WO2014151837A1 - A bacillus subtilis strain for use in maintaining or improving the paw health of poultry - Google Patents

Abstract

The present invention relates to a method of maintaining or improving the paw health of poultry comprising administering to the poultry a composition comprising a Bacillus subtilis strain or a mutant thereof. The present invention also provides methods of maintaining or improving the paw health of poultry comprising applying to poultry litter an effective amount of a Bacillus subtilis strain to reduce the production of ammonia and/or the growth of pathogenic bacteria in the poultry litter.

Description

A BACILLUS SUBTILIS STRAIN FOR USE IN MAINTAINING OR

IMPROVING THE PAW HEALTH OF POULTRY

RELATED APPLICATIONS

[0001] This present application claims the benefit of U.S. Provisional Patent

Application No. 61/793,931, filed on March 15, 2013, in accordance with 35 U.S.C. Section 119. The foregoing application is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

[0002] The present invention relates to the field of probiotics and their ability to enhance animal health or the general physical condition of animals.

BACKGROUND OF INVENTION

[0003] 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. Although 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.

[0004] Footpad dermatitis is a condition that causes necrotic lesions on the surface of the footpads in growing broiler chickens. This condition not only causes downgrades and condemnations of saleable chicken paws, the portion of the leg below the spur, but is also an animal welfare concern. Recently, chicken paw prices have escalated due to an increasing demand for high-quality paws in the export markets. This demand has turned paws into the third most important economic part of the chicken behind the breast and wings, with paws accounting for approximately $280 million a year (US Poultry and Egg Export Council, 2009). As such, harvesting large, unblemished paws has become a priority to poultry companies all over the world.

[0005] Previous research demonstrated that litter moisture is a significant factor in the onset of footpad dermatitis. By continually standing on wet litter, the footpad of broiler chickens softens and become more prone to lesions and damage. Protein level and source also affects the incidence and severity of footpad dermatitis. Birds reared on low-protein diet and fed a diet based on vegetable and animal proteins showed low incidence of footpad dermatitis while birds fed a high-protein diet consisting of only plant-based proteins showed high incidence and severe cases of footpad dermatitis. This can be attributed to the high levels of indigestible proteins in the feces, resulting in increased ammonia levels in the litter, and predisposing birds to footpad dermatitis. There is a need for effective poultry probiotics that improve the digestibility of dietary proteins and reduce the occurrence and severity of footpad dermatitis in broiler chickens.

SUMMARY OF INVENTION

[0006] The present invention provides a strain of Bacillus subtilis that, when administered to an animal, enhances the health of such animal. Specifically, the present invention relates to methods for maintaining or improving the paw health of poultry by feeding to such animals, in feed or drinking water (and not through gavage), compositions comprising a Bacillus subtilis strain such as (i) Bacillus subtilis QST713, (ii) a mutant of (i), a cell-free preparation of (i) or (ii), or a metabolite of (i) or (ii). In one embodiment, the composition of the present invention comprises Bacillus subtilis QST713 or mutants thereof and metabolites produced by the bacteria. In another embodiment, the composition comprises Bacillus subtilis QST713 mainly in its spore form.

[0007] In some embodiments of the present invention, the compositions are administered to animals such as poultry in feed over multiple days throughout the animal's life or during particular stages or portions of the animal's life. For example, in some embodiments the compositions are administered only in a starter diet or only in a finisher diet of farm animals.

[0008] 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. In one embodiment 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.

[0009] In one embodiment, the composition of the present invention comprises a Bacillus subtilis strain such as, but not limited to, Bacillus subtilis QST713 or mutants thereof, and is administered to an animal at a rate of about 1 x 10³ CFU/g feed or mL drinking water, or about 1 x 10⁴ CFU/g feed or mL drinking water, or about 1 x 10^s CFU/g feed or mL drinking water, or about 1 x 10⁶ CFU/g feed or mL drinking water, or about 1 x 10⁷ CFU/g feed or mL drinking water, or about 1 x 10^s CFU/g feed or mL drinking water, or about 1 x 10⁹ CFU/g feed or mL drinking water, or about 1 x 10¹⁰ CFU/g feed or mL drinking water, or about 1 x 10¹¹ CFU/g feed or mL drinking water.

[0010] In another embodiment, the compositions of the present invention are administered or fed to an animal in an amount effective to decrease the growth of pathogenic bacteria in the animal gut. Such pathogenic bacteria include Clostridia, Listeria, Salmonella, Campylobacter, Escherichia coli, and Vibrio. Relatedly, the methods of the present invention may be used to decrease the amount of pathogenic bacteria shed in animal feces. The methods of the present invention may also be used to maintain or increase the growth of beneficial bacteria, such as lactic acid bacteria, in the animal gut. By decreasing pathogenic bacteria and/or increasing or maintaining beneficial bacteria, the compositions of the present invention are able to maintain an overall healthy gut microflora.

[0011] 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. In one embodiment, the animals are farm animals, which are raised for consumption or as food-producers, such as broilers and egg-producing chickens.

[0012] This invention also provides compositions that are adapted to enhancing the animal's health or improving the animal's physical condition. Thus, the compositions of the present invention may include Bacillus subtilis strains such as Bacillus subtilis QST713 or its mutants, cell-free preparations thereof or metabolites thereof and carriers that make these compositions suitable for feeding to animals as a feed additive or as an additive for drinking water. Alternatively, the Bacillus subtilis QST713 or its mutants, cell-free preparations thereof or metabolites thereof may be formulated with animal feed ingredients, including feed protein and/or feed carbohydrates. Such combinations may be in the form of pellets that are extruded through standard pelleting processes.

[0013] Compositions of the present invention also comprise combinations of Bacillus subtilis QST713 or its mutants, cell-free preparations of QST713 or its mutants and metabolites of QST713 and its mutants with other probiotics and/or with prebiotics.

[0014] The present invention also encompasses a method for preparing animal feed containing a direct fed microbial comprising adding Bacillus subtilis QST713 spores in an amount effective to enhance animal health upon feeding to animals standard feed components, such as carbohydrates and proteins, prior to the pelleting process.

[0015] In another embodiment, the present invention provides a method for maintaining or improving the paw health of poultry, the method comprising applying to poultry litter an effective amount of a Bacillus subtilis strain to reduce the production of ammonia and/or the growth of pathogenic bacteria in the poultry litter. The poultry litter may be covering the floor of a cage where the poultry are grown at the time of application. In other aspects, the method comprises applying to poultry litter an effective amount of a Bacillus subtilis strain to reduce the production of ammonia and/or the growth of pathogenic bacteria in the poultry litter; and covering the floor of cages where the poultry are grown with the poultry litter.

[0016] The Bacillus subtilis strain may be B. subtilis TISTR10 (BIOTEC 7123), Bacillus subtilis QST713 or a mutant thereof, Bacillus subtilis QST30002, B. subtilis QST30004, or a mutant of B. subtilis QST713, B. subtilis QST30002 or B. subtilis QST30004. The Bacillus subtilis strain may be were applied to poultry litter at a rate of about 1 x 10⁴ CFU/g of litter, about 1 x 10⁵ CFU/g of litter, about 1 x 10⁶ CFU/g of litter, about 1 x 10⁷ CFU/g of litter, about 1 x 10^s CFU/g of litter, about 1 x 10⁹ CFU/g of litter, or about 1 x 10¹⁰ CFU/g of litter. In certain aspects, applying the Bacillus subtilis strain to the poultry litter comprises spraying, coating, mixing, and/or blending the poultry litter with the Bacillus subtilis strain.

[0017] Pathogenic bacteria inhibited by the present compositions include, but are not limited to, Salmonella spp., Clostridia spp., Listeria spp., Campylobacter spp., Escherichia spp., Pseudomonas spp., Enterococcus spp., Vibrio spp., and Staphylococcus spp.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows results of a test of a cell-free preparation of Bacillus subtilis QST713 for efficacy against various isolates of Clostridia. OD 600 nm is indicated on the y- axis.

[0019] FIG. 2 represents results of a test of a heat-treated cell-free preparation of Bacillus subtilis QST713 for efficacy against various isolates of Clostridia. OD 600 nm is indicated on the y-axis.

[0020] FIG. 3 shows results of a test of a cell-free preparation of Bacillus subtilis

QST713 for efficacy against various isolates of Listeria. OD 600 nm is indicated on the y-axis.

[0021] FIG. 4 represents results of a test of a heat-treated cell-free preparation of Bacillus subtilis QST713 for efficacy against various isolates of Listeria. OD 600 nm is indicated on the y-axis.

[0022] FIG. 5 shows results of a test of a cell-free preparation of Bacillus subtilis

QST713 for efficacy against various isolates of Salmonella. OD 600 nm is indicated on the y- axis.

[0023] FIG. 6 represents results of a test of a heat-treated cell-free preparation of Bacillus subtilis QST713 for efficacy against various isolates of Salmonella. OD 600 nm is indicated on the y-axis. [0024] FIG. 7 shows agar plates on which Bacillus subtilis QST713 (vertical) and various isolates of Clostridium perfringens (horizontal) were cross streaked in order to test efficacy of QST713 against the pathogens.

[0025] FIG. 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.

[0026] FIG. 9 shows paw necrotic lesion scores of broiler chickens fed with the control, Bacillus subtilis QST713, and Bacitracin Methylene Disalicylate (BMD) supplemented diets on Day 26. Percent improvement over the control is indicated by the values marked with an asterisk.

[0027] FIG. 10 shows paw necrotic lesion scores of broiler chickens fed with the control, Bacillus subtilis QST713, and BMD supplemented diets on Day 40. Percent improvement over the control is indicated by the values marked with an asterisk.

[0028] FIG. 11 shows paw necrotic lesion scores of broiler chickens fed with the control, Bacillus subtilis QST713, BMD, CALSPORIN^®, or OPTI-BAC^® L supplemented diets on Day 26.

[0029] FIG. 12 shows paw necrotic lesion scores of broiler chickens fed with the control, Bacillus subtilis QST713, BMD, CALSPORIN^®, or OPTI-BAC^® L supplemented diets on Day 40.

DETAILED DESCRIPTION OF INVENTION

[0030] All publications, patents and patent applications, including any drawings and appendices, herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0031] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0032] The present invention relates to a novel use of Bacillus subtilis strains that are effective to enhance animal health as a probiotic. The Bacillus subtilis strain can be any suitable strain or its metabolites that are effective to enhance paw health of poultry. Examples of suitable Bacillus subtilis strains include, but are not limited to commercially available strains such as Bacillus subtilis TISTR10 (also known as Bacillus subtilis BIOTEC 7123, and available from Nippon Biotec, see also Visessanguan et al., Journal of Food Biochemistry (2005) 349-366), or Bacillus subtilis strain QST713 and/or its metabolites. Other examples of suitable Bacillus subtilis strains include Bacillus subtilis strain AQ30002 (aka QST30002) or the strain AQ30004 (aka QST30004), deposited as Accession Numbers NRRL B-50421 and NRRL B-50455, respectively, which are described in international patent publication number WO 2012/087980 or mutants of these B. subtilis strains having all of the physiological and morphological characteristics of B. subtilis strain AQ30002 (aka QST30002) or AQ30004 (aka QST30004). Yet other Bacillus subtilis strains include the strain described by or isolates/strains that are described by Zachary Thomas Williams, "Effects of Management on the Bacterial Community Present in Poultry Litter", Dissertation submitted to the Graduate Faculty of Auburn University, August 2012, that are chosen based on in vitro inhibition of several S. enterica serovars and ammonia utilization as described in this dissertation. Such probiotics can be 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. There are many mechanisms by which probiotics are thought to maintain healthy gut microflora: competitive exclusion of pathogenic bacteria, reduction of pathogenic bacteria through production of antimicrobial substances, enhancing growth and viability of beneficial gut microflora, and stimulating a systemic immune response in the animal.

[0033] The present invention encompasses a method for maintaining or improving paw health of poultry by administering to poultry a composition comprising (i) a Bacillus subtilis strain, (ii) mutants of a given Bacillus subtilis strain, (iii) cell-free preparations of (i) or (ii), or (iv) metabolites of (i) or (ii). In one embodiment the composition comprises (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).

[0034] By "improving the paw health of poultry" is meant in one aspect that the paws (feet) of poultry such as chicken, turkey, ducks, etc. are in a better condition than the paws of poultry that is keep under the same conditions without being administered a composition comprising a Bacillus subtilis strain as described herein. By "improving" is also meant that the paw of poultry is in a better condition (health) than the same group of poultry before the administration of a composition comprising a Bacillus subtilis strain as described herein. The term "maintaining the paw health of poultry" is meant in one aspect that healthy paws of poultry are kept as the same disease free level as was observed earlier, regardless of whether the poultry had been subjected to administration of a composition comprising a Bacillus subtilis strain as described herein at this earlier point of time of not.

[0035] The health (status) or paw quality can be assessed herein by the occurrence or presence of food pad dermatitis (FPD) in the poultry in question. Footpad dermatitis (FPD) is a condition that causes necrotic lesions on the plantar surface of the footpads in growing broilers and turkeys, for example. This condition not only causes downgrades and condemnations of saleable chicken paws, the portion of the leg below the spur, but is also an animal welfare concern in both the United States and in Europe. Paw quality or paw health as used herein refers to the overall health of the foot, including toes and footpad. Paw quality has been shown to be affected by a myriad of factors including genetics, environmental factors, nutrition, and bedding materials. Paw quality can be judged both in the field (at the poultry farm/grower) or in the processing plant. In the field, several scales have been used to determine lesion severity including a 3-point scale that ranges from 0 to 2 (Bilgili, S. F., M. A. Alley, J. B. Hess, and M. Nagaraj, 2006, "Influence of Age and Sex on Footpad Quality and Yield in Broiler Chickens Reared on Low and High Density Diets," J. Appl. Poult. Res. 15:433-44), a 7-point scale that ranges from 0 to 6 (Ekstrand, C, B. Algers, and J. Svedberg, 1997, "Rearing Conditions and Foot-pad Dermatitis in Swedish Broiler Chickens," Prev. Vet. Med. 31 :167-174), or the modified Ekstrand score (Ekstrand, C, T. E. Carpenter, I. Andersson, and B. Algers, 1998, "Prevalence and Control of Footpad Dermatitis in Broilers in Sweden," Br. Poult. Sci. 39:318- 324), which uses a l-to-3 scale (cf. the review of Shepherd & Fairchild, 2010, "Footpad

Dermatitis in Poultry," Poultry Science 89: 2043-2051.) The highest number represents the most severe lesion in all 3 scoring systems. The paw (foot) quality or health can be evaluated herein using any of these three scoring systems. Thus, in illustrative terms, "maintaining the paw health/quality" means that, for example, the score of 1 on the modified Ekstrand score is maintained in a poultry group over a certain period of time such as 26 or 42 days after start of administration of a composition comprising a Bacillus subtilis strain as described herein, while "improving the paw health/quality" means that the score drops from 3 to 1 on the modified Ekstrand score within a given time period after administering of a composition comprising a Bacillus subtilis strain as described herein. The administration can be done continuously or in intervals.

[0036] Bacillus subtilis QST713, its mutants, its supernatants, and its lipope tide 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 B- 21661. Any references in this specification to QST713 refer to Bacillus subtilis QST713.

[0037] 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. Without wishing to be limited by any particular theory, it is thought that Bacillus subtilis QST713 enhances animal health by a multifaceted mode of action, including producing antibacterial metabolites and competing with pathogens by using more nutrients and attachment spaces than the pathogens, thereby preventing effective establishment of pathogenic bacteria in the gut.

[0038] In one aspect of the invention, compositions administered to animals comprise mutants of Bacillus subtilis QST713. The term "mutant" refers to a genetic variant derived from QST713. In one embodiment, the mutant has all the identifying characteristics of QST713. In a particular instance, the mutant maintains or improves the health of paws of poultry at least as well as the parent QST713 strain. In another particular instance, the mutant maintains or improves the health of paws of poultry better than the parent QST713 strain. In another embodiment, mutants are genetic variants having a genomic sequence that has greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% sequence identity to the QST713 strain. Mutants may be obtained by treating QST713 cells with chemicals or irradiation or by selecting spontaneous mutants from a population of QST713 cells (such as phage resistant mutants) or by other means well known to those practiced in the art. In some embodiments, mutants are spontaneous mutants. The term 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.

[0039] In one embodiment, the mutants (spore-forming bacterial cells used in the present invention) are Bacillus subtilis QST713 bacterial cells having a mutation in the swrA gene and compositions thereof as described in international patent publication number WO 2012/087980. In one such aspect, the Bacillus subtilis QST713 bacterial cells comprise at least one nucleic acid base pair change in a start codon and/or at least one nucleic acid base pair insertion or deletion in a swrA gene. In yet another aspect, the swrA^~ cells (mutants) of Bacillus subtilis QST713 are selected from the group consisting of the strain AQ30002 (aka QST30002) and the strain AQ30004 (aka QST30004), deposited as Accession Numbers NRRL B-50421 and NRRL B-50455, respectively, which are described in international patent publication number WO 2012/087980 or mutants of these B. subtilis strains having all of the physiological and morphological characteristics of B. subtilis strain AQ30002 (aka QST30002) or AQ30004 (aka QST30004). These strains can be used in the method of the invention, either alone or in mixture with Bacillus subtilis QST713. In still another aspect of the invention, the Bacillus subtilis QST713 having the mutation in the swrA gene is wild type for epsC, sfp and degQ. In another aspect the Bacillus subtilis QST713 having the mutation is otherwise isogenic to Bacillus subtilis QST713.

[0040] All references in this application to Bacillus subtilis strains, including, for example, 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.

[0041] Cells of any Bacillus subtilis strain including Bacillus subtilis QST713 cells and cells of mutants of Bacillus subtilis QST713 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. In some embodiments, the composition comprises mainly spores.

[0042] 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.

[0043] 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 U.S. Patent No. 6,060,051. Conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture. 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 (CFU) 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. In some embodiments, the concentrated fermentation broth is washed, for example via a diafiltration process, to remove residual fermentation broth and metabolites.

[0044] 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.

[0045] 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.

[0046] Metabolites of QST713 can be obtained according to the methods set forth in U.S. 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 bactericidal metabolites of QST713 are between 600 kilodaltons and 100 daltons. Therefore, in some embodiments, after a cell-free preparation is made by centrifugation of fermentation broth of QST713, 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.

[0047] 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. Such carriers may be added individually or in combination. In some embodiments, the carriers are anti-caking agents, anti-oxidation agents, bulking agents and/or protectants. Examples of 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. In some embodiments, the carriers are added after concentrating fermentation broth and during and/or after drying.

[0048] In embodiments in which the compositions are formulated as feed additives, the concentration on a weight by weight basis (w/w) of (i) Bacillus subtilis 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%. In some embodiments, for example, where the concentrated formulation broth has been washed and dried without heat, such as via freeze drying, the concentration of Bacillus subtilis QST713 or its mutants in the final composition may be from about 90% to about 100%.

[0049] 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. Thus, in line with the above, 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 QST713, a mutant of Bacillus subtilis QST713, a cell-free preparation derived from Bacillus subtilis QST713 or its mutant, or metabolites of QST713 or its mutants. [0050] In some embodiments in which 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. As used herein, 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. In one aspect of this 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. A reduction of this ratio relates to increased feed efficiency. 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.

[0051] 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. Without wishing to be bound by any particular theory, it is thought that 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 dificile), Listeria spp. (such as money togenes, seeligeri and welshimeri), Salmonella spp. (such as enterica, arizonae, typhirium, enteritidis and bonglori), E. coli, Enterococcus spp. (such asfaecalis andfaecium), Campylobacter, Aeromonas spp.,

Staphylococcus aureus and Vibrio spp. In some embodiments, 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. [0052] The above pathogenic bacteria lead to various diseases in animals. For example, in poultry, feed contaminated with Clostridium perfringens has been implicated in outbreaks of necrotic enteritis (or necrotic lesions in the gut wall) in chicken. Interestingly, although this bacteria is commonly found in the intestinal tract of chickens, it does not always result in necrotic enteritis, although increased levels have been linked to the disease. Reduction of this pathogen through use of a probiotic results in enhanced health and weight gain, as shown in the examples, below. Thus, control of these bacteria by decreasing their ability to grow in the gut reduces incidence of disease caused by such bacteria. Table 1 , below, shows various microorganisms and the diseases or conditions to which they are linked.

Table 1

[0053] Maintenance of healthy gut microflora and, in particular, reduction of one or more of the above-described detrimental bacteria, also causes a reduction in pathogen shedding through animal feces. 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. [0054] 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. The term "therapeutic amount" refers to an amount sufficient to ameliorate or reverse a disease state in an animal.

[0055] 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.

[0056] Provided herein is a method of inhibiting bacterial growth on an animal and/or surrounding environment, such as litter in a chicken house or coop. As used herein, the term "cage" refers to a house, coop, corral, hutch, pen, cote, or other enclosure for growing poultry.

[0057] 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. In one embodiment 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. In another embodiment, the method may be applied to mammals such as swine. In yet another embodiment the method may be applied to polygastric animals, such as cattle, goat and sheep, also referred to herein as ruminants. In one embodiment, 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.

[0058] In one aspect, the compositions of the present invention are feed additives that are added to the subject animal's feed or drinking water prior to feeding. In such case, the compositions may be formulated with a carrier, such as calcium carbonate or whey protein, as described above. In one aspect of the invention, such carrier is hygrophobic. [0059] In another aspect, 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. In some embodiments, 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.

[0060] In another aspect, 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 U.S. 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 U.S. Patent No. 4,919,936, and deposited as FERM BP-1096 with the Fermentation Research Institute, Agency of Industrial Science and Technology, in Japan), sold by Calpis as CALSPORIN^®, or a mixture of Bacillus licheniformis and Bacillus subtilis spores sold by Chr. Hansen under the trademark BIOPLUS2B^®, Bacillus coagulans, including those strains described in U.S. Patent No. 6,849,256, Bacillus licheniformis, Bacillus lentus, Bacillus pumilus, Bacillus laterosporus, and Bacillus alevi. Other non-Bacillus probiotics, such as Saccharomyces cerevisiae, may also be used in compositions of the present invention. If such other probiotics are not formulated as part of the compositions of the present invention, they may be administered with (either at the same time or at different times) the compositions of the present invention.

[0061] In another aspect, the 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.

[0062] In embodiments in which the compositions comprise QST713 or its mutants, 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 10⁴ CFU Bacillus subtilis per gram feed or mL drinking water to about 1 x 10¹⁰ Bacillus subtilis per gram feed or mL drinking water. In another embodiment from about 1 x 10^s CFU Bacillus subtilis per gram feed or mL drinking water to about 1 x 10⁹ Bacillus subtilis per gram feed or mL drinking water should be administered. In yet another from about 1 x 10^s CFU Bacillus subtilis per gram feed or mL drinking water to about 1 x 10⁹ Bacillus subtilis per gram feed or mL drinking water should be administered. In yet another from about 1 x 10⁶ CFU

Bacillus subtilis per gram feed or mL drinking water to about 1 x 10⁸ Bacillus subtilis per gram feed or mL drinking water should be administered. In some embodiments the inclusion rate is about 1 x 10³ CFU Bacillus subtilis per gram feed or mL drinking water, or about 1 x 10⁴ or about 1 x 10⁵ or about 1 x 10⁶ or about 1 x 10⁷ or about 1 x 10⁸ or about 1 x 10⁹ or about 1 x 10¹⁰ or about 1 x 10¹¹ CFU Bacillus subtilis per gram feed or mL drinking water. In embodiments in which 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.

[0063] 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. In this aspect, if 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. In some embodiments the pelleting process involves temperatures of at least about 65° C. In others, pelleting temperatures are between about 65° C and about 120° C. In still others, pelleting temperatures are between about 80° 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.

[0064] The 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 (iii) enhance animal health at various doses.

[0065] For aquatic animals, including salmon, trout, shrimp and ornamental fish, in one embodiment, the 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. Such effective amounts can be between about 10⁴ and about 10¹⁰ CFU Bacillus subtilis QST713 per mL of rearing water, or in another embodiment between about 10^s and about 10⁹ CFU Bacillus subtilis QST713 per mL of rearing water, or in yet another embodiment between about 10⁶ and about 10⁸ CFU Bacillus subtilis QST713 per mL of rearing water.

[0066] The following examples are given for purely illustrative and non-limiting purposes of the present invention.

EXAMPLES

Example 1 - In vitro Studies of Efficacy of QST713 Cell-Free Preparations against Animal Pathogens

[0067] Cell-free preparations of QST713 were tested for antimicrobial activity against Clostridia (Clostridia perfringens ATCC13124 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); and E. coli, using Kirby-Bauer and minimal inhibitory concentration (MIC) techniques.

[0068] 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, centrifuging the culture for 15 minutes at 3000 rpm at 23° C and filtering it through a 0.45μιη Nalgene filter unit. To test for heat stability, a portion of the cell-free preparation was heated to 50° C for one hour before each of the Kirby-Bauer and MIC tests

Table 2

Genus Species/ATCC Growth Media Conditions for Growth

Listeria seeligeri Same as above Same as above

ATCC 35968

Listeria welshimeri Same as above Same as above

ATCC 35897

Salmonella enterica Trypticase Soy Broth Same as above

ATCC 10398

Salmonella arizonae Same as above Same as above

ATCC 13314

Salmonella bongori Same as above Same as above

ATCC 43975

[0069] In the Kirby-Bauer experiments, 2 mm sterile filter paper disks were immersed in QST713 supernatant and air-dried under sterile conditions. These disks were then placed on lawns of the target pathogen, incubated overnight and zones of inhibition measured. Zones of inhibition were observed for the Clostridia and Listeria targets.

[0070] In the MIC technique, wells of microtiter plates were inoculated with 75 μΤ of each target pathogen, diluted to 1 x 10⁵. The above-described cell-free preparation was added to each well at final dilutions of 1 :2, 1 :10 and 1:50. Plates were incubated overnight at 37° C and OD600 and read with a Wallach microtiter reader. The cell-free preparation (both heat-treated and non-heat treated) was significantly effective against the Clostridia and Listeria targets and inhibited growth of Salmonella and E. coli, although no zones of inhibition were observed for these last two pathogens on Kirby-Bauer plates. Data for Clostridia, Listeria and Salmonella are shown in Figures 1-6. Example 2 - In vitro Studies of Efficacy of QST713 Against Various Bacteria

[0071] A powder formulation of Bacillus subtilis QST713 was tested for efficacy against various environmental isolates of the following bacteria: Clostridium perfringens, Escherichia coli, Salmonella 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 10⁹ CFU Bacillus subtili Ί 'gram and at a maximum approximately 1 x 10¹⁰ CFU Bacillus subtilislgm . 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 1 x 10⁹ 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. The Clostridium perfringens and Campylobacter jejuni isolates were incubated in a Campy gas atmosphere (10% CO2, 5% O2, 8% N2) at 41 ± 2 overnight. The other isolates, which are aerobic, were incubated in 36 ± 2 ⁰ C 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. In addition, in some cases Bacillus subtilis QST713 showed aggressive competitive overgrowth of the pathogenic bacteria. Results are summarized in the table below. The zones of inhibition reported in the table were measured from the edge of the Bacillus growth to the beginning of growth of the test organism. In addition, photographs of the Clostridium perfringens and Campylobacter jejuni plates are shown in FIGS. 7 and 8, respectively.

Table 3

Atmosphere Zone of

and inhibition

Culture Name Isolate ID Temperature (mm) Comments

SE 22 0

Campylobacter Cj-1 1 Bacillus swarming

jejuni Slight inhibition of growth

Cj-2 Campy gas, 0 although no zone, Bacillus

41° C swarming

NCjl 0 Bacillus swarming

NCj2 1

Listeria Aerobic,

LM 1 2

monocytogenes 36° C

Example 3 - In vivo Studies of QST713 in Broilers

[0072] 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.

Basal diet only - control

> Basal diet + 0.05% CALSPORIN^® (0.5g/kg; 10⁶ CFU/g) (designated as CS in the table below)

> Basal diet + 0.05% Composition 1 (0.5g/kg; 10⁶ CFU/g) (designated as Comp.1-10⁶ in the table below)

> Basal diet + 0.0005% Composition 1 (0.5 mg/kg; 10³ CFU/g) (designated as Comp. 1-10³ 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.

Table 4: Ingredient Composition of Starter (d 1 to 21) and Finisher (d 22 to 42) Basal Diets for Broiler Chickens

[0073] The below results in Table 5 show that Composition 1 improved the weight gain of the birds at the 10⁶ CFU/g level. Feed efficiency was improved for the 21-42 day period and for the overall growth period (1-42 days). In the chart below, ADG refers to average daily gain, ADFI refers to average daily feed intake.

Table 5: Effect of Dietary Treatment on Performance of Broiler Chickens

Treatment

Item Control cs CompJ-lO Comp. 1-10

d 1 - 42 0.50 + 0.022 0.51 + 0.020 0.52 + -0.022 0.48 + 0.022

Mortality, n 1.2 + 0.51 1.2+ 0.46 1.4 + 0.51 1.4 + 0.51

Example 4 - Stability of QST713 in Feed Pelleting Process

[0074] To determine the stability of Bacillus subtilis QST713 during the animal feed pelleting process, animal feed pellets containing Composition 1 were prepared and samples tested at various temperatures. Control feed contained the ingredients shown in Table 6, while experimental feed was supplemented with 8% Composition 1.

Table 6

Ingredient %

Corn 68.94

Soybean Meal 20.40

Fishmeal 5.50

Monocalcium Phosphate 0.51

Limestone 0.58

Salt 0.33

DL-Methionine 0.31

L-Lysine 98% 0.18

Poultry Vit/Min Premix 0.25

Soybean Oil 3.00

TOTAL 100.000 [0075] 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 ¼" 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 lb. batch.

[0076] Mixer samples were diluted and allowed to sit for five minutes to fully wet.

Pellet samples were soaked for 30 minutes in phosphate buffer in order to recover QST713 cells. Diluted samples were plated to determine colony forming units. Colony forming units decreased insignificantly from the mixer to the pelleting stages, as shown in Table 7, below. Table 7

Example 5 - Use of Composition 2 for Various Studies

[0077] 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 10¹⁰ CFU Bacillus subtilis! 'gram 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. Example 6 - In vivo Studies in Poultry to Improve or Maintain Paw Health

[0078] In this study the experimental house is divided into pens of equal size, arranged along a central aisle. The birds are kept in 48 pens of a house equipped with 50 floor pens, each having an area of 4 x 10 = 40 ft² (3.72 m²) , with built up wood shavings as bedding, with thickness of approximately 4 inches. The stocking density, after subtracting out for equipment, is approximately 0.81 ft²/bird. Standard floor pen management practices are used throughout the experiment. The temperature of the building is monitored and environmental conditions during the trial (temperature) are optimum to the age of the animals. The experiment consists of 48 pens starting with 45 male broiler chickens. The treatments are replicated in eight blocks, randomized within blocks of six pens each.

[0079] Unmedicated commercial type chicken starter, grower, and finisher rations are formulated with feedstuffs commonly used in the United States. These rations are fed ad libitum from the date of chick arrival until Day 42 of the study. Experimental treatment feeds are prepared from these basal feeds. Birds receive feed appropriate to the treatment from Day 0 to 42. A change from starter to grower in which all previous feed is removed and weighed occurred on Day 21. Grower diet is removed and weighed from each pen on Day 35 and replaced with the finisher diet. On the final day of the study (Day 42), finisher is weighed back.

[0080] Two thousand one hundred and sixty (2, 160) Cobb X Cobb chicks are allocated to the study. All chicks are spray vaccinated using a Spraycox machine with the label recommended dosage of Coccivac-B on day of hatch.

[0081] At study initiation forty-five males are allocated to each treatment pen by blocks. No birds are replaced during the course of the study. Feed and watering method are ad libitum. All pens have approximately 4 inches of built up litter with a coating of fresh pine shavings. Poultry production in the United States routinely uses built up litter. Litter ammonia levels are monitored using GASTEC Ammonia Pasive Dosi-Tubes per the manufacturer's instructions for safe operation. Dosimeters are used to collect samples from individual pens on a weekly basis. Timed confined monitoring is used to estimate litter ammonia levels in randomized locations within each pen.

[0082] Quantitative detection of QST713, and commensal Enterobacteriacea from pen litter are enumerated using litter samples that are collected from individual pens on a weekly basis. Randomized timed monitoring is used to collect multiple samples from 5 locations within each pen. Pooled samples are flash frozen and held at -80° C for analysis. Quantitative Real Time PCR (QrtPCR) assays using a proprietary probe based on contig 00530 for QST713 are carried our using materials and methods provided by IDEXX Laboratories. Commensal flora are monitored based on published 16S probes, using the Syber green system. Internal positive controls and Pan Bakt SYBER internal controls insure probe and PCR specificity.

[0083] Foot/paw quality is evaluated on Day 42 using the modified Exstrand score (Ekstrand, C, T. E. Carpenter, I. Andersson, and B. Algers, "Prevalence and Control of Footpad Dermatitis in Broilers in Sweden," 1998, Br. Poult. Sci. 39:318-324). Expected Results

[0084] Birds fed with diets containing supplementary Bacillus subtilis QST713 are expected to show improvements in paw quality. Pen litter from birds receiving continuous fed QST713 should have reduced gaseous ammonia production. Furthermore the litter samples are expected to show alterations in the quality and composition of the flora found in the litter from pens of birds that received diet amended with DFM when compared to the untreated controls. Not all Bacillus are expected to perform equally. This study illustrates the need to modify the commensal flora continuously with a Bacillus strain like QST713 to affect the litter flora in a way that insures improved environmental quality through reduced gaseous ammonia, and optimum production of high quality chicken paws.

Example 7 - Supplementation with Bacillus subtilis QST713 Improves Paw Necrotic Lesion Score in Broiler Chickens Experimental Design

[0085] Day-old male broiler chicks (Ross x Ross 708) were obtained from the hatchery facility of the commercial integrator research farm where the study was conducted. At the hatchery, birds were sexed and received routine vaccination (Marek's, IBD, and NC/IBV). Upon arrival at the study site, 480 chicks were randomly placed in 24 litter floor pens (built-up litter covered with pine shavings ~4 inches) with 20 birds placed per pen. A total of 3 treatment groups were evaluated (8 pens of 20 birds/treatment). The treatments were: 1) Basal diet (Control); 2) Basal diet + GROBIG BS™ Bacillus subtilis QST713 (100 g/ton, 1 x 10⁶ CFU/g); 3) Basal diet + Bacitracin Methylene Disalicylate (BMD) (50 g/ton). GROBIG BS™ is a commercially available feed additive containing Bacillus subtilis QST713.

[0086] A non-medicated commercial type chicken starter (crumble, 0 to 14 days), grower (pelleted, 15 to 28 days), and finisher rations (pelleted, 29 to 40 days) were formulated with feedstuffs commonly used in the United States. From these basal feeds, the experimental treatment feeds were prepared and fed ad libitum to the birds from day-old until Day 40 of the study. Composite and pelleted samples of feeds were assayed to confirm B. subtilis QST713 levels.

[0087] The severity of footpad dermatitis was assessed by scoring the paw necrotic lesions at day 26 and 40. The collected chicken paws from all birds in each treatment were assessed macroscopically using the modified Ekstrand score (Ekstrand, C, T. E. Carpenter, I. Andersson, and B. Algers, "Prevalence and Control of Footpad Dermatitis in Broilers in Sweden," 1998, Br. Poult. Sci. 39:318-324). Scores were 0 = no lesion, slight discoloration of the skin, or healed lesion; 1 = mild lesion, superficial discoloration of the skin, and

hyperkeratosis; 2 = severe lesion, epidermis is affected, blood scabs, hemorrhage, and severe swelling of the skin. Results

[0088] On Day 26, broiler chickens fed with GROBIG BS™ Bacillus subtilis QST713 showed less severe paw necrotic lesion score compared to birds fed the control and the BMD supplemented diets. Relative to the control, birds supplemented with GROBIG BS™ Bacillus subtilis QST713 experienced reduced severity of paw necrotic lesions (from 0.539 to 0.462), resulting in a +16.7% improvement in paw necrotic lesion score. In contrast, birds supplemented with BMD experienced an increased severity of paw necrotic lesions (from 0.539 to 0.620), resulting in a -13.1% decline in paw necrotic lesion score. Therefore, compared to BMD, the positive impact of GROBIG BS™ Bacillus subtilis QST713 on chickens paw quality can be manifested as early as 26 days in the production period (see FIG. 9).

[0089] The improvement in paw necrotic lesion score in birds supplemented with

GROBIG BS™ Bacillus subtilis QST713 extended up to the harvesting of broiler chickens (Day 40). Relative to the control, birds supplemented with GROBIG BS™ Bacillus subtilis QST713 experienced a reduced severity of paw necrotic lesions (from 0.626 to 0.439), resulting in a +42.6% improvement in paw necrotic lesion score. The supplementation of GROBIG BS™ Bacillus subtilis QST713 during the entire production cycle of broiler chickens resulted in consistent improvements in paw necrotic lesion score. In comparison to birds supplemented with BMD, the severity of paw necrotic lesions in GROBIG BS™ Bacillus subtilis QST713 fed birds was consistently reduced in the first and second halves of the production period. Therefore, GROBIG BS™ Bacillus subtilis QST713 appears to be a more reliable dietary intervention than BMD in improving chicken paw quality. Without wishing to be bound to any theory, the improvement in dietary protein digestibility as a result of Bacillus subtilis QST713

supplementation may have contributed in lowering fecal and litter ammonia level, thus reducing the severity of paw necrotic lesion. Example 8 - Supplementation with Bacillus subtilis QST713 Increases the Number of

Marketable Chicken Paws Compared to Supplementation with Other Bacillus Probiotics

[0090] The objective of this study was to evaluate the efficacy of Bacillus subtilis QST713 in preventing footpad dermatitis lesions in chickens in comparison to other Bacillus probiotics. CALSPORIN^® is a probiotic feed additive containing viable spores of Bacillus subtilis C-3102), and OPTI-BAC^® L is a feed additive with an active ingredient of Bacillus licheniformis.

[0091] The following treatment groups were evaluated:

1. Untreated control;

2. Bacillus subtilis QST713 454 g/short ton (2.5 x 10⁹ cfu/g = 1.0 x 10⁶ cfu/g of feed);

3. Bacillus subtilis QST713 227g/short ton QRD159.4 (2.5 x 10⁹cfu/g = 5.0 x 10^s cfu/g of feed);

4. 50 g BMD;

5. CALSPORIN^® at commercial rate (3.0 x 10⁵ cfu/g of feed);

6. OPTI-BAC^® L 1 lb/short ton (3.2 x 10⁹ cfu/g = 1.0 x 10⁶ cfu/g of feed); and

7. Bacillus subtilis QST713 90g/short ton (1.0 x 10¹⁰cfu/g = 1.0 x 10⁶ cfu/g of feed). Note that treatment groups 2, 3, and 7 received Bacillus subtilis QST713 feed additives that were prepared as described in Example 5 but had different concentrations of Bacillus subtilis QST713 cells.

[0092] Eight replicates were evaluated for each treatment group using the modified Ekstrand score (Ekstrand, C, T. E. Carpenter, I. Andersson, and B. Algers, "Prevalence and Control of Footpad Dermatitis in Broilers in Sweden," 1998, Br. Poult. Sci. 39:318-324). A score of zero indicates no paw lesions or healed paw lesions while increasing scores denote a greater incidence and severity of paw lesions. 45 broiler chickens were kept in each pen, and 360 broiler chickens were evaluated in each treatment group. Each treatment group consumed approximately 2 tons of feed. Generally, only those chicken paws with a score of zero are marketable.

[0093] Footpad lesion evaluations were made on Day 26 and Day 40 of the treatments {see FIGS. 11 and 12). At Day 26, supplementation with Bacillus subtilis QST713 at 1.0 x 10⁶ cfu/g of feed (treatment groups 2 and 7) resulted in a numerically greater number of marketable chicken paws than BMD, CALSPORIN^®, or OPTI-BAC^® L (treatment groups 4, 5, and 6, respectively). At Day 40, supplementation with Bacillus subtilis QST713 at each of the amounts tested (treatment groups 2, 3, and 7) resulted in a markedly greater number of marketable chicken paws than CALSPORIN^® and OPTI-BAC^® L (treatment groups 5 and 6, respectively).

[0094] The results demonstrate that supplementation of broiler chicken feed with Bacillus subtilis QST713 allows for the production of a greater number of marketable chicken paws compared to supplementation with either CALSPORIN^® or OPTI-BAC^® L.

[0095] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.

[0096] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0097] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims

CLAIMS We claim:

1. A method for maintaining or improving the paw health of poultry comprising administering to the poultry a composition comprising a Bacillus subtilis strain wherein the composition is administered in an amount effective to maintain or improve paw health of the poultry.

2. The method of Claim 1, wherein the Bacillus subtilis strain is B. subtilis TISTR10 (BIOTEC 7123), B. subtilis QST713, B. subtilis QST30002, B. subtilis QST30004, a mutant of B. subtilis QST713, a mutant of B. subtilis QST30002, or a mutant of B. subtilis QST30004.

3. The method of Claim 2, wherein the mutant of B. subtilis QST713 is selected from B. subtilis AQ30002 (aka QST30002) and B. subtilis AQ30004 (aka QST30004).

4. The method of any of Claims 1 to 3 wherein the poultry is selected from the group consisting of a chicken, a duck and a turkey.

5. The method of Claim 4, wherein the poultry is an egg -producing chicken or a broiler chicken.

6. The method of any of the foregoing Claims wherein the composition further comprises a carrier.

7. The method of any of the foregoing Claims wherein the composition is administered with animal feed.

8. The method of Claim 7 wherein the animal feed ingredients comprise feed protein.

9. The method of Claim 7 wherein the animal feed ingredients further comprise feed carbohydrate.

10. The method of any of the foregoing Claims wherein the composition is administered at a rate of about 1 x 10³ colony forming units (CFU) Bacillus subtilis QST713 per gram of the animal feed to about 1 x 10^s CFU Bacillus subtilis QST713 per gram of the animal feed.

11. A method for maintaining or improving the paw health of poultry, the method comprising applying to poultry litter an effective amount of a Bacillus subtilis strain to reduce the production of ammonia and/or the growth of pathogenic bacteria in the poultry litter, wherein the poultry litter covers the floor of cages where the poultry are grown.

12. A method for maintaining or improving the paw health of poultry, the method comprising: applying to poultry litter an effective amount of a Bacillus subtilis strain to reduce the production of ammonia and/or the growth of pathogenic bacteria in the poultry litter; and

covering the floor of cages where the poultry are grown with the poultry litter.

13. The method of Claim 11 or Claim 12, wherein applying the Bacillus subtilis strain to the poultry litter comprises spraying, coating, mixing, and/or blending the poultry litter with the Bacillus subtilis strain.

14. The method of any of Claim 11 to 13, wherein the Bacillus subtilis strain is B. subtilis TISTR10 (BIOTEC 7123), B. subtilis QST713, B. subtilis QST30002, B. subtilis QST30004, a mutant of B. subtilis QST713, a mutant of B. subtilis QST30002, or a mutant of B. subtilis QST30004.

15. The method of Claim 14, wherein the mutant of Bacillus subtilis QST713 is selected from B. subtilis AQ30002 (aka QST30002) and B. subtilis AQ30004 (aka QST30004).

16. The method of any of Claims 11 to 15 wherein the poultry is selected from the group consisting of a chicken, a duck and a turkey.

17. The method of Claim 16, wherein the poultry is an egg-producing chicken or a broiler chicken.

18. The method of any of Claims 11 to 17, wherein the pathogenic bacteria are selected from the group consisting of Salmonella spp., Clostridia spp., Listeria spp.,

Campylobacter spp., Escherichia spp., Pseudomonas spp., Enterococcus spp., Vibrio spp., and Staphylococcus spp.

19. A composition comprising poultry litter and a Bacillus subtilis strain, wherein the Bacillus subtilis strain is B. subtilis TISTR10 (BIOTEC 7123), B. subtilis QST713, B. subtilis QST30002, B. subtilis QST30004, a mutant of B. subtilis QST713, a mutant of B. subtilis QST30002, or a mutant of B. subtilis QST30004.