Methods for gut beneficial fastidious bacterial production and treatment in early life

Abstract

Methods of inoculating an animal with a non-cultivable spore-forming bacteria, such as segmented filamentous bacteria (SFB), are provided. Also provided are methods of preparing a litter enriched with the bacteria, as are methods of using the enriched litter to inoculate animals with the bacteria. The disclosure further relates to the use of non-cultivable spore-forming bacteria to improve gastrointestinal health, reduce bacterial pathogens, and stimulate host immune function in animals.

Description

PATENT APPLICATION Docket No. P14804WQ00TITLE: METHODS FOR GUT BENEFICIAL FASTIDIOUS BACTERIALPRODUCTION AND TREATMENT IN EARLY LIFECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 to provisional patent application U.S. Serial No. 63 / 685,821, filed August 22, 2024. The provisional patent application is herein incorporated by reference in its entirety, including without limitation, the specification, claims, and abstract, as well as any figures, tables, appendices, or drawings thereof.TECHNICAL FIELD

[0002] The present disclosure relates generally to methods of mass production of fastidious or non-cultivable gut spore-forming beneficial bacteria, such as segmented filamentous bacteria (SFB) by inoculating subjects in a controlled environment, like pathogen-free, with an enriched bacterial inoculum (U.S. Patent Pub. No. 2021 / 0235722) to let bacteria grow and sporulate in the subject gut, which then shed through feces to bedding during a period of time. The disclosure also relates to the use of these inoculated subjects or their litter to horizontally spread bacterial spores to other subjects to mature the subjects’ gastrointestinal tract, improve health, nutrients absorption, reduce bacterial pathogens, stimulate host immune function, and increase productivity.BACKGROUND

[0003] The gastrointestinal tract (GIT) plays a pivotal role in health and diseases. However, GIT is immature in early life and host-specific bacteria are involved in gut immune maturation after hatch / birth. Thus, treatment strategies maximizing GIT maturation in early life highly benefit poultry health and productivity. The traditional probiotics used are cultivable indigenous gut microbes that are considered key players in maintaining GIT integrity and some of these bacteria (e.g., Lactobacillus acidophilus, Enterococcus faecium, Bifidobacterium spp.). They are produced by fermentation and then routinely used as feed probiotics to improve poultry health. However, many of these probiotics are not host-specific nor involved in gut maturation early life, must be given daily to maintain an effect, and could serve as reservoirs for antibiotic-resistance genes.PATENT APPLICATION Docket No. P14804WQ00

[0004] The majority of intestinal bacteria are uncultivable or have fastidious growth (require complex media, grow slow and cannot be produced by fermentation) and the majority of them form spores to be transmitted between individuals Among these, Segmented filamentous bacteria (SFB), non-pathogenic endospore-forming bacteria that commonly attach to the intestinal of animals. These bacteria have recently emerged as keystone bacterial species playing a major beneficial role in shaping a healthy GIT in early life. Recent improvements in high-throughput sequencing and imaging have demonstrated the role of SFB in triggering protective immune response as mainly demonstrated in mice and recently confirmed by our team in poultry animals (Redweik et al., Front Vet Sci 2020; Meinen-Jochum et al., Front Microbiol. 2023). They are host specific and SFB from one species cannot colonize the digestive tract of another species to provide a health benefit.

[0005] SFB has a unique lifecycle. Spores formed from vegetative SFB filaments travel from the environment to the gastrointestinal tract of the host organism in which they germinate in the distal small intestine to form small holdfast cells that intimately bind to the epithelium of the ileum and propagate into differentiating filaments that in turn release intracellular offspring that can directly bind to the host or travel through the large intestine to exit as spores or in some cases bind to areas like the ceca. The germination of holdfast cells to a replicative filament is solely reliant on the intimate binding with the host.

[0006] Animals receive these host-specific bacteria mainly from their progenitures to whom they were in contact during and after the birth / hatching. However, in some circumstances, like in commercial farms, chicks are hatched in an almost sterile environment, or some animals are bom by caesarian, thus not in contact with their mothers. The first microbiota they inherit is from the environment and may lack host-specific bacteria, like SFB. Animals from mothers with dysbiotic microbiota that lack key microbes are also at risk.

[0007] Because of the nature of SFB cycle and fastidious nature of the bacteria outside of host, passage in a host is important for SFB growth and spore production. Mass production through fermentation and sporulation is difficult and not cost effective for some animal industries, like the poultry industry. Moreover, present methods of inoculating poultry with SFB are timeconsuming and not practical for large numbers of animals.

[0008] Thus, there exists a need in the art for cost effective and practical methods of preparing and administering fastidious growth beneficial growth bacteria like SFB spore-based treatments to animal and poultry populations.PATENT APPLICATION Docket No. P14804WQ00SUMMARY

[0009] Methods of preparing a litter enriched with a fastidious or non-cultivable spore-forming bacteria are provided. In some embodiments, the methods comprise inoculating an animal with the bacteria or their spores; housing the inoculated animal for an amount of time sufficient for colonization of the inoculated animal by the bacteria; and collecting the litter produced by the inoculated animal, wherein the litter comprises viable spores of the bacteria. In certain embodiments, the non-cultivable spore-forming bacteria is a segmented filamentous bacteria (SFB). In certain embodiments, the animal is a poultry animal.

[0010] Methods of improving the gastrointestinal health of an animal by inoculating an animal are also provided. In some embodiments, the methods comprise exposing the animal to a litter (excreta, spilled feed, feathers, and material used as bedding in poultry operations) comprising viable spores of a non-cultivable spore-forming bacteria so that the bacteria are horizontally transferred to the animal. In some embodiments, the non-cultivable spore-forming bacteria is a segmented filamentous bacteria (SFB). In some embodiments, said exposure can be through feeding the animal the litter and / or through housing the animal with inoculated animals of the same species that are colonized with the bacteria. In some embodiments, the animal exhibits gut maturation, improved intestinal health and nutrients absorption, and / or increased resistance to a bacterial infection as compared to an animal not exposed to the litter.

[0011] These and / or other objects, features, advantages, aspects, and / or embodiments will become apparent to those skilled in the art after reviewing the following brief and detailed descriptions of the drawings. The present disclosure encompasses (a) combinations of disclosed aspects and / or embodiments and / or (b) reasonable modifications not shown or described.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of thePATENT APPLICATION Docket No. P14804WQ00 invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0013] FIG. 1 shows a general schematic of the processing and testing of SFB-enriched litter as described in Example 1.

[0014] FIG. 2A-B show results following testing of SFB-enriched litter pre- and post-heat treatment as described in Example 1. 2 A is a bar graph showing the amount of SFB in the litter pre- and post-heat treatment. 2B shows microbial gram staining of ground SFB-enriched litter pre- and post-heat treatment. 2A and 2B demonstrate that SFB spores survive heat treatment.

[0015] FIG. 3 shows a general schematic of the generation of an SFB-enriched litter inoculum as described in Example 1.DETAILED DESCRIPTION

[0016] So that the present invention may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation; the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.

[0017] Numeric ranges recited within the specification, including ranges of “greater than,” "at least", or "less than" a numeric value, are inclusive of the numbers defining the range and include each integer within the defined range. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4- 5”, “1-3 & 5”, and the like.

[0018] The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The word "or" means any one member of a particular list and also includes any combination of members of that list.

[0019] The term “about” as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, and time. Further, given solidPATENT APPLICATION Docket No. P14804WQ00 and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. The term “about” also encompasses these variations. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[0020] As used herein the term “agriculturally acceptable excipient” is a natural or synthetic substance formulated alongside the active ingredient of a formulation included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, increasing viscosity, or enhancing solubility.

[0021] The term “animal” as used herein, includes any animal that may be benefitted by the methods disclosed herein. Animals can include, but are not limited to, livestock animals such as cows, pigs, sheep, goats, horses, and the like; poultry animals, such as chickens, ducks, turkeys, and the like; and companion animals, such as dogs, cats, rabbits, gerbils, and the like.

[0022] The terms “effective amount” or “therapeutically effective amount” describes a quantity of a composition, such as the litters of the present disclosure, sufficient to achieve a desired effect in the animal being treated with that composition. For example, this can be the amount of litter comprising SFB necessary to prevent and / or treat a disease, disorder or condition capable of being prevented and / or treated, at least in part, by the SFB.

[0023] The term "intestinal microbiota", as used herein, refers to the population of microorganisms inhabiting the gastrointestinal tract.

[0024] The term “isolated” refers to a material that is substantially or essentially free from components that normally accompany it in its native state. For example, isolated SFB spores may refer to SFB spores that have been purified or removed from naturally or non-naturally occurring components that are present in their naturally occurring environment.

[0025] The term “litter”, as used herein, refers to a mixture of poultry excreta, spilled feed, feathers, and material used as bedding in poultry operations. The litter can comprise bedding materials such as wood shavings, sawdust, peanut hulls, leaves, grass clippings, shredded sugar cane, sand, straw, and other dry, absorbent, low-cost organic material.PATENT APPLICATION Docket No. P14804WQ00

[0026] The term "microbiome", as used herein, refers to a population of microorganisms from a particular environment, including the environment of the body or a part of the body. The term is interchangeably used to address the population of microorganisms itself (sometimes referred to as the microbiota), as well as the collective genomes of the microorganisms that reside in the particular environment.

[0027] As used herein the term “poultry” relates to the class of domesticated fowl (birds) used for food or for their eggs. Poultry includes wildfowl, waterfowl, and game birds. Examples of poultry include, but are not limited to, chicken, broilers, bantams, turkey, duck, geese, guinea fowl, peafowl, quail, dove, pigeon (squab), and pheasant.

[0028] The term “probiotic” is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism.

[0029] As used herein, “spore” or “spores” refer to structures produced by bacteria that are adapted for survival and dispersal. Spores are generally characterized as dormant structures; however, spores are capable of differentiation through the process of germination. Germination is the differentiation of spores into vegetative cells that are capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single bacterial vegetative cell. Bacterial spores are structures for surviving conditions that may ordinarily be nonconductive to the survival or growth of vegetative cells.

[0030] The gastrointestinal tract (GIT) is considered the central site for optimizing health and performance of production in animals. In chickens, for example, this complex tissue system must absorb nutrients to energize functions like growth and egg production while simultaneously serving as a barrier against pathogenic bacteria like Salmonella. At birth, a subject’s gut is immature, and its development depends on the external factors they are exposed to, e.g., environmental microbes and diet. Thus, interventions at the GIT are imperative for maximizing health and production potential. However, modern farming practices often interfere with gut maturation. For example, in the poultry industry chicks are hatched in a hatchery and not in contact with hens or other individuals from their own species that are capable of transferring host specific bacteria to them at the time of hatching. A similar problem is encountered when animals of other species, such as pigs, cows, horses, and the like, are delivered via cesarean section, or bom from a mother with a dysbiotic microbiota, or treatedPATENT APPLICATION Docket No. P14804WQ00 with antibiotics in early life. Thus, animals hatched or bom under these circumstances often need supplementation for gut maturation.

[0031] Some members of the gut microbiota, termed “keystone taxa”, have disproportionate effects on the host in comparison to other microbes. Many of these are “non-cultivable”, i.e. resistant to culture, due to their fastidious nature making traditional inoculation via supplementation difficult. Very few intestinal spore-forming bacteria, for example, have been successfully grown in culture. Among these include segmented filamentous bacteria (SFB), otherwise known as Candidatus Savagella. These bacteria intimately colonize the distal ileum, ceca tonsil and loops in the poultry intestine. Interestingly, SFB directly attach to the epithelium without damaging the gut barrier nor causing excessive inflammation, when they are inherited at early life. In fact, high-performing turkeys were shown to be consistently colonized by SFB versus their lower-performing counterparts, suggesting they may improve feed efficiency. The most impressive characteristic of SFB is their immunomodulatory activities in their respective host. This intimate colonization of intestinal epithelium enables potent stimulation of the homeostasis immunity, promoting T cell differentiation that improves epithelial barrier functions. This interaction results in a more immuno-competent host, as mice colonized with SFB are demonstrably more-resistant to enteric infections versus SFB-absent counterparts.

[0032] Importantly, SFB induce resistance to intestinal and extra-intestinal bacteria. In the gut it provides resistance to Enteric bacteria and particularly to Salmonella, Campylobacters. Minimizing Salmonella and Campylobacter in poultry would be extremely beneficial, as poultry products are the primary vehicle for these bacteria contamination and costing the U.S. billions annually. Additionally, a high level of other Enteric bacteria “Enterobacteriaceae” in the gut at early life is not desirable due their Lipopolysaccharide (LPS) that trigger inflammation; reducing this group of bacteria in the gut will lead to a reduced risk of inflammation and dysbiosis in animals. Outside of the gut, it can also provide resistance to microbes, such as, avian pathogenic E. coll (APEC) that cause colibacillosis (Meinen-Jochum et al., 2024 in preparation) and even to viruses as demonstrated in mice. Lastly, SFB induce these many effects early in its host’s life. Mammals are typically colonized by SFB post- weaning / post-hatch, when maternal IgA is no longer available from the mother’s milk. Soon after, SFB become reduced in the host once the animal increases its own IgA production (resulting via SFB-stimulation). On the contrary, egg yolk serves as a poor source of IgA for young chicks. Thus, without IgA in the young chicken gut, it is feasible SFB can bePATENT APPLICATION Docket No. P14804WQ00 successfully-inoculated and colonize young birds and promote resistance against pathogens. Collectively, these characteristics support SFB as a potential probiotic that can dramatically improve production in the poultry industry.

[0033] Although SFB are present in several animal species (including humans and mice), they are host-specific, as SFB from one animal cannot colonize another. However, SFB are sporeformers and may be transmitted horizontally (via feces). Thus, protocols to induce sporeformation and inoculate naive hosts via spores can be established. Furthermore, given that microbiota between animal farms are vastly different and factors like overcrowding and lack of bedding may decrease SFB abundance, SFB could be used to “standardize” gut microbial activities in poultry farms.

[0034] The present disclosure provides methods of mass producing non-cultivable sporeforming beneficial bacteria, such as segmented filamentous bacteria (SFB), by inoculating subjects with, and exposing subjects to, an enriched culture of these bacteria. The disclosure also relates to the use of these inoculated subjects or their litter to horizontally spread bacterial spores to other subjects. These bacteria will grow and sporulate in the host with no need for fermenters and spore-specific media. These hosts then become “super-shedders” of these bacteria. These super-shedders can directly spread the bacterial spores to other animals by cohousing. In other embodiments, litters from these super-shedders can be collected and processed and then use as a treatment to newly hatched birds or recently bom animals.Enriched Litter and Methods of Preparation

[0035] The present disclosure provides a novel and cost-effective strategy for preparing a non- cultivable bacteria spore-based treatment for poultry production using live poultry and their litter for the inoculation of naive poultry.

[0036] Non-cultivable spore-forming bacteria include segmented filamentous bacteria (SFB), otherwise known as Candidatus Savagella, as well as other uncultured species of the gut microbiota, including, for example, members of the genus Ruminococcus, Bacteroides, Prevotella, Clostridium, and / or Collinsella. See also Koopman, Ini. J. Mol. Sci. 2022, 25(6), 3405; available at world wide web at doi.org / 10.3390 / ijms23063405.

[0037] In some aspects of the disclosure, methods of preparing a litter enriched with a non- cultivable spore-forming bacteria are provided. In certain embodiments, the method comprises inoculating an animal with the bacteria. Initial inoculation can be through any method knownPATENT APPLICATION Docket No. P14804WQ00 in the art, such as those described in U.S. Patent Pub. No. 2021 / 0235722, which is herein incorporated by reference in its entirety. Inoculation is further described in Meinen-Jochum et al., FRONT MICROBIOL. 2023;14: 1231837 and Redweik et al., FRONT VET SCI. 2020;7:629. Initial inoculation can also be through exposure to litter comprising non-cultivable bacteria spores, as described herein.

[0038] Following inoculation, the animal can be housed for an amount of time sufficient for bacterial colonization to occur in the inoculated animal. This can be about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, or any range or timeframe therein.

[0039] In embodiments, the inoculated animal is housed on bedding material. The bedding material can be any material suitable for the housing of animals. In embodiments, the bedding material is plant-based, such as, for example, wood shavings, leaves, glass clippings, sawdust, and / or straw. In embodiments, during this time the inoculated animal is isolated from noninoculated animals.

[0040] In embodiments, the inoculated animal may be housed in a manner which allows for collection of only the excreta without mixture with bedding material. As a non-limiting example, this may comprise housing the animal on a grid floor elevated over a surface, such as a pan, base, board, or similar, that allows for passage of the excreta through the grid onto the surface below. The surface may be configured to allow for easy removal so that the excreta can be collected without disruption of the animals.

[0041] After colonization of the inoculated animal by the bacteria, which typically peaks at around 2 weeks in chickens, the litter produced by the animal can be collected. The litter can comprise the bedding material along with excreta. If the animal is housed in a manner that prevents mixture of the excreta with bedding material, the litter may comprise only excreta. In embodiments, the liter comprises viable spores of the bacteria. The amount of spores in the liter will depend on the number of inoculated animals producing the litter and the level of colonization of the animals by the bacteria.Litter Processing and End-Use Litter

[0042] In certain embodiments, the litter is further processed after collection. The litter may be ground-up, pulverized, heat-treated, and / or chemically treated. Grinding is useful for making the liter more uniform and easily dispersible or administrable. Heat-treatment and / or chemicalPATENT APPLICATION Docket No. P14804WQ00 treatment can be used to reduce the unintended passage of pathogenic microorganisms to humans or other animals whilst retaining viable bacterial spores. Suitable chemical treatments can include, for example, alcohol, chloroform, and the like. In some embodiments, the litter is not heat-treated and / or chemically treated.

[0043] After collection, the litter can be mixed with other suitable materials. Suitable materials can include, as non-limiting examples, vitamins, minerals, trace elements, emulsifiers, aromatizing products, binders, colorants, odorants, thickening agents, and the like. In some embodiments, the litter can include one or more of an ionophore; vaccine; antibiotic; antihelmintic; virucide; nematicide; amino acids such as methionine, glycine, and arginine; fish oil; oregano; and biologically active molecules such as enzymes.

[0044] In some embodiments, processed litters of the present disclosure are solid when in their end-use formulation. Where solid compositions are used, it may be desired to include one or more carrier materials including, but not limited to: mineral earths such as silicas, talc, kaolin, limestone, chalk, clay, dolomite, diatomaceous earth; calcium sulfate; magnesium sulfate; magnesium oxide; zeolites, calcium carbonate; magnesium carbonate; trehalose; chitosan; shellac; albumins; starch; skim-milk powder; sweet-whey powder; maltodextrin; lactose; inulin; dextrose; products of vegetable origin such as cereal meals, tree bark meal, wood meal, and nutshell meal.

[0045] In some embodiments, processed litters of the present disclosure are liquid when in their end-use formulation. In some embodiments, the liquid comprises a solvent that may include water or an alcohol or a saline or carbohydrate solution, and other animal-safe solvents. In some embodiments, the litters can include binders such as animal-safe polymers, carboxymethylcellulose, starch, polyvinyl alcohol, and the like.

[0046] In some embodiments, the processed litters can further comprise thickening agents such as silica, clay, natural extracts of seeds or seaweed, synthetic derivatives of cellulose, guar gum, locust bean gum, alginates, and methylcelluloses. In some embodiments, the litters comprise anti-settling agents such as modified starches, polyvinyl alcohol, xanthan gum, and the like.

[0047] In some embodiments, the litters can be formulated to comprise colorants including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. In some embodiments, the littersPATENT APPLICATION Docket No. P14804WQ00 comprise trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc. In some embodiments, the litters comprise dyes, both natural and artificial.

[0048] In some embodiments, the litters of the present disclosure are formulated to comprise an animal-safe virucide, bacteriocide, or nematicide.

[0049] In some embodiments, the litters can be formulated to comprise saccharides (e.g., monosaccharides, disaccharides, trisaccharides, polysaccharides, oligosaccharides, and the like), polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides, proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acid polymers, silica, inorganic salts and combinations thereof. In a further embodiment, the litters comprise polymers of agar, agarose, gelrite, and gellan gum, and the like. In some embodiments, the litters comprise plastic capsules, emulsions (e.g., water and oil), membranes, and artificial membranes.

[0050] In some embodiments, litters of the present disclosure comprise 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 once the litter is mixed with food and / or water to be administered to the fowl. In one embodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators are not chemically active when administered to the fowl.

[0051] In some embodiments, processed litters of the present disclosure are formulated to comprise one or more preservatives. The preservatives may be in liquid or gas formulations. The preservatives may be selected from one or more of monosaccharide, disaccharide, trisaccharide, polysaccharide, acetic acid, ascorbic acid, calcium ascorbate, erythorbic acid, iso-ascorbic acid, erythrobic acid, potassium nitrate, sodium ascorbate, sodium erythorbate, sodium iso-ascorbate, sodium nitrate, sodium nitrite, nitrogen, benzoic acid, calcium sorbate, ethyl lauroyl arginate, methyl-p-hydroxy benzoate, methyl paraben, potassium acetate, potassium benzoiate, potassium bisulphite, potassium diacetate, potassium lactate, potassium metabisulphite, potassium sorbate, propyl-p-hydroxy benzoate, propyl paraben, sodium acetate, sodium benzoate, sodium bisulphite, sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite, sodium salt of methyl-p-hydroxy benzoic acid, sodium salt of propyl- p-hydroxy benzoic acid, sodium sulphate, sodium sulfite, sodium dithionite, sulphurous acid, calcium propionate, dimethyl dicarbonate, natamycin, potassium sorbate, potassium bisulfite, potassium metabisulfite, propionic acid, sodium diacetate, sodium propionate, sodium sorbate,PATENT APPLICATION Docket No. P14804WQ00 sorbic acid, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, butylated hydro-xyanisole, butylated hydroxytoluene (BHT), butylated hydroxyl anisole (BHA), citric acid, citric acid esters of mono- and / or diglycerides, L-cysteine, L-cysteine hydrochloride, gum guaiacum, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate, monoisopropyl citrate, propyl gallate, sodium metabisulphite, tartaric acid, tertiary butyl hydroquinone, stannous chloride, thiodipropionic acid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin, sulfur dioxide, formic acid, or tocopherol(s).

[0052] In some embodiments, the processed litters comprise sodium bicarbonate.

[0053] In some embodiments, the processed litter can be mixed with animal feed. In some embodiments, animal feed may be present in various forms such as pellets, capsules, granulated, powdered, mash, liquid, or semi-liquid.

[0054] In some embodiments, processed litter of the present disclosure is mixed into the premix or mash at the feed mill, alone as a standalone premix, and / or alongside other feed additives. In one embodiment, the processed litter of the present disclosure is mixed into or onto the feed at the feed mill. In another embodiment, processed litter of the present disclosure is mixed into the feed itself.

[0055] In some embodiments, feed comprising processed litter of the present disclosure may be supplemented with water, premix or premixes, forage, fodder, beans (e.g., whole, cracked, or ground), grains (e.g., whole, cracked, or ground), bean- or grain-based oils, bean- or grainbased meals, bean- or grain-based haylage or silage, bean- or grain-based syrups, fatty acids, sugar alcohols (e.g., polyhydric alcohols), commercially available formula feeds, oyster shells and those of other bivalves, and mixtures thereof.

[0056] In some embodiments, forage encompasses hay, haylage, and silage. In some embodiments, hays include grass hays (e.g., sudangrass, orchardgrass, or the like), alfalfa hay, and clover hay. In some embodiments, haylages include grass haylages, sorghum haylage, and alfalfa haylage. In some embodiments, silages include maize, oat, wheat, alfalfa, clover, and the like.

[0057] In some embodiments, premix or premixes may be utilized in the feed. Premixes may comprise micro-ingredients such as vitamins, minerals, amino acids; chemical preservatives; pharmaceutical compositions such as antibiotics and other medicaments; fermentation products, and other ingredients. In some embodiments, premixes are blended into the feed.PATENT APPLICATION Docket No. P14804WQ00

[0058] In some embodiments, the feed comprising processed litter may include feed concentrates such as soybean hulls, soybean oils, sugar beet pulp, molasses, high protein soybean meal, ground com, shelled corn, wheat midds, distiller grain, cottonseed hulls, and grease. See Anderson et al. (U.S. Pat. No. 3,484,243), Iritani et al. (U.S. Pat. No. 6,090,416), Axelrod et al. (U.S. Publication US20060127530A1), and Katsumi et al. (U.S. Pat. No. 5,741,508) for animal feed and animal feed supplements capable of use in the present compositions and methods.

[0059] In some embodiments, feed comprising processed litter occurs as a compound, which includes, in a mixed composition capable of meeting the basic dietary needs, the feed itself, vitamins, minerals, amino acids, and other necessary components. Compound feed may further comprise premixes.

[0060] In some embodiments, processed litter of the present disclosure may be mixed with animal feed, premix, and / or compound feed. Individual components of the animal feed may be mixed with the processed litter prior to feeding to the animal. The processed litter of the present disclosure may be applied into or on a premix, into or on a feed, and / or into or on a compound feed.Methods of Inoculating Animals Using Enriched Litter

[0061] Methods of inoculating animals with a non-cultivable spore-forming bacteria, such as SFB, are provided herein. In certain embodiments, the method comprises exposing the animal to litters of the present disclosure comprising viable spores of the bacteria.

[0062] The exposure can be through feeding of the litter. In some embodiments, the litter collected from inoculated animals can be further processed as described herein and fed as a dietary supplement to non-inoculated animals. In some embodiments, the litter is not further processed before feeding to non-inoculated animals.

[0063] In some embodiments, the animal is exposed to the enriched litter through addition of the litter to drinking water, feed, spraying the enriched litter on bedding material in which the animal is in contact with, mixing the enriched litter with medications or vaccines, and gavage. In some embodiments, the enriched litter is sprayed directly on the animal. In some embodiments, the enriched litter is sprayed on and / or sprayed in feed, and the feed is administered to the animal. In further embodiments, the animal ingests the bacteria through the preening of feathers that have come into contact with the enriched litter.PATENT APPLICATION Docket No. P14804WQ00

[0064] In some embodiments, the animal is exposed to the enriched litter through co-housing with an inoculated animal of the same species that is already colonized with the bacteria. Beneficially, housing with an inoculated poultry animal allows for horizontal transmission of the bacteria through exposure to litter produced by the inoculated animals, which comprises bacterial spores shed by the inoculated animals. Thus, through this method, it is possible to manually inoculate a relatively small population of animals, house those animals with noninoculated animals, and passively inoculate the rest of the flock.

[0065] In some embodiments, the ratio of inoculated animals to non-inoculated animals in a housed flock is about 1:1, 1:2, 1:3, 1:4, 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, 1:25, 1:30, 1:35, 1:40, 1 :45, 1:50, 1:55, 1:60, 1:65, 1:70, 1 :75, 1:80, 1:85, 1:90, 1:95, 1:100, about 1:1 to about 1:50, about 1:5 to about 1:20, about 1:10 to about 1:20, about 1:10 to about 1:30, about 1:10 to about 1:40, or any range or integer therein.

[0066] In some embodiments, animals are exposed to the enriched litters of the present disclosure on day 0, 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, or 31 post-hatching or post-birth. In some embodiments, the animals are exposed prior to hatching or birth, at the time of hatching or birth, or within 24 hours of hatching or birth. In some embodiments, the enriched litters are administered to the exterior surface of an egg as a liquid, semi-liquid, or solid on day 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 pre-hatching. In some embodiments, animals are exposed to enriched litters of the present disclosure in multiple dosing sessions in week(s) 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, and / or 30 week(s) post-hatching or post-birth. In some embodiments, animals are exposed to the enriched litters within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of hatching or birth.

[0067] In some embodiments, the animals are exposed to the enriched litters at a dose comprising a total of, or at least, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 5000, or 10000 bacterial spores. In some embodiments, the administered dose of the probiotic composition comprises about 10 to about 10000, about 50 to about 10000, about 100 to about 10000, about 500 to about 10000, about 1000 to about 10000, about 5000 to about 10000, about 10 to about 5000, about 50 to about 5000, about 100 to about 5000, about 500 to about 5000, about 1000 to about 5000, about 10 to about 1000, about 50 to about 1000, about 100 to about 1000, about 500 to about 1000, about 10 to about 500, about 50 to about 500,PATENT APPLICATION Docket No. P14804WQ00 about 100 to about 500, about 10 to about 200, about 50 to about 200, about 100 to about 200, about 10 to about 100, or about 50 to about 100 bacterial spores.Inducing gut maturation, nutrients absorption, Resistance to Pathogens, and Improving Gut and overall Health

[0068] In some aspects, the present disclosure is drawn to inoculating an animal with a non- cultivable spore-forming bacteria, such as segmented filamentous bacteria (SFB), as described herein to induce resistance to bacterial infection by a pathogenic microbe. In some embodiments, the present disclosure is further drawn to inoculating an animal with the bacteria to prevent colonization of pathogenic microbes in the gastrointestinal tract. In some embodiments, the present disclosure is further drawn to inoculating a poultry animal with the bacteria to improve gastrointestinal health. The use of the enriched litters described herein result in reduced colonization of the gastrointestinal tracts of animals by bacterial pathogens, including but not limited to Salmonella spp., Campylobacter spp., and Clostridium spp.

[0069] In some embodiments, inoculation with the bacteria as described herein reduces lower gut permeability and reduces microbial leakage from the gastrointestinal tract, further providing for a reduced risk of extraintestinal pathogens including for bacterial sepsis from pathogens like Escherichia coli.

[0070] Pathogenic microbes of poultry include, but are not limited to, the following: Mycoplasma gallisepticum, Mycoplasma meleagridis, Mycoplasma synoviae, Pasteur ell a multocida, Clostridium perfringens, Clostridium colinum, Clostridium botulinum, Salmonella Typi, Salmonella Typhimurium, Salmonella enterica, Salmonella pullorum, Salmonella gallinarum, Hemophilus gallinarum, Erysipelothrix insidiosa, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Listeria monocytogenes, Arcobacter butzleri, Mycobacterium avium, and pathogenic strains of Escherichia coli and Staphylococcus aureus. In some embodiments, the pathogenic microbes are pathogenic to both poultry and humans. In some embodiments, the pathogenic microbes are pathogenic to either poultry or humans.

[0071] In some embodiments, the inoculation with bacteria as described herein modulates the makeup of the gastrointestinal microbiome such that the administered microbes outcompete microbial pathogens present in the gastrointestinal tract. In some embodiments, inoculation of animals harboring microbial pathogens outcompetes the pathogens and clears the animal of the pathogens. In some embodiments, the inoculation with bacteria as described herein stimulatesPATENT APPLICATION Docket No. P14804WQ00 host immunity, and aids in clearance of the microbial pathogens. In some embodiments, the inoculation with bacteria as described herein improves food safety by preventing colonization of pathogenic microbes that are pathogenic to both animals and humans.

[0072] In some embodiments, challenging animals with a microbial colonizer or microbial pathogen after exposure to non-cultivable spore-forming bacteria as described herein prevents the microbial colonizer 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 animals with a microbial colonizer or microbial pathogen after exposure to non-cultivable spore-forming bacteria prevents the microbial colonizer or microbial pathogen from colonizing poultry.

[0073] The following examples are intended only to further illustrate the disclosure and are not intended to limit the scope of the subject matter which is defined by the claims.EXAMPLESExample 1. Litter-sourced SFB spore inoculum preparation and testing in pre- and post- ncwly hatched chicks.

[0074] The horizontal transfer of segmented filamentous bacteria (SFB) through SFB-enriched litter will be tested. The litter from SFB-treated birds will be processed and tested as a source of inoculum for pre- and post-hatched chicks. The treatment will be tested as a feed supplement. For cost optimization, we will test whether treating only a few birds of the same flock will naturally shed the SFB in the environment and inoculate the non-treated birds housed in the same environment.

[0075] SFB are spore-forming bacteria that reside mainly in the ileum of animals and Eire keystone bacteria for gut maturation in early life. SFB have a unique lifecycle described primarily in mice studies. Spores formed from vegetative SFB filaments travel from the environment to the gastrointestinal tract of the host organism in which they germinate in the distal small intestine to form small holdfast cells that intimately bind to the epithelium of the ileum and propagate into differentiating filaments, which in turn release intracellular offspring that can directly bind to the host or travel through the large intestine to exit as spores or in some cases bind to areas like the ceca. The germination of holdfast cells to a replicative filament solely relies on the intimate binding with the host.PATENT APPLICATION Docket No. P14804WQ00

[0076] SFB spores orally inoculated to day-of-hatch chicks germinate to filamentous SFB in the ileum and feces as early as 5 day-post-inoculation (dpi). This indicates that SFB had already bound and progressed through their lifecycle inside of the host. It is an objective of this example to 1) test the levels of SFB in litter, 2) process the SFB-enriched litter to eliminate germinative bacteria, and 3) evaluate the litter-sourced SFB inoculum for their probiotic potential in chickens.Experimental Approaches

[0077] SFB-enriched litter inoculum preparation. Day-old Specific-pathogen-free (SPF) chicks will be inoculated with SFB-enriched ileal spores as described in U.S. Patent Pub. No. 2021 / 0235722, which is herein incorporated by reference in its entirety. Chickens will be housed in open pens and in a controlled environment. Plant-based bedding (e.g., wood shavings, leaves, grass clippings, etc.) will be used. At around SFB peak colonization (~ 2 weeks), litters will be collected from pens, ground, and heat-treatment or chemically treated, e.g., alcohol, chloroform, to eliminate vegetative bacteria, as shown generally in FIG. 1. The level of SFB in litters will be evaluated by RT-qPCR and microscopy (FIG. 2A). The absence of bacteria will be verified by plating the litter material on different selective and non-selective media in anaerobic and aerobic conditions (FIG. 2B). Chicks and litter microbiota will be analyzed using high-throughput 16S rRNA sequencing and bioinformatic-based approaches. At about 3 weeks, birds will be euthanized; ileal scraping will be collected for SFB inoculum preparation for the next group. A general outline of this procedure is depicted in FIG. 3.

[0078] Treatment of pre-hatched chicks. The chicken incubation time is around 21 days. At around the prehatching period (Days 18-21), SPF and conventional fertilized eggs will be spread with litter-sourced SFB (-102, 10s, or IO4 spores), so the newly hatched chicks will be in contact with SFB spores. At different times, 3, 7, 21 days post-hatch, chicks will be euthanized. The level of SFB in the ileum will be evaluated by RT-qPCR. Gut immune maturation and resistance to pathogens will be tested.

[0079] Treatment of post-hatched chicks. Based on our preliminary studies, the litter-sourced SFB will be added to the feed at a proportion of - 1 / 10s. At different times (3, 7, 14 days), chicks will be euthanized and evaluated for SFB colonization as described above. The potential vertical transfer will be assessed by testing the presence of SFB by PCR in the reproductive tract of hens, e.g., oviduct and ovary (follicles), and eggs from conventional hens.PATENT APPLICATION Docket No. P14804WQ00

[0080] Use ofSFB super-shedder birds to transfer SFB to non-treated birds in poultry housing. For the development of a cost-effective treatment, it will be tested whether treating only a few birds from the same flocks will lead to the spread ofSFB to non-treated chicks. From the same flock of day-old chicks, few birds will be SFB treated as above, tagged, and cohoused with the non-treated birds from the same flock at different ratios (treated to non-treated chicks) such as 1 / 5, 1 / 10, 1 / 20, 1 / 30, etc. At various times post hatched (3, 7, 21 days), chicks will be euthanized. Ileal SFB colonization and immune maturation will be tested as previously described. A second option is using the super-shedder to propagate the SFB spores on the bedding of pens that will house the newly hatched chicks.Expectations

[0081] The litter-sourced SFB treatment will first be tested at a ratio of -1 / 105 (10 mg of litter- sourced SFB to 1 kg of feed). This calculation is based on the amount of feed chicks eat in their first week of life (-15 g), the amount of SFB we detected in litter by qPCR (~ 108 gene copies / g), and the amount of SFB previously tested in day-old chicks by qPCR (-104 gene copies / bird). Data will show whether this ratio should be adjusted for optimal effect. Once birds are treated with SFB, the spores of these bacteria are shed into the environment through feces. Horizontal transmission of SFB will occur when birds consume litter, feed, or water contaminated with SFB. Testing both pre-hatch and post-hatch and different bedding will determine the best strategy for optimal SFB colonization in newly hatched chicks.

[0082] Commercial farms need an easy, cost-effective strategy to transfer SFB to newly hatched chicks. At the end of these experiments, it will be determined how long the newly hatched chicks get colonized with SFB from the shedder birds. The percentage of SFB shedder chicks needed for the maximal coverage of the non-treated birds will also be determined.Example 2. Intestinal and Extra-Intestinal Immune Protection

[0083] In vitro assays and chicken bacterial challenge models will be used to evaluate the efficacy of litter-sourced SFB treatment in chickens. Preventing pathogen colonization in chickens is important for maintaining food safety and increasing productivity. From Example 1, the optimal inoculum will be selected to treat conventional day-old chicks. We will evaluate whether litter-sourced SFB treatment increases resistance to both intestinal and extra-intestinal pathogens. These studies will provide new insights into the potential protective capabilities ofPATENT APPLICATION Docket No. P14804WQ00SFB treatment respiratory infections. Overall, this study will contribute to food safety and could greatly benefit poultry producers.SFB Intestinal Protection

[0084] Gut immune assessment. Small intestinal scrapings (SISs), tissues (ceca and ilea), content, and feces will be collected from treated and non-treated groups. Samples will be analyzed for in vitro bactericidal effects (SISs); immune effectors (IgA, immune cells, cytokines), histopathology (EM, FISH), and microbiota sequencing (ceca). Intestinal pathology scoring and goblet cell enumeration will determine the pathological inflammation and proliferation of cecal goblet cells in response to treatment and challenge. To determine underlying mechanisms responsible for improved antimicrobial responses upon SFB treatment, we will measure changes in gene expression and transcription via RT-qPCR, e.g., IL-10 and IL-2 cytokines; CTLA-4 (Treg); antimicrobial peptides (AMPs) (BD-12, BD-14, and Fowl-1).

[0085] Assess intestinal broad protection In vitro. For total Enterobacteriaceae testing, fresh feces will be collected from different groups at 3, 7, and 21 days and serially diluted and plated onto MacConkey agar. Broad protection against a large number of intestinal pathogens will be evaluated using in vitro challenge. Small intestinal scraping (SIS) from both control and SFB- treated birds will be tested for bactericidal ability against a large number of virulent and antibiotic-resistant Salmonella enterica serovars from our collection, e.g. Typhimurium, Kentucky, Albert, Enteritidis, etc.

[0086] Salmonella oral challenges. At two to three days, treated and non-treated birds will be orally challenged with Salmonella (S.) Typhimurium or S'. Enteritidis (0, IO3, 10e, and IO9 CFU / 200 pl of PBS) (Table 1). At different times post-challenge, birds will be euthanized, and ceca contents will be collected and plated onto MacConkey agar. Chicken weights will be collected regularly throughout the study. From the pilot studies, we will select an optimal Salmonella challenge dose for a medium-scale study. Pre-necropsy, fresh feces will be collected from different groups at 3, 7, and 21 days, serially diluted, and plated onto MacConkey agar for Enterobacteriaceae and Salmonella enumeration. Post-necropsy, intestinal (Ileum, ceca, bursa) tissues and extra-intestinal organs (spleen, liver) will be aseptically collected for bacterial enumeration.SFB Extra-Intestinal Protection

[0087] Extra-intestinal immune assessment. The immune response to SFB treatment will be evaluated by testing biological markers in blood and lungs, such as IL-22, complementPATENT APPLICATION Docket No. P14804WQ00 components 1 q (Clq), and the lung macrophage receptor NOTCH4 that exhibits antiinflammatory activity in activated macrophages.

[0088] Assess extra-intestinal broad protection In vitro. The bactericidal ability of the blood serum and the phagocytosis ability of the lung macrophages will be tested using APEC strains.

[0089] APEC airsac challenge. At 2 weeks old, SFB-treated and non-treated birds will be air sac inoculated with 105CFU of washed stationary phase APEC strain in 100 L of saline. Gross pathological lesions will be scored, and bacterial loads will be enumerated in lung, heart, and blood.Expectations

[0090] Avian chronic diseases, an economic burden to the poultry industry, are mainly caused by pathogens, e.g., the Avian influenza virus and APEC, that infect the respiratory tract. Here, the effect of SFB treatment on the phagocytosis of lung macrophages will be tested. Lung macrophages are a key defense against bacteria and viruses. At the end of these experiments, we will demonstrate the APEC protection in vivo and the SFB extra-intestinal immune protection mechanism. Future studies will test protection against viruses.

[0091] Statistical analyses. All results will be analyzed using GraphPad Prism software version 6.0. Data quality and distributional assumptions will be assessed using summary statistics (means, standard errors, medians, and frequency counts for discrete variables).

Claims

1. PATENT APPLICATION Docket No. P14804WQ00CLAIMSWhat is claimed is:

1. A method of preparing a litter enriched with a non-cultivable spore-forming bacteria comprising: inoculating an animal with the bacteria; housing the inoculated animal for an amount of time sufficient for colonization of the inoculated animal by the bacteria; and collecting the litter produced by the inoculated animal, wherein the litter comprises viable spores of the bacteria.

2. The method of claim 1, wherein the non-cultivable spore-forming bacteria is a segmented filamentous bacteria (SFB).

3. The litter produced by the method of claim 1.

4. The method of claim 1, further comprising grinding, heat-treating, and / or chemically treating the litter.

5. The method of claim 1, wherein the amount of time sufficient for bacterial colonization of the inoculated animal is about 1 week to about 4 weeks.

6. The method of claim 1, wherein the litter comprises excreta from the inoculated animal.

7. The method of claim 1 , wherein the litter comprises a plant-based bedding material.

8. The method of claim 6, wherein the plant-based bedding material comprises wood shavings, leaves, glass clippings, sawdust, and / or straw.

9. The method of claim 1, wherein the animal is a poultry animal.PATENT APPLICATION Docket No. P14804WQ0010. A method of inoculating an animal with a non-cultivable spore-forming bacteria comprising: exposing the animal to the litter produced by the method of claim 1.

11. The method of claim 10, wherein the animal exhibits improved intestinal health and / or increased resistance to a bacterial infection as compared to an animal not exposed to the litter.

12. The method of claim 11 , wherein the bacterial infection is caused by one or more of Salmonella spp., Campylobacter spp., Clostridium spp., or Escherichia coli.

13. An inoculant composition comprising the litter of claim 1 and an excipient.

14. A method of improving gastrointestinal health of an animal by inoculating an animal comprising: exposing the animal to a litter comprising viable spores of a non-cultivable sporeforming bacteria so that the bacteria is horizontally transferred to the animal.

15. The method of claim 14, wherein the non-cultivable spore-forming bacteria is a segmented filamentous bacteria (SFB).

16. The method of claim 14, wherein the litter comprises excreta from an inoculated poultry animal colonized with the bacteria.

17. The method of claim 14, wherein the litter is ground, heat-treated, and / or chemically treated prior to exposure to the animal.

18. The method of claim 17, wherein the exposing step comprises feeding the animal the ground, heat-treated, and / or chemically treated litter.

19. The method of claim 14, wherein the exposing step comprises housing the animal with inoculated animals of the same species that Eire colonized with the bacteria.PATENT APPLICATION Docket No. P14804WQ0020. The method of claim 19, wherein the ratio of inoculated animals to non-inoculated animals is about 1:1 to about 1:50.

21. The method of claim 19, wherein the ratio of inoculated animals to non-inoculated animals is about 1:10 to about 1:20.

22. The method of claim 14, wherein the exposing step is performed within 24 hours of hatching or birth.

23. The method of claim 14, wherein the animal is a poultry animal.

24. The method of claim 23, wherein the poultry animal is a chicken.

25. The method of claim 14, wherein the animal exhibits improved intestinal health and / or increased resistance to a bacterial infection as compared to an animal not exposed to the litter.

26. The method of claim 25, wherein the bacterial infection is caused by one or more of Salmonella spp., Campylobacter spp., Clostridium spp., or Escherichia coli.

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