Classifications

• • 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
  • 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
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the Bacillus genus comprises numerous endospore-forming bacteria that have myriad uses in the agricultural and animal nutrition fields, among others.
• Several strain of Bacillus are currently marketed for use as probiotics in animal feed as an alternative to antibiotics. These probiotics enhance animal health, including improving animal growth and feed efficiency, by modulating the gastrointestinal flora.
• Use of such probiotics has increased due to concerns about antibiotic residues in animal products for human consumption and the development of resistance to antibiotics. Work has been conducted in recent years to screen spore- forming bacteria for use as probiotics.
• various commercial products contain strains of Bacillus subtilis, Bacillus licheniformis, and Bacillus coagulans, such screening reveals that not all Bacillus strains are effective feed additives.
• the present invention provides a strain of Bacillus subtilis that, when administered to an animal, enhances the health of such animal.
• the present invention relates to methods for enhancing the health of non-insect and non-human animals or for improving the general physical condition of such animals by feeding to such animals, in feed or drinking water (and not through gavage), compositions comprising (i) Bacillus subtilis QST 713, (ii) a mutant of (i), a cell-free preparation of (i) or (ii), or a metabolite of (i) or (ii).
• the composition of the present invention comprises Bacillus subtilis 713 or mutants thereof and metabolites produced by the bacteria.
• the composition comprises Bacillus subtilis QST713 mainly in its spore form.
• the compositions are administered to animals in feed over multiple days throughout the animal's life or during particular stages or portions of the animal's life.
• the compositions are administered only in a starter diet or only in a finisher diet of farm animals.
• the methods of the present invention may be used to increase weight gain of an animal, to increase feed utilization efficiency, to reduce morbidity, to increase disease resistance, to increase survival rates, to increase the immune response of the animal and to maintain healthy gut microflora.
• the methods of this invention are used to assist with re-establishing a healthy balance of gut microflora after administration of a course of antibiotics for therapeutic purposes.
• the composition of the present invention comprises Bacillus subtilis QST713 or mutants thereof and is administered to an animal at a rate of about 1x10 CFU/g feed or ml drinking water, or about IxIO 4 CFU/g feed or ml drinking water or about IxIO 5 CFU/g feed or ml drinking water, or about IxIO 6 CFU/g feed or ml drinking water, or about 1x10 7 CFU/g feed or ml drinking water, or about 1x10 CFU/g feed or ml drinking water, or about 1x10 CFU/g feed or ml drinking water, or about IxIO 10 CFU/g feed or ml drinking water, or about 1x10 11 CFU/g feed or ml drinking water.
• the methods of the present invention may be used for all non-human and non-insect animals.
• Animals that may benefit from methods of the present invention include but are not limited to birds, swine, ruminants, pets and exotic animals, zoo animals, aquatic animals, and horses, among others.
• the animals are farm animals, which are raised for consumption or as food-producers, such as broilers and egg-producing chickens.
• Figure 2 represents results of a test of a heat-treated cell-free preparation of Bacillus subtilis QST713 for efficacy against various isolates of Clostridia.
• Figure 3 shows results of a test of a cell-free preparation of Bacillus subtilis QST713 for efficacy against various isolates of Listeria.
• Figure 5 shows results of a test of a cell-free preparation of Bacillus subtilis QST713 for efficacy against various isolates of Salmonella.
• Figure 6 represents results of a test of a heat-treated cell-free preparation of Bacillus subtilis QST713 for efficacy against various isolates of Salmonella.
• Figure 8 shows agar plates on which Bacillus subtilis QST713 (vertical) and various isolates of Campylobacter jejuni (horizontal) were cross streaked in order to test efficacy of QST713 against the pathogens.
• the present invention relates to a novel use of Bacillus subtilis strain QST 713 and/or its metabolites that are effective to enhance animal health as a probiotic.
• Probiotics are used in animal health applications in order to maintain healthy gut microflora, including a reduction in detrimental bacteria such as Clostridia and Campylobacter and an increase in beneficial bacteria such as Lactobacillus spp. and Bifidobacterium.
• Probiotics are well-suited to maintaining a healthy balance between pathogenic and beneficial bacteria because, unlike antibiotics, they do not destroy bacteria indiscriminately nor do they lead to antibiotic resistant strains of pathogenic bacteria.
• the present invention encompasses a method for enhancing animal health by administering to an animal a composition comprising (i) Bacillus subtilis QST713, (ii) mutants of Bacillus subtilis QST713, (iii) cell-free preparations of (i) or (ii), or (iv) metabolites of (i) or (ii).
• Bacillus subtilis QST713, its mutants, its supernatants, and its lipopeptide metabolites, and methods for their use to control plant pathogens and insects are fully described in U.S. Patent Nos. 6,060,051, 6,103,228, 6,291,426, 6,417,163, and 6,638,910. In these patents, the strain is referred to as AQ713.
• Bacillus subtilis QST713 has been deposited with the NRRL on May 7, 1997 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure under Accession Number B21661. Any references in this specification to QST713 refer to Bacillus subtilis QST713.
• the Bacillus subtilis QST713 strain has certain properties, which, surprisingly, have been found to make the strain well-suited for enhancing animal health. Spores of QST713 are viable at low pHs and cells of QST713 grow (given conducive nutrient conditions) at pHs as low as 4.5. In addition, as described in Examples 8 and 4, respectively, below, QST713 is able to grow in high salt conditions for at least ten days and can survive the high temperatures necessary for pelleting animal feed. QST713 also has the ability to aggregate, or swarm, as shown in Example 2, thereby outcompeting and reducing pathogenic bacteria.
• compositions administered to animals comprise mutants of Bacillus subtilis QST713 having all the identifying characteristics of QST713.
• Such mutants may have DNA sequence identity to QST713 of at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
• mutants are spontaneous mutants.
• spontaneous mutant refers to mutants that arise from QST713 without the intentional use of mutagens. Such spontaneous mutants may be obtained by classical methods, such as growing the Bacillus subtilis strain in the presence of a certain antibiotic to which the parent is susceptible and testing any resistant mutants for improved biological activity or, in this application, improved ability to enhance one or more of the indicia of animal health described below. Other methods for identifying spontaneous mutants will be known to those of ordinary skill in the art.
• Bacillus subtilis QST713 or its mutants refer to bacteria that have been isolated from nature and are grown by humans, for example, in the laboratory or under industrial conditions.
• Bacillus subtilis QST713 cells may be present in the compositions of the present invention as spores (which are dormant), as vegetative cells (which are growing), as transition state cells (which are transitioning from growth phase to sporulation phase) or as a combination of all of these types of cells.
• the composition comprises mainly spores.
• Metabolites of QST713 or its mutants include lipopeptides, such as iturins, surfactins, plipastatins, and agrastatins and other compounds with antibacterial properties. Lipopeptide metabolites of QST713 are described in detail in U.S. Patent Nos. 6,291,426 and 6,638,910.
• compositions of the present invention can be obtained by culturing Bacillus subtilis QST713 or its mutants according to methods well known in the art, including by using the media and other methods described in US Patent No. 6,060,051.
• Conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture.
• Bacillus subtilis QST713 cells Towards the end of fermentation, as nutrients are depleted, Bacillus subtilis QST713 cells begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of this Bacillus subtilis and is generally initiated by the cell in response to nutrient limitation.
• Fermentation is configured to obtain high levels of colony forming units of Bacillus subtilis QST713 and to promote sporulation.
• the bacterial cells, spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential-flow filtration, depth filtration, and evaporation.
• the concentrated fermentation broth is washed, for example via a diafiltration process, to remove residual fermentation broth and metabolites.
• the fermentation broth or broth concentrate can be dried with or without the addition of carriers using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation.
• the resulting dry products may be further processed, such as by milling or granulation, to achieve a specific particle size or physical format. Carriers, described below, may also be added post-drying.
• Cell-free preparations of fermentation broth of QST713 can be obtained by any means known in the art, such as extraction, centrifugation and/or filtration of fermentation broth. Those of skill in the art will appreciate that so-called cell-free preparations may not be devoid of cells but rather are largely cell-free or essentially cell-free, depending on the technique used (e.g., speed of centrifugation) to remove the cells.
• the resulting cell-free preparation may be dried and/or formulated with components that aid in its administration to animals. Concentration methods and drying techniques described above for fermentation broth are also applicable to cell-free preparations.
• Metabolites of QST713 can be obtained according to the methods set forth in US Patent No. 6,060,051.
• the term "metabolites” as used herein may refer to semi-pure and pure or essentially pure metabolites, or to metabolites that have not been separated from Bacillus subtilis QST713.
• the lipopeptides and other bacteriacidal metabolites of QST713 are between 600 kilodaltons and 100 daltons.
• the metabolites may be purified by size exclusion filtration that groups metabolites into different fractions based on molecular weight cut-off, such as molecular weight of less than 600 kDa, less than 500 kDa, less than 400 kDa and so on. Concentration methods and drying techniques described above for formulation of fermentation broth are also applicable to metabolites.
• compositions of the present invention may include carriers, which are inert formulation ingredients added to compositions comprising cells, cell-free preparations or metabolites to improve recovery, efficacy, or physical properties and/or to aid in packaging and administration.
• carriers may be added individually or in combination.
• the carriers are anti-caking agents, anti-oxidation agents, bulking agents, and/or protectants.
• useful carriers include polysaccharides (starches, maltodextrins, methylcelluloses, proteins, such as whey protein, peptides, gums), sugars (lactose, trehalose, sucrose), lipids (lecithin, vegetable oils, mineral oils), salts (sodium chloride, calcium carbonate, sodium citrate), and silicates (clays, amorphous silica, fumed/precipitated silicas, silicate salts).
• Suitable carriers for animal feed additives are set forth in the American Feed Control Officials, Inc.' s Official Publication, which publishes annually. See, for example Official Publication of American Feed Control Officials, Sharon Krebs, editor, 2006 edition, ISBN 1-878341-18-9.
• the carriers are added after concentrating fermentation broth and during and/or after drying.
• the concentration on a weight by weight basis (w/w) of (i) Bacillus siibtilis QST713 or its mutants, (ii) cell-free preparations of QST713 or its mutants, (iii) metabolites of QST713 or its mutants or (iv) combinations of cells and metabolites in the formulated composition may be about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17% about 18%, about 19%, about 20% about 25%, about 30%, about, 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
• the concentration of Bacillus subtilis QST 713 or its mutants in the final composition may be from about 90% to about 100%.
• the compositions of the present invention may be administered/fed to non-insect and non-human animals to improve animal health or the general overall physical condition of such animals.
• the compositions can be administered both for therapeutic and non-therapeutic applications.
• An effective amount of a composition is an amount effective to enhance the health of an animal in comparison to an animal that has not been administered the composition but otherwise has been administered the same diet (including feed and other compounds) as has the animal receiving the compositions of the present invention.
• Indicia of enhanced health include one or more of the following: increase in weight gain, which may include an increase in weight of a specific part of the animal or an increase in overall weight; maintenance of gut microflora; increase in feed utilization efficiency; reduction in risk of mortality; increase in disease resistance; reduction in morbidity; increase in immune response; decrease in occurrence of diarrhea, increase in productivity; and/or reduction of pathogen shedding.
• embodiments of the present application are directed to non-therapeutic methods such as increasing the weight of the animal, the maintenance of gut microflora, or an increase in feed utilization efficiency by administering/feeding to the animal a composition comprising Bacillus subtilis QST 713, a mutant of Bacillus subtilis QST 713, a cell-free preparation derived from Bacillus subtilis QST713 or its mutant, or metabolites of QST713 or its mutants.
• the compositions of the present invention are administered/fed to farm animals
• the compositions are administered in order to improve growth performance of the farm animal.
• improvements to growth performance refer to increased growth (weight or length) and/or feed utilization efficiency and/or decreased mortality/increased survival rate compared to animals that have not been administered the compositions of the present invention.
• weight increases of between about 1% and about 20%, or between about 1% and about 15%, or between about 1% and about 9% are achieved.
• the method of the present invention also may increase feed utilization efficiency in animals as compared to animals to which the compositions of the present invention have not been administered. Feed efficiency is typically evaluated using the feed conversion ratio, which is the ratio of feed consumption to weight gain.
• Feed efficiency may be improved by between about 1% and 15%, between about 2% and about 10% and between about 3% and about 8%.
• the methods of the present invention may also reduce mortality. Survival rate improvements of between about 1% and about 20%, or between about 2% and 17% or between about 4% and about 13% or between about 5% and about 10% may be achieved.
• Increased growth, feed efficiency improvements and decreased mortality may be determined individually compared to averages known in the animal husbandry field or by comparing averages of growth performance data from a group of farm animals of about the same age, typically raised together and/or under similar conditions, some of which do not receive the compositions of the present invention.
• Maintenance of gut microflora refers to decreasing (by killing or inhibiting the growth of) harmful, disease-causing microorganisms of public health concern and/or increasing beneficial bacteria, such as Lactobacilli and Bifidobacteria, as compared to an animal to which the methods of this invention have not been applied.
• beneficial bacteria such as Lactobacilli and Bifidobacteria
• increases to beneficial bacteria may be caused by stimulating growth of such bacteria or simply by selectively decreasing pathogenic bacteria, thereby giving the beneficial bacteria more space to grow and to attach to the gut wall.
• Harmful, disease-causing bacteria that may be decreased by the methods of this invention include Clostridia spp. (such as perfringens and perate), Listeria spp.
• harmful, disease-causing microorganisms may be reduced by about 0.5 log, about 1 log, about 2 log, about 3 log, about 4 log, or about 5 log.
• Pathogen quantities may be determined by several methods known to those of skill in the art, including analyzing pathogen shed in animal feces or by sacrificing animals during studies and analyzing the populations of bacteria (beneficial and pathogenic) in their gut.
• the methods of the present invention may also be used to restore normal intestinal balance after administration of therapeutic amounts of antibiotics by inhibiting growth of pathogenic bacteria and/or increasing or maintaining growth of beneficial bacteria.
• therapeutic amount refers to an amount sufficient to ameliorate or reverse a disease state in an animal.
• Increased productivity obtained through the methods of the present invention refers to any of the following: production of more or higher quality eggs, milk or meat or increased production of weaned offspring.
• the methods of the present invention may be applied to any animal, including vertebrates, such as mammals and aquatic animals, and crustaceans, such as shrimp, but excluding insects and humans.
• Mammals that may be treated with the composition of the present invention include farm animals; animals used for sports, recreation or for work, such as horses, including race horses; domestic household pets, including dogs, cats, birds and exotics; and zoo animals. Farm animals refer to animals raised for consumption or as food-producers.
• the method is applied to monogastric animals such as poultry and game birds. Poultry may include chicken, turkey, duck, geese, guinea fowl and ratite, such as ostrich and emu.
• Game birds may include quail, chukkar, pheasant, grouse, Cornish hens, and partridge.
• Chicken refers to meat-bearing chicken, which encompass chickens which are raised for slaughter, which are also called broilers, and egg-producing chickens, which are those that are used to produce eggs for human consumption.
• the method may be applied to mammals such as swine.
• the method may be applied to polygastric animals, such as cattle, goat and sheep, also referred to herein as ruminants.
• the compositions of this invention may be fed to preruminants to enhance their health and, in particular, to decrease the incidence of diarrhea in these animals.
• Preruminants are ruminants, including calves, ranging in age from birth to about twelve weeks.
• the compositions of the present invention may be administered to preruminants in conjunction with milk replacers.
• Milk replacers refer to formulated feed intended to replace colostrum during milk feeding stages of the preruminant.
• compositions of the present invention are feed additives that are added to the subject animal's feed or drinking water prior to feeding.
• the compositions may be formulated with a carrier, such as calcium carbonate or whey protein, as described above.
• a carrier such as calcium carbonate or whey protein, as described above.
• such carrier is hygrophobic.
• compositions comprising Bacillus subtilis QST713, its mutants, cell-free preparations of QST713 and its mutants, and metabolites of QST713 and its mutants can be formulated in combination with animal feed ingredients needed to promote and maintain growth of an animal.
• Such animal feed ingredients may include one or more of the following: protein, carbohydrate, fats, vitamins, minerals, coccidiostats, acid-based products and/or medicines, such as antibiotics.
• carriers such as those described above, will also be present.
• Protein and carbohydrates needed to promote and maintain growth shall be referred to as feed protein and feed carbohydrate to distinguish them from any residual proteins and/or carbohydrates that may remain from the bacterial fermentation process.
• compositions of the present invention comprising Bacillus subtilis QST713, its mutants, cell-free preparations of QST713 and its mutants and metabolites of QST713 and its mutants may further include other probiotics, such as other species and strains of Bacillus that are fed to animals to enhance animal health or to improve the general physical condition of the animal.
• Exemplary strains include Bacillus subtilis PB6 (as described in US Patent No. 7,247,299 and deposited as ATCC Accession No. PTA-6737), which is sold by Kemin under the trademark CLOSTAT ® or Bacillus subtilis C-3102 (as described in US Patent No.
• compositions of the present invention may include or be administered with (either at the same time or at different times) enzymes that aid in digestion of feed, such as amylase, glucanase, glucoamylase, cellulase, xylanase, glucanase, amylase and pectinase; immune modulators, such as antibodies, cytokines, spray-dried plasma; interleukins, interferons; and/or oligosaccharides, such as fructooligosaccharides, mannanoligosaccharides, galactooligosacharides, inulin, oligofructose enriched inulin, tagatose, and polydextrose.
• enzymes that aid in digestion of feed
• immune modulators such as antibodies, cytokines, spray-dried plasma
• interleukins, interferons such as antibodies, cytokines, spray-dried plasma
• oligosaccharides such as fructo
• the bacteria should be added to feed or drinking water and fed to animals in an amount that is effective to enhance the animals' health. In one embodiment, it can be added at an inclusion rate of from about 1 x 10 4 CFU Bacillus subtilis per gram feed or ml drinking water to about 1 x 10 10 Bacillus subtilis per gram feed or ml drinking water. In another embodiment from about 1 x 10 5 CFU Bacillus subtilis per gram feed or ml drinking water to about 1 x 10 9 Bacillus subtilis per gram feed or ml drinking water should be administered.
• the inclusion rate is about 1 x 10 3 CFU Bacillus subtilis per gram feed or ml drinking water, or about 1 x 10 4 or about IxIO 5 or about 1 x 10 6 or about 1 x 10 7 or about 1 x 10 8 or about 1 x 10 9 or about 1 x 10 1 or about 1 x 10 1 CFU Bacillus subtilis per gram feed or ml drinking water.
• compositions containing QST713 or its mutants are provided as feed additives, such compositions should have a CFU count that allows for dilution to the above-described ranges upon addition to the animal feed or drinking water.
• compositions comprising Bacillus subtilis QST713 or its mutants, cell-free preparations thereof, or metabolites thereof, can be added to animal feed prior to the pelleting process, such that the composition used in the above-described method forms part of animal feed pellets.
• bacterial cells are used in the composition they are typically added in spore form to other components of the animal feed prior to the pelleting process.
• Standard pelleting processes known to those of skill in the art may be used, including extrusion processing of dry or semi-moist feeds.
• the pelleting process involves temperatures of at least about 65° C. In others, pelleting temperatures are between about 65° C and about 120° C.
• pelleting temperatures are between about 80 0 C and about 100° C. In yet others, the pelleting temperature is about 60° C, about 65° C, about 70° C, about 75° C, about 80° C, about 85° C, about 90° C or about 100° C.
• compositions of the present invention can also be administered orally as a pharmaceutical in combination with a pharmaceutically acceptable carrier.
• Optimal dosage levels for various animals can easily be determined by those skilled in the art, by evaluating, among other things, the composition's ability to (i) inhibit or reduce pathogenic bacteria in the gut at various doses, (ii) increase or maintain levels of beneficial bacteria and /or (ii) enhance animal health at various doses.
• compositions of the present invention may be added to fish rearing waters (rather than or in addition to fish feed) in an amount effective to enhance the health of the fish.
• effective amounts can be between about 10 4 and about 10 10 CFU Bacillus subtilis QST713 per ml of rearing water, or in another embodiment between about 10 5 and about 10 9 CFU Bacillus subtilis QST713 per ml of rearing water, or in yet another embodiment between about 10 and about 10 CFU Bacillus subtilis QST713 per ml of rearing water.
• Clostridia Clostridia perfringens ATCC 13124 and two environmental isolates of Clostridia perfringens
• Listeria Listeria moncytogenes ATCC 19116 and 19111, Listeria seeligeri ATCC 35968 and Listeria welshimeri ATCC 35897
• Salmonella Salmonella enterica ATCC 10398, Salmonella arizonae ATCC 13314 and Salmonella bongori ATCC 43975
• E. coli using Kirby-Bauer and minimal inhibitory concentration (MIC) techniques.
• Cell-free preparations were prepared by growing QST713 in media corresponding to media in which the target pathogen was grown, as shown in Table 1 , below, centriruging the culture for 15 minutes at 3000 rpm at 23 C and filtering it through a 0.45 ⁇ m Nalgene filter unit. To test for heat stability, a portion of the cell-free preparation was heated to 50 0 C for one hour before each of the Kirby-Bauer and MIC tests Table 2
• a powder formulation of Bacillus subtilis QST713 was tested for efficacy against various environmental isolates of the following bacteria: Clostridium perfringens, Escherichia coli, Samonella enteritidis, Campylobacter jejuni, and Listeria monocytogenes.
• This powder formulation was prepared by fermenting Bacillus subtilis QST713, concentrating the fermentation broth, and drying, as described above in the Detailed Description of Invention. It had 14.6% concentrated, dried broth and 85.4% formulation inerts (chosen from the possibilities described above) and contained at a minimum approximately 7.3 x 10 9 CFU Bacillus subtilis/g ⁇ am and at a maximum approximately Ix 10 10 CFU Bacillus subtilis i 'gram.
• Composition 1 This formulation shall be referred to herein as Composition 1.
• Stock solutions of Composition 1 were prepared by adding 0.2 gram of the formulated powder to 1.8 ml of sterile distilled water, such that the solution contained roughly IxIO 9 CFU Bacillus subtilis per ml. Test organisms were streaked to trypticase soy agar with 5% sheep blood with up to four organisms streaked to a single agar plate each in a single line that bisects the agar plate. The organisms were allowed to dry overnight. Then, the inoculated plates were streaked with the suspension of formulated QST713 described above, which was swabbed perpendicular to the test organisms.
• Clostridium perfringens and Campylobacter jejuni isolates were incubated in a Campy gas atmosphere (10% CO2, 5% 02, 8% N2) at 41 ⁇ 2 C overnight.
• the other isolates, which are aerobic, were incubated in 36 ⁇ 2 overnight without Campy gas.
• QST713 caused inhibition of several of the isolates of Clostridium perfringens, Salmonella enteritidis, Campylobacter jejuni and Listeria monocytogenes, although no inhibition of E. coli.
• Bacillus subtilis QST713 showed aggressive competitive overgrowth of the pathogenic bacteria. Results are summarized in the table below.
• Composition 1 was added to starter and finisher diets of broiler chickens and weight gain and feed efficiency observed. 252 Jumbo Cornish Cross broiler chicks were randomly separated into four groups and fed one of the diets listed below.
• Composition 1 (0.5g/kg; 10 6 CFU/g) (designated as Comp.l- I O 6 in the table below)
• Composition 1 (0.5 mg/kg; 10 3 CFU/g) (designated as Comp. 1-10 3 in the table below)
• the basal diet consisted of the following starter diet for days 1-22 and the following finisher diet for days 22-42.
• TOTAL 100.000 Ingredients were mixed in a Foberg mixer at ambient temperatures and then heated to various target temperatures at which they were maintained for about 30 s before being pelleted at about 2000 lbs/hour through a 5/32" x 1 !4" pellet die. Ten samples were taken from ten different places throughout the mixer. Pellet samples were taken at target temperatures of 65° C, 75° C, 80° C, and 85° C within the same 750 Ib. batch.
• a trial using QST713 in feed pellet form was conducted using 750 pigs in a nursery pig setting. Composition 1 was added to feed prior to its pelletization by standard processes. Studies of CFU of QST713 before and after the standard pelletization process were consistent with the results obtained in Example 4, above, and showed that CFU of QST713 did not decrease dramatically after pelleting.
• Approximate starting weight/pig was 10 lbs., and goal was for pigs to grow to approximately 40 lbs.
• a control treatment consisting of a standard diet without any antibiotics or Bacillus was fed to approximately 250 pigs.
• Another 250 pigs received the standard diet plus IxIO 6 CFU Bacillus subtilis QST713 per g of feed.
• a third group of 250 pigs was fed 1x10 7 CFU Bacillus subtilis QST713 per gram feed. The number of culled pigs in the third group was significantly reduced compared to the control group.
• the practice of culling involves removing less healthy or undersized pigs from feeding to be given therapeutic products or euthanized.
• Composition 1 was added to standard feed for poultry (not containing other probiotics or antibiotics) at a rate of 91 grams Composition 1 per ton feed (approximately 6.64 x l ⁇ " CFU/ton).
• One of the control groups and the group of birds administered feed supplemented with Composition 1 were challenged with Clostridium perfringens on days, 19, 20 and 21 of the study. Weight was recorded throughout the study and is shown in Table 8, below. Feed efficiency was determined and reported as the feed conversion ratio in Table 9, below. The feed conversion ratio was adjusted with the weights of dead and removed birds.
• NE Necrotic Enteritis
• Feed Conversion Ratio feed to gain ratio
• Composition 1 CP 1.765 B 1.964 B 2.128 ⁇ 0.77 B
• Trials are conducted to test growth performance improvement using a second formulation of Bacillus subtilis QST713.
• This powder formulation is prepared by fermenting, concentrating the fermentation broth, drying it and washing it via a diafiltration process to remove residual fermentation medium and metabolites, all as described above, such that the composition is comprised essentially of cells - mainly spores and some vegetative cells.
• This composition contains 14.6% concentrated, dried, washed culture and 85.4% formulation inerts (chosen from the possibilities described above in the Detailed Description of Invention) and about 1.0 x 10 10 CFU Bacillus subtilis/g ⁇ am and shall be referred to herein as Composition 2.
• Composition 2 is substituted for Composition 1 in the trials described in Examples 3-6 and results are expected to be the same as those achieved with Composition 1.
• Dilution bottles of l%Nutrient Broth and 1% NaCl were prepared (99ml per bottle) and sterilized by autoclaving.
• Nutrient Agar was prepared according to the manufacturer's directions; 15x100mm plates were prepared for the plate count testing, one plate per test plus uninoculated controls.
• Turbidity readings were obtained using a Spectronic 20D+ instrument, 12x75 polystyrene fraction collector tubes as cuvettes, and 660 wave length by measuring the absorbency of each sample.
• the test samples were made on day 0 by preparing a 1: 10 dilution from each tube (0.5ml in 4.5ml 1% saline and on days 2 and 10 by testing 5 ml of the 24hr subcultures of thelO "2 and 10 "4 dilutions).
• turbidity readings were obtained on the 8 th day of incubation of the day 2 dilutions.
• the culture plates were read at 24 and 48hr. At 24 hr the 10 "3 plates were all confluent with bacterial growth. The 10 "5 plates were lighter, but still had -1000 colonies per plate. By 48hrs all plates had heavy confluent growth, thus over 10 8 cfu per ml in the original tube. This number was maintained at days 0, 2, and 10. There were no differences observed in cultures grown with a range of concentrations of seawater or NaCl. The culture of Composition 1 grew well in all ranges tested. There was no growth at any time in the uninoculated sea waters or nutrient broth with NaCl.
• Composition 1 was tested in three concentrations of artificial seawater (10ppt,30ppt, and 50ppt), and three concentrations of NaCl in nutrient broth (1%, 3%, and 5%), for persistence.
• the probiotic grew in all concentrations and was viable for at least ten days in high numbers >10 /ml.
• the concentration of salt in the sea water or in the broth did not affect the ability of the spores to germinate and grow.
• compositions 1 and 2 are tested against three bacterial pathogens isolated from cultivated shrimp: Vibrio parahaemolyticus, V. alginolyticus, and V. vulnificus and a pathogenic fungus from shrimp, Fusarium solani.
• Stock solutions of Composition 1 and Composition 2 are prepared by adding 0.2 gram of the formulated powder to 1.8 ml of sterile distilled water, such that each solution contains about IxIO 9 CFU Bacillus subtilis per ml.
• Condiospores from 5 day-old cultures of Fusarium solani grown on Sabouraud-Dextrose agar are harvested in sterile 2% NaCl. Vibrio are grown on Luria Broth medium at 37°C. Marine bacterial substrains are enriched on Mueller Hinton agar or marine broth 2216 medium at 30 0 C and then cultured in tryptic soy broth (TSB), supplemented with 1-3% NaCl at30°C. V. parahaemolyticus strains are selectively grown on Thiosulfate Citrate Bile Salts agar at 42° C and then cultured on TSB supplemented with 3% NaCl at 30oC.
• test organisms Vibrio and Fusarium
• a single supportive agar plate each in a single line that bisects the agar plate.
• the organisms are allowed to dry overnight.
• two sets of the inoculated plates are streaked with either the suspension of Composition 1 or of Composition 2 described above, which is swabbed perpendicular to the test organisms.
• the streaked plates are incubated in 36 0 C ⁇ 2 overnight.
• QST713 is expected to cause inhibition of several of the isolates of pathogenic Vibrio and Fusarium.
• Bacillus subtilis QST713 is expected to show aggressive competitive overgrowth of the pathogenic bacteria.

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