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WO2025166034A1 - Utilisation de microbes en alimentation directe dans la prévention et/ou le traitement d'infections à base d'e. coli chez l'animal - Google Patents

Utilisation de microbes en alimentation directe dans la prévention et/ou le traitement d'infections à base d'e. coli chez l'animal

Info

Publication number
WO2025166034A1
WO2025166034A1 PCT/US2025/013832 US2025013832W WO2025166034A1 WO 2025166034 A1 WO2025166034 A1 WO 2025166034A1 US 2025013832 W US2025013832 W US 2025013832W WO 2025166034 A1 WO2025166034 A1 WO 2025166034A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
feed
bacillus
animal
feed additive
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/013832
Other languages
English (en)
Inventor
Chong SHEN
Joshua WALKER
Pia Bilde RASMUSSEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
International N&H Denmark ApS
Danisco US Inc
Original Assignee
International N&H Denmark ApS
Danisco US Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by International N&H Denmark ApS, Danisco US Inc filed Critical International N&H Denmark ApS
Publication of WO2025166034A1 publication Critical patent/WO2025166034A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the field relates to the use of direct-fed microbials in preventing and/or treating animals having an E. co/z-based infection.
  • Escherichia coli is a gram-negative, rod-shaped bacterium that normally inhabits the intestinal microflora or ecosystem of most mammalian and bird species. E. coli is classified into 150 to 200 serotypes or serogroups based on somatic (O), capsular (K), fimbrial (F) and flagellar (H) antigens. Most E. coli are commensals, that is, they reside in the intestine but are not harmful for the host animal. Only a small proportion of strains are pathogenic, producing virulence factors permitting them to cause disease. Some E. coli possess virulence genes in combinations not known to be associated with disease and may be considered as potentially pathogenic. All E. coli may carry genes for resistance to antimicrobial agents.
  • E. coli In animals, virulent strains of E. coli are responsible of a variety of diseases, among others septicemia and diarrhea in newborn calves, acute mastitis in dairy cows, colibacillosis also associated with chronic respiratory disease with Mycoplasma where it causes perihepatitis, pericarditis, septicemic lungs, peritonitis etc. in poultry, and Alabama rot in dogs.
  • E. coli bacteria are constantly being shed into the immediate environment of the animals via the feces, and contaminate the pens, litter, and floor of animals being housed indoors and the soil for outdoor animals. They can persist for long periods, possibly more than 10 weeks, and be spread via slurry and manure to fertilized fields and crops, and to ground and surface water. E. coli is transmitted to other animals via contaminated feed, handlers, and drinking water, and possibly farm to farm by vehicles such as transport trucks. Infection occurs by the oral route or via inhalation of contaminated dust in the case of birds. E. coli from animals may also be transmitted to humans by direct contact, or ingestion of food or water contaminated following spread of manure, or ingestion of meat following contamination of carcasses at the slaughterhouse.
  • ETEC Enterotoxigenic Escherichia
  • pigs and calves Intestinal infection due to Enterotoxigenic Escherichia (E.) coli
  • ETEC Enterotoxigenic Escherichia
  • pigs and calves Intestinal infection due to Enterotoxigenic Escherichia (E.) coli
  • ETEC in pigs is often contagious, the same strain being found in high numbers and in several sick pigs and from one batch to another. These strains are usually only shed for a few days after infection, probably due to the development of immunity.
  • a composition for preventing and/or treating an E.coli-based infection in an animal wherein said composition comprises a direct-fed microbial Bacillus-based component comprising Bacillus strains 2084 (NRRL B-50013); LSSA01 (NRRL B-50I04), and 15AP4 (PTA-6507) either alone or in combination with a culture supernatant derived from these strains.
  • the E. coli-based infection can be Enterotoxigenic Escherichia (E.) coli (ETEC).
  • the animal can be swine.
  • the infection can be neonatal and/or post-weaning diarrhea caused by ETEC.
  • the composition disclosed herein can produce one or more performance benefits in the animal (such as swine), the performance benefit being selected from the group consisting of increased body weight gain, improved gut barrier integrity, reduced mortality, positive modulation of cytokine profile, improved innate immunity, improved wound healing, reduced E. coli shedding in feces, and decreased neonatal and/or post-weaning diarrhea caused by ETEC.
  • the E. coli can be Enterotoxigenic Escherichia (E.) coli (ETEC).
  • the direct-fed microbial is in the form of an endospore.
  • any of the compositions described herein further comprise at last one enzyme which, optionally, may be encapsulated.
  • At least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase and beta-glucanase.
  • any of the compositions described herein can be a feed additive composition or a premix.
  • feed comprising any of the feed additive compositions disclosed herein.
  • kits comprising any of the feed additive compositions disclosed herein and instructions for administration.
  • a method for preventing and/or treating an E. coZZ-based infection in an animal which comprises administering an effective amount of a composition comprising a direct- fed microbial comprising Bacillus strains 2084 (NRRL B- 50013); LSSA01 (NRRL B-50104), and 15AP4 (PTA-6507).
  • the composition so administered can produce one or more performance benefits in the animal, the performance benefit being selected from the group consisting of increased bodyweight gain, improved gut barrier integrity, reduced mortality, positive modulation of cytokine profile, improved innate immunity, improved wound healing, and reduced E. coli shedding in feces.
  • the E. coli-based infection can be Enterotoxigenic Escherichia (E.) coli (ETEC).
  • the animal can be swine.
  • the infection can result in neonatal and/or post-weaning diarrhea caused by ETEC.
  • composition can encompass any of the features described above or elsewhere in this disclosure.
  • FIG. 2 shows fimbriae receptor expression of IPEC-J2 cells in a three Bacillus strain CFS coculture.
  • FIG. 4 depicts wound-healing of IPEC-J2 cells in Bacillus strain CFS coculture.
  • the data represent 2 experiments with 6 replicates. *: P ⁇ 0.05; **: PcO.Ol; ***: PcO.OOl, LSSA01 or Enviva EO vs TSB control.
  • FIG. 5A, FIG. 5B, and FIG. 5C depict three Bacillus strains (competitively) excluded ETECs adhesion to epithelial cells.
  • FIG. 5A depicts an exclusion assay: pretreatment of IPEC- J2 cells with three Bacillus strains (LSSA01, 15AP4 and 2084) prevent ETECs (EC-88, EC-23 and EC-90) adhesion.
  • the data represent 2 experiments with 6 replicates.
  • FIG. 5B shows a competitive exclusion assay: simultaneous treated IPEC-J2 cells with three Bacillus strains (LSSA01, 15AP4 and 2084) prevent ETECs (EC-88, EC-23 and EC-90) adhesion.
  • the data represent 2 experiments with 6 replicates.
  • FIG. 6 is a bar graph showing diarrhea score (upper) and percentage reduction in diarrhea score in pigs fed the experimental diets from day 1 to day 14 of study.
  • FIG. 7 is a bar graph depicting diarrhea frequency in weaned piglets fed experimental diets with a score above 3 from dl- 14 of the study
  • the term “about” refers to a range of +/- 0.5 of the numerical value, unless the term is otherwise specifically defined in context.
  • the phrase a “pH value of about 6” refers to pH values of from 5.5 to 6.5, unless the pH value is specifically defined otherwise.
  • an animal includes all non-ruminant (including humans) and ruminant animals.
  • the animal is a non-ruminant animal, such as a horse and a mono-gastric animal.
  • monogastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, sows; poultry such as turkeys, ducks, chicken, broiler chicks, layers; fish such as salmon, trout, tilapia, catfish and carps; and crustaceans such as shrimps and prawns.
  • the animal can be multigastric, such as a ruminant animal, including, but not limited to, cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and nilgai.
  • a ruminant animal including, but not limited to, cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and nilgai.
  • ruminant refers to a mammal that is able to acquire nutrients from plant-based food by fermenting it in a specialized stomach prior to digestion, principally, through microbial actions.
  • the process typically requires the fermented ingesta (known as cud) to be regurgitated and chewed again.
  • cud fermented ingesta
  • the process of rechewing the cud to further break down plant matter and stimulate digestion is called rumination.
  • Roughly 150 species of ruminants include both domestic and wild species. Ruminating animals include, but are not limited to, cattle, cows, goats, sheep, giraffes, yaks, deer, elk, antelope, buffalo and the like.
  • CFU colony forming units
  • DFM direct-fed microbial
  • a DFM can comprise one or more of such naturally occurring microorganisms such as bacterial strains. Categories of DFMs include spore-forming bacteria such Bacillus and Clostridium as well non-spore forming bacteria such as Eactic Acid Bacteria, Yeasts and Fungi. Thus, the term DFM encompasses one or more of the following: direct fed bacteria, direct fed yeast, direct fed yeast or fungi and combinations thereof.
  • Bacillus is a unique, gram-positive rod that forms spores. These spores are very stable and can withstand environmental conditions such as heat, moisture and a range of pH. These spores germinate into active vegetative cells when ingested by an animal and can be used in meal and pelleted diets.
  • Bacillus-based component refers to (i) a Bacillus -based direct fed microbial comprising the Bacillus bacterial strains described herein, (ii) a supernatant obtained from a Bacillus culture made from these strains or (iii) a combination of (i) and (ii).
  • a “feed” and a “food”, respectively, means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by a nonhuman animal and a human being, respectively.
  • the term "food” is used in a broad sense - and covers food and food products for humans as well as food for non-human animals (i.e. a feed).
  • feed is used with reference to products that are fed to animals in the rearing of livestock.
  • feed and “animal feed” are used interchangeably.
  • the food or feed is for consumption by monogastric and multigastric animals.
  • probiotic as used herein defines live microorganisms (including bacteria or yeasts for example) which, when for example ingested or locally applied in sufficient numbers, beneficially affects the host organism, i.e. by conferring one or more demonstrable health benefits on the host organism. Probiotics may improve the microbial balance in one or more mucosal surfaces.
  • the mucosal surface may be the intestine, the urinary tract, the respiratory tract or the skin.
  • probiotic as used herein also encompasses live microorganisms that can stimulate the beneficial branches of the immune system and at the same time decrease the inflammatory reactions in a mucosal surface, for example the gut.
  • At least 10 6 - 10 12 Whilst there are no lower or upper limits for probiotic intake, it has been suggested that at least 10 6 - 10 12 , preferably at least 10 6 - IO 10 , preferably 10 8 - 10 9 , cfu as a daily dose will be effective to achieve the beneficial health effects in a subject.
  • prebiotic means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of beneficial bacteria.
  • pathogen means any causative agent of disease.
  • causative agents can include, but are not limited to, bacterial, viral, fungal causative agents and the like.
  • E.coli -based infection means a disease or infection, such as diarrhea caused by E. coli bacteria (such as ETEC).
  • ETEC E. coli bacteria
  • the terms “entertoxigenic Escherichia coli” or “ETEC” are used interchangeably to reference a major pathogen responsible for illnesses, such as intestinal disease and/or diarrhea in man and farm animals (for example, swine).
  • This E. coli strain is the principal causal agent of traveller’s diarrhea in humans.
  • neonatal and post-weaning diarrhea caused by ETEC is one of the most economically important porcine diseases.
  • ETEC strains are believed to be responsible for the death of 10.8% of all pre-weaned pigs and up to more than 3% of all weaned pigs.
  • derived from and “obtained from” refer to not only a protein produced or producible by a strain of the organism in question, but also a protein encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protein which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protein in question.
  • the term “effective amount” means a sufficient amount of the specified component administered to an animal to achieve the desired effect.
  • compositions for preventing and/or treating an E. coli-based infection such as ETEC
  • said composition comprises a direct- fed microbial Bacillus-based component comprising Bacillus strains 2084 (NRRL B-50013); LSSA01 (NRRL B-50104), and 15AP4 (PTA-6507).
  • the composition disclosed herein can produce one or more performance benefits in the animal, the performance benefit being selected from the group consisting of increased bodyweight gain, improved gut barrier integrity, reduced mortality, positive modulation of cytokine profile, improved innate immunity, improved wound healing, and reduced E. coli shedding in feces.
  • the Bacillus-based DFM component described herein may comprise viable bacteria or may comprise supernatant or be a combination of viable bacteria and culture supernatant.
  • the preferred Bacillus-based DFM component is viable bacteria.
  • the DFM may be a spore forming bacterial strain and hence the term DFM may be comprised of or contain spores, e.g. bacterial spores.
  • the term “viable bacteria” as used herein may include bacterial spores, such as endospores or conidia.
  • the DFM in a feed additive composition described herein may not comprise of or may not contain bacterial spores, e.g. endospores or conidia.
  • the Bacillus -based DFM component described herein is a combination of the following strains:
  • Bacillus strain LSSA01 (a.k.a. Bacillus strain BS8) Accession No. NRRL B-50104, and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507.
  • Bacillus strains 2084 (NRRL B-50013); LSSA01 (NRRL B-50104), and 15AP4 (PTA-6507) is commercially available as Enviva® PRO from Danisco Animal Nutrition & Health.
  • the Bacillus-based DFM component be heat tolerant, i.e., is thermotolerant e.g., spore-forming. This is particularly the case when feed is pelleted. Bacilli are able to form stable endospores when conditions for growth are unfavorable and are very resistant to heat, pH, moisture and disinfectants. If the bacterium/DFM is not a spore-former then it should be protected to survive feed processing as is described hereinbelow.
  • a Bacillus-based component as described herein may be prepared as culture(s) and carrier(s) (where used) and can be added to a ribbon or paddle mixer and mixed for about 15 minutes, although the timing can be increased or decreased. The components are blended such that a uniform mixture of the cultures and carriers result.
  • the final product is preferably a dry, flowable powder.
  • a Bacillus -based component can comprise a: a Bacillus-based direct fed microbial comprising the three Bacillus bacterial strains described herein or a supernatant obtained from a Bacillus culture or a combination of both Bacillus bacterial strain or strains and supernatant.
  • Such a Bacillus-based component can then be added to animal feed or a feed premix. It can be added to the top of the animal feed (“top feeding”) or it can be added to a liquid such as the animal’s drinking water.
  • Suitable dosages of the Bacillus -based component as described herein in animal feed may range from about IxlO 3 CFU/g feed to about IxlO 10 CFU/g feed, suitably between about IxlO 4 CFU/g feed to about IxlO 8 CFU/g feed, suitably between about 7.5xl0 4 CFU/g feed to about I lO 7 CFU/g feed.
  • Animal feeds may include plant material such as com, wheat, sorghum, soybean, canola, sunflower or mixtures of any of these plant materials or plant protein sources for poultry, pigs, ruminants, aquaculture and pets.
  • animal feed can comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as com gluten meal, Distillers Dried Grains with Solubles (DDGS) (particularly com based Distillers Dried Grains with Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; and/or e)
  • cereals such as small grains (e.g., wheat, barley,
  • a Bacillus-based component as described herein may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient.
  • At least one component selected from the group consisting of a protein, a peptide, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben and propyl paraben.
  • a Bacillus -based component as described herein may be admixed with a feed component to form a feedstuff.
  • feed component means all or part of the feedstuff. Part of the feedstuff may mean one constituent of the feedstuff or more than one constituent of the feedstuff, e.g. 2 or 3 or 4 or more.
  • the term "feed component” encompasses a premix or premix constituents.
  • the feed may be a fodder, or a premix thereof, a compound feed, or a premix thereof.
  • a feed additive composition comprising a Bacillus-based component as described herein may be admixed with a compound feed or to a premix of a compound feed or to a fodder, a fodder component, or a premix of a fodder.
  • fodder means any food which is provided to an animal (rather than the animal having to forage for it themselves). Fodder encompasses plants that have been cut.
  • fodder includes hay, straw, silage, compressed and pelleted feeds, oils and mixed rations, and also sprouted grains and legumes.
  • Fodder may be obtained from one or more of the plants selected from: alfalfa (lucerne), barley, birdsfoot trefoil, brassicas, Chau moellier, kale, rapeseed (canola), rutabaga (swede), turnip, clover, alsike clover, red clover, subterranean clover, white clover, grass, false oat grass, fescue, Bermuda grass, brome, heath grass, meadow grasses (from naturally mixed grassland swards, orchard grass, rye grass, Timothy-grass, corn (maize), millet, oats, sorghum, soybeans, trees (pollard tree shoots for tree -hay), wheat, and legumes.
  • alfalfa lucerne
  • barley birdsfoot trefoil
  • brassicas Chau moellier
  • kale kale
  • rapeseed canola
  • rutabaga rutabag
  • compound feed means a commercial feed in the form of a meal, a pellet, nuts, cake or a crumble.
  • Compound feeds may be blended from various raw materials and additives. These blends are formulated according to the specific requirements of the target animal.
  • Compound feeds can be complete feeds that provide all the daily required nutrients, concentrates that provide a part of the ration (protein, energy) or supplements that only provide additional micronutrients, such as minerals and vitamins.
  • the main ingredients used in compound feed are the feed grains, which include com, soybeans, sorghum, oats, and barley.
  • a premix as referred to herein may be a composition composed of microingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients. Premixes are usually compositions suitable for blending into commercial rations.
  • Any feedstuff described herein may comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as com gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins.
  • cereals such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large
  • feedstuff may contain at least 30%, at least 40%, at least 50% or at least 60% by weight com and soybean meal or corn and full fat soy, or wheat meal or sunflower meal.
  • a feedstuff may comprise at least one high fibre feed material and/or at least one by-product of the at least one high fibre feed material to provide a high fibre feedstuff.
  • high fibre feed materials include: wheat, barley, rye, oats, by products from cereals, such as com gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp.
  • Some protein sources may also be regarded as high fibre: protein obtained from sources such as sunflower, lupin, fava beans and cotton.
  • feed may be one or more of the following: a compound feed and premix, including pellets, nuts or (cattle) cake; a crop or crop residue: com, soybeans, sorghum, oats, barley, corn stover, copra, straw, chaff, sugar beet waste; fish meal; freshly cut grass and other forage plants; meat and bone meal; molasses; oil cake and press cake; oligosaccharides; conserved forage plants: hay and silage; seaweed; seeds and grains, either whole or prepared by crashing, milling etc.; sprouted grains and legumes; yeast extract.
  • a compound feed and premix including pellets, nuts or (cattle) cake
  • a crop or crop residue including pellets, nuts or (cattle) cake
  • a crop or crop residue including pellets, nuts or (cattle) cake
  • a crop or crop residue including pellets, nuts or (cattle) cake
  • a crop or crop residue including pellets, nuts or (cattle) cake
  • feed as used herein also encompasses in some embodiments pet food.
  • a pet food is plant or animal material intended for consumption by pets, such as dog food or cat food.
  • Pet food, such as dog and cat food may be either in a dry form, such as kibble for dogs, or wet canned form.
  • Cat food may contain the amino acid taurine.
  • the term feed may also encompass in some embodiments fish food.
  • a fish food normally contains macro nutrients, trace elements and vitamins necessary to keep captive fish in good health. Fish food may be in the form of a flake, pellet or tablet. Pelleted forms, some of which sink rapidly, are often used for larger fish or bottom feeding species.
  • Some fish foods also contain additives, such as beta carotene or sex hormones, to artificially enhance the color of ornamental fish.
  • bird food including food that is used both in birdfeeders and to feed pet birds.
  • bird food comprises of a variety of seeds, but may also encompass suet (beef or mutton fat).
  • the term "contacted" refers to the indirect or direct application of the feed additive composition to the product (e.g. the feed).
  • the application methods include, but are not limited to, treating the product in a material comprising the feed additive composition, direct application by mixing the feed additive composition with the product, spraying the feed additive composition onto the product surface or dipping the product into a preparation of the feed additive composition.
  • the Bacillus -based component may be preferably admixed with the product (e.g. feedstuff). Alternatively, it may be included in the emulsion or raw ingredients of a feedstuff.
  • the Bacillus-based component may be applied to intersperse, coat and/or impregnate a product (e.g. feedstuff or raw ingredients of a feedstuff) with a controlled amount of a Bacillusbased component.
  • a product e.g. feedstuff or raw ingredients of a feedstuff
  • the DFMs described herein can be added in suitable concentrations, for example, in concentrations in the final feed product which offer a daily dose of between about 2xl0 3 CFU/g of feed to about 2xlO n CFU/g of feed, suitably between about 2xl0 6 to about IxlO 10 , suitably between about 3.75xl0 7 CFU/g of feed to about IxlO 10 CFU/g of feed.
  • the Bacillus-based component will be thermally stable to heat treatment up to about 70 °C; up to about 85°C; or up to about 95°C.
  • the heat treatment may be performed from about 30 seconds up to several minutes.
  • thermally stable means that at least about 50% of Bacillus-based component that was present/active before heating to the specified temperature are still present/active after it cools to room temperature.
  • the Bacillus-based component is homogenized to produce a powder.
  • the Bacillus-based component is formulated to granules as described in W02007/044968 (referred to as TPT granules) incorporated herein by reference.
  • the granules comprise a hydrated barrier salt coated over the protein core.
  • the advantage of such salt coating is improved thermo-tolerance, improved storage stability and protection against other feed additives otherwise having adverse effect on the at least one protease and/or DFM comprising one or more bacterial strains.
  • the salt used for the salt coating has a water activity greater than 0.25 or constant humidity greater than 60% at 20°C.
  • the salt coating comprises a Na2SO4.
  • Feed containing the Bacillus-based component may be produced using a feed pelleting process.
  • the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets.
  • the mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection.
  • the mixture is heated in the conditioner up to a specified temperature, such as from 60-100°C, typical temperatures would be 70°C, 80°C, 85°C, 90°C or 95 °C.
  • the residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minutes 2 minutes., 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.
  • At least one coating may comprise a moisture hydrating material that constitutes at least 55% w/w of the granule; and/or at least one coating may comprise two coatings.
  • the two coatings may be a moisture hydrating coating and a moisture barrier coating.
  • the moisture hydrating coating may be between 25% and 60% w/w of the granule and the moisture barrier coating may be between 2% and 15% w/w of the granule.
  • the moisture hydrating coating may be selected from inorganic salts, sucrose, starch, and maltodextrin and the moisture barrier coating may be selected from polymers, gums, whey and starch.
  • the granule may be produced using a feed pelleting process and the feed pretreatment process may be conducted between 70°C and 95 °C for up to several minutes, such as between 85°C and 95°C.
  • the Bacillus-based component may be formulated to a granule for animal feed comprising: a core; an active agent, the active agent of the granule retaining at least 80% activity after storage and after a steam-heated pelleting process where the granule is an ingredient; a moisture barrier coating; and a moisture hydrating coating that is at least 25% w/w of the granule, the granule having a water activity of less than 0.5 prior to the steam-heated pelleting process.
  • the granule may have a moisture barrier coating selected from polymers and gums and the moisture hydrating material may be an inorganic salt.
  • the moisture hydrating coating may be between 25% and 45% w/w of the granule and the moisture barrier coating may be between 2% and 10% w/w of the granule.
  • a granule may be produced using a steam-heated pelleting process which may be conducted between 85 °C and 95°C for up to several minutes.
  • the composition is in a liquid formulation suitable for consumption preferably such liquid consumption contains one or more of the following: a buffer, salt, sorbitol and/or glycerol.
  • the feed additive composition may be formulated by applying, e.g. spraying, the Bacillus-based component onto a carrier substrate, such as ground wheat for example.
  • such feed additive composition comprising a Bacillus -based component as described herein may be formulated as a premix.
  • the premix may comprise one or more feed components, such as one or more minerals and/or one or more vitamins.
  • the composition is in a liquid formulation suitable for consumption preferably such liquid consumption contains one or more of the following: a buffer, salt, sorbitol and/or glycerol.
  • the feed additive composition may be formulated by applying, e.g., spraying, the Bacillus -based component onto a carrier substrate, such as ground wheat for example.
  • such Bacillus-based component as described herein may be formulated as a premix.
  • the premix may comprise one or more feed components, such as one or more minerals and/or one or more vitamins.
  • Bacillus-based component as disclosed herein is suitable for addition to any appropriate feed material.
  • feed material refers to the basic feed material to be consumed by an animal. It will be further understood that this may comprise, for example, at least one or more unprocessed grains, and/or processed plant and/or animal material such as soybean meal or bone meal.
  • the feedstuff may comprise feed materials comprising maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/ or any of the by-products, as well as protein rich components like soybean mean, rape seed meal, canola meal, cotton seed meal, sunflower seed mean, animal-by-product meals and mixtures thereof. More preferably, the feedstuff may comprise animal fats and I or vegetable oils.
  • the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins.
  • additional minerals such as, for example, calcium and/or additional vitamins.
  • the feedstuff is a com soybean meal mix.
  • Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives.
  • the feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and enzyme may be added.
  • Production of feedstuff may also involve an additional step that includes extrusion or expansion prior to pelleting, in particular, by suitable techniques that may include at least the use of steam.
  • the feedstuff may be a feedstuff for a monogastric animal, such as poultry (for example, broiler, layer, broiler breeders, turkey, duck, geese, waterfowl), swine (all age categories), a pet (for example dogs, cats) or fish, preferably the feedstuff is for poultry.
  • poultry for example, broiler, layer, broiler breeders, turkey, duck, geese, waterfowl
  • swine all age categories
  • a pet for example dogs, cats
  • fish preferably the feedstuff is for poultry.
  • the Bacillus -based component described herein may be placed on top of the animal feed, i.e., top fed. Alternatively, the Bacillus -based component described herein may be added to a liquid such as in the drinking water of the animal.
  • contacted refers to the indirect or direct application of a Bacillus -based component as described herein to a product (e.g. the feed).
  • application methods include, but are not limited to, treating the product in a material comprising the Bacillus-based component, direct application by mixing a feed additive composition Bacillus-based component as described herein with the product, spraying such feed additive composition onto the product surface or dipping the product into a preparation of the feed additive composition.
  • a feed additive composition Bacillus-based component as described herein is preferably admixed with the product (e.g. feedstuff).
  • the feed additive composition may be included in the emulsion or raw ingredients of a feedstuff. This allows the composition to impart a performance benefit.
  • a method of preparing the Bacillus -based component as described herein may also comprise the further step of pelleting the powder.
  • the powder may be mixed with other components known in the art.
  • the powder, or mixture comprising the powder may be forced through a die and the resulting strands are cut into suitable pellets of variable length.
  • the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets.
  • the mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection.
  • the mixture is heated in the conditioner up to a specified temperature, such as from 60-100°C, typical temperatures would be 70°C, 80°C, 85°C, 90°C or 95°C.
  • the residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.
  • the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins.
  • the feedstuff is a corn soybean meal mix.
  • Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives.
  • the feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and enzyme may be added.
  • Production of feedstuff may also involve an additional step that includes extrusion or expansion prior to pelleting, in particular by suitable techniques that may include at least the use of steam.
  • Bacillus -based component and/or a feedstuff comprising the same may be used in any suitable form. It may be used in the form of solid or liquid preparations or alternatives thereof.
  • solid preparations include powders, pastes, boluses, capsules, pellets, tablets, dusts, and granules which may be wettable, spray-dried or freeze-dried.
  • liquid preparations include, but are not limited to, aqueous, organic or aqueous- organic solutions, suspensions and emulsions.
  • the feed additive compositions may be mixed with feed or administered in the drinking water.
  • a Bacillus-based component comprising admixing a Bacillus -based component as described herein with a feed acceptable carrier, diluent or excipient, and (optionally) packaging.
  • the feedstuff and/or Bacillus -based component may be combined with at least one mineral and/or at least one vitamin.
  • the compositions thus derived may be referred to herein as a premix.
  • the feedstuff may comprise at least 0.0001 % by weight of Bacillus -based component.
  • the feedstuff may comprise at least 0.0005%; at least 0.0010%; at least 0.0020%; at least 0.0025%; at least 0.0050%; at least 0.0100%; at least 0.020%; at least 0.100% at least 0.200%; at least 0.250%; at least 0.500% by weight of the Bacillus-based component.
  • a food or Bacillus -based component may further comprise at least one physiologically acceptable carrier.
  • the physiologically acceptable carrier is preferably selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na2SO4, Talc, PVA and mixtures thereof.
  • the food or feed may further comprise a metal ion chelator.
  • the metal ion chelator may be selected from EDTA or citric acid.
  • a Bacillus -based component as described herein can be formulated with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na SCM, Talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate and mixtures thereof.
  • a Bacillus-based component as described herein will be in a physiologically acceptable carrier.
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient. Once formulated, the can be administered directly to the subject.
  • the subjects to be treated can be animals.
  • composition disclosed herein can produce one or more performance benefits in the animal (i.e., animal performance), the performance benefit being selected from the group consisting of increased bodyweight gain, improved gut barrier integrity, reduced mortality, positive modulation of cytokine profile, improved innate immunity, improved wound healing, and reduced E. coli shedding in feces.
  • animal performance is determined by feed efficiency and/or weight gain of the animal and/or by the feed conversion ratio.
  • improved animal performance it is meant that there is increased feed efficiency, and/or increased weight gain and/or reduced feed conversion ratio and/or improved gut barrier integrity and/or decreased mortality and/or reduced E. coli shedding in feces and/or positive modulation of cytokine profile (for example, decreased expression of IL6, increased expression of IL10, and/or increased expression of plgR,), and/or improved innate immunity, and/or improved wound healing, and/or reduced E. coli shedding in feces in comparison to feed or a feed additive composition which does not comprise the Bacillus strains disclosed herein.
  • the improvement is any one of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or greater (including percentages falling within these values) improved animal performance in comparison to feed or a feed additive composition which does not comprise the Bacillus strains disclosed herein.
  • feed efficiency refers to the amount of weight gain in an animal that occurs when the animal is fed ad- libitum or a specified amount of food during a period of time.
  • feed additive composition in feed results in an increased weight gain per unit of feed intake compared with an animal fed without said feed additive composition being present.
  • feed conversion ratio refers to the amount of feed fed to an animal to increase the weight of the animal by a specified amount.
  • An improved feed conversion ratio means a lower feed conversion ratio.
  • lower feed conversion ratio or “improved feed conversion ratio” it is meant that the use of a feed additive composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the feed does not comprise a feed additive composition as disclosed herein.
  • Gut integrity and microbiota appear to be helpful in maintaining gut health. Supporting the intestinal barrier helps to decrease the risk of infection and inflammation. For example, tight junctions are closely associated areas of two cells whose membranes join together forming a barrier virtually impermeable to fluid. The ability to protect tight junction integrity can improve the health of an animal. Tight junctions also need to be maintained to avoid a “leaking gut” which can result in further cell damage. Thus, improving gut integrity by helping or increasing the ability of animal to maintain a well-regulated barrier function that hinder bacteria from entering the animal’s body unintentionally is desirable.
  • Tight junctions also known as occluding junctions are the closely associated areas of two cells whose membranes join together forming a barrier virtually impermeable to fluid.
  • tight junctions are the channels between the gut epithelial cells that can lead to either good or poor gut integrity.
  • tight junction proteins such as Claudin 3 and Occludin are expressed between two epithelial cells at higher levels, helping to provide a barrier that can prevent the translocation of pathogens from the gut lumen into the systemic circulation (reduced permeability).
  • Claudins are the most important family of tight junction proteins and claudin 3 is one of the genes that encodes for these tight junction proteins.
  • Occludin is another important tight junction protein.
  • Claudins and Occludin were the first tight junctional integral membrane proteins identified. Measurement of tight junction protein RNA can serve as an indicator of barrier integrity and gut health because if tight junctions are not maintained in the gastrointestinal tract of an animal, it can result in permeability that may allow the translocation of pathogens and toxins from the gut lumen into the systemic circulation and, thus compromise animal health or even result in death of the animal (for example sepsis).
  • Reduced E. coli shedding in feces by using the composition as taught herein can reduce further spreading of an E.coZz-based infection (such as ETEC), and improve animal performance as well.
  • E.coZz-based infection such as ETEC
  • survival means the number of subjects remaining alive.
  • improved survival may be another way of saying “reduced mortality”.
  • An “increased weight gain” refers to an animal having increased body weight on being fed feed comprising a feed additive composition compared with an animal being fed a feed without said feed additive composition being present.
  • compositions and methods disclosed herein include: .
  • composition of embodiment 1 wherein the said composition produces one or more performance benefits selected from the group consisting of of increased body weight gain, improved gut barrier integrity, reduced mortality, positive modulation of cytokine profile, improved innate immunity, improved wound healing, and reduced E. coli shedding in feces. .
  • the composition of embodiments 1 or 2 wherein the direct-fed microbial is in the form of an endo spore. .
  • the composition of embodiments 1 or 2 wherein said composition further comprises at last one enzyme which, optionally, may be encapsulated. .
  • composition of embodiment 3 wherein said composition further comprises at last one enzyme which, optionally, may be encapsulated. .
  • composition of embodiment 4 wherein the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase and beta-glucanase. .
  • Feed comprising the feed additive composition of embodiments 8 or 9.
  • a kit comprising the feed additive composition of embodiment 7 and instructions for administration.
  • a method for preventing and/or treating an E. co/z-based infection such as ETEC
  • an E. co/z-based infection such as ETEC
  • an animal which comprises administering an effective amount of a composition comprising a Bacillus -based direct-fed microbial component Bacillus strains 2084 (NRRL B-50013); LSSA01 (NRRL B-50104), and 15AP4 (PTA-6507).
  • the method of embodiment 14 wherein the composition produces one or more performance benefits selected from the group consisting of increased body weight gain, improved gut barrier integrity, reduced mortality, positive modulation of cytokine profile, improved innate immunity, improved wound healing, and reduced E. coli shedding in feces.
  • the method of embodiments 14 or 15 wherein the direct-fed microbial is in the form of an endo spore.
  • composition further comprises at last one enzyme which, optionally, may be encapsulated.
  • composition further comprises at last one enzyme which, optionally, may be encapsulated.
  • the method of embodiment 17 wherein the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase and beta-glucanase.
  • the method of embodiment 18 wherein the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase and beta-glucanase.
  • composition is a feed additive composition or a premix.
  • compositions 1-9 or the method of any one of embodiments 14-22 wherein said composition is a feed additive composition or a premix.
  • ETEC Enterotoxigenic Escherichia
  • Example 1 B. velezensis prevent pathogen growth and host colonization in pig production
  • a cell free supernatant contains metabolites resulting from microbial growth and residual nutrition of the medium used.
  • CFS cell free supernatant
  • Lactobacillus spp. and Pediococcus spp. (Drummond MM, 2023; Kaewchomphunuch T, 2022) have antimicrobial activity against Escherichia coli or Pseudomonas aeruginosa. This is due to its content of organic acids, fatty acids, and proteinaceous compounds.
  • CFS from 3 strains of Bacillus was investigated to determine if the combination has any growth inhibitory effect on bacteria pathogens commonly seen in post weaning diarrhea (PWD) in pigs. These include Enterotoxigenic Escherichia coli (ETECs), Clostridium perfringens (CP) and Salmonella.
  • ETECs Enterotoxigenic Escherichia coli
  • CP Clostridium perfringens
  • Salmonella Salmonella
  • B. velezensis 27 was included in the study for comparison.
  • ETECs, CP and Salmonella are from the IFF Danisco Animal Nutrition collection which have been previously genetically characterized.
  • Bacteria CFS was prepared by growing B. velezensis in TSB medium at 32°C till OD 0.25-0.3. The bacterial culture was then centrifuged and sterile filtered and stored at -20°C till use. Inoculum plates were prepared by growing ETEC and Salmonella grew in TSB and incubated aerobically, while CP in BHI+YS+LC media and incubated anaerobically. All plates were cultured at 37 °C for 4 hours and CFS were then added and cultured for another 16 hours before OD600 reading.
  • the CFS of the three Bacillus strains significantly inhibited F4 ETEC growth. This was ensembled by all 3 strains (LSSA01, 38.019 ⁇ 7.277 %; 15AP4, 47.209 ⁇ 4.595 %; 2084, 51.405 ⁇ 4.216 %). All of them were significantly better compared to strain 27. Similarly, the inhibitory effect could be observed in F18 ETEC and Salmonella. In addition, LSSAOl also hindered the growth of CP.
  • Example 2 Bacillus strains have higher binding affinity to IPEC-J2 cells
  • IPEC-J2 cells are porcine intestinal epithelial cells that were isolated from neonatal piglet mid-jejunum. IPEC-J2 cells differentiate and exhibit enterocytic features, including microvilli and tight junctions (Bresnahan AJ, 2012). Enterotoxigenic Escherichia coli (ETEC) has been reported to bind via fimbriae (mainly F4, or F18) and their receptors (F4 receptor, F4R, or F18R, Luppi A, 2016; Xia P, 2016). In this Example, the ability of the three Bacillus strains to bind to IPEC-J2 cells was investigated.
  • ETEC Enterotoxigenic Escherichia coli
  • IPEC-J2 pig intestinal epithelial cell line
  • DSMZ Steunschweig, Germany
  • Cells were maintained in DMEM supplemented with 20% FBS, at 37°C with 5% CO2 atmosphere.
  • Cell cultures were supplemented with antibiotics (Penicillin and Streptomycin, lOOx). Normocin were added every three months (Invivogen, Toulouse France).
  • the three Bacillus strains utilized in this study were from fi. velezensis (LSSA01, 15AP4 and 2084).
  • ETEC was from the IFF Danisco Animal Nutrition collection. All bacteria were grown on Tryptic Soy Broth (TSB) broth at 37°C under an anaerobic atmosphere (Anaerocult, Merck, Darmstadt, Germany).
  • Bacteria strains were grown for 2 days, and then concentrations adjusted respectively before loading onto pig IPEC-I2 epithelial cells and cocultured for 30 mins. The cell monolayer was then gently washed and lysed with cold Triton X-100. The lysates containing total cell- associated bacteria were diluted serially in PBS and plated onto Tryptic Soy Agar (TSA agar) plates at 37 °C for the enumeration of adherent bacteria (Bacteria adhered). In parallel, the bacteria suspension was separately diluted serially and plated onto TSA agar plates (Bacteria loaded).
  • TSA agar Tryptic Soy Agar
  • Example 3 Bacillus strains down-regulate ETEC fimbriae binding receptors of epithelial cells.
  • ETECs utilize fimbriae receptor to bind and colonize in the gut. Consequently, downregulation of receptor expression provides a rationale to mitigate their onset.
  • ETEC fimbriae binding receptors in epithelial cells was investigated. Materials and Methods
  • IPEC-J2 cells were prepared and maintained as described in Example 2.
  • Cell-free supernatants (CFS) from B. velezensis (LSSA01, 15AP4 and 2084 respectively) were prepared from cultures grown in TSB for 48 h at 37°C. The absorbance of the resulting suspension was read at 600 nm and the concentration adjusted to an OD of 1 by the addition of culture medium, as necessary. The bacterial suspensions were then plated on agar, grown and colonies counted. It was determined that an OD of 1 was equivalent to a bacterial concentration of IxlO 7 CFU/ml. CFS was added to reach final IxlO 7 CFU/ml and coculture with IPEC-J2 cells for 6 hours. Cells were then harvested for RT-PCR analysis.
  • Example 4 Three Bacillus strains positively modulate cytokine profile and enhance innate immunity
  • Gut mucosa maintains a delicate homeostasis as evidenced by a balance of pro- and antiinflammatory cytokines. Modulation of cytokine production is a common approach to reestablish the balance in gut infectious disease (Liu Y, 2021). Insufficient IgA plays a role in gut infectious diseases including post weaning diarrhea in piglets (Johansen FE, 2011). plgR specifically transports IgA from submucosal to luminal side. plgR up-regulation may enhance luminal IgA content and facilitate gut health. In this Example, the ability of three Bacillus strains to positively modulate cytokine profile and enhance innate immunity was investigated.
  • CFS of the three Bacillus strains reduced IL-6 production of IPEC- J2 cells, from 15.751 ⁇ 0.018 (TSB control) to 11.826 ⁇ 0.020 (LSSA01), 14.007 ⁇ 1.009 (15AP4) and 12.758 ⁇ 0.068 (2084) respectively; IL-10 was enhanced by LSSA01 (0.300 ⁇ 0.028) and 15AP4 (0.435 + 0.041, vs TSB control, 0.159 + 0.018).
  • plgR was improved by all 3 strains consistently.
  • Gut health relies heavily on the gut barrier, including an intact epithelia cell layer. Exposure to toxin, pathogens and stress frequently results in “wound” formation in the gut. A quick healing thus is critical to reduce the exposure to these detrimental factors and prevent the host from a further infection (Leoni G, 2015). In this Example, Bacillus strain LSSAOl’s ability to facilitate wound healing is examined.
  • IPEC-J2 were seeded in a culture-insert 2 Well 35 mm p-Dish and grown for 3 days until confluent. A “wound” was then made by removing the 2 “well-insert” in the middle, which covers an 8 mm x 8 mm square area. Bacillus strain LSSA01 supernatant was added (l*10 6 CFU/ml). “Healing” was recorded after 24 hours microscope observation. Enviva® EO (100 ng/ml) is an IFF phytogenic product and was introduced into the assay and run in parallel.
  • Bacillus strain LSSA01 CFS facilitated healing in 48 hours.
  • the healing area is significantly higher with Bacillus strain LSSA01 with 43.067 ⁇ 4.288% compared to 32.333 ⁇ 6.271% in TSB control.
  • Example 6 Three Bacillus strains competitively exclude ETECs adhesion to epithelial cells
  • IPEC-J2 cells three Bacillus strains (Bacillus velenzensis LSSA01, 2084 and 1584) and ETECs were grown and prepared as described previously in Example 2.
  • pig IPEC-J2 cells were first loaded with individual Bacillus strains and then washed and further challenged with ETEC (1:10 ratio by OD);
  • ETEC 1:10 ratio by OD
  • For the Competitive Exclusion assay cells were loaded with individual Bacillus strains and ETEC simultaneously. Cells were then washed and lysed. The lysate was placed on TSA agar (for Bacillus and ETEC) and MacConkey agar (for ETEC) respectively. Colony was counted 24-48h later and data are from 2 experiments with 6 duplicates. Bacteria alone without using in cell assay were also plated on agar plate in parallel, used for CFU count. The final calculation is based on CFU counts.
  • Bacillus strain LSSA01 and 15AP4 were further investigated with multiple ETECs farm isolates.
  • Bacillus strain LSSA01 showed a strong exclusion capability as evidenced by >75% ETECs elimination while strain 15AP4 varies from 28.145% till 95.042% (FIG. 5C).
  • Bacillus strains LSSA01 and 15AP4 both exhibited >50% ETECs exclusion rate.
  • Example 7 Efficacy of three Bacillus strains on growth performance in weaned piglets
  • This example demonstrates the efficacy of Bacillus strains LSSA01, 15AP4 and 2084 (Enviva® PRO) when supplemented in postweaning diets in maintaining or improving piglet growth performance and reducing the incidence of diarrhoea when fed diets without antimicrobials and pharmacological levels of Zinc.
  • Dietary treatments Pigs were fed nutrient- adequate control diet (CON) and CON supplemented with a 3-strain Bacillus direct fed microbial (Enviva® PRO @ Ikg/MT of feed) providing 3.0 x 10 8 cfu/kg feed. Diets were fed ad lib as mash in 1 feeding phase (1-42 days). The 2 dietary treatments tested are as detailed in Table 2. Control diet composition is given in Table 3.
  • vitamin A 12,000 IU; vitamin D3, 2,500 IU; vitamin E, 30 IU; vitamin K3, 3 mg; D-pantothenic acid, 15 mg; nicotinic acid, 40 mg; choline, 400 mg; and vitamin B12, 12
  • Feed intake was calculated biweekly as the total amount of feed distributed per pen minus residual feed, divided by the number of animals per pen during each period.
  • Feed conversion ratio was calculated biweekly as feed intake divided by body weight gain per pen corrected for mortality using pig days.
  • Microbial profile Fecal samples were collected from pigs on days 0 and 21 of the study (2 pigs per pen and from 8 replicate pens replicates; 16 samples/treatment) to determine the microbial population using 16S amplicon sequencing.
  • Table 4 Growth performance in weaned pigs fed diets supplemented with Enviva PRO
  • ADG Average Daily Gain
  • ADFI Average Daily Feed Intake
  • the fecal consistency score and diarrhea frequency are presented in FIG. 6 and FIG. 7.
  • Example 8 Efficacy of three Bacillus strains on growth performance in weaned piglets
  • This example demonstrates the efficacy of Bacillus strains LSSA01, 15AP4 and 2084 (Enviva® PRO) when supplemented in postweaning diets in improving piglet growth performance and promoting beneficial microbiome communities when fed diets without antimicrobials and pharmacological levels of Zinc.
  • Experimental design and housing The experiment was carried out as a randomized complete block design with 2 dietary treatments, 20 replicates and 10 pigs per pen. A total of 400 mixed sex (1:1) pigs (LW x LR x D) weaned at 28 days of age with an average BW of 7.2 + 1.5 kg were blocked by body weight and sex, and randomly assigned to the 2 dietary treatments from weaning until 6 weeks post-weaning. The pens were housed in a temperature and ventilation- controlled facility.
  • Dietary treatments Pigs were fed nutrient adequate control diet (CON) and CON supplemented with a 3-strain Bacillus direct-fed microbial (Enviva® PRO @ Ikg/MT of feed) providing 3.0 x 10 8 cfu/kg feed. Diets were fed ad lib as mash in 2 feeding phases (1-14 and 14- 42 days). The 2 dietary treatments tested are as detailed in Table 5. The control diet composition is given in Table 6.
  • Table 5 Description of 2 dietary treatments evaluated in the study.
  • vitamin A 20,000 IU; vitamin D3, 3,500 IU; vitamin E, 50 IU; vitamin K3, 3.75 mg; vitamin Bl, 2.5 mg; Vitamin B2, 5 mg; Vitamin B6, 5 mg; Vitamin B12, 0.05 mg; D-pantothenic acid, 12.5 mg; nicotinic acid, 25 mg; choline, 312 mg; and folic acid, 0.63 mg; Fe, 170 mg; Cu, 125 mg ; Zn, 130 mg; Mn, 37 mg; I, 1.1 mg mg; Se, 0.50 mg
  • Table 7 Growth performance in weaned pigs fed diets supplemented with Enviva® PRO
  • the microbiome analysis showed that the microbial communities started with similar composition on day 0 (for both treatments), then gradually shifted over time, and became significantly different (beneficial taxa) on day 21 and 42.
  • Differential abundance analysis revealed that Segatella copri and Clostridium butyricum on day 21, Clostridium saudiense, Faecalibacterium prausnitzii and Xylanibacter sp. on day 42, were more abundant (P ⁇ 0.05) in the Enviva PRO diets compared to control.

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Abstract

L'invention concerne une composition et une méthode de prévention et/ou de traitement d'une infection à base d'E. coli chez l'animal.
PCT/US2025/013832 2024-01-31 2025-01-30 Utilisation de microbes en alimentation directe dans la prévention et/ou le traitement d'infections à base d'e. coli chez l'animal Pending WO2025166034A1 (fr)

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