of interest in a multiplexed reaction, allowing each sequence to be bioinformatically assigned to a sample.

[0328] Illumina / Solexa sequencing generates average read lengths ranging from approximately 25 base pairs (bp) to approximately 300 bp (Bennett et al. (2005) Pharmacogenomics, 6:373-382; Lange et al. (2014) BMC Genomics, 15, p. 63; Fadrosh et al. (2014) Microbiome, 2, p. 6; Caporaso et al. (2012) ISME J, 6, pp. 1621-1624; Bentley et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature, 456:53-59). This sequencing technology is also a sequencing by synthesis technique, but uses reversible dye terminators and a flow cell with an oligo-attachment region. DNA fragments to be sequenced are washed onto a flow cell filled with specific oligonucleotides that hybridize to the ends of the fragments and have specific adapters on either end. Each fragment is then replicated to create clusters of identical fragments. Reversible dye terminator nucleotides are then washed onto the flow cell and attached for a predetermined period of time. Excess nucleotides are washed away, the flow cell is imaged, and the reversible terminators are removed, making the process repeatable, allowing nucleotides to continue to be added in subsequent cycles. Paired-end reads of 300 bases each can be achieved. The Illumina platform can generate 4 billion paired-end fragments, each with 125 bases per read, in a single run. Barcodes can also be used for sample multiplexing, but with indexing primers.

[0329] The SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) process is a "sequencing-by-ligation" approach that can be used with the methods described herein to detect the presence and abundance of a first marker and / or a second marker (Figure 1, 1003-1004; Figure 2, 2003-2004) (Peckham et al. SOLiD™ Sequencing and 2-Base Encoding. San Diego, CA: American Society of Human Genetics, 2007; Mitra et al. (2013) Analysis of the intestinal microbiota using SOLiD 16S rRNA gene sequencing and SOLiD shotgun sequencing. BMC Genomics, 14(Suppl 5):S16; Mardis (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 9:387-402, each of which is incorporated herein by reference in its entirety. A library of DNA fragments is prepared from the sample to be sequenced and used to prepare a clonal bead population, in which only one type of fragment is present on the surface of each magnetic bead. The fragments attached to the magnetic beads have a universal P1 adapter sequence, so the starting sequence of all fragments is known and identical. Primers hybridize to the P1 adapter sequence within the library template. A set of four fluorescently labeled dibase probes competes for ligation to the sequencing primer. Specificity of the dibase probes is achieved by interrogating every first and second base in each ligation reaction. Multiple cycles of ligation, detection, and cleavage are performed, with the number of cycles determining the final read length. The SOLiD platform can generate up to 3 billion reads per run, with reads up to 75 bases long.Paired-end sequencing is available and can be used here, but the second read of the pair is only 35 bases long. Samples can be multiplexed through a system similar to that used by Illumina, allowing for separate indexing runs.

[0330] The Ion Torrent system, similar to 454 sequencing, is suitable for use with the methods described herein to detect the presence and abundance of a first marker and / or a second marker (Figure 1, 1003-1004; Figure 2, 2003-2004). It uses a plate of microwells containing beads with DNA fragments attached. However, it differs from all other systems in that it detects base incorporation. When a base is added to a growing DNA strand, a proton is liberated, which slightly changes the surrounding pH. pH-sensitive microdetectors associated with the wells on the plate record these changes. By sequentially washing different bases (A, C, G, T) through the wells, the sequence from each well can be inferred. The Ion Proton platform can generate up to 50 million reads per run, with a read length of 200 bases. The Personal Genome Machine platform has a longer read length of 400 bases. Bidirectional sequencing is available. Multiplexing is possible via standard in-line molecular barcode sequencing.

[0331] Pacific Biosciences (PacBio) SMRT sequencing uses a single-molecule, real-time sequencing approach, which, in one embodiment, is used in conjunction with the methods described herein to detect the presence and abundance of a first marker and / or a second marker (Figure 1, 1003-1004; Figure 2, 2003-2004). The PacBio sequencing system does not require an amplification step, setting it apart from other major next-generation sequencing systems. In one embodiment, sequencing is performed on a chip containing many zero-mode waveguide (ZMW) detectors. DNA polymerase attaches to the ZMW detectors, which image the incorporation of phosphorylated, dye-labeled nucleotides in real time as the DNA strands are synthesized. The PacBio system produces very long read lengths (average approximately 4,600 bases) and a very high number of reads per run (approximately 47,000). The typical "paired-end" approach is not used with PacBio. This is because the reads are usually long enough that fragments can be coated multiple times via CCS without being sequenced independently from each end. Multiplexing with PacBio does not involve independent reads, but rather follows the standard "in-line" barcode model.

[0332] In one embodiment, where the first unique marker is an ITS genomic region, automated ribosomal intergenic spacer analysis (ARISA) is used to determine the number and identity of microbial strains in a sample (Figure 1, 1003; Figure 2, 2003) (Ranjard et al. (2003). Environmental Microbiology 5, pp. 1111-1120, incorporated by reference in its entirety for all purposes). ITS regions exhibit significant heterogeneity in both length and nucleotide sequence. The use of fluorescently labeled forward primers and automated DNA sequencers allows for high-resolution and high-throughput separation. The inclusion of an internal standard in each sample allows for accurate determination of common fragment sizes.

[0333] In another embodiment, fragment length polymorphism (RFLP) of PCR-amplified rDNA fragments, known as amplified ribosomal DNA restriction enzyme cleavage analysis (ARDRA), is used to characterize unique first markers and their abundance in samples (Figure 1, 1003; Figure 2, 2003) (For details, see Massol-Deya et al. (1995). Mol. Microb. Ecol. Manual. 3.3.2, pp. 1-18, incorporated herein by reference in its entirety for all purposes). rDNA fragments are generated by PCR using universal primers, digested with restriction enzymes, electrophoresed in agarose or acrylamide gels, and stained with ethidium bromide or silver nitrate.

[0334] One fingerprinting technique used to detect the presence and abundance of unique first markers is single-strand conformation polymorphism (SSCP) (Lee et al. (1996). Appl Environ Microbiol 62, pp. 3112-3120; Scheinert et al. (1996). J. Microbiol. Methods 26, pp. 103-117; Schwieger and Tebbe (1998). Appl. Environ. Microbiol. 64, pp. 4870-4876, each of which is incorporated herein by reference in its entirety). In this technique, DNA fragments, such as PCR products obtained using primers specific to the 16S rRNA gene, are denatured and electrophoresed directly on a non-denaturing gel. Separation is based on differences in the size and folding conformation of single-stranded DNA, which affect electrophoretic mobility. Reannealing of DNA strands during electrophoresis can be prevented by several strategies, including using one phosphorylated primer in PCR followed by specific digestion of the phosphorylated strand with lambda exonuclease, and using one biotinylated primer to perform magnetic separation of one strand after denaturation. To assess the identity of the dominant population in a given microbial composition, in one embodiment, bands can be excised and sequenced, or SSCP patterns can be hybridized with specific probes. Electrophoresis conditions, such as the gel matrix, temperature, and the addition of glycerol to the gel, can affect separation.

[0335] In addition to sequencing-based methods, other methods for quantifying the expression (e.g., gene, protein expression) of a second marker are suitable for use with the methods provided herein for determining the expression levels of one or more second markers (Figure 1, 1004; Figure 2, 2004). For example, linear amplification techniques such as quantitative RT-PCR, microarray analysis, and nucleic acid sequence-based amplification (NASBA) are all suitable for use in the methods described herein and can be performed according to methods known to those of skill in the art.

[0336] In another embodiment, the sample, or a portion thereof, is subjected to quantitative polymerase chain reaction (PCR) to detect the presence and abundance of the first marker and / or the second marker (Figure 1, 1003-1004; Figure 2, 2003-2004). Specific microbial strain activity is measured by reverse transcription of transcribed ribosomal and / or messenger RNA (rRNA and mRNA) into complementary DNA (cDNA) followed by PCR (RT-PCR).

[0337] In another embodiment, the sample, or a portion thereof, is subjected to a PCR-based fingerprinting technique to detect the presence and abundance of the first and / or second markers (Figure 1, 1003-1004; Figure 2, 2003-2004). PCR products can be separated by electrophoresis based on their nucleotide composition. Sequence variation among different DNA molecules affects their melting behavior, so molecules with different sequences stop migrating at different positions within the gel. Thus, the electrophoretic profile can be defined by the positions and relative intensities of different bands or peaks and converted into numerical data for the calculation of diversity indices. Bands can also be excised from the gel and subsequently sequenced to reveal the phylogenetic affiliation of community members. Electrophoretic methods include, but are not limited to, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), single-strand conformation polymorphism analysis (SSCP), restriction fragment length polymorphism analysis (RFLP) or amplified ribosomal DNA restriction cleavage analysis (ARDRA), terminal restriction fragment length polymorphism analysis (T-RFLP), automated ribosomal intergenic spacer analysis (ARISA), random amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF), and Bb-PEG electrophoresis.

[0338] In another embodiment, a sample, or a portion thereof, is subjected to a chip-based platform such as a microarray or microfluidics to determine the abundance of a unique first marker and / or the presence / abundance of a unique second marker (Figure 1, 1003-1004; Figure 2, 2003-2004). PCR products are amplified from the total DNA in the sample and directly hybridized to known molecular probes immobilized on the microarray. After hybridization of fluorescently labeled PCR amplicons to the probes, positive signals are scored using confocal laser scanning microscopy. Microarray technology allows for rapid evaluation of samples in duplicate, which is a major advantage in microbial community analysis. Generally, hybridization signal intensity on the microarray can be directly proportional to the abundance of the target organism. Universal high-density 16S microarrays (e.g., PHYLOCHIP) contain approximately 30,000 probes of the 16S rRNA gene targeting several cultured microbial species and "candidate phyla." These probes target all 121 defined prokaryotic orders, enabling the simultaneous detection of 8,741 bacterial and archaeal species. Another microarray used for profiling microbial communities is the Functional Gene Array (FGA). Unlike PHYLOCHIP, FGA is primarily designed to detect specific bacterial metabolic groups. Therefore, FGA not only reveals community structure but also sheds light on the community's metabolic potential in situ. Because FGA contains probes derived from genes with known biological functions, it is useful for linking microbial community composition to ecosystem function. The FGA, referred to as GEOCHIP, contains >24,000 probes derived from all known metabolic genes involved in various biogeochemical, ecological, and environmental processes, such as ammonia oxidation, methane oxidation, and nitrogen fixation.

[0339] Protein expression assays, in one embodiment, are used in conjunction with the methods described herein to determine the expression level of one or more second markers (FIG. 1, 1004; FIG. 2, 2004). For example, in one embodiment, mass spectrometry or an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA), is utilized to quantify the expression level of one or more specific second markers, where the one or more specific second markers are proteins.

[0340] In one embodiment, the sample, or a portion thereof, is subjected to bromodeoxyuridine (BrdU) incorporation to determine the level of a second specific marker (Figure 1, 1004; Figure 2, 2004). BrdU, a synthetic nucleoside analog of thymidine, can be incorporated into newly synthesized DNA of replicating cells. Antibodies specific for BrdU can then be used to detect the base analog. Thus, BrdU incorporation identifies cells that are actively replicating DNA, which is a measure of microbial activity according to one embodiment of the methods described herein. BrdU incorporation can be used in combination with FISH to provide the identity and activity of target cells.

[0341] In one embodiment, the sample, or a portion thereof, is subjected to microautoradiography (MAR) combined with FISH to determine the level of a second specific marker (Figure 1, 1004; Figure 2, 2004). MAR-FISH is based on the incorporation of a radioactive substrate into cells, the detection of active cells using autoradiography, and the identification of the cells using FISH. Detection and identification of active cells at single-cell resolution is performed microscopically. MAR-FISH provides information on the percentage of total cells, probe-targeted cells, and cells that have incorporated a given radiolabeled substance. This method provides an assessment of the in situ function of target microorganisms and is an effective approach for studying the in vivo physiology of microorganisms. The technique developed for the quantification of cell-specific substrate uptake combined with MAR-FISH is known as quantitative MAR (QMAR).

[0342] In one embodiment, the sample, or a portion thereof, is subjected to stable isotope Raman spectroscopy combined with FISH (Raman FISH) to determine the level of a second specific marker (Figure 1, 1004; Figure 2, 2004). This technique combines stable isotope probing, Raman spectroscopy, and FISH to associate metabolic processes with specific organisms. The rate of stable isotope incorporation by cells affects light scattering, resulting in measurable peak shifts for labeled cellular components, including proteins and mRNA components. Raman spectroscopy can be used to identify whether cells have synthesized compounds including, but not limited to, oils (such as alkanes), lipids (such as triacylglycerols (TAGs)), certain proteins (e.g., hemoproteins, metalloproteins), cytochromes (e.g., P450, cytochrome c), chlorophyll, chromophores (such as light-harvesting pigments carotenoids and rhodopsin), organic polymers (such as polyhydroxyalkanoates (PHAs), polyhydroxybutyrates (PHBs)), hopanoids, steroids, starches, sulfides, sulfates, and secondary metabolites (such as vitamin B12).

[0343] In one embodiment, the sample, or a portion thereof, is subjected to DNA / RNA stable isotope probing (SIP) to determine the level of a second unique marker (Figure 1, 1004; Figure 2, 2004). SIP allows for the determination of microbial diversity associated with a particular metabolic pathway and is commonly applied to the study of microorganisms involved in the utilization of carbon and nitrogen compounds. The substrate of interest is probed with a stable isotope (e.g., 13 C or 15 The substrate is labeled with 1N and added to the sample. Only microorganisms that can metabolize the substrate will incorporate it into their cells. 13 C-DNA and 15 N-DNA can be isolated by density gradient centrifugation and used for metagenomics analysis. Because RNA itself reflects cellular activity, RNA-based SIPs can be a responsive biomarker for use in SIP studies.

[0344] In one embodiment, the sample, or a portion thereof, is subjected to an isotope array to determine the level of a second unique marker (Figure 1, 1004; Figure 2, 2004). The isotope array allows for functional and phylogenetic screening of active microbial communities in a high-throughput manner. This technique uses a combination of SIP to monitor substrate uptake profiles and microarray technology to determine the taxonomic identity of the active microbial community. The sample is then subjected to isotope array analysis to determine the level of the second unique marker that becomes incorporated into the microbial biomass during growth. 14 Incubate with C-labeled substrate. 14 C-labeled rRNA is separated from unlabeled rRNA and then labeled with a fluorescent dye. The fluorescently labeled rRNA is hybridized to a phylogenetic microarray, followed by scanning for radioactive and fluorescent signals. This technique therefore enables the simultaneous study of microbial community composition and the consumption of specific substrates by metabolically active microorganisms in complex microbial communities.

[0345] In one embodiment, the sample, or a portion thereof, is subjected to a metabolomics assay to determine the level of a second specific marker (Figure 1, 1004; Figure 2, 2004). Metabolomics studies the metabolome, which represents the collection of all metabolites, the end products of cellular processes, in a biological cell, tissue, organ, or organism. This method allows specific metabolite profiles to be associated with different microorganisms and can therefore be used to monitor the presence of microorganisms and / or microorganism-mediated processes. Intracellular and extracellular metabolite profiles associated with microbial activity can be obtained using techniques such as gas chromatography-mass spectrometry (GC-MS). Complex mixtures of metabolomic samples can be separated by techniques such as gas chromatography, high-performance liquid chromatography, and capillary electrophoresis. Metabolite detection can be performed by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, ion mobility spectrometry, electrochemical detection (combined with HPLC), and radiolabeling (when combined with thin-layer chromatography).

[0346] According to embodiments described herein, the presence and respective numbers of one or more active microbial strains in a sample are determined (FIG. 1, 1006; FIG. 2, 2006). For example, the strain identity information obtained by assaying the number and presence of first markers is analyzed to determine the occurrence of unique first markers, thereby representing unique microbial strains (e.g., by counting the number of sequence reads in a sequencing assay). In one embodiment, this value can be expressed as a percentage of all sequence reads for the first manufacturer to provide the percentage of unique microbial strains of a particular microbial type. In a further embodiment, this percentage is multiplied by the number of microbial types (obtained in step 1002 or 2002, see FIGS. 1 and 2) to provide the absolute abundance of one or more microbial strains in a sample and a given volume.

[0347] As described above, one or more microbial strains are considered active if the level of expression of the second specific marker is at or above a threshold level, e.g., at least about 5%, at least about 10%, at least about 20%, or at least about 30% higher than the control level.

[0348] In another embodiment of the present disclosure, the method for determining the absolute abundance of one or more microbial strains is determined in multiple samples (see Figure 2, especially 2007). To be classified as active, a microbial strain needs to be active in only one of the samples. The samples may be taken from the same source over multiple time points, or may be from different environmental sources (e.g., different animals).

[0349] In one embodiment, the absolute abundance values ​​in the sample are used to associate one or more active microbial strains with an environmental parameter (Figure 2, 2008). In one embodiment, the environmental parameter is the presence of a second active microbial strain. Associating one or more active microbial strains with an environmental parameter is, in one embodiment, performed by determining the co-occurrence of strains and parameters by correlation or network analysis.

[0350] In one embodiment, determining the co-occurrence of one or more active microbial strains with environmental parameters includes network and / or cluster analysis methods to measure the connectivity of multiple or single strains with environmental parameters in a network, where a network is a collection of two or more samples that share common or similar environmental parameters. In another embodiment, network and / or cluster analysis methods may be applied to determine the co-occurrence of two or more active microbial strains in a sample (Figure 2, 2008). In another embodiment, network analysis includes non-parametric approaches, including mutual information, to establish connectivity between variables. In another embodiment, network analysis includes linkage analysis, modularity analysis, robustness measures, betweenness measures, connectivity measures, transitivity measures, centrality measures, or combinations thereof (Figure 2, 2009). In another embodiment, the cluster analysis method involves building a connectivity model, a subspace model, a distribution model, a density model, or a centroid model, and / or using a crowd detection algorithm such as the Louvain, Bron-Kerbosch, Girvan-Newman, Clauset-Newman-Moore, Pons-Latapy, and Wakita-Tsurumi algorithms (Figure 2, 2010).

[0351] In one embodiment, the cluster analysis method is a heuristic method based on modularity optimization. In a further embodiment, the cluster analysis method is the Louvain method (see, e.g., Blondel et al. (2008) Fast unfolding of communities in large networks. Journal See the methods described in "Statistical Mechanics: Theory and Experiment," Volume 2008, October 2008, which is incorporated herein by reference in its entirety for all purposes.

[0352] In another embodiment, the network analysis comprises predictive modeling of networks through link mining and prediction, population classification, link-based clustering, relationship similarity, or a combination thereof. In another embodiment, the network analysis comprises differential equation-based population modeling. In another embodiment, the network analysis comprises Lotka-Volterra modeling.

[0353] In one embodiment, relating one or more active microbial strains in a sample to environmental parameters (e.g., determining co-occurrence) includes creating a linkage matrix showing the association between the environmental parameters and the microbial strains.

[0354] In one embodiment, the multiple sample data obtained in step 2007 (e.g., across two or more samples that may be collected at two or more time points, where each time point corresponds to an individual sample) is aggregated. In a further embodiment, the cell counts of each of the one or more microbial strains in each sample are stored in an association matrix (which in some embodiments may be an abundance matrix). In one embodiment, the association matrix is ​​used to identify associations between active microbial strains in specific time point samples using a rule mining approach weighted with the association (e.g., abundance) data. In one embodiment, a filter is applied to remove insignificant rules.

[0355] In one embodiment, the absolute abundance of one or more active microbial strains, or two or more active microbial strains, is related to one or more environmental parameters (Figure 2, 2008), for example, via co-occurrence determination. The environmental parameters are selected by the user depending on the sample being analyzed and are not limited by the methods described herein. The environmental parameters can be parameters of the sample itself, such as pH, temperature, or the amount of protein in the sample. Alternatively, the environmental parameters can be parameters that affect changes in the identity of the microbial community (i.e., when the "identity" of a microbial community is characterized by the type of microbial strain and / or the number of specific microbial strains within the community) or parameters that are affected by changes in the identity of the microbial community. In one embodiment, the environmental parameter is the presence, activity, and / or abundance of a second microbial strain within the microbial community that is present in the same sample.

[0356] In some embodiments described herein, the environmental parameters are referred to as metadata parameters.

[0357] Other examples of metadata parameters include, but are not limited to, genetic information (e.g., DNA mutation information) from the host from which the sample was obtained, sample pH, sample temperature, expression of specific proteins or mRNA, nutritional conditions of the surrounding environment / ecosystem (e.g., the level and / or identity of one or more nutrients), susceptibility or resistance to disease, disease onset or progression, susceptibility or resistance of the sample to toxins, efficacy of xenobiotic compounds (pharmaceuticals), biosynthesis of natural products, or combinations thereof.

[0358] For example, according to one embodiment, changes in microbial strain abundance are calculated across multiple samples according to the method of FIG. 2 (i.e., 2001-2007). The strain abundance changes of one or more active strains over time are tallied (e.g., one or more strains initially identified as active according to step 2006), and the direction of the change is noted (i.e., negative values ​​indicate a decrease, positive values ​​indicate an increase). The number of cells over time is represented as a network, with microbial strains representing nodes and abundance-weighted rules representing edges. Markov chains and random walks are utilized to determine connectivity between nodes and define clusters. In one embodiment, the clusters are filtered using metadata to identify clusters associated with desired metadata (FIG. 2, 2008).

[0359] In a further embodiment, the microbial strains are ranked according to importance by integrating cell count changes over time with the strains present in the target cluster, with the largest cell count changes being ranked highest.

[0360] Canine pathogen resistance and elimination In some aspects, the present disclosure is directed to administering one or more microbial compositions described herein to a dog to eliminate pathogenic microorganisms in the gastrointestinal tract. In some embodiments, the present disclosure is directed to administering a microbial composition described herein to further prevent colonization of the gastrointestinal tract by pathogenic microorganisms. In some embodiments, administration of a microbial composition described herein further eliminates pathogens from the dog's coat and respiratory tract and / or prevents pathogen colonization on the coat and in the respiratory tract. In some embodiments, administration of a microbial composition described herein reduces leaky gut / intestinal permeability, histamine levels, lipopolysaccharide (LPS) production, inflammation, bloating, diarrhea, GI dysbiosis, GI enteropathy, bloody diarrhea, and / or the incidence of GI pathogen-induced disease.

[0361] In some embodiments, the microorganisms of the present disclosure colonize the gastrointestinal tract of a dog, which may prevent colonization by pathogenic microorganisms. In some embodiments, the microorganisms of the present disclosure colonize the hindgut of a dog, which may prevent colonization by pathogenic microorganisms.

[0362] In some embodiments, the microbial compositions of the present disclosure comprise one or more microorganisms present in the gastrointestinal tract of a dog at a relative abundance of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01%.

[0363] In some embodiments, after administration of a microbial composition of the present disclosure, one or more microorganisms are present in the gastrointestinal tract of a dog at a relative abundance of at least 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0364] Pathogenic microorganisms in dogs include: Clostridium perfringens, Clostridium botulinum, Salmonella typi, Salmonella typhimurium, Salmonella enterica, Salmonella pullorum, and Erysipelothrix. insidiosa, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Listeria monocytogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Clostridium difficile, Corynebacterium bovis, Mycoplasma sp., Citrobacter sp., Enterobacter sp., Pseudomonas aeruginosa, Pasteurella sp., Bacillus cereus, Bacillus licheniformis, Streptococcus uberis, Staphylococcus aureus, and enteropathogenic, enteroinvasive, or enterohemorrhagic pathogenic strains of Escherichia coli, Staphylococcus aureus, Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Proteus sp., Klebsiella sp., Shigella sp., and Aspergillus sp.

[0365] In some embodiments, the pathogenic microorganism comprises a viral pathogen. In some embodiments, the pathogenic microorganism is pathogenic to both dogs and humans. In some embodiments, the pathogenic microorganism is pathogenic to either dogs or humans.

[0366] In some embodiments, administration of a composition of the present disclosure to a dog modulates the composition of the gastrointestinal microbiome such that the administered microorganisms outcompete microbial pathogens present in the gastrointestinal tract. In some embodiments, administration of a composition of the present disclosure to a dog carrying a microbial pathogen outcompetes the pathogen and eliminates the pathogen in the dog. In some embodiments, administration of a composition of the present disclosure stimulates host immunity and aids in the elimination of the microbial pathogen. In some embodiments, administration of a composition of the present disclosure introduces microorganisms that produce bacteriostatic and / or bactericidal components that reduce or eliminate microbial pathogens in the dog. (U.S. Patent No. 8,345,010).

[0367] In some embodiments, the microorganisms of the present disclosure outcompete pathogens, preventing them from colonizing the GI tract and preventing them from attaching to attachment sites on the GI epithelium or GI mucosa. In some embodiments, the microorganisms of the present disclosure prevent pathogens from colonizing due to the production of antimicrobials that inhibit pathogen growth. In some embodiments, the microorganisms of the present disclosure reduce the inflammatory state of the GI tract. In some embodiments, the microorganisms of the present disclosure reduce the inflammatory state of the epithelium of the GI tract. In some embodiments, the microorganisms of the present disclosure reduce the inflammatory state of subepithelial cells and tissues of the gastrointestinal tract.

[0368] In some embodiments, administration of the microorganisms of the present disclosure results in protection of the mucosal layer in the GI tract. In some embodiments, administration of the microorganisms of the present disclosure results in an increase in mucus produced by the GI tract. In some embodiments, administration of the microorganisms of the present disclosure results in a decrease in the incidence of biochemical degradation of the GI mucosa. In some embodiments, administration of the microorganisms of the present disclosure results in a decrease in the ability of pathogens to penetrate the mucosal layer of the GI tract and localize in GI endothelial cells. In some embodiments, the microorganisms of the present disclosure create a mucosal shield that prevents or reduces the occurrence of pathogens by gaining access to GI endothelial cells.

[0369] In some embodiments, the microorganisms of the present disclosure outcompete one or more canine GI pathogens for binding sites within the GI tract, thereby preventing pathogen colonization or intracellular or intercellular access of the pathogen.

[0370] In some embodiments, the microorganisms of the present disclosure can correct or maintain the typical physiological pH of various subcompartments or sections of the gastrointestinal tract to prevent pH shifts that would favor pathogen colonization. In some embodiments, the microorganisms of the present disclosure include carbon dioxide and / or hydrogen-utilizing microorganisms that contribute to the pH / redox balance of the GI tract. In some embodiments, the microorganisms of the present disclosure include VFA-producing microorganisms that contribute to the pH / redox balance of the GI tract.

[0371] In some embodiments, challenging a dog with a microbial engraftment or microbial pathogen after administering one or more compositions of the present disclosure prevents the microbial engraftment or microbial pathogen from growing to a relative abundance of greater than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01%. In further embodiments, challenging a dog with a microbial engraftment or microbial pathogen after administering one or more compositions of the present disclosure prevents the microbial engraftment or microbial pathogen from establishing in the dog.

[0372] In some embodiments, clearance of microbial engraftment or microbial pathogens occurs in less than 25 days, less than 24 days, less than 23 days, less than 22 days, less than 21 days, less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, or less than 2 days after administration of one or more compositions of the present disclosure.

[0373] In some embodiments, clearance of microbial engraftment or microbial pathogens occurs 1 to 30 days, 1 to 25 days, 1 to 20 days, 1 to 15 days, 1 to 10 days, 1 to 5 days, 5 to 30 days, 5 to 25 days, 5 to 20 days, 5 to 15 days, 5 to 10 days, 10 to 30 days, 10 to 25 days, 10 to 20 days, 10 to 15 days, 15 to 30 days, 15 to 25 days, 15 to 20 days, 20 to 30 days, 20 to 25 days, or 25 to 30 days or more after administration of one or more compositions of the present disclosure.

[0374] Improved traits A diverse population of microorganisms inhabits the gastrointestinal tract of dogs. The enzymatic activity of microorganisms in the gastrointestinal tract is important for breaking down chemicals and molecules in feed into simple sugars and volatile fatty acids. This enzymatic activity is important for extracting energy from feed; more efficient breakdown ultimately provides more energy to the animal. Some of the soluble sugars present in feed are converted to volatile fatty acids, such as butyric acid, propionic acid, and acetic acid. Volatile fatty acids result from the digestion of both fibrous and non-fibrous components of the feed.

[0375] In some embodiments, the present disclosure is directed to administering the microbial compositions described herein to dogs to improve one or more traits through modulation of aspects of body weight, gastrointestinal health, digestive chemistry, feed digestibility, fecal excretion, prevention of pathogenic microbial colonization, and clearance of pathogenic microbial organisms. In some embodiments, the present disclosure is further directed to achieving a reduced incidence of gastrointestinal dysbiosis, a reduced severity of gastrointestinal dysbiosis, a reduced incidence of diarrhea, a reduced severity of diarrhea, a reduced incidence of irritable bowel disease (IBD), a reduced severity of irritable bowel disease, a reduced severity of enteropathy, a reduced incidence of enteropathy, a reduced incidence of gastrointestinal pathogen colonization, a reduced incidence of gastrointestinal pathogen-induced illness, a reduced frequency of gastrointestinal pathogen carriage, a reduced amount of primary bile acids present in the feces, an increased amount of secondary bile acids present in the feces, and / or a combination thereof, by administering the microbial compositions described herein to dogs.

[0376] In some embodiments, administration of the microbial compositions described herein improves at least one trait in dogs. In some embodiments, the at least one improved trait is selected from the group consisting of: a reduced incidence of gastrointestinal dysbiosis, a reduced severity of gastrointestinal dysbiosis, a reduced incidence of diarrhea, a reduced severity of diarrhea, a reduced incidence of irritable bowel disease (IBD), a reduced severity of irritable bowel disease, a reduced severity of enteropathy, a reduced incidence of enteropathy, a reduced incidence of gastrointestinal pathogen colonization, a reduced incidence of gastrointestinal pathogen-induced illness, a reduced frequency of gastrointestinal pathogen carriage, a reduced amount of primary bile acids present in the feces, an increased amount of secondary bile acids present in the feces, an increased production of fatty acids in the GI tract, an increased breakdown of polysaccharides and lignin, fat, starch, and and / or increased protein digestion, increased pH balance, increased vitamin availability, decreased likelihood or incidence of mortality, decreased likelihood or incidence of morbidity, increased production of antimicrobial agents, increased mammalian and / or microbial synthesis of vitamins, decreased alpha diversity of the gastrointestinal microbiome, improved stool consistency, increased regularity of bowel movements, reduced straining during bowel movements, improved oral hygiene, reduced side effects from antibiotics, livelier eyes, increased energy, increased appetite, improved coat and coat quality, increased lifespan and / or combinations thereof, wherein the increase or decrease is determined by comparing the increase or decrease to animals not administered the composition.

[0377] In some embodiments, the number of dysbiosis events in the dog is reduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to animals not administered a composition of the present disclosure.

[0378] In some embodiments, the incidence of GI bloody diarrhea is reduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to animals not administered a composition of the present disclosure.

[0379] In some embodiments, the generation of secondary bile acids is increased by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to animals not administered a composition of the present disclosure. In some aspects, secondary bile acids include deoxycholic acid and lithocholic acid.

[0380] In some embodiments, the occurrence of primary bile acids is reduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to animals not administered a composition of the present disclosure. In some aspects, primary bile acids include cholic acid and chenodeoxycholic acid.

[0381] In some embodiments, dogs administered one or more microorganisms of the present disclosure experience at least a 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% increase in buffering capacity in the GI tract compared to animals not administered a composition of the present disclosure.

[0382] In some embodiments, the incidence of irritable bowel disease is reduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to animals not administered a composition of the present disclosure.

[0383] In some embodiments, microbial pathogen colonization of the GI tract is reduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to animals not administered a composition of the present disclosure.

[0384] In some embodiments, the alpha diversity of microorganisms in the gastrointestinal tract is reduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an animal not administered a composition of the present disclosure.

[0385] In some embodiments, production of antibacterial agents in the gastrointestinal tract is increased by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an animal not administered a composition of the present disclosure.

[0386] In some embodiments, production of the vitamin in the gastrointestinal tract is increased by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an animal not administered a composition of the present disclosure.

[0387] In some embodiments, the incidence of bloating is reduced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to animals not administered a composition of the present disclosure.

[0388] In some embodiments, the synthesis of one or more volatile fatty acids is increased by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an animal not administered a composition of the present disclosure.

[0389] In some embodiments, the animal's body weight is increased by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,...

Claims

[Claim 1] The invention described in this specification.

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