Patent Description:
The present invention relates to a non-therapeutic method for improving the performance of an animal, improving the environment of the animal and a combination thereof, wherein the animal is a porcine species, a non-therapeutic method for improving the performance of an animal, wherein the animal is a poultry species, and an animal feed composition, as defined in the appended claims. These improvements enhance commercial value of animal populations. An animal's gastrointestinal tract is constantly challenged by large numbers of bacteria, viruses, and protozoa found in feed, bedding, and the environment. The gastrointestinal tract has a sophisticated system to counter these potential pathogens consisting of physical, chemical, and immunological lines of defense. Beneficial bacteria are an important part of this system. Pathogens, stress, metabolic upset, the use of antimicrobials, and other causes can upset the balance of intestinal bacteria, which may impair digestion and make the animal more susceptible to disease. Thus, providing the animal with bacteria that assist in establishment (or reestablishment) of a normal bacterial profile can help maintain optimal animal performance.

Direct-fed microbial products are products that contain live (viable) microorganisms (e.g., bacteria). Over time, many of the direct-fed microbial products previously considered useful for improving animal performance, either directly via feed conversion improvements or indirectly via environmental improvements, have lost overall efficacy. The change in efficacy may be associated with the increased use of dried distillers grain solubles (DDGS), or similar feed components, in the diets of animals. The use of DDGS in the diets of animals also affects manure waste pit systems. DDGS, along with any feed component that is high in nitrogen, lipids, and fiber, is difficult for the animal to digest and is often released into the manure pit, affecting manure handling, storage, and decomposition. Feeding animals high levels of DDGS, or other high fiber and/or lipid-containing diets, results in increased solids accumulation, as well as increased ammonia, methane, phosphine, and hydrogen sulfide gas in manure pits. Currently, commercially available products do not appear to help control the negative environmental effects associated with DDGS, or other high fiber and/or lipid-containing diets (e.g., corn-soy diets, and the like). Thus, direct-fed microbial strains are needed that work both in the animal and in the manure waste pit system. Microbial strains are also needed that will improve animal performance, including average daily feed intake, average daily gain, and feed conversion, which have been reduced in DDGS-fed animals.

<CIT> provides microbial strains that improve performance of DDGS fed animals. <CIT> relates to the use of Bacillus strains to treat or prevent Clostridium-based disease in animals. <CIT> relates to Bacillus strains that inhibit pathogenic E. coli in swine; and <CIT> relates to Bacillus strains that improve gastrointestinal health of animals by reducing the growth and presence of bacterial pathogens, such as Salmonella, Clostridium and Campylobacter.

Applicants have developed a direct-fed microbial composition that results in increased average daily gain, increased average daily feed intake, and improved feed conversion in an animal, improved metabolizable energy due to breakdown of difficult to digest feed components in the diet (e.g., DDGS), reduced negative environmental effects on animals due to ammonia volatilization, reduced disease concerns from animal pathogens (e.g., E. coli, Salmonella, and Clostridia), improved manure nitrogen value due to reduction in NH<NUM> ammonia volatilization, reduced disease spreading and nuisance manure pit foaming, and reduced explosive gases (e.g., methane, hydrogen, phosphine) in barns due to reduction in long chain fatty acid-containing foams. The direct-fed microbial compositions described herein offer a commercial benefit by providing all of these properties, or a combination thereof, in a single direct-fed microbial composition. In addition, the direct-fed microbial compositions described herein result in a reduction in the use of antibiotics, and an increase in feed efficiency, which reduces the overall cost of animal feed.

Methods and compositions disclosed herein for improving the performance of an animal, improving the health of the animal, improving the environment of the animal, and combinations thereof. In various aspects of the disclosure, the animal can be selected from the group consisting of a poultry species, a porcine species, a bovine species, an ovine species, an equine species, and a companion animal. In the aspect of the disclosure where the animal is a poultry species, the poultry species can be a broiler chicken. In the aspect of the disclosure where the animal is a porcine species, the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

In various aspects of the disclosure, the compositions for use in the methods described herein can be a commercial package, a feed additive for an animal feed composition, an additive for the drinking water of an animal, or an animal feed composition (e.g., a complete feed), each comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof.

In one aspect of the methods described herein, a method is provided of feeding an animal. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, wherein the Bacillus strain causes an effect selected from the group consisting of improving the performance of the animal, improving the health of the animal, improving the environment of the animal, and combinations thereof.

In another aspect of the methods described herein, a method is provided of controlling detrimental environmental effects of manure. The method comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, and controlling the detrimental environmental effects of the manure.

In yet another illustrative aspect of the methods described herein, a method is provided of controlling detrimental environmental effects of manure. The method comprises the step of applying to manure, litter, a pit, or a manure pond a composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, and controlling the detrimental environmental effects of the manure.

Methods and compositions are disclosed herein for improving the performance of an animal, improving the health of the animal, improving the environment of the animal, and combinations thereof. The compositions for use in the methods described herein can be a commercial package, a feed additive for an animal feed composition, an additive for the drinking water of an animal, or an animal feed composition (e.g., a complete feed), each comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof.

In one aspect of the methods described herein, a method is disclosed of feeding an animal. The method comprises the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, wherein the Bacillus strain causes an effect selected from the group consisting of improving the performance of the animal, improving the health of the animal, improving the environment of the animal, and combinations thereof.

In another aspect of the methods described herein, a method is disclosed of controlling detrimental environmental effects of manure. The method comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, and controlling the detrimental environmental effects of the manure.

In yet another illustrative aspect of the methods described herein, a method is disclosed of controlling detrimental environmental effects of manure. The method comprises the step of applying to manure, litter, a pit, or a manure pond a composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, and controlling the detrimental environmental effects of the manure.

In various aspects of the disclosure, the animal to which a feed additive, a feed composition, or drinking water as described herein is administered can be selected from the group consisting of a poultry species, a porcine species, a bovine species, an ovine species, an equine species, and a companion animal. In the aspect where the animal is a companion animal, the companion animal can be, for example, a canine species or a feline species. In the aspect where the animal is a porcine species, the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig. In various exemplary aspects of the disclosure, the animal can be selected from the group consisting of a chicken (e.g., a broiler or a layer), a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish (e.g., a tilapia, a catfish, a flounder, or a salmon), a crustacean (e.g., a shrimp or a crab), and combinations thereof.

In one aspect of the disclosure, an effective amount of the Bacillus strain can be administered to improve the performance of the animal, improve the health of the animal, improve the environment of the animal, or combinations thereof. By "effective amount" is meant an amount of the Bacillus strain (e.g., strain <NUM> or <NUM>) capable of improving the performance of the animal, improving the health of the animal, improving the environment of the animal, or combinations thereof, by any mechanism, including those described herein.

In aspect of the disclosure described herein wherein the compositions of the present invention comprising Bacillus strains <NUM> and/or <NUM> are administered to an animal, the compositions are preferably administered to animals orally in a feed composition or in drinking water, but any other effective method of administration known to those skilled in the art may be utilized. In one illustrative aspect of the disclosure, the Bacillus strains <NUM> and/or <NUM> are provided in the form of an additive for addition to the drinking water of an animal.

In another illustrative aspect of the disclosure, the Bacillus strains <NUM> and/or <NUM> are provided in the form of a feed additive for addition to a feed composition. The feed composition may contain Bacillus strain <NUM> and/or <NUM> in a mixture with an animal feed blend, including any art-recognized animal feed blend or any animal feed blend described herein. As used herein, "feed composition" or "animal feed composition" means a feed composition comprising Bacillus strain <NUM> and/or Bacillus strain <NUM> in a mixture with an animal feed blend, and, optionally any other components that could be used in a feed composition, including other bacterial strains, such as other Bacillus strains or Lactobacillus strains.

Any animal feed blend, including those known in the art and those described herein, may be used in accordance with the methods and compositions described in this patent application, such as rapeseed meal, cottonseed meal, soybean meal, cornmeal, barley, wheat, silage, and haylage. In various aspects of the disclosure, the animal feed blend can be supplemented with Bacillus strain <NUM> and/or Bacillus strain <NUM>, but other ingredients may optionally be added to the animal feed blend, including other bacterial strains, such as other Bacillus strains or Lactobacillus strains.

In various illustrative aspects of the disclosure, optional ingredients of the animal feed blend include sugars and complex carbohydrates such as both water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides. Other optional ingredients include dried distillers grain solubles, fat (e.g., crude fat), phosphorous, sodium bicarbonate, limestone, salt, phytate, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, ash, fish oil, an oil derived from fish meal, raw seed (e.g., flaxseed), an antioxidant, and starch. In another aspect of the disclosure, minerals may be added in the form of a mineral premix.

Optional amino acid ingredients that may be added to the animal feed blend are arginine, histidine, isoleucine, leucine, lysine, cysteine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HCl, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ethyl HCl, and analogs, and salts thereof. Vitamins that may be optionally added are thiamine HCl, riboflavin, pyridoxine HCl, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like. In another aspect of the disclosure, vitamins may be added in the form of a vitamin premix. In yet another aspect of the disclosure, protein ingredients may be added to the animal feed blend and include protein obtained from meat meal, bone meal, or fish meal, liquid or powdered egg, fish solubles, crude protein, and the like.

In another illustrative aspect, any medicament ingredients known in the art may be added to the animal feed blend or to an additive for the drinking water of the animal, such as antibiotics. In various aspects of the disclosure, the antibiotic is selected from the group consisting of ampicillin, chloramphenicol, ciprofloxacin, clindamycin, tetracycline, chlortetracycline, Denagard™, BMD™, Carbadox™, Stafac™, and combinations thereof. In one aspect of the methods described herein wherein a feed composition or drinking water is administered to the animal, the method may further comprise the step of avoiding administering to the animal an antibiotic selected from the group consisting of erythromycin, levofloxacin, trimethoprim/ sulfamethoxazole, trimethoprim, daptomycin, rifampicin, Tylan™, Pulmotil™, vancomycin, and combinations thereof. In another aspect of the disclosure, the animal feed blend, the feed composition, the feed additive, or the additive for the drinking water of the animal may contain no antibiotics.

In another illustrative aspect of the disclosure, one or more enzymes may be added to the animal feed blend. In various aspects of the disclosure, the enzymes that may be added include a galactosidase, a phytase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, combinations thereof, and any other enzyme that improves the effectiveness of the feed composition for improving the performance or health of the animal or that is effective for improving the environment of the animal. In yet another aspect of the disclosure, yeast, fungi (e.g., Aspergillus or Trichoderma), or micronutrients may be added to the animal feed. Any of the ingredients described above that are suitable for addition to an additive for the drinking water of the animal may be added as a component of the additive for the drinking water of the animal as described herein.

In various illustrative aspect of the disclosure, the Bacillus strain (e.g., Bacillus strain <NUM> and/or <NUM>), or any other bacterial strains added in addition to Bacillus strain <NUM> and/or <NUM>, can be administered in the feed composition at a dose of about <NUM> × <NUM><NUM> CFU/gram of the feed composition to about <NUM> × <NUM><NUM> CFU/gram of the feed composition or at a dose of about <NUM> × <NUM><NUM> CFU/gram of the feed composition to about <NUM> × <NUM><NUM> CFU/gram of the feed composition. In other aspects of the disclosure, the Bacillus strain (e.g., Bacillus strain <NUM> and/or <NUM>) is administered in the feed composition at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition, or at a dose greater than about <NUM> × <NUM><NUM> CFU/gram of the feed composition. In yet another aspect of the disclosure, the Bacillus strain (e.g., Bacillus strain <NUM> and/or <NUM>) is administered in the feed composition at a dose of about <NUM> × <NUM><NUM> CFU/gram of the feed composition.

In various aspects of the disclosure, the Bacillus strain (e.g., Bacillus strain <NUM> and/or <NUM>) for use in accordance with the methods and compositions described herein can be selected from the group consisting of Bacillus strain <NUM>, a strain having all of the identifying characteristics of Bacillus strain <NUM>, Bacillus strain <NUM>, and a strain having all of the identifying characteristics of Bacillus strain <NUM>. Bacillus strain MDG86 and Bacillus strain MDG300 were deposited on March <NUM>, <NUM> at the Agricultural Research Service Culture Collection (NRRL), National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, <NUM> North University Street, Peoria, Illinois <NUM>-<NUM>, and were given accession numbers B-<NUM> and B-<NUM>, respectively. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The NRRL strain designations are MDG86 and MDG300, which are equivalent to Bacillus strain <NUM> and <NUM>, respectively, as referred to in the application.

Any of these strains can be administered alone or in combination in the form of a feed composition (e.g., a complete feed comprising an animal feed blend) or drinking water for an animal. In one aspect of the disclosure, multiple strains are administered in combination in a single composition. In another aspect of the disclosure, multiple strains are administered in combination in separate compositions. In one illustrative aspect of the disclosure, any of these strains is isolated from a high performing grow finish pig.

In another aspect of the disclosure, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strain <NUM> and/or Bacillus strain <NUM>) can be administered to the animal along with another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof. One or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strain <NUM> and/or Bacillus strain <NUM>) can be administered to the animal along with any other bacterial strain effective to improve the performance or health of the animal or that is effective to improve the environment of the animal.

As used herein "a strain having all of the identifying characteristics of' Bacillus strain <NUM> or Bacillus strain <NUM> can be a mutant strain having all of the identifying characteristics of Bacillus strain <NUM> or Bacillus strain <NUM> (e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerprint of Bacillus strain <NUM> or Bacillus strain <NUM>, enzyme activities that correspond to Bacillus strain <NUM> or Bacillus strain <NUM>, antimicrobial activity that corresponds to Bacillus strain <NUM> or Bacillus strain <NUM>, antibiotic sensitivity and tolerance profiles that correspond to Bacillus strain <NUM> or Bacillus strain <NUM>, or combinations thereof). In alternate aspects of the disclosure, the mutation can be a natural mutation, or a genetically engineered mutation. In another aspect of the disclosure, "a strain having all of the identifying characteristics of" Bacillus strain <NUM> or Bacillus strain <NUM> can be a strain, for example, produced by isolating one or more plasmids from Bacillus strain <NUM> or Bacillus strain <NUM> and introducing the one or more plasmids into another bacterium, such as another Bacillus strain, as long as the one or more plasmids contain DNA that provides the identifying characteristics of Bacillus strain <NUM> or Bacillus strain <NUM> (e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerprint of Bacillus strain <NUM> or Bacillus strain <NUM>).

The feed composition or drinking water comprising Bacillus strain <NUM> and/or <NUM> may be administered to the animal for any time period that is effective to improve the performance of the animal, improve the health of the animal, improve the environment of the animal, or combinations thereof. For example, in one aspect of the disclosure the feed composition or drinking water may be provided to the animal daily. In an alternate aspect of the disclosure, the feed composition or drinking water may be administered to the animal during lactation and/or during gestation. The time periods for administration of the feed composition or drinking water described above are non-limiting examples and it should be appreciated that any time period or administration schedule determined to be effective to improve the performance of the animal, improve the health of the animal, improve the environment of the animal, or combinations thereof, may be used.

As described herein, one of the method disclosed herein is a method of feeding an animal by administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, wherein the Bacillus strain causes an effect selected from the group consisting of improving the performance of the animal, improving the health of the animal, improving the environment of the animal, and combinations thereof.

In the aspect of the disclosure where the effect is improving the performance of the animal, the improvement in animal performance is selected from the group consisting of decreasing feed conversion (e.g., reducing the feed to gain ratio (F/G)), increasing average daily feed intake (ADFI), increasing average daily gain (ADG), improving consistency of performance, improving digestibility of a diet, improving the metabolizable energy to gross energy ratio, and combinations thereof. In one aspect of the disclosure, Bacillus strain <NUM> and/or Bacillus strain <NUM> can increase the digestibility of a diet by producing enzymes that increase the digestibility of consumed nutrients where the enzymes are selected from the group consisting of an α-galactosidase, a protease, a phytase, a lipase, an amylase, a xylanase, a cellulase, and combinations thereof. The enzyme can also be any other enzyme that degrades long chain fatty acids, such as enzymes that degrade stearic, palmitic, and/or oleic acid, but not limited to these fatty acids. Such an increase in digestibility of a diet leads to improvements in animal performance selected from the group consisting of decreasing feed conversion (e.g., reducing the feed to gain ratio (F/G)), increasing average daily feed intake (ADFI), increasing average daily gain (ADG), improving consistency of performance of the animal (e.g., reducing variation in performance such as reducing variation and increasing uniformity in F/G, ADFI, and ADG), improving the metabolizable energy to gross energy ratio, and combinations thereof.

In the aspect of the disclosure where the effect is improving the health of the animal, the improvement can result from a mechanism including, but not limited to, antimicrobial activity of Bacillus strain <NUM> and/or Bacillus strain <NUM>. In various aspects of the disclosure, the antimicrobial activity is against bacteria selected from the group consisting ofE. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, and combinations thereof. Thus, Bacillus strain <NUM> and Bacillus strain <NUM> can improve gut health of the animal, and reduce pathogens in the animal, and the animal's environment. In yet another aspect of the disclosure, the animal is a chicken and the improvement in the health of the chicken results in an increase in the number of eggs laid by the chicken, an increase in the number of chicks born to the chicken, or an increase in the number of live chicks born to the chicken.

Bacillus strain <NUM> and Bacillus strain <NUM> can also reduce high ammonia and high pH in manure. The reduction in high ammonia and high pH in manure reduces ammonia toxicity to the animal and toxicity to natural flora that degrade long chain fatty acids. Ammonia toxicity to the animal is caused, in part, by NH<NUM> dissociation to NH<NUM> to produce ammonia gas in the environment of the animal. NH<NUM> is more toxic than NH<NUM> and can lead to respiratory diseases in the animal. Thus, Bacillus strain <NUM> and Bacillus strain <NUM> help to reduce toxicity to the animal, help to maintain the normal microbial balance in the animal, and help to reduce diseases related to environmental toxicity in the animal.

In the aspect of the disclosure where the effect is improving the environment of the animal, the improvement to the environment is selected from the group consisting of reducing the pH of manure, reducing the explosive gas entrapping long chain fatty acid content of manure, reducing ammonia in manure, reducing ammonia volatilization, reducing manure pit foaming, reducing explosive gases in manure, and combinations thereof.

In these aspects of the disclosure, high ammonia and high pH in manure may inhibit the growth of bacteria that degrade long chain fatty acids (e.g., lactic acid bacteria and Syntrophomonas). Long chain fatty acids are typically found in the manure of animals fed diets high in lipids where the lipids breakdown to long chain fatty acids (e.g., diets containing dried distillers grain solubles or diets containing soybeans, such as a soybean meal diet). A build up of long chain fatty acids in manure can be detrimental to the environment of the animal because long chain fatty acids entrap explosive gases leading to, for example, pit foaming and explosions in barns. Long chain fatty acids can also inhibit natural flora which would degrade the long chain fatty acids to produce less detrimental products.

Bacillus strain <NUM> and Bacillus strain <NUM> not only reduce high ammonia and high pH in manure, but these strains also degrade long chain fatty acids. The reduction in explosive gas entrapping long chain fatty acid content in the manure caused by Bacillus strain <NUM> and Bacillus strain <NUM> causes a reduction in explosive gases in the manure (e.g., methane gas, hydrogen gas, phosphine gas, and combinations thereof), and, thus, reduces explosions in barns. As discussed above, the reduction in high ammonia and high pH in manure improves the health of the animal by reducing NH<NUM> in the environment of the animal. The reduction in high ammonia and high pH in manure also inhibits ammonia flashing in barns (e.g., when NH<NUM> flashes off) which results in loss of nitrogen and a decrease in the value of the manure as a fertilizer. Thus, Bacillus strain <NUM> and Bacillus strain <NUM> increase the value of manure as a fertilizer.

As described herein, another method aspect is a method of controlling detrimental environmental effects of manure. The method comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, and controlling the detrimental environmental effects of the manure.

In this aspect of the disclosure, Bacillus strain <NUM> and/or Bacillus strain <NUM> causes an effect selected from the group consisting of reducing the pH of manure, reducing the long chain fatty acid content of manure, reducing ammonia in manure, reducing manure pit foaming, reducing explosive gases in manure, reducing ammonia volatilization, and combinations thereof. In these aspects of the disclosure, as already discussed, high ammonia and high pH in manure may inhibit the growth of bacteria that degrade long chain fatty acids (e.g., lactic acid bacteria and Syntrophomonas). Long chain fatty acids are typically found in the manure of animals fed diets high in lipids where the lipids breakdown to long chain fatty acids. A build up of long chain fatty acids in manure can be detrimental to the environment of the animal because long chain fatty acids entrap explosive gases leading to explosions in barns. Long chain fatty acids can also inhibit natural flora which would degrade the long chain fatty acids to less detrimental products.

Bacillus strain <NUM> and Bacillus strain <NUM> not only reduce high ammonia and high pH in manure, but these strains also degrade long chain fatty acids. The reduction in long chain fatty acid content in the manure caused by Bacillus strain <NUM> and Bacillus strain <NUM> causes a reduction in explosive gases in the manure, and, thus, reduces explosions in barns. The reduction in high ammonia and high pH in manure inhibits ammonia flashing in barns which results in loss of nitrogen and a decrease in the value of the manure as a fertilizer. Thus, Bacillus strain <NUM> and Bacillus strain <NUM> increase the value of manure as a fertilizer.

In another method aspect, a method of controlling detrimental environmental effects of manure is provided. The method comprises the step of applying to manure, litter, a pit, or a manure pond a composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, and controlling the detrimental environmental effects of the manure.

In this aspect, Bacillus strain <NUM> and Bacillus strain <NUM> can be applied to the manure, litter, the pit (e.g., a swine pit), or the manure pond by any suitable method. For example, Bacillus strain <NUM> and Bacillus strain <NUM> can be applied to the manure, litter, pit, or the manure pond by spraying or in the form of a powder, liquid, or pellet or by adding a powder or a pumpable liquid. In the aspect where Bacillus strain <NUM> and/or Bacillus strain <NUM> is applied to manure, the manure can be in an anaerobic digester, an anaerobic lagoon, or a grease trap. The strains can be applied to the anaerobic digester, an anaerobic lagoon, or a grease trap, for example, in the form of a powder, liquid, or a pellet. In this aspect, the application of the Bacillus strain to the litter can result in ammonia reduction in the litter.

In this method aspect, the method can improve the health of the animal by improving the animal's environment by effects selected from the group consisting of reducing respiratory problems of the animal, improving gut health of the animal, improving consistency of performance of the animal, reducing diseases related to environmental toxicity in the animal, and reducing pathogens in the animal. In an aspect of the disclosure where the animal is a poultry species, the method can improve the health of the animal by an effect selected from the group consisting of reducing respiratory problems of the poultry species, reducing breast blisters of the poultry species, improving consistency of performance of the poultry species, and reducing damage to the feet of the poultry species. These mechanisms of improvement to the health of the animal are non-limiting examples.

In additional aspects of the disclosure, compositions comprising Bacillus strain <NUM> and/or Bacillus strain <NUM> are provided. In one aspect, a commercial package is described comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof.

In another aspect of the disclosure, a feed additive for an animal feed is discribed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof.

In yet another aspect of the disclosure, an additive for the drinking water of an animal is described comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof.

In yet another illustrative aspect of the disclosure, an animal feed composition is described comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), a strain having all of the identifying characteristics of Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof.

In one aspect of the disclosure the feed additive for addition to an animal feed blend to produce a complete feed composition can be mixed with the animal feed blend, for example, with an automated micro-nutrient delivery system, or, for example, by hand-weighing and addition to achieve any of the doses of Bacillus strain <NUM> and Bacillus strain <NUM> described herein, for administration to the animal in the form of a complete feed composition. The mixing can also be done by any other suitable method known in the art for combining direct-fed microbials with an animal feed blend to obtain a uniform mixture. In various aspects of the disclosure, the mixing can be done for any suitable time period (e.g., about <NUM> to about <NUM> minutes). In the aspect where Bacillus strain <NUM> and/or Bacillus strain <NUM> is in the form of an additive for the drinking water of the animal, the Bacillus strain <NUM> and/or Bacillus strain <NUM> can be in the form of, for example, a powder, a liquid, or pellets, and can be mixed with the drinking water using any suitable method known in the art to achieve any of the doses of Bacillus strain <NUM> and Bacillus strain <NUM> described herein, for administration to the animal in the drinking water of the animal. Bacillus strain <NUM> and/or Bacillus strain <NUM> can also be fed directly to the animal orally (i.e., by oral insertion) in the form of a powder, a liquid, or a pellet.

In any of the composition aspects described herein, the Bacillus strain <NUM> and/or Bacillus strain <NUM> can cause an effect selected from the group consisting of improving the performance of the animal, improving the health of the animal, improving the environment of the animal, and combinations thereof. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal described herein can also inhibit a pathogen selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, and combinations thereof. These effects are non-limiting examples of the types of effects Bacillus strain <NUM> and/or Bacillus strain <NUM> can cause.

In one illustrative aspect, the feed additive, additive for the drinking water of the animal, or the feed composition can be in the form of a commercial package. In another illustrative aspect of the disclosure, the feed additive or additive for the drinking water of the animal, or the Bacillus strain <NUM> and/or Bacillus strain <NUM> in the commercial package can be in the form of a concentrate (e.g., about <NUM> × <NUM><NUM> to about <NUM> × <NUM><NUM> CFU/g) or a superconcentrate (e.g., about <NUM> × <NUM><NUM> to about <NUM> × <NUM><NUM> CFU/g), or can be in the same form but can be for use in treatment of manure, litter, a pit, or a manure pond. In another aspect of the disclosure, the feed additive, feed composition, or additive for the drinking water of the animal, or the Bacillus strain <NUM> and/or Bacillus strain <NUM> in a composition in a commercial package, can be in a dry form (e.g., a powder), a pelleted form, a liquid form, in the form of a top-dressing, or in the form of a gel, or any other suitable form.

In yet another aspect of the disclosure, the strains in the form of a commercial package can be in a form for use in treatment of manure, in a form for treatment of a pit or in a form for use in treatment of litter. In this aspect of the disclosure, the Bacillus strain <NUM> and/or Bacillus strain <NUM> can be, for example, in a dry form (e.g., a powder or freeze-dried form), in a pelleted form, or in a liquid form.

In another illustrative aspect of the disclosure, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise a carrier for the Bacillus strain <NUM> and/or Bacillus strain <NUM>. The carrier can be selected from the group consisting of a bran, rice hulls, a salt, mineral oil, a dextrin (e.g., maltodextrin), whey, sugar, limestone, dried starch, sodium silico aluminate, vegetable oil, and combinations thereof. In another aspect of the disclosure, the carrier can be any suitable carrier known in the art for a direct-fed microbial. In another aspect of the disclosure, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise a binder such as clay, yeast cell wall components, aluminum silicate, glucan, or other known binders.

In yet other aspects of the disclosure, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition comprising Bacillus strain <NUM> and/or Bacillus strain <NUM> is in a container for commercial use. In various aspects of the disclosure the container can be, for example, a bag (e.g., a <NUM>-pound bag, a <NUM>-pound bag, a <NUM>-ounce bag, a <NUM>-pound bag, or a <NUM>-kilogram bag), a pouch, a drum, a bottle, or a box. In illustrative aspects, the container for the commercial package, feed additive, additive for the drinking water of the animal, or feed composition comprising Bacillus strain <NUM> and/or Bacillus strain <NUM> can comprise plastic, metal, foil, paper, fiber, or cardboard (e.g., a plastic pail, a paper bag, a foil bag, a fiber drum, etc.). The commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise instructions for use of one or more of the Bacillus strains.

The following examples are for illustrative purposes only.

The objective of the instant example was to determine the efficacy of several Bacillus species strain combinations for promoting weight gain and feed/energy conversion in broiler chickens. The growth performance responses of broiler chickens fed diets supplemented with various strain combinations (DFMs) were evaluated.

For this investigation, a total of <NUM> pens were utilized, with a group size of <NUM> birds per pen. The litter utilized in the pens was wood shavings that were previously used (but had no previous DFM use).

Approximately <NUM> hatch chickens (Cobb <NUM> genetics) were evaluated, including roosters and hens in mixed-sex pens. Chickens received an anti-coccidia vaccine at hatch (mist application). The start weight of the chickens was about <NUM> grams, and the end weight of the chickens was about <NUM>.

In the investigation, the chickens were divided into four groups as shown in Table <NUM>:.

For the allotment of chickens to the experiment, birds were weighed by pen, and pens were grouped into replicates of six (<NUM>) similar-weight pens. Pens were randomly allotted to dietary treatment from within replicate and immediately started on the study, and remained on dietary treatments until the end of the experimental period. Daily management of the animals followed standard operating procedures on farms. Birds requiring medicinal treatment for <NUM> straight days were removed from the experiment. The day of the investigation and body weight of the bird at removal were recorded for proper accounting in the performance of that pen.

Measurements of litter compositions were obtained and evaluated. Samples of litter were taken from four (<NUM>) replicates (two in each half of the barn) prior to chick placement, during week <NUM> of the investigation, and at the end of the investigation. Litter samples underwent analyses for coliforms, pH, moisture, and ammonia, and were retained for possible future analyses.

Measurements of live performance were also obtained and evaluated. Total pen weights were recorded at the beginning of the investigation and at biweekly intervals, corresponding to dietary phase changes, thereafter until the end of the experimental period. Experimental feeds were manufactured, delivered, and recorded by hand. The feed-in-feeder at the end of the experimental period was removed and weighed, and that weight was used to calculate total feed disappearance by pen. Pen weights, feed delivered, and feed-in-feeder for each pen were used to calculate body weights, feed intakes, and feed conversion ratios (grams feed/grams body weight). Caloric efficiency (kcal/lb gain) was calculated from the total feed intake per pen, metabolizable energy (ME) content of each diet, and the total live gain per bird. All morbidity and mortality events were recorded, along with any major health issues. Any bird that became morbid was removed from the study and weighed and its individual identification number, gender, room, pen, date of removal, and reason of removal were recorded.

Evaluations of economic performance were also assessed. The cost of feed per pen was calculated as the sum-product of total feed consumed per pen and the cost per each diet. Feed cost per bird was calculated as the total feed cost per pen divided by the total number of birds present at the end of the experiment. Feed cost per <NUM> pound gain was calculated as the product of feed/gain (F/G) and the cost/<NUM> pound feed (total feed cost /total carcass weight (cwt) feed). Revenue per bird was calculated as the product of carcass weight (cwt) and meat price ($/pound). Margin-over-feed per bird was calculated as the difference between revenue and feed cost per bird.

The experimental diet formulation used in the instant example included yellow dent corn (Producers Cooperative, Bryan, TX) and poultry fat (Griffin Industries). The test materials DFM strains were provided by Microbial Discovery Group (Franklin, WI). The control diet contained supplemental fat (poultry), <NUM>% DDGS, and <NUM>% meat-and-bone meal. Identification information of DFM materials was as follows:.

Chickens were fed diets in three phases according to the following age ranges: Phase <NUM> - Week <NUM>-<NUM>; Phase <NUM> - Week <NUM>-<NUM>; and Phase <NUM> - Week <NUM>-<NUM>.

All diets were formulated to be adequate in SID Lys (%, NRC, <NUM>) and the other essential amino acids (AA), available phosphorus (P), calcium (Ca), and sodium (Na) (see Table <NUM>).

Diets were devoid of feed-grade antibiotics and coccidiostats. The test DFM materials were supplemented to the final diets at the expense of corn (see Table <NUM>).

Diet components were mixed in a horizontal mixer. An amount of corn was added to the mixer for purposes of flushing the system between each batch of DFM-supplemented diets. Each diet was pelleted at <NUM> (<NUM>°F) following <NUM> seconds of conditioning. The diets for Week <NUM>-<NUM> were crumbled following pelleting. Experimental diets were delivered to each pen by hand, and the addition was recorded manually.

Samples of corn, SBM, and DDGS were taken at each time of diet manufacture. Final experimental diets were sampled at the time of manufacture. Samples of both feedstuffs (pooled) and experimental diets were split into five (<NUM>) samples, of which one was retained and the remaining four were available for submission for the following analyses: proximate components and minerals, amino acids, microbial counts, and mycotoxins.

Initial data analysis was performed for all metrics to determine homogeneity of variance, normal distribution, and outliers (± > <NUM> standard deviations in difference from the grand mean).

Table <NUM> shows the mortality-corrected feed conversion ratio (FCR) of straight-run broilers in the various treatment groups at the starter, grower, and finisher phases.

In the starter phase, the inclusion of DFM in all treatments yielded similar results to the control, except for MDG-<NUM> (<NUM><NUM>), which resulted in a significant decrease compared to the control. Throughout the growth and finisher phase, no significant differences in FCR were observed.

Table <NUM> shows the mortality-corrected feed conversion ratio (FCR) of straight-run broilers in the various treatment groups from Days <NUM>-<NUM> of the treatment phase and for Days <NUM>-<NUM> of the treatment phase.

Through day <NUM>, the inclusion of MDG-<NUM> and MDG-<NUM> (<NUM><NUM>) yielded similar FCR to the control diet. A significant decrease in FCR was observed with the inclusion of MDG-<NUM> (<NUM><NUM>) compared to the control diet. At the conclusion of the trial (i.e., through day <NUM>), no significant differences were observed between treatments.

Table <NUM> shows the calculated body weights of straight-run broilers fed a standard corn-soy diet in the various treatment groups at day <NUM>, day <NUM>, day <NUM>, and day <NUM> of treatment.

As shown in Table <NUM>, no significant differences were observed in body weight associated with treatment throughout the duration of the trial.

Table <NUM> shows the mortality of straight-run broilers fed standard corn-soy diet in the various treatment groups at the starter, grower, and finisher phases.

As shown in Table <NUM>, no differences in mortality were observed between treatments throughout the trial.

In conclusion for the experiments in this investigation, DFM products resulted in improved feed conversion ratio in coccidiosis vaccinated broilers. The improvements reached the level of significance through <NUM> days of age and were <NUM> points improved at day <NUM> with the inclusion of MDG-<NUM> (<NUM><NUM>).

<FIG> shows the growth performance of finishing pigs fed various DFM strain combinations. <FIG> suggests that strain <NUM> alone performed better than in combination with other strains in relation to ADFI and ADG. However, an additional trial described at the end of Example <NUM> suggests that a combination of strain <NUM> plus strain <NUM> performed better than strain <NUM> alone on ADFI and ADG. The ratios for <NUM> + <NUM> + <NUM> were <NUM>%, <NUM>%, and <NUM>%, respectively. The ratios for <NUM> + <NUM> and <NUM> + <NUM> were <NUM>:<NUM>.

The objective of this investigation was to determine the efficacy of several Bacillus species DFM strain combinations for promoting weight gain and growth performance in finishing pigs.

Three different groups were evaluated in the instant example: <NUM>-F040, <NUM>-F071, and <NUM>-F072. In the example, PIC <NUM> × Camborough <NUM> pigs were evaluated, including barrows and gilts in mixed-sex pens. The parameters for each group are shown in Table <NUM> as follows:.

In this investigation, the pigs were divided into three groups as shown in Table <NUM>:.

At the start of each experiment, mixed-sex pens of pigs were sorted by weight into replicates based on body weight. Pens were then randomly allotted to dietary treatment from within replicates and immediately started on the study. Pens remained on dietary treatments until the end of the experimental period.

Daily management followed standard operating procedures within each farm. Pigs were visually inspected daily to ensure that individual pigs were meeting the standard for body condition and criteria for the standard operating procedures of the farm. Pigs not meeting such criteria were medicated per standards of the farm and recommendations of the attending veterinarian. Pigs requiring medicinal treatment for <NUM> consecutive days and not showing signs of improvement were immediately removed from the experiment.

All morbidity and mortality events were recorded, along with any major health issues. Any pig that became morbid and was removed from the study was weighed and its gender, room, pen, date of removal, and reason for removal was recorded. Pigs that were removed from trial were classified as a nutritional or a non-nutritional reason for removal (see Table <NUM>).

Measurements of carcass performance were also obtained and evaluated. Carcass weights, back fat and loin depths at the 10th rib, calculated lean content, sort discounts, and lean premiums were collected. Carcass yield was calculated as the carcass weight divided by live weight as measured at the plant, multiplied by <NUM>. Carcass ADG was calculated as the difference between carcass weight and the weight of the carcass at the beginning of the experiment, defined as start weight × <NUM>; carcass F/G was calculated as ADFI/carcass ADG. Carcass caloric efficiency (kcal/pound carcass gain) was calculated from the total ingredient intake per pen, ME content of each ingredient used, and the total carcass gain per pen.

Evaluations of economic performance were also assessed. The cost of feed per pen was calculated as the sum-product of total ingredient consumed per pen and the cost per unit of each ingredient. Feed cost per pig was calculated as the total feed cost per pen divided by the total number of pigs present at the end of the experiment. Feed cost per pound gain (both live and carcass) was calculated as the product of F/G and the cost/pound feed (total feed cost /total pound feed). Revenue per pig was calculated as the product of carcass weight (cwt) and the sum of base price and lean premium ($/cwt). Margin-over-feed (MOF) per pig was calculated as the difference between revenue and feed cost per pig.

The experimental diet formulation used in the instant example included feedstuffs such as yellow dent corn (Co-Alliance, Frankfort, IN), soybean meal (ADM, Frankfort, IN), DDGS (The Andersons, Clymers, IN), choice white grease (IPC, Delphi, IN), and basemixes (JBS United, Inc. , Sheridan, IN). Loading values for corn, soy bean meal (SBM), and DDGS are shown in Table <NUM>.

Each of the Bacillus (Bs) strain combinations was manufactured and provided by Microbial Discovery Group (Franklin, WI). Treatment B comprised Bs strains <NUM> and <NUM>, while Treatment C comprised Bs strains <NUM>, <NUM>, and <NUM>. Each strain combination was premixed with corn prior to addition to experimental diets. Pigs were fed experimental diets by weight range according to the budgets for each experiment outlined in Table <NUM>.

All diets were formulated to be adequate in essential amino acids, available P, Ca, and Na using recommended values. Diets were constructed using corn and soybean meal, and included DDGS at <NUM>% of the diet within each experiment. Diets did not contain supplemental fat.

Diet manufacture and delivery parameters are shown in Table <NUM>. For the <NUM>-F040 and <NUM>-F071 groups, diet components were mixed and delivered to each feeder through an electronic feed mixing, delivery, and recording system. For the <NUM>-F072 group, final experimental diets were mixed in a feed mill and were delivered to each feeder through an electronic feed mixing, delivery, and recording system.

Each feedstuff was sampled for nutrient and mycotoxin analyses according to standard protocol. Final experimental diets were sampled bi-weekly from a minimum of <NUM> feeders per dietary treatment and pooled together for each treatment. Feedstuff and experimental diet samples were submitted for the following analyses if deemed necessary: proximate components and minerals, amino acids, and mycotoxins.

Each experiment was statistically analyzed separately from the others, as well as through a meta-analysis, and within each, data from the three experiments were combined using an additional class variable of 'Exp'. Initial data analysis was performed for all metrics for both the individual and combined datasets to determine normality of distribution and outliers (± > <NUM> standard deviations in difference from the grand mean).

The grand means of performance metrics across experiments is shown in Table <NUM>.

Growth performance of pigs fed different DFM combinations in late-finishing is shown in Table <NUM>. Comparison of strain <NUM> alone and in combination with two other strains in a three strain combination shows that strain <NUM> alone was significantly improved over the control on average daily gain. Strain <NUM> alone was trending toward significance with a p= <NUM>, but significant when starting weight is considered as covariable. Ending weight of strain <NUM> was not considered significant compared to the control, but was considered significant in consideration of starting weight as a covariable. Carcass weight of strain <NUM> alone was trending, with p=<NUM> but considered significant with starting weight as a co-variable.

Accounting for start weight and days on feed differences across experiments, the effect of DFM addition on average daily gain (Trt P = <NUM>) was dependent on DFM formulation, as the Bs strain <NUM> alone increased (P = <NUM>) ADG <NUM>% over the control, but the <NUM>-strain combination had no effect. Similarly, DFM addition affected feed/gain ratio (Trt P = <NUM>) in a formulation-dependent manner, with the Bs strain <NUM> alone reducing (P = <NUM>) feed/gain ratio <NUM>% and the <NUM>-strain formulation having no impact.

<FIG> shows a meta-analysis of DFM strain effect on growth performance across three experiments. A total of three (<NUM>) separate trials were conducted using DFM strains <NUM> and <NUM> in combination, individually or in combination with other Bacillus or lactic acid bacteria strains. A universal dose of <NUM> × <NUM><NUM> CFU/gram was utilized.

As shown in <FIG>, treatment with DFM strain <NUM> alone increased gain approximately <NUM>% (P = <NUM>) and reduced F/G approximately <NUM>% (P = <NUM>). The combination of DFM strains <NUM> and <NUM> in experiment <NUM>-F072 (<NUM>% strain <NUM> and <NUM>% strain <NUM>) was numerically better than DFM strain <NUM> alone for ADG.

This example describes the use of plate media screening methods to detect enzymatic activity in DFM strains <NUM>, <NUM>, and other related isolates. Enzyme assay media plates were prepared by supplementing tryptic soy agar with between <NUM>% and <NUM>% of various substrates, including polysaccharides (corn starch, carboxymethylcellulose, or xylan), proteins (casein), and lipids (tributyrin). Bacillus strains of interest (including DFM strains <NUM> and <NUM>) obtained from fresh overnight cultures were spotted onto plates (<NUM>µL) and incubated at <NUM> for up to <NUM> hours. For protein and lipid agar plates, zones of clearing around enzyme-producing colonies were visible without further treatment. Polysaccharide-containing plates were stained with Gram's iodine for <NUM> minute to visualize zones of clearing. DFM strains <NUM> and <NUM> were both observed to be positive for protease, amylase, and carboxymethylcellulose (CMCase) activity after <NUM> hours and strong positives on tributyrin agar (lipase) after <NUM> hours were observed.

As shown in <FIG>, the Bacillus strains have enzymatic activity including but not limited to amylase, carboxymethylcellulose, protease, xylanase and lipase.

In the instant example, a comparison of DFM strain <NUM> and DFM strain <NUM> was performed to determine the variations in substrate utilization. A number of parameters were tested, including enzyme activity, pathogen antimicrobial activity, digestion of DDGS, and tolerance of certain production antibiotics.

A list of carbohydrate and carboxylic acid carbon sources utilized for DFM strain <NUM> and DFM strain <NUM> is presented in Table <NUM>.

A list of phosphate and sulfur sources utilized by DFM strain <NUM> and DFM strain <NUM> is presented in Table <NUM>.

A list of unique nutritional utilization observed for DFM strain <NUM> and DFM strain <NUM> is presented in Table <NUM>.

As shown in Tables <NUM>-<NUM>, both DFM strains appeared to utilize an extremely wide array of amino acids and dipeptide bonds. DFM strain <NUM> and DFM strain <NUM> utilized <NUM> and <NUM> amino acids and dipeptide bonds, respectively. DFM strain <NUM> does appear to be more versatile, as this strain used <NUM> more amino acids and dipeptide bonds than DFM strain <NUM>, including several D-amino acids which are not often found in microbes.

Both strains use a very wide variety of carbon sources, but DFM strain <NUM> appears to use a number of α-galactosides such as meliobiose, stachyose and raffinose. Furthermore, DFM strain <NUM> is able to utilize maltose and maltotriose, which was not observed for DFM strain <NUM>.

In addition, DFM strain <NUM> and strain <NUM> are both able to use a variety of phosphate and sulfur sources, including phytate. Both strains are able to use the phytate breakdown product m-inositol. DFM strain <NUM> appears to be broader regarding the array of phosphate compound utilization.

Finally, DFM strain <NUM> and strain <NUM> are both able to use a variety of Tween compounds, which is indicative of the ability to breakdown long chain fatty acids such as palmitic, oleic and stearic acids. Other data suggest that the observed activities on long chain fatty acids may differ depending on the presence in an aerobic or an anaerobic environment.

This example describes the use of the cross-streak plating method and the stab-streak method to screen DFM strains of interest, including DFM strain <NUM> and DFM strain <NUM>, for antimicrobial activity against a range of other organisms.

Bacillus strains of interest (including DFM strain <NUM> and DFM strain <NUM>) obtained from frozen glycerol stocks were inoculated in a single <NUM> wide streak (cross-streak) across the center of plates of a suitable nutrient medium. Tryptic soy agar was used when screening for activity against most non-fastidious organisms (for screening against Clostridium strains, reinforced clostridial agar was found to support satisfactory growth of the Bacillus strains as well as the Clostridium strains tested). Bacillus-streaked plates were incubated for <NUM> hours at <NUM>, until a heavy streak of growth was present.

Organisms to be tested for susceptibility, such as Clostridium, Salmonella, and E. coli (from frozen glycerol stocks) were streaked in lines perpendicular to the Bacillus streak and incubated for <NUM> hours under their optimal growth conditions. After incubation, plates were examined for zones of inhibition around the initial Bacillus streak, and the width of each zone of inhibition was measured. As shown in <FIG>, DFM strains <NUM> and <NUM> produce antimicrobial substances which inhibit pathogenic organisms including, but not limited to, Staphylococcus, Enterococcus, E. coli, Salmonella, and Clostridium. As shown in <FIG>, DFM strains <NUM> and <NUM> produce antimicrobial substances which inhibit additional pathogenic organisms. This example describes the stab streak plating method used to screen the DFM strains of interest, including DFM strain <NUM> and DFM strain <NUM>, for antimicrobial activity against other organisms. Bacillus strains of interest (including DFM strain <NUM> and DFM strain <NUM>) obtained from frozen glycerol stocks were inoculated by puncturing the center of a nutrient rich medium plate with an inoculum needle. Brain heart infusion agar was utilized for the simultaneous growth of both Bacillus species and anaerobic or microaerophilic microorganisms (Clostridium perfringens, Clostridium difficile, Campylobacter jejuni). Bacillus inoculated plates were incubated for <NUM>-<NUM> hours at <NUM> depending on the colony size and proliferation tendencies of each Bacillus strain. Once incubation had concluded, lids were removed and plates were inverted over <NUM> of concentrated chloroform for <NUM> minutes to kill the Bacillus colonies. The chloroform was contained via an absorbent material which also served as the platform for the plates to rest upon. After <NUM> minutes, the plates were placed face up in a biological safety cabinet in order to allow excess chloroform to evaporate while maintaining plate sterility for an additional <NUM> minutes. BHI (<NUM>%) agar was prepared in <NUM> aliquots and utilized for top agar overlays of recently killed Bacillus strains. These tubes were inoculated with 100µl of a pathogenic liquid media suspension, vortexed, applied topically to plates inoculated with Bacillus, and allowed to solidify. Plates were then inverted and incubated under varying pathogen specific growth conditions (<NUM>° C with atmospheric growth paks provided by BD). After incubation, zones of inhibition were observed around the Bacillus colonies and measured in mm for relative quantitative purposes. As shown in <FIG>, strains <NUM> and <NUM> produce antimicrobial substances which inhibit pathogenic organisms including, but not limited to, Staphylococcus, Enterococcus, E. coli, Salmonella, Clostridium, Vibrio, and Campylobacter.

Bacillus strains of interest (including DFM strains <NUM> and <NUM>) from <NUM> hour-old cultures in tryptic soy broth were combined with molten Muller-Hinton agar II, poured into petri plates, and allowed to solidify. After plates containing Bacillus were hardened, an Etest strip (Biomerieux) containing a concentration gradient of an antibiotic was placed on the surface of each Bacillus-inoculated plate. After incubation for <NUM> hours at <NUM>, plates were covered with a uniform lawn of bacterial growth, with zones of inhibition surrounding the Etest strips. A minimum inhibitory concentration of each antibiotic was determined by reading the test strips according to the Etest protocol.

As shown in <FIG>, DFM strains <NUM> and <NUM> were tested for antibiotic sensitivity using various antibiotics and they are susceptible to a number of antibiotics.

Antibiotic media plates were prepared with five antibiotics of agricultural importance at either 1x, <NUM>/10x, or <NUM>/100x their recommended dose rates (Pulmotil™, <NUM>/ton; BMD™, <NUM>/ton; Stafac™, <NUM>/ton; Tylan™, <NUM>/ton, chlortetracycline (CTC) + Denagard™, <NUM>/ton CTC + <NUM>/ton Denagard™). Bacillus isolates were screened by spotting <NUM>µl of fresh overnight culture onto antibiotic media plates and incubating for <NUM> hours. The presence or absence of colony growth was noted and used as a rough measure of antibiotic susceptibility for screening purposes.

As shown in <FIG>, the presence of CTC + Denagard™ at the recommended dose, BMD™ at <NUM>/<NUM> the recommended dose, and Stafac™ at <NUM>/<NUM> the recommended dose caused minimal sized zones of inhibition against DFM strains <NUM> and <NUM>. Therefore, these antibiotics should not interfere with the growth of DFM strains <NUM> and <NUM>. These strains were also tolerant to Formaldehyde (Sal CURB, Termin-<NUM>) and minimal inhibition was observed with Carbadox™ (<NUM>/ton).

To screen Bacillus isolates (including DFM strains <NUM> and <NUM>) for their ability to utilize dried distiller's grains with solubles (DDGS) as a nutritional source, a minimal medium was prepared with DDGS as the main carbon and nitrogen source: <NUM>% K<NUM>HPO<NUM>, <NUM>% MgSO<NUM>*<NUM><NUM>O, <NUM>% trace element stock, <NUM>% yeast extract, <NUM>% MnCl<NUM>*<NUM><NUM>O, <NUM>% FeCl<NUM>, and <NUM>% DDGS. Sterile <NUM> tubes of this medium were inoculated with these Bacillus strains, incubated <NUM> hours at <NUM>, and scored for growth. Tubes with strong growth were used for 10x serial dilutions in the same medium, incubated for <NUM> hours at <NUM>, and scored again for growth, with turbidity at later dilution steps serving as a rough indicator of initial cell count.

As shown in <FIG>, with DDGS as a sole carbon source, both DFM strains <NUM> and <NUM> exhibited ability to grow and digest DDGS. DFM strain <NUM> was among the strains with the highest growth, with turbidity in the <NUM>-<NUM> dilution tube after the secondary screening. DFM strain <NUM> showed moderate growth at <NUM> hours with turbidity in the <NUM>-<NUM> dilution tube after the secondary screening.

This example describes the use of Bacillus DFM strains <NUM> and <NUM> to decrease the pH and free ammonia of liquid hog manure in vitro. Liquid manure collected from deep pit systems in several swine production facilities was divided into <NUM> portions and inoculated with freeze-dried cultures of DFM strains <NUM>, <NUM>. All freeze-dried cultures were enumerated by the plate count method, and inocula were prepared by suspending spores in a solution of <NUM>% peptone.

Inoculated manure sample tubes were capped loosely and incubated at <NUM> without shaking under stagnant, non-aerated conditions for a minimum of <NUM> hours. At <NUM> hours (and in some cases, at additional <NUM>-hour intervals afterward), the pH was determined using a pH electrode and total ammonia nitrogen (TAN) was determined by diluting a portion of the manure sample <NUM>/<NUM> in Millipore water for analysis with the Hach ISENH4 ammonium ion-sensitive electrode. All measurements were taken at <NUM>. The fraction and concentration of free ammonia were derived from the pH, temperature, and TAN of the sample.

As shown in <FIG> and <FIG>, treatment with strain blends containing DFM strains <NUM> and <NUM> was associated with lowered pH and lowered free ammonia in multiple trials. The size of the effect varied between manure types.

To determine whether DFM strains <NUM> and <NUM> were able to utilize long-chain fatty acids as an energy source, minimal media were prepared with either no carbon source, or oleic, palmitic, or stearic acid as the sole carbon source (2x mineral stock, <NUM> phosphate buffer, and <NUM>% fatty acid, adjusted to pH <NUM>, <NUM>, <NUM>. ) DFM strains <NUM> and <NUM> obtained from frozen glycerol stocks were inoculated into each type of minimal medium and incubated at <NUM> for <NUM> hours with or without shaking. Growth was determined by measuring optical density at <NUM> with a spectrophotometer.

As shown in <FIG>, under aerobic conditions, DFM strain <NUM> showed substantial growth (change in A<NUM>><NUM>) with palmitic acid and stearic acid at different pHs and DFM strain <NUM> showed substantial growth with palmitic acid and stearic acid at pHs of <NUM> and <NUM>. As shown in <FIG>, under anaerobic conditions, DFM strain <NUM> showed substantial growth (change in A<NUM>><NUM>) with palmitic acid and oleic acid at pHs of <NUM>, <NUM> and <NUM> and DFM strain <NUM> showed substantial growth with palmitic acid and oleic acid at pHs of <NUM> and <NUM>.

In order to obtain genetic barcodes or "fingerprints" of the Bacillus strains collected in the strain library, RAPD (randomly amplified polymorphic DNA) analysis was performed with each strain from the Bacillus library. Each isolate was cultured overnight in <NUM> of tryptic soy broth at <NUM>. Thereafter, DNA was extracted from these cultures using Qiagen's DNeasy Mini kit, following the protocol provided for Gram-positive bacteria. AGE Healthcare Illustra Ready-To Go RAPD kit was used to perform RAPD-PCR with each DNA sample to amplify genetic fragments of arbitrary length. PCR products were separated via gel electrophoresis, and banding patterns were detected and matched using BioNumerics software. As shown in <FIG>, although some bands were observed to be shared by the DFM strains, a common DNA fingerprint was not observed between the two DFM strains. Therefore, <FIG> demonstrates that DFM Strains <NUM> and <NUM> are different strains, each having a unique DNA fingerprint.

In the instant example, growth of DFM strains <NUM> and <NUM> can be achieved by culturing. On a small scale, TSB or nutrient broth can be utilized to culture DFM strains <NUM> and <NUM>.

Agar medium may be produced using <NUM> grams Nutrient Agar (BD <NUM>) and <NUM> of DI water, followed by autoclaving at <NUM>. Broth medium may be produced using <NUM> grams of Nutrient Broth (BD <NUM>) and <NUM> of DI water, followed by autoclaving at <NUM>.

For culturing DFM strain <NUM>, a pure culture of DFM strain <NUM> was streaked on a nutrient agar plate and allowed to grow for <NUM> hours at <NUM>. Thereafter, a single colony was inoculated in nutrient broth medium. The single colony was incubated at <NUM> and at <NUM>-<NUM> rpm, for <NUM> to <NUM> hours. Finally, the culture was streaked on a nutrient agar plate to check morphology.

The objective of the instant example was to determine the response in weight gain, caloric efficiency, and survivability of broiler chickens to multiple utilization strategies of a DFM. The effect of selected DFM combinations on increased weight gain, reduced feed conversion ratio, and improved survival of broiler chickens reared to <NUM> weeks (<NUM> days) of age was investigated in which growth and carcass data was analyzed using a randomized complete-block design, live weight (by pen) as the replication factor, and <NUM> replicates.

For this investigation, a total of <NUM> pens (<NUM> × <NUM> ft, <NUM> ft<NUM>/pen - subtracting <NUM> sq ft for feeder space) were utilized, with a group size of <NUM> birds per pen. The pens were equipped with a dry tube feeder (<NUM>-lb feed capacity) with a total feeder space of <NUM> in. /bird) and <NUM> nipple drinkers/pen (<NUM> birds/nipple).

Approximately <NUM> hatch rooster chickens (Cobb <NUM> genetics) were evaluated. The start weight of the chickens was about <NUM> grams, and the end weight of the chickens was about <NUM>.

In the investigation, the chickens were divided into six groups as shown in Table <NUM>:.

Experimental procedures were as follows:
Animal care protocol: Care was provided following an approved Animal Use Protocol. Environmental conditions were monitored <NUM> times daily. Age appropriate temperature was provided and regulated by a Rodem Plantium Junior. Heat was provided with multiple force draft heaters. Houses were tunnel ventilated with cool pads on one end and <NUM> - <NUM> inch fans on the other end.

Allotment of animals to the experiment: Birds were assigned to pen based on day old chick weight. Initial pen weight of all replicate pens had a maximum of range of <NUM> grams. Pens were then randomly allotted to dietary treatment from within replicate and immediately started on the study. Pens remained on dietary treatments until the end of the experiment.

Carcass performance: A subset of each replicate-pen was processed for the determination of carcass, fat pad, and breast meat yield. Six broilers per replicate pen were randomly selected for yield determination.

Feedstuffs were: Corn - yellow-dent, soybean meal, corn low-oil DDGS, and porcine meat and bone meal (Producers Cooperative, Bryan, TX) and fat was poultry fat (Griffin Industries).

Experimental test materials: MDG. DFM (<NUM>% strain <NUM> and <NUM>% strain <NUM>) <NUM> × <NUM><NUM> CFU/g; MDG. DFM-v2 (<NUM>% strain <NUM> and <NUM>% strain <NUM>) <NUM> × <NUM><NUM> CFU/g.

Experimental diet specifications: Four dietary phases - wk <NUM>-<NUM>, wk <NUM>-<NUM>, wk <NUM>-<NUM>, wk <NUM>. The control diet contained at least <NUM>% supplemental fat (poultry fat), <NUM>% DDGS, and <NUM>% meat-and-bone meal. All diets were formulated to be adequate in SID Lys (%, NRC, <NUM>) and the other essential AA, available P, and Ca. The test materials were supplemented to the final diets at the expense of corn (Table <NUM>). Diet components were mixed in a horizontal mixer.

Each diet was pelleted at <NUM> (<NUM>°F) following <NUM> of conditioning. The diets for Wk <NUM>-<NUM> were crumbled following pelleting. In the starter phase the pellet stability of the DFM formulation was determined. The samples were obtained as follows and sent for analysis: A <NUM> (<NUM> lb) sample of Mash was obtained at the start and <NUM> samples (<NUM> (<NUM> lb) per sample) of pelleted feed were obtained during the course of each pelleting run for each DFM-containing treatment. Samples of pelleted feed were collected following a brief acclimation in each new pelleting run and <NUM> (<NUM> lb) from each <NUM> (<NUM> lb) sample combined to form a pooled sample for each treatment. Pelleted feed samples were allowed to cool to room temperature before they were sealed for shipping. Both Mash & Pelleted feed samples were sent for analysis.

Diet sampling: Each feedstuff was sampled for nutrient and mycotoxin analyses at each point of manufacturing of experimental diets, and final experimental diets were sampled.

Statistical Procedures were carried out as follows: Prior to analysis, all data was checked for outliers. Any observation > <NUM> standard deviations in difference from the grand mean for that metric was removed from the dataset. Cumulative body weight, growth, carcass, and economic performance were analyzed as a RCBD with six (<NUM>) treatments and <NUM> replicates. Morbidity, mortality, and other health-related metrics were analyzed as non-parametric data. Data were subjected to a one-way ANOVA and separated using Fisher's LSD.

Dietary formulations for experimental diets are shown in Table <NUM>.

Results are shown in following Tables <NUM>-<NUM> based on Treatments A-F of Table <NUM>, summarized as below:.

For D14 body weight, no significant differences were observed.

For D28 body weight, treatments B and D showed significantly higher average body weights when compared to the control diet with all remaining treatments being intermediate.

For D42 body weight, treatments B, D, and F had significantly higher average body weight when compared to the control and treatment E, with treatment C being intermediate.

On D48, treatments D and F yielded higher body weights when compared to the control diet with all remaining treatments being intermediate.

For feed consumed during the starter phase, no significant differences were observed.

During the grower phase, treatment F consumed the most feed per bird, which was significantly higher than the control diet. All other treatments were intermediate.

For feed consumed during the finisher phase, treatment D had the highest feed consumption rate out of all treatments and was significantly higher than the control and treatment E diets with all remaining diets being intermediate.

During the finisher II phase, treatments D and F had the highest feed consumption rates which were significantly higher than the control, treatment C and E diets with treatment B remaining intermediate.

For feed consumed of broilers fed different DFMs, no considerable differences were observed.

For FCR of broilers fed different DFMs, no significant differences were observed during the starter, grower, and finisher II.

Inclusion of the DFM at <NUM> × <NUM><NUM> in treatment D during the finisher phase significantly reduced FCR when compared to treatment E with all other treatments being intermediate.

For cumulative FCR on day <NUM>-<NUM>, broilers fed the treatment B diet had significantly improved cumulative FCR compared to the control and treatment F fed broilers with all other treatments being intermediate.

For cumulative FCR of broilers fed different DFMs on day <NUM>-<NUM>, treatment D had a significantly lower FCR when compared to the control diet with all other treatments remaining intermediate. Cumulative FCR of broilers fed different DFMs on day <NUM>-<NUM> resulted in no significant differences observed.

For uniformity of broilers fed different DFMs on D14, D28, and D42, no significant differences were observed.

Uniformity of broilers fed different DFMs on D48, treatment E had a significantly higher uniformity when compared to treatment F with all other diets being intermediate.

For Litter pH, treatment C had a significantly lower pH when compared to treatment F with all other diets being intermediate.

Ammonium ion concentration resulted in no significant differences being observed.

For processing weights of broilers fed different DFMs, treatment D had the highest average live weight. Treatments B, C, and F were significantly higher than the control. Treatment E was not different form the control.

For WOG weights, treatments B, D, and F were significantly higher than the control with treatment D being the largest. Treatment C and E were greater than the control but not at a significant level.

For fat pad weights, no significant differences were observed.

For filet weights (breast meat weights), treatments D and F were significantly higher when compared to the control, treatment C, and E with all other treatments being intermediate.

For processing yield of broilers fed different DFMs, no differences were observed between treatment groups on WOG and fat pad yield.

For filet yield (breast meat yield), treatment F was significantly higher when compared to the control, treatment C and E with all other treatment groups being intermediate.

For mortality rate of broilers fed different DFMs, no significant differences were observed on days <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, and <NUM>-<NUM>.

For days <NUM>-<NUM>, treatment F had a significantly higher mortality rate when compared to the control, treatment B and C with all other treatments being intermediate.

In conclusion for the experiments in this investigation, the addition of DFMs to broiler diets in this study improved overall broiler performance and processing parameters in some capacity, particularly at the inclusion level of <NUM> × <NUM><NUM> continuously throughout the trial. Significant differences in performance parameters occurred in later phases of the trial with no differences being observed in the starter phase.

The object of the instant example was to compare the performance in a <NUM> day floor pen trial broilers fed with and without <NUM>/<NUM> DFM (<NUM>% strain <NUM> and <NUM>% strain <NUM>) to compare broiler performance to those fed the antibiotic growth promotant (AGP) program of bacitracin MD (BMD) shuttled to virginiamycin (VM) and to those not receiving any supplements.

The feed supplements tested were as follows: <NUM>/<NUM> DFM test material with <NUM>% strain <NUM> and <NUM>% strain <NUM> DFM strains was provided by Microbial Discovery Group (Franklin, WI). BMD (bacitracin methylene disalicylate). VM (virginiamycin).

For this investigation, a total of <NUM> pens were utilized, with an initial group size of <NUM> birds per pen. The experiment was conducted in a building which is a wood and cinder block structure with a metal roof and clay floor. Each study pen contained <NUM> water fountain and a <NUM> lb. capacity feed tube. The dimensions of the pens were <NUM>' × <NUM>' which provided a stocking density of <NUM> ft<NUM> per bird when there were <NUM> birds per pen. Each pen had a polymer slatted floor covered with a <NUM> mil thick plastic sheet topped with approximately <NUM> inches of new wood shavings at Day <NUM>. On Day <NUM>, each pen was top dressed with <NUM> pounds of previously used broiler from a flock having no disease issues or exposure to direct fed microbials. Continuous lighting was provided.

All pens were checked at least daily during the study. Observations included availability of feed and water, and brooder control for maintaining desired temperatures.

Approximately <NUM> hatch straight-run broiler chickens (Cobb <NUM> genetics) were evaluated. The initial age of the chickens at Day <NUM> was one day.

Diets: A <NUM> phase custom broiler diet program of which all were formulated to be consistent with current industry specifications was used and are located in Appendix A.

Study Design: There were <NUM> treatment groups with each group replicated <NUM> times for a total of <NUM> pens (<NUM> chicks/pen at placement) as shown in Table <NUM>. Salinomycin <NUM>/ton was present in all starter and grower feeds.

Animal Placement: On the first day of the trial, <NUM> stacks of <NUM> hatchery crates containing at least <NUM> chicks in each crate were set on a row of tables. Thirty (<NUM>) chicks from each of the <NUM> stacks were placed into a transport crate, those <NUM> chicks were group weighed and then placed into the pen <NUM>. This was repeated for all <NUM> pens. Remaining chicks were used to replace any unthrifty or lame chicks found during days <NUM> through <NUM>.

Data Collected: Pen body weights and feed weights (for feed conversions) at Days <NUM>, <NUM>, <NUM> and <NUM>. At Day <NUM> all birds were individually weighed and each bird's gender determined by phenotype. All deaths and removed birds (mortality) were documented. No feeds were removed from pens until Day <NUM> to ensure all pens received the same plane of nutrition throughout the study. Each response variable was evaluated by One-Way ANOVA and means separated by Tukey HSD Test (Statistix <NUM>, Analytical Software, Tallahassee, FL).

Live Bird Weights: All birds were weighed by pen at Days <NUM> (by pen), <NUM> (by pen), and <NUM> (by gender, by pen) as shown in Table <NUM>.

DAY <NUM> Live Weight/Bird (lb) was heavier (p<<NUM>) for BMD/VM, and <NUM>/<NUM> in starter or continuously, compared to nCON. The <NUM>/<NUM> in starter only gave heavier (p≤<NUM>) weight than the BMD/VM diets. The treatments containing <NUM>/<NUM> (starter or continuously) gave highest body weights and were in the same statistical grouping.

DAY <NUM> Live Weight/Bird (lb): The <NUM>/<NUM> in starter only gave heavier (p<<NUM>) weights than nCON diets.

DAY <NUM> Live Weight/Bird (lb) averages for males and females combined were heavier (p≤<NUM>) for BMD/VM and <NUM>/<NUM> in starter only compared to nCON treatment, with <NUM>/<NUM> continuously treatment being intermediate. This same statistical pattern was evident for the male body weights. For females, diets with BMD/VM, <NUM>/<NUM> in starter only or continuously gave heavier (p<_0. <NUM>) broilers than nCON diets.

Feed Conversion Ratio: Feed conversion and Feed Conversion Adjusted for Mortality (Table <NUM>) were calculated using the following equations: <MAT> <MAT>.

DAY <NUM>-<NUM> Feed Conversion Ratio (unadjusted) of <NUM>/<NUM> starter only or continuously was lower (p<_0. <NUM>) than nCON, with BMD/VM intermediate.

DAY <NUM>-<NUM> Feed Conversion Ratio (unadjusted) results were not significantly different (p><NUM>).

DAY <NUM>-<NUM> Mortality Adjusted Feed Conversion Ratios followed the same statistical pattern as described for the unadjusted Feed Conversion Ratios.

Mortality (%): Mortality (%) results by treatment group are shown in Table <NUM>.

DAY <NUM>-<NUM> Mortality (%) was not significantly different (p><NUM>) between treatment groups. No mortality was observed in any treatments during the first <NUM> weeks.

DAY <NUM>-<NUM> Mortality (%) was not significantly different (p><NUM>) between treatment groups. Overall average mortality % from <NUM>-<NUM> days was <NUM>%.

Carcass and Breast Yields (%Live Weight): Results for carcass yield and breast yield as a % of live weight by gender and by sexes combined are shown in Table <NUM> and Table <NUM>, which shows combined average weights and per cent yields.

As a side point, all broilers for processing were carefully observed for breast blisters. Only one of the birds processed was found with blisters (a female from the <NUM>/<NUM> Starter Only group) while all other broilers' breasts were found to be without blemishes.

Carcass Yield (%Live Weight) was significantly (p≤<NUM>) greater for BMD/VM fed broilers than for nCON broilers, with other treatment values being intermediate. Female carcass yield results were statistically similar to combined sexes results. There were no significant differences between treatments for male carcass yield (p><NUM>).

Breast Yields (%Live Weight) average for combined sexes was significantly greater (p≤<NUM>) for birds in the <NUM>/<NUM> continuously treatment group than for nCON birds, with other groups intermediate. By individual sexes, no significant differences (p><NUM>) were detected in breast yield.

In conclusion for the experiments in this <NUM>-day pen trial with <NUM> treatments and <NUM> replicate pens (with <NUM> chicks/pens), the DFM supplemented groups <NUM>/<NUM> in starter only or continuously gave final body weights statistically equivalent to BMD/VM and greater (p≤<NUM>) than nCON group. Therefore, BMD/VM or <NUM>/<NUM> (either in starter or all phases) broilers had the most improved <NUM>-day body weight of broiler chickens on this study. No significant treatment effects on mortality (%) were found. Diets supplemented with BMD/VM had greater (p≤<NUM>) carcass yield as a % of live weight than nCON diets, with other experimental diets being intermediate. For combined sexes, breast yield as a % of live weight was greater (p≤<NUM>) for DFM product <NUM>/<NUM> continuously than for nCON, with other treatment results being intermediate.

To confirm the presence of galactomannan-degrading enzymes in DFM strains via the dinitrosalicylic acid (DNS) reducing sugar assay, this example describes the use of plate media screening methods to detect enzymatic activity in DFM strains <NUM>, <NUM>, <NUM>, and <NUM> and other related isolates.

The DNS reducing sugar assay was performed as in the manufacturer's protocol, inoculating the <NUM>% guar gum solution with overnight cultures of MDG strains <NUM>,<NUM>, <NUM>, and <NUM> grown in TSB + <NUM>% guar gum. The reducing sugar assay was performed with samples taken after <NUM> minutes and <NUM> hours of incubation at <NUM>. Standard curves were prepared with solutions of D-mannose and D-galactose at concentrations between <NUM> and <NUM> to determine the relation between free reducing sugar reacted and OD <NUM>. Negative control samples (NC) consisted of guar gum substrate inoculated with buffer only or with sterile media (TSB+guar gum).

As shown in <FIG>, Bacillus strains <NUM>,<NUM>, <NUM>, and <NUM> showed mannanase activity in the range of about <NUM> mmol to about <NUM> mmol in <NUM> hours.

In the instant example, a comparison of DFM strains was performed to determine the variations in substrate utilization. DFM strains <NUM>, <NUM>, <NUM>, and <NUM> were individually tested, and were tested with various strain combinations. For example, two-strain combinations included, but were not limited to, a combination of strain <NUM>/strain <NUM> as follows: <NUM>/<NUM>; <NUM>/<NUM>; <NUM>/<NUM>; <NUM>/<NUM>; <NUM>/<NUM>, and <NUM>/<NUM>. Three-strain combinations included, but were not limited to, a combination of strain <NUM>/strain <NUM>/strain <NUM> as follows: <NUM>/<NUM>/<NUM>; <NUM>/<NUM>/<NUM>; <NUM>/<NUM>/<NUM>; and <NUM>/<NUM>/<NUM>. A combination of all four strains (i.e., <NUM>/<NUM>/<NUM>/<NUM>) was also tested. A negative control that included no Bacillus was also assessed with the strains.

In particular, a substrate utilization assay was conducted to determine whether the Bacillus strains exhibited growth in a minimal medium with linoleic acid as the only carbon source.

In performing the substrate utilization assay, each microtiter plate well contained <NUM>µl of linoleic acid and <NUM>µl of minimal medium inoculated with a respective Bacillus strain or strain combination. The substrate utilization test was run at <NUM> for <NUM> days with medium shaking. As shown in <FIG>, The most effective strains or strain combinations for using linoleic acid as the only carbon source were: <NUM>/<NUM>; <NUM>/<NUM>/<NUM>; <NUM>/<NUM>/<NUM>; <NUM>/<NUM>; <NUM>/<NUM>/<NUM>; <NUM>/<NUM>; and <NUM>.

In order to obtain genomic "fingerprints" of Bacillus strains <NUM> and <NUM> collected in the strain library, Next Generation Sequencing analysis was performed according to the manufacturer's instructions with each Bacillus strain. The raw sequencing data was assembled de novo into contigs and consensus sequences. Gene prediction using the microbial genome was then followed by functional annotation of novel sequences.

<FIG> and <FIG> show genomic annotation of DFM strains <NUM> and <NUM>, respectively. As shown in <FIG>, strain <NUM> comprises approximately <NUM>,<NUM>,<NUM> bp (i.e., about <NUM> kb) of genomic sequence. DFM strain <NUM> was slightly smaller comprising about <NUM>,<NUM>,<NUM> bp (i.e., about <NUM> kb) of genomic sequence. Therefore, <FIG> and <FIG> demonstrate that DFM Strains <NUM> and <NUM> are different strains, each having a unique DNA fingerprint.

In addition, sequencing of DFM strains <NUM> and <NUM> identified many different genes within the strains. Genes identified in strains <NUM> and <NUM> included, but were not limited to, antimicrobial genes, such as bacilysin, bacilyosin, antlisterial subtilisin, sufactin, Lantibiotic lichenidicin, streptomycin, lactococcin, polyketides, plipastatin, and macrolactin. Sequencing also identified additional genes that were incorporated into strains <NUM> and <NUM> including, but not limited to antimicrobial tolerance genes, such as bacitracin, β-lactams, penicillin, erythromycin, cephalosporin, lincomycin, tetracycline, tunicamycin, fosmidomycin, and fosmomycin. Sequencing also identified five unnamed bacteriocins, genetic redundancies within the strains, multi-drug efflux transporters, and different compounds.

This multi-experiment test was conducted to determine the growth performance of nursery pigs fed different direct-fed microbial diet combinations. One Experiment (Experiment <NUM> below) was performed to assess different direct-fed microbial diet treatments on nursery pigs. Specific parameters regarding the experimental methods and details, and the statistical procedures are described below, along with the results of the experiment as shown in Table <NUM>.

Allotment of animals to the experiment: At the start of each experiment, mixed-sex pens of pigs were sorted by weight into replicates based on body weight. Pens were then randomly allotted to dietary treatment from within replicate and immediately started on the study. Pens remained on dietary treatments until the end of the experimental period.

Daily management: Daily management followed standard operating procedures within each farm. Pigs were visually inspected daily to ensure that individual pigs were meeting the standard for body condition and criteria for the standard operating procedures of the farm. Pigs not meeting such criteria were medicated per standards of the farm and recommendations of the attending veterinarian. Any pig requiring medicinal treatment for <NUM> consecutive days and not showing signs of improvement was immediately removed from the experiment.

Measurements of Live performance and Morbidity Rates: Total pen weights were recorded at allotment of the experiment and at regular intervals during the experimental period.

Period feed intakes corresponded with pen weight periods. Pen weights, feed delivered, and feed-in-feeder for each pen were used to calculate ADG, ADFI, and F/G ratio. All morbidity and mortality was recorded, along with any major health issues.

Statistical Procedures: Each experiment was statistically analyzed individually. Initial data analysis was performed for all metrics to determine normality of distribution and outliers (± > <NUM> standard deviations in difference from the grand mean) using the Univariate procedure of SAS. Body weights and cumulative growth rates, feed intakes, and feed/gain ratios were analyzed according to randomized complete-block designs using the Mixed procedure of SAS, with the main effect of diet and random effect of replicate. Serial body weights, growth rates, feed intakes, and feed/gain ratios were analyzed similarly, with week or period included as a repeated measure. Morbidity, mortality, and other health-related metrics were analyzed as nonparametric data using the NParlway procedure of SAS.

Conclusions: MDG Bacillus strains <NUM> & <NUM> supplemented to diets at <NUM> lb/ton increased ADG and reduced F/G.

Claim 1:
A non-therapeutic method for improving the performance of an animal, improving the environment of the animal and a combination thereof, the method comprising the step of administering to the animal a feed composition comprising, at a dose of <NUM> × <NUM><NUM> CFU/gram of the feed composition to <NUM> × <NUM><NUM> CFU/gram of the feed composition, an isolated Bacillus strain selected from the group consisting of Bacillus strain <NUM> (NRRL No. B-<NUM>), Bacillus strain <NUM> (NRRL No. B-<NUM>), and combinations thereof, wherein the animal is a porcine species, wherein the improvement in animal performance is selected from the group consisting of promoting weight gain, increasing final body weight and increasing growth rate, and wherein the improvement of the environment of the animal is selected from the group consisting of reducing the pH of manure and reducing ammonia in the manure.