WO2024254478A2 - Microbis pour inhibition bactérienne - Google Patents

Microbis pour inhibition bactérienne Download PDF

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Publication number
WO2024254478A2
WO2024254478A2 PCT/US2024/033048 US2024033048W WO2024254478A2 WO 2024254478 A2 WO2024254478 A2 WO 2024254478A2 US 2024033048 W US2024033048 W US 2024033048W WO 2024254478 A2 WO2024254478 A2 WO 2024254478A2
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nrrl
strain
bacillus strain
animal
bacillus
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WO2024254478A3 (fr
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Sona SON
Michael R. King
Jessica EDWARD
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Microbial Discovery Group LLC
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Microbial Discovery Group LLC
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Priority to CN202480042253.7A priority Critical patent/CN121712518A/zh
Publication of WO2024254478A2 publication Critical patent/WO2024254478A2/fr
Publication of WO2024254478A3 publication Critical patent/WO2024254478A3/fr
Priority to MX2025014302A priority patent/MX2025014302A/es
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the invention relates to direct-fed microbials for use in E. coli, Salmonella, and/or Clostridium inhibition in animals. More particularly, the invention relates to isolated Bacillus strains CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), and MDG1607 (NRRL No. B-67666), and strains having all of the identifying characteristics of these strains, for a use comprising the above-mentioned use. The invention also relates to the use of isolated Bacillus strains CM256 (NRRL No. B-67706), CM857 (NRRL No.
  • CM978 NRRL No. B-67703
  • CM1004 NRRL No. B-67708
  • the present invention relates to direct-fed microbial (DFM) compositions and methods for E. coli, Salmonella, and/or Clostridium inhibition in an animal.
  • An animal’s gastrointestinal tract is constantly challenged by large numbers of bacteria, viruses, fungi, and protozoa found in feed, bedding, and the environment.
  • the gastrointestinal tract has a sophisticated system to counter these potential pathogens consisting of physical, chemical, and immunological lines of defense.
  • Beneficial bacteria are an important part of this system because they provide animals with bacteria that assist in establishment (or reestablishment) of a normal bacterial profile, they strengthen the animal’s immune system, and they help to fight disease (e.g., disease caused by E. coli, Salmonella, and/or Clostridium in animals).
  • Applicants have developed direct-fed microbials that result in E. coli, Salmonella, and/or Clostridium inhibition.
  • the direct-fed microbials and compositions comprising the direct-fed microbials described herein offer a commercial benefit by providing E. coli, Salmonella, and/or Clostridium inhibition in animals, such as agricultural animals.
  • the direct-fed microbial compositions described herein result in a reduction or elimination in the use of antibiotics which reduces the overall cost of animal feed and reduces the development of antibiotic resistance.
  • these microbial strains, and strains having all of the identifying characteristics of these strains can be used to treat plants having diseases caused by E. coli, Salmonella, and/or Clostridium.
  • the animal can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.
  • the poultry species can be selected from the group consisting of a broiler, a chicken, a layer, a breeder, a turkey, a turkey poult, a gosling, a duckling, a guineakeet, a pullet, a hen, a rooster, a cockerel, and a capon.
  • the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.
  • a method of feeding an animal 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 CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Bacillus strain CM978 a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, wherein the Bacillus strain causes E. coli, Salmonella, and/or Clostridium inhibition in the animal.
  • a method of controlling a detrimental effect of E. coli, Salmonella, and/or Clostridium comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B- 67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • a commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), and combinations thereof.
  • a feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • an additive for the drinking water of an animal comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B- 67706 a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), and combinations thereof.
  • an animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B- 67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B- 67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • a method of feeding an animal 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 CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • a method of treating a plant to inhibit a bacterial disease caused by E. coli, Salmonella, and/or Clostridium comprises contacting the plant with a composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No.
  • a method of feeding an animal comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B- 67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Bacillus strain CM978 a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, wherein the Bacillus strain causes E. coli, Salmonella, and/or Clostridium inhibition in the animal.
  • CM857 (NRRL No. B-67705)
  • CM978 (NRRL No. B-67703)
  • CM1004 (NRRL No. B-67708)
  • MDG1607 (NRRL No. B-67666)
  • a method of controlling a detrimental effect of E. coli, Salmonella, and/or Clostridium 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 CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B-67706 a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, and controlling the detrimental effect of E. coli, Salmonella, and/or Clostridium in the animal.
  • Salmonella, and/or Clostridium inhibition prevents E. coli, Salmonella, and/or Clostridium disease in the animal.
  • Salmonella, and/or Clostridium inhibition reduces E. coli, Salmonella, and/or Clostridium disease in the animal.
  • CM256 (NRRL No. B-67706)
  • CM857 (NRRL No. B-67705)
  • CM978 (NRRL No. B-67703)
  • CM1004 (NRRL No. B-67708)
  • MDG1607 (NRRL No. B-67666) are all administered.
  • NSPase NSPase
  • a phytase NSPase
  • combinations thereof NSPase, a phytase, and combinations thereof.
  • a commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B- 67708), and combinations thereof.
  • a feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B- 67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B- 67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • An animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • a method of feeding an animal comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B- 67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Salmonella, and/or Clostridium inhibition prevents E. coli, Salmonella, and/or Clostridium disease in the animal.
  • Salmonella, and/or Clostridium inhibition reduces E. coli, Salmonella, and/or Clostridium disease in the animal.
  • CM256 (NRRL No. B-67706)
  • CM857 (NRRL No. B-67705)
  • CM978 (NRRL No. B-67703)
  • CM1004 (NRRL No. B-67708), and MDG1607 (NRRL No. B-67666) are all administered.
  • NSPase NSPase
  • a phytase NSPase
  • combinations thereof NSPase, a phytase, and combinations thereof.
  • a method of treating a plant to inhibit a bacterial disease caused by E. coli, Salmonella, and/or Clostridium comprising contacting the plant with a composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B- 67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B-67706 a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, and inhibiting E. coli, Salmonella, and/or Clostridium in the plant.
  • CM857 (NRRL No. B-67705)
  • CM978 (NRRL No. B-67703)
  • CM1004 (NRRL No. B-67708)
  • MDG1607 (NRRL No. B-67666) are all used to treat the plant.
  • MDG1607 (NRRL No. B-67666), or a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), is used to treat the plant in combination with one or more of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • FIGS. 1A-1C show the results of whole cell preparations of Bacillus strains CM256, CM857, CM978, CM1004, and MDG1607 for efficacy against various bacterial isolates.
  • Fig. 1A shows the results of whole cell preparations of Bacillus strains CM256, CM857, CM978, CM1004, and MDG1607 for efficacy against various isolates of E. coli.
  • FIG. IB show the results of whole cell preparation of Bacillus strains CM256, CM857, CM978, CM1004, and MDG1607 for efficacy against various isolates of Clostridium.
  • FIG. 1C shows the results of whole cell preparation of Bacillus strains CM256, CM857, CM978, CM1004, and MDG1607 for efficacy against various isolates of Salmonella.
  • FIGS. 2A-2E show the RAPD testing results for Bacillus strains of the present disclosure.
  • FIG. 2A shows the RAPD testing results for Bacillus strain CM256.
  • FIG. 2B shows the RAPD testing results for Bacillus strain CM857.
  • FIG. 2C shows the RAPD testing results for Bacillus strain CM978.
  • FIG. 2D shows the RAPD testing results for Bacillus strain CM1004.
  • FIG. 2E shows the RAPD testing results for Bacillus strain MDG1607.
  • FIG. 3 shows the results of whole cell preparations of Bacillus strains CM256, CM857, CM978, CM1004, and MDG1607 for enzymatic activity of lipase, amylase, protease, xylanase, and cellulase.
  • Methods and compositions are provided for inhibiting E. coli, Salmonella, and/or Clostridium in animals.
  • the animal can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.
  • the poultry species can be selected from the group consisting of a broiler, a chicken, a layer, a breeder, a turkey, a turkey poult, a gosling, a duckling, a guineakeet, a pullet, a hen, a rooster, a cockerel, and a capon.
  • the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.
  • a method of feeding an animal 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 CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Bacillus strain CM978 a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, wherein the Bacillus strain causes E. coli, Salmonella, and/or Clostridium inhibition in the animal.
  • a method of controlling a detrimental effect of E. coli, Salmonella, and/or Clostridium comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B- 67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • the method comprises the steps of administering to an animal a feed composition or drinking water that comprises an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), and CM1004 (NRRL No. B-67708), or any combination thereof.
  • an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), and CM1004 (NRRL No. B-67708), or any combination thereof.
  • a commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), and combinations thereof.
  • a feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • a feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), and CM1004 (NRRL No. B-67708), or any combination thereof.
  • an additive for the drinking water of an animal comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • an additive for the drinking water of an animal comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B- 67705), CM978 (NRRL No. B-67703), and CM1004 (NRRL No. B-67708), or any combination thereof.
  • an animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B- 67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B- 67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • an animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), and CM1004 (NRRL No. B-67708), or any combination thereof.
  • a method of feeding an animal 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 CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • a method of treating a plant to inhibit a bacterial disease caused by E. coli, Salmonella, and/or Clostridium comprises contacting the plant with a composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No.
  • a method of feeding an animal comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B- 67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Bacillus strain CM978 a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, wherein the Bacillus strain causes E. coli, Salmonella, and/or Clostridium inhibition in the animal.
  • CM857 (NRRL No. B-67705)
  • CM978 (NRRL No. B-67703)
  • CM1004 (NRRL No. B-67708)
  • MDG1607 (NRRL No. B-67666)
  • a method of controlling a detrimental effect of E. coli, Salmonella, and/or Clostridium 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 CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B-67706 a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, and controlling the detrimental effect of E. coli, Salmonella, and/or Clostridium in the animal.
  • Salmonella, and/or Clostridium inhibition prevents E. coli, Salmonella, and/or Clostridium disease in the animal.
  • Salmonella, and/or Clostridium inhibition reduces E. coli, Salmonella, and/or Clostridium disease in the animal.
  • the animal is a porcine species and the porcine species is selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.
  • CM256 (NRRL No. B-67706)
  • CM857 (NRRL No. B-67705)
  • CM978 (NRRL No. B-67703)
  • CM1004 (NRRL No. B-67708)
  • MDG1607 (NRRL No. B-67666) are all administered.
  • NSPase NSPase
  • a phytase NSPase
  • combinations thereof NSPase, a phytase, and combinations thereof.
  • a commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B- 67708), and combinations thereof.
  • a feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B- 67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B- 67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • An animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • strains are in a form selected from the group consisting of a powder, a liquid, and a pellet form.
  • a method of feeding an animal comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B- 67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Salmonella, and/or Clostridium inhibition prevents E. coli, Salmonella, and/or Clostridium disease in the animal.
  • Salmonella, and/or Clostridium inhibition reduces E. coli, Salmonella, and/or Clostridium disease in the animal.
  • CM256 (NRRL No. B-67706)
  • CM857 (NRRL No. B-67705)
  • CM978 (NRRL No. B-67703)
  • CM1004 (NRRL No. B-67708), and MDG1607 (NRRL No. B-67666) are all administered.
  • a method of treating a plant to inhibit a bacterial disease caused by E. coli, Salmonella, and/or Clostridium comprising contacting the plant with a composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B- 67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B-67706 a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, and inhibiting E. coli, Salmonella, and/or Clostridium in the plant.
  • CM857 (NRRL No. B-67705)
  • CM978 (NRRL No. B-67703)
  • CM1004 (NRRL No. B-67708)
  • MDG1607 (NRRL No. B-67666) are all used to treat the plant.
  • MDG1607 (NRRL No. B-67666), or a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), is used to treat the plant in combination with one or more of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • the animal to which a feed additive, a feed composition, or drinking water as described herein is administered can be selected from the group consisting of a poultry species, a porcine species, a caprine species, a bovine species, an ovine species, an equine species, and a companion animal.
  • the companion animal can be, for example, a canine species or a feline species.
  • the porcine species can be selected from the group consisting of a grow finish pig, a nursery pig, a sow, and a breeding stock pig.
  • the animal can be selected from the group consisting of a chicken (e.g., a broiler or a layer), a pig, a horse, a pony, a cow, a turkey, a goat, a sheep, a quail, a pheasant, an ostrich, a duck, a fish (e.g., a tilapia, a catfish, a flounder, or a salmon), a crustacean (e.g., a shrimp or a crab), and combinations thereof.
  • the feed additive, feed composition, or drinking water as described herein could be administered to a human.
  • isolated Bacillus strains selected from the group consisting of CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B- 67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No.
  • Bacillus strain CM 1004 NRRL No. B-67708
  • a strain having all of the identifying characteristics of Bacillus strain MDG1607 can reduce contamination by detrimental E. coli, Salmonella, and/or Clostridium in meat products produced from the animal, or can be used in food processing to reduce detrimental effects of E. coli, Salmonella, and/or Clostridium in foods.
  • the Bacillus strain can have an effect selected from the group consisting of maintaining microbial balance in the gut of the animal, preventing or reducing E. coli, Salmonella, and/or Clostridium disease in the animal, improving animal performance or health, maintaining gut health in the animal, reducing detrimental pathogens in the gut of the animal, odor reduction, reducing detrimental pathogens in the urine or feces of the animal, and preserving the growth of beneficial bacteria in the gut of the animal.
  • the Bacillus strain can be a Bacillus subtilis strain (e.g., strains CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), and CM 1004 (NRRL No. B-67708)), or a Bacillus amyloliquefaciens strain (e.g., strain MDG1607).
  • Bacillus subtilis strain e.g., strains CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), and CM 1004 (NRRL No. B-67708)
  • a Bacillus amyloliquefaciens strain e.g., strain MDG1607.
  • the Bacillus strains can be administered alone or in any combination, or can be in the form of any composition described herein so that the strains are alone or in any combination in the compositions described herein.
  • the Bacillus strains described herein can also be used in combination with other microbial strains, including other Bacillus strains or Lactobacillus strains. Exemplary combinations include all of strains CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), and MDG1607 (NRRL No. B-67666) used together in combination.
  • Another exemplary combination includes Bacillus strain MDG 1607 (NRRL No. B-67666) in combination with one of more of Bacillus strains CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), and/or CM1004 (NRRL No. B- 67708).
  • Another embodiment is one or more of Bacillus strains CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM 1004 (NRRL No. B-67708), and MDG1607 (NRRL No. B-67666) in any combination.
  • a method of feeding an animal 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 CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • a method of feeding an animal 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 CM256 (NRRL No. B-67706), CM857 (NRRL No.
  • an effective amount of the Bacillus strain can be administered to inhibit E. coli, Salmonella, and/or Clostridium in the animal.
  • “inhibit E. coli, Salmonella, and/or Clostridium'' can mean reducing E. coli, Salmonella, and/or Clostridium disease, preventing E. coli, Salmonella, and/or Clostridium disease, maintaining the normal microbial balance in the animal, reducing the number of detrimental E. coli, Salmonella, and/or Clostridium in the animal, reducing the activity of E. coli, Salmonella, and/or Clostridium in the animal, or reducing the symptoms of E.
  • an amount of the Bacillus strain e.g., strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B- 67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), and/or MDG1607 (NRRL No. B-67666)) capable of E. coli, Salmonella, and/or Clostridium inhibition or capable of controlling a detrimental effect of E. coli, Salmonella, and/or Clostridium, as described below, by any mechanism.
  • the animal has a disease caused by E. coli, Salmonella, and/or Clostridium.
  • a method of controlling a detrimental effect of E. coli, Salmonella, and/or Clostridium comprises the steps of administering to an animal a feed composition or drinking water comprising an effective amount of an additive comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B- 67706 a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, and controlling the detrimental effect of E. coli, Salmonella, and/or Clostridium in the animal.
  • compositions of the present invention comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B- 67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No.
  • an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No.
  • CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM 1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No.
  • B- 67666 and combinations thereof, are provided in the form of an additive for addition to the drinking water of an animal.
  • the strains may be used as a litter treatment to control detrimental effects of E. coli, Salmonella, and/or Clostridium.
  • an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B- 67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No.
  • the feed composition may contain an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No.
  • B-67666 a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, in a mixture with an animal feed blend, including any art-recognized animal feed blend or any animal feed blend described herein.
  • feed composition or “animal feed composition” means a feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B- 67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B- 67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No.
  • the feed composition may be in the form of a ground meal.
  • any animal feed blend including those known in the art and those described herein, may be used in accordance with the methods and compositions described in this patent application, such as rapeseed meal, cottonseed meal, soybean meal, cornmeal, barley, wheat, silage, and haylage.
  • the animal feed blend can be supplemented with an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No.
  • B-67666 a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, but other ingredients may optionally be added to the animal feed blend, including other bacterial strains, such as other Bacillus strains or Lactobacillus strains.
  • optional ingredients of the animal feed blend include sugars and complex carbohydrates such as both water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides.
  • Other optional ingredients include dried distillers grain solubles, fat (e.g., crude fat), phosphorous, sodium bicarbonate, limestone, salt, phytate, calcium, sodium, sulfur, magnesium, potassium, copper, iron, manganese, zinc, ash, fish oil, an oil derived from fish meal, raw seed (e.g., flaxseed), an antioxidant, and starch.
  • minerals may be added in the form of a mineral premix.
  • Optional amino acid ingredients that may be added to the animal feed blend are arginine, histidine, isoleucine, leucine, lysine, cysteine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HC1, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ethyl HC1, and analogs, and salts thereof.
  • Vitamins that may be optionally added are thiamine HC1, riboflavin, pyridoxine HC1, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like.
  • vitamins may be added in the form of a vitamin premix.
  • protein ingredients may be added to the animal feed blend and include protein obtained from meat meal, bone meal, or fish meal, liquid or powdered egg, fish solubles, crude protein, and the like.
  • any medicament ingredients known in the art may be added to the animal feed blend or to an additive for the drinking water of the animal, such as antibiotics.
  • the antibiotic is selected from the group consisting of ampicillin, chloramphenicol, ciprofloxacin, clindamycin, tetracycline, chlortetracycline, DenagardTM (i.e., tiamulin), BMDTM (i.e., bacitracin methylene disalicylate), CarbadoxTM (i.e., carbadox), StafacTM (i.e., virginiamycin), erythromycin, levofloxacin, trimethoprim/ sulfamethoxazole, trimethoprim, daptomycin, rifampicin, TylanTM (i.e., tylosin), PulmotilTM (i.e., tilmicosin), vancomycin, and combinations
  • one or more enzymes may be added to the animal feed blend.
  • the enzymes that may be added include a galactosidase, a phytase, a protease, a lipase, an amylase, a hemicellulase, an arabinoxylanase, a xylanase, a cellulase, an NSPase, combinations thereof, and any other enzyme that improves the effectiveness of the feed composition for E. coli, Clostridium, and/or Salmonella inhibition or controlling a detrimental effect of E. coli, Clostridium, and/or Salmonella.
  • yeast, fungi e.g., Aspergillus or Trichoderma'
  • micronutrients may be added to the animal feed. Any of the ingredients described above that are suitable for addition to an additive for the drinking water of the animal may be added as a component of the additive for the drinking water of the animal as described herein.
  • an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B- 67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No.
  • a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, can be administered in the feed composition at a dose of about 1.0 x 10 3 CFU/gram of the feed composition to about 5.0 x 10 12 CFU/gram of the feed composition or at a dose of about 1.0 x 10 3 CFU/gram of the feed composition to about 1.0 x 10 7 CFU/gram of the feed composition.
  • the Bacillus strain is administered in the feed composition at a dose greater than about 1.0 x 10 3 CFU/gram of the feed composition, at a dose greater than about 1.1 x 10 3 CFU/gram of the feed composition, at a dose greater than about 1.25 x 10 3 CFU/gram of the feed composition, at a dose greater than about 1.5 x 10 3 CFU/gram of the feed composition, at a dose greater than about 1.75 x 10 3 CFU/gram of the feed composition, at a dose greater than about 1.0 x 10 4 CFU/gram of the feed composition, at a dose greater than about 2.0 x 10 4 CFU/gram of the feed composition, at a dose greater than about 3.0 x 10 4 CFU/gram of the feed composition, at a dose greater than about 4.0 x 10 4 CFU/gram of the feed composition, at a dose greater than about 5.0 x 10 4 CFU/gram of the feed composition, at a dose greater than about 6.0 x 10 4 CFU/gram of the feed composition
  • the Bacillus strain is administered in the feed composition at a dose of about 7.3 x 10 4 CFU/gram of the feed composition.
  • an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B- 67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • any of the dosages described herein can be in CFU/ml of drinking water in embodiments where the strains are administered in the drinking water of the animal.
  • the isolated Bacillus strains described herein are selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No.
  • Bacillus strains CM256, CM857, CM978, and CM1004 were deposited on November 29, 2018 at the Agricultural Research Service Culture Collection (NRRL), National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, 1815 North University Street, Peoria, Illinois 61604-3999, and were given accession numbers B-67706, B-67705, B-67703, and B-67708, respectively.
  • NRRL strain designations are B- 67706, B-67705, B-67703, and B-67708, which are equivalent to Bacillus strain CM256, CM857, CM978, and CM1004, respectively, as referred to in the application.
  • Bacillus strain MDG1607 (NRRL No. B-67666) was deposited on August 14, 2018 at the Agricultural Research Service Culture Collection (NRRL), International Depository Authority, 1815 North University Street, Peoria, Illinois 61604, and was given accession number B-67666.
  • the NRRL strain designation is MDG1607, which is equivalent to Bacillus strain MDG1607 or strain 1607, as referred to in the application. Any of these strains can be administered alone or in combination in the form of a feed composition (e.g., a complete feed comprising an animal feed blend) or drinking water for an animal. In one embodiment, multiple strains are administered in combination in a single composition. In another embodiment, multiple strains are administered in combination in separate compositions.
  • one or more of the Bacillus strains including isolated Bacillus strains selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • Bacillus strain CM978 a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, can be administered to the animal along with another bacterial strain selected from the group consisting of another Bacillus strain, a lactic acid bacterial strain, and combinations thereof.
  • CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, can be administered to the animal along with any other bacterial strain effective to inhibit or control detrimental effects of E. coli, Salmonella, and/or Clostridium in the animal.
  • a strain having all of the identifying characteristics of’ Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B- 67703), CM1004 (NRRL No. B-67708), or MDG1607 (NRRL No. B-67666) can be a mutant strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B- 67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B- 67708), or MDG1607 (NRRL No.
  • B-67666 e.g., a DNA fingerprint based on DNA analysis that corresponds to the DNA fingerprint of Bacillus strain Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), or MDG1607 (NRRL No. B-67666), enzyme activities that correspond to Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B- 67703), CM1004 (NRRL No. B-67708), or MDG1607 (NRRL No.
  • B-67666 antimicrobial activity that corresponds to Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), or MDG1607 (NRRL No. B-67666), antibiotic sensitivity and tolerance profiles that correspond to Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B- 67703), CM1004 (NRRL No. B-67708), or MDG1607 (NRRL No. B-67666), or combinations of these identifying characteristics).
  • the mutation can be a natural mutation, or a genetically engineered mutation.
  • a strain having all of the identifying characteristics of’ Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), or MDG1607 (NRRL No. B-67666) can be a strain, for example, produced by isolating one or more plasmids from Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B- 67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No.
  • the feed composition or drinking water comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B-67706 a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, may be administered to the animal for any time period that is effective to inhibit E. coli, Salmonella, and/or Clostridium or control a detrimental effect of E. coli, Salmonella, and/or Clostridium in the animal, or combinations thereof.
  • the feed composition or drinking water may be provided to the animal daily.
  • the feed composition or drinking water may be administered to the animal during lactation and/or during gestation.
  • the time periods for administration of the feed composition or drinking water described above are non-limiting examples and it should be appreciated that any time period or administration schedule determined to be effective to inhibit E. coli, Salmonella, and/or Clostridium or control a detrimental effect of E. coli, Salmonella, and/or Clostridium, or combinations thereof, may be used.
  • controlling a detrimental effect of E. coli, Salmonella, and/or Clostridium can mean reducing E. coli, Salmonella, and/or Clostridium disease, preventing E. coli, Salmonella, and/or Clostridium disease, maintaining the normal microbial balance in the animal, reducing the number of detrimental E. coli, Salmonella, and/or Clostridium in the animal, reducing the activity of E. coli, Salmonella, and/or Clostridium in the animal, or reducing the symptoms of E. coli, Salmonella, and/or Clostridium disease in the animal, or combinations thereof.
  • effective amount is meant an amount of the Bacillus strain capable of controlling a detrimental effect of E. coli, Salmonella, and/or Clostridium, as described above, by any mechanism.
  • compositions comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B- 67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B- 67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No.
  • a commercial package comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B- 67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B- 67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), and combinations thereof.
  • a feed additive for an animal feed comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • an additive for the drinking water of an animal comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B- 67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • B- 67705 a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), and combinations thereof.
  • an animal feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B- 67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • the feed additive for addition to an animal feed blend to produce a complete feed composition can be mixed with the animal feed blend, for example, with an automated micro-nutrient delivery system, or, for example, by hand-weighing and addition to achieve any of the doses of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B- 67666), a strain having all of the identifying characteristics of Bacillus strain MDG 1607 (NRRL No.
  • B-67666 a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), and combinations thereof, described herein, for administration to the animal in the form of a complete feed composition.
  • the mixing can also be done by any other suitable method known in the art for combining direct-fed microbials with an animal feed blend to obtain a uniform mixture.
  • the mixing can be done for any suitable time period (e.g., about 1 to about 4 minutes).
  • an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B- 67666), a strain having all of the identifying characteristics of Bacillus strain MDG 1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • Bacillus strain CM857 a strain having all of the identifying characteristics of Bacillus strain CM857
  • a strain having all of the identifying characteristics of Bacillus strain CM978 a strain having all of the identifying characteristics of Bacillus strain CM1004
  • the isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No.
  • B-67666 a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No.
  • B-67708), and combinations thereof can be in the form of, for example, a powder, a liquid, or pellets, and can be mixed with the drinking water using any suitable method known in the art to achieve any of the doses of the Bacillus strains described herein, for administration to the animal in the drinking water of the animal.
  • the isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No.
  • B-67705 a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B- 67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), and combinations thereof, can also be fed directly to the animal orally (e.g., by oral insertion) in the form of a powder, a liquid, or a pellet.
  • the feed additive, additive for the drinking water of the animal, or the feed composition can be in the form of a commercial package.
  • the feed additive or additive for the drinking water of the animal, or the isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No.
  • the feed additive, feed composition, or additive for the drinking water of the animal, or the isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No.
  • B-67666 a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B- 67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No.
  • B-677078 can be in a dry form (e.g., a powder), a pelleted form, a liquid form, in the form of a top-dressing, or in the form of a gel, or any other suitable form.
  • the strains in the form of a commercial package can be, for example, in a dry form (e.g., a powder or freeze-dried form), in a pelleted form, or in a liquid form.
  • a dry form e.g., a powder or freeze-dried form
  • a pelleted form e.g., a pellet or in a liquid form.
  • the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise a carrier for the isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM 1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • the carrier can be selected from the group consisting of a bran, rice hulls, a salt, mineral oil, a dextrin (e.g., maltodextrin), whey, sugar, sucrose, limestone, yeast culture, dried starch, sodium silico aluminate, silicon dioxide, polypropylene glycol, polysorbate 80, vegetable oil, and combinations thereof.
  • the carrier can be any suitable carrier known in the art for a direct-fed microbial.
  • the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise a binder such as clay, yeast cell wall components, aluminum silicate, glucan, or other known binders.
  • the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise inorganic/organic binders, essential oils, and/or organic acids.
  • the commercial package, feed additive, additive for the drinking water of the animal, or feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • the container can be, for example, a bag (e.g., a 20-pound bag, a 50-pound bag, a 2-ounce bag, a 1- pound bag, or a 1 -kilogram bag), a pouch, a drum, a bottle, or a box.
  • a bag e.g., a 20-pound bag, a 50-pound bag, a 2-ounce bag, a 1- pound bag, or a 1 -kilogram bag
  • a pouch e.g., a 20-pound bag, a 50-pound bag, a 2-ounce bag, a 1- pound bag, or a 1 -kilogram bag
  • a pouch e.g., a drum, a bottle, or a box.
  • the container for the commercial package, feed additive, additive for the drinking water of the animal, or feed composition comprising an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B- 67666), a strain having all of the identifying characteristics of Bacillus strain MDG 1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • the commercial package, feed additive, additive for the drinking water of the animal, or feed composition can further comprise instructions for use of one or more of the Bacillus strains.
  • isolated Bacillus strain MDG1607 (NRRL No. B-67666), or a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), can be used in combination with one or more of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • a method of treating a plant to inhibit a bacterial disease caused by E. coll, Salmonella, and/or Clostridium comprises contacting the plant with a composition comprising an effective amount of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), MDG1607 (NRRL No. B-67666), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B-67706 a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), and combinations thereof, and inhibiting E. coli, Salmonella, and/or Clostridium in the plant.
  • Bacillus strains CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), and MDG1607 (NRRL No. B-67666) can all used to treat the plant, or any of Bacillus strains CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), and/or MDG1607 (NRRL No. B-67666) can be used in any combination.
  • isolated Bacillus strain MDG1607 (NRRL No. B- 67666), or a strain having all of the identifying characteristics of Bacillus strain MDG1607 (NRRL No. B-67666), can be used to treat the plant in combination with one or more of an isolated Bacillus strain selected from the group consisting of Bacillus strain CM256 (NRRL No. B-67706), CM857 (NRRL No. B-67705), CM978 (NRRL No. B-67703), CM1004 (NRRL No. B-67708), a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No.
  • B-67708 a strain having all of the identifying characteristics of Bacillus strain CM256 (NRRL No. B-67706), a strain having all of the identifying characteristics of Bacillus strain CM857 (NRRL No. B-67705), a strain having all of the identifying characteristics of Bacillus strain CM978 (NRRL No. B-67703), a strain having all of the identifying characteristics of Bacillus strain CM1004 (NRRL No. B-67708), and combinations thereof, can be used in the same methods and compositions and for the same purpose as described herein for the whole cell preparations of the strains.
  • FIG. 1A shows antimicrobial screening utilizing the agar cross-streak method against various isolates of E. coli.
  • FIG. IB shows antimicrobial screening utilizing the agar cross-streak method against various isolates of Clostridium.
  • FIG. 1C shows antimicrobial screening utilizing the agar cross-streak method against various isolates of Salmonella.
  • RAPD-PCR Randomly Amplified Polymorphic DNA PCR method
  • RAPD-PCR The Randomly Amplified Polymorphic DNA PCR method
  • Preparation of the DNA to be used in the RAPD-PCR reaction was done by using the QIAGEN® Tissue and Blood single column kit (QIAGEN®, Venlo, The Netherlands). To obtain DNA, an overnight culture was prepared, struck for purity, pelleted, and DNA was extracted following the manufacturer’s protocol.
  • the RAPD-PCR reaction was performed with the following run conditions in a thermal cycler: 95°C 5 min, followed by 45 cycles of (95°C 1 min, 36°C 1 min, 72°C 2 min), followed by 72°C 7 min, and finished with a 4°C indefinite hold to preserve the product.
  • the RAPD-PCR product was analyzed by gel electrophoresis.
  • FIG. 2A illustrates RAPD-PCR results for CM256, where the first lane is primer 1 (Pl), followed by primer 2 (P2), the molecular weight ladder (MW), then primer 3 (P3), primer 4 (P4), primer 5 (P5), and primer 6 (P6).
  • FIG. 2B illustrates RAPD-PCR results for CM857, where the first lane is primer 1 (Pl), followed by primer 2 (P2), primer 3 (P3), primer 4 (P4), primer 5 (P5), primer 6 (P6), and the molecular weight ladder (MW) in the last lane.
  • FIG. 2A illustrates RAPD-PCR results for CM256, where the first lane is primer 1 (Pl), followed by primer 2 (P2), primer 3 (P3), primer 4 (P4), primer 5 (P5), primer 6 (P6), and the molecular weight ladder (MW) in the last lane.
  • FIG. 2C illustrates RAPD-PCR results for CM978, where the first lane is a molecular weight ladder (MW) and each subsequent lane is the banding pattern for primer 1 (Pl), primer 2 (P2), primer 3 (P3), primer 4 (P4), primer 5 (P5), and primer 6 (P6) in the last lane.
  • FIG. 2D illustrates RAPD-PCR results for CM1004, where the first lane is primer 1 (Pl), followed by primer 2 (P2), the molecular weight ladder (MW), then primer 3 (P3), primer 4 (P4), primer 5 (P5), and primer 6 (P6).
  • 2E illustrates RAPD-PCR results for MDG1607, where the first lane is a molecular weight ladder (MW) and each subsequent lane is the banding pattern for primer 1 (Pl), primer 2 (P2), primer 3 (P3), primer 4 (P4), primer 5 (P5), and primer 6 (P6) in the last lane.
  • MW molecular weight ladder
  • strain specific enzyme activity for lipids, carbohydrates, proteins, xylan plant fibers, and cellulose was tested using agar methods known in the art and described below. Briefly, strains CM256, CM857, CM978, CM1004, and MDG1607 were cultured from a frozen stock by using a sterile inoculating loop to iso-streak the cells on tryptic soy agar media and incubated overnight at 37°C. When the colonies were visible the next day, they were morphologically examined for purity and a sterile inoculating loop was used to collect a single colony. The single colony was cultured to a higher density by inoculating it into tryptic soy broth and incubated overnight at 37°C, 230 rpm.
  • Agar plates for lipase, amylase, protease, xylanase, and cellulase were prepared as follows.
  • tryptic soy agar containing 5% tween 80 and 0.25% Spirit Blue (solvent blue 38, phthalocyanine metal complex) was used.
  • tryptic soy agar containing 1% corn starch was used.
  • tryptic soy agar containing 1% casein was used.
  • For xylan plant fibers tryptic soy agar containing 1% xylan was used.
  • tryptic soy agar containing 0.5% carboxymethylcellulose was used.
  • ingredients were dissolved in the TSA media on a hot plate stirrer and subsequently autoclaved at 121 °C for 30 minutes.
  • agar was poured into sterile petri dishes and allowed to dry inside a biological safety cabinet.
  • the agar plates for each respective recipe were inoculated with lOpL spots of culture, in triplicate, dried, and incubated overnight at 37°C. The following day, results were analyzed by observation of clearing zones around the spotted colonies indicating positive enzymatic activity, or no clearing zone for negative enzymatic activity.

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Abstract

La présente invention concerne des microbes d'alimentation directe destinés à être utilisés dans l'inhibition d'E. coli, de Salmonell, et/ou de Clostridium chez l'animal. Plus particulièrement, l'invention concerne des souches isolées de Bacillus CM256 (NRRL N° B-67706), CM857 (NRRL N° B-67705), CM978 (NRRL N° B-67703), CM1004 (NRRL N° B-67708) et MDG1607 (NRRL N° B-67666), ainsi que des souches possédant toutes les caractéristiques d'identification de ces souches, pour une utilisation comprenant l'utilisation susmentionnée. L'invention concerne également l'utilisation de souches isolées de Bacillus CM256 (NRRL N° B-67706), CM857 (NRRL N° B-67705), CM978 (NRRL N° B-67703) et CM1004 (NRRL N° B-67708), ainsi que des souches possédant toutes les caractéristiques d'identification de ces souches, pour traiter des plantes atteintes de maladies provoquées par E. coli, Salmonella et/ou Clostridium .<u />
PCT/US2024/033048 2023-06-08 2024-06-07 Microbis pour inhibition bactérienne Pending WO2024254478A2 (fr)

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