DIRECT-FED MICROBIALS AND METHODS OF THEIR USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional

Application Serial No. 61/992607, filed May 13, 2014 and U.S. Provisional Application Serial No. 62/078665, filed November 12, 2014, in which all of which are incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

[0002] The invention relates to direct-fed microbials for use in improving the performance of an animal, improving the health of an animal, improving the environment of an animal, and combinations thereof. More particularly, the invention relates to isolated Bacillus strains 86 and 300, and strains having all of the identifying characteristics of these strains, for uses comprising the above-mentioned uses.

BACKGROUND AND SUMMARY OF THE INVENTION

[0003] The present invention relates to direct-fed microbial (DFM) compositions and methods for improving the performance of an animal, improving the health of an animal, improving the environment of an animal, and combinations thereof. These improvements enhance commercial value of animal populations.

[0004] 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.

[0005] 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.

[0006] 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 N¾ 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.

[0007] Methods and compositions are provided for improving the performance of an animal, improving the health of the animal, improving the environment of the animal, and combinations thereof. In various embodiments, 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 embodiment where the animal is a poultry species, the poultry species can be a broiler chicken. In the embodiment 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. [0008] In various embodiments, 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[0009] In one embodiment 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), 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.

[0010] In another embodiment 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure.

[0011] In yet another illustrative embodiment 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure. [0012] The following clauses, and combinations thereof, provide various additional illustrative aspects of the invention described herein. The various embodiments described in any other section of this patent application, including the section titled "DETAILED

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS" and the EXAMPLES are applicable to any of the following embodiments of the invention described in the numbered clauses below.

[0013] 1. A method of feeding an animal, the method comprising 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), 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.

[0014] 2. The method of clause 1 wherein the animal is 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.

[0015] 3. The method of clause 2 wherein the poultry species is a broiler chicken.

[0016] 4. The method of clause 3 wherein the effect is improving the performance of the animal.

[0017] 5. The method of clause 4 wherein the improvement in animal performance is selected from the group consisting of decreasing feed conversion, increasing average daily feed intake, increasing average daily gain, improving consistency of performance, and combinations thereof.

[0018] 6. The method of clause 4 wherein the improvement in animal performance is increased breast meat weight.

[0019] 7. The method of clause 4 wherein the improvement in animal performance is increased breast meat yield as a per cent of live weight.

[0020] 8. The method of clause 2 wherein the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.

[0021] 9. The method of any one of clauses 1 to 8 wherein the effect is improving the performance of the animal.

[0022] 10. The method of clause 9 wherein the improvement in animal performance is selected from the group consisting of decreasing feed conversion, increasing average daily feed intake, increasing average daily gain, improving consistency of performance, and combinations thereof.

[0023] 11. The method of clause 9 wherein the improvement in animal performance is selected from the group consisting of improving digestibility of a diet, improving the metabolizable energy to gross energy ratio, and combinations thereof.

[0024] 12. The method of any one of clauses 1 to 11 wherein the Bacillus strain produces an enzyme selected from the group consisting of an a-galactosidase, a protease, a lipase, an amylase, a xylanase, a cellulase, and combinations thereof.

[0025] 13. The method of clause 1 wherein the effect is improving the health of the animal.

[0026] 14. The method any one of clauses 1 to 13 wherein at least one of the

Bacillus strains has antimicrobial activity.

[0027] 15. The method of clause 14 wherein the antimicrobial activity is against bacteria selected from the group consisting of E. coli, Salmonella, Staphylococcus,

Enterococcus, Clostridia, Campylobacter, and combinations thereof.

[0028] 16. The method of clause 1 wherein the effect is improving the environment.

[0029] 17. The method of clause 16 wherein the improvement to the environment is 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.

[0030] 18. The method of clause 17 wherein the reduction in ammonia in the manure causes a reduction selected from the group consisting of a reduction in ammonia toxicity in the animal and a reduction in ammonia toxicity to natural flora in the environment of the animal.

[0031] 19. The method of clause 17 wherein the reduction in ammonia in the manure reduces ammonia flashing in a barn.

[0032] 20. The method of clause 17 wherein the reduction in long chain fatty acid content in the manure causes a reduction in explosive gases in the manure wherein the gases are selected from the group consisting of methane gas, hydrogen gas, phosphine gas, and combinations thereof.

[0033] 21. The method of clause 17 wherein the reduction in ammonia in the manure improves nitrogen value in the manure wherein the manure is used as a fertilizer.

[0034] 22. The method of any one of clauses 1 to 21 further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof. [0035] 23. The method of any one of clauses 1 to 22 wherein the strain administered is Bacillus strain 86 (NRRL No. B-50944) or a strain having all of the identifying

characteristics of Bacillus strain 86 (NRRL No. B-50944).

[0036] 24. The method of any one of clauses 1 to 22 wherein the strain administered is Bacillus strain 300 (NRRL No. B-50943) or a strain having all of the identifying

characteristics of Bacillus strain 300 (NRRL No. B-50943).

[0037] 25. The method of any one of clauses 1 to 22 wherein Bacillus strain 86

(NRRL No. B-50944), or a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), and Bacillus strain 300 (NRRL No. B-50943), or a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), are administered in combination in a single composition.

[0038] 26. The method of any one of clauses 1 to 22 wherein Bacillus strain 86

(NRRL No. B-50944), or a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), and Bacillus strain 300 (NRRL No. B-50943), or a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), are administered in combination in separate compositions.

[0039] 27. The method of any one of clauses 1 to 26 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 5.0 x 10 12 CFU/gram of the feed composition.

[0040] 28. The method of any one of clauses 1 to 27 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 1.0 x 10 CFU/gram of the feed composition.

[0041] 29. The method of any one of clauses 1 to 28 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10 ⁴ CFU/gram of the feed composition.

[0042] 30. The method of any one of clauses 1 to 29 wherein the Bacillus strain is administered in the feed composition at a dose of about 7.3 x 10 ⁴ CFU/gram of the feed composition.

[0043] 31. The method of any one of clauses 1 to 30 wherein the Bacillus strain is isolated from a high performing grow finish pig.

[0044] 32. The method of any one of clauses 1 to 31 further comprising the step of administering an antibiotic to the animal wherein the antibiotic is selected from the group consisting of Denagard™, BMD™, Carbadox™, and Stafac™.

[0045] 33. The method of any one of clauses 1 to 32 further comprising 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™, and vancomycin.

[0046] 34. The method of any one of clauses 1 to 33 further comprising the step of administering to the animal an enzyme selected from the group consisting of a galactosidase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, and combinations thereof.

[0047] 35. The method of any one of clauses 1 to 34 wherein the microbial balance in the animal is maintained.

[0048] 36. The method of clause 2 wherein the animal is a companion animal.

[0049] 37. The method of clause 36 wherein the animal is a canine species or a feline species.

[0050] 38. The method of clause 2 wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0051] 39. The method of clause 2 wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0052] 40. The method of any one of clauses 1 to 39 wherein the feed composition is administered daily to the animal.

[0053] 41. The method of clause 1 wherein the animal is selected from the group consisting of a chicken, a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish, a crustacean, and combinations thereof.

[0054] 42. A method of controlling detrimental environmental effects of manure, the method comprising 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure.

[0055] 43. The method of clause 42 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a bovine species, an ovine species, and an equine species.

[0056] 44. The method of clause 42 wherein the Bacillus strain 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. [0057] 45. The method of clause 44 wherein the reduction in ammonia in the manure reduces ammonia flashing in a barn.

[0058] 46. The method of clause 44 wherein the reduction in long chain fatty acid content in the manure results in a reduction in explosive gases in the manure wherein the gases are selected from the group consisting of methane gas, hydrogen gas, phosphine gas, and combinations thereof.

[0059] 47. The method of clause 44 wherein the reduction in ammonia in the manure improves nitrogen value in the manure wherein the manure is used as a fertilizer.

[0060] 48. The method of any one of clauses 42 to 47 further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[0061] 49. The method of any one of clauses 42 to 48 wherein the strain

administered is Bacillus strain 86 (NRRL No. B-50944), or a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944).

[0062] 50. The method of any one of clauses 42 to 48 wherein the strain

administered is Bacillus strain 300 (NRRL No. B-50943), or a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943).

[0063] 51. The method of any one of clauses 42 to 50 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 5.0 x 10 12 CFU/gram of the feed composition.

[0064] 52. The method of any one of clauses 42 to 51 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 1.0 x 10 CFU/gram of the feed composition.

[0065] 53. The method of any one of clauses 42 to 52 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10 ⁴ CFU/gram of the feed composition.

[0066] 54. The method of any one of clauses 42 to 53 wherein the Bacillus strain is administered in the feed composition at a dose of about 7.3 x 10 ⁴ CFU/gram of the feed composition.

[0067] 55. A method of controlling detrimental environmental effects of manure, the method comprising 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure.

[0068] 56. The method of clause 55 wherein the method improves the health of the animal 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.

[0069] 57. The method of clause 55 wherein the animal is a poultry species and the method improves 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.

[0070] 58. The method of clause 55 wherein the manure is in an anaerobic digester.

[0071] 59. The method of clause 55 wherein the manure is in an anaerobic lagoon.

[0072] 60. The method of any one of clauses 55 to 59 wherein the applying step comprises spraying or adding a powder or a pumpable liquid.

[0073] 61. The method of any one of clauses 55 to 60 wherein the pit is a swine pit.

[0074] 62. The method of clause 32 wherein the antibiotic is Denagard™ and the

Denagard™ is administered in combination with chlortetracycline.

[0075] 63. The method of clause 11 wherein the diet includes a component selected from the group consisting of dried distillers grain solubles, corn, soy, and combinations thereof.

[0076] 64. A commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[0077] 65. A feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[0078] 66. An additive for the drinking water of an animal comprising an isolated

Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[0079] 67. An animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[0080] 68. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 64 to 67 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.

[0081] 69. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 64 to 68, wherein the Bacillus strain inhibits a pathogen selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Campylobacter, and Clostridium.

[0082] 70. The feed additive or additive for the drinking water of the animal of clause 65 or 66 in the form of a concentrate.

[0083] 71. The feed additive or additive for the drinking water of the animal of clause 65 or 66 in the form of a superconcentrate.

[0084] 72. The feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 65 to 67 in dry form.

[0085] 73. The feed composition of clause 67 in pelleted form.

[0086] 74. The commercial package of clause 64 comprising the Bacillus strains in a form for use in treatment of a pit, a manure pond, or for use in treatment of litter.

[0087] 75. The commercial package of clause 74 wherein the strains are in a form selected from the group consisting of a powder, a liquid, and a pellet form.

[0088] 76. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 73 further comprising a carrier for the Bacillus strains.

[0089] 77. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 76 wherein the carrier is selected from the group consisting of a bran, rice hulls, a salt, a dextrin, and combinations thereof.

[0090] 78. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 77 in a bag. [0091] 79. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 78 wherein the bag is a plastic bag.

[0092] 80. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 79 further comprising instructions for use of one or more of the Bacillus strains.

[0093] 81. The commercial package, feed additive, or additive for the drinking water of the animal of any one of clauses 78 to 80 in a 20-pound bag.

[0094] 82. The commercial package, feed additive, or additive for the drinking water of the animal of any one of clauses 78 to 80 in a 50-pound bag.

[0095] 83. The feed additive or additive for the drinking water of the animal of any one of clauses 65, 66, 68, 69 to 72, or 76 to 82 in powder form.

[0096] 84. The feed additive or additive for the drinking water of the animal of any one of clauses 65, 66, 68 to 71, or 78 to 80 in liquid form.

[0097] 85. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 84 in a container for commercial use.

[0098] 86. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 85 wherein the container comprises plastic.

[0099] 87. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 85 wherein the container comprises paper.

[00100] 88. The method of clause 55 wherein the Bacillus strain is applied to litter and reduces ammonia in the litter.

[00101] 89. The method of clause 1 wherein the animal is a chicken and the improvement in 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.

[00102] 90. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 87 further comprising a binder.

[00103] 91. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 90 wherein the binder is selected from the group consisting of clay, yeast cell wall components, aluminum silicate, and glucan, or combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[00104] Fig. 1 shows growth performance of finishing pigs fed various DFM strain combinations, including Bacillus strains 86 and 300. Bs 86+552 denotes a 50:50 (1: 1) mixture of Bs strains 86 and 552; Bs 300+552 denotes a 50:50 (1: 1) mixture of Bs strains 300 and 552; Bs 86+300+552 denotes a 33.3:33.3:33.3 (1: 1: 1) mixture of Bs strains 86, 300 and 552.

In 13-F072, treatment 3 (Bs 86+300) is 80% strain 86 and 20% strain 300.

[00105] Fig. 2 shows meta-analysis of DFM strains, including Bacillus strain 86, for three (3) experiments. Three-strain denotes a 33.3:33.3:33.3 (1: 1: 1) mixture of Bs strains 86, 300 and 552.

[00106] Fig. 3 shows enzymatic screening for strains 86 and 300 for amylase, CMCase, protease, xylanase, and lipase activities (strain 86 is the first bar in each set of two bars and strain 300 is the second bar).

[00107] Figs. 4A and B show antimicrobial activities against various bacterial strains for

Bacillus strains 86 and 300.

[00108] Fig. 5 shows antibiotic susceptibility of Bacillus strains 86 and 300.

[00109] Fig. 6 shows susceptibility and tolerance of Bacillus strains 86 and 300 to antibiotics.

[00110] Fig. 7 shows the ability of Bacillus strains 86 and 300 to utilize dried distillers grain solubles.

[00111] Figs. 8 and 9 show the ability of Bacillus strains 86 and 300 to reduce pH and free ammonia in manure.

[00112] Figs. 10 and 11 show the ability of Bacillus strains 86 and 300 to digest long chain fatty acids (the four legends at the top of the graphs from left to right correspond to the bars from left to right in each group of four bars).

[00113] Fig. 12 shows a photograph of a gel displaying RAPD PCR profiles (Primer 1 to

6) for Bacillus strains 86 and 300. Strain 86 has the top profile and strain 300 has the bottom profile. The leftmost and rightmost lanes have markers and each set of two consecutive lanes between the markers corresponds to Primers 1 to 6 going from left to right.

[00114] Fig. 13 shows mannanase enzymatic activity in DFM strains 86, 101, 290, and

300.

[00115] Fig. 14 shows linoleic acid utilization as the sole carbon source by DFM strains

86, 101, 290, and 300, as well as linoleic acid utilization by various combinations of those DFM strains. The strain combinations with the highest linoleic acid utilization were (top curve to bottom) 86/101/300, 86/101/290, 101/290, 86/290, 101/300, 300, and 86/300. All other combinations had similar utilization. [00116] Figs. 15 and 16 show genomic annotation of DFM strains 86 and 300, respectively.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[00117] Methods and compositions are provided for improving the performance of an animal, improving the health of the animal, improving the environment of the animal, and combinations thereof. In various embodiments, 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[00118] In one embodiment 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), 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.

[00119] In another embodiment 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure.

[00120] In yet another illustrative embodiment 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure.

[00121] The following clauses, and combinations thereof, provide various additional illustrative aspects of the invention described herein. The various embodiments described in this section titled "DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS" are applicable to any of the following embodiments of the invention described in the numbered clauses below.

[00122] 1. A method of feeding an animal, the method comprising 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), 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.

[00123] 2. The method of clause 1 wherein the animal is 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.

[00124] 3. The method of clause 2 wherein the poultry species is a broiler chicken.

[00125] 4. The method of clause 3 wherein the effect is improving the performance of the animal.

[00126] 5. The method of clause 4 wherein the improvement in animal performance is selected from the group consisting of decreasing feed conversion, increasing average daily feed intake, increasing average daily gain, improving consistency of performance, and combinations thereof.

[00127] 6. The method of clause 4 wherein the improvement in animal performance is increased breast meat weight.

[00128] 7. The method of clause 4 wherein the improvement in animal performance is increased breast meat yield as a per cent of live weight.

[00129] 8. The method of clause 2 wherein the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig. [00130] 9. The method of any one of clauses 1 to 8 wherein the effect is improving the performance of the animal.

[00131] 10. The method of clause 9 wherein the improvement in animal performance is selected from the group consisting of decreasing feed conversion, increasing average daily feed intake, increasing average daily gain, improving consistency of performance, and combinations thereof.

[00132] 11. The method of clause 9 wherein the improvement in animal performance is selected from the group consisting of improving digestibility of a diet, improving the metabolizable energy to gross energy ratio, and combinations thereof.

[00133] 12. The method of any one of clauses 1 to 11 wherein the Bacillus strain produces an enzyme selected from the group consisting of an a-galactosidase, a protease, a lipase, an amylase, a xylanase, a cellulase, and combinations thereof.

[00134] 13. The method of clause 1 wherein the effect is improving the health of the animal.

[00135] 14. The method any one of clauses 1 to 13 wherein at least one of the

Bacillus strains has antimicrobial activity.

[00136] 15. The method of clause 14 wherein the antimicrobial activity is against bacteria selected from the group consisting of E. coli, Salmonella, Staphylococcus,

Enterococcus, Clostridia, Campylobacter, and combinations thereof.

[00137] 16. The method of clause 1 wherein the effect is improving the environment.

[00138] 17. The method of clause 16 wherein the improvement to the environment is 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.

[00139] 18. The method of clause 17 wherein the reduction in ammonia in the manure causes a reduction selected from the group consisting of a reduction in ammonia toxicity in the animal and a reduction in ammonia toxicity to natural flora in the environment of the animal.

[00140] 19. The method of clause 17 wherein the reduction in ammonia in the manure reduces ammonia flashing in a barn.

[00141] 20. The method of clause 17 wherein the reduction in long chain fatty acid content in the manure causes a reduction in explosive gases in the manure wherein the gases are selected from the group consisting of methane gas, hydrogen gas, phosphine gas, and combinations thereof. [00142] 21. The method of clause 17 wherein the reduction in ammonia in the manure improves nitrogen value in the manure wherein the manure is used as a fertilizer.

[00143] 22. The method of any one of clauses 1 to 21 further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[00144] 23. The method of any one of clauses 1 to 22 wherein the strain administered is Bacillus strain 86 (NRRL No. B-50944) or a strain having all of the identifying

characteristics of Bacillus strain 86 (NRRL No. B-50944).

[00145] 24. The method of any one of clauses 1 to 22 wherein the strain administered is Bacillus strain 300 (NRRL No. B-50943) or a strain having all of the identifying

characteristics of Bacillus strain 300 (NRRL No. B-50943).

[00146] 25. The method of any one of clauses 1 to 22 wherein Bacillus strain 86

(NRRL No. B-50944), or a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), and Bacillus strain 300 (NRRL No. B-50943), or a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), are administered in combination in a single composition.

[00147] 26. The method of any one of clauses 1 to 22 wherein Bacillus strain 86

(NRRL No. B-50944), or a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), and Bacillus strain 300 (NRRL No. B-50943), or a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), are administered in combination in separate compositions.

[00148] 27. The method of any one of clauses 1 to 26 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 5.0 x 10 12 CFU/gram of the feed composition.

[00149] 28. The method of any one of clauses 1 to 27 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 1.0 x 10 CFU/gram of the feed composition.

[00150] 29. The method of any one of clauses 1 to 28 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10 ⁴ CFU/gram of the feed composition.

[00151] 30. The method of any one of clauses 1 to 29 wherein the Bacillus strain is administered in the feed composition at a dose of about 7.3 x 10 ⁴ CFU/gram of the feed composition.

[00152] 31. The method of any one of clauses 1 to 30 wherein the Bacillus strain is isolated from a high performing grow finish pig. [00153] 32. The method of any one of clauses 1 to 31 further comprising the step of administering an antibiotic to the animal wherein the antibiotic is selected from the group consisting of Denagard™, BMD™, Carbadox™, and Stafac™.

[00154] 33. The method of any one of clauses 1 to 32 further comprising 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™, and vancomycin.

[00155] 34. The method of any one of clauses 1 to 33 further comprising the step of administering to the animal an enzyme selected from the group consisting of a galactosidase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, and combinations thereof.

[00156] 35. The method of any one of clauses 1 to 34 wherein the microbial balance in the animal is maintained.

[00157] 36. The method of clause 2 wherein the animal is a companion animal.

[00158] 37. The method of clause 36 wherein the animal is a canine species or a feline species.

[00159] 38. The method of clause 2 wherein the animal is a sow and the Bacillus strain is administered during lactation.

[00160] 39. The method of clause 2 wherein the animal is a sow and the Bacillus strain is administered during gestation.

[00161] 40. The method of any one of clauses 1 to 39 wherein the feed composition is administered daily to the animal.

[00162] 41. The method of clause 1 wherein the animal is selected from the group consisting of a chicken, a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish, a crustacean, and combinations thereof.

[00163] 42. A method of controlling detrimental environmental effects of manure, the method comprising 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure. [00164] 43. The method of clause 42 wherein the animal is selected from the group consisting of a poultry species, a porcine species, a bovine species, an ovine species, and an equine species.

[00165] 44. The method of clause 42 wherein the Bacillus strain 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.

[00166] 45. The method of clause 44 wherein the reduction in ammonia in the manure reduces ammonia flashing in a barn.

[00167] 46. The method of clause 44 wherein the reduction in long chain fatty acid content in the manure results in a reduction in explosive gases in the manure wherein the gases are selected from the group consisting of methane gas, hydrogen gas, phosphine gas, and combinations thereof.

[00168] 47. The method of clause 44 wherein the reduction in ammonia in the manure improves nitrogen value in the manure wherein the manure is used as a fertilizer.

[00169] 48. The method of any one of clauses 42 to 47 further comprising the step of administering to the animal another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.

[00170] 49. The method of any one of clauses 42 to 48 wherein the strain

administered is Bacillus strain 86 (NRRL No. B-50944), or a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944).

[00171] 50. The method of any one of clauses 42 to 48 wherein the strain

administered is Bacillus strain 300 (NRRL No. B-50943), or a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943).

[00172] 51. The method of any one of clauses 42 to 50 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 5.0 x 10 12 CFU/gram of the feed composition.

[00173] 52. The method of any one of clauses 42 to 51 wherein the Bacillus strain is administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 1.0 x 10 CFU/gram of the feed composition.

[00174] 53. The method of any one of clauses 42 to 52 wherein the Bacillus strain is administered in the feed composition at a dose greater than about 7.0 x 10 ⁴ CFU/gram of the feed composition. [00175] 54. The method of any one of clauses 42 to 53 wherein the Bacillus strain is administered in the feed composition at a dose of about 7.3 x 10 ⁴ CFU/gram of the feed composition.

[00176] 55. A method of controlling detrimental environmental effects of manure, the method comprising 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure.

[00177] 56. The method of clause 55 wherein the method improves the health of the animal 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.

[00178] 57. The method of clause 55 wherein the animal is a poultry species and the method improves 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.

[00179] 58. The method of clause 55 wherein the manure is in an anaerobic digester.

[00180] 59. The method of clause 55 wherein the manure is in an anaerobic lagoon.

[00181] 60. The method of any one of clauses 55 to 59 wherein the applying step comprises spraying or adding a powder or a pumpable liquid.

[00182] 61. The method of any one of clauses 55 to 60 wherein the pit is a swine pit.

[00183] 62. The method of clause 32 wherein the antibiotic is Denagard™ and the

Denagard™ is administered in combination with chlortetracycline.

[00184] 63. The method of clause 11 wherein the diet includes a component selected from the group consisting of dried distillers grain solubles, corn, soy, and combinations thereof.

[00185] 64. A commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof. [00186] 65. A feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[00187] 66. An additive for the drinking water of an animal comprising an isolated

Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[00188] 67. An animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[00189] 68. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 64 to 67 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.

[00190] 69. The commercial package, feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 64 to 68, wherein the Bacillus strain inhibits a pathogen selected from the group consisting of E. coli, Salmonella, Staphylococcus, Enterococcus, Campylobacter, and Clostridium.

[00191] 70. The feed additive or additive for the drinking water of the animal of clause 65 or 66 in the form of a concentrate.

[00192] 71. The feed additive or additive for the drinking water of the animal of clause 65 or 66 in the form of a superconcentrate.

[00193] 72. The feed additive, feed composition, or additive for the drinking water of the animal of any one of clauses 65 to 67 in dry form.

[00194] 73. The feed composition of clause 67 in pelleted form.

[00195] 74. The commercial package of clause 64 comprising the Bacillus strains in a form for use in treatment of a pit, a manure pond, or for use in treatment of litter.

[00196] 75. The commercial package of clause 74 wherein the strains are in a form selected from the group consisting of a powder, a liquid, and a pellet form. [00197] 76. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 73 further comprising a carrier for the Bacillus strains.

[00198] 77. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 76 wherein the carrier is selected from the group consisting of a bran, rice hulls, a salt, a dextrin, and combinations thereof.

[00199] 78. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 77 in a bag.

[00200] 79. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 78 wherein the bag is a plastic bag.

[00201] 80. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 79 further comprising instructions for use of one or more of the Bacillus strains.

[00202] 81. The commercial package, feed additive, or additive for the drinking water of the animal of any one of clauses 78 to 80 in a 20-pound bag.

[00203] 82. The commercial package, feed additive, or additive for the drinking water of the animal of any one of clauses 78 to 80 in a 50-pound bag.

[00204] 83. The feed additive or additive for the drinking water of the animal of any one of clauses 65, 66, 68, 69 to 72, or 76 to 82 in powder form.

[00205] 84. The feed additive or additive for the drinking water of the animal of any one of clauses 65, 66, 68 to 71, or 78 to 80 in liquid form.

[00206] 85. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 84 in a container for commercial use.

[00207] 86. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 85 wherein the container comprises plastic.

[00208] 87. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 85 wherein the container comprises paper.

[00209] 88. The method of clause 55 wherein the Bacillus strain is applied to litter and reduces ammonia in the litter.

[00210] 89. The method of clause 1 wherein the animal is a chicken and the improvement in 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. [00211] 90. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of any one of clauses 64 to 87 further comprising a binder.

[00212] 91. The commercial package, feed additive, additive for the drinking water of the animal, or feed composition of clause 90 wherein the binder is selected from the group consisting of clay, yeast cell wall components, aluminum silicate, and glucan, or combinations thereof.

[00213] In various embodiments, 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 embodiment where the animal is a companion animal, the companion animal can be, for example, a canine species or a feline species. In the embodiment 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 embodiments, 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.

[00214] In one embodiment of the invention, 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 86 or 300) 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.

[00215] In embodiments described herein wherein the compositions of the present invention comprising Bacillus strains 86 and/or 300 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 embodiment, the Bacillus strains 86 and/or 300 are provided in the form of an additive for addition to the drinking water of an animal.

[00216] In another illustrative embodiment, the Bacillus strains 86 and/or 300 are provided in the form of a feed additive for addition to a feed composition. The feed

composition may contain Bacillus strain 86 and/or 300 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 86 and/or Bacillus strain 300 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.

[00217] 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 embodiments, the animal feed blend can be supplemented with Bacillus strain 86 and/or Bacillus strain 300, but other ingredients may optionally be added to the animal feed blend, including other bacterial strains, such as other Bacillus strains or Lactobacillus strains.

[00218] In various illustrative embodiments, 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 embodiment, minerals may be added in the form of a mineral premix.

[00219] 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 HC1, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ethyl HC1, and analogs, and salts thereof. Vitamins that may be optionally added are thiamine HC1, riboflavin, pyridoxine HC1, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like. In another embodiment, vitamins may be added in the form of a vitamin premix. In yet another embodiment, 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.

[00220] 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 embodiments, the antibiotic is selected from the group consisting of ampicillin, chloramphenicol, ciprofloxacin, clindamycin, tetracycline, chlortetracycline, Denagard™, BMD™, Carbadox™, Stafac™, and combinations thereof. In one embodiment 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 embodiment, the animal feed blend, the feed composition, the feed additive, or the additive for the drinking water of the animal may contain no

antibiotics.

[00221] In another illustrative embodiment, one or more enzymes may be added to the animal feed blend. In various embodiments, 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 embodiment, 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.

[00222] In various illustrative embodiments, the Bacillus strain (e.g., Bacillus strain 86 and/or 300), or any other bacterial strains added in addition to Bacillus strain 86 and/or 300, can be administered in the feed composition at a dose of about 1.0 x 10 CFU/gram of the feed composition to about 5.0 x 10 12 CFU/gram of the feed composition or at a dose of about 1.0 x

10 3 CFU/gram of the feed composition to about 1.0 x 107 CFU/gram of the feed composition. In other embodiments, the Bacillus strain (e.g., Bacillus strain 86 and/or 300) is administered in the feed composition at a dose greater than about 1.0 x 10 CFU/gram of the feed composition, at a dose greater than about 1.1 x 10 CFU/gram of the feed composition, at a dose greater than about 1.25 x 10 3 CFU/gram of the feed composition, at a dose greater than about 1.5 x 103 CFU/gram of the feed composition, at a dose greater than about 1.75 x 10 CFU/gram of the feed composition, at a dose greater than about 1.0 x 10 ⁴ CFU/gram of the feed composition, at a dose greater than about 2.0 x 10 ⁴ CFU/gram of the feed composition, at a dose greater than about 3.0 x 10 ⁴ CFU/gram of the feed composition, at a dose greater than about 4.0 x 10 ⁴ CFU/gram of the feed composition, at a dose greater than about 5.0 x 10 ⁴ CFU/gram of the feed composition, at a dose greater than about 6.0 x 10 ⁴ CFU/gram of the feed composition, at a dose greater than about 7.0 x 10 ⁴ CFU/gram of the feed composition, at a dose greater than about 8.0 x 10 ⁴ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10 ⁵ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10 ⁶ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10 CFU/gram of the feed composition, at a dose greater than about 1.0 x 10 8 CFU/gram of the feed composition, at a dose greater than about 1.0 x 109 CFU/gram of the feed composition, at a dose greater than about 1.0 x 10 ¹⁰ CFU/gram of the feed composition, at a dose greater than about 1.0 x 10 ¹¹ CFU/gram of the feed composition, or at a dose greater than about 1.0 x 1012 CFU/gram of the feed composition. In yet another embodiment, the Bacillus strain (e.g., Bacillus strain 86 and/or 300) is administered in the feed composition at a dose of about 7.3 x 10 ⁴ CFU/gram of the feed composition.

[00223] In various embodiments, the Bacillus strain (e.g., Bacillus strain 86 and/or 300) for use in accordance with the methods and compositions described herein can be selected from the group consisting of Bacillus strain 86, a strain having all of the identifying characteristics of Bacillus strain 86, Bacillus strain 300, and a strain having all of the identifying characteristics of Bacillus strain 300. Bacillus strain MDG86 and Bacillus strain MDG300 were deposited on March 14, 2014 at the Agricultural Research Service Culture Collection (NRRL), National Center for Agricultural Utilization Research, Agricultural Research Service, USD A, 1815 North University Street, Peoria, Illinois 61604-3999, and were given accession numbers B-50944 and B-50943, 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 86 and 300, respectively, as referred to in the application.

[00224] 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 embodiment, multiple strains are administered in combination in a single composition. In another embodiment, multiple strains are administered in combination in separate compositions. In one illustrative embodiment, any of these strains is isolated from a high performing grow finish pig.

[00225] In another embodiment, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strain 86 and/or Bacillus strain 300) 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. In yet another embodiment, one or more of the Bacillus strains described in the preceding paragraphs (e.g., Bacillus strain 86 and/or Bacillus strain 300) 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.

[00226] As used herein "a strain having all of the identifying characteristics of Bacillus strain 86 or Bacillus strain 300 can be a mutant strain having all of the identifying

characteristics of Bacillus strain 86 or Bacillus strain 300 (e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerprint of Bacillus strain 86 or Bacillus strain 300, enzyme activities that correspond to Bacillus strain 86 or Bacillus strain 300, antimicrobial activity that corresponds to Bacillus strain 86 or Bacillus strain 300, antibiotic sensitivity and tolerance profiles that correspond to Bacillus strain 86 or Bacillus strain 300, or combinations thereof). In alternate embodiments, the mutation can be a natural mutation, or a genetically engineered mutation. In another embodiment, "a strain having all of the identifying

characteristics of Bacillus strain 86 or Bacillus strain 300 can be a strain, for example, produced by isolating one or more plasmids from Bacillus strain 86 or Bacillus strain 300 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 86 or Bacillus strain 300 (e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerprint of Bacillus strain 86 or Bacillus strain 300).

[00227] The feed composition or drinking water comprising Bacillus strain 86 and/or

300 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 embodiment the feed composition or drinking water may be provided to the animal daily. In an alternate embodiment, 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.

[00228] As described herein, one of the method embodiments 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-

50943) , a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-

50944) , a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), 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.

[00229] In the embodiment 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 embodiment, Bacillus strain 86 and/or Bacillus strain 300 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

a-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.

[00230] In the embodiment 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 86 and/or Bacillus strain 300. In various embodiments, the antimicrobial activity is against bacteria selected from the group consisting of E. coli, Salmonella,

Staphylococcus, Enterococcus, Clostridia, Campylobacter, and combinations thereof. Thus, Bacillus strain 86 and Bacillus strain 300 can improve gut health of the animal, and reduce pathogens in the animal, and the animal's environment. In yet another embodiment, 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.

[00231] Bacillus strain 86 and Bacillus strain 300 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 ₄ dissociation to NH ₃ to produce ammonia gas in the environment of the animal. NH is more toxic than NH ₄ and can lead to respiratory diseases in the animal. Thus, Bacillus strain 86 and Bacillus strain 300 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.

[00232] In the embodiment 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. [00233] In these embodiments, 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.

[00234] Bacillus strain 86 and Bacillus strain 300 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 86 and Bacillus strain 300 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 ₃ 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 ₃ flashes off) which results in loss of nitrogen and a decrease in the value of the manure as a fertilizer. Thus, Bacillus strain 86 and Bacillus strain 300 increase the value of manure as a fertilizer.

[00235] As described herein, another method embodiment 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure.

[00236] In this embodiment, Bacillus strain 86 and/or Bacillus strain 300 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 embodiments, 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.

[00237] Bacillus strain 86 and Bacillus strain 300 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 86 and Bacillus strain 300 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 86 and Bacillus strain 300 increase the value of manure as a fertilizer.

[00238] In another method embodiment, 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 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof, and controlling the detrimental environmental effects of the manure.

[00239] In this embodiment, Bacillus strain 86 and Bacillus strain 300 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 86 and Bacillus strain 300 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 embodiment where Bacillus strain 86 and/or Bacillus strain 300 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 embodiment, the application of the Bacillus strain to the litter can result in ammonia reduction in the litter.

[00240] In this method embodiment, 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 embodiment 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.

[00241] In additional embodiments of the invention, compositions comprising Bacillus strain 86 and/or Bacillus strain 300 are provided. In one embodiment, a commercial package is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[00242] In another embodiment, a feed additive for an animal feed is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[00243] In yet another embodiment, an additive for the drinking water of an animal is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[00244] In yet another illustrative aspect of the invention, an animal feed composition is provided comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 86 (NRRL No. B-50944), Bacillus strain 300 (NRRL No. B-50943), a strain having all of the identifying characteristics of Bacillus strain 86 (NRRL No. B-50944), a strain having all of the identifying characteristics of Bacillus strain 300 (NRRL No. B-50943), and combinations thereof.

[00245] In one embodiment, 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 86 and Bacillus strain 300 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 embodiments, the mixing can be done for any suitable time period (e.g., about 1 to about 4 minutes). In the embodiment where Bacillus strain 86 and/or Bacillus strain 300 is in the form of an additive for the drinking water of the animal, the Bacillus strain 86 and/or Bacillus strain 300 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 86 and Bacillus strain 300 described herein, for administration to the animal in the drinking water of the animal. Bacillus strain 86 and/or Bacillus strain 300 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.

[00246] In any of the composition embodiments described herein, the Bacillus strain 86 and/or Bacillus strain 300 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 86 and/or Bacillus strain 300 can cause.

[00247] 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 embodiment, the feed additive or additive for the drinking water of the animal, or the Bacillus strain 86 and/or Bacillus strain 300 in the commercial package can be in the form of a concentrate (e.g., about 1 x 10 8 to about 5 x 109 CFU/g) or a superconcentrate (e.g., about 1 x 10 ¹⁰ to about 5 x 10 ¹² 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 embodiment, the feed additive, feed composition, or additive for the drinking water of the animal, or the Bacillus strain 86 and/or Bacillus strain 300 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.

[00248] In yet another embodiment, 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 embodiment, the Bacillus strain 86 and/or Bacillus strain 300 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.

[00249] In another illustrative embodiment, 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 86 and/or Bacillus strain 300. 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 embodiment, the carrier can be any suitable carrier known in the art for a direct-fed microbial. In another embodiment, 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.

[00250] In yet other embodiments, the commercial package, feed additive, additive for the drinking water of the animal, or feed composition comprising Bacillus strain 86 and/or Bacillus strain 300 is in a container for commercial use. In various embodiments the container can be, for example, a bag (e.g., a 20-pound bag, a 50-pound bag, a 2-ounce bag, a 1-pound bag, or a 1 -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 86 and/or Bacillus strain 300 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.

[00251] The following examples are for illustrative purposes only. The examples are non-limiting, and are not intended to limit the invention in any way.

EXAMPLE 1

Efficacy of Strain Combinations for Increasing Live Growth Performance of Broiler Chickens

[00252] 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.

[00253] For this investigation, a total of 60 pens were utilized, with a group size of 35 birds per pen. The litter utilized in the pens was wood shavings that were previously used (but had no previous DFM use).

[00254] Approximately 2100 hatch chickens (Cobb 500 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 50 grams, and the end weight of the chickens was about 2.75 kg. [00255] In the investigation, the chickens were divided into four groups as shown in

Table 1 :

Table 1. Experimental Treatment Groups

[00256] For the allotment of chickens to the experiment, birds were weighed by pen, and pens were grouped into replicates of six (6) 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 3 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.

[00257] Measurements of litter compositions were obtained and evaluated. Samples of litter were taken from four (4) replicates (two in each half of the barn) prior to chick placement, during week 3 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.

[00258] 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.

[00259] 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 100 pound gain was calculated as the product of feed/gain (F/G) and the cost/100 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.

[00260] 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), 5% DDGS, and 3% meat-and-bone meal. Identification information of DFM materials was as follows:

DFM additive CFU/g

Combo MDG-1 8.30 x 10 ⁸

Combo MDG-3 1.82 x 10 ¹⁰

[00261] Chickens were fed diets in three phases according to the following age ranges:

Phase 1 - Week 1-2; Phase 1 - Week 3-4; and Phase 3 - Week 5-6.

[00262] All diets were formulated to be adequate in SID Lys ( , NRC, 1994) and the other essential amino acids (AA), available phosphorus (P), calcium (Ca), and sodium (Na) (see Table 2). Table 2. Dietary nutrient formulation constraints of experimental diets by phase of production.

Nutrient Wk 1-2 Wk 3-4 Wk 5-6

DDGS, % 5.0 5.0 5.0

Meat-and-bone meal (pork),

3.0 3.0 3.0

%

ME, kcal/lb 1,385 1,410 1,443

SID AA, %

Lys 1.18 1.04 0.90

Met 0.57 0.53 0.43

Total AA, %

SAA 0.92 0.88 0.75

Thr 0.72 0.62 0.55

Trp 0.22 0.20 0.18

Arg 1.40 1.25 1.10

Phosphorus, available, % 0.45 0.40 0.38

Calcium, % 0.92 0.80 0.75

Sodium, % 0.20 0.20 0.20

[00263] 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 3).

Table 3. Dietary formulations for experimental diets.

Ingredient (lbs/ton) Starter (wk 1-2) Grower (wk 3-4) Finisher (wk 5-6)

Corn 1,150.65 1,259.89 1,347.92

Soybean meal 592.10 491.67 396.71

DDGS 100.00 100.00 100.00

Meat & bone meal 60.00 60.00 60.00

Fat, poultry 44.75 43.82 54.71

Limestone 20.44 16.52 15.04

Salt 8.11 5.76 3.54

Monocalcium phosphate 7.65 3.53 2.28

Sodium bicarbonate 0.20 3.47 6.57

DL-methionine 5.23 4.86 3.27

L-Lysine.HCl 4.62 4.24 3.72

Trace mineral premix 1.00 1.00 1.00

Vitamin premix 5.00 5.00 5.00

Phytase (OptiPhos 2000) 0.25 0.25 0.25

Nutrient composition

ME, kcal/lb 1,385 1,410 1,443 Crude protein, % 22.5 20.5 18.5

Crude fat, % 5.45 5.56 6.23

NDF, % 8.2 8.2 8.2

Phosphorus

Total, % 0.57 0.51 0.48

Phytate-P, %

Available, % 0.45 0.40 0.38

Phytase, FTU/kg 250 250 250

Calcium, % 0.92 0.80 0.75

Sodium, % 0.20 0.20 0.20

Lysine, total, % 1.31 1.16 1.01

Lysine, SID, % 1.18 1.04 0.90

Methionine, SID, % 0.57 0.53 0.43

TSAA, SID, % 0.85 0.79 0.67

Threonine, SID, % 0.70 0.63 0.56

Tryptophan, SID, % 0.21 0.19 0.16

Arginine, SID, % 1.31 1.16 1.02

Isoleucine, SID, % 0.81 0.72 0.64

Valine, SID, % 0.91 0.83 0.75

[00264] 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 185°F following 30 seconds of conditioning. The diets for Week 1-2 were crumbled following pelleting. Experimental diets were delivered to each pen by hand, and the addition was recorded manually.

[00265] 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 (5) 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.

[00266] Initial data analysis was performed for all metrics to determine homogeneity of variance, normal distribution, and outliers (+ > 3 standard deviations in difference from the grand mean).

[00267] Feed Conversion Ratio (FCR):

[00268] Table 4 shows the mortality-corrected feed conversion ratio (FCR) of straight- run broilers in the various treatment groups at the starter, grower, and finisher phases. Table 4. Mortality corrected feed conversion ratio (FCR) of straight-run broilers fed standard corn-soy diets with the inclusion of a direct fed microbial.

Treatment Starter Grower Finisher

Control (C) 1.370 ^a 1.541 1.919

C + MDG-1 (1.84 ⁵ ) 1.346 ^ab 1.540 1.934

C + MDG-3 (7.35 ⁴ ) 1.309 ^b 1.500 1.907

C + MDG-3 (1.84 ⁵ ) 1.376 ^a 1.521 1.903

Pooled SEM 0.006 0.006 0.011 ^l ' Means with different groupings differ significantly at P < 0.05.

[00269] In the starter phase, the inclusion of DFM in all treatments yielded similar results to the control, except for MDG-3 (7.35 ⁴ ), which resulted in a significant decrease compared to the control. Throughout the growth and finisher phase, no significant differences in FCR were observed.

[00270] Table 5 shows the mortality-corrected feed conversion ratio (FCR) of straight- run broilers in the various treatment groups from Days 1-28 of the treatment phase and for Days 1-41 of the treatment phase.

Table 5. Mortality corrected cumulative feed conversion ratio (FCR) of straight-run broilers fed standard corn-soy diet with DFM inclusion.

Treatment Day 1-28 Day 1-41

Control (C) 1.499 ^a 1.702

C + MDG-1 (1.84 ⁵ ) 1.492 ^ab 1.707

C + MDG-3 (7.35 ⁴ ) 1.457 ^c 1.673

C + MDG-3 (1.84 ⁵ ) 1.486 ^abc 1.690

Pooled SEM 0.004 0.005 ^l ' Means with different groupings differ significantly at P < 0.05.

[00271] Through day 28, the inclusion of MDG- 1 and MDG-3 (1.84 ⁵ ) yielded similar

FCR to the control diet. A significant decrease in FCR was observed with the inclusion of MDG-3 (7.35 ⁴ ) compared to the control diet. At the conclusion of the trial (i.e., through day 41), no significant differences were observed between treatments.

Body Weights:

[00272] Table 6 shows the calculated body weights of straight-run broilers fed a standard corn-soy diet in the various treatment groups at day 1, day 14, day 28, and day 41 of treatment. Table 6. Body weights (kg) of straight-run broilers fed a standard corn-soy diet with the inclusion of a direct fed microbial.

Treatment Day 1 Day 14 Day 28 Day 41

Control (C) 0.034 0.338 1.274 2.460

C + MDG-1 (1.84 ⁵ ) 0.034 0.324 1.236 2.413

C + MDG-3 (7.35 ⁴ ) 0.034 0.340 1.280 2.438

C + MDG-3 (1.84 ⁵ ) 0.033 0.316 1.239 2.419

Pooled SEM 0.000 0.003 0.008 0.013

[00273] As shown in Table 6, no significant differences were observed in body weight associated with treatment throughout the duration of the trial.

Mortality:

Table 7 shows the mortality of straight-run broilers fed standard corn- soy diet in the various treatment groups at the starter, grower, and finisher phases.

Table 7. Mortality (%) of straight-run broilers fed standard corn- soy diet with the inclusion of a direct fed microbial.

Treatment Starter Grower Finisher Total

Control (C) 5.1 0.6 0.6 6.3

C + MDG-1 (1.84 ⁵ ) 5.1 1.1 0.9 7.1

C + MDG-3 (7.35 ⁴ ) 4.3 1.1 1.1 6.6

C + MDG-3 (1.84 ⁵ ) 6.0 0.6 0.9 7.4

Pooled SEM 0.4 0.2 0.2 0.4

[00274] As shown in Table 7, no differences in mortality were observed between treatments throughout the trial.

[00275] 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 28 days of age and were 3 points improved at day 41 with the inclusion of MDG-3 (7.35 ⁴ ).

EXAMPLE 2

Growth Performance Studies of Finishing Pigs Used to Determine Ideal DFM Strain

Combinations [00276] Fig. 1 shows the growth performance of finishing pigs fed various DFM strain combinations. Fig. 1 suggests that strain 86 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 3 suggests that a combination of strain 86 plus strain 300 performed better than strain 86 alone on ADFI and ADG. The ratios for 86 + 300 + 552 were 33.3%, 33.3%, and 33.3%, respectively. The ratios for 86 + 552 and 300 + 552 were 50:50.

EXAMPLE 3

Growth Performance of Finishing Pigs Fed Various DFM Strain Combinations

[00277] 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.

[00278] Three different groups were evaluated in the instant example: 13-F040, 13-

F071, and 13-F072. In the example, PIC 337 x Camborough 29 pigs were evaluated, including barrows and gilts in mixed- sex pens. The parameters for each group are shown in Table 8 as follows:

Table 8. Parameters for Treatment Protocols

[00279] In this investigation, the pigs were divided into three groups as shown in Table

9: Table 9. Experimental Treatment Groups

[00280] 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.

[00281] 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 3 consecutive days and not showing signs of improvement were immediately removed from the experiment.

[00282] 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 10).

Table 10. Identification of causes for removals.

Cause Nutritional Non-nutritional

Respiratory Disease ¹ X

Gastrointestinal Disease ² X

Other Disease X

Injury ⁴ X

Structural Defects ⁵ X

Hernia ⁶ X

Emaciation ⁷ X

NANI ⁸ X

Can't find feed or water ⁹ X

Low body weight ¹⁰ X

Other ¹¹ X ¹ Respiratory disease = PRRS; pneumonia; influenza; or thumping.

Gastrointestinal disease = ileitis; hemorrhagic bowel; obstructed bowel; or ulcers.

Other disease = strep or greasy pig.

⁴ Injury = broken bones; cannibalism; abscesses; or swollen joints

⁵ Structural defects = broken top; leg soundness issues; or spraddle legs

⁶ Hernia = scrotal or umbilical hernia

Emaciation = fall-off; anorexia; or general unthrifitiness

Non- Ambulatory, Non-Injured (NANI) = downers and stress related issues

⁹ Can't find feed or water

¹⁰ Low body weight

uOther reason not listed

[00283] 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 100. 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 x 0.75; 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.

[00284] 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.

[00285] 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 11. Table 11. Nutrient loading values and analyses of feedstuff s.

Nutrient Corn SBM DDGS

Exp: 40 71/72 An. 40 71/72 An. 40 71/72 An.

DM, % 84.3 84.9 85.1 88.1 88.2 88.3 89.5 88.2 88.2

ME, kcal/lb 1,474 1,487 — 1,500 1,461 — 1,475 1,439 —

Fat, % 3.25 3.33 3.30 1.90 1.71 1.95 8.08 7.37 6.12

CP, % 7.63 7.98 8.18 47.50 47.88 46.88 27.94 30.98 31.26

He, total, % 0.27 0.28 0.28 2.12 2.13 2.14 1.03 1.15 1.21

He, SID, % 0.21 0.22 — 1.91 1.92 — 0.78 0.87 —

Lys, total, % 0.24 0.24 0.24 2.92 2.95 2.98 0.89 0.98 1.06

Lys, SID, % 0.17 0.17 — 2.63 2.66 — 0.56 0.62 —

SAA, total, % 0.33 0.34 0.34 1.37 1.38 1.37 1.00 1.11 1.12

SAA, SID, % 0.27 0.27 — 1.19 1.20 — 0.71 0.87 —

Thr, total, % 0.28 0.29 0.29 1.84 1.85 1.85 0.97 1.14 1.14

Thr, SID, % 0.21 0.21 — 1.61 1.62 — 0.69 0.81 —

Trp, total, % 0.06 0.06 0.06 0.65 0.65 0.65 0.21 0.21 0.22

Trp, SID, % 0.05 0.05 — 0.59 0.59 — 0.14 0.15 —

Val, total, % 0.36 0.38 0.38 2.23 2.23 2.24 1.33 1.45 1.57

Val, SID, % 0.28 0.30 — 1.97 1.97 — 1.00 1.09 —

P, % 0.26 0.26 0.25 0.71 0.70 0.71 0.83 0.85 0.89

P, avail, % 0.04 0.04 — 0.18 0.17 — 0.58 0.60 —

Ca, % 0 0.01 0 0.31 0.34 0.35 0.01 0.02 0.03

Na, % 0 0 0 0.02 0.02 0.04 0.17 0.23 0.19

S, % 0.09 — 0.09 0.40 — 0.39 — — 0.75

K, % 0.36 0.31 0.29 2.28 2.26 2.26 0.87 0.99 1.03

Cu, mg/kg 2 3 3 15 15 15 4 6 6

Zn, mg/kg 18 21 19 48 50 52 54 63 65

ADF, % 2.4 2.4 2.2 5.2 5.3 4.9 9.9 11.7 12.3

NDF, % 6.6 7.0 6.6 7.5 6.9 7.2 21.6 23.7 27.8

Ash, % 0.96 0.80 1.0 5.75 6.00 6.0 4.3 4.1 4.2

Starch, % 59.7 60.8 60.1 1.2 0.8 0.9 4.9 2.3 2.4

DON, mg/kg — — 2.24 — — — — — 1.62

Zer, mg/kg — — 0.02 — — — — — 0

AfBl, ug/kg — — 0 — — — — — 0

FumBl, mg/kg — — 1.57 — — — — — 6.25

[00286] Each of the Bacillus (Bs) strain combinations was manufactured and provided by Microbial Discovery Group (Franklin, WI). Treatment B comprised Bs strains 86 and 300, while Treatment C comprised Bs strains 86, 300, and 552. 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 12. Table 12. Dietary feedstuff and formulation constraints for experimental diets.

Nutrient 160-200 lbs 200-240 lbs 240-280 lbs

Basemix R&D DG40L R&D DG40L R&D DG40L

ID code (JBS United, Inc.) 30881 30881 30881

Inclusion rate, lbs/ton 42 40 40

DDGS, % 30 30 30

SID Lysine, g/Mcal ME 2.12 1.91 1.80

Minimum ratios to SID Lys, %

SAA 57 57 57

Thr 63 64 65

Trp 17 17 17

Val 65 65 65

lie 54 54 54

P, available, % 0.24 0.22 0.20

Ca, % 0.48 0.45 0.45

Na, % 0.22 0.21 0.21

[00287] 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 30% of the diet within each experiment. Diets did not contain

supplemental fat.

[00288] Diet manufacture and delivery parameters are shown in Table 13. For the

13-F040 and 13-F071 groups, diet components were mixed and delivered to each feeder through an electronic feed mixing, delivery, and recording system. For the 13-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.

Table 13. Formulations of experimental diets.

13-F040 13-F071 & 13-F072

Ingredients 200-240 lbs 240-280 lbs 160-200 lbs 200-240 lbs 240-280 lbs

Corn to 2,000 to 2,000 to 2,000 to 2,000 to 2,000

Soybean meal, dehulled 129.2 100.7 193.1 145.6 117.4

DDGS 600.0 600.0 600.0 600.0 600.0

Basemix 40.0 40.0 42.0 40.0 40.0

DFM premixes + / - + / - + / - + / - + / -

Nutrient composition

ME, kcal/lb 1,448 1,448 1,449 1,451 1,451 Crude protein, % 16.15 15.58 18.56 17.63 17.06

Crude fat, % 4.54 4.57 4.32 4.36 4.38

NDF, % 11.5 11.5 11.9 11.9 12.0

Lysine

Total, % 0.78 0.74 0.85 0.78 0.74

SID, % 0.61 0.57 0.68 0.61 0.58

SID, g/Mcal ME 1.91 1.80 2.12 1.91 1.80

SID AA/Lys, %

SAA 82 85 78 83 86

Thr 77 78 75 78 79

Trp 19 18 19 19 19 lie 85 86 83 85 86

Val 105 107 99 104 106

Phosphorus, %

Total 0.49 0.48 0.47 0.46 0.46

Bioavailable 0.27 0.27 0.27 0.26 0.26

Phytase, FTU/kg 100 100 106 100 100

Calcium, % 0.45 0.44 0.48 0.45 0.44

Sodium, % 0.23 0.23 0.22 0.21 0.21

[00289] Each feedstuff was sampled for nutrient and mycotoxin analyses according to standard protocol. Final experimental diets were sampled bi-weekly from a minimum of 5 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.

[00290] 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 Έχρ' . Initial data analysis was performed for all metrics for both the individual and combined datasets to determine normality of distribution and outliers (+ > 3 standard deviations in difference from the grand mean).

[00291] The grand means of performance metrics across experiments is shown in

Table 14.

Table 14. Grand means of performance metrics across experiments.

Performance metrics 13-F040 13-F071 13-F072

Live weights, lbs

Start 232.2 171.1 209.5

Final 286.6 275.8 275.5

ADG, lbs 1.75 1.82 1.73

ADFI, lbs 6.47 5.49 5.68

F/G, lb/lb 3.73 3.03 3.28

Carcass weight, lbs 211.0 205.0 206.0

Lean, % 55.6 56.4 55.9

Carcass yield, % 74.9 75.3 75.7

Carcass ADG, lbs 1.27 1.35 1.29

Carcass F/G, lb/lb 5.14 4.08 4.41

Feed costs, $/lb

Live

Carcass

[00292] Growth performance of pigs fed different DFM combinations in late-finishing is shown in Table 15. Comparison of strain 86 alone and in combination with two other strains in a three strain combination shows that strain 86 alone was significantly improved over the control on average daily gain. Strain 86 alone was trending toward significance with a p= 0.057, but significant when starting weight is considered as covariable. Ending weight of strain 86 was not considered significant compared to the control, but was considered significant in consideration of starting weight as a covariable. Carcass weight of strain 86 alone was trending, with p=0.051 but considered significant with starting weight as a co-variable.

Table 15. Growth performance of pigs fed different DFM combinations in late-finishing. Treatment Pooled P-value Covariable P-value

Response criteria ¹ Control Bs86 3-strain SEM Trt B S6 StWt DOF

Live weights, lbs

Start 204.3 204.6 204.9 9.2 0.573 0.544 — < 0.001

End 279.3 280.2 280.0 2.1 0.524 0.273 < 0.001 0.01 1

ADG, lbs 1.75 1.80 1.76 0.02 0.053 0.018 0.263 0.458

ADFI, lbs 5.85 5.91 5.92 0.15 0.318 0.201 0.004 0.806

F/G, lb/lb 3.36 3.31 3.36 0.07 0.089 0.057 < 0.001 0.103

Carcass

Wt, lbs 207.8 208.0 207.6 0.6 0.784 0.821 < 0.001 < 0.001

ADG, lbs 1.30 1.31 1.31 0.02 0.819 0.558 0.224 0.603

F/G, lb/lb 4.53 4.53 4.53 0.13 0.998 0.969 0.003 0.395

Yield, % 75.4 75.3 75.3 0.3 0.786 0.541 0.698 0.486

Feed cost, $/lb gain ³

Live 0.361 0.363 0.368 0.003 0.058 0.570 < 0.001 0.092

Carcass 0.486 0.497 0.496 0.005 0.087 0.051 < 0.001 0.354

Income, $/pig ⁴ 166.27 166.38 166.05 0.51 0.784 0.821 < 0.001 < 0.001

Income over feed, $/pig 139.22 139.21 138.39 0.39 0.083 0.972 < 0.001 < 0.001

Data are means from three experiments combined from which all three listed treatments appeared.

² Data were analyzed as a randomized complete-block design, including the main effect of treatment, random effects of experiment and replicate within experiment, and covariables start wt and days on feed.

³ Feed costs were calculated based on the following feedstuff costs: corn, $4.25 bu; soybean meal, $500/ton; DDGS, $225/ton; basemix, $900/ton; and the DFMs were valued at a cost equivalent to $5/treated ton of feed.

4Income was calculated as the carcass wt (cwt/pig) multiplied by a base meat price of $80/cwt.

[00293] Accounting for start weight and days on feed differences across experiments, the effect of DFM addition on average daily gain (Trt P = 0.053) was dependent on DFM formulation, as the Bs strain 86 alone increased (P = 0.018) ADG 3% over the control, but the 3-strain combination had no effect. Similarly, DFM addition affected feed/gain ratio (Trt P = 0.089) in a formulation-dependent manner, with the Bs strain 86 alone reducing (P = 0.057) feed/gain ratio 1.5% and the 3-strain formulation having no impact.

[00294] Fig. 2 shows a meta-analysis of DFM strain effect on growth performance across three experiments. A total of three (3) separate trials were conducted using DFM strains 86 and 300 in combination, individually or in combination with other Bacillus or lactic acid bacteria strains. A universal dose of 7.35 x 10 ⁴ CFU/gram was utilized.

[00295] As shown in Fig. 2, treatment with DFM strain 86 alone increased gain approximately 3% (P = 0.05) and reduced F/G approximately 2% (P = 0.06). The combination of DFM strains 86 and 300 in experiment 13-F072 (80% strain 86 and 20% strain 300) was numerically better than DFM strain 86 alone for ADG.

EXAMPLE 4

Enzymatic Activity Screenin

[00296] This example describes the use of plate media screening methods to detect enzymatic activity in DFM strains 86, 300, and other related isolates. Enzyme assay media plates were prepared by supplementing tryptic soy agar with between 0.5% and 1% of various substrates, including polysaccharides (corn starch, carboxymethylcellulose, or xylan), proteins (casein), and lipids (tributyrin). Bacillus strains of interest (including DFM strains 86 and 300) obtained from fresh overnight cultures were spotted onto plates (5 μί) and incubated at 32°C for up to 48 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 1 minute to visualize zones of clearing. DFM strains 86 and 300 were both observed to be positive for protease, amylase, and carboxymethylcellulose (CMCase) activity after 48 hours and strong positives on tributyrin agar (lipase) after 72 hours were observed.

[00297] As shown in Fig. 3, the Bacillus strains have enzymatic activity including but not limited to amylase, carboxymethylcellulose, protease, xylanase and lipase. EXAMPLE 5

Substrate Testing Summary

[00298] In the instant example, a comparison of DFM strain 86 and DFM strain 300 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.

[00299] A list of carbohydrate and carboxylic acid carbon sources utilized for DFM strain 86 and DFM strain 300 is presented in Table 16.

Table 16. Utilized Carbohydrate and Carboxylic Acid Carbon Sources.

DFM strain 86 DFM strain 300

2-deoxy-D-ribose X X

3-0-b-galactopyranosyl-d-arabinose X X

5-keto-d-gluconic aid X X

a-D-glucose X X

a-D-lactose X

Amydalin X X

Arbutin X X

b-methyl-d-glucoside X X

Bromosuccinic acid X X

Citric acid X

D,L-a-glycerol phosphate X X

D,L-Malic acid X X

D-arabinose X X

D-cellobiose X X

Dextrin X

D-fructose X X

D-fructose-6-phosphate X X

D-galacturonic acid X X

D-glucosamine X X

D-glucuronic acid X X

D-mannitol X X

D-mannose X X D-melibiose X

D-Psicose X X

D-raffinose X

D-ribose X X

D-saccharic acid X

D-sorbitol X X

D-tagatose X X

D-trehalose X X

D-xylose X X

Fumaric acid X X

Gelatin X X

Gentiobiose X X

Glycerol X

Glycogen X X

L-arabinose X X

L-lactic acid X X

L-lyxose X X

L-Malic acid X X

L-rhamnose X

Maltose X

Maltotriose X

Methylpyruvate X X

Mono-methylsuccinate X

Mucic acid X

N-acetyl-d-glucosamine X

Palatinose X X

Pectin X X

Pyruvic acid X X

Salicin X X

Stachyose X

Starch X X

Succinic acid X

Sucrose X X

Thymidine X

Uridine X X

[00300] A list of phosphate and sulfur sources utilized by DFM strain 86 and DFM strain 300 is presented in Table 17.

Table 17. Utilized Phosphate and Sulfur Sources.

DFM strain 86 DFM strain 300

Adenosine 2- monophosphate X

Adenosine 2,3 cyclic monophosphate X X

Adenosine 5 monophosphate X

Adenosinee 3,5 cyclic monophosphate X X

B-glycerol phosphate X

Cysteamine-S-Phosphate X X

Cytidine 2 monophosphate X

Cytidine 2,3 cyclic monophosphate X

Cytidine 3 monophosphate X

Cytidine 5 monophosphate X

D,L-lipoamide X X

D-glucosamine 6 phosphate X

D-glucose-6-phosphate X

Dithiophosphate X X

D-l-a-glycerol phosphate X

D-Mannose-l-phosphate X

D-mannose-6-phosphate X

Guanosine 2 monophosphate X

Inositol hexaphosphate (phytate) X X

L-cysteine X

O-phospho-L-serine X

Phosphate X X

Phosphoenol pyruvate X X

Phospho-glycolic acid X

Phosphoryl choline X

Thiophosphate X X

Thiosulfate X X [00301] A list of unique nutritional utilization observed for DFM strain 86 and DFM strain 300 is presented in Table 18.

Table 18. Unique Nutritional Utilization.

[00302] As shown in Tables 16-18, both DFM strains appeared to utilize an extremely wide array of amino acids and dipeptide bonds. DFM strain 86 and DFM strain 300 utilized 123 and 149 amino acids and dipeptide bonds, respectively. DFM strain 300 does appear to be more versatile, as this strain used 26 more amino acids and dipeptide bonds than DFM strain 86, including several D-amino acids which are not often found in microbes.

[00303] Both strains use a very wide variety of carbon sources, but DFM strain 86 appears to use a number of a-galactosides such as meliobiose, stachyose and raffinose.

Furthermore, DFM strain 86 is able to utilize maltose and maltotriose, which was not observed for DFM strain 300.

[00304] In addition, DFM strain 86 and strain 300 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 300 appears to be broader regarding the array of phosphate compound utilization.

[00305] Finally, DFM strain 86 and strain 300 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.

EXAMPLE 6

Antimicrobial Screening with the Cross-Streak Method and Stab-Streak Method

[00306] 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 86 and DFM strain 300, for antimicrobial activity against a range of other organisms.

[00307] Bacillus strains of interest (including DFM strain 86 and DFM strain 300) obtained from frozen glycerol stocks were inoculated in a single 1 cm 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 24 hours at 32°C, until a heavy streak of growth was present.

[00308] 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 24 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. 4A, DFM strains 86 and 300 produce

antimicrobial substances which inhibit pathogenic organisms including, but not limited to, Staphylococcus, Enterococcus, E. coli, Salmonella, and Clostridium. As shown in Fig. 4B, DFM strains 86 and 300 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 86 and DFM strain 300, for antimicrobial activity against other organisms. Bacillus strains of interest (including DFM strain 86 and DFM strain 300) 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 micro aerophilic

microorganisms (Clostridium perfringens, Clostridium difficile, Campylobacter jejuni). Bacillus inoculated plates were incubated for 16-24 hours at 37°C depending on the colony size and proliferation tendencies of each Bacillus strain. Once incubation had concluded, lids were removed and plates were inverted over 1ml of concentrated chloroform for 15 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 15 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 15 minutes. BHI (0.7%) agar was prepared in 5 ml aliquots and utilized for top agar overlays of recently killed Bacillus strains. These tubes were inoculated with ΙΟΟμΙ 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 (37° 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. 4B, strains 86 and 300 produce antimicrobial substances which inhibit pathogenic organisms including, but not limited to, Staphylococcus, Enterococcus, E. coli, Salmonella, Clostridium, Vibrio, and Campylobacter.

EXAMPLE 7

Antibiotic Susceptibility and Tolerance Screening

[00309] Bacillus strains of interest (including DFM strains 86 and 300) from 24 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 Ztod/Zws-inoculated plate. After incubation for 24 hours at 32°C, 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.

[00310] As shown in Fig. 5, DFM strains 86 and 300 were tested for antibiotic sensitivity using various antibiotics and they are susceptible to a number of antibiotics. EXAMPLE 8

Antibiotic Susceptibility and Tolerance Screening

[00311] Antibiotic media plates were prepared with five antibiotics of agricultural importance at either lx, 1/lOx, or 1/lOOx their recommended dose rates (Pulmotil™, 181g/ton; BMD™, 30g/ton; Stafac™, lOg/ton; Tylan™, 20g/ton, chlortetracycline (CTC) + Denagard™, 400g/ton CTC + 35 g/ton Denagard™). Bacillus isolates were screened by spotting 5 μΐ of fresh overnight culture onto antibiotic media plates and incubating for 24 hours. The presence or absence of colony growth was noted and used as a rough measure of antibiotic susceptibility for screening purposes.

[00312] As shown in Fig. 6, the presence of CTC + Denagard™ at the recommended dose, BMD™ at 1/10 the recommended dose, and Stafac™ at 1/10 the recommended dose caused minimal sized zones of inhibition against DFM strains 86 and 300. Therefore, these antibiotics should not interfere with the growth of DFM strains 86 and 300. These strains were also tolerant to Formaldehyde (Sal CURB, Termin-8) and minimal inhibition was observed with Carbadox™ (113.4g/ton).

EXAMPLE 9

Distilled Grain Minimal Medium Screenin

[00313] To screen Bacillus isolates (including DFM strains 86 and 300) 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: 0.023% Κ ₂ ΗΡ0 ₄ , 0.01% MgS0 ₄ *7H ₂ 0, 0.1% trace element stock, 0.05% yeast extract, 0.0002% MnCl ₂ *4H ₂ 0, 0.002% FeCl ₃ , and 0.2% DDGS. Sterile 10 ml tubes of this medium were inoculated with these Bacillus strains, incubated 72 hours at 25°C, and scored for growth. Tubes with strong growth were used for lOx serial dilutions in the same medium, incubated for 24 hours at 25 °C, and scored again for growth, with turbidity at later dilution steps serving as a rough indicator of initial cell count.

[00314] As shown in Fig. 7, with DDGS as a sole carbon source, both DFM strains 86 and 300 exhibited ability to grow and digest DDGS. DFM strain 86 was among the strains with the highest growth, with turbidity in the 10 - ^" 8 dilution tube after the secondary screening. DFM strain 300 showed moderate growth at 72 hours with turbidity in the 10 ^"4 dilution tube after the secondary screening. EXAMPLE 10

Evaluation of DFM Strains Relative to Manure pH

[00315] This example describes the use of Bacillus DFM strains 86 and 300 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 50 ml portions and inoculated with freeze-dried cultures of DFM strains 86, 300. All freeze-dried cultures were enumerated by the plate count method, and inocula were prepared by suspending spores in a solution of 0.1% peptone.

[00316] Inoculated manure sample tubes were capped loosely and incubated at 25°C without shaking under stagnant, non-aerated conditions for a minimum of 24 hours. At 24 hours (and in some cases, at additional 24-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 1/20 in Millipore water for analysis with the Hach ISENH4 ammonium ion-sensitive electrode. All measurements were taken at 25°C. The fraction and concentration of free ammonia were derived from the pH, temperature, and TAN of the sample.

[00317] As shown in Figs. 8 and 9, treatment with strain blends containing DFM strains

86 and 300 was associated with lowered pH and lowered free ammonia in multiple trials. The size of the effect varied between manure types.

EXAMPLE 11

Digestion of Long Chain Fatty Acids by DFM Strains 86, 300

[00318] To determine whether DFM strains 86 and 300 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, lOmM phosphate buffer, and 0.1% fatty acid, adjusted to pH 7.0, 7.5, 8.0.) DFM strains 86 and 300 obtained from frozen glycerol stocks were inoculated into each type of minimal medium and incubated at 37°C for 48 hours with or without shaking. Growth was determined by measuring optical density at 600 nm with a spectrophotometer.

[00319] As shown in Fig. 10, under aerobic conditions, DFM strain 86 showed substantial growth (change in Α ₆ οο>0.1) with palmitic acid and stearic acid at different pHs and DFM strain 300 showed substantial growth with palmitic acid and stearic acid at pHs of 7.5 and 8.0. As shown in Fig. 11, under anaerobic conditions, DFM strain 86 showed substantial growth (change in A ₆ oo>0.1) with palmitic acid and oleic acid at pHs of 7.0, 7.5 and 8.0 and DFM strain 300 showed substantial growth with palmitic acid and oleic acid at pHs of 7.5 and 8.0.

EXAMPLE 12

RAPD (Randomly Amplified Polymorphic DNA) Analysis of

DFM Strains 86 and 300

[00320] 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 10 ml of tryptic soy broth at 32°C. Thereafter, DNA was extracted from these cultures using Qiagen's DNeasy Mini kit, following the protocol provided for Gram-positive bacteria. A GE 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. 12, 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. 12 demonstrates that DFM Strains 86 and 300 are different strains, each having a unique DNA fingerprint.

EXAMPLE 13

Culture Growth of DFM Strains 86 and 300

[00321] In the instant example, growth of DFM strains 86 and 300 can be achieved by culturing. On a small scale, TSB or nutrient broth can be utilized to culture DFM strains 86 and 300.

[00322] Agar medium may be produced using 23 grams Nutrient Agar (BD 213000) and

1000 ml of DI water, followed by autoclaving at 121°C. Broth medium may be produced using 8.0 grams of Nutrient Broth (BD 234000) and 1000 ml of DI water, followed by autoclaving at 121°C.

[00323] For culturing DFM strain 86, a pure culture of DFM strain 86 was streaked on a nutrient agar plate and allowed to grow for 48 hours at 32°C. Thereafter, a single colony was inoculated in nutrient broth medium. The single colony was incubated at 37°C and at 230-240 rpm, for 16 to 24 hours. Finally, the culture was streaked on a nutrient agar plate to check morphology. [00324] For culturing DFM strain 300, a pure culture of DFM strain 300 was streaked on a nutrient agar plate and allowed to grow for 48 hours at 32°C. Thereafter, a single colony was inoculated in nutrient broth medium. The single colony was incubated at 37°C and at 230- 240 rpm, for 16 to 24 hours. Finally, the culture was streaked on a nutrient agar plate to check morphology.

EXAMPLE 14

Growth Performance Responses to Varying Strategies of

Utilizing DFM in Broilers

[00325] 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 7 weeks (49 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 10 replicates.

[00326] For this investigation, a total of 60 pens (6 x 6 ft, 35 ft ² /pen - subtracting 1 sq ft for feeder space) were utilized, with a group size of 35 birds per pen. The pens were equipped with a dry tube feeder (30-lb feed capacity) with a total feeder space of 50 in. (1.7 in./bird) and 5 nipple drinkers/pen (7 birds/nipple).

[00327] Approximately 2100 hatch rooster chickens (Cobb 500 genetics) were evaluated. The start weight of the chickens was about 40 grams, and the end weight of the chickens was about 3.2 kg.

[00328] In the investigation, the chickens were divided into six groups as shown in

Table 19:

Table 19. Experimental Treatment Groups

MDG.DFM denotes 80% strain 86 and 20% strain 300; MDG.DMF-v2 denotes 50% strain 86 and 50% strain 300 (Microbial Discovery Group, Franklin, WI).

[00329] Experimental procedures were as follows:

Animal care protocol: Care was provided following an approved Animal Use Protocol. Environmental conditions were monitored 3 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 3 - 60 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 30 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.

Measurements:

Live performance:

Total pen weights - start, wk 2, wk 4, wk 6, & wk 7 post-hatch.

Feed disappearance - wk 2, wk 4, wk 6, & wk 7 post-hatch.

Feed/gain ratio was adjusted for mortality by the following equation:

Total feed consumed/ (pen weight gain + mortality weight).

Caloric efficiency (kcal/lb gain): - wk 2, wk 4, wk 6, & wk 7 post-hatch. Morbidity and mortality.

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. Experimental diet formulation:

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 (80% strain 86 and 20% strain 300) 9.8 x 10 ⁸ CFU/g; MDG.DFM-v2 (50% strain 86 and 50% strain 300) 6.55 x 10 ⁸ CFU/g.

Experimental diet specifications: Four dietary phases - wk 1-2, wk 3-4, wk 5-6, wk 7. The control diet contained at least 2% supplemental fat (poultry fat), 5% DDGS, and 5% meat-and-bone meal. All diets were formulated to be adequate in SID Lys (%, NRC, 1994) and the other essential AA, available P, and Ca. The test materials were supplemented to the final diets at the expense of corn (Table 20). Diet components were mixed in a horizontal mixer.

Each diet was pelleted at 185°F following 30 s of conditioning. The diets for Wk 1-2 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 2- lb sample of Mash was obtained at the start and 5 samples (1-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 1.0 lb from each 2.0 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 > 3 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 (6) treatments and 10 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.

[00330] Dietary formulations for experimental diets are shown in Table 20. Table 20. Dietary formulations for experimental diets.

Feed

Finisher Composition Starter Grower Finisher

II

(%)

Corn 58.306 63.228 67.590 71.277

Soybean Meal 27.005 23.093 18.759 16.369

DL - Methionine 0.263 .250 .165 .159

Lysine HCL 0.265 .233 .202 .248

Fat, Blended 2.705 2.710 3.131 2.238

Limestone .675 .639 .629 .630

Biofos 16/21% .047 N/A N/A N/A

Salt .322 .239 .141 .105

Sodium

.100 .228 .369 .424 Bicarbonate

Trace Minerals .050 .050 .050 .050

Vitamins .250 .250 .250 .250

LO - DDGS 5.000 5.000 5.000 5.000

MBM 5.000 4.069 3.702 3.237

Phytase .013 .013 .012 .013

[00331] Results are shown in following Tables 21-30 based on Treatments A-F of

Table 19, summarized as below:

T T TREATMENT

A Control

B C + 7.35 x l0 ⁴ MDGDFM starter only

C C + 7.35 x l0 ⁴ MDGDFM starter and grower only

D C + 7.35 x l0 ⁴ MDGDFM continuously

E C + 5.50 x l0 ⁴ MDGDFM continuously

F C + 7.35 x l0 ⁴ MDGDFM-V2 continuously

Table 21. Body weight of male broilers fed different DFMs.

TRT DAY 0 (g) DAY 14 (g) DAY 28 (kg) DAY 42 (kg) DAY 48 (kg)

A 41.8 366.0 + 3.1 1.432 + 0.011 ^b 2.682 + 0.053 ^b 3.057 + 0.079 ^b

B 42.0 1.468 + 0.015 ³ 2.802 + 0.059 ³ 3.215 + 0.097 ^ab

C 42.0 2.749 + 0.048 ^ab 3.173 + 0.088 ^ab

D 42.1 370.2 + 2.5 1.462 + 0.010 ³ 2.840 + 0.051 ³ 3.297 + 0.072 ³

E 41.8 365.4 + 3.3 1.453 + 0.009 ^ab 2.707 + 0.042 ^b 3.075 + 0.080 ^b

F 41.8 368.9 + 4.9 1.460 + 0.013 ^ab 2.815 + 0.055 ³ 3.279 + 0.096 ³

Pooled SEM 0.1 0.002 0.005 0.022 0.033

Pooled CV 1.1 3.4 2.8 6.2 7.8

l' ^b Means with different groupings differ significantly at P < 0.05. 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.

Table 22. Feed consumed (g/bird/day) of male broilers fed different DFMs.

TRT STARTER GROWER FINISHER FINISHER II

A 30.1 ± 0.3 116.7 ± 1.6 178.0 ± 4.2 142.1 ± 7.7

B 118.4 ± 1.3 ^a 185.2 ± 5.0 ^a 151.3 ± 9.0 ^a

C 181.5 ± 3.7 ^a 146.5 ± 8.0

D 30.1 ± 0.2 118.9 ± 1.0 ^a 189.5 ± 3.4 ^a 162.4 ± 6.3 ^a

E 30.2 ± 0.3 118.6 ± 1.3 ^a 180.1 ± 3.3 139.3 ± 7.9

F 29.9 ± 0.3 120.4 ± 1.2 ^a 186.3 ± 4.2 ^a 163.6 ± 8.6 ^a

Pooled SEM 0.1 0.5 1.7 3.3

Pooled CV 3.1 3.7 6.8 12.8

l' ^b Means with different groupings differ significantly at P < 0.05.

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. Table 23. Feed consumed (g/bird/day) of male broilers fed different DFMs.

TRT TREATMENT Day 1-28 Day 1-42 Day 1-48

A Control 73.2 ±0.9 107.8 ± 1.9 111.4 ±2.5

B C + 7.35 x 10 ⁴ MDGDFM starter only 73.5 ±0.8 109.6 ±2.0 112.9 ±3.0

C C + 7.35 x 10 ⁴ MDGDFM starter and grower only 109.6 ± 1.7 110.8 ±2.7

D C + 7.35 x 10 ⁴ MDGDFM continuously 73.7 ±0.7 111.2 ± 1.6 114.9 ±2.4

E C + 5.50 x 10 ⁴ MDGDFM continuously 74.2 ±0.8 109.1 ± 1.3 111.9 ±2.2

F C + 7.35 x 10 ⁴ MDGDFM-V2 continuously 74.9 ±0.7 111.3 ± 1.8 116.7 ±2.7

Pooled SEM 0.3 0.7 1.1

Pooled CV 3.7 4.9 7.0 ^a ' ^b Means with different groupings differ significantly at P < 0.05.

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

Table 24. Feed conversion ratio of male broilers fed different DFMs.

TRT STARTER GROWER FINISHER FINISHER II

A 1.31310.012 1.53810.013 2.01010.050 ^ab 2.99810.230

B 1.54010.014 1.94910.047 ^ab 3.12110.240

C 1.96310.049 ^ab 2.86010.252

D 1.31610.008 1.54410.009 1.91110.036 ^b 2.73610.103

E 1.32010.013 1.52910.016 2.02610.033 ³ 3.10010.222

F 1.29210.015 1.55510.009 1.92710.049 ^ab 2.64310.137

Pooled SEM 0.006 0.005 0.019 0.090

Pooled CV 3.3 2.6 7.0 21.0

a' ^b Means with different groupings differ significantly at P < 0.05.

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

Inclusion of the DFM at 7.35 X 10 ⁴ in treatment D during the finisher phase significantly reduced FCR when compared to treatment E with all other treatments being intermediate. Table 25. Cumulative feed conversion ratio of male broilers fed different DFMs.

TRT TREATMENT DAY 0-28 DAY 0-42 DAY 0-48

A Control 1.480 ± 0.008 ³ 1.737 ±0.019 ^a 1.86410.017

B C + 7.35 x 10 ⁴ MDGDFM starter only 1.457 ± 0.009 ^b 1.699 ±0.021 ^ab 1.83310.016

C C + 7.35 x 10 ⁴ MDGDFM starter and grower only 1.716 ±0.022 ^ab 1.83210.018

D C + 7.35 x 10 ⁴ MDGDFM continuously 1.477 ± 0.006 ^ab 1.697 ±0.014 ^b 1.82110.014

E C + 5.50 x 10 ⁴ MDGDFM continuously 1.477 ±0.010 ^ab 1.739 ±0.015 ^a 1.86710.008

F C + 7.35 x 10 ⁴ MDGDFM-V2 continuously 1.487 ± 0.007 ³ 1.707 ±0.016 ^ab 1.82310.015

Pooled SEM 0.004 0.006 0.008

Pooled CV 2.1 2.8 3.0 ^a ' ^b Means with different groupings differ significantly at P < 0.05.

For cumulative FCR on day 0-28, 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 0-42, 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 0-48 resulted in no significant differences observed.

Table 26. Uniformity of male broilers (Coefficient of Variation) fed different DFMs.

TRT DAY 14 DAY 28 DAY 42 DAY 48

A 9.8310.52 3.5210.37 9.1110.49 10.1510.55 ^ab

B 9.2010.33 9.1910.42 9.5110.65 ^ab

C 9.2410.34 9.8710.68 ^ab

D 10.1510.20 10.2910.56 8.5410.42 9.2510.57 ^ab

E 10.6110.32 9.9610.39 8.9810.52 10.4310.71 ^a

F 9.9810.47 9.8910.38 8.4710.48 8.8310.35 ^b

Pooled SEM 0.16 0.23 0.18 0.26

Pooled CV 12.3 17.2 15.6 18.7

a' ^b Means with different groupings differ significantly at P < 0.05.

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. Table 27. Litter pH and ammonium ion concentration collected on day 44 from broilers fed different DFMs. Parameters were determined by dilution of 12 g of litter material into 60 mL of distilled water. Duplicate samples were measured for each replicate pen.

Ammonium Ion

TRT TREATMENT PH (mg/L)

A Control 8.73 + 0.08 ^ab 125.5 + 5.8

B C + 7.35 x 10 ⁴ MDGDFM starter only 8.83 + 0.08 ^ab 112.8 + 10.0

C C + 7.35 x 10 ⁴ MDGDFM starter and grower only 8.64 + 0.08 ^b 126.7 + 6.5

D C + 7.35 x 10 ⁴ MDGDFM continuously 8.78 + 0.07 ^ab 120.5 + 8.3

E C + 5.50 x 10 ⁴ MDGDFM continuously 8.82 + 0.04 ^ab 111.1 + 8.4

F C + 7.35 x 10 ⁴ MDGDFM-V2 continuously 8.86 + 0.05 ³ 113.7 + 8.0

Pooled SEM 0.16 0.23

Pooled CV 2.6 19.3

a' ^b Means with different groupings differ significantly at P < 0.05.

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. Table 28. Processing weights (g) of male broilers fed different DFMs.

TRT TREATMENT Live Wt WOG Wt Fat Pad Wt Filet Wt

A Control 3143 + 46 ^d 2485 + 35 ^d 61.9 + 2.6 615.2 + 12.8 ^b

B C + 7.35 x 10 ⁴ MDGDFM starter only 3303 + 44 ^abc 2594 + 32 ^abc 59.9 + 2.4 648.0 + 11.5 ^ab

C C + 7.35 x 10 ⁴ MDGDFM starter and grower 3289 + 39 ^bc 2572 + 31 ^bcd 62.4 + 2.3 637.9 + 12.0 ^b

D C + 7.35 x 10 ⁴ MDGDFM continuously 3418 + 44 ^a 2682 + 35 ^a 67.7 + 2.3 676.5 + 11.9 ^a

E C + 5.50 x 10 ⁴ MDGDFM continuously 3213 + 43 ^cd 2537 + 33 ^cd 64.4 + 2.4 630.8 + 11.7 ^b

F C + 7.35 x 10 ⁴ MDGDFM-V2 continuously 3378 + 47 ^ab 2650 + 36 ^ab 66.9 + 2.2 680.5 + 13.2 ^a

Pooled SEM 18 14 1.0 5.1

Pooled CV 10.4 10.2 2.8 14.5 ^a ' ^b ' ^c Means with different groupings differ significantly at P < 0.05.

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.

Table 29. Processing yield ( ) of male broilers fed different DFMs

TRT TREATMENT WOG Yield Fat Pad Yield Filet Yield

A Control 79.1110.32 2.5010.11 24.6610.24 ^b

B C + 7.35 x 10 ⁴ MDGDFM starter only 78.6310.26 2.3210.09 24.9210.22 ^ab

C C + 7.35 x 10 ⁴ MDGDFM starter and grower 78.2210.25 2.4210.08 24.7410.27 ^b

D C + 7.35 x 10 ⁴ MDGDFM continuously 78.4910.30 2.5210.08 25.1810.22 ^ab

E C + 5.50 x 10 ⁴ MDGDFM continuously 78.9810.24 2.5410.09 24.8010.23 ^b

F C + 7.35 x 10 ⁴ MDGDFM-V2 continuously 78.4710.25 2.5110.07 25.6010.26 ^a

Pooled SEM 0.11 0.04 0.10

Pooled CV 2.6 26.3 7.2 ^a ' ^b Means with different groupings differ significantly at P < 0.05.

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.

Table 30. Mortality rate ( ) of male broilers fed different DFMs.

TRT DAY 0-14 DAY 15-28 DAY 29-42 DAY 43-48 DAY 0-48

A 3.4311.40 1.2210.69 0.6010.40 0.9410.48 ^b 6.1912.02

B 0.0010.0 2.0510.62 0.6010.40 ^b 4.4911.05

C 1.8210.69 0.8910.63 ^b 7.2311.48

D 2.3410.43 1.3410.40 1.8010.79 1.5110.50 ^ab 8.2711.61

E 2.0010.63 1.1810.78 2.9811.00 1.5110.67 ^ab 7.8311.93

F 1.4010.64 2.8112.49 0.6210.41 3.0910.63 ^a 8.0412.92

Pooled SEM 0.35 0.46 0.29 0.24 0.77

a' ^b Means with different groupings differ significantly at P < 0.05.

For mortality rate of broilers fed different DFMs, no significant differences were observed on days 0-14, 15-28, 29-42, and 0-48.

For days 43-48, treatment F had a significantly higher mortality rate when compared to the control, treatment B and C with all other treatments being intermediate. [00332] 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 7.35 x 10 ⁴ 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.

EXAMPLE 15

Growth Performance Effects of a Direct-Fed Microbial (86/300)

in the Diets of Broiler Chickens

[00333] The object of the instant example was to compare the performance in a 35 day floor pen trial broilers fed with and without 86/300 DFM (80% strain 86 and 20% strain 300) 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.

[00334] The feed supplements tested were as follows: 86/300 DFM test material with

80% strain 86 and 20% strain 300 DFM strains was provided by Microbial Discovery Group (Franklin, WI). BMD (bacitracin methylene disalicylate). VM (virginiamycin).

[00335] For this investigation, a total of 56 pens were utilized, with an initial group size of 30 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 1 water fountain and a 75 lb. capacity feed tube. The dimensions of the pens were 4' X 5' which provided a stocking density of 0.67 ft per bird when there were 30 birds per pen. Each pen had a polymer slatted floor covered with a 3 mil thick plastic sheet topped with approximately 3 inches of new wood shavings at Day 0. On Day 7, each pen was top dressed with 4 pounds of previously used broiler from a flock having no disease issues or exposure to direct fed microbials. Continuous lighting was provided.

[00336] All pens were checked at least daily during the study. Observations included availability of feed and water, and brooder control for maintaining desired temperatures.

[00337] Approximately 2520 hatch straight-run broiler chickens (Cobb 500 genetics) were evaluated. The initial age of the chickens at Day 0 was one day.

[00338] Diets: A 2 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. [00339] Study Design: There were 4 treatment groups with each group replicated 14 times for a total of 56 pens (30 chicks/pen at placement) as shown in Table 31. Salinomycin 60 g/ton was present in all starter and grower feeds.

Table 31. Study Design

1. BMD in Starter/VM in Grower - Positive control with BMD (50 g/ton d 1-28) and virginiamycin (20 g/ton d 29-35).

2. nCON - Negative control w/o feed-grade antibiotic addition.

3. 86/300 Continuously - Same as Group 2 + 7.35 x 10 ⁴ CFU/g 86/300 DFM continuously.

4. 86/300 Starter Only - Same as Group 2 + 7.35 x 10 ⁴ CFU/g 86/300 DFM in starter (d 1- -14) only.

[00340] Animal Placement: On the first day of the trial, 10 stacks of 3 hatchery crates containing at least 100 chicks in each crate were set on a row of tables. Thirty (30) chicks from each of the 10 stacks were placed into a transport crate, those 30 chicks were group weighed and then placed into the pen 1. This was repeated for all 56 pens. Remaining chicks were used to replace any unthrifty or lame chicks found during days 1 through 7.

[00341] Data Collected: Pen body weights and feed weights (for feed conversions) at

Days 0, 14, 28 and 35. At Day 35 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 35 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 10, Analytical Software, Tallahassee, FL).

[00342] Live Bird Weights: All birds were weighed by pen at Days 14 (by pen), 28 (by pen), and 35 (by gender, by pen) as shown in Table 32. Table 32. Live Bird Weights (lb)

NOTE: Column data having different superscripts are different (p<0.05). 1. Individual D35 body weights by pen and gender for a pen within a treatment group were used for statistical analysis.

DAY 14 Live Weight/Bird (lb) was heavier (p<0.05) for BMD/VM, and 86/300 in starter or continuously, compared to nCON. The 86/300 in starter only gave heavier (p<0.05) weight than the BMD/VM diets. The treatments containing 86/300 (starter or continuously) gave highest body weights and were in the same statistical grouping.

DAY 28 Live Weight/Bird (lb): The 86/300 in starter only gave heavier (p<0.05) weights than nCON diets.

DAY 35 Live Weight/Bird (lb) averages for males and females combined were heavier (p<0.05) for BMD/VM and 86/300 in starter only compared to nCON treatment, with 86/300 continuously treatment being intermediate. This same statistical pattern was evident for the male body weights. For females, diets with BMD/VM, 86/300 in starter only or continuously gave heavier (p<0.05) broilers than nCON diets.

[00343] Feed Conversion Ratio: Feed conversion and Feed Conversion Adjusted for

Mortality (Table 33) were calculated using the following equations:

Day 0-14, 0-28, or 0-35 Feed Conversion (Unadjusted)

= Total Pen Feed Intake ÷ Total Pen Live Weight Day 0-35 Feed Conversion (Adjusted for Mortality)

= Total Pen Feed Intake ÷ (Total Pen Live Weight + Mortality Weight)

Table 33. Feed Conversion Ratios

80/300 Starter Only 1,153 1.812 1.737 1.709

NOTE: Column data having different superscripts are different (p<0.05).

DAY 0-14 Feed Conversion Ratio (unadjusted) of 86/300 starter only or continuously was lower (p<0.05) than nCON, with BMD/VM intermediate.

DAY 0-28 Feed Conversion Ratio (unadjusted) results were not significantly different (p>0.05).

DAY 0-35 Mortality Adjusted Feed Conversion Ratios followed the same statistical pattern as described for the unadjusted Feed Conversion Ratios.

[00344] Mortality (%): Mortality (%) results by treatment group are shown in Table 34. Table 34. Mortality (%)

D2B 035

TREATMENT DO ^» 1 mm a A ta Start«r VM kt Grower 4,20% a a

f CON 5,48% 738% a A

80 300 Continuously SJ1% a A

O6 00 Start r nly 0.-00% 4,29% 5,24%

NOTE: Column data having different superscripts are different (p<0.05).

DAY 0-14 Mortality (%) was not significantly different (p>0.05) between treatment groups. No mortality was observed in any treatments during the first 2 weeks.

DAY 0-28 Mortality (%) was not significantly different (p>0.05) between treatment groups. Overall average mortality % from 0-28 days was 4.73%.

DAY 0-35 Mortality (%) was not significantly different (p>0.05) between treatment groups. Overall average mortality % from 0-28 days was 5.95%.

[00345] 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 35 and Table 36, which shows combined average weights and per cent yields.

[00346] 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 86/300 Starter Only group) while all other broilers' breasts were found to be without blemishes. Table 35. Carcass and Breast Yields ( Live Weight)

NOTE: Column data having different superscripts are different (p<0.05).

Table 36. Combined Average Carcass and Breast Yields

Carcass Yield ( Live Weight) was significantly (p<0.05) 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>0.05).

Breast Yields ( Live Weight) average for combined sexes was significantly greater (p<0.05) for birds in the 86/300 continuously treatment group than for nCON birds, with other groups intermediate. By individual sexes, no significant differences (p>0.05) were detected in breast yield.

In conclusion for the experiments in this 35-day pen trial with 4 treatments and replicate pens (with 30 chicks/pens), the DFM supplemented groups 86/300 in starter only or continuously gave final body weights statistically equivalent to BMD/VM and greater (p<0.05) than nCON group. Therefore, BMD/VM or 86/300 (either in starter or all phases) broilers had the most improved 35-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≤0.05) 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<0.05) for DFM product 86/300 continuously than for nCON, with other treatment results being intermediate.

Example 15, Appendix A: Feed Formulas

Broiler Starter (0-21 Days)

11QGEJBD1E PERCEN WEIGHT CO: ST

Cors, Yel ? .5% 59.555 1191.77 104 .29

Soy Meal 4? .5% 26.250 525,00 600 .00

Meat&Bone 50% 5,000 100.00 550 .00 .00 00

Corn D0?S 261 5,000 100, 0 2 3 .00 5 ,00 5, 00

Soyfo¾an Oil 1.895 37, 90 703 .00

Li.s-estone Fine 0.55 13,07 SO .86

Die¾l 21/1 S.5% 0,328 6, 56 685 .00

Salt 0.249 4.98 1 5 .03 0. 50

Choline Ci €0% 0.080 1.60 1240 .00 0 .08 0. 08

YDRp Yit temix 0.040 0.00 3730 ,00 0 ,04 0, 04

Sodium Bicar 0.2 SO 5.00 320 .00 0 .25 0. 25

YD -p TraMi.fi P.x 0.085 1-70 1.750 .00 0 .03 0. 5

L~ Lysine HCi 0.245 4.91 2220 .30 0 ,

DL-Heth 99% 0.264 5.28 3354 .00 0. 35

L~ a:eord.ne90% 0.054 1,07 2340 .00 0. 10

0.018 0.36 1530 .03 0 ,02 0, 02

TOTALS 1 0.000 2000. 0 334 .93 10 .47

O NUTRIENT OMITS MIN ACT

1 WEIGHT LEG 100. 000 2000.000* 100.080

CR PROTEIN PCI 21 , 300 21.300*

3 CR FAT PCT 5, 397

4 CR FIBER PCT 2 ,938

6 POCLKE ECAL/LB 1400, 000 1398,982*

7 DIG RGIB PCT 1, 202 1,203

8 PIG LYSINE PCI 1 < 160 1.180*

a DIG ME7H PCT 0, 511 0.578

10 BIG E TOYS PCT 0. 861 0,862*

11 DIG NISTIP PCI 0.50?

12 BIG LE ^' OCI PCT 1, 233 1.633

13 RIG I LEU PCI 0, 800 0.326

14 BIG TBRSON PCT 0, 756 0.752 ^s

15 BIG VALINE PCT 0, 228 0.048

16 AESIN1NE PCI 1.422

1? LOOS IRE PCT L 303

IS METHIONINE PCT 0.609

19 METROES PCI 0.961

20 TRYPTOPHAN PCT 0.241

21 GLYCINE PCT 1.1.24

HI SUPINE PCI 0.542

23 LEUCINE PCT 1.875

24 ISOLEDCTNE PCT 0.210

25 PHENYI2YL22N PCT 1.015

26 PHE8TYR PCI 1,730

27 THREONINE PCI 0.8/0

23 vRLINS PCT 1.004

29 GLY&SER PCI 2.212

3 ASH PCT 4.838

32 CALCIUM OT , 850 0.350* 0.850

33 PROS- OTAL PCT 0,631

3 BROS~A iL PCT 0. 420 0.420·* 0.420

35 POTASSIUM CI 0.818

36 SODIP3M PCT 0, 230 0.230* 0,230

37 CHLORIPE OT » 230 0.306

3S MA ESIU PCT 0.168

32 IRON PPM 160.152

0 SING PPM 97.533

41 MA GAR EG E PPM 107.421

42 COPPER PPM 1.4,534

43 ADD Els S« PPM 0, 300 44 100IME PPM 1, 009

45 LXNOLEXC PCX 665

46 XSMTBO MG/LB 4, 005

47 SOLFUR FCT 0. 265

48 vIT ¾ KIG/LB , 000

4 VIT £ IIF/LB 16. 800

50 VIT B12 MGG/LB 000

51 RIBOFLAV MG/LB 3. 600

.52 P¾ETO AG MG/LB 70 200

53 MIACIK MG/LB 82. 000

54 CHOLINE MG/LB 712. 033

55 FOLIC AO MG/LB 2, 830

56 PYBIDOX!f MG/LB 1.. 380

5? THIAMINE MG/LB 1. 280

58 BIOTI^ MG/LB 0» 800

55 VI D3 TIO/LB 1. 800

60 MEHADION MG/LB 0. 800

81 BIG TRYPT POT 304 0. 206

82 DIG CBFRO FCT 18, 420

Broiler Grower (21-35 Days)

INGREDIEN PERL :SHC WEIGH COS H r a

Corn, Ysi 7.5% 63. ,372 1267 .44 164.29

Soy Mes.1 47,9% ^• 0 .036 440 .75 600.60

Meat&Bone 50% 5, .0 1 0 .00 550.00 . v U

Cor DOGS 26% 5 , .000 100 .00 243,00 U3 3 Π

Soybean Oil .5S3 51 .65 780.00

Limesstone Fine 0, .506 11 ,76 50.06

Dicai 21/10.5% 0. .251 5 .03 685.00

S lt 0. ,22 4 , 47 1.45.00 o . 50

Choline Cl 60% 0. _f 080 1 , 60 1240.00 0 , 00 0. 08

VDRp Vit?remix 0. ,040 0 , 0 3700,00 Q ,04 0 04

SodiusS Bi arb 0. ,250 5 > 00 320.00 0 ,25 0. 25

VDRp ra ii: lx 0. .085 I . ?0 1750.00 6 , 00 0, 3

L-Lysi e HCi 0. .216 4 .33 .2220.0 0. ■··;■

DL- «t¾ 02% 0. .22? 4 .53 3354.00 0. 35

L--- hreonin 90% 0 , .026 0 .57 2340.00 0. 10

Konosy-¾e?5000 0, .016 0 .36 1500.00 0 .02 0. 02

TOTJ1LS 100, .000 2000 .00 317.05 0 .47 Ki800RIE6fT UNIT;? MIM ACT MAX

1 WEIGHT LBS 100. 000 2000, 000*- 108.000

CR PPOTE1U PCT 18. 500 1.9.500*

3 CP FAT POT 6,184

4 CR FIBER PCT 2.858

€ POULME RCAL/LB 1435. 000 14340883 ^S

? DIG ARGIN FGT i. 110 1.147

8 DIG LYSINE PCT I . 032 1,032*

8 DIG METE PCT 0 > 498 CO 521

0 DIG ETCYS PCT 0 , 785 8.785*

1 DIG HIS ID PCT CO 466

2 DIG LEGC1N PCT ^' I 104 1,5.24

3 DIG 13CLSU PCT 0 > 732 CO 744

4 DIG THRBOH PCT 0. 678 0, 8 0*

5 DIG VALINE POT 8. 826 0.866

6 A imm PCT 1,288

7 LYSINE PCT 1.168

8 E EIOBIHE PCT 0.549

8 METSCYS PCT 0.575

0 TRYPTOPHAN PCT 0,216

1 GLYCIME PCT 1,0 5

KISTIDIBE PCT 0.503

3 LEUCINE PCT 1.754

4 ISOLEOCIHE PCT 0.827

5 PHEHYLALAN PCT 0.329

6 .PHE&TYB PCT 1.581

THREOEI E PCT 0.775

8 VALINE PCT 0.874

8 GL SSER ^¬ PCT 2.039

0 ASE PCT 4.501

CALCIUM PCT 0. .800 0.860*

3 FHCC-TOTAL PCT 0.599

PBOS-AVAIL PCT 0 < .400 0 ,480* 0 4085 POTASSIUM PCT 0.747

6 SODIUM PCT 0. 0.220* 0 .

CHLORIDE P 2 0.220 0.281

MAGN SIU P 2 0.162

IRO RRM 145.333

3INO RRM 85,917

MANGA1ESE RRM 105.605

COPPER PPM 13..785

ADDED $¾ PPM 0.300

IDDIME PPM 1.704

2INODEIC ROT 3.068

XANTHO MG/LB 5.090

SO ^' LEOR ROT 0.242

7IT A K.XU/LB 4.080

VIT E 2/LB X€, 000

PIT 812 MCG/LB 6.080

RIBOFLAY MG/LB 3.660

PANTO AO MG/LB 7.200

OXACID ^" MG/LB 32.000

CHOLINE MG/LB 668 , 266

FOLIO AC MG/LB 0.880

PYRTDOXM MG/LB 1,880

THI MIME MG/LB 1,200

BIO IM MG/LB 0..088

VIT D3 KID/LB 1.806

ME ADIOH MG/LB 8.800

DIG TRYFT PC2 y , i ; 6 0.1 S3

DIG CRPROT POT 16 855

EXAMPLE 16

Enzymatic Activity Screening

[00348] To confirm the presence of galactomannan-degrading enzymes in DFM strains via the dinitro salicylic acid (DNS) reducing sugar assay, this example describes the use of plate media screening methods to detect enzymatic activity in DFM strains 86, 101, 290, and 300 and other related isolates.

[00349] The DNS reducing sugar assay was performed as in the manufacturer's protocol, inoculating the 0.5% guar gum solution with overnight cultures of MDG strains 86,101, 290, and 300 grown in TSB + 0.5% guar gum. The reducing sugar assay was performed with samples taken after 30 minutes and 18 hours of incubation at 37°C. Standard curves were prepared with solutions of D-mannose and D-galactose at concentrations between 1M and ΙμΜ to determine the relation between free reducing sugar reacted and OD 575. Negative control samples (NC) consisted of guar gum substrate inoculated with buffer only or with sterile media (TSB+guar gum).

[00350] As shown in Fig. 13, Bacillus strains 86,101, 290, and 300 showed mannanase activity in the range of about 0.467 mmol to about 0.720 mmol in 24 hours.

EXAMPLE 17

Substrate Testing Summary

[00351] In the instant example, a comparison of DFM strains was performed to determine the variations in substrate utilization. DFM strains 86, 101, 290, and 300 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 1/strain 2 as follows: 86/101; 86/290; 86/300; 101/290; 101/300, and 290/300. Three-strain combinations included, but were not limited to, a combination of strain 1/strain 2/strain 3 as follows: 86/101/290; 101/290/300; 86/290/300; and 86/101/300. A combination of all four strains (i.e.,

86/101/290/200) was also tested. A negative control that included no Bacillus was also assessed with the strains.

[00352] 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.

[00353] In performing the substrate utilization assay, each microtiter plate well contained 2 μΐ of linoleic acid and 200 μΐ of minimal medium inoculated with a respective Bacillus strain or strain combination. The substrate utilization test was run at 37°C for 4 days with medium shaking. As shown in Figure 14, The most effective strains or strain

combinations for using linoleic acid as the only carbon source were: 86/300; 86/101/290;

86/101/300; 101/290; 101/290/300; 101/300; and 300.

EXAMPLE 18

Genomic Analysis of DFM Strains 86 and 300

[00354] In order to obtain genomic "fingerprints" of Bacillus strains 86 and 300 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.

[00355] Figs. 15 and 16 show genomic annotation of DFM strains 86 and 300, respectively. As shown in Fig. 15, strain 86 comprises approximately 4,055,712 bp (i.e., about 4056 kb) of genomic sequence. DFM strain 300 was slightly smaller comprising about 3,957,082 bp (i.e., about 3957 kb) of genomic sequence. Therefore, Figs. 15 and 16

demonstrate that DFM Strains 86 and 300 are different strains, each having a unique DNA fingerprint.

[00356] In addition, sequencing of DFM strains 86 and 300 identified many different genes within the strains. Genes identified in strains 86 and 300 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 86 and 300 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.

Example 19

Analysis of Growth Performance of Nursery Pigs fed Different Direct-Fed Microbial

Combinations.

[00357] This multi-experiment test was conducted to determine the growth performance of nursery pigs fed different direct-fed microbial diet combinations. One Experiment (Experiment 1 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 38.

[00358] Experiment 1:

1. Genetics: PIC 337 x C29

2. Study Animal Start Weight: 13.9 + 2.3 lb

3. Replicates: 12 replicates of 23-26 mixed-sex pigs/pen (6.7 ft /pig stocking density)

4. Feeder: 3-space dry box feeder and 2 swinging water nipples

5. Four (4) treatments

i. Non-DFM control

ii. As 1 + 0.75 lb/ton Bs 86/300 combo (80/20%) (5.5 x 10 ⁴ CFU/g) iii. As 1 + 1 lb/ton Bs 86/300 combo (80/20%) (7.35 x 10 ⁴ CFU/g) iv. As 1 + 1 lb/ton Bs 86/300 combo (50/50%) (7.35 x 10 ⁴ CFU/g)

6. Corn-SBM-DDGS experimental diets fed from weaning to 6 wk postweaning (Table 37)

[00359] Experimental Procedures:

[00360] 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.

[00361] 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 3 consecutive days and not showing signs of improvement was immediately removed from the experiment.

[00362] 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.

[00363] 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. [00364] Statistical Procedures: Each experiment was statistically analyzed individually.

Initial data analysis was performed for all metrics to determine normality of distribution and outliers (+ > 3 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.

[00365] Results:

[00366] Experiment 1 RESULTS (see Table 38):

1. Final body weights and growth rates were increased (P < 0.05) with the supplementation of MDG Bs 86/300, with the 50/50 proportion effecting the greatest response (P < 0.01; 8% compared to 3% for the 80/20 proportion).

2. Feed/gain ratios were higher (Diet, P = 0.34; 1 lb/ton vs. 0.75 lb/ton, P < 0.05) in the group fed the lower dose of 86/300 by 3.4%.

[00367] Conclusions: MDG Bacillus strains 86 & 300 supplemented to diets at 1 lb/ton increased ADG and reduced F/G.

Table 37. Animal Diets for Experiment #1.

Ingredient Phase 1 Phase 2 Phase 3 Phase 4

Corn to 2,000 to 2,000 to 2,000 to 2,000

Soybean meal 332.64 498.09 524.96 380.12

Flex Start 935.00 450.00 200.00

Flex Start Add Pak 110.00 25.00

DDGS 150.00 300.00 450.00

R&D HD190 99.76 151.23 190.00

Choice white grease 50.00

DFM +/- +/- +/-

Total 2,000 2,000 2,000 2,000

Budget (wks fed) 1 2 3 4-6

Nutrient composition

ME, kcal/lb 1,475 1,437 1,418 1,461

Crude protein, % 21.67 23.15 24.19 22.44

Crude fat, % 3.45 3.26 3.26 5.83

NDF, % 4.72 7.39 7.39 11.07

P, bioavailable, % 0.56 0.48 0.46 0.44

Ca, % 0.88 0.80 0.75 0.68

Na, % 0.54 0.40 0.36 0.35

Lysine

Total, % 1.49 1.51 1.53 1.37

SID, % 1.34 1.34 1.34 1.19

SID, g/Mcal ME 4.12 4.23 4.29 3.70

AA/Lys ratios, SID, %

SAA 55 57 59 64

Thr 64 69 73 80

Trp 19 19 19 18 lie 54 60 63 62

Val 72 72 73 75

Table 38. Growth performance responses of nursery pigs to dietary supplementation of experimental Bacillus subtilis strain combinations at different concentrations and strain proportions Experiment #l \

Body weights (lbs) ADG ADFI F/G Feed cost ⁴ Margin ⁴

Dietary treatment ² ' ³ d 1 d 41 (lbs) (lbs) (lb/lb) (S/lb gain) (S/pig)

1. Non-DFM control 13.9 44.7 0.74 1.08 1.45 0.246 28.17

2. As 1 + 1 lb/ton 86/300 (80/20) 13.9 45.2 0.76 1.09 1.44 0.245 28.41

3. As 1 + 0.75 lb/ton 86/300 (80/20) 13.9 45.1 0.76 1.14 1.50 0.260 27.61

4. As 1 + 1 lb/ton 86/300 (50/50) 13.9 46.7 0.80 1.15 1.45 0.247 29.02

Pooled SEM 0.7 1.9 0.04 0.06 0.03 0.004 1.14

Levels of significance

Dietary treatment 0.520 0.028 0.024 0.038 0.337 0.019 0.029

86/300 (80/20) dose (trt 2 vs 3) 0.615 0.946 0.783 0.055 0.025 0.001 0.032

86/300 strain proportion (trt 2 vs 4) 0.336 0.011 0.008 0.022 0.668 0.602 0.097

Data are means of 11 replicates of 23-26 mixed-sex pigs per pen fed experimental diets starting at d 1 post-weaning and continuing to the end of the experiment.

Experimental diets were based on corn & SBM, and included DDGS at 0, 7.5, 15, & 22.5% in Phases 1-4, respectively. The DFM materials were added to the experimental diets at the expense of corn.

The experiment was designed with a treatment where the pigs were fed 1 lb/ton of 86/300 (80/20 proportion) during the nursery period. In this table, the treatments fed the common diets have been combined, resulting in Trt 1 & 2 made up of 22 pens each, and Trt 3 & 4 made up of 11 pens each.

⁴ Feed costs were calculated using common corn, soybean meal, and DDGS costs, and a cost of $3.75/treated ton for 86/300 DFM. Margin over feed was calculated as the difference between pig value ($0.80/lb live weight) and feed cost ($/pig).