Combinations of bacterial strains to inhibit pathogens in animals

Abstract

This disclosure relates to combinations of Bacillus, essentially consisting of or comprising Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. The disclosure also includes compositions of Bacillus combinations further comprising additional components, such as carriers, chemicals, feeds, feed supplements, plant compounds and / or vitamins, and any combination thereof, and methods for administering such combinations. In some embodiments, the compositions may comprise dried spores, liquid cultures, and / or aqueous solutions, and combinations thereof. In some embodiments, the compositions of this disclosure treat, prevent, and / or inhibit outbreaks of microbial infections in livestock. For example, some embodiments include methods and compositions for treating, prevent, and / or inhibiting pathogenic infections in animals.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of the earlier filing date of U.S. Provisional Patent Application No. 63 / 506,527, filed on June 6, 2023, which is incorporated herein by reference in its entirety.

[0002] This application relates to combinations and / or compositions comprising three Bacillus species (Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis) for administration to animals, particularly birds. This application also relates to the use of these combinations and / or compositions for the prevention, inhibition, and / or treatment of infections caused by pathogens such as Enterococcus cecorum and / or Clostridium species. [Background technology]

[0003] The gastrointestinal tract (GI) of animals is home to a diverse community of microbiota. The GI tract can contain hundreds of different species, and this community profile can change over time based on the individual's age and health status. A healthy microbiome in a subject offers numerous benefits, including pathogen resistance, nutrient absorption, and immune system performance. The gut microbiota also plays a crucial role in the transmission of intestinal pathogenic infections, significantly impacting quality of life. The composition of an animal's gastrointestinal microbiota is substantially dependent on ingested substances. Therefore, direct-feed microorganism (DFM) compositions are commonly administered to influence physiological health.

[0004] DFMs can limit the adhesion of pathogenic microorganisms to mucosal surfaces and stimulate immune responses or the growth of other endogenous beneficial microorganisms. Furthermore, certain DFMs produce and secrete other beneficial compounds or compositions, such as antimicrobial substances. Comparable results have been shown between feeding DFM products and the preventive levels for antibiotic growth. [Overview of the project]

[0005] This disclosure includes a combination of Bacillus essentially comprising Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. This combination of Bacillus may be useful for treating, preventing, or inhibiting pathogens in animals, such as Enterococcus cecorum infection in poultry. Another aspect of this disclosure is a combination of Bacillus comprising Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. Bacillus licheniformis may be or may contain strain OBT618, Bacillus pumilus may be or may contain strain OBT13216, and / or Bacillus subtilis may be or may contain strain PR104.

[0006] The relative amount of each Bacillus species in a Bacillus combination can be 1% to 99% of the combination such that the total amount of Bacillus species in the composition is 100%. In some embodiments, the combination includes 25% to 75% of B. licheniformis, 25% to 75% of B. pumilus, and 25% to 75% of B. subtilis, such that the sum of each species is 100% of the other species. In other embodiments, the combination includes about 33.3% of B. licheniformis, about 33.3% of B. pumilus, and about 33.3% of B. subtilis. In further embodiments, the combination includes about 20% of B. licheniformis, about 30% of B. pumilus, and about 50% of B. subtilis.

[0007] In some embodiments, this combination is 10 2 ~10 11 CFU / g B. licheniformis, 10 2 ~10 11CFU / g of B. pumilus, and 10 2 ~10 11 Includes B. subtilis at CFU / g.

[0008] The disclosure also includes compositions comprising a combination of Bacillus species and additional components. The additional components in the composition may be carriers, vitamins, copper salts, allicin, alliinase, algae, polyphenols, or plant materials containing polyphenols, feed supplements, additional DFM, feed, or combinations thereof. In some embodiments, the composition does not include additional Bacillus species. In some embodiments, the composition does not include additional DFM. In other embodiments, the additional components may be yucca, quillaja, silica, mineral clay, glucan, inulin, mannan, or endoglucanohydrolase, or any combination thereof.

[0009] In addition, this specification discloses compositions comprising a combination of Bacillus and water. In some embodiments, the composition comprising a combination of Bacillus and water may further contain an acid. In some embodiments, the acid includes acetic acid.

[0010] Another embodiment is a feed composition for administration to poultry, comprising a combination of Bacillus and poultry feed. This feed composition may further contain additional components. In some embodiments, this poultry feed comprises plant materials, carbonates, sulfates, lactates, oxides, propionates, stearates, phosphates, minerals, copper seeds, sugars, salts, animal protein products, feed products, cereal products, plant protein products, processed cereal products, roughage products, molasses products, or combinations thereof. In further embodiments, the poultry feed may include beet pulp, ground corn, corn syrup solids, vegetable fiber, rice husks, soluble vegetable fiber, wheat flour, microcrystalline cellulose, calcium carbonate, potassium carbonate, potassium sulfate, sodium sulfate, calcium lactate, calcium oxide, calcium propionate, calcium stearate, dicalcium phosphate dehydrated, monocalcium phosphate, sodium tripolyphosphate, tetrasodium pyrophosphate, dolomite, silicon dioxide, silica, limestone, vermiculite, bentonite, montmorillonite, kaolin, glucose, sucrose, dextrose, fructose, maltodextrin, sodium chloride, carrageenan, cellulose, guar gum, polyol, sodium aluminosilicate, urea, biotin, folic acid, sodium sesquicarbonate, a source of methionine, a source of lysine, L-threonine, or a combination thereof. The poultry feed may further include copper sulfate.

[0011] In addition, this specification discloses methods for administering the disclosed combinations of Bacillus to subjects, which may be, for example, livestock and / or aquatic species. In some embodiments, livestock are poultry and / or ruminants. In some examples, aquatic species are tilapia. This disclosure also includes methods for reducing avian mortality, lesion scores, incidence of Enterococcus cecorum, Salmonella species, Esherichia coli, and / or Clostridium perfingens, and / or oocysts in fecal matter, and / or administering an effective amount of any one of the combinations disclosed herein to poultry.

[0012] The aforementioned and other purposes, features, and advantages of the embodiments will become more apparent from the following detailed description, which proceeds with reference to the attached figures. [Brief explanation of the drawing]

[0013] [Figure 1] This graph shows the inhibition zone for Bacillus species, specifically the average inhibition zone for Bacillus species against E. cecorum strain. Error bars indicate the standard deviation. The raw data is shown in Table 1. [Figure 2] This graph shows the inhibition zone for Bacillus species, specifically the average inhibition zone for Bacillus species and strains relative to E. cecorum strain. Error bars indicate the standard deviation. Raw data is presented in Tables 2-5. [Modes for carrying out the invention]

[0014] I. Terminology The following explanations of terms and abbreviations are provided to better describe this disclosure and to guide those skilled in the art in implementing it. Where used herein, unless the context clearly indicates otherwise, “comprising” means “including,” and the singular forms “a,” “an,” or “the” include multiple referenced objects. The term “or” refers to a single element or combination of two or more elements of the alternative elements described, unless the context clearly indicates otherwise.

[0015] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure relates. Similar or equivalent methods and materials may be used in the implementation or testing of this disclosure, but suitable methods and materials are described below. Materials, methods, and examples are illustrative and not intended to be limiting. Other features of the present invention are evident from the following detailed description and claims.

[0016] Unless otherwise indicated, all figures used herein or in the claims, representing component amounts, molecular weights, percentages, temperatures, times, etc., should be understood to be modified by the term “approximately.” Therefore, unless implicitly or explicitly indicated otherwise, the numerical parameters shown are approximations and may depend on the desired properties and / or detection limits under standard test conditions / methods. Unless otherwise indicated in the context, “approximately” refers to plus or minus 5% of a baseline value. For example, “approximately” 100 refers to 95–105. Where an aspect is directly and explicitly distinguished from the prior art discussed, the aspect number is not an approximation unless the word “approximately” is enumerated.

[0017] Administration: Administer via any route to subjects such as poultry, ruminants, or aquatic species.

[0018] Antimicrobial agents: Agents that kill and / or inhibit the growth of microorganisms. As used herein, antimicrobial agents include antibiotics, antifungal agents, antiviral agents, and antiparasitic agents, including anticoccidial agents, or combinations thereof.

[0019] Carrier: A substance used as an additive in (or together with) any combination, composition, or component disclosed herein. Where used herein, the carrier may be incorporated into the particles of the combination, composition, or component, or physically mixed with the particles of the combination, composition, or component. Carriers can be used to modify non-biological properties of the combination or composition, such as fluidity, stability during storage, or exposure to moisture. Examples of carriers are included herein.

[0020] Colony-forming unit (CFU): A "colony-forming unit" refers to an individual bacterial colony. A colony is a cluster of individual bacteria growing together. In certain aspects, a colony may, but not necessarily, contain substantially the same species and substantially the same strain. CFU is a measure of the number of bacteria present in or on a sample surface. However, since colonies can be formed from a single cell or spore, or a cluster of cells or spores, CFU is not necessarily a measure of individual cells or spores.

[0021] Combinations: A combination comprises two or more components administered such that the shelf life of at least one component overlaps with the shelf life of at least one other component. The combination, or its components, may be a composition. In some embodiments, the shelf lives of all administered components overlap with each other. In an exemplary embodiment of a combination containing three components, the shelf life of the first administered component may overlap with the shelf lives of the second and third components, but the shelf lives of the second and third components may overlap with each other independently, or they may not. In another exemplary embodiment of a combination containing three components, the shelf life of the first administered component overlaps with the shelf life of the second component but not with the shelf life of the third component, and the shelf life of the second component overlaps with the shelf lives of the first and third components. A combination may be a composition containing components, a composition containing one or more components and another distinct component (or more components), or a composition (or more) containing the remaining components, or a combination may be two or more individual components. In some embodiments, the two or more components may include the same component administered at two or more different times, two or more different components administered substantially simultaneously or sequentially in any order, or a combination thereof.

[0022] Bacillus combination: refers to a combination or composition of DFM containing three Bacillus species selected from Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. A Bacillus combination essentially consisting of Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis does not contain any other Bacillus species, but in some embodiments it may include, for example, additives, carriers, compounds, chemicals, foods, nutritional supplements, or vitamins. In some disclosed embodiments, “Bacillus combination” refers to a composition for administration to a subject, in particular to an animal, for example, birds such as chickens and turkeys, and consisting of, or essentially consisting of, Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. In other embodiments, “Bacillus combination” refers to Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis administered in combination without any other DFM. Those skilled in the art will understand that a Bacillus combination may include additional residues carried over from the production of any or all of the three Bacillus species, such as dried dairy products and / or carriers, which do not substantially affect the structure, function, novel features and / or basic characteristics of the Bacillus species.

[0023] Direct feeding microorganisms: Compositions containing living and / or viable microorganisms, typically bacteria and / or yeast, that provide beneficial effects to animals.

[0024] Feed conversion ratio: A measure of the efficiency with which an animal converts feed mass into weight gain. Typically, the feed conversion ratio can be expressed as a dimensionless number, as it is calculated by dividing pounds of feed by pounds of weight gain. In the art, the feed conversion ratio is also known as the feed conversion ratio or feed efficiency.

[0025] Mannan: A class of polysaccharides containing the sugar mannose. The mannan family includes pure mannan (i.e., the polymer backbone contains a mannose monomer), glucomannan (the polymer backbone contains mannose and glucose), and galactomannan (a mannan or glucomannan in which a single galactose residue is linked to the polymer backbone). Mannan is found in the cell walls of several plant species and yeasts and may be supplied as extracts of such plant species and / or yeasts.

[0026] Mineral clay: The term "mineral clay" refers to hydrated aluminum silicate. Mineral clay typically contains small amounts of impurities such as potassium, sodium, calcium, magnesium, and / or iron.

[0027] Saponins are a class of compounds and one of many secondary metabolites found in natural sources. Saponins are particularly abundant in various plant species, such as Quillaja and Yucca. More specifically, saponins are amphiphilic glycosides, classified structurally by their composition. In certain embodiments, saponins contain one or more hydrophilic glycoside moieties, such as lipophilic triterpenes or triterpene derivatives, steroids or steroid derivatives, or combinations thereof.

[0028] A strain refers to two members of the same species that share distinguishable phenotypic and / or genetic differences.

[0029] Subject: Any animal, but especially domesticated animals (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), and aquatic species (e.g., species used in aquaculture, such as tilapia). Most typically, “subject” in this specification refers to birds, including poultry such as chickens and turkeys.

[0030] Effective amount: The amount or concentration of a specified compound, composition, or combination sufficient to achieve the desired effect in the target area.

[0031] Vitamins: Vitamin A, Vitamin B1 (Thiamine), Vitamin B2 (Riboflavin), Vitamin B3 (Niacin or Niacinamide), Vitamin B5 (Pantothenic Acid), Vitamin B6 (Pyridoxine, Pyridoxal, or Pyridoxamine, or Pyridoxine Hydrochloride), Vitamin B7 (Biotin), Vitamin B9 (Folic Acid), Vitamin B 12 This includes various cobalamins (cyanocobalamins commonly found in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, K1 and K2 (i.e., MK-4, MK-7), folic acid and biotin, as well as their derivatives and analogues.

[0032] Further disclosures are presented in U.S. Patent Application No. 14 / 699,740, U.S. Patent Application No. 13 / 566,433, U.S. Patent Application No. 13 / 872,935, U.S. Patent Publication No. 2013 / 0017211, U.S. Patent Publication No. 2012 / 0156248, U.S. Patent Publication No. 2007 / 0253983, U.S. Patent Publication No. 2007 / 0202092, U.S. Patent Publication No. 2007 / 0238120, U.S. Patent Publication No. 2006 / 0239992, U.S. Patent Publication No. 2005 / 0220846, U.S. Patent Publication No. 2005 / 0180964, and Austrian Patent Application No. 2011 / 201420, each of which is incorporated herein by reference in its entirety.

[0033] II. Overview Enterococcus cecorum is an emerging pathogen in poultry worldwide. While traditionally considered a symbiotic bacterium in the digestive tract of animals, the proportion of pathogenic E. cecorum strains in poultry is increasing. E. cecorum is a Gram-positive, facultative anaerobic bacterium and is increasingly recognized as a causative agent of enterococcal spondylitis in chickens. The main symptom of enterococcal spondylitis is impaired spontaneous movement resulting from necrosis of the femoral head. Outbreaks result in high morbidity and mortality rates, leading to mass culling, carcass disposal, and significant economic losses for the poultry industry. Because E. cecorum has only recently emerged as a devastating pathogen, best practices for its control have not yet been established.

[0034] Clostridium species are other bacterial pathogens that pose a problem in agricultural industries, including ruminant production. These bacteria are Gram-positive anaerobic and are generally considered opportunistic pathogens. Infections can develop in animals after they consume contaminated feed or through open wounds. Symptoms of C. perfringens infection in poultry include diarrhea, intestinal lesions, decreased nutrient absorption, and weight loss, and can cause mortality of up to 50%. Symptoms of Clostridium perfringens infection in cattle include intestinal disorders, hemorrhagic enterocolitis, calf growth retardation, blindness, and sudden death. Effective control measures to prevent Clostridium infections are important for agriculture and livestock farming.

[0035] III. Bacillus combinations A Bacillus combination is essentially composed of, or a combination or composition of, three Bacillus species selected from Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. In some embodiments, a Bacillus combination is essentially composed of Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. That is, a Bacillus combination does not contain any additional Bacillus species other than Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. In other embodiments, a Bacillus combination consists of Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis. In some embodiments, a Bacillus combination does not contain additional DFM.

[0036] Certain aspects of this disclosure relate to the finding that administering the disclosed combination of Bacillus to a subject provides a substantial benefit to the subject compared to a subject that does not receive the combination. In particular with respect to poultry, the disclosed combination of Bacillus provides a substantial benefit to poultry with respect to the incidence of Enterococcus cecorum, Salmonella species, Escherichia coli, and / or Clostridium perfingens (CP), and / or feed conversion ratio, mean body weight, mean body weight gain, coefficient of weight variation, poultry mortality, lesion score, and / or oocysts in fecal matter, compared to poultry that do not receive any of these Bacillus, poultry that receive one, or poultry that receive two of any combination.

[0037] A. bacillus strain Those skilled in the art will understand that any strain or combination of strains of Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis may be used in a Bacillus combination. Where used herein, the terms “Bacillus licheniformis,” “Bacillus pumilus,” and “Bacillus subtilis” may independently refer to a single strain of each Bacillus species, or to multiple strains of each Bacillus species, such as two, three, four, five, six, seven, eight, nine, ten, or more strains. In certain embodiments, a Bacillus combination includes a single strain of Bacillus licheniformis, a single strain of Bacillus pumilus, and a single strain of Bacillus subtilis. Specific acceptable exemplary strains of each Bacillus species are listed below, for illustrative purposes only and without limitation.

[0038] Bacillus licheniformis strain Bacillus licheniformis (Weigmann) Chester ATCC (Registered Trademark) 6598 (Trademark) strain NRS745, Bacillus licheniformis (Weigmann) Chester ATCC (Registered Trademark) 6634 (Trademark) strain NRS304, Bacillus licheniformis (Weigmann) Chester ATCC (Registered Trademark) 8480 (Trademark) strain NRS1128, Bacillus licheniformis (Weigmann) Chester ATCC (Registered Trademark) 9259 (Trademark), Bacillus licheniformis (Weigmann) Chester ATCC(Registered Trademark) 9789(Trademark) Co. AMNH723, ATCC102, ATCC4527, ATCC8243, ATCC9800, NCTC2586, NCTC6346, NRS243, NRS978, W.Ford1, DSM8785, DSM46308, BU171, CCDBb-30, CCEB631, CCM2205, CN1060, HNCMB101012, IFO12195, IFO12196, IMET11025, NBRC12195, NBRC12196, NCDO735, NCDO835, NCIB6346, NCIB8059, NCIB8061, OUT8367, OUT8368, Smith243, Smith978, Bacillus by (Ehrenberg) Cohn HankeyB13, Bacillus licheniformis (Weigmann) Chester ATCC (Registered Trademark) 9945 (Trademark) strain NRS712, NCIB8062, Bacillus licheniformis (Weigmann) Chester ATCC (Registered Trademark) 9945a (Trademark) strain CD-2, NCIB11709, Bacillus licheniformis (Weigmann) ChesterATCC(Registered Trademark) 10716(Trademark) Stock ATCC11944, BS2181, Boots1343, CCM2181, FDABT1, NCIB8874, NRS1330, TracyI, DSM603, IFO12199, NBRC12199, Bacillus licheniformis (Weigmann) Chester ATCC(Registered Trademark) 11945(Trademark) Stock 1331, FDABT3, Bacillus licheniformis (Weigmann) Chester ATCC(Registered Trademark) 11946(Trademark) Stock 1333, B-1001, Bacillus licheniformis (Weigmann) Chester deposited with ATCC as Bacillus subtilis by (Ehrenberg) Cohn ATCC(Registered Trademark) 12139(Trademark) Stock CSC, Bacillus licheniformis (Weigmann) Chester ATCC® 12713(TM) strain PRLB479, NRRL B-1001, Bacillus licheniformis (Weigmann) Chester ATCC(R) 12759(TM) strain ATCC11560, Damodaron P-8, LMG7560, NRS1415, Vitek#200148, NCIB8549, HankeyB133, P8, Bacillus licheniformis (Weigmann) Chester ATCC(R) 12759-MINI-PACK(TM) strain ATCC11560, Damodaron P-8, LMG7560, NRS1415, Vitek#200148, Bacillus licheniformis (Weigmann) Chester ATCC® 13438® Strain NCTC8233, M.II Strain, deposited with ATCC as Bacillus abysseus by ZoBell and Upham, Bacillus licheniformis (Weigmann) Chester; ATCC® 14409® Strain 620, NRS1114, NCIB1042, Bacillus licheniformis (Weigmann) ChesterATCC(registered trademark) 14580(trademark) strain (Gibson) 46, NCIB9375, NCTC10341, NRS1264, DSM13, CCM2145, IFO12200, NBRC12200, WDCM00068, Bacillus licheniformis (Weigmann) Chester ATCC(registered trademark) 14580D-5(trademark) strain name: Genomic DNA derived from Bacillus licheniformis strain 46 [ATCC(registered trademark) 14580(trademark)], Bacillus licheniformis (Weigmann) Chester ATCC(registered trademark) 14594(trademark), Bacillus licheniformis (Weigmann) Chester ATCC(registered trademark) 21038(trademark) strain L-065, Bacillus licheniformis (Weigmann) Chester ATCC(registered trademark) 21039(trademark), Bacillus licheniformis (Weigmann) Chester deposited with ATCC as Bacillus subtilis by (Ehrenberg) Cohn ATCC(registered trademark) 21415(trademark) strain NS1, Bacillus licheniformis (Weigmann) Chester deposited with ATCC as Bacillus subtilis by (Ehrenberg) Cohn ATCC(registered trademark) 21417(trademark) strain M, Bacillus licheniformis (Weigmann) Chester deposited with ATCC as Bacillus subtilis by (Ehrenberg) Cohn ATCC(registered trademark) 21418(trademark), Bacillus licheniformis (Weigmann) Chester ATCC(registered trademark) 21424(trademark) strain DSM1969, Bacillus by (Ehrenberg) Cohn Bacillus licheniformis (Weigmann) Chester, deposited with ATCC as a subtilis, ATCC (Registered Trademark) 21610 (Trademark), Stock B-201-7, Bacillus licheniformis (Weigmann) ChesterATCC(Registered Trademark) 21667(Trademark) Stock FD23612, Bacillus licheniformis (Weigmann) Chester deposited with ATCC as Bacillus subtilis by (Ehrenberg) Cohn, ATCC(Registered Trademark) 21733(Trademark) Stock DSM1913, Bacillus licheniformis (Weigmann) Chester, ATCC(Registered Trademark) 25972(Trademark) Stock 749 / C, DSM8782, DSM46217, IMET10723, NCIB9443, Bacillus licheniformis (Weigmann) Chester, ATCC(Registered Trademark) 27326(Trademark) Stock OM-81, Bacillus licheniformis (Weigmann) Chester, ATCC(Registered Trademark) 27811(Trademark) Stock 584, FERM-P1038, Bacillus licheniformis (Weigmann) Chester ATCC(R) 31667(TM) strain DG14, Bacillus licheniformis(Weigmann) Chester ATCC(R) 31972(TM) strain PM-3, Bacillus licheniformis(Weigmann) Chester ATCC(R) 33632(TM) strain (IOC) 2390, NCIB11672, Bacillus licheniformis(Weigmann) Chester ATCC(R) 39326(TM), Bacillus licheniformis(Weigmann) Chester ATCC(R) 53757(TM) strain PWD-1, Bacillus licheniformis(Weigmann) Chester ATCC® 53926(TM) strain E312, Bacillus licheniformis (Weigmann) Chester ATCC (Registered Trademark) 55768 (Trademark) Strain OWU138B [OWU138B], Bacillus licheniformis (Weigmann) Chester strain DSM15, C, Bacillus licheniformis (Weigmann) Chester strain DSM392, BacillusBacillus licheniformis (Weigmann) Chester strain DSM394, Bacillus licheniformis (Weigmann) Chester strain DSM7259, NRRL-NRS1263, Bacillus licheniformis (Weigmann) Chester strain DSM7459, Bacillus licheniformis (Weigmann) Chester strain DSM11258, Bacillus licheniformis (Weigmann) Chester strain DSM11259, Bacillus licheniformis (Weigmann) Chester strain DSM12369, Bacillus licheniformis (Weigmann) Chester strain DSM12370, Bacillus licheniformis (Weigmann) Chester strain DSM26543, Bacillus licheniformis (Weigmann) Chester strain DSM28096, Bacillus licheniformis (Weigmann) Chester strain DSM28591, Bacillus licheniformis (Weigmann) Chester strain DSM30523, Bacillus licheniformis (Weigmann) Chester strain DSM30535, Bacillus licheniformis (Weigmann) Chester strain DSM30542, Bacillus licheniformis (Weigmann) Chester strain DSM30585, Bacillus licheniformis (Weigmann) Chester strain DSM30615, Bacillus licheniformis (Weigmann) Chester strain DSM30620, Bacillus licheniformis (Weigmann) Chester strain DSM30624, Bacillus licheniformis (Weigmann) Chester strain DSM30643, Bacillus licheniformis (Weigmann) Chester strain DSM30654, Bacillus licheniformis (Weigmann) Chester strain DSM30724, BacillusBacillus licheniformis (Weigmann) Chester strain DSM30766, Bacillus licheniformis (Weigmann) Chester strain DSM30769, Bacillus licheniformis (Weigmann) Chester strain DSM30778, Bacillus licheniformis (Weigmann) Chester strain DSM30779, Bacillus licheniformis (Weigmann) Chester strain DSM30865, Bacillus licheniformis (Weigmann) Chester strain DSM30926, Bacillus lich Bacillus eniformis (Weigmann) Chester strain DSM30959, Bacillus licheniformis (Weigmann) Chester strain DSM30960, Bacillus licheniformis (Weigmann) Chester strain DSM30961, Bacillus licheniformis (Weigmann) Chester strain DSM30976, Bacillus licheniformis (Weigmann) Chester strain DSM31019, Bacillus licheniformis (Weigmann) Chester strain DSM100653, Bacillus licheniformis (Weigmann) Chester strain DSM100655, Bacillus licheniformis (Weigmann) Chester strain DSM103059, Bacillus licheniformis (Weigmann) Chester strain NCIB1525, 1229, Bacillus licheniformis (Weigmann) Chester strain NCIB6816, Glaxo417, Bacillus licheniformis (Weigmann) Chester strain NCIB7224, Loos, Bacillus licheniformis (Weigmann) Chester strain NCIB8536, P1, Bacillus licheniformis (Weigmann) Chester strain NCIB8537, Ho, Bacillus licheniformis (Weigmann) Chester strain NCIB9536, Gibson1319, NRS1553, Bacillus licheniformis (Weigmann) Chester strain NCIB9667, 1, Bacillus licheniformis (Weigmann) Chester strain NCIB9668, 2, Bacillus licheniformis (Weigmann) Chester strain NCIB9669, 3, Bacillus licheniformis (Weigmann) Chester strain NCIB10689, Bacillus licheniformis (Weigmann) Chester strain NCIB11143, BacillusBacillus licheniformis (Weigmann) Chester strains NCIB11643, YNS7712R, Bacillus licheniformis (Weigmann) Chester strains NCIB13497, Bacillus licheniformis (Weigmann) Chester strains NCIB14014, DA33, Bacillus licheniformis B1 (NRRL deposit number B-50907), Bacillus subtilis B2 (deposit number B-50908), Bacillus licheniformis RW25 (NRRL deposit number B-50911), Bacillus licheniformis RW32 (NRRL deposit number B-50912), and Bacillus licheniformis RW41 (NRRL deposit number B-50913), Bacillus licheniformis BL21 (NRRL Hansen Bio Systems), and Bacillus licheniformis OBT618 (NRRL-B-23318).

[0039] Bacillus pumilus strain Bacillus pumilus OBT13216 (ATCC number or NRRL-B-68101).

[0040] Bacillus subtilis strain Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 82(Trademark) strain AMC, ATCC8037, NRS315, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 82D-5(Trademark) strain name: Genomic DNA derived from Bacillus subtilis strain AMC [ATCC(Registered Trademark) 82(Trademark)], Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 465(Trademark) strain NRS743, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 4529(Trademark) strain 3 deposited with ATCC as Bacillus vulgatus by Trevisan, ATCC8013, NCTC2588, NRS1004, Bacillus subtilis(Ehrenberg)Cohn deposited with ATCC as Bacillus nigrificans by Fabian and Nienhuis ATCC(registered trademark) 4925(trademark) strain NRS740, Bacillus parvus, deposited with ATCC as Bacillus subtilis(Ehrenberg) Cohn. ATCC(registered trademark) 4944(trademark) strain NCTC, NRS1106, Bacillus subtilis(Ehrenberg) Cohn, a subspecies of Bacillus subtilis(Ehrenberg) Cohn, deposited with ATCC as Bacillus subtilis(Ehrenberg) Cohn. ATCC (registered trademark) 6051 (trademark) strain Marburg strain, ATCC6051-U, CCM2216, CCRC10255, CCUG163B, CFBP4228, CIP52.65, DSM10, IAM12118, IFO12210, IFO13 719, IFO16412, IMET10758, JCM1465, LMG7135, NCAIMB.01095, NCCB32009, NCCB53016, NCCB70064, NCFB1769, NCIB3610, NCTC3610, NRRL B-4219, NRS1315, NRS744, VKMB-501, NBRC13719, Bacillus subtilis (Ehrenberg) Cohn ATCC(R) 6051a(TM) strain P31K6, phiThe Bacillus subtilis bacteriophage phi-e, ATCC® 6051-B1® strain Phi-e, deposited with ATCC as e; the Bacillus subtilis (Ehrenberg) Cohn, ATCC® 6460® strain NRS259, deposited with ATCC by Lehmann and Neumann as Bacillus aterrimus; the Bacillus subtilis (Ehrenberg) Cohn, ATCC® 6461® strain NRS275, CN2192, NCIB8055, deposited with ATCC as Bacillus aterrimus; and the Bacillus subtilis subspecies spizizenii Nakamura et al., deposited with ATCC as Bacillus subtilis (Ehrenberg) Cohn. ATCC(registered trademark) 6633(trademark) strains NRS231, DSM347, CCM1999, IAM1069, NCIB8054, NCTC10400, WDCM00003, and Bacillus subtilis subspecies spizizenii Nakamura et al. deposited with ATCC as Bacillus subtilis (Ehrenberg) Cohn. ATCC(registered trademark) 6633D-5(trademark) strain name: Genomic DNA derived from Bacillus subtilis subspecies spizizenii strain NRS231 [ATCC(registered trademark) 6633(trademark)], and Bacillus subtilis subspecies spizizenii Nakamura et al. deposited with ATCC as Bacillus subtilis (Ehrenberg) Cohn. ATCC(registered trademark) CRM-6633(trademark) strain NRS231, and Bacillus subtilis (Ehrenberg) Cohn deposited with ATCC as Bacillus The subspecies *spizizenii* Nakamura et al. ATCC(registered trademark) 6633-MINI-PACK(trademark) strain NRS231, and the subspecies *hydrolyticus* of *Bacillus vulgatus* deposited with ATCC as *Bacillus subtilis* (Ehrenberg) CohnATCC(Registered Trademark) 6984(Trademark) Stock NRS747, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 7003(Trademark) Stock NRS730, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 7058(Trademark) Stock NRS351, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 7059(Trademark) Stock NRS352, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 7060(Trademark) Stock NRS659, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 7067(Trademark) Stock NRS238, ATCC7974, ATCC8012, Bacillus subtilis(Ehrenberg) Cohn deposited with ATCC as Bacillus endoparasiticus by Benedek ATCC(registered trademark) 7480(trademark) strain NRS1107, deposited with ATCC as Tyrothrix minimus, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 8188(trademark) strain ATCC8450, NRS773, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 8473(trademark) strain NRS762, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 9466(trademark) strain name: FDA strain PCI220 [BUCSAV170, NCIB8159, NRRL B-558, NRS1088], Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 9524(trademark) strain 3R9675, NRS1109, Bacillus subtilis (Ehrenberg) Cohn ATCC(R) 9799(TM) Strain NCTC6276, NRS1125, Bacillus subtilis (Ehrenberg) Cohn ATCC(R) 9858(TM) Strain NRS237, NCIB8063, Bacillus subtilis(Ehrenberg) Cohn ATCC(R) 9943(TM) Strain NRS979, BacillusBacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 10774(Trademark) Stocks BU169, NCIB8872, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 10783(Trademark) Stocks NRRL B-543, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 11774(Trademark) Stocks NCTC8236, DSM2109, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 11838(Trademark) Stocks AMC46-A-6(Stock I), NCIB8850, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 12100(Trademark) Stocks NCA1558, ND957, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 12432(Trademark) Stock MB32, 56R188, ATCC13597, NCIB8993, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 12695(Trademark) Stock 51-52, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 12711(Trademark) Stock PRL B92, Ra, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 13542(Trademark), Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 13933(Trademark) Stock NRRL B-1471, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 13952(Trademark) Stock 1346, Bacillus deposited with ATCC as Bacillus borborokoites by ZoBell and Upham Bacillus subtilis(Ehrenberg)Cohn ATCC (Registered Trademark) 14410 (Trademark) Strain 625, NRS 1115 Bacillus, deposited with ATCC as Bacillus submarinus by ZoBell and Upham. Bacillus subtilis(Ehrenberg)Cohn ATCC (Registered Trademark) 14415 (Trademark) Strain 569, NRS 1120, by ZoBell and Upham.Bacillus subtilis(Ehrenberg)Cohn, deposited with ATCC as thalassokoites, ATCC(registered trademark) 14416(trademark) stock 576, NRS1121, Bacillus subtilis(Ehrenberg)Cohn, ATCC(registered trademark) 14593(trademark) stock IAM1145, Bacillus subtilis(Ehrenberg)Cohn, ATCC(registered trademark) 14617(trademark) stock A-1625, Bacillus subtilis(Ehrenberg)Cohn, ATCC(registered trademark) 14660(trademark) stock C30-1, Bacillus subtilis(Ehrenberg)Cohn, ATCC(registered trademark) 14662(trademark) stock C30-109, Bacillus subtilis(Ehrenberg)Cohn, ATCC(registered trademark) 14807(trademark) stock MB-155, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 15040(Trademark) Stock SX-67, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 15041(Trademark) Stock SX-92, Bacillus subtilis(Ehrenberg) Cohn deposited with ATCC by Mann as Bacillus uniflagellatus ATCC(Registered Trademark) 15134(Trademark), Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 15183(Trademark) Stock 309, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 15244(Trademark) Stock 3369, Bacillus subtilis(Ehrenberg) Cohn deposited with ATCC by Sawamura as Bacillus natto ATCC(Registered Trademark) 15245(Trademark) Stock 3349, IAM1-3, Bacillus subtilis(Ehrenberg) Cohn ATCC (Registered Trademark) 15476 (Trademark) Stock M-4-45, Bacillus subtilis (Ehrenberg) Cohn deposited with ATCC as Bacillus pumilus by Meyer and Gottheil.ATCC(registered trademark) 15477(trademark) strain M-24-1, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 15561(trademark) strain KX-1, A-1, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 15563(trademark) strain Marburg, Bacillus subtilis deposited with ATCC as SP8 bacteriophage s bacteriophage SP8 ATCC(registered trademark) 15563-B1(trademark) strain SP8, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 15575(trademark) strain SB19, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 15811(trademark) strain 5380, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 15818(trademark) strain RIA445, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 15819(trademark) strain RIA447, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 15841(trademark), Sa bacteriophage deposited with ATCC as Bacillus subtilis bacteriophage Sa ATCC(Registered Trademark) 15841-B1(Trademark) Stock Sa, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 19659(Trademark) Stock PRD66, IFO13722, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 19659-MINI-PACK(Trademark) Stock PRD66, IFO13722, Bacillus subtilis(Ehrenberg) Cohn deposited with ATCC as Bacillus pumilus by Meyer and Gottheil ATCC(Registered Trademark) 21008(Trademark) Stock 182-H-86, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 21183(Trademark) Stock 5221, Bacillus subtilis(Ehrenberg) Cohn ATCC(registered trademark) 21228(trademark) strain SC8548, SO-4, DSM1970, Bacillus subtilis (Ehrenberg) Cohn; ATCC(registered trademark) 21331(trademark) strain IFO35, Bacillus subtilis (Ehrenberg) Cohn; ATCC(registered trademark) 21332(trademark) strain IAM1213, Bacillus subtilis (Ehrenberg) Cohn deposited with ATCC as Bacillus subtilis subspecies sakainensis.ATCC(Registered Trademark) 21394(Trademark) Strain 4-3-Ky, DSM1971, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 21555(Trademark) Strain Y13, Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 21556(Trademark), Bacillus subtilis(Ehrenberg) Cohn ATCC(Registered Trademark) 21742(Trademark) Strain AHr-5, Bacillus subtilis(Ehrenberg) Cohn deposited with ATCC as Bacillus cereus by Frankland and Frankland ATCC(Registered Trademark) 21770(Trademark) Strain SP-3, Bacillus subtilis(Ehrenberg) Cohn deposited with ATCC as Bacillus pumilus by Meyer and Gottheil ATCC(registered trademark) 21951(trademark) strain 716, IFO13322, Bacillus subtilis(Ehrenberg) Cohn ATCC(registered trademark) 23059(trademark) strain W23, Bacillus subtilis(Ehrenberg) Cohn ATCC(registered trademark) 23856(trademark) strain EMG50, SB19, Bacillus subtilis(Ehrenberg) Cohn ATCC(registered trademark) 23857(trademark) strain 168, Bacillus subtilis(Ehrenberg) Cohn ATCC(registered trademark) 23857D-5(trademark) strain name: Genomic DNA derived from Bacillus subtilis strain 168 [ATCC(registered trademark) 23857(trademark)], Bacillus subtilis(Ehrenberg) Cohn ATCC(registered trademark) 23858(trademark) strain EMG52, Bacillus subtilis(Ehrenberg) Cohn ATCC(registered trademark) 23859(trademark) strain EMG53, deposited with ATCC as Bacillus pulvifaciens by Nakamura, Bacillus subtilis(Ehrenberg)Cohn ATCC(registered trademark) 25369(trademark) strain 24028, Bacillus subtilis(Ehrenberg)CohnATCC(registered trademark) 27328(trademark) strain C, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 27370(trademark) strain 168M, Bacillus subtilis bacteriophage SPO1 deposited with ATCC as SPO1 ATCC(registered trademark) 27370-B1(trademark) strain SPO1, Bacillus subtilis (Ehrenberg) Cohn deposited with ATCC as Bacillus subtilis subspecies amyloliquefaciens ATCC(registered trademark) 27505(trademark) strain K49, HER1346, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 27689(trademark) strain SB168(trp-), Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 29056(trademark) strain SB100, Bacillus Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 29233(Trademark) strain X6, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 31002(Trademark) strain Ahr.AUr-9, FERM-1998, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 31028(Trademark) strain FD6404 deposited with ATCC as Bacillus globigii by Migula, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 31091(Trademark) strain 1054, IFO13586, Bacillus subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 31094(Trademark) strain 1097, IFO13621, Bacillus deposited with ATCC as Bacillus pumilus by Meyer and Gottheil subtilis(Ehrenberg)Cohn ATCC(Registered Trademark) 31098(Trademark) Strain 1027, IFO13585, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. ATCC(Registered Trademark) 31578(Trademark) Strain DSM6223, RUB331, Bacillus subtilis(Ehrenberg)CohnATCC(R) 31954(TM) strain MO7S-16 / 11, Bacillus subtilis(Ehrenberg) Cohn ATCC(R) 33234(TM) strain NCIB10106, Bacillus subtilis(Ehrenberg) Cohn ATCC(R) 35021(TM) strain 5230, NRS6, Bacillus subtilis(Ehrenberg)Cohn ATCC(R) 35854(TM) strain NRRL B-3411, Bacillus subtilis (Ehrenberg) Cohn ATCC(R) 35946(TM) strain OSU75, Bacillus subtilis subspecies subtilis(Ehrenberg) Nakamura et al. ATCC(R) 37014(TM) strain DSM6224, BD170, pSA2100, Bacillus Bacillus subtilis subtilis(Ehrenberg) Nakamura et al. ATCC(Registered Trademark) 37015(Trademark) strains DSM4514, BD170, NCIB11624, pUB110, Bacillus subtilis subtilis(Ehrenberg) Nakamura et al. ATCC(Registered Trademark) 37108(Trademark) strains DSM4873, BGSC1E32, BR151, pPL608, Bacillus subtilis subtilis(Ehrenberg) Nakamura et al. ATCC(Registered Trademark) 37128(Trademark) strains DSM4554, BGSC1E18, pE194, Bacillus subtilis subtilis(Ehrenberg) Nakamura et al. ATCC(Registered Trademark) 39090(Trademark) strains DSM6198, BGSC1S53, Bacillus Bacillus subtilis (Ehrenberg) Cohn ATCC® 39320 (Trademark) strain MB4488, Bacillus subtilis (Ehrenberg) Cohn ATCC® 39374 (Trademark) strain MB3575, Bacillus subtilis (Ehrenberg) Cohn ATCC® 39706 (Trademark) strain B1-20, Bacillus subtilis (Ehrenberg) CohnATCC(Registered Trademark) 43223(Trademark) Stock ABM261, Bacillus subtilis (Ehrenberg) Cohn ATCC(Registered Trademark) 49343(Trademark) Stock IMVS0101, Bacillus subtilis (Ehrenberg) Cohn deposited with ATCC by Migula as Bacillus globigii ATCC(Registered Trademark) 49760(Trademark), Bacillus subtilis (Ehrenberg) Cohn deposited with ATCC by Migula as Bacillus globigii ATCC(Registered Trademark) 49822(Trademark), Bacillus subtilis (Ehrenberg) Cohn ATCC(Registered Trademark) 55033(Trademark) Stock SMS274, Bacillus subtilis (Ehrenberg) Cohn ATCC(Registered Trademark) 55060(Trademark) Stock MB4974, Bacillus subtilis (Ehrenberg) Cohn ATCC(registered trademark) 55405(trademark) strain 300, Bacillus subtilis subspecies inaquosorum deposited with ATCC as Bacillus licheniformis (Weigmann) Chester, ATCC(registered trademark) 55406(trademark) strain DA33, Bacillus subtilis (Ehrenberg) Cohn, ATCC(registered trademark) 55422(trademark) strain SC15257, Bacillus subtilis (Ehrenberg) Cohn, ATCC(registered trademark) 55614(trademark) strain 1.2, AQ153, Bacillus subtilis (Ehrenberg) Cohn, ATCC(registered trademark) 55675(trademark) strain BP01, Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al. strain DSM402, BRC111470, NCIB10106, Bacillus subtilis subspecies spizizeni Nakamura et al. al. strain DSM618, Bacillus subtilis subspecies spizizeni Nakamura et al. strain DSM1087, Bacillussubtilis(Ehrenberg)Cohn strain DSM1088, IFO13169, NBRC13169, OUT8353, Bacillus subtilis(Ehrenberg)Cohn strain DSM1089, IFO3026, NBRC3026, OUT8350, Bacillus subtilis subtilis(Ehrenberg)Nakamura et al. strain DSM1090, OUT8424, Bacillus subtilis subtilis( Ehrenberg) Nakamura et al. strain DSM1091, OUT8425, Bacillus subtilis (Ehrenberg) Cohn strain DSM1092, IFO3009, NBRC3009, OUT8235, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM3256, IAM1213, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM3257, IAM1259, Bacillus subtilis (Ehrenberg) Cohn strain DSM3258, IAM1260, Bacillus subtilis (Ehrenberg) Cohn strain DSM4181, NCA72-52, SA22, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM4393, pC194, SB202, Bacillus subtilis (Ehrenberg) Cohn strain DSM4449, natto3335UM4, Bacillus subtilis (Ehrenberg) Cohn strain DSM4450, natto3335UM8, pLS20, pBC16, Bacillus subtilis (Ehrenberg) Cohn strain DSM4451, Bacillus subtilis (Ehrenberg) Cohn strain DSM4515, DB163, pGR71, Bacillus subtilis (Ehrenberg) Cohn strain DSM4608, BR157, pMW1, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM4750, 1E7, BGSC1E7, pE194-cop6, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM4751, 1E34, BGSC1E34, pAM77, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM4871, BD426, BGSC1E21, pBD8, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura etal. Strain DSM4872, BD466, BGSC1E24, pBD10, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. Strain DSM4874, BGSC1E38, pMK3, YB886, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM5213, BGSC1A40, BR151, Bacillus subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM5214, BD393, BGSC1A511, Bacillus subtilis subsp. subtilis subsp. subtilis (Ehrenberg) Nakamura et al. strain DSM5547, Bacillus Bacillus subtilis (Ehrenberg) Cohn strain DSM5552, Bacillus subtilis (Ehrenberg) Cohn strain DSM5611, NRRL B-360, Bacillus subtilis subspecies subtilis (Ehrenberg) Nakamura et al. strain DSM5660, NRRL B-362, Bacillus subtilis subspecies spizizenii Nakamura et al. strain DSM6395, BGSC2A2, W232A2, WB672, Bacillus subtilis (Ehrenberg) Cohn strain DSM6397, BGSC1A2, SB491, Bacillus subtilis subspecies spizizenii Nakamura et al. strain DSM6399, BGSC2A1, SB623, Bacillus subtilis subspecies spizizenii Nakamura et al. strain DSM6405, BGSC2A3, W23SR, Bacillus subtilis subtilis(Ehrenberg)Nakamura et al. strain DSM6887, BGSC1A309, NP40, Bacillus subtilis subtilis(Ehrenberg)Nakamura et al.al. strain DSM6889, 1A658, BGSC1A658, DA65 Bacillus subtilis subsp. spizizenii Nakamura et al. strain DSM8439, CCM2268, IAM12021, Bacillus subtilis (Ehrenberg) Cohn strain DSM13019, SSI MK1, Bacillus subtilis subsp. spizizenii Nakamura et al. strain DSM15029, NRRL B-23049, Bacillus subtilis subsp. inaquosorum Rooney et al. strain DSM21200, Bacillus subtilis (Ehrenberg) Cohn strain DSM21393, Bacillus subtilis subsp. inaquosorum Rooney et al. strain DSM22148, KCTC13429, Bacillus subtilis (Ehrenberg) Cohn strain DSM23521, Bacillus subtilis (Ehrenberg) Cohn strain DSM23778, Bacillus subtilis (Ehrenberg) Cohn strain DSM25152, Bacillus subtilis (Ehrenberg) Cohn strain DSM28592, Bacillus subtilis (Ehrenberg) Cohn strain DSM30512, Bacillus subtilis (Ehrenberg) Cohn strain DSM30529, Bacillus subtilis (Ehrenberg) Cohn strain DSM30533, Bacillus subtilis (Ehrenberg) Cohn strain DSM30534, Bacillus subtilis (Ehrenberg) Cohn strain DSM30540, Bacillus subtilis (Ehrenberg) Cohn strain DSM30541, Bacillus subtilis (Ehrenberg) Cohn strain DSM30551, Bacillus subtilis (Ehrenberg) Cohn strain DSM30558, Bacillus subtilis (Ehrenberg) Cohn strain DSM30562, Bacillus subtilis (Ehrenberg) Cohn strain DSM30570, BacillusBacillus subtilis (Ehrenberg) Cohn strain DSM30581, Bacillus subtilis (Ehrenberg) Cohn strain DSM30597, Bacillus subtilis (Ehrenberg) Cohn strain DSM30642, Bacillus subtilis (Ehrenberg) Cohn strain DSM30651, Bacillus subtilis (Ehrenberg) Cohn strain DSM30652, Bacillus subtilis (Ehrenberg) Cohn strain DSM30671, Bacillus subtilis (Ehrenberg) Cohn strain DSM30676, Bacillus subtilis (Ehrenberg) Cohn strain DSM30677, Bacillus subtilis (Ehrenberg) Cohn strain DSM30682, Bacillus subtilis (Ehrenberg) Cohn strain DSM30711, Bacillus subtilis (Ehrenberg) Cohn strain DSM30723, Bacillus subtilis (Ehrenberg) Cohn strain DSM30801, Bacillus subtilis (Ehrenberg) Cohn strain DSM30924, Bacillus subtilis (Ehrenberg) Cohn strain DSM30925, Bacillus subtilis (Ehrenberg) Cohn strain DSM30927, Bacillus subtilis (Ehrenberg) Cohn strain DSM30928, Bacillus subtilis (Ehrenberg) Cohn strain DSM30929, Bacillus subtilis (Ehrenberg) Cohn strain DSM30941, D1, Bacillus subtilis (Ehrenberg) Cohn strain DSM30942, D-FC1, Bacillus subtilis (Ehrenberg) Cohn strain DSM31008, Bacillus subtilis (Ehrenberg) Cohn strain DSM31009, Bacillus subtilis (Ehrenberg) Cohn strain DSM31010, Bacillus subtilis (Ehrenberg) Cohn strain DSM31020, BacillusBacillus subtilis (Ehrenberg) Cohn strain DSM31021, Bacillus subtilis (Ehrenberg) Cohn strain DSM31033, Bacillus subtilis (Ehrenberg) Cohn strain DSM100605, Bacillus subtilis (Ehrenberg) Cohn strain DSM100612, Bacillus subtilis (Ehrenberg) Cohn strain DSM100613, Bacillus subtilis (Ehrenberg) Cohn strain DSM100614, Bacillus subtilis (Ehrenberg) Cohn strain DSM103044, Bacillus subtilis (Ehrenberg) Cohn strain DSM103047, Bacillus subtilis (Ehrenberg) Cohn strain DSM103051, Bacillus subtilis (Ehrenberg) Cohn strain DSM103758, Bacillus subtilis AM0904 (NRRL deposit number B-50914), Bacillus subtilis AM0911 (NRRL deposit number B-50915), Bacillus subtilis NP122 (NRRL deposit number B-50910), Bacillus subtilis NP119B (NRRL deposit number B-50909), Bacillus subtilis BS18 (NRRL B-50633), Bacillus subtilis BS278 (NRRL 50634), Bacillus subtilis 4-7d (NRRL B-50505), Bacillus subtilis 3-5h (NRRL B-50507), Bacillus subtilis AGTP BS3BP5 (NRRL B-50510), Bacillus subtilis BS918 (NRRL B-50508), Bacillus subtilis AGTP BS1013 (NRRL-50509), Bacillus subtilis AGTP 944 (NRRL B-50548), Bacillus subtilis AGTP BS442 (NRRL B-50542), Bacillus subtilis AGTP BS1069 (NRRL B-50544), Bacillus subtilis AGTPBS521 (NRRL B-50545), Bacillus subtilis B27 (NRRL B-50105), Bacillus subtilis 3A-P4 (PTA-6506), Bacillus subtilis 22C-P1 (PTA-6508), Bacillus subtilis BL21 (NRRL B-50134), Bacillus subtilis strain GB03, Bacillus subtilis strain QST713, Bacillus subtilis DSM5750 (BioPlus® 2B, Chr. Hansen Bio Systems), and Bacillus subtili PR104 (NRRL-B-68096).

[0041] IV. Amounts of Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis in combinations of Bacillus The relative amounts of Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis present in the combination of Bacillus are selected to obtain the desired result. In certain embodiments, the combination of Bacillus comprises from about 10 2 to about 10 12 CFU / gram, more typically from about 10 2 to about 10 11 CFU / gram, or from about 10 3 to 10 10 CFU / gram of each Bacillus species in the combination of Bacillus. In some embodiments, the combination of Bacillus comprises from about 10 4 to about 10 6 CFU / gram of each Bacillus species and / or may comprise a combination of Bacillus of from about 10 5 to about 10 7 [[ID=二十九]]CFU / gram total per gram of feed.

[0042] In some embodiments, a combination of Bacillus may be formulated and / or administered to provide different CFU ratios for each Bacillus species contained therein. For example, one embodiment provides 10 per gram of finished feed. 5 Bacillus licheniformis of CFU, 10 5 Bacillus pumilus, and 10 5 This includes Bacillus subtilis from CFU.

[0043] In another example, the configuration is 1 to 5 × 10 per gram of final feed. 5 Bacillus licheniformis in CFU, 1-5 x 10 5 CFU Bacillus pumilus, and 1-5 x 10 5 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 10 per gram of finished food. 6 This will be a CFU of the Bacillus species. In other embodiments, the formulation will be 1-5 × 10 per gram of final feed. 5 Bacillus licheniformis in CFU, 1-5 x 10 5 Bacillus pumilus in CFU, and 2-8 × 10 5 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 10 per gram of finished food. 6 This is a CFU (Concentrated Fungal Unit) Bacillus species.

[0044] In another example, the configuration is 3 × 10 per gram of final feed. 5 Bacillus licheniformis from CFU, 2x10 5 Bacillus pumilus of CFU, and 5x10 5 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 6 Becomes CFU (Chief Football Unit).

[0045] Another aspect is 5 × 10 per gram of final feed. 5Bacillus licheniformis from CFU, 2x10 5 Bacillus pumilus of CFU, and 3x10 5 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 6 Becomes CFU (Chief Football Unit).

[0046] Another aspect is 2 × 10 per gram of final feed. 5 Bacillus licheniformis from CFU, 3x10 5 Bacillus pumilus of CFU, and 5x10 5 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 6 Becomes CFU (Chief Football Unit).

[0047] Another aspect is 2 × 10 per gram of final feed. 5 Bacillus licheniformis from CFU, 5x10 5 Bacillus pumilus of CFU, and 3x10 5 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 6 Becomes CFU (Chief Football Unit).

[0048] Another aspect is 5 × 10 per gram of final feed. 5 Bacillus licheniformis from CFU, 3x10 5 Bacillus pumilus of CFU, and 2×10 5 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 6 Becomes CFU (Chief Football Unit).

[0049] Another aspect is 3 × 10 per gram of final feed. 5 Bacillus licheniformis from CFU, 2x10 5 Bacillus pumilus of CFU, and 5x105 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 6 Becomes CFU (Chief Football Unit).

[0050] Another aspect is 3 × 10 per gram of final feed. 6 Bacillus licheniformis from CFU, 2x10 6 Bacillus pumilus of CFU, and 5x10 6 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 7 Becomes CFU (Chief Football Unit).

[0051] Another aspect is 5 × 10 per gram of final feed. 6 Bacillus licheniformis from CFU, 2x10 6 Bacillus pumilus of CFU, and 3x10 6 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 7 Becomes CFU (Chief Football Unit).

[0052] Another aspect is 2 × 10 per gram of final feed. 6 Bacillus licheniformis from CFU, 3x10 6 Bacillus pumilus of CFU, and 5x10 6 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 7 Becomes CFU (Chief Football Unit).

[0053] Another aspect is 2 × 10 per gram of final feed. 6 Bacillus licheniformis from CFU, 5x10 6 Bacillus pumilus of CFU, and 3x10 6 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food.7 Becomes CFU (Chief Football Unit).

[0054] Another aspect is 5 × 10 per gram of final feed. 6 Bacillus licheniformis from CFU, 3x10 6 Bacillus pumilus of CFU, and 2×10 6 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 7 Becomes CFU (Chief Football Unit).

[0055] Another aspect is 3 × 10 per gram of final feed. 6 Bacillus licheniformis from CFU, 2x10 6 Bacillus pumilus of CFU, and 5x10 6 It can contain CFU of Bacillus subtilis, and the final total concentration is approximately 1 × 10⁶ per gram of finished food. 7 Becomes CFU (Chief Football Unit).

[0056] In some embodiments, a combination of Bacillus may be formulated and / or administered to provide different concentrations or percentages of each Bacillus species contained therein. For example, with respect to other Bacillus species in a combination of Bacillus, the total amount of Bacillus subtilis compared to other Bacillus species may be greater than zero to 99%, e.g., 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 35% to 65%, 45% to 55%, or substantially 50%, such that the total percentage of all Bacillus species is 100%. For example, the total amount of Bacillus licheniformis relative to other Bacillus species is greater than zero to 99%, e.g., 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 35% to 65%, 45% to 55%, 10% to 50%, 10% to 40%, 15% to 30%, effectively 20%, or effectively 50%, so that the total percentage of all Bacillus species is 100%. For example, the total amount of Bacillus pumilus relative to other Bacillus species is greater than zero to 99%, e.g., 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 35% to 65%, 45% to 55%, 10% to 50%, 15% to 40%, 20% to 40%, substantially 30%, or substantially 50%, such that the total percentage of all Bacillus species is 100%. In one embodiment, the total amount of Bacillus subtilis is approximately 50% compared to other Bacillus species, the total amount of B. licheniformis is approximately 30% compared to other Bacillus species, and the total amount of B. pumilus is approximately 20% compared to other Bacillus subtilis, such that the total percentage of all Bacillus species is 100%.

[0057] In another embodiment, the total amount of Bacillus subtilis is approximately 50% compared to other Bacillus species, the total amount of B. licheniformis is approximately 20% compared to other Bacillus species, the total amount of B. pumilus is approximately 30% compared to other Bacillus subtilis, and the total percentage of all Bacillus species is 100%.

[0058] V. Additional ingredients (multiple ingredients allowed) Bacillus combinations can also be administered in combination with one or more additional components or compositions. The additional components or compositions may be any components or compositions that can be administered to subjects, particularly animals such as birds, including poultry, in combination with the Bacillus species in the Bacillus combination. Some particular disclosed Bacillus combinations are formulated specifically for administration to poultry and therefore can be combined with any other components or compositions currently known or hereafter developed for administration to poultry. Exemplary additional components include carriers, vitamins, copper salts, allicin, alliinase, algae, polyphenols or plant materials containing polyphenols, feed supplements (e.g., yucca, quillaja, silica, mineral clay, glucan, mannan, and / or additional DFMs), feeds (e.g., poultry feed, ruminant feed, or aquatic species feed), or combinations thereof. Typically, if an additional component includes an additional DFM, such additional DFM is not a Bacillus species. The additional components may comprise 1 wt% to 99 wt% of the total weight of the combination, and the Bacillus combination may comprise 99 wt% to 1 wt% of the total weight of the combination. In some embodiments, the additional components may comprise 10 wt% to 90 wt% of the total weight of the combination, and the Bacillus combination may comprise 90 wt% to 10 wt% of the total weight of the combination. In some embodiments, the additional components may comprise 20 wt% to 80 wt% of the total weight of the combination, and the Bacillus combination may comprise 80 wt% to 20 wt% of the total weight of the combination. The Bacillus combination may be administered together with the other components, or optionally, in mixtures with other components, such as poultry feed and / or feed supplements, in an amount sufficient to provide the desired amount of Bacillus species in a particular combination.

[0059] A. Carrier In some embodiments, Bacillus combinations may be mixed with and / or dispersed in a carrier to form a dispersed composition. The carrier(s) may be selected to provide non-biological advantages to the composition compared to Bacillus combinations without a carrier. These advantages include, but are not limited to, achieving or improving a readily fluid state and / or improving stability during storage and / or transport. Suitable carriers that may be used in combination with Bacillus combinations include, but are not limited to, plant materials such as beet pulp, ground corn, corn syrup solids, vegetable fiber, rice husks, soluble vegetable fiber, wheat flour, microcrystalline cellulose; carbonates such as metal carbonates such as calcium carbonate and potassium carbonate; sulfates such as metal sulfates such as potassium sulfate and sodium sulfate; lactates containing metal lactates such as calcium lactate; oxides containing metal oxides such as calcium oxide; metal propionic acids such as calcium propionate; stearates containing metal stearates such as calcium stearate; dicalcium phosphate, monocalcium phosphate, tripolyphosphate Phosphates such as sodium phosphate and tetrasodium pyrophosphate; minerals such as dolomite, silicon dioxide, silica, limestone, and vermiculite; clays such as bentonite, montmorillonite, and kaolin; sugars such as glucose, sucrose, dextrose, and fructose, or combinations thereof; salts such as maltodextrin and sodium chloride; carrageenan; cellulose; guar gum; polyols; sodium aluminosilicate; urea; animal protein products; feed products; cereal products; plant protein products; processed cereal products; roughage products; molasses products; oils such as mineral oil, vegetable oil, corn oil, and soybean oil, or combinations thereof; or combinations thereof. In some embodiments, the carrier is or contains calcium carbonate. In certain embodiments, the carrier is or contains calcium carbonate, mineral oil, vegetable oil, dextrose, maltodextrin, or any combination thereof.

[0060] Animal protein products include blood meal; animal-derived powders; buttermilk including concentrated and dried buttermilk; casein; dried hydrolyzed casein; cheese rind; crab meal; fish by-products, fish liver and gland meal, fish meal, fish protein concentrates, fish residue meal, fish products including dried and / or concentrated fish soluble substances; meat hydrolysates; hydrolyzed hair; hydrolyzed leather powder; hydrolyzed poultry by-product bone material; hydrolyzed poultry feathers; leather hydrolysates; meat and bone meal; dried meat and bone meal; meat meal; dried meat meal; dried meat soluble substances ;Dried lactalbumin;dried feed milk;dried milk protein;poultry by-products and / or poultry-derived powders;poultry hatchery by-products;shrimp powder;concentrated skim milk, concentrated cultured skim milk, dried skim milk, or skim milk containing dried cultured skim milk;concentrated whey, concentrated cultured whey, concentrated hydrolyzed whey, dried whey, or whey containing dried hydrolyzed whey;concentrated whey products and / or dried whey products;concentrated whey and / or dried whey-soluble substances;or combinations thereof may be included, but are not limited to these.

[0061] Feed products may include, but are not limited to, alfalfa products such as dried feed (optionally in pellet form), ground hay, or sun-dried feed (optionally in pellet form); coastal Bermuda grass hay; dried corn plants; dried silage; flax products; ground grass; bush clover powder and / or stem powder; ground soybean hay; or combinations thereof.

[0062] Grain products may include, but are not limited to, barley, corn, grain sorghum, mixed feed oats, oats, rye, wheat, ground brown rice, broken or chipped rice, sake rice, rye, or combinations thereof. Grain products can take any suitable form, such as whole, ground, crushed, sifted, flaked, coarsely ground, toasted, and / or heat-processed.

[0063] Plant-based protein products may include, but are not limited to, dried beans; canola flour; coconut flour; cottonseed (flakes, cakes, flour, low-gossypol flour, and / or whole-grain pressed cottonseed); guar meal; dried kelp; flaxseed flour; peanut flour; peas; potato protein; dried seaweed flour; safflower flour; soy protein concentrate; soy feed; ground soybeans; soy flour (optionally crushed); heat-treated soybeans; ground and extruded whole-grain soybeans; soy flour, soy grits; sunflower flour (optionally hulled); yeast such as active dried yeast, brewer's yeast, cultured yeast, dried yeast, primary dried yeast, Torula dried yeast, and / or Candida dried yeast; or combinations thereof.

[0064] Processed grain by-products may include aspirated grain fractions; beer-dried grains; buckwheat flour; concentrated distilled solubles; concentrated fermented corn extract; corn bran; corn flour; corn germ flour; corn gluten feed and / or flour; corn grits; optionally distilled dried grains containing solubles; distilled dried solubles, wheat flour, cereal sorghum germ cake, flour, grits, and / or milled feed; powdered hominy feed; malt germ; oat grains; feed oatmeal; pearl barley by-products; peanut hulls; rice bran; rice polish; rye flour; gelatinized or partially aspirated sorghum grain flour; wheat bran, wheat flour, short, germ flour, defatted germ flour, med flour, mill run and / or red dog; or combinations thereof.

[0065] Roughage products may include, but are not limited to, almond hulls; dried apple pectin pulp; dried apple pomace; bagasse; barley hulls; barley milling by-products; dried plain beet pulp; buckwheat hulls; dried citrus meal; dried citrus pulp; citrus seed meal; corn cob fragments; cottonseed hulls; flax straw by-products; ground corn cobs; plantain seed hulls; malt hulls; harvested oat by-products; oat hulls; oat milling by-products; peanut hulls; rice hulls; rice milling by-products; rye mill run; soybean hulls, milling feed, and / or mill run; sunflower hulls; ground straw; dried tomato pomace; or combinations thereof.

[0066] Molasses products may include beet molasses, dried beet molasses products, dried beet pulp molasses, sugarcane molasses, citrus molasses, molasses yeast concentrate soluble, concentrate separator by-products, concentrated molasses fermentation soluble, starch molasses, molasses distillation concentrate soluble, molasses distillation dry soluble, or combinations thereof.

[0067] B. Copper species The disclosed combinations may be mixed with copper species, such as copper species that provide copper ions. The copper species may be copper salts. Exemplary copper species that may be combined with the Bacillus combinations may include, but are not limited to, copper chloride, copper bromide, copper iodide, copper sulfate, copper sulfite, copper bisulfite, copper thiosulfate, copper phosphate, monobasic copper phosphate, dibasic copper phosphate, copper hypophosphate, copper dihydrogen pyrophosphate, copper tetraborate, copper borate, copper carbonate, copper bicarbonate, copper metasilicate, copper citrate, copper malate, copper methionate, copper succinate, copper lactate, copper formate, copper acetate, copper butyrate, copper propionate, copper benzoate, copper tartrate, copper ascorbate, copper gluconate, or combinations thereof. In certain embodiments, the copper salt is copper chloride, copper sulfate, copper phosphate, copper oxide, copper glycinate, copper hydroxide, basic copper chloride, copper dihydrogen pyrophosphate, copper carbonate, copper citrate, copper acetate, copper gluconate, or a combination thereof, for example, basic copper chloride, copper carbonate, copper glycinate, copper oxide, copper sulfate, or a combination thereof. Copper species such as copper salts may be provided separately or individually, or as part of a composition such as a feed or feed supplement. Certain disclosed embodiments include, essentially consist of, Bacillus licheniformis, Bacillus pumilis, Bacillus subtilis, and copper species. In any embodiment, the copper species may be a salt capable of providing copper ions, for example, a copper salt such as copper sulfate.

[0068] C. Vitamins (multiple selections possible) The disclosed combinations may be mixed with vitamins. Exemplary vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (including folic acid), and vitamin B 12 Examples include, but are not limited to, various cobalamins (cyanocobalamin in vitamin supplements), vitamin C (ascorbic acid or its salts, e.g., sodium ascorbate or calcium sorbate), vitamin D (vitamin D1, vitamin D2, vitamin D3, vitamin D4, vitamin D5, 25-hydroxyvitamin D3, 25-dihydroxyvitamin D3, or combinations thereof), vitamin E, vitamin K (K1 and K2 (i.e., MK-4, MK-7)), and one or more derivatives, salts, and / or analogues thereof.

[0069] D. Feed The feed may be any feed suitable for administration to an animal. The Bacillus combination may be administered in combination with the feed, such as by forming a mixture of the Bacillus combination and the feed, or by administering the Bacillus combination and the feed sequentially in any order. In a particular disclosed embodiment, the animal is poultry, and the Bacillus combination may be used in combination with and mixed with poultry feed, such as poultry base diet. The feed may include corn, alfalfa, peas, soybean meal, soybean oil, wheat, oats, sorghum, barley, rye, rice husks, canola, corn oil, limestone, salt (e.g., sodium chloride), distilled dry grains (DDGS) containing soluble substances, dicalcium phosphate, sodium sesquicarbonate, methionine source, lysine source, L-threonine, mineral oil, biotin, folic acid, kelp, menadione dimethylpyrimidinol bisulfite, calcium aluminosilicate, or any combination thereof. The feed may also include one or more additional components. Additional ingredients may be used for any desired purpose, such as fillers or substantially biologically inert materials added to provide a desired beneficial effect. For example, feed may contain carbonates (including metal carbonates such as calcium carbonate), trace minerals (such as chlorides, fluorides, iodides, chromium, copper, zinc, iron, magnesium, manganese, molybdenum, phosphorus, potassium, sodium, sulfur, selenium, or combinations thereof), bulking agents, carriers, colorants, flavor enhancers, preservatives, one or more vitamins, or combinations thereof.Preservatives may include benzoic acid or its salts (e.g., sodium benzoate), lactic acid or its salts (e.g., sodium lactate, potassium lactate, or calcium lactate), propionic acid or its salts (e.g., sodium propionate), ascorbic acid or its salts (e.g., sodium ascorbate), gallic acid or its salts (e.g., sodium gallate), sulfur dioxide and / or sulfites, nitrites, nitrates, choline or its salts (e.g., choline anionic salts, halogenated choline such as chlorides, bromides, iodides, fluorides, or choline hydroxide), or any combination thereof. One or more vitamins may include vitamin A, vitamin B1 (e.g., thiamine mononitrate), vitamin B2 (e.g., riboflavin-5-phosphate), vitamin B3 (e.g., niacin or niacinamide), vitamin B5 (e.g., pantothenic acid or d-calcium pantothenate), vitamin B6 (e.g., pyridoxine or pyridoxine hydrochloride), and vitamin B. 12 Examples include vitamin C (e.g., ascorbic acid, sodium ascorbate, or calcium sorbate), vitamin D, vitamin E, vitamin K, or combinations thereof. Vitamin D may include vitamin D1, vitamin D2, vitamin D3, vitamin D4, vitamin D5, 25-hydroxyvitamin D3, 25-dihydroxyvitamin D3, or combinations thereof.

[0070] Feeds such as poultry feed may also contain blended animal and vegetable fats, which may include: fats and / or oils such as tallow obtained by refining cattle entrails; lard obtained by refining pig entrails; poultry fat obtained by refining poultry entrails; feed-grade animal fats obtained by refining rendered beef, pork, and / or poultry raw materials; yellow fats obtained by refining restaurant fats and / or cooking oils; and / or blends of animal and vegetable fats and vegetable oils from restaurant grease. Furthermore, or instead, feeds may contain protein sources, such as canola, fish meal, peas, meat and bone meal, soybeans, and / or grain by-products.

[0071] E. Allicin, alliin, and / or alliinase Allicin (diallylthiosulfate; 2-propen-1-sulfinothioate S-2-propenyl ester) is a compound found in garlic, such as raw garlic. Allicin is usually produced in damaged garlic cells from alliin ((2R)-2-amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid) by the action of the enzyme alliinase. Allicin, alliin, and / or alliinase may be provided as whole garlic cloves or bulbs, mashed or chopped garlic, garlic extract, and / or synthesized or isolated compounds.

[0072] F. Polyphenol compounds Polyphenols can be provided by plant extracts from polyphenol-containing plant materials. Plant materials may also contain non-polyphenolic compounds, including polyphenol degradation products such as gallic acid and trans-caphthalic acid. Degradation may occur, for example, through oxidative and / or biological processes. Both polyphenolic and non-polyphenolic compounds may be biologically active. Plant extracts may be prepared from a single plant material or a combination of plant materials. Suitable plant materials from which plant extracts can be obtained include, but are not limited to, apples, blackberries, black chokeberries, blackcurrants, black elderberries, blueberries, cherries, cranberries, grapes, green tea, hops, onions, quillaja, plums, pomegranates, raspberries, strawberries, and yucca.

[0073] In some embodiments, plant extracts are prepared from pressed plant materials such as grape pomace, dried plant materials such as tea, or combinations thereof. Pomace can be obtained substantially immediately after pressing, or as a siloed product, i.e., pomace collected after pressing and stored for up to several months. Suitable plants have multiple polyphenols and / or other non-polyphenol compounds, including but not limited to non-polyphenolic organic acids (such as gallic acid and / or trans-caphthalic acid), flavanols, gallic acid esters, flavanodiols, phloroglucinol, pyrogallol, and catechol. In some embodiments, plant extracts are prepared from Pinot Noir pomace, Pinot Gris pomace, or green tea.

[0074] In some embodiments, the pressed or dried plant material is ground into a fine powder before or during extraction. The pressed plant material may be frozen to facilitate grinding. Polyphenols and other non-polyphenol compounds may be extracted for administration. For example, polyphenols and other non-polyphenol compounds can be extracted from the powder using a solution containing a polar solvent such as water, alcohol, ester, or a combination thereof. In some embodiments, the solution contains a water-miscible alcohol, ester, or a combination thereof, e.g., a lower alkyl alcohol, a lower alkyl ester, or a combination thereof. In some embodiments, the solution is an aqueous solution containing water, or a solvent of 25-99%, such as 25-95% solvent, 30-80% solvent, or 50-75% solvent. In certain embodiments, the solution is an aqueous solution containing methanol, ethanol, isopropanol, ethyl acetate, or a combination thereof. The solution may be acidified by the addition of an acid. The acid can prevent or minimize the oxidative degradation of the bioactive polyphenols and other non-polyphenol compounds in the extract. The acid may be any suitable acid, such as a mineral acid (e.g., hydrochloric acid), or an organic acid, such as citric acid or acetic acid. In some embodiments, the solution contains 0.01% to 1%, for example, 0.02 to 0.5%, 0.025 to 0.25%, or 0.05 to 0.15% of the acid. In some examples, the solution contains 0.1% hydrochloric acid.

[0075] Extraction can be carried out at temperatures ranging from 0 to 100°C. In some embodiments, extraction can be carried out at temperatures ranging from 20 to 70°C, or at ambient temperature. Extraction can be carried out over a period ranging from several minutes to several days. To increase extraction efficiency, the plant material and solution may be mixed or stirred during extraction by methods such as grinding the plant material, stirring the mixture, shaking the mixture, or homogenizing the mixture. In some embodiments, extraction may be repeated one or more times with fresh solution to increase the recovery rate of polyphenols and other non-polyphenol compounds from the plant material. The liquid phases from each extraction cycle are then combined for further processing.

[0076] The liquid phase can be recovered, and any remaining solids or pulp can be discarded. Recovery of the liquid phase may involve decanting the liquid from the remaining solids and / or filtering the liquid phase to remove any remaining solids. The solvent (alcohol, ester, or a combination thereof) can be removed from the solution by any suitable means, such as evaporation (e.g., rotational evaporation), to produce an aqueous extract containing the biologically active components in a weakly acidic solution.

[0077] In certain embodiments where the plant material contains a substantial amount of oil or lipids, an initial extraction of nonpolar components may be performed before the extraction of polyphenols and other polar non-polyphenolic compounds. Nonpolar components can be extracted by homogenizing the plant material in a nonpolar solvent (e.g., hexane, heptane, or a combination thereof). The solvent layer containing the extracted nonpolar components is separated from the plant material and discarded.

[0078] Aqueous plant extracts may be further purified by suitable means, such as extraction, chromatography, or distillation, in order to remove non-polyphenol compounds and / or to increase the concentration of polyphenols compared to other compounds in the extract.

[0079] The aqueous plant extract may be dried, for example, by freeze-drying or other low-temperature drying methods, or it may be ground into a powder to obtain a dried plant extract. In some embodiments, the dried plant extract contains 0.01 wt% to 25 wt% of total polyphenols, for example, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.01 wt% to 2.5 wt%, 0.01 wt% to 1 wt%, 0.01 wt% to 0.5 wt%, 0.02 to 0.25 wt%, or 0.03 to 0.1 wt% of total polyphenols. In certain embodiments, the dried plant extract further contains non-polyphenol compounds. For example, a dried plant extract may contain 0.01 to 1 mg / g of gallic acid, e.g., 0.05 to 0.5 mg / g or 0.09 to 0.25 mg / g of gallic acid, and / or 0.001 to 0.1 mg / g of trans-caphthalic acid, e.g., 0.005 to 0.05 mg / g or 0.01 to 0.025 mg / g of trans-caphthalic acid.

[0080] Aqueous plant extracts may be concentrated to a smaller volume, for example, by evaporation, and used as aqueous plant extracts. In other embodiments, aqueous plant extracts are mixed with a carrier before drying and grinding. Suitable carriers include, for example, diatomaceous earth, silica, maltodextrin, ground grains (e.g., corn), crude flours (e.g., soybean or cottonseed flour), by-products (e.g., grains dried in a distiller, rice husks, wheat myrrun), clay (e.g., bentonite), and combinations thereof. Plant extracts may be combined with carriers in a weight ratio ranging from 10:1 to 1:10, for example, from 5:1 to 1:5. For example, plant extracts may be mixed with diatomaceous earth in a weight ratio of 3:1.

[0081] G. Feed Supplements The Bacillus combination may be used in combination with one or more feed supplements. In some embodiments, the Bacillus combination is mixed with a feed supplement to form a mixture or composition containing the Bacillus combination and the feed supplement(s). In other embodiments, the Bacillus combination is administered in combination with a feed supplement.

[0082] 1. Yucca and / or Quillaja, or their extracts. The disclosed Bacillus combinations may be administered in combination with yucca and / or quillaja plant materials or extracts thereof. Examples of yucca include Yucca aloifolia, Yucca angustissima, Yucca arkansana, Yucca baccata, Yucca baileyi, Yucca brevifolia, Yucca campestris, Yucca capensis, Yucca carnerosana, Yucca cernua, Yucca coahuilensis, Yucca constricta, Yucca decipiens, Yucca declinata, Yucca de-smetiana, Yucca elata, Yucca endlichiana, Yucca faxoniana, Yucca filamentosa, Yucca filifera, Yucca flaccida, Yucca gigantean, Yucca glauca, Yucca gloriosa, Yucca grandiflora, Yucca harrimaniae, Yucca intermedia, Yucca jaliscensis, Yucca lacandonica, Yucca linearifolia, Yucca Examples include, but are not limited to, Yucca luminosa, Yucca madrensis, Yucca mixtecana, Yucca necopina, Yucca neomexicana, Yucca pallida, Yucca periculosa, Yucca potosina, Yucca queretaroensis, Yucca reverchonii, Yucca rostrata, Yucca rupicola, Yucca schidigera, Yucca schottii, Yucca sterilis, Yucca tenuistyla, Yucca thompsoniana, Yucca treculeana, Yucca utahensis, Yucca valida, or combinations thereof. In certain embodiments, Yucca is or includes Yucca schidigera.

[0083] Examples of Quillaya include, but are not limited to, Quillaya brasiliensis, Quillaya lanceolata, Quillaya lancifolia, Quillaya molinae, Quillaya petiolaris, Quillaya poeppigii, Quillaya saponaria, Quillaya sellowiana, Quillaya smegmadermos, or combinations thereof. In certain embodiments, Quillaya is or includes Quillaya saponaria.

[0084] Those skilled in the art will understand that, as used herein, a plant name may refer to the whole plant, or to any part of the plant, such as roots, stems or trunks, bark, leaves, flowers, flower stalks, seeds, or combinations thereof. These plant parts may be used in their unprocessed or dried state, and may be used whole, powdered, or ground. A plant name may also refer to extracts from any part(s) of the plant, such as chemical extracts, or extracts obtained by pressing, or any other method of concentrating or extracting oils or other extracts known to those skilled in the art or hereafter discovered. Plant extracts may include compounds such as saponins, triterpenoids, polyphenols, antioxidants, or resveratrol, or combinations thereof.

[0085] This combination may include a composition containing yucca and / or quillaja, which may also include carriers and binders suitable for formulating yucca and / or quillaja for administration to animals. In certain embodiments, such a composition may be a commercially available product, such as a composition containing Yucca schidigera and Quillaja saponaria, which is sold by Desert King International under the trademark NUTRAFITO plus® and / or by Phibro Animal Health Corporation under the trademark MAGNI-PHI®. Such compositions may include 99% or more of Quillaja saponaria and 1% or less of Yucca schidigera, ranging from 75% of Quillaja saponaria and 25% of Yucca schidigera, for example, 95% of Quillaja saponaria and 5% of Yucca schidigera, to 80% of Quillaja saponaria and 20% of Yucca schidigera, in certain embodiments, 85% of Quillaja saponaria and 15% of Yucca schidigera, or 90% to 95% of Quillaja saponaria and 5% to 10% of Yucca schidigera, for example, 92% to 93% of Quillaja saponaria and 7% to 8% of Yucca schidigera, or about 92.5% of Quillaja saponaria and about 7.5% of Yucca schidigera.

[0086] 2. Silica, mineral clay, glucan, and mannan Furthermore, or alternatively, the Bacillus combination can be administered in combination with a feed supplement containing silica, mineral clay, glucan, and mannan. The feed supplement may further contain endoglucanohydrolase, either endogenously or as an actively added component. As used herein, the weight percent of endoglucanohydrolase is based on a 70,000 units / gram endoglucanohydrolase product. The endoglucanohydrolase may be β-1,3(4)-endoglucanohydrolase.

[0087] In any embodiment disclosed herein, the feed supplement may include, essentially consist of, or consist of glucans (e.g., β-1,3(4) glucans), silica, mineral clay, and mannan. In some embodiments, the feed supplement includes, essentially consists of, or consists of glucans (e.g., β-1,3(4) glucans), silica, mineral clay, mannan, and endoglucanohydrolase. In any embodiment disclosed herein, glucans and mannan may be provided at least partially by yeast cell walls or extracts thereof. Thus, in some embodiments, the feed supplement may include, essentially consist of, or consist of silica, mineral clay, and yeast cell walls or extracts thereof, or the feed supplement may include, essentially consist of, or consist of silica, mineral clay, yeast cell walls or extracts thereof, and endoglucanohydrolase. Similarly, in certain disclosed embodiments, endoglucanohydrolase may be provided by yeast cell walls or yeast cell wall extracts.

[0088] Suitable silica sources include, but are not limited to, sand, diatomaceous earth, and synthetic silica. In one embodiment, quartz may be used. In certain embodiments, the mannan includes glucomannan.

[0089] The components of the feed supplement can be prepared by methods commonly known in the art and obtained from commercial sources. β-1,3(4)-endoglucanohydrolase can be produced from the immersion fermentation of Trichoderma longibrachiatum strain. Diatomaceous earth is available as a commercial product and contains 70% to 95% silica (SiO2), with the remaining components not analyzed, but according to the definition of the Association of Analytical Chemists (AOAC, 2002), it is mainly ash (minerals). The mineral clay (e.g., aluminosilicate) used in this feed supplement can be any of the various commercially available clays, including but not limited to montmorillonite clay, bentonite, and zeolite. Glucans, mannans, and / or endoglucanohydrolases can be obtained from plant cell walls, yeast or yeast cell walls or extracts thereof (e.g., Saccharomyces cerevisiae, Candida utilis), certain fungi (e.g., mushrooms), algae, and bacteria. In certain embodiments, yeast can be actively administered to endogenously provide glucans, mannans, and endoglucanohydrolases.

[0090] In one embodiment, the feed supplement comprises, in interrelated amounts, 1 to 40 wt% silica, 0.5 to 25 wt% glucan and mannan, and 40 to 92 wt% mineral clay, or consists thereof. In another embodiment, the feed supplement comprises, in interrelated amounts, 5 to 40 wt% silica, 0.5 to 15 wt% glucan and mannan, and 40 to 80 wt% mineral clay, or consists thereof. In yet another embodiment, the feed supplement comprises, in interrelated amounts, 20 to 40 wt% silica, 0.5 to 10 wt% glucan and mannan, and 50 to 70 wt% mineral clay, or consists thereof. In another embodiment, the feed supplement contains, essentially consists of, or comprises, 15-40 wt% silica, over 0-15 wt% glucan, over 0-10 wt% mannan, and 50-81 wt% mineral clay in interrelated amounts. In another embodiment, the feed supplement contains, essentially consists of, or comprises, 15-40 wt% silica, 0.5-5.0 wt% glucan, 0.5-8.0 wt% mannan, and 50-81 wt% mineral clay in interrelated amounts. In another embodiment, the feed supplement contains, essentially consists of, or comprises, 20-30 wt% silica, 0.5-3.5 wt% glucan, 0.5-6.0 wt% mannan, and 60-70 wt% mineral clay in interrelated amounts.

[0091] In some embodiments, β-glucan and mannan are obtained from yeast or yeast cell wall or an extract thereof. The feed supplement comprises, in relative amounts, 1 to 40 wt% silica, 1 to 30 wt% yeast cell wall or an extract thereof, and 40 to 92 wt% mineral clay, or comprises essentially these, or may comprise them. In one embodiment, the feed supplement comprises, in relative amounts, 10 to 40 wt% silica, 5 to 20 wt% yeast cell wall or an extract thereof, and 40 to 80 wt% mineral clay, or comprises essentially these, or comprises them. In another embodiment, the feed supplement comprises, in relative amounts, 15 to 30 wt% silica, 5 to 15 wt% yeast cell wall or an extract thereof, and 50 to 70 wt% mineral clay, or comprises essentially these, or comprises them.

[0092] In any of the above embodiments, the feed supplement may further contain endoglucanohydrolase, for example, β-1,3(4)-endoglucanohydrolase. The feed supplement may contain 0.025 wt% to 5 wt% of endoglucanohydrolase, or more, for example, 0.05 wt% to 3 wt% of β-1,3(4)-endoglucanohydrolase, relative to the amount of silica, mineral clay, glucan, mannan, and / or yeast, yeast cell wall, or yeast cell wall extract present in the feed supplement. In one embodiment, the feed supplement contains, or essentially consists of, 0.1 to 3 wt% of β-1,3(4)-endoglucanohydrolase, 20 to 40 wt% of silica, 0.5 to 20 wt% of glucan and mannan, and 50 to 70 wt% of mineral clay, in relative amounts to each other. In another embodiment, the feed supplement contains, in relative amounts, 0.1 to 3 wt% β-1,3(4)-endoglucanohydrolase, 20 to 40 wt% silica, 0.5 to 10 wt% glucan and mannan, and 50 to 70 wt% mineral clay, or is essentially composed of them, or is composed of them. Alternatively, the feed supplement contains, in relative amounts, 0.1 to 3 wt% β-1,3(4)-endoglucanohydrolase, 1 to 40 wt% silica, 5 to 30 wt% yeast cell wall or its extract, and 40 to 92 wt% mineral clay, or may be essentially composed of them. In one embodiment, the feed supplement contains, in relative amounts, 0.1 to 3 wt% β-1,3(4)-endoglucanohydrolase, 10 to 40 wt% silica, 5 to 20 wt% yeast cell wall or extract thereof, and 40 to 80 wt% mineral clay, or consists essentially of them, or consists of them. In another embodiment, the feed supplement contains, in relative amounts, 0.1 to 3 wt% β-1,3(4)-endoglucanohydrolase, 15 to 30 wt% silica, 5 to 15 wt% yeast cell wall or extract thereof, and 50 to 70 wt% mineral clay, or consists essentially of them, or consists of them.

[0093] In any of the above embodiments, silica may be provided by diatomaceous earth. In any of the above embodiments, glucan may be β-glucan. In some embodiments, β-glucan may be obtained from yeast or other materials, such as fungi, algae, bacteria, etc. In any of the above embodiments, mannan may include glucomannan.

[0094] Glucans and mannans (or yeast or yeast cell walls or extracts thereof) can be prepared by methods known to those skilled in the art, and by methods disclosed in patent documents incorporated herein by reference. Yeast cell walls or extracts thereof may have a feed supplement containing 0-15% moisture and 85-100% dry matter. The dry matter may contain 10-65% protein, 0-25% fat, 0-3% phosphorus, 5-30% β-glucan, 5-35% mannan, and 0-15% ash. In an independent embodiment, commercial sources of β-1,3(4) glucan and glucomannan derived from primary inactivated yeast (Saccharomyces cerevisiae) using the following chemical feed supplements may be used. Moisture 2-5%; protein 40-50%, fat 3-8%, phosphorus 0-2%, mannan 10-16%, β-1,3-(4) glucan 10-20%, and ash 2-12%.

[0095] In another independent embodiment, the yeast cell wall or its extract may contain 1-7% moisture and 93-99% dry matter, the dry matter of which may contain 18-28% protein, 10-17% fat, 0-2% phosphorus, 20-30% mannan, 18-28% β-1,3-(4) glucan, and 2-5% ash.

[0096] In independent embodiments of the feed supplement, silica, glucan, and mannan, as well as mineral clay, are combined in weight ratios of 1-40%, 0.5-25%, and 40-92%, respectively. In independent embodiments of the feed supplement and / or combination, β-1,3(4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall or extract, and mineral clay are combined in weight ratios of 0.05-3%, 1-40%, 1-20%, and 40-92%, respectively. In independent embodiments of the feed supplement and / or combination, β-1,3(4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall or extract, and mineral clay are combined in weight ratios of 0.1-3%, 5-40%, 2-15%, and 40-80%, respectively. In another independent embodiment of the feed supplement and / or combination, β-1,3(4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall or extract thereof, and mineral clay are combined in weight ratios of 0.1-3%, 30-40%, 4-15%, and 50-65%, respectively.

[0097] Feed supplements may also contain one or more additional components. These additional components may be used for any desired purpose, such as fillers or substantially biologically inert materials added to provide a desired beneficial effect. For example, feed supplements may include carbonates (including metal carbonates such as calcium carbonate), trace minerals (such as chlorides, fluorides, iodides, chromium, copper, zinc, iron, magnesium, manganese, molybdenum, phosphorus, potassium, sodium, sulfur, selenium, or combinations thereof), fillers, microtracers (such as dye-coated iron particles), yeast, allicin, alliin, alliinase, algae, polyphenols or plant materials containing polyphenols, carriers, colorants, flavor enhancers, preservatives, oils, vitamins, sorbic acid or salts thereof, or combinations thereof. Yeast may be yeast culture, active yeast, live yeast, dead yeast, yeast extract, or combinations thereof. Preservatives may include benzoic acid or its salts (e.g., sodium benzoate), lactic acid or its salts (e.g., sodium lactate, potassium lactate, or calcium lactate), propionic acid or its salts (e.g., sodium propionate), ascorbic acid or its salts (e.g., sodium ascorbate), gallic acid or its salts (e.g., sodium gallate), sulfur dioxide and / or sulfites, nitrites, nitrates, choline or its salts (e.g., choline anionic salts, such as choline halogenated salts like chlorides, bromides, iodides, fluorides, or choline hydroxide), or any combination thereof. Oils may be mineral oil, corn oil, soybean oil, or a combination thereof. Sorbic acid or its salts may be potassium sorbate, sodium sorbate, ammonium sorbate, or a combination thereof. Vitamins may include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B 12 This could be vitamin C, vitamin D, vitamin E, vitamin K, or a combination thereof.

[0098] Furthermore, or alternatively, additional ingredients may include corn, soybean meal, wheat, wheat fiber, barley, rye, rice husks, canola, limestone, salt, distilled dry grains (DDGS) containing soluble substances, dicalcium phosphate, sodium sesquicarbonate, methionine source, lysine source, L-threonine, biotin, folic acid, kelp, menadione dimethylpyrimidinol bisulfite, calcium aluminosilicate, or any combination thereof.

[0099] Further information relating to feed supplements and / or additional ingredients can be found in PCT application PCT / US2015 / 053439, and in U.S. applications 15 / 359,342, 14 / 699,740, 14 / 606,862, and 62 / 449,959, each of which is incorporated herein by reference in whole.

[0100] In some embodiments, the feed supplement does not contain additional components. In other embodiments, the feed supplement contains additional components ranging from more than 0 to 40% by weight, for example, 0.1% to 40% by weight, or 0.2% to 35% by weight. In certain embodiments, the feed supplement contains additional components ranging from 0.1% to 5% by weight, for example, 0.2% to 3% by weight. In other embodiments, the feed supplement contains additional components ranging from 5% to 20% by weight, for example, 10% to 15% by weight. Furthermore, in even more embodiments, the feed supplement contains additional components ranging from 20% to 40% by weight, for example, 30% to 35% by weight.

[0101] In some embodiments, the feed supplement is silica, mineral clay, glucan, mannan, and endoglucanohydrolase; silica, mineral clay, glucan, mannan, endoglucanohydrolase, microtracer, and mineral oil; silica, mineral clay, glucan, mannan, endoglucanohydrolase, microtracer, mineral oil, and vitamins; silica, mineral clay, glucan, mannan, endoglucanohydrolase, microtracer, mineral oil, vitamins, and potassium sorbate; silica, mineral clay, glucan, mannan, endoglucanohydrolase, vitamins, and active yeast; Silica, mineral clay, glucan, mannan, endoglucanohydrolase, microtracer, mineral oil, and active yeast; silica, mineral clay, glucan, mannan, endoglucanohydrolase, and mineral oil; silica, mineral clay, glucan, mannan, endoglucanohydrolase, vitamins, and calcium carbonate; silica, mineral clay, glucan, mannan, endoglucanohydrolase, microtracer, and wheat fiber; or a composition comprising, or comprising, silica, mineral clay, glucan, mannan, endoglucanohydrolase, and microtracer. In any of these embodiments, glucan and mannan may be provided by yeast, yeast cell wall, or yeast cell wall extract.

[0102] In some embodiments, the feed supplement does not contain peroxide compounds. In some embodiments, the feed supplement does not contain hydrogen peroxide. In some embodiments, the feed supplement does not contain urea peroxide. In some embodiments, the feed supplement does not contain urea. In some embodiments, the feed supplement does not contain hydrogen peroxide and urea.

[0103] In certain embodiments, the feed supplement is a powdered supplement. In other embodiments, the feed supplement is a granular supplement. The granular feed supplement may contain silica, mineral clay, glucan and / or mannan, and optionally, the above-mentioned endoglucanohydrolase. The granular feed supplement is 40 lb / ft 3 ~150 lb / ft 3It may have a bulk density of . In some embodiments, each granule in the granular composition contains silica, mineral clay, glucan and / or mannan, and optionally endoglucanohydrolase, in substantially the same relative amount as the relative amount of each component in the whole composition. Each granule in the granular composition may contain, essentially consist of, or consist of silica, mineral clay, glucan, mannan, and endoglucanohydrolase. Alternatively or additionally, each granule may contain a substantially homogeneous blend of silica, mineral clay, glucan and mannan, and optionally endoglucanohydrolase. The composition may contain more than 40% by weight of granules having at least one dimension between 0.149 mm (100 mesh, US standard mesh size) and 4.76 mm (4 mesh), and in some embodiments, the composition may contain more than 90% by weight of granules having at least one dimension between 0.149 mm (100 mesh) and 2 mm (10 mesh). And / or, the composition may comprise granules ranging from more than 0% to 100% by weight and particles ranging from 0% to 60% by weight or less, for example, 10% or less, wherein the granules have at least one dimension between 10 mesh (2.00 mm) and 100 mesh (0.149 mm), and the particles have at least one dimension less than 100 mesh (0.149 mm) (i.e., smaller than 100 mesh (0.149 mm)). In any embodiment, the granular composition comprises a plurality of granules, each granule comprising silica, mineral clay, glucan, and mannan, and the granules, when administered to animals, have a size that increases the expression of interleukin 10 receptor β (IL10RB) for a certain period after administration, such as after the start of administration, compared to animals not administered the composition. In some embodiments, the period may be from 28 days to at least 42 days from the start of administration. And / or, the composition may have a mineral coefficient of variation of 0% to 10%, or an approximate coefficient of variation of 0% to 20%, or both. Further information regarding granular feed supplements can be found in WO2018 / 140450, which is incorporated herein by reference in its entirety.

[0104] In some embodiments, the feed supplement is administered to the animal daily at time intervals that are considered effective or deemed effective in achieving beneficial results. The feed supplement may be administered in a once-daily dose or in divided doses throughout the day. The amount may range from more than 0 grams to 500 grams per animal per day, for example, from 0.5 grams to 250 grams, 5 grams to 200 grams, or from 10 grams to 70 grams per animal per day. Alternatively, the feed supplement may be fed or administered in an amount greater than 0 and greater than 1000 mg per kilogram of animal body weight per day, for example, from more than 0 and greater than 500 mg per kilogram of body weight. In other embodiments, the feed supplement is fed or administered in proportion to the weight of the animal feed. Feed supplements can be fed or administered in amounts of more than 0 kg to 150 kg per ton (2000 pounds) of feed, for example, 0.1 kg to 100 kg per ton of feed. Alternatively, feed supplements can be fed or administered in amounts of more than 0 grams to 20 grams per kilogram of feed, for example, more than 0 grams to 10 grams per kilogram of feed.

[0105] 3. Additional DFMs (multiple options possible) The disclosed Bacillus combinations can be administered to animals in the absence of additional DFMs or in combination with one or more additional DFMs. Additional DFMs may be any DFM suitable for administration to a particular animal. In some embodiments, the animal is poultry, particularly chickens or turkeys, and the additional DFMs are DFMs that benefit poultry. In other embodiments, the animal is a ruminant or aquatic species. Additional DFMs may, but are not limited to, additional Lactobacillus, Enterococcus, Bifidobacterium, Propionibacterium, Streptococcus, Pediococcus, yeast, or combinations thereof.

[0106] Exemplary additional DFMs include, but are not limited to, Lactobacillus acidophilis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus gallinarum, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus bulgaricus, Bifidobacterium pseudolongum, Bifidobacterium thermophilium, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Streptococcus bovis, Streptococcus faecium, Enterococcus faecium, Enterococcus faecalis, Enterococcus diacetylactis, Saccharomyces cerevisiae, Saccharomyces boulardii, Aspergillus oryzae, Aspergillus niger, Selenomonas ruminantium, Megasphaera elsdenii, Propionibacterium freudenreichii, Propionibacterium shermanii, Propionibacterium acidipropionici, Propionibacterium fensenii, Prevotella bryantii, Pediococcus acidilactici, Pediococcus cerevisiae, or combinations thereof.

[0107] In some embodiments, the additional DFM may be an additional Bacillus species, such as, but not limited to, Bacillus alcalophilus, Bacillus alvei, Bacillus aminovorans, Bacillus aneurinolyticus, Bacillus anthracis, Bacillus aquaemaris, Bacillus atrophaeus, Bacillus boroniphilus, Bacillus brevis, Bacillus caldolyticus, Bacillus centrosporus, Bacillus cereus, Bacillus circulans, Bacillus firmus, Bacillus flavothermus, Bacillus fusiformis, Bacillus galliciensis, Bacillus globigii, Bacillus infernus, Bacillus larvae, Bacillus laterosporus, Bacillus lentus, Bacillus megaterium, Bacillus mesentericus, Bacillus mucilaginosus, Bacillus mycoides, Bacillus pantothenticus, Bacillus polymyxa, Bacillus pseudoanthracis, Bacillus schlegelii, Bacillus sphaericus, Bacillus sporothermodurans, Bacillus stearothermophilus, Bacillus thermoglucosidasius, Bacillus thuringiensis, Bacillus vulgatis, or Bacillus Weihenstephanensis etc. can be included.

[0108] F. liquid In some embodiments, the Bacillus combination is mixed with a liquid, such as water. When mixed with water, the Bacillus combination can take the form of spores or liquid cultures. In further embodiments, the Bacillus combination with water is further combined with an acid, such as acetic acid or sorbic acid, and optionally with glycerol. In one embodiment, the Bacillus combination is mixed with water and acetic acid. In another embodiment, the Bacillus combination is mixed with water and sorbic acid. In yet another embodiment, the Bacillus combination is mixed with water, sorbic acid, and glycerol.

[0109] In some embodiments, Bacillus combined with water is further combined with alkaline compounds.

[0110] The liquid Bacillus combination composition may have any suitable pH range, for example, 2-8, 3-8, 4-8, 5-8, or 5-7.

[0111] Beneficial results from the administration of Bacillus combinations VI. Administration of the disclosed Bacillus combination to poultry may reduce pathogenic Enterococcus cecorum in the poultry. The reduction in pathogenic Enterococcus cecorum may be greater than zero and greater than 30%, such as a 5%–30%, 10%–30%, or 15%–30% reduction compared to the amount of pathogenic Enterococcus cecorum present in poultry not administered the combination. This reduction may be specified at various time points, such as 21, 28, 30, 35, and / or 42 days of age, for specific disclosed modes of operation.

[0112] Administration of the disclosed Bacillus combination to poultry may reduce the infiltration of pathogenic Enterococcus cecorum into the spleen and liver in the poultry. The reduction in pathogenic Enterococcus cecorum in the spleen and / or liver may be greater than 0 and greater than 30%, such as a 5%–30%, 10%–30%, or 15%–30% reduction compared to the amount of pathogenic Enterococcus cecorum present in the liver or spleen not administered with the combination. This reduction may be specified at various time points, such as 21, 28, 30, 35, and / or 42 days of age, for specific disclosed modes of operation.

[0113] Administration of the disclosed Bacillus combination to poultry may reduce the infiltration of pathogenic Enterococcus cecorum in the spine of the poultry. The reduction in pathogenic Enterococcus cecorum in the spine may be greater than 0 and greater than 30%, such as a 5%–30%, 10%–30%, or 15%–30% reduction compared to the amount of pathogenic Enterococcus cecorum present in spines not administered with the combination. This reduction may be specified at various time points, such as 21, 28, 30, 35, and / or 42 days of age, for specific disclosed modes of operation.

[0114] The combined administration of Bacillus species to animals such as poultry or cattle has yielded substantially beneficial results compared to the administration of each Bacillus species individually or in combinations of only two species. These beneficial results are determined by considering, for example, feed conversion ratio, mean body weight, mean body weight gain, coefficient of variation in body weight, breast meat yield, poultry mortality, lesion score, incidence of Salmonella / E. coli / Clostridium perfingens (CP), and / or oocysts in feces at various time points during chick rearing.

[0115] Administration of the disclosed Bacillus combination to poultry may reduce E. coli in the poultry. The reduction in E. coli may be greater than zero and greater than 25%, such as a 5%–25% or 10%–22% reduction compared to the amount of E. coli present in poultry not administered the combination. This reduction may be specified at various time points, such as 21, 28, 30, 35, and / or 42 days of age, for specific disclosed modes of operation.

[0116] Administration of the disclosed Bacillus combination to poultry may reduce the aerobic bacterial count (APC) in the poultry. The reduction in APC may be greater than zero and greater than 20%, such as a 5%–20% or 10%–18% reduction compared to the amount of APC present in poultry not administered the combination. This reduction may be specified at various time points, such as 21 and / or 42 days of age, for specific disclosed modes of operation.

[0117] Administration of the disclosed Bacillus combination to poultry may reduce Salmonella levels in the poultry. The reduction in Salmonella levels may be greater than zero and greater than 65%, such as a 25%–65%, 35%–65%, or 45%–65% reduction compared to the amount of Salmonella present in poultry not administered the combination. This reduction may be specified at various time points, such as 21, 28, 30, 35, and / or 42 days of age, for specific disclosed modes of operation.

[0118] Administration of the disclosed Bacillus combination to poultry may reduce Clostridium perfringens in the poultry. The reduction in Clostridium perfringens may be greater than zero and greater than 30%, such as a 5%–30%, 10%–30%, or 15%–30% reduction compared to the amount of Clostridium perfringens present in poultry not administered the combination. This reduction may be specified at various time points, such as 21, 28, 30, 35, and / or 42 days of age, for specific disclosed modes of operation.

[0119] Administration of the disclosed Bacillus combination to poultry may reduce fecal oocysts in the poultry. The reduction in oocysts may be greater than zero and greater than 90%, such as a 50%–90% or 75%–90% reduction compared to the amount of oocysts present in poultry not administered the combination. This reduction may be specified at various time points, such as 21, 28, 30, 35, and / or 42 days of age, for specific disclosed work modes.

[0120] Administration of the disclosed Bacillus combination to poultry may improve lesion scores in the poultry. Lesion scores may improve (i.e., decrease) by more than 0 and 75%, e.g., 25% to 75%, or 30% to 75%, compared to lesion scores in poultry not administered the combination. This improvement may be specified at various time points, such as 21 and / or 42 days of age, for specific disclosed work models.

[0121] Administering the disclosed Bacillus combination to poultry may improve their feed conversion ratio. The feed conversion ratio may improve (i.e., decrease) by more than 0 and 10%, e.g., 2%–8% or 4%–8%, compared to poultry not administered the combination. Improvements may be observed at various time points, such as at 14, 21, 28, 30, 35, and / or 42 days of age.

[0122] Administration of the disclosed Bacillus combination to poultry may reduce mortality in the poultry. Mortality may decrease by more than 0% and more than 95%, e.g., 50%–95%, 75%–95%, or 80%–95%, compared to the mortality of poultry not administered the combination. Improvement may be observed at various time points, such as when the poultry are 14, 21, 28, 30, 35, and / or 42 days old. [Examples]

[0123] Example 1 In vitro inhibition of Enterococcus cecorum by Bacillus strains - inoculation into liquid culture overview An in vitro inhibition assay determines whether, and to what extent, one cultureable microorganism can inhibit or prevent the growth of a second and / or third microorganism. To perform the assay, two or more different microorganisms are cultured close to each other on the same petri dish containing culture media that can support the growth of each microorganism. After incubation of the plate, the degree of inhibition of growth by the first microorganism against the second and / or third microorganism can be observed by measuring the radius from the colony edge of the first microorganism to the colony edge of the inhibited microorganism. This halo-shaped area, or radius, is called the "inhibition zone" and may be an area where neither microorganism grows on the medium, or where growth is inhibited. Therefore, the larger the inhibition zone, the more effectively the first microorganism can prevent the growth of the second microorganism. The inhibition zone may be caused by secondary metabolites secreted by the first microorganism and leached into the surrounding medium. In vitro inhibition assays can be used to determine whether, and to what extent, beneficial microorganisms can prevent or inhibit the growth of pathogenic microorganisms. Examples 1 and 2 of this specification demonstrate, by in vitro inhibition assays, the ability of beneficial Bacillus species to inhibit the growth of various Enterococcus cecorum strains.

[0124] A. Materials and Methods From the frozen cultures, each Bacillus and wild-isolated Enterococcus cecorum strain was streaked onto plates containing either triptycase soybean agar (TSA) or blood agar (BA), respectively. The seeded plates were then incubated overnight at 36±2°C, aerobically for Bacillus and in 5±2% CO2 for E. cecorum. The cultures were then maintained at 2–8°C for up to two weeks. Next, four spots of Bacillus were inoculated onto Brain Heart Infusion Agar (BHIA) plates, yielding two plates with four strains each. Each Bacillus spot was approximately 5 mm. These plates were then incubated overnight aerobically at 36±2°C. One colony of each Enterococcus cecorum strain to be tested was inoculated into approximately 5 mL of BHI broth. Next, the E. cecorum culture was incubated in 5±2% CO2 at 36±2°C for 16-18 hours to obtain a theoretical final concentration of 1 × 10⁻⁶. 9 The concentration was adjusted to CFU / mL. After incubation overnight, a very thin layer of molten BHIA was sprayed onto a BHIA plate containing the grown Bacillus spots and dried. Each cultured Enterococcus cecorum culture was diluted 1:1,000 with 0.1% peptone to a theoretical concentration of 1× 10 Prepared to a concentration of 6 CFU / mL. Three tubes containing 9 mL of molten BHIA were seeded with 1 mL of diluted Enterococcus cecorum in each tube, and 1 × 10⁶ of each tube was used. 5CFU / mL was obtained and gently vortexed. 3 mL of seeded molten BHIA was removed. The remaining 7 mL of suspension was slowly poured into two BHIA plates containing Bacillus spots, taking care not to pour directly onto the Bacillus spots. For control, a third seeded BHIA was poured onto the surface of an unseeded BHIA plate and allowed to dry. Once the plates were dry, a disk containing 20 μL of 200 μg / mL enrofloxacin solution and a disk containing 20 μL of sterile distilled water were placed on the surface of the solidified culture medium. The plates were then incubated overnight in 5 ± 2% CO2 at 36 ± 2°C. The Enterococcus cecorum inhibition zone was measured radially from the Bacillus spots.

[0125] B. Results [Table 1]

[0126] Example 2 In vitro inhibition of Enterococcus cecorum by Bacillus strains - spray test A. Materials and Methods Frozen liquid stocks of Bacillus and Enterococcus cecorum strains were thawed to room temperature. Loops of inoculum from each Bacillus species were individually plated onto triptycase soybean agar (TSA) plates and grown aerobically overnight at 36°C. Simultaneously, liquid stocks of E. cecorum wild isolates were thawed to room temperature and plated onto Brain Heart Infusion Agar (BHIA) plates overnight at 36°C in a 5% CO2 atmosphere. Bacillus or Enterococcus cecorum strains were simultaneously seeded on separate BHIA plates and incubated overnight at 36°C in an aerobic atmosphere (Bacillus) or a 5% CO2 atmosphere (E. cecorum).

[0127] After incubation overnight, the diluted E. cecorum culture was added to a liquid atomizer equipped with a spray nozzle. The liquid atomizer was decontaminated and washed with peroxyacetic acid before use, followed by rinsing with sterile water. The lid was removed from the BHIA plate containing the Bacillus species that had been grown overnight. Using the atomizer, a thin layer of E. cecorum diluted with sterile water was sprayed onto visible Bacillus colonies. The lid was replaced, and the BHIA plate was placed in an incubator overnight at 36°C in an atmosphere containing 5% CO2. After incubation, the plate was visually inspected for Bacillus colony size and inhibition zone size, and the sizes were reported in millimeters.

[0128] B. Results Tables 2-5. Zones of inhibition (mm) of various Bacillus species and strains against various Enterococcus cecorum strains. [Table 2] [Table 3] [Table 4] [Table 5]

[0129] Example 3 In vivo inhibition of E. cecorum by Bacillus combination therapy A.Purpose In this example, we evaluate the ability of the Bacillus combination DFM to prevent E. cecorum from systemically establishing itself in broiler chickens.

[0130] B. Materials and Methods Experimental ration The feed consists of non-medicinal, commercially available broiler starter and grower diets. Feeding is free from the date of arrival of the chicks, as follows: Starter from DOT0 to DOT22, grower from DOT22 to DOT36, and finisher from DOT36 to DOT41 (end of experiment). All feeds are labeled with the processing code, dietary stage, and manufacturing date. Throughout the experiment, the feed is given as a mash. Experimental processed feeds are prepared from the base feed formulation. The processed feeds are mixed to ensure a uniform distribution of each test substance.

[0131] Animal information On the day of hatching, chicks of the Ross x Ross strain were obtained from the hatchery and their sex was determined. All birds were vaccinated (by spray cabinet) with commercially available, approved coccidia vaccine at the normally recommended dose. Only chicks that appeared healthy were used in this study.

[0132] housing Upon arrival, the chicks are placed in a barn with a dirt floor and hard walls, in a 5x5 foot (1.5m x 1.5m) floor pen (1.0 foot per chick). 2 The birds were raised within a specified stocking density, on fresh litter (at the time of placement), and under ambient humidity. The litter was not changed or modified during the study period. Feed and water were provided freely throughout the experiment. Each pen contained one tube feeder and a Plasson drinker (bird-to-feeder / drinker ratio of 25).

[0133] Where necessary, thermostat-controlled gas heaters are the primary heat source for the chicken coop. One heat lamp per pen provides supplemental heat during brooding. Fans and evaporative cooling pads are used to cool the birds. The birds are provided with a lighting program according to the primary breeder's recommendations.

[0134] Experimental design and processing Number of processing groups and targets Between the two chicken coops, there are approximately 32 pens containing a total of about 800 chicks, with each pen containing about 25 chickens. There are 4 processing groups and 8 replication blocks.

[0135] Type of processing The study includes four treatments: (1) a control group with no preventative treatment, (2) a Bacillus combination treatment with a disclosed feed content of 500,000 CFU / g per gram of finished feed, (3) a Bacillus combination treatment with a disclosed feed content of 1,000,000 CFU / g per gram of finished feed, and (4) a Bacillus combination treatment with a disclosed feed content of 1,500,000 CFU / g per gram of finished feed. Each of these four treatment groups will be challenged with Enterococcus cecorum on day 14. Data collection will be carried out during the starter, grower, and finisher periods. The Bacillus combinations consist of Bacillus pumilus (30%), Bacillus subtilis (50%), and Bacillus licheniformis (20%).

[0136] Bird allocation and pen randomization 800 birds are assigned to four treatment groups, each containing 8 replicated pens and 25 birds per pen. Treatment groups are assigned to pens within a block using randomized complete blocks. Complete randomization is performed, and a random permutation table is used for assigning treatment groups to pens. The experiment begins when the birds are placed (hatching day, DOT0), at which point the birds are assigned to experimental pens by random allocation, and the weight of the pens is recorded. Only healthy birds are selected. At DOT0, the weight of each group is recorded per pen. No birds are exchanged during the experiment.

[0137] Challenge administration, sample collection, and analysis 1. Challenge Method: In DOT14, administer 0.1 ml of Enterococcus cecorum SA3 (1.0 x 10) to all birds during processing. 7 CFU / Tri) is administered orally by forced administration. 2. Sepsis samples. At 28 days of age, four birds per pen were necropsed, and the spleens were aseptically collected in individual Whirlpak bags and immediately placed on ice for culture of challenge strains of E. cecorum. 3. Spondylitis Samples: For each treatment, 100 birds were necrotized using DOT41, and free thoracic vertebrae (FTVs) were cultured sterile using Stuart swabs. The spleens were also removed sterile, placed in individual Whirlpak bags, and frozen. 4. Mortality. After DOT28, autopsy is performed on any mortality, the spleen is removed and frozen, and vertebral sections are immersed in 10% buffered formalin.

[0138] Feed changes Feed the birds feed appropriate for processing from DOT0 to DOT41. At DOT22, remove the remaining starter feed, weigh it, and replace it with grower feed up to DOT36 and finisher feed up to DOT41. At DOT41, remove the remaining finisher feed and weigh it again.

[0139] Body weight and feed weight Weigh all birds in each pen at DOT 0, 22, 36, and 41. Measure the weight of the feed to be added to each pen's feeder at the start of each feeding period at DOT 0, 22, 36, and 41 (starter and grower). Weigh (and record) any additional feed bags for each pen (as needed) during each feeding period. Dispense the feed into the feeder as needed from the pre-weighed bags (assigned to each pen) throughout each period. Weigh and dispose of any remaining feed in the feeder (and feed bags, if applicable) at DOT 22, 36, and 41. Record the weight of the empty feeder before the start of the test. The test ends at DOT 41.

[0140] management Disease control: No adjunctive drug therapies will be used during the trial.

[0141] Monitoring: Monitor all birds for general flock conditions, temperature, lighting, water, feed, litter conditions, and any unexpected conditions / events in the poultry house.

[0142] Mortality Rate: Check the pen daily to confirm the mortality rate. Birds are euthanized only to alleviate suffering. Record the date and weight (kg) of all birds euthanized (or found dead). Perform a macroscopic autopsy on all dead or euthanized birds to determine their sex and presumed cause of death.

[0143] Data Management Data management and statistical analysis will be performed on the results of culture and lesions related to weight gain, feed intake, and feed conversion ratio, as well as lesion scores.

[0144] Performance: Calculate the average performance of weight gain, feed intake, and feed conversion ratio (adjusted for mortality: feed intake / final live weight + death weight) in the pen.

[0145] The number of spleen cultures positive for E. cecorum will be compared using a significance test performed with Fisher's exact test, with a cutoff of P=0.05.

[0146] Osteochondrosis (OCD) and spinal cord lesions. Mean OCD scores were compared between treatment groups using the nonparametric Kruskal-Wallis test, and lesion prevalence was compared to the control group using Fisher's exact test. Significance was set at P=0.05.

[0147] Example 4 Prevention of Enterococcus cecorum-induced spondylitis (kinky back) using microorganisms directly administered. Experimental Design Enterococcus cecorum has been demonstrated to be the leading bacterium most frequently isolated from cases of free thoracic spondylitis (FTV). Commonly known as "kinkyback" in broilers, E. cecorum can be a normal flora bacterium found in the intestines of broiler chickens. This study evaluated the ability of directly fed microorganisms and autovaccines to prevent systemic colonization of broilers by E. cecorum.

[0148] Materials and methods Experimental ration The feed consisted of non-medicinal, commercially available broiler starter and grower feeds formulated according to NRC guidelines, including feeds commonly used in the United States. Feeding was free-flowing from the day of arrival of the chicks, as follows: Starter from DOT0 to DOT22, grower from DOT22 to DOT32, and finisher from DOT32 to DOT42 (end of the study). Throughout the study, the feed was given as crumble (starter) or pellets (grower and finisher). The diet formulation was included in the source data. Experimental treated feeds were prepared from the basal feed formulation. The amounts of all basal feeds and test substances in each treatment batch were documented and included in the source data file.

[0149] Animal information Chicks were acquired from the hatchery on the day of hatching, and their sex was determined. Upon arrival, all birds were vaccinated (by spray cabinet) with commercially available, approved coccidia vaccine at the normally recommended dose. Only chicks that appeared healthy were used in this study.

[0150] housing Upon arrival, the chicks are placed in a barn with a dirt floor and hard walls, in a 5x5 foot (1.5m x 1.5m) floor pen (1.0 foot per chick). 2 The birds were reared at ambient humidity on fresh litter (at the time of placement) within a specified stocking density. The litter was not changed or modified during the study period. Feed and water were freely available during the study period. Each pen contained one tube feeder and a Plasson drinker (bird-to-feeder / drinker ratio of 25).

[0151] Where necessary, thermostat-controlled gas heaters were the primary heat source for the chicken coop. One heat lamp per pen provided supplemental heat during brooding. Fans and evaporative cooling pads were used to cool the birds. The birds were provided with a lighting program according to the primary breeder's recommendations. Pen diagrams were documented and included in the final report along with source data. [Table 6]

[0152] The disclosed composition used in this study contained Bacillus pumilus (30%), Bacillus subtilis (50%), and Bacillus licheniformis (20%) as the sole active (DFM) component.

[0153] Bird allocation and pen randomization The birds were assigned to four treatment groups, each containing 8 replicated pens and 25 birds per pen. Treatment groups were assigned to pens within their blocks using randomized complete blocks. SPRG completed the randomization of treatment groups and pen assignments using a random permutation table. The experiment began when the birds were placed (hatching day, DOT0), at which point the birds were assigned to experimental pens by random assignment, and the pen weights were recorded. Only healthy birds were selected. At DOT0, group weights were recorded per pen. No birds were exchanged during the experiment.

[0154] Challenge administration, sample collection, and analysis Challenge Method: In DOT04, administer 0.1 ml of Enterococcus cecorum SA3 (4.0 x 10) to all birds during processing. 7 CFU / Tri) was administered orally by forced administration.

[0155] Sepsis samples: At 22 days of age, four birds were randomly selected from the first acquired birds, necropsied, and their spleens were aseptically collected in individual Whirlpak bags and immediately placed on ice for culture of challenge strains of E. cecorum.

[0156] For the DOT42 sample of spondylitis, 100 birds were initially selected, and 12 birds were sampled from 6 pens and 14 birds from the remaining 2 pens after each treatment. These samples were then necrotized, and free thoracic vertebrae (FTVs) were aseptically cultured using Stuart swabs. Additionally, the spleens were aseptically removed, placed in individual Whirlpak bags, and frozen.

[0157] Mortality. After DOT22, any mortality was autopsied, the spleen was removed and frozen, and vertebral sections were immersed in 10% buffered formalin.

[0158] Feed changes The birds were fed feed suitable for processing from DOT0 to DOT42. At DOT22, the remaining starter feed was removed, weighed, and replaced with grower feed up to DOT32, then weighed, removed, and replaced with finisher feed at DOT42. At DOT42, the remaining finisher feed was removed and weighed again. The weight of all unused feed was measured and discarded in the SPRG waste bit.

[0159] Body weight and feed weight The weight of all birds was weighed in each pen at DOT 0, 22, 32, and 42. The weight of feed to be added to each pen's feeder was measured at the start of each feeding period at DOT 0, 22, 32, and 42 (starter and grower). Any additional feed bags were weighed (and recorded) for each pen (as needed) during each feeding period. Feed was distributed as needed from pre-weighed bags (assigned to each pen) into the feeders throughout each period. Any remaining feed in the feeders (and feed bags, if applicable) was weighed and disposed of at DOT 22, 32, and 42. The weight of the empty pan feeders was recorded before the start of the experiment. The experiment ended at DOT 42.

[0160] management Disease control: No adjunctive drug therapies were used during the study. Study personnel who needed to enter the pen (e.g., collecting birds for the study procedure) wore disposable plastic boots. To prevent fecal matter from being scattered around the facility, disposable plastic boots were removed when people left the pen. Disposable plastic boots were properly disposed of after use.

[0161] Monitoring: All chickens were monitored for general flock conditions, temperature, lighting, water, feed, litter conditions, and unexpected conditions / events in the chicken coop. Results were recorded twice daily during normal working hours (one observation was recorded on the final day of the survey). One observation was recorded on Saturdays, Sundays, and compensatory holidays.

[0162] Mortality Rate: The mortality rate was checked daily using a pen. Birds were euthanized only to alleviate suffering. The date and weight (kg) of all birds euthanized (or found dead) were recorded. Gross autopsies were performed on all dead or euthanized birds to determine their sex and presumed cause of death.

[0163] Euthanasia and Disposal: Birds requiring euthanasia were euthanized by designated personnel according to facility procedures and disposed of according to facility procedures.

[0164] Disposal of birds and feed: All birds were disposed of in an appropriate manner.

[0165] Scale: Procedures for maintaining and standardizing the scale were performed before use.

[0166] Source Data Management and Handling: Data was recorded using non-volatile ink. Entries were legible, and source data sheets were signed (or initially signed) and dated for each individual record entry. All source data errors and / or changes were initially and dated, and a brief explanation (or error code) was written directly on the form.

[0167] Performance: The mean performance metrics for weight gain, feed intake, and feed conversion ratio (adjusted for mortality: feed intake / final live weight + mortality weight) in pens were calculated. Statistical evaluation of the data was performed using the Statistix for Windows program. The procedure used was a standard linear procedure using ANOVA comparing means with a minimum significance level (t-test) (LSD) (T) at a significance level of 0.05.

[0168] The number of spleen cultures positive for E. cecorum was compared using significance testing with Fisher's exact test at a cutoff of P=0.05.

[0169] Osteochondrosis (OCD) and spinal cord lesions. Mean OCD scores were compared between treatment groups using the nonparametric Kruskal-Wallis test, and lesion prevalence was compared to the control group using Fisher's exact test. Significance was set at P=0.05.

[0170] result Results from d0 to d22 are summarized in Table 7, and the pre-planned comparison of the effects of administration is summarized in Table 8. No overall significant effect of the treatment was observed with respect to feed intake (P=0.19), adjusted FCR (P=0.51), unadjusted FCR (P=0.52), or body weight gain (P=0.30). [Table 7] [Table 8]

[0171] Results from d0 to d32 are summarized in Table 9, and the pre-planned comparison of the effects of administration is summarized in Table 10. No overall significant effect of the treatment was observed with respect to feed intake (P=0.093), adjusted FCR (P=0.45), or unadjusted FCR (P=0.50), but a significant effect of the treatment was observed with respect to body weight gain (P=0.037). [Table 9] [Table 10]

[0172] Results from d0 to d42 are summarized in Table 11, and the pre-planned comparisons of the effects of administration are summarized in Table 12. No overall significant effect of the treatment was observed for adjusted FCR (P=0.19) or unadjusted FCR (P=0.32), but significant effects were observed for both feed intake (P=0.019) and body weight gain (P=0.044). While the overall effect of the treatment was significantly higher for body weight gain, none of the individual pairwise comparisons between the treatments were statistically significant when the overall probability of a Type I error was controlled at 5% using the Tukey method. [Table 11] [Table 12]

[0173] Overall mortality and FHN mortality. The percentages of overall mortality and FHN mortality are summarized in Table 13. There were no significant differences between the treatments in terms of the percentage of overall mortality (P=0.50) or the percentage of FHN mortality (P=0.45). [Table 13]

[0174] Prevalence of E. cecorum in the spleen. The prevalence of E. cecorum in the spleen is summarized in Table 14. Factor analysis showed a significant interaction between the effect of treatment and day (P<0.001). There was no significant difference between treatments on day 22 (P=0.41), but a significant difference was observed on day 42 (P<0.001). On day 42, the prevalence at both T2 and T5 was significantly lower than the prevalence at T4.

Table 14

[0175] Incidence rate of free thoracic vertebra (FTV) E. cecorum. The incidence rate of FTV E. cecorum is summarized in Table 15. On the 42nd day, there was a significant difference between treatments (P = 0.002), and the incidence rate of T2 was significantly lower than either T1 or T4.

Table 15

[0176] Conclusion In this study, the disclosed compositions (500,000 CFU / g, 1,000,000 CFU / g, and 1,500,000 CFU / g feed) in the starter diet in an Enterococcus cecorum (EC) challenge were evaluated. Each group was represented by eight replicate pens of 25 male Ross broiler chickens. On Day of Trial 4 (DOT4), E. cecorum was force-fed orally (4.0×10 7 CFU / bird). At DOT22, spleens were collected from four birds within each pen and subjected to culture. At DOT42, spleens and free thoracic vertebra swabs were collected from 100 birds per treatment. Bird and feed weights were measured at DOT22, 32, and 42 to evaluate performance metrics.

[0177] Performance results In previous E. cecorum challenge models, this bacterium has been shown to have an adverse effect on growth and performance. Given the DOT4 challenge, birds may have experienced an impact on performance during the starter phase. At DOT22, the weight gain in the 500,000 CFU / g group (T2) was numerically greater (0.860 A ) than the challenge control (0.816 A(Table 7). The disclosed composition at 500,000 CFU / g also resulted in higher feed intake than the challenge control. Feed intake and weight gain in the other groups were intermediate between the lowest content of the disclosed composition and the challenge control group (Table 7). As the birds gained more weight, the stress on the leg joints increased, which was evident from the increase in birds with leg disabilities in all difficult processes. At DOT32, the disclosed composition at 500,000 CFU / g showed significantly greater weight gain than the challenge control (Table 9). The disclosed 1,000,000 CFU / g composition was statistically intermediate between these groups. Feed intake reflected the weight data at day 32 (Table 9). At day 42, the disclosed composition at 500,000 CFU / g showed a weight gain (2.601) compared to the challenge control (2.601 B Compared to (2.773), they maintained a greater weight gain. A ) (Table 11). 1,000,000CFU / g(2.698 A The disclosed composition showed moderate weight gain. The disclosed composition maintained significantly the highest feed intake throughout the study period when it was present in the lowest amount. At the end of the study, the disclosed composition of 1,000,000 CFU / g had the lowest unadjusted FCR (Table 11). The overall mortality rate in this study was relatively low at 3.5% in the challenge group. Both the 500,000 CFU / g and 1,000,000 CFU / g disclosed compositions had numerically low mortality rates of 2.0% (Table 13).

[0178] Culture results of E. cecorum Spleen samples from day 22 indicated that the challenge was progressing normally. There was no significant difference in splenic E. cecorum positivity (Table 14). On day 42, the prevalence of E. cecorum in the 500,000 CFU / g treatment group was lowest in the splenic E. cecorum area.

[0179] Preventing the colonization of free thoracic vertebrae (FTVs) can reduce the incidence of kinky back in broilers. On day 42, the 500,000 CFU / g treatment group showed a significant reduction in E. cecorum culture-positive vertebrae from challenge controls (Table 15).

[0180] whole The 500,000 CFU / g composition of this disclosure consistently improved the performance of birds in the Enterococcus cecoum challenge. This 500,000 CFU / g content more effectively prevented the migration of E. cecorum to the spleen and spine.

[0181] Example 5 Prevention of Enterococcus cecorum-induced spondylitis (kinky back) in broilers from breeders that have received in-house vaccination and broilers fed with direct-feed microorganisms.

[0182] Experimental Design Enterococcus cecorum has been demonstrated to be the leading bacterium most frequently isolated from cases of free thoracic spondylitis (FTV). Commonly known as "kinkyback" in broilers, E. cecorum can be a normal flora bacterium found in the intestines of broiler chickens. It has been demonstrated that microfractures occur in broiler FTV, and these are often infected with E. cecorum. In this study, we evaluated the ability of directly fed microorganisms and maternal antibodies derived from autovaccines to prevent systemic colonization of broilers by E. cecorum.

[0183] Materials and methods Experimental ration Broiler feed consisted of non-medicinal, commercially available broiler starter and grower feeds formulated according to NRC guidelines, including feeds commonly used in the United States. Subsequently, hen feed was formulated to meet Cobb breeder guidelines. Feeding was free-flowing from the day of chick arrival, as follows: Starter from DOT0 to DOT28, grower from DOT28 to DOT35, and finisher from DOT35 to DOT42 (end of study). All feeds were labeled with the treatment code, dietary stage, and manufacturing date. During the study period, hens were fed mashed feed and broilers were fed pelletized feed. Experimental treatments were prepared from the basal feed formulations. The amounts of all basal feeds and test substances in each treatment batch were documented and included in the source data file. Treatment feeds were mixed to ensure uniform distribution of each test substance.

[0184] Animal information Experiments on young hens For the experiment, 240 Cobb hens and 30 Cobb roosters, all 10 weeks old, were selected. Upon placement, 120 hens (T3 and T4) were vaccinated on the left breast and tagged with colored ear tags on their wings. Hens (T1 and T2, control) and DFM-only hens were tagged with different colored wings and were not vaccinated. The chamber was divided in half. The birds were housed on a concrete floor covered with fresh pine shavings. Water was provided in either bell or nipple form, depending on the source farm. Feeders were trough feeders with space according to breeder guidelines. Subsequently, when the hens reached 18 weeks of age, T3 and T4 hens received a second vaccination on the right breast. At 17 weeks of age, the day length was extended by 15 minutes per week to stimulate egg production. After 18 weeks of vaccination, manually collected nests were placed in each chamber. From week 23 (6 weeks after the second vaccination), eggs were collected from each chamber for the following three weeks and processed in two Jamesway incubators according to T1 / T2 and T3 / T4. After hatching, the chicks were positioned according to Table 18. [Table 16]

[0185] The DFM composition used in this study contained Bacillus pumilus (30%), Bacillus subtilis (50%), and Bacillus licheniformis (20%) as the sole active (DFM) component.

[0186] Kinkyback test The chicks were acquired from the hatchery on the day of hatching. The strain was Cobb x Cobb. The sex of the birds was determined at the hatchery. Upon arrival, all birds were vaccinated (by spray cabinet) with commercially available, approved coccidia vaccine at the normally recommended dose. Only chicks that appeared healthy were used in this study.

[0187] housing Upon arrival, the chicks are placed in a barn with a dirt floor and hard walls, in a 5x5 foot (1.5m x 1.5m) floor pen (1.0 foot per chick). 2 The birds were reared at ambient humidity on fresh litter (at the time of placement) within a specified stocking density. The litter was not changed or modified during the study period. Feed and water were freely available during the study period. Each pen contained one tube feeder and a Plasson drinker (bird-to-feeder / drinker ratio of 25).

[0188] Where necessary, thermostat-controlled gas heaters were the primary heat source for the chicken coop. One heat lamp per pen provided supplemental heat during brooding. Fans and evaporative cooling pads were used to cool the birds. The birds were provided with a lighting program according to the recommendations of the primary breeder. [Table 17]

[0189] Bird allocation and pen randomization 800 straight-line (male and female) birds were assigned to four treatment groups, each containing 8 replica pens and 25 birds per pen. Treatment groups were assigned to pens within their blocks using randomized complete blocks. The experiment began when the birds were placed (hatching day, DOT0), at which point the birds were randomly assigned to experimental pens, and the weight of the pens was recorded. Only healthy birds were selected. At DOT0, the group weight was recorded per pen. No bird changes were made during the experiment.

[0190] Challenge administration, sample collection, and analysis 1. Challenge Method: In DOT01, all birds undergoing treatment were force-administered 0.1 ml of Enterococcus cecorum SA3 (1.0 × 10⁷ CFU / bird) orally.

[0191] 2. Sepsis samples. At 13 days of age, four birds were randomly selected from each pen by the first birds obtained, necropsied, and the spleens were aseptically collected in individual Whirlpak bags and immediately placed on ice for culture of challenge strains of E. cecorum.

[0192] 3. Spondylitis Samples. In DOT42, 100 birds were initially selected, and 12 birds were sampled from 6 pens and 14 birds from the remaining 2 pens after each treatment. These samples were necrotized, and free thoracic vertebrae (FTVs) were aseptically cultured using Stuart swabs. The spleens were also aseptically removed, placed in individual Whirlpak bags, and frozen.

[0193] 4. Mortality. Deceased animals were autopsied after DOT28, and if inflammation or abscess was observed, the spleen or infected joint or vertebrae were cultured.

[0194] Feed changes The birds were fed feed suitable for processing from DOT0 to DOT42. At DOT28, the remaining starter feed was removed, weighed, and replaced with grower feed up to DOT35. The remaining grower feed was weighed, removed, and replaced with finisher feed at DOT42. At DOT43, the remaining finisher feed was removed and weighed again. All unused feed was weighed and discarded.

[0195] Body weight and feed weight The weight of all birds was weighed in each pen at DOT 0, 28, 35, and 42. The weight of feed to be added to the feeder in each pen was measured at the start of each feeding period at DOT 0, 28, 35, and 42 (starter and grower). Any additional feed bags were weighed (and recorded) for each pen (as needed) during each feeding period. Feed was distributed as needed from pre-weighed bags (assigned to each pen) into the feeder throughout each period. Any remaining feed in the feeder (and feed bags, if applicable) was weighed and disposed of at DOT 28, 35, and 42. The weight of the empty pan feeder was recorded before the start of the experiment. The experiment ended at DOT 42.

[0196] management Disease control: No adjunctive drug therapies were used during the study. Study personnel who needed to enter the pen (e.g., collecting birds for the study procedure) wore disposable plastic boots. To prevent fecal matter from being scattered around the facility, disposable plastic boots were removed when people left the pen. Disposable plastic boots were properly disposed of after use.

[0197] Monitoring: All chickens were monitored for general flock conditions, temperature, lighting, water, feed, litter conditions, and unexpected conditions / events in the chicken coop. Results were recorded twice daily during normal working hours (one observation was recorded on the final day of the survey). One observation was recorded on Saturdays, Sundays, and compensatory holidays.

[0198] Mortality: Pens were checked daily to confirm mortality. Birds were euthanized only for the purpose of relieving suffering. The date and removal weight (kg) of all birds that were disposed of (or found dead) were recorded. Gross necropsies were performed on all dead or disposed birds to identify the sex of the birds and the presumed cause of death.

[0199] Euthanasia and disposal: Birds requiring euthanasia were euthanized by designated personnel following the facility's procedures and disposed of following the facility's procedures.

[0200] Disposal of birds and feed: All birds were disposed of in an appropriate manner. All dead animals and remaining feed (including mixer flushes) were buried.

[0201] Scale: Procedures for scale maintenance and standardization were performed before use.

[0202] Source data management and handling: Data were recorded in non-volatile ink. Entries were legible, and source data sheets were signed (or initialed) and dated for each individual recording entry. Initials and dates were attached to all source data errors and / or changes, and a brief explanation (or error code) was written directly on the form.

[0203] Performance. Performance averages for weight gain, feed intake, and feed requirement ratio (adjusted for mortality: feed intake / final live weight + dead weight) in pens were calculated. Statistical evaluation of the data was performed using the Statistix for Windows program. The statistical model included the main effects of vaccine and DFM and their interactions. For response criteria where the vaccine × DFM interaction was significant, means were separated by the PDIFF option with Tukey-Kramer adjustment at a significance level of 0.05 and a trend level of 0.10.

[0204] Spleen cultures. The number of E. cecorum-positive spleen cultures was compared using a significance test performed using Fisher's exact test with a cutoff set at P = 0.05.

[0205] Osteochondrosis (OCD) and spinal cord lesions. The mean OCD scores were compared between treatment groups using the non-parametric Kruskal-Wallis test, and the lesion prevalence was compared with the control using Fisher's exact test. Significance was set at P = 0.05.

[0206] Statistical methods The prevalence of E. cecorum in the spleen and FTV samples was compared between treatments using generalized estimating equations (GEE) logistic regression with an exchangeable working correlation structure to account for the correlation between the responses of chickens from the same pen. Pairwise comparisons between treatments were made using the Bonferroni method, limiting the overall type I error probability to 5%. All statistical tests assumed two-sided hypotheses and considered P < 0.05 significant. Analyses were performed using commercially available statistical software (Stata version 18.0, StataCorp LLC, College Station, TX).

[0207] Results Prevalence of E. cecorum in the spleen. The prevalence of E. cecorum in the spleen is summarized in Table 18. In the factor analysis, there was no significant effect of treatment (P = 0.39), but there was a significant effect of day (P < 0.001), and the prevalence on day 42 was higher than that on day 13. There was a significant interaction between the effect of treatment and day (P < 0.12).

Table 18

[0208] Prevalence of E. cecorum in free thoracic vertebrae (FTV). The prevalence of E. cecorum in FTV is summarized in Table 19. There was no significant difference between treatments on day 42 (P = 0.098).

Table 19

[0209] Spleen E. faecalis prevalence. The prevalence of spleen E. faecalis is summarized in Table 20. Since there were no E. faecalis-positive samples in T2, it was not possible to compare treatments using logistic regression. Using Fisher's exact test, which does not consider correlations between responses in birds within the same pen, the overall effect of treatment was not significant (P=0.15), but there was a significant effect with respect to days (P=0.001), with the prevalence at day 13 being higher than at day 42. When comparing treatments individually at each day, no significant effect of treatment was observed at either day 13 (P=0.21) or day 42 (P=1.00). [Table 20]

[0210] Prevalence of E. faecalis / E. faecium in free thoracic vertebrae (FTV). E. faecalis was identified in 1 / 100 (1%) of T4 FTV samples, and E. faecium was also identified in 1 / 100 (1%) of T4 FTV samples. Neither E. faecalis nor E. faecium were identified in any T1, T2, or T3 FTV samples. [Table 21] [Table 22] [Table 23]

[0211] Performance Data Analysis Weight gain, feed intake, feed conversion ratio (adjusted for mortality: feed intake / final live weight + mortality weight), and mean causes of death were calculated for each pen. Mortality was assessed by macroscopic lesions at autopsy. Statistical evaluation of the data was performed using the STATISTIX for Windows program (Analytical Software, Tallahassee, FL). The procedure used was a standard linear procedure using ANOVA comparing means with a minimum significance level (t-test) (LSD) (T) at a significance level of 0.05.

[0212] Interpretation of the research This study evaluated the ability of broilers to resist Enterococcus cecorum infection when they were directly fed microorganisms and / or when they were descendants of offspring whose parents were vaccinated with an autologous E. cecorum vaccine. Treatments consisted of a challenge control, DFM alone, hen vaccine alone, and DFM plus hen vaccine. Each treatment consisted of eight replicate pens of 25 straight-line Cobb broilers. Vaccinated parents were intramuscularly vaccinated with an autologous E. cecorum vaccine at 12 and 18 weeks of age. The feed microorganism product (disclosed DFM composition) was included at 0.25 lbs / ton (500,000 CFU / g of finished feed) throughout all stages of the DFM-fed treatment. All broilers were vaccinated with a commercially available coccidiosis vaccine (single dose) by coarse spray. After vaccination, chicks were force-administered 0.1 mL of E. cecorum orally. (Dosage). To monitor the progression of E. cecorum sepsis, spleens were collected from four birds in each repeated study on day 13. On day 42, 100 swab samples of spleens and free thoracic vertebrae were collected from each treatment. These samples were submitted to NCSU for prevalence analysis. To monitor outcome indicators, the weight of the birds and feed was measured on days 0, 28, 35, and 42.

[0213] Prevalence data The prevalence of EC-positive spleen was 13% overall on day 13. a(17 / 128 chickens), and increased significantly to 57% by day 42 b (226 / 400 chickens) (Table 18). There was no significant difference in the prevalence of Enterococcus in spleen samples among treatments. However, both treatments with DFM numerically had lower prevalence values compared to their respective control groups (same vaccination status). The free thoracic swabs in the challenge control group were 22% positive on day 41 で . There was no significant difference in the prevalence of FTV swabs on day 42. Similar to the spleen data, both DFM groups numerically had lower prevalence compared to their respective control groups. Specifically, the challenge control (22% a ) was numerically greater than DFM alone (20% a ), and mendel vaccine alone (37% a ) was numerically greater than DFM plus mendel vaccine (23% a ).

[0214] Enterococcus faecalis was also identified in some of the collected samples. The prevalence was significantly higher on day 13 (5% a ) compared to the 42-day samples (0.5% b ) (Table 20). There was no significant difference in the spleen prevalence of E. faecalis among treatments. The challenge control accounted for more than half of the total positive spleen samples, 5 out of 9 E. faecalis-positive samples, and was numerically the largest. Two chickens were positive for other Enterococcus species (E. faecalis and E. faecium) in the free thoracic samples. Both chickens had received vaccination and DFM treatment.

[0215] Performance results There were no significant differences in the performance of broilers from day 0 to day 28 (Table 21). The adjusted feed requirement rate was compared to the challenge control (1.538 B ) at 35 days, and DFM alone (1.494 B) was low (Table 22). The DFM alone group also had a heavier body weight at the 35-day time point than the challenge control. By 42 days, no significant differences in feed requirement rate or weight gain were observed between treatments (Table 23). The groups administered DFM had numerically lower feed requirement rates, greater weight gain, and lower mortality compared to their respective control groups.

[0216] Overall, there was no significant difference in the prevalence of Enterococcus cecorum among the treatments. However, a numerical decrease in prevalence was seen in the groups receiving DFM in all tissues evaluated. Similarly, the DFM treatment reduced the feed requirement rate at each interval compared to the treatment without the product. No significant change was observed in the ability of Enterococcus cecorum SA3 from vaccinated breeders to migrate and colonize tissues in broilers. Also, the treatment with DFM alone had the lowest mortality from keel break and femoral head necrosis throughout the study (Table 23). Based on the consistent numerical decrease in prevalence and the improvement in broiler performance, DFM appears to affect Enterococcus infections.

[0217] From the perspective of many possible aspects to which the principles of the present disclosure can be applied, it should be recognized that the exemplified aspects are only preferred examples of the present disclosure and should not be construed as limiting the scope of the present disclosure. Rather, the scope of the present disclosure is defined by the following claims. Therefore, the inventors claim as the present disclosure all that falls within the scope and spirit of these claims.

Claims

1. A combination of Bacilus, essentially consisting of Bacilus licheniformis, Bacilus pumilus, and Bacilus subtilis.

2. The combination of Bacillus according to claim 1, comprising Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis.

3. The aforementioned Bacillus licheniformis includes strain OBT618. The aforementioned Bacillus pumilus includes strain OBT13216. The aforementioned Bacillus subtilis includes strain PR104, or Those combinations, A combination of Bacillus according to claim 1 or 2.

4. The aforementioned Bacillus licheniformis is strain OBT618. The Bacillus pumilus mentioned above is strain OBT13216. The Bacillus subtilis mentioned above is strain PR104, or Those combinations, A combination of Bacillus according to claim 1 or 2.

5. A combination of Bacillus according to any one of claims 1 to 3, wherein the relative amounts of Bacillus species in the combination of Bacillus are such that the total amount of Bacillus species is 100%, and each combination includes 1% to 99% of Bacillus lichenformis, 1% to 99% of Bacillus pumilus, and 1% to 99% of Bacillus subtilis in relative amounts.

6. The above combination is 10 ２ ~10 １１ B. lichenformis, 10 CFU / g ２ ~10 １１ B. pumilus at CFU / g, and 10 ２ ~10 １１ A combination of Bacillus according to any one of claims 1 to 3, comprising B. subtilis in CFU / g.

7. The combination of Bacilus according to any one of claims 1 to 6, wherein the combination comprises about 50% Bacilus subtilis, about 20% Bacilus lichenformis, and about 30% Bacilus pumilus in relative amounts such that the relative amounts of Bacilus species are 100% of the total amount of Bacilus species.

8. The combination of Bacillus according to any one of claims 1 to 6, wherein the aforementioned combination is a composition.

9. A combination of Bacillus according to any one of claims 1 to 8, Additional ingredients, A composition containing the following:

10. The composition according to claim 9, wherein the additional components include a carrier, a vitamin, a copper salt, allicin, alliin, alliinase, algae, polyphenols, or plant raw materials containing polyphenols, a feed supplement, additional DFM, feed, or a combination thereof.

11. The composition according to claim 10, wherein the copper salt is copper chloride, copper bromide, copper iodide, copper sulfate, copper sulfite, copper bisulfite, copper thiosulfate, copper phosphate, monobasic copper phosphate, dibasic copper phosphate, copper hypophosphate, copper dihydrogen pyrophosphate, copper tetraborate, copper borate, copper carbonate, copper bicarbonate, copper metasilicate, copper citrate, copper malate, copper methionate, copper succinate, copper lactate, copper formate, copper acetate, copper butyrate, copper propionate, copper benzoate, copper tartrate, copper ascorbate, copper gluconate, or a combination thereof.

12. The composition according to any one of claims 9 to 11, wherein the additional component comprises a vitamin.

13. The vitamin is vitamin A, vitamin B １ , vitamin B ２ , vitamin B ３ , vitamin B ５ , vitamin B ６ , vitamin B １２ , vitamin C, vitamin D, vitamin E, vitamin K, or a combination thereof, the composition according to claim 12.

14. The composition according to any one of claims 9 to 13, further comprising additional direct feeding microorganisms.

15. The composition according to any one of claims 9 to 14, wherein the additional components include one or more of inulin, yucca, quillaja, silica, mineral clay, glucan, mannan, endoglucanohydrolase, or a combination thereof.

16. The composition according to claim 15, wherein the additional component comprises Yucca schidigera, Quillaja saponaria, or a combination thereof.

17. The composition according to claim 15 or 16, wherein the additional components include silica, mineral clay, glucan, and mannan.

18. A combination of Bacillus according to any one of claims 1 to 8, Water and, A composition containing the following:

19. The composition according to claim 18, further comprising an acid.

20. The composition according to claim 19, wherein the acid is acetic acid.

21. A composition for administration to poultry, A combination of Bacillus according to any one of claims 1 to 8, Poultry feed and The composition comprising the above.

22. A composition for administration to poultry, A composition according to any one of claims 1 to 20, Poultry feed and The composition comprising the above.

23. The composition according to claim 21 or 22, wherein the poultry feed comprises plant raw materials, carbonates, sulfates, lactates, oxides, propionates, stearates, phosphates, minerals, copper seeds, sugars, salts, animal protein products, feed products, grain products, plant protein products, processed grain products, roughage products, molasses products, or a combination thereof.

24. The poultry feed is a composition according to any one of claims 21 to 23, comprising beet pulp, ground corn, corn syrup solids, vegetable fiber, rice husks, soluble vegetable fiber, wheat flour, microcrystalline cellulose, calcium carbonate, potassium carbonate, potassium sulfate, sodium sulfate, calcium lactate, calcium oxide, calcium propionate, calcium stearate, dicalcium phosphate dehydrated product, monocalcium phosphate, sodium tripolyphosphate, tetrasodium pyrophosphate, dolomite, silicon dioxide, silica, limestone, vermiculite, bentonite, montmorillonite, kaolin, glucose, sucrose, dextrose, fructose, maltodextrin, sodium chloride, carrageenan, cellulose, guar gum, polyol, sodium aluminosilicate, urea, biotin, folic acid, sodium sesquicarbonate, a methionine source, a lysine source, L-threonine, or a combination thereof.

25. The poultry feed is the composition according to any one of claims 21 to 24, comprising copper sulfate.

26. The composition, 10 ２ ~10 １１ B. licheniformis, 10 CFU / g ２ ~10 １１ B. pumilus at CFU / g, and 10 ２ ~10 １１ A composition according to any one of claims 21 to 25, comprising B. subtilis at CFU / g.

27. The composition, 10 ４ ~10 ６ B. licheniformis, 10 CFU / g ４ ~10 ６ B. pumilus at CFU / g, and 10 ４ ~10 ６ A composition according to any one of claims 21 to 25, comprising B. subtilis at CFU / g.

28. The aforementioned composition, about 2 × 10 ５ Bacillus licheniformis from CFU, approximately 3 x 10 ５ Bacillus pumilus, and approximately 5 x 10 ５ It contains CFU Bacillus subtilis, with a final total concentration of approximately 1 × 10⁻¹⁶ per gram of feed. ６ A composition according to any one of claims 21 to 25, which is a CFU.

29. A method comprising administering the combination of Bacillus described in any one of claims 1 to 8 to a target.

30. The method according to claim 29, wherein the subject is livestock.

31. The method according to claim 30, wherein the livestock is poultry.

32. The method according to claim 30, wherein the livestock is a ruminant.

33. The method according to claim 29, wherein the subject is an aquatic species.

34. A method for reducing poultry mortality, lesion score, incidence of Enterococcus cecorum, Salmonella species, Esherichia coli, and / or Clostridium perfingens (CP), and / or oocysts in fecal matter, comprising administering an effective amount of the composition according to any one of claims 1 to 28 to poultry.

35. The composition according to any one of claims 1 to 28, used in a method for reducing bird mortality, lesion scores, the incidence of Enterococcus cecorum, Salmonella species, Esherichia coli, and / or Clostridium perfingens (CP), and / or oocysts in fecal matter.

36. Use of the composition according to any one of claims 1 to 28 in the manufacture of an agent for reducing bird mortality, lesion scores, the incidence of Enterococcus cecorum, Salmonella species, Esherichia coli, and / or Clostridium perfingens (CP), and / or oocysts in fecal matter.

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