Microbial strains and antibiotics

Abstract

The present invention relates to direct-fed microorganisms for use in combination with an antibiotic selected from the group consisting of ceftiofur, oxytetracycline, tolamectin, and lincomycin for the inhibition of E. coli in animals. More particularly, the present invention relates to isolated Bacillus strains 2, 3, 4, 5, 9, 57, 71, and 126, and strains having all of the identifying characteristics of these strains, for use in the uses described above.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 63 / 068,668, filed on August 21, 2020, the disclosure of which is expressly incorporated herein by reference. Technical Field

[0003] The present invention relates to direct-fed microorganisms for use in combination with an antibiotic selected from the group consisting of ceftiofur, oxytetracycline, tulathromycin, and lincomycin for the inhibition of Escherichia coli (E. coli) in animals. More particularly, the present invention relates to isolated Bacillus strains 2, 3, 4, 5, 9, 57, 71, and 126, and strains having all of the identifying characteristics of these strains, for use in combination with an antibiotic selected from the group consisting of ceftiofur, oxytetracycline, tulathromycin, and lincomycin.

[0004] Background and Overview of the Invention

[0005] The present invention relates to direct-fed microbial (DFM) compositions and methods for E. coli inhibition in animals. The gastrointestinal tract of animals is constantly under attack by a large number of bacteria, viruses, fungi, and protozoa found in feed, bedding, and the environment. The gastrointestinal tract has a complex system of physical, chemical, and immune defenses against these potential pathogens. Beneficial bacteria are an important part of this system because they provide bacteria that help establish (or re-establish) a normal bacterial profile for the animal, they strengthen the animal's immune system, and they help fight disease.

[0006] Antibiotic resistance is an increasingly important issue in human medicine and the livestock industry. The U.S. Food and Drug Administration reported that in 2016, the U.S. swine industry purchased approximately 6.9 million pounds of medically important antibiotics, highlighting the importance of preventing the development of antibiotic-resistant organisms in the livestock industry. While methods are being developed to combat antibiotic resistance, little has been done to utilize synergistic direct-fed microorganisms, such as probiotics, as a tool to combat antibiotic resistance.

[0007] Applicants have developed direct-fed microorganisms that, in combination with an antibiotic selected from the group consisting of ceftiofur, oxytetracycline, tolamectin, and lincomycin, result in E. coli inhibition in animals. The direct-fed microorganisms and antibiotics, and compositions comprising the direct-fed microorganisms and antibiotics, described herein, provide commercial benefits by providing E. coli inhibition in animals, such as agricultural animals.

[0008] Provided are methods for inhibiting Escherichia coli in animals. In various embodiments, the animal can be selected from poultry species, porcine species, goat species, bovine species, ovine species, equine species, and companion animals. In embodiments where the animal is a poultry species, the poultry species can be selected from broilers, chickens, laying hens, breeders, turkeys, turkey chicks, goslings, ducklings, guinea fowl, pullets, hens, roosters, cockerels, and capons. In embodiments where the animal is a porcine species, the porcine species can be selected from growing and finishing pigs, nursery pigs, sows, and breeding pigs.

[0009] In one embodiment, a method of feeding an animal is provided. The method comprises the steps 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 10 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 11 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 12 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 13 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 14 (NRRL No. B-67714), a strain having all the identifying characteristics of Bacillus strain 15 (NRRL No. B-67715), a strain having all the identifying characteristics of Bacillus strain 16 (NRRL No. B-67716), a strain having all the identifying characteristics of Bacillus strain Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to an animal, wherein the Bacillus strain and the antibiotic cause E. coli inhibition in the animal, and wherein the antibiotic is selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0010] In another embodiment, a method of controlling the deleterious effects of E. coli is provided. The method comprises the steps 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 10 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 11 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 12 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 13 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 14 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 15 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 16 (NRRL No. B-6786 Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering antibiotics to animals and controlling the deleterious effects of Escherichia coli in the animals, and wherein the antibiotic is selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0011] In yet another embodiment, a method of feeding an animal is provided. The method comprises the steps 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 10 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 11 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 12 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 13 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 14 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 15 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 16 (NRRL No. B-6786 Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to the animal, wherein the antibiotic is selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[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 sections and examples entitled "Detailed Description of Illustrative Embodiments") are applicable to any of the following embodiments of the invention described in the following numbered clauses.

[0013] 1. A method of feeding an animal, the method comprising the steps 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 10 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 11 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 12 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 13 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 14 (NRRL No. B-67714), a strain having all the identifying characteristics of Bacillus strain 15 (NRRL No. B-67715), a strain having all the identifying characteristics of Bacillus strain 16 (NRRL No. B-67717), a strain having all the identifying characteristics of Bacillus strain 17 (NRRL No. B-67718), a strain having all the identifying characteristics of Bac Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to an animal, wherein the Bacillus strain and the antibiotic cause E. coli inhibition in the animal, and wherein the antibiotic is selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0014] 2. The method of clause 1, wherein the animal is selected from the group consisting of poultry species, porcine species, caprine species, bovine species, ovine species, equine species, and companion animals.

[0015] 3. The method of any one of clauses 1 to 2, wherein the E. coli inhibition prevents E. coli disease in animals.

[0016] 4. The method of any one of clauses 1 to 2, wherein the E. coli inhibition reduces E. coli disease in the animal.

[0017] 5. The method of any one of clauses 1 to 4, wherein the animal is a porcine species, and the porcine species is selected from the group consisting of grower-finisher pigs, nursery pigs, sows, and breeding pigs.

[0018] 6. The method of any one of clauses 1 to 5, wherein the Bacillus strain produces an enzyme selected from the group consisting of α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0019] 7. The method of any one of clauses 1 to 6, further comprising the step of administering to the animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, and combinations thereof.

[0020] 8. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 2 (NRRL No. B-67709).

[0021] 9. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 3 (NRRL No. B-67710).

[0022] 10. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 4 (NRRL No. B-67711).

[0023] 11. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 5 (NRRL No. B-67712).

[0024] 12. The method of any one of clauses 1 to 11, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 5.0×10 12 A dose of CFU / gram of feed composition is administered in the feed composition.

[0025] 13. The method of any one of clauses 1 to 11, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 1.0×10 7 A dose of CFU / gram of feed composition is administered in the feed composition.

[0026] 14. The method of any one of clauses 1 to 11, wherein the Bacillus strain is present at a concentration greater than about 7.0×10 4 A dose of CFU / gram of feed composition is administered in the feed composition.

[0027] 15. The method of any one of clauses 1 to 14, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0028] 16. The method of any one of clauses 1 to 15, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0029] 17. The method of any one of clauses 1 to 15, wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0030] 18. The method of any one of clauses 1 to 17, wherein the feed composition is administered to the animal daily.

[0031] 19. The method of clause 1, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0032] 20. A method for controlling the deleterious effects of Escherichia coli, 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67714), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67715), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67716), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67717), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67718), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to an animal and controlling the deleterious effects of Escherichia coli in the animal, wherein the antibiotic is selected from the group consisting of ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0033] 21. The method of clause 20, wherein the animal is selected from the group consisting of poultry species, porcine species, caprine species, bovine species, ovine species, equine species, and companion animals.

[0034] 22. The method of any one of clauses 20 to 21, wherein controlling the deleterious effects of E. coli is suppressing E. coli disease in animals.

[0035] 23. The method of any one of clauses 20 to 21, wherein controlling the deleterious effects of E. coli is reducing E. coli disease in animals.

[0036] 24. The method of any one of clauses 20 to 23, wherein the animal is a porcine species, and the porcine species is selected from the group consisting of grower-finisher pigs, nursery pigs, sows, and breeding pigs.

[0037] 25. The method of any one of clauses 20 to 24, wherein the Bacillus strain produces an enzyme selected from the group consisting of α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0038] 26. The method of any one of clauses 20 to 25, further comprising the step of administering to the animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, and combinations thereof.

[0039] 27. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 2 (NRRL No. B-67709).

[0040] 28. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 3 (NRRL No. B-67710).

[0041] 29. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 4 (NRRL No. B-67711).

[0042] 30. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 5 (NRRL No. B-67712).

[0043] 31. The method of any one of clauses 20 to 30, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 5.0×10 12 A dose of CFU / gram of feed composition is administered in the feed composition.

[0044] 32. The method of any one of clauses 20 to 30, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 1.0×10 7 A dose of CFU / gram of feed composition is administered in the feed composition.

[0045] 33. The method of any one of clauses 20 to 30, wherein the Bacillus strain is present at a rate greater than about 7.0×104 A dose of CFU / gram of feed composition is administered in the feed composition.

[0046] 34. The method of any one of clauses 20 to 33, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0047] 35. The method of any one of clauses 20 to 34, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0048] 36. The method of any one of clauses 20 to 34, wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0049] 37. The method of any one of clauses 20 to 36, wherein the feed composition is administered to the animal daily.

[0050] 38. The method of clause 20, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0051] 39. A method of feeding an animal, the method comprising the steps 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a Bacillus strain 9 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-6771 ... No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to the animal, wherein the antibiotic is selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0052] 40. The method of clause 39, wherein the animal is selected from the group consisting of poultry species, porcine species, caprine species, bovine species, ovine species, equine species, and companion animals.

[0053] 41. The method of any one of clauses 39 to 40, wherein the animal is a porcine species, and the porcine species is selected from the group consisting of grower-finisher pigs, nursery pigs, sows, and breeding pigs.

[0054] 42. The method of any one of clauses 39 to 41, wherein the Bacillus strain produces an enzyme selected from the group consisting of α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0055] 43. The method of any one of clauses 39 to 42, further comprising the step of administering to the animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, and combinations thereof.

[0056] 44. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 2 (NRRL No. B-67709).

[0057] 45. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 3 (NRRL No. B-67710).

[0058] 46. ​​The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 4 (NRRL No. B-67711).

[0059] 47. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 5 (NRRL No. B-67712).

[0060] 48. The method of any one of clauses 39 to 47, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 5.0×10 12 A dose of CFU / gram of feed composition is administered in the feed composition.

[0061] 49. The method of any one of clauses 39 to 47, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 1.0×10 7 A dose of CFU / gram of feed composition is administered in the feed composition.

[0062] 50. The method of any one of clauses 39 to 47, wherein the Bacillus strain is present at a concentration greater than about 7.0×10 4 A dose of CFU / gram of feed composition is administered in the feed composition.

[0063] 51. The method of any one of clauses 39 to 50, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0064] 52. The method of any one of clauses 39 to 51 wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0065] 53. The method of any one of clauses 39 to 51 wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0066] 54. The method of any one of clauses 39 to 53, wherein the feed composition is administered to the animal daily.

[0067] 55. The method of clause 39, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0068] 56. The method of any one of clauses 1 to 55, wherein the Bacillus strain has an effect selected from the group consisting of maintaining microbial balance in the intestinal tract of an animal, improving animal performance or health, maintaining intestinal health of an animal, reducing harmful pathogens in the intestinal tract of an animal, reducing odor, reducing harmful pathogens in the urine or feces of an animal, and maintaining the growth of beneficial bacteria in the intestinal tract of an animal.

[0069] 57. The method of any one of clauses 1 to 7, 12 to 26, 31 to 43, or 48 to 56, wherein the strain applied is Bacillus strain 9 (NRRL No. B-67866).

[0070] 58. The method of any one of clauses 1 to 7, 12 to 26, 31 to 43, or 48 to 56, wherein the strain applied is Bacillus strain 57 (NRRL No. B-67870).

[0071] 59. The method of any one of clauses 1 to 7, 12 to 26, 31 to 43, or 48 to 56, wherein the strain applied is Bacillus strain 71 (NRRL No. B-67867).

[0072] 60. The method of any one of clauses 1 to 7, 12 to 26, 31 to 43, or 48 to 56, wherein the strain applied is Bacillus strain 126 (NRRL No. B-67868). BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1A -B shows the percent inhibition values ​​from 3 of the 12 E. coli strains that showed improved inhibitory responses when the Bacillus supernatant and ceftiofur were combined.

[0075] Figure 2A -B shows the percent inhibition values ​​from 3 of the 12 E. coli strains that showed improved inhibitory responses when Bacillus supernatant and oxytetracycline were combined.

[0076] Figure 3A -B shows the percent inhibition values ​​from 3 of the 8 E. coli strains that showed improved inhibitory responses when Bacillus supernatant and tolamectin were combined.

[0077] Figure 4A -B shows the percent inhibition values ​​from 3 of the 12 E. coli strains that showed improved inhibitory responses when Bacillus supernatant and lincomycin were combined.

[0078] Figure 5 Figure 2 is a heat map of the inhibition percentage values ​​from all 12 lincomix-resistant E. coli strains, showing effective inhibition when eight Bacillus supernatants were used instead of lincomycin.

[0079] Figure 6 The photographs show RAPD PCR patterns of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 6 (NRRL No. B-67714), and Bacillus strain 7 (NRRL No. B-67713).

[0080] Figure 7 These are photographs showing RAPD PCR patterns of Bacillus strain 9 (NRRL No. B-67866) and Bacillus strain 57 (NRRL No. B-67870).

[0081] Figure 8 These are photographs showing RAPD PCR patterns of Bacillus strain 71 (NRRL No. B-67867) and Bacillus strain 126 (NRRL No. B-67868).

[0082] Detailed Description of Illustrative Embodiments

[0083] Methods for inhibiting Escherichia coli in animals are provided. In various embodiments, the animal can be selected from poultry species, porcine species, goat species, bovine species, ovine species, equine species, and companion animals. In embodiments where the animal is a poultry species, the poultry species can be selected from broilers, chickens, laying hens, breeders, turkeys, turkey chicks, goslings, ducklings, guinea fowl, pullets, hens, roosters, cockerels, and capons. In embodiments where the animal is a porcine species, the porcine species can be selected from growing and finishing pigs, nursery pigs, sows, and breeding pigs.

[0084] In one embodiment, a method of feeding an animal is provided. The method comprises the steps 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67861 ... coli in the animals, and administering an antibiotic to an animal, wherein the Bacillus strain and the antibiotic cause E. coli inhibition in the animal.

[0085] In another embodiment, a method of controlling the deleterious effects of E. coli is provided. The method comprises the steps 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 10 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 11 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 12 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 13 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 14 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 15 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 16 (NRRL No. B-6786 coli), and combinations thereof, as well as administering antibiotics to animals and controlling the deleterious effects of Escherichia coli in animals.

[0086] In yet another embodiment, a method of feeding an animal is provided. 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67861 ...2), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67863), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0087] The following clauses and combinations thereof provide various additional illustrative aspects of the inventions described herein.The various embodiments described in this section entitled "Detailed Description of Illustrative Embodiments" are applicable to any of the following embodiments of the invention described in the following numbered clauses.

[0088] 1. A method of feeding an animal, the method comprising the steps 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 10 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 11 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 12 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 13 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 14 (NRRL No. B-67714), a strain having all the identifying characteristics of Bacillus strain 15 (NRRL No. B-67715), a strain having all the identifying characteristics of Bacillus strain 16 (NRRL No. B-67717), a strain having all the identifying characteristics of Bacillus strain 17 (NRRL No. B-67718), a strain having all the identifying characteristics of Bac Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to an animal, wherein the Bacillus strain and the antibiotic cause E. coli inhibition in the animal, and wherein the antibiotic is selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0089] 2. The method of clause 1, wherein the animal is selected from the group consisting of poultry species, porcine species, caprine species, bovine species, ovine species, equine species, and companion animals.

[0090] 3. The method of any one of clauses 1 to 2, wherein the E. coli inhibition prevents E. coli disease in animals.

[0091] 4. The method of any one of clauses 1 to 2, wherein the E. coli inhibition reduces E. coli disease in the animal.

[0092] 5. The method of any one of clauses 1 to 4, wherein the animal is a porcine species, and the porcine species is selected from the group consisting of grower-finisher pigs, nursery pigs, sows, and breeding pigs.

[0093] 6. The method of any one of clauses 1 to 5, wherein the Bacillus strain produces an enzyme selected from the group consisting of α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0094] 7. The method of any one of clauses 1 to 6, further comprising the step of administering to the animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, and combinations thereof.

[0095] 8. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 2 (NRRL No. B-67709).

[0096] 9. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 3 (NRRL No. B-67710).

[0097] 10. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 4 (NRRL No. B-67711).

[0098] 11. The method of any one of clauses 1 to 7, wherein the strain applied is Bacillus strain 5 (NRRL No. B-67712).

[0099] 12. The method of any one of clauses 1 to 11, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 5.0×10 12 A dose of CFU / gram of feed composition is administered in the feed composition.

[0100] 13. The method of any one of clauses 1 to 11, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 1.0×10 7 A dose of CFU / gram of feed composition is administered in the feed composition.

[0101] 14. The method of any one of clauses 1 to 11, wherein the Bacillus strain is present at a concentration greater than about 7.0×10 4 A dose of CFU / gram of feed composition is administered in the feed composition.

[0102] 15. The method of any one of clauses 1 to 14, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0103] 16. The method of any one of clauses 1 to 15, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0104] 17. The method of any one of clauses 1 to 15, wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0105] 18. The method of any one of clauses 1 to 17, wherein the feed composition is administered to the animal daily.

[0106] 19. The method of clause 1, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0107] 20. A method for controlling the deleterious effects of Escherichia coli, 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67714), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67715), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67716), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67717), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67718), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to an animal and controlling the deleterious effects of Escherichia coli in the animal, wherein the antibiotic is selected from the group consisting of ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0108] 21. The method of clause 20, wherein the animal is selected from the group consisting of poultry species, porcine species, caprine species, bovine species, ovine species, equine species, and companion animals.

[0109] 22. The method of any one of clauses 20 to 21, wherein controlling the deleterious effects of E. coli is suppressing E. coli disease in animals.

[0110] 23. The method of any one of clauses 20 to 21, wherein controlling the deleterious effects of E. coli is reducing E. coli disease in animals.

[0111] 24. The method of any one of clauses 20 to 23, wherein the animal is a porcine species, and the porcine species is selected from the group consisting of grower-finisher pigs, nursery pigs, sows, and breeding pigs.

[0112] 25. The method of any one of clauses 20 to 24, wherein the Bacillus strain produces an enzyme selected from the group consisting of α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0113] 26. The method of any one of clauses 20 to 25, further comprising the step of administering to the animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, and combinations thereof.

[0114] 27. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 2 (NRRL No. B-67709).

[0115] 28. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 3 (NRRL No. B-67710).

[0116] 29. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 4 (NRRL No. B-67711).

[0117] 30. The method of any one of clauses 20 to 26, wherein the strain applied is Bacillus strain 5 (NRRL No. B-67712).

[0118] 31. The method of any one of clauses 20 to 30, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 5.0×10 12 A dose of CFU / gram of feed composition is administered in the feed composition.

[0119] 32. The method of any one of clauses 20 to 30, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 1.0×10 7 A dose of CFU / gram of feed composition is administered in the feed composition.

[0120] 33. The method of any one of clauses 20 to 30, wherein the Bacillus strain is present at a concentration greater than about 7.0×104 A dose of CFU / gram of feed composition is administered in the feed composition.

[0121] 34. The method of any one of clauses 20 to 33, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0122] 35. The method of any one of clauses 20 to 34, wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0123] 36. The method of any one of clauses 20 to 34, wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0124] 37. The method of any one of clauses 20 to 36, wherein the feed composition is administered to the animal daily.

[0125] 38. The method of clause 20, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0126] 39. 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, wherein the additive comprises an isolated Bacillus strain selected from the group consisting of: Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67714), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67715), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67716), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67717), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67718), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67719), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof, and administering an antibiotic to the animal, wherein the antibiotic is selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0127] 40. The method of clause 39, wherein the animal is selected from the group consisting of poultry species, porcine species, caprine species, bovine species, ovine species, equine species, and companion animals.

[0128] 41. The method of any one of clauses 39 to 40, wherein the animal is a porcine species, and the porcine species is selected from the group consisting of grower-finisher pigs, nursery pigs, sows, and breeding pigs.

[0129] 42. The method of any one of clauses 39 to 41, wherein the Bacillus strain produces an enzyme selected from the group consisting of α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

[0130] 43. The method of any one of clauses 39 to 42, further comprising the step of administering to the animal another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, and combinations thereof.

[0131] 44. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 2 (NRRL No. B-67709).

[0132] 45. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 3 (NRRL No. B-67710).

[0133] 46. ​​The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 4 (NRRL No. B-67711).

[0134] 47. The method of any one of clauses 39 to 43, wherein the strain applied is Bacillus strain 5 (NRRL No. B-67712).

[0135] 48. The method of any one of clauses 39 to 47, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 5.0×10 12 A dose of CFU / gram of feed composition is administered in the feed composition.

[0136] 49. The method of any one of clauses 39 to 47, wherein the Bacillus strain is present at a concentration of approximately 1.0×10 3 CFU / g feed composition to about 1.0×10 7 A dose of CFU / gram of feed composition is administered in the feed composition.

[0137] 50. The method of any one of clauses 39 to 47, wherein the Bacillus strain is present at a concentration greater than about 7.0×10 4 A dose of CFU / gram of feed composition is administered in the feed composition.

[0138] 51. The method of any one of clauses 39 to 50, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0139] 52. The method of any one of clauses 39 to 51 wherein the animal is a sow and the Bacillus strain is administered during lactation.

[0140] 53. The method of any one of clauses 39 to 51 wherein the animal is a sow and the Bacillus strain is administered during gestation.

[0141] 54. The method of any one of clauses 39 to 53, wherein the feed composition is administered to the animal daily.

[0142] 55. The method of clause 39, wherein the animal is selected from the group consisting of chicken, pig, horse, pony, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish, crustacean, and combinations thereof.

[0143] 56. The method of any one of clauses 1 to 55, wherein the Bacillus strain has an effect selected from the group consisting of maintaining microbial balance in the intestinal tract of an animal, improving animal performance or health, maintaining intestinal health of an animal, reducing harmful pathogens in the intestinal tract of an animal, reducing odor, reducing harmful pathogens in the urine or feces of an animal, and maintaining the growth of beneficial bacteria in the intestinal tract of an animal.

[0144] 57. The method of any one of clauses 1 to 7, 12 to 26, 31 to 43, or 48 to 56, wherein the strain applied is Bacillus strain 9 (NRRL No. B-67866).

[0145] 58. The method of any one of clauses 1 to 7, 12 to 26, 31 to 43, or 48 to 56, wherein the strain applied is Bacillus strain 57 (NRRL No. B-67870).

[0146] 59. The method of any one of clauses 1 to 7, 12 to 26, 31 to 43, or 48 to 56, wherein the strain applied is Bacillus strain 71 (NRRL No. B-67867).

[0147] 60. The method of any one of clauses 1 to 7, 12 to 26, 31 to 43, or 48 to 56, wherein the strain applied is Bacillus strain 126 (NRRL No. B-67868).

[0148] In various embodiments, the animal using feed additives described herein, feed compositions or drinking water and antibiotic can be selected from poultry species, pig species, goat species, cattle species, sheep species, horse species and companion animals. In the embodiment where animal is a companion animal, companion animal can be such as dog species or cat species. In the embodiment where animal is a pig species, pig species can be selected from growing and fattening pigs, nursery pigs, sows and breeding pigs. In various exemplary embodiments, animal can be selected from chicken (such as broiler chickens or laying hens), pig, horse, pony, dairy cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish (such as tilapia, catfish, flounder or salmon), crustaceans (such as shrimp or crab) and combination thereof. In another embodiment, feed additives described herein, feed compositions or drinking water and antibiotic can be applied to animal, and animal is people.

[0149] In various embodiments of the methods or compositions described herein, the Bacillus strain and the antibiotic selected from ceftiofur, oxytetracycline, tolamycin and lincomycin can have an effect selected from the following: maintaining microbial balance in the intestinal tract of an animal, preventing or reducing E. coli disease in an animal, improving animal performance or health, maintaining intestinal health of an animal, reducing harmful pathogens in the intestinal tract of an animal, reducing odor, reducing harmful pathogens in the urine or feces of an animal, and maintaining the growth of beneficial bacteria in the intestinal tract of an animal. In any one of the embodiments described herein, the Bacillus strain can be a Bacillus subtilis strain (e.g., strains 2, 3, 9, 57, 71 and / or 126) or a Bacillus licheniformis strain (e.g., strains 4 and 5).

[0150] In any of the embodiments described herein, the Bacillus strain can be used in any combination with an antibiotic selected from ceftiofur, oxytetracycline, tolamectin and lincomycin, or can be in the form of any composition described herein. In one embodiment, one or more strains and an antibiotic selected from ceftiofur, oxytetracycline, tolamectin and lincomycin are used in the same composition or in separate compositions. The Bacillus strain described herein and an antibiotic selected from ceftiofur, oxytetracycline, tolamectin and lincomycin can also be used in combination with other different microbial strains (including other Bacillus strains or Lactobacillus (Lactobacillus) strains).

[0151] In one embodiment of the present invention, an effective amount of a Bacillus strain can be administered to inhibit E. coli in an animal. As used herein, "inhibiting E. coli" can refer to reducing E. coli disease, preventing E. coli disease, maintaining normal microbial balance in an animal, reducing the number of harmful E. coli organisms in an animal, reducing the activity of E. coli in an animal, or reducing the symptoms of E. coli disease in an animal, or a combination thereof. An "effective amount" refers to an amount of a Bacillus strain (e.g., strains 2, 3, 4, 5, 9, 57, 71, and 126) that can inhibit E. coli or control the harmful effects of E. coli by any mechanism as described below.

[0152] In embodiments described herein in which a composition of the invention comprising Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126, or strains having their identifying characteristics, and an antibiotic selected from the group consisting of ceftiofur, oxytetracycline, tolamycin, and lincomycin is administered to an animal, the composition is preferably administered orally to the animal in a feed composition or in drinking water, but any other effective administration method known to those skilled in the art may be employed, such as in a paste, gel, liquid drench, top dress, powder, liquid, or capsule. In one illustrative embodiment, Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126, or strains having their identifying characteristics, and an antibiotic selected from the group consisting of ceftiofur, oxytetracycline, tolamycin, and lincomycin are provided as an additive for addition to the animal's drinking water.

[0153] In another illustrative embodiment, Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126, or strains having their identifying characteristics, and an antibiotic selected from ceftiofur, oxytetracycline, tolamycin and lincomycin are provided in the form of an additive for addition to a feed composition. The feed composition may contain Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126, or strains having their identifying characteristics, mixed with an animal feed blend (including any animal feed blend known in the art or any animal feed blend described herein). As used herein, "feed composition" or "animal feed composition" refers to a feed composition comprising Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126, or strains having their identifying characteristics, mixed with an animal feed blend and, optionally, any other components that can be used in a feed composition, including other bacterial strains, such as other different Bacillus strains or Lactobacillus strains. In one embodiment, the feed composition may be in the form of ground meal.

[0154] Any animal feed blend can be used according to the methods and compositions described in this patent application, including those known in the art and those described herein, such as rapeseed meal, cottonseed meal, soybean meal, corn meal, barley, wheat, silage, and semi-dry silage. In various embodiments, the animal feed blend can be supplemented with Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having their identifying characteristics, and an antibiotic selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin, or the antibiotic can be administered alone, but other ingredients can optionally be added to the animal feed blend, including other different bacterial strains, such as other Bacillus strains or Lactobacillus strains.

[0155] In various illustrative embodiments, optional ingredients of the animal feed blend include sugars and complex carbohydrates, such as water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides. Other optional ingredients include dried distiller's grains solubles, fat (e.g., crude fat), phosphorus, sodium bicarbonate, limestone, salt, phytate, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, ash, fish oil, oil derived from fish meal, raw seeds (e.g., flaxseed), antioxidants, and starch. In another embodiment, minerals can be added in the form of a mineral premix.

[0156] Optional amino acid components that can be added to the animal feed blend are arginine, histidine, isoleucine, leucine, lysine, cysteine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl ester hydrochloride, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ​​ethyl ester hydrochloride, and analogs and salts thereof. Optional vitamins that can be added are thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, etc. In another embodiment, vitamins can be added in the form of a vitamin premix. In yet another embodiment, a protein component can be added to the animal feed blend and include protein obtained from meat meal, bone meal or fish meal, liquid or powdered eggs, fish solubles, crude protein, etc.

[0157] In various embodiments, the antibiotics used in the methods and compositions described herein are selected from the group consisting of ceftiofur, oxytetracycline, tolamectin, and lincomycin.

[0158] In various embodiments, the dosage of the antibiotic can be from about 0.1 mg / lb feed to about 1000 mg / lb feed, from about 0.1 mg / lb feed to about 900 mg / lb feed, from about 0.1 mg / lb feed to about 800 mg / lb feed, from about 0.1 mg / lb feed to about 700 mg / lb feed, from about 0.1 mg / lb feed to about 600 mg / lb feed, from about 0.1 mg / lb feed to about 500 mg / lb feed, from about 0.1 mg / lb feed to about 40 0 mg / lb feed, about 0.1 mg / lb feed to about 300 mg / lb feed, about 0.1 mg / lb feed to about 200 mg / lb feed, about 0.1 mg / lb feed to about 100 mg / lb feed, about 0.1 mg / lb feed to about 90 mg / lb feed, about 0.1 mg / lb feed to about 80 mg / lb feed, about 0.1 mg / lb feed to about 70 mg / lb feed, about 0.1 mg / lb feed to about 60 mg / lb feed, about 0.1 mg / lb feed to about 50 mg / lb feed, about 0.1 mg / lb feed to about 40 mg / lb feed, about 0.1 mg / lb feed to about 30 mg / lb feed, about 0.1 mg / lb feed to about 20 mg / lb feed, about 0.1 mg / lb feed to about 10 mg / lb feed, about 0.1 mg / lb feed to about 9 mg / lb feed, about 0.1 mg / lb feed to about 8 mg / lb feed, about 0.1 mg / lb feed to about 7 mg / lb feed, about 0.1 mg / lb feed to about 6 mg / lb feed, about 0.1 mg / lb feed to about 5 mg / lb feed, about 0.1 mg / lb feed to about 4 mg / lb feed, about 0.1 mg / lb feed to about 3 mg / lb feed, about 0.1 mg / lb feed to about 2 mg / lb feed, about 0.1 mg / lb feed to about 1 mg / lb feed, about 0.1 mg / lb feed to about 0.5 mg / lb feed, about 0.1 mg / lb feed to about 0.3 mg / lb feed, about 0.5 mg / lb feed to about 1 mg / lb feed, about 1 mg / ml to about 1000 mg / ml drinking water, about 1 mg / ml to about 900 mg / ml drinking water, about 1 mg / ml to about 800 mg / ml drinking water, about 1 mg / ml to about 700 mg / ml drinking water, about 1 mg / ml to about 600 mg / ml drinking water, about 1 mg / ml to about 500 mg / ml drinking water, about 1 mg / ml to about 400 mg / ml drinking water, about 1 mg / ml to about 300 mg / ml drinking water, about 1 mg / ml to about 200 mg / ml drinking water, about 1 mg / ml to about 100 mg / ml drinking water, about 50 mg / ml to about 100 mg / ml drinking water, about 50 mg / ml to about 90 mg / ml drinking water, about 50 mg / ml to about 80 mg / ml drinking water, about 50 mg / ml to about 70 mg / ml drinking water, about 50 mg / ml to about 60 mg / ml drinking water, about 1000 ppm, about 900 ppm, about 800 ppm, about 700 ppm, about 600 ppm, about 500 ppm, about 400 ppm, about 300 ppm, about 200 ppm, about 100 ppm, about 90 ppm, about 80 ppm, about 70 ppm, about 60 ppm, about 50 ppm, about 40 ppm, about 30 ppm, about 20 ppm, or about 10 ppm.

[0159] In another illustrative embodiment, one or more enzymes can be added to the animal feed blend or feed composition or feed additive or bacterial strain. In various embodiments, the enzymes that can be added include galactosidase, phytase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSP enzyme, combinations thereof, and any other enzyme that improves the effectiveness of the E. coli inhibition or control of the harmful effects of E. coli of the feed composition or feed additive. In yet another embodiment, yeast, fungi (e.g., Aspergillus or Trichoderma) or trace nutrients can be added to the animal feed, feed composition, or feed additive, or bacterial strain. Any of the above ingredients suitable for addition to an additive for animal drinking water can be added as a component of the additive for animal drinking water described herein.

[0160] In various illustrative embodiments, a Bacillus strain (e.g., Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having their identifying characteristics), or any other different bacterial strain added in addition to Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having their identifying characteristics, can be added at a rate of approximately 1.0×10 3 CFU / g feed composition to about 5.0×10 12 CFU / g of feed composition, or at a dose of approximately 1.0×10 3 CFU / g feed composition to about 1.0×10 7 In other embodiments, the Bacillus strain (e.g., Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having identifying characteristics thereof) is administered in a dose of greater than about 1.0×10 3 CFU / g of feed composition, at a dose greater than about 1.1×10 3 CFU / g feed composition, at a dose greater than about 1.25×10 3 CFU / g feed composition, at a dose greater than about 1.5×10 3 CFU / g feed composition, at a dose greater than about 1.75×10 3 CFU / g feed composition, at a dose greater than about 1.0×10 4 CFU / g feed composition, at a dose greater than about 2.0×10 4 CFU / g feed composition, at a dose greater than about 3.0×10 4 CFU / g feed composition, at a dose greater than about 4.0×10 4 CFU / g feed composition, at a dose greater than about 5.0×10 4 CFU / g feed composition, at a dose greater than about 6.0×10 4 CFU / g feed composition, at a dose greater than about 7.0×10 4 CFU / g feed composition, at a dose greater than about 8.0×10 4 CFU / g feed composition, at a dose greater than about 1.0×10 5 CFU / g feed composition, at a dose greater than about 1.0×10 6 CFU / g feed composition, at a dose greater than about 1.0×10 7 CFU / g feed composition, at a dose greater than about 1.0×10 8 CFU / g feed composition, at a dose greater than about 1.0×109 CFU / g feed composition, at a dose greater than about 1.0×10 10 CFU / g feed composition, at a dose greater than about 1.0×10 11 CFU / g feed composition, or at a dose greater than about 1.0×10 12 In another embodiment, a Bacillus strain (e.g., Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having their identifying characteristics), or any other different bacterial strain added in addition to Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having their identifying characteristics, may be administered in a dose of approximately 1.0×10 2 CFU / g feed composition to about 5.0×10 12 CFU / g feed composition, or at a dose of approximately 1.0×10 2 CFU / g feed composition to about 1.0×10 7 CFU / g feed composition, or at a dose greater than about 1.0×10 2 The dosage of CFU / gram of feed composition is administered in the feed composition. In another embodiment, in embodiments where the strain is administered in the animal's drinking water, any of the dosages described herein may be in units of CFU / ml of drinking water.

[0161] In various embodiments, the Bacillus strain for use according to the methods or compositions described herein (e.g., Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having their identifying characteristics) can be selected from Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having Bacillus strain 5 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67714), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67715), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67716), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67717), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67718), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67719), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof. Bacillus strains 2, 3, 4, and 5 were deposited on November 29, 2018, at the Agricultural Research Service Culture Collection (NRRL), USDA, 1815 North University Street, Peoria, Illinois 61604-3999, and were given accession numbers. Deposits were made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The NRRL strain names are NRRL No. B-67709, NRRL No. B-67710, NRRL No. B-67711, and NRRL No. B-67712, respectively. NRRL No. B-67709, NRRL No. B-67710, NRRL No. B-67711, and NRRL No. B-67712 are equivalent to Bacillus strains 2, 3, 4, and 5, respectively, mentioned in this application.

[0162] Bacillus strains 9, 57, 71, and 126 were deposited on October 10, 2019, at the Agricultural Research Service Culture Collection (NRRL), Agricultural Research Institute, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604-3999, and were assigned accession numbers B-67866, B-67870, B-67867, and B-67868, respectively. Deposits were made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. NRRL No. B-67866, NRRL No. B-67870, NRRL No. B-67867, and NRRL No. B-67868 are equivalent to Bacillus strains 9, 57, 71, and 126, respectively, referred to in this application. The deposit certificates refer to the strains as MDG9, MDG57, MDG71, and MDG126, respectively.

[0163] On the one hand, any one of these strains can be administered alone, or in the form of a feed composition (e.g., a complete feed comprising an animal feed blend) or animal drinking water. 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 yet another embodiment, any one of these strains or a combination thereof is administered in combination with an antibiotic selected from ceftiofur, oxytetracycline, tolamectin, and lincomycin as described herein in the same composition or in separate compositions.

[0164] In another embodiment, one or more of the Bacillus strains described in the previous paragraph (e.g., Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having their identifying characteristics) can be administered to an animal together with another different bacterial strain selected from another Bacillus strain, a lactic acid bacteria strain, and combinations thereof. In yet another embodiment, one or more of the Bacillus strains described in the previous paragraph (e.g., Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126, or strains having their identifying characteristics) can be administered to an animal together with any other bacterial strain that is effective in inhibiting E. coli or controlling its deleterious effects in an animal.

[0165] As used herein, a "strain having all the identifying characteristics" of Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126 can be a mutant having all the identifying characteristics of Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126 (e.g., a DNA fingerprint based on DNA analysis corresponding to the DNA fingerprint of Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126, an enzyme activity corresponding to Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126, an antimicrobial activity corresponding to Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126, an antibiotic sensitivity and tolerance profile corresponding to Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126, or a combination of these identifying characteristics). In alternative embodiments, the mutation can be a natural mutation or a genetically engineered mutation. In another embodiment, a strain "having all the identifying characteristics of" Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126 can be, for example, a strain produced by isolating one or more plasmids from Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126 and introducing the one or more plasmids into another bacterium (such as another Bacillus strain), so long as the one or more plasmids contain DNA that provides the identifying characteristics of Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126 (e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerprint of Bacillus strain 2, 3, 4, 5, 9, 57, 71, or 126).

[0166] The feed composition or drinking water comprising Bacillus strain 2, 3, 4, 5, 9, 57, 71 or 126 or a strain with their identification characteristics and an antibiotic described herein can be administered to an animal within any time period that is effective to inhibit E. coli or control the harmful effects of E. coli or a combination thereof. For example, in one embodiment, a feed composition or drinking water can be provided to the animal every day. In an alternative embodiment, the feed composition or drinking water can be administered to the animal during lactation and / or during pregnancy. The time periods for administering the feed composition or drinking water are non-limiting examples, and it should be understood that any time period or administration regimen determined to be effective for inhibiting E. coli or controlling the harmful effects of E. coli or a combination thereof can be employed.

[0167] In embodiments directed to "controlling the deleterious effects of E. coli," controlling the deleterious effects can refer to reducing E. coli disease, preventing E. coli disease, maintaining a normal microbial balance in an animal, reducing the number of deleterious E. coli in an animal, reducing the activity of E. coli in an animal, or reducing the symptoms of E. coli disease in an animal, or a combination thereof. An "effective amount" refers to an amount of a Bacillus strain (e.g., Bacillus strains 2, 3, 4, 5, 9, 57, 71, and / or 126) that is capable of controlling the deleterious effects of E. coli by any mechanism as described herein.

[0168] In one embodiment, the feed additive for adding to the animal feed blend to produce a complete feed composition can be mixed with the animal feed blend, for example, using an automated micronutrient delivery system, or for example, by manual weighing and addition, to achieve any dosage of Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126 or strains having their identifying characteristics for administration to animals in the form of a complete feed composition as described herein. Mixing can also be performed by any other suitable method known in the art for combining direct-fed microorganisms with animal feed blends to obtain a homogenous mixture. In various embodiments, mixing can be performed over any suitable time period (e.g., about 1 minute to about 4 minutes). In embodiments where Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126 or strains having their identifying characteristics are in the form of an additive for use in drinking water for animals, the Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126 or strains having their identifying characteristics can be in the form of, for example, a powder, liquid, or pellets and can be mixed with drinking water using any suitable method known in the art to achieve any dosage of Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126 or strains having their identifying characteristics described herein for administration to animals in their drinking water. Bacillus strains 2, 3, 4, 5, 9, 57, 71 and / or 126 or strains having their identifying characteristics can also be administered directly orally (i.e., by oral placement) to animals in the form of a powder, freeze-dried composition, gel, top-dressing, liquid, capsule, paste, drench, or pellets. Any of these embodiments may also be applied to the admixture and administration of an antibiotic selected from the group consisting of ceftiofur, oxytetracycline, tolamectin, and lincomycin as described herein.

[0169] In another embodiment of the present invention, a composition is provided that comprises Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67861). Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0170] In one embodiment, a commercial package is provided comprising an isolated Bacillus strain selected from the group consisting of: Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67861). Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0171] In another embodiment, a feed additive for animal feed is provided, comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67861). Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0172] In yet another embodiment, an additive for animal drinking water is provided, comprising an isolated Bacillus strain selected from the group consisting of: Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67861). Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0173] 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 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67869), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67861), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67861). Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof.

[0174] In one illustrative aspect, the strain for addition to a commercial package, a feed additive, an additive for animal drinking water, or a feed composition can be a concentrate (e.g., approximately 1×10 8 to approximately 5×10 9 CFU / g) or super concentrates (e.g., approximately 1×10 10 to approximately 5×10 12 In another embodiment, the strain for addition to commercial packaging, feed additives, feed compositions, or additives for animal drinking water can be in dry form (e.g., powder), granular form, liquid form, drenching solution, powder, freeze-dried composition, external application, paste form, or gel form, or any other suitable form.

[0175] In another illustrative embodiment, commercial packaging, feed additives, one or more bacterial strains in the additive for animal drinking water or feed composition can further include a carrier for one or more bacillus bacterial strains.Carrier can be selected from wheat bran, rice husk, salt, mineral oil, dextrin (for example maltodextrin), whey, sugar, sucrose, limestone, yeast culture, dry starch, sodium aluminosilicate, silicon dioxide, polypropylene glycol, polysorbate 80, vegetable oil and combination thereof.In another embodiment, carrier can be any suitable carrier known in the art for directly feeding microorganisms.Carrier exogenous addition in bacterial strain (i.e. not naturally occurring or not present in nature with bacterial strain).In another embodiment, feed additives, additive for animal drinking water or feed composition can further include adhesive, such as clay, yeast cell wall component, aluminum silicate, dextran or other known adhesives.In another embodiment, commercial packaging, feed additives, additive for animal drinking water or feed composition can further include inorganic / organic adhesive, essential oil and / or organic acid. The binder is exogenously added to the bacterial strain (ie, does not occur naturally or with the bacterial strain in nature).

[0176] In still other embodiments, the present invention comprises Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 9 (NRRL No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67867), Bacillus strain 57 (NRRL No. B-67868), Bacillus strain 57 (NRRL No. B-67869), Bacillus strain 57 (NRRL No. B-67870). No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof in commercial packages, feed additives, additives for animal drinking water, or feed compositions in containers for commercial use.

[0177] In various embodiments, the container can be, for example, a bag (e.g., a 20 lb bag, a 50 lb bag, a 2 oz bag, a 1 lb bag, or a 1 kg bag), a pouch, a bucket, a bottle, or a box. In illustrative aspects, a container for containing Bacillus strain 2 (NRRL No. B-67709), a strain having all the identifying characteristics of Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 5 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67710), a strain having all the identifying characteristics of Bacillus strain 10 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 11 (NRRL No. B-67711), a strain having all the identifying characteristics of Bacillus strain 12 (NRRL No. B-67712), a strain having all the identifying characteristics of Bacillus strain 13 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 14 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 15 (NRRL No. B-67713), a strain having all the identifying characteristics of Bacillus strain 16 (NRRL No. B-67711), a strain having all the identifying No. B-67866), a strain having all the identifying characteristics of Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), a strain having all the identifying characteristics of Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), a strain having all the identifying characteristics of Bacillus strain 71 (NRRL No. B-67867), Bacillus strain 126 (NRRL No. B-67868), a strain having all the identifying characteristics of Bacillus strain 126 (NRRL No. B-67868), and combinations thereof. Containers for commercial packaging, feed additives, additives for animal drinking water, or feed compositions can comprise plastic, metal, foil, paper, fiber, or cardboard (e.g., plastic buckets, paper bags, foil bags, fiberboard buckets, etc.). The commercial package, feed additive, additive for animal drinking water, or feed composition may further comprise instructions for use of the one or more Bacillus strains.

[0178] In one aspect, the commercial packages, feed additives, additives for animal drinking water, or feed compositions described herein can further comprise exogenously added nutrients (i.e., nutrients that do not occur in nature with the bacterial strain) selected from the group consisting of vitamins, antibiotics, enzymes, water-soluble or water-insoluble monosaccharides, disaccharides, or polysaccharides, fats, phosphorus, sodium bicarbonate, limestone, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, fish oils, raw seeds, antioxidants, and starch.

[0179] In one embodiment, the exogenously added nutrient is an enzyme, and the enzyme is selected from the group consisting of galactosidase, protease, lipase, amylase, hemicellulase, arabinoxylanase, xylanase, cellulase, NSPase, phytase, and combinations thereof.

[0180] In one embodiment, any of the compositions described herein may be a dietary nutrient composition (eg, a probiotic composition).

[0181] In some embodiments of the methods and compositions described herein, a synergistic effect occurs between the Bacillus strain and the antibiotic.

[0182] The following examples are for illustrative purposes only. The examples are non-limiting and are not intended to limit the present invention in any way.

[0183] Example 1

[0184] Resistant to Excede (12), Draxxin (8), Cofetin (12) and Liquamycin (11) Bacillus spore testing for medicinal Escherichia coli Four Bacillus strains were tested using the broth AST method against 12 Estrogen-resistant E. coli, 8 Recosine-resistant E. coli, 12 Cofetin-resistant E. coli, and 11 Liquamycin-resistant E. coli. Four additional Bacillus strains were identified and tested in the same manner against the same lincomycin-resistant E. coli.

[0185] When the same concentrations of antibiotics were administered in conjunction with Bacillus supernatants, Bacillus strain 2 (NRRL No. B-67709), Bacillus strain 3 (NRRL No. B-67710), Bacillus strain 4 (NRRL No. B-67711), Bacillus strain 5 (NRRL No. B-67712), Bacillus strain 9 (NRRL No. B-67866), Bacillus strain 57 (NRRL No. B-67870), Bacillus strain 71 (NRRL No. B-67867), and Bacillus strain 126 (NRRL No. B-67868) consistently improved and effectively inhibited (>50% inhibition) antibiotic-resistant pathogens.

[0186] Among 12 Estrogen-resistant E. coli strains: When combined with the antibiotic, Bacillus strains 9, 57, and 71 effectively inhibited 11 of the 12 isolates. When combined with the antibiotic, Bacillus strain 126 effectively inhibited 10 of the 12 isolates.

[0187] Among 12 liquamycin-resistant E. coli: Bacillus strains 9, 57, 71, and 126 improved inhibition in 12 of the 12 isolates when combined with the antibiotic.

[0188] Among eight recosine-resistant E. coli: Bacillus strains 9, 57, 71, and 126 improved inhibition in eight of the eight isolates when combined with the antibiotic.

[0189] Among 12 chloramphenicol-resistant E. coli: When combined with the antibiotic, Bacillus strain 9 improved inhibition in 11 of the 12 isolates. When combined with the antibiotic, Bacillus strains 57, 71, and 96 improved inhibition in 12 of the 12 isolates. When compared to chloramphenicol alone, Bacillus strains 9, 57, 71, and 126 significantly inhibited 12 of the 12 isolates. When compared to chloramphenicol alone, Bacillus strains 2, 3, 4, and 5 significantly inhibited 11 of the 12 isolates.

[0190] The goal of this project was to identify Bacillus strains that exhibit strong inhibitory potential against target pathogens in combination with antibiotics. This synergy can be defined as a higher percentage of inhibition against antibiotic-resistant pathogens achieved when a Bacillus supernatant is combined with an antibiotic than when the supernatant and antibiotic are tested separately against the same isolates. Eight Bacillus strains demonstrated consistent and strong inhibitory activity against Escherichia coli strains resistant to Estradiol, Recosin, Cofetin, and Liquamycin.

[0191] Example 2

[0192] RAPD-PCR DNA profile

[0193] The random amplified polymorphic DNA PCR method (hereinafter referred to as RAPD-PCR) was used to identify the genetic variability of each strain. Tissue and Blood single column kit ( The DNA to be used in RAPD-PCR reactions was prepared by using DNA from the laboratory of ELISA (Venlo, The Netherlands). Figure 6 、 7 Figures 2 and 8 show the RAPD-PCR results for strains 2, 3, 4, 5, 6, 7, 9, 57, 71, and 126, where the unlabeled lanes or lanes labeled "PC" are molecular weight ladder markers. The results showed that all strains were unique to each other.

Claims

1. Use of an additive in the preparation of a feed composition or drinking water for use in a method of feeding an animal the additive to enhance the efficacy of antibiotic treatment and inhibit the growth of antibiotic-resistant Escherichia coli, the method comprising the steps of: i) administering to the animal a feed composition or drinking water comprising an effective amount of the additive, wherein the additive comprises an isolated Bacillus strain selected from the group consisting of Bacillus strain 9 having accession number NRRL No. B-67866, Bacillus strain 57 having accession number NRRL No. B-67870, Bacillus strain 71 having accession number NRRL No. B-67867, and Bacillus strain 126 having accession number NRRL No. B-67868, and combinations thereof, and administering an antibiotic to the animal, wherein the antibiotic is selected from the group consisting of ceftiofur, oxytetracycline, tulathromycin, and lincomycin, wherein administration of the additive in combination with the antibiotic enhances the efficacy of the antibiotic relative to administration of the antibiotic in the absence of the additive; or ii) administering to the animal a feed composition or drinking water comprising an effective amount of the additive, wherein the additive comprises an isolated Bacillus strain selected from the group consisting of Bacillus strain 2 having accession number NRRL No. B-67709, Bacillus strain 3 having accession number NRRL No. B-67710, Bacillus strain 4 having accession number NRRL No. B-67711, Bacillus strain 5 having accession number NRRL No. B-67712, and combinations thereof, and administering cortisone to the animal, wherein the additive in combination with administration of the cortisone enhances the efficacy of the cortisone relative to administration of the cortisone in the absence of the additive.

2. The use of claim 1, wherein the animal is a poultry species.

3. The use of claim 1, wherein the animal is a porcine species.

4. The use of claim 1, wherein the animal is a goat species.

5. The use of claim 1, wherein the animal is a bovine species or an ovine species.

6. The use of claim 1, wherein the animal is of the equine species.

7. The use of claim 3, wherein the animal is a growing-finishing pig or a nursery pig.

8. The use of claim 3, wherein the animal is a sow.

9. The method of claim 3, wherein the animal is a sow.

10. The use of claim 1, wherein the Bacillus strain produces an enzyme selected from the group consisting of α-galactosidase, protease, lipase, amylase, xylanase, cellulase, and combinations thereof.

11. The use as claimed in claim 1, wherein the strain used in ii) is Bacillus strain 2 having accession number NRRL No. B-67709.

12. The use as claimed in claim 1, wherein the strain used in ii) is Bacillus strain 3 having accession number NRRL No. B-67710.

13. The use as claimed in claim 1, wherein the strain used in ii) is Bacillus strain 4 having accession number NRRL No. B-67711.

14. The use as claimed in claim 1, wherein the strain used in ii) is Bacillus strain 5 having accession number NRRL No. B-67712.

15. The use according to claim 1, wherein the Bacillus strain is expressed at a concentration of 1.0×10 3 CFU / g of the feed composition to 5.0×10 12 A dosage of CFU / gram of the feed composition is administered in the feed composition.

16. The use according to claim 1, wherein the Bacillus strain is expressed at a concentration of 1.0×10 3 CFU / g of the feed composition to 1.0×10 7 A dosage of CFU / gram of the feed composition is administered in the feed composition.

17. The use of claim 1, wherein the Bacillus strain has a concentration greater than 7.0×10 4 A dosage of CFU / gram of the feed composition is administered in the feed composition.

18. The use of claim 1, further comprising the step of administering to the animal an enzyme selected from the group consisting of galactosidase, protease, lipase, amylase, phytase, and combinations thereof.

19. The use of claim 1, further comprising the step of administering NSPase to the animal.

20. The use of claim 1, further comprising the step of administering to the animal an enzyme selected from the group consisting of hemicellulases, xylanases, and cellulases.

21. The use of claim 1, wherein the animal is a sow and the Bacillus strain is administered during lactation.

22. The use of claim 1, wherein the animal is a sow and the Bacillus strain is administered during gestation.

23. The use of claim 1, wherein the animal is selected from the group consisting of chicken, pig, horse, cow, turkey, goat, sheep, quail, pheasant, ostrich, duck, fish and crustaceans.

24. The use of claim 1, wherein the Bacillus strain has an effect selected from the group consisting of maintaining microbial balance in the intestinal tract of the animal, improving animal performance or health, maintaining intestinal health of the animal, reducing harmful pathogens in the intestinal tract of the animal, reducing odor, reducing harmful pathogens in the urine or feces of the animal, and maintaining the growth of beneficial bacteria in the intestinal tract of the animal.

25. The use as claimed in claim 1, wherein in i), the strain used is Bacillus strain 9 having accession number NRRL No. B-67866.

26. The use as claimed in claim 1, wherein in i), the strain used is Bacillus strain 57 having accession number NRRL No. B-67870.

27. The use as claimed in claim 1, wherein in i), the strain used is Bacillus strain 71 having accession number NRRL No. B-67867.

28. The use as claimed in claim 1, wherein in i), the strain used is Bacillus strain 126 having accession number NRRL No. B-67868.

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