BR112017015585B1 - COMPOSITION CONTAINING BACILLUS STRAIN AND USE OF THE COMPOSITION - Google Patents

Abstract

BACILLUS STRAIN, BIOLOGICALLY PURE CULTURE, AND COMPOSITION. The present invention relates to Bacillus strains that improve the health and performance of farm animals. The invention further relates to compositions comprising Bacillus strains.

Description

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

[001] This application contains a reference to a deposit of biological material, which deposit is incorporated into this document by way of reference.

REFERENCE TO SEQUENCE LISTING

[002] This request contains a Sequence Listing in computer-readable form. The machine-readable form is incorporated herein by reference.

[003] Index for sequence listing: SEQ ID NO: 1 is 16S rDNA from DSM 29869. SEQ ID NO: 2 is 16S rDNA from DSM 29870. SEQ ID NO: 3 is 16S rDNA from DSM 29871. SEQ ID NO: 4 is 16S rDNA from DSM 29872. SEQ ID NO: 5 is 16S rDNA from Bacillus vallismortis from AB021198. SEQ ID NO: 6 is 16S rDNA from Bacillus subtilis of AJ276351. SEQ ID NO: 7 is 16S rDNA from Bacillus amyloliquefaciens of AB255669. SEQ ID NO: 8 to SEQ ID NO: 13: PCR and sequencing primers.

BACKGROUND OF THE INVENTION FIELD OF INVENTION

[004] The present invention relates to strains of Bacillus that improve the health and performance of production animals. The invention further relates to compositions comprising Bacillus strains.

BACKGROUND OF THE INVENTION

[005] Clostridium perfringens (C. Perfringens)) is a spore-forming, anaerobic, rod-shaped, Gram-positive bacterium of the genus Clostridium. C. perfringens is widely present in nature and can be found as a component of decaying vegetation, marine sediment, in the intestinal tracts of humans and other vertebrates, insects, and in soil.

[006] Infections due to C. perfringens show evidence of tissue necrosis, bacteremia, emphysematous cholecystitis and gas gangrene, which is also known as clostridial myonecrosis. C. perfringens can also result in anaerobic polymicrobial infections.

[007] The incidence of necrotic enteritis associated with Clostridium perfringens in poultry has increased in countries that have stopped using antibiotic growth promoters. Necrotic enteritis is an enterotoxemic disease that results in significant economic losses in the poultry industry.

[008] There is a need to develop tools and strategies for the prevention and control of C. perfringens in monogastric animals such as poultry. Although vaccinating animals has been suggested, there are challenges associated with vaccinating millions of animals. Therefore, the discovery of a solution that could be administered as an additive to animal feed would be advantageous.

[009] It is, therefore, an objective of the invention to provide solutions that prevent and/or control C. perfringens in monogastric animals, such as poultry, through the use of an animal feed comprising one or more bacteria with activity against Clostridium perfringens.

[0010] An additional objective of the invention is to provide a solution that does not present a risk to the animal's health. The solution to this problem is the use of non-hemolytic Bacillus strains with activity against Clostridium perfringens.

[0011] A challenge of applying Bacillus spp. in feed is the common use of antibiotics as growth promoters in feed. Therefore, it is necessary to determine strain compatibility with commonly used feed antibiotics such as monensin in order to identify any personal conflicts with use as a direct feed microorganism. The present invention relates, in one embodiment, to Bacillus strains with high compatibility with monensin.

DESCRIPTION OF RELATED TECHNIQUE

[0012] Knap et al. (2010) describes that Bacillus licheniformis has an effect on necrotic enteritis in broiler chickens (Knap, Lund, Kehlet, Hofacre and Mathis; Bacillus licheniformis Prevents Necrotic Enteritis in Broiler Chickens; Avian Diseases 54(2): 931 to 935. 2010) . This strain of Bacillus licheniformis has no effect on Clostridium perfringens in vitro as demonstrated in Example 2.

[0013] Clostat (alias Bacillus PB6) is described in document WO2007/064741. Bacillus PB6 has an antagonistic effect against C. perfringens. Bacillus PB6 is hemolytic as described in Example 1.

SUMMARY OF THE INVENTION

[0014] It was surprisingly concluded that the addition of direct feed microbes (DFM) of the Bacillus species to animal feed can be used to prevent and/or control C. perfringens infections and/or necrotic enteritis in monogastric animals such as pigs and/or or poultry. Bacillus species may also improve body weight gain and/or feed conversion rate (e.g. in both Clostridium perfringens challenged and non-challenged chickens).

[0015] In a first aspect, the invention relates to a strain of Bacillus distinguished by the fact that: i) the strain of Bacillus is non-hemolytic, ii) the strain of Bacillus has antimicrobial activity against Clostridium perfringens and iii) the strain of Bacillus improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.

[0016] In a second aspect, the invention relates to a strain of Bacillus distinguished by the fact that: i) the strain of Bacillus is non-hemolytic, ii) the strain of Bacillus has antimicrobial activity against Clostridium perfringens and iii) the strain of Bacillus is sensitive to at least seven, as well as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.

[0017] In a third aspect, the invention relates to a strain of Bacillus distinguished by the fact that: i) the strain of Bacillus is non-hemolytic, ii) the strain of Bacillus is sensitive to at least seven, such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline and iii) the Bacillus strain enhances body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.

[0018] In a fourth aspect, the invention relates to a Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and optionally iii) the strain of Bacillus has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyze one or more of the substrates selected from the group consisting of Amylose, Arabinane, Arabinoxylan, Casein and Xylan.

[0019] The invention further relates to the strain having deposit accession number DSM 29869; a strain that has all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof.

[0020] The invention further relates to the strain having deposit accession number DSM 29870; a strain that has all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.

[0021] The invention further relates to the strain having deposit accession number DSM 29871; a strain that has all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof.

[0022] The invention further relates to the strain having deposit accession number DSM 29872; and a strain that has all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.

[0023] The present invention also relates to compositions comprising one or more of the Bacillus strains, according to the invention, which improve the health and performance of production animals.

[0024] In a preferred embodiment, the invention refers to a composition, for example, comprising a carrier and a strain of Bacillus, in which: i. the Bacillus strain is selected from the group consisting of: the strain having deposit accession number DSM 29869; wherein a strain has all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof, the strain having deposit accession number DSM 29870; wherein a strain has all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof, the strain having deposit accession number DSM 29871; wherein a strain has all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof, the strain having deposit accession number DSM 29872; and wherein a strain has all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof, and ii. the Bacillus strain has antimicrobial activity against Clostridium perfringens (such as Clostridium perfringens strains 23 or 48 [Gholamiandekhordi AR, Ducatelle R, Heyndrickx M, Haesebrouck F, Van Immerseel F. 2006. Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different diseasestatus. Vet. Microbiol. 113:143 to 152] and/or E. coli (such as ATCC10536 or ATCC25922).

[0025] In a preferred embodiment, the Bacillus strain has a high compatibility with monensin as being compatible with at least 2.4 μg/ml of monensin as determined in Example 12.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Alignment of 16 S rDNA sequences. SEQ ID NO: 1 (DSM 29869), SEQ ID NO: 2 (DSM 29870), SEQ ID NO: 3 (DSM 29871), SEQ ID NO: 4 (DSM 29872), SEQ ID NO: 5 (Bacillus vallismortis AB021198) , SEQ ID NO: 6 (Bacillus subtilis of AJ276351), and SEQ ID NO: 7 (Bacillus amyloliquefaciens AB255669) were aligned. The coverage position of region 481 to 1200 is not shown in Figure 1 (in this region all sequences are identical).

DEFINITIONS

[0027] In general, the terms and expressions used in this document have their meaning recognized in the art, which can be found by reference to standard texts, references to publications and context known to those skilled in the art. The following definitions are provided to clarify their specific use in contesting disclosure.

[0028] As used herein, the singular forms "a", "an" and "the" and "a" are intended to also include the plural forms, unless the context clearly indicates otherwise.

[0029] ANTIMICROBIAL ACTIVITY AGAINST CLOSTRIDIUM PERFRINGENS: The term "Antimicrobial activity against Clostridium perfringens" means that the growth of Clostridium perfringens is inhibited and/or that some or all Clostridium perfringens are killed. This can be determined by the assay described in Example 2.

[0030] BODY WEIGHT GAIN: An animal's Body Weight Gain is the increase in the animal's body weight in a specific period of time. An example of determining Body Weight Gain is given in Example 8.

[0031] COMPOSITION: The term "composition" refers to a composition comprising a carrier and at least one bacterial strain as described herein. The compositions described herein can be mixed with an animal feed (or feeds) and called "puree feed".

[0032] CONTROL OF INFECTIONS BY C. PERFRINGENS AND/OR NECROTICAL ENTERITIS: The term "control of infections by C. perfringens and/or necrotic enteritis" means a method and/or composition that partially or completely inhibits infections by C. perfringens and /or necrotic enteritis in an animal. Accordingly, the term "control of C. perfringens infections and/or necrotic enteritis" means that C. perfringens infections and/or necrotic enteritis are reduced or completely eliminated or prevented.

[0033] DIRECT-FEEDING MICRO-ORGANISM: The term "direct-feeding microorganism" means live microorganisms that include spores that, when administered in adequate quantities, confer a benefit, such as improved digestion or health, on the host.

[0034] ENZYME ACTIVITY UNDER AEROBIC CONDITIONS: The term "enzyme activity under aerobic conditions" means the activity of enzymes produced by a strain of Bacillus during growth under aerobic conditions as described in Example 6.

[0035] ENZYMATIC ACTIVITY UNDER ANAEROBIC CONDITIONS: The term "enzymatic activity under anaerobic conditions" means the activity of enzymes produced by a strain of Bacillus during growth under anaerobic conditions as described in Example 6.

[0036] EUROPEAN PRODUCTION EFFECTIVENESS FACTOR (EPEF): The European Production Efficiency Factor is a way of comparing the performance of live birds in flocks. This single figure facilitates comparison of performance within and between farms and can be used to evaluate environmental, climatic and management variables. EPEF is calculated as [(habitability (%) x Live weight (kg)) / (Age at depletion (days) x FCR)] x 100, where habitability is the percentage of birds alive at slaughter, Live weight is the average weight of birds at slaughter, depletion age is the age of birds at slaughter and FCR is the feed conversion ratio at slaughter.

[0037] FED: The term "fed" means any type of oral administration such as administration through animal feed or through drinking water.

[0038] FCR (FEED CONVERSION RATE): FCR is a measurement of an animal's efficiency in converting feed mass into increases in the desired product. In animals raised for meat - such as pigs, poultry and fish - the product is the mass gained by the animal. Specifically, the FCR is the mass of the food eaten divided by the product, over a specified period. FCR can be determined as described in Example 8. Improvement in FCR means reducing the FCR value. An improvement in FCR of 2% means that the FCR has been reduced by 2%.

[0039] ISOLATED: The term "isolated" means that the one or more bacterial strains described herein are in a form or environment that does not occur in nature, that is, the one or more bacterial strains are at least partially removed from a or more or all of the naturally occurring constituents with which they are associated in nature.

[0040] NON-HEMOLYTIC: Hemolysis is the breakdown of erythrocytes. The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify bacterial strains into hemolytic and nonhemolytic strains. In this context, hemolysis is defined as described in EFSA Journal 2011;9(11):2445, with the use of Bacillus subtilis 168 (BGSC-1A1, Bacillus Genetic Stock Center) as a negative control. A Bacillus strain can be classified as non-hemolytic using the assay described in Example 1.

[0041] FARMING BIRDS: The term "farming birds" means domesticated birds kept by humans for the eggs they produce and/or for their meat and/or their feathers. Farmed birds include broilers and layers. Farmed birds include members of the superorder Galloanserae (chickens), especially the order Galliformes (which includes chickens, guinea fowl, quail and turkeys) and the family Anatidae, in the order Anseriformes, commonly known as "waterfowl", including ducks domestic and domestic geese. Farmed birds also include other birds that are killed for their meat, such as young pigeons. Examples of farmed poultry include chickens (including layers, broilers and chicks), ducks, geese, pigeons, turkeys and quail.

[0042] PREVENTION OF INFECTIONS BY C. PERFRINGENS AND/OR NECROTICAL ENTERITIS: The term "prevention of infections by C. perfringens and/or necrotic enteritis" means a method and/or composition that prevents the development of an infection by C. perfringens and/or necrotic enteritis in an animal.

[0043] ANTIBIOTICS SENSITIVE: The term "antibiotic sensitive" means the phenotypic property of a bacterial strain, the growth of which said bacterial strain is inhibited under conditions in which the bacterial strain would otherwise develop. In this context, antibiotic sensitivity is tested following the CLSI guidelines (M07-A9 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 2012). S. aureus from ATCC 29213 is used as the reference strain, meaning that it would be included in the test, and that the results are only valid if S. aureus from ATCC 29213 shows results that meet the CLSI guideline breakpoints (see Example 5, Table 5.5) (M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014). A strain of Bacillus is considered sensitive if growth is detected at or below the appropriate antibiotic breakpoint concentration in EFSA journal 2012; 10(6):2740.

[0044] SPORE: The terms "spore" and "endospore" are interchangeable and have their normal meaning that is well known and understood by those skilled in the art. As used herein, the term spore refers to a microorganism in its dormant and protected state.

[0045] STABLE: The term "stable” is a term that is known in the art and, in a preferred aspect, stable means the ability of the microorganism to remain in a spore form until it is administered to an animal to improve the animal health.

[0046] SWINE: The term "swine" or "pigs" means domesticated pigs kept by humans for their food, such as their meat. Swine includes members of the genus Sus such as Sus scrofa domesticus or Sus domesticus and include piglets, developing pigs, and sows.

DETAILED DESCRIPTION OF THE INVENTION

[0047] It was surprisingly concluded that the addition of direct feed microbes (DFM) of the Bacillus species to animal feed can be used to prevent and/or control C. perfringens infections and/or necrotic enteritis in monogastric animals such as pigs. and/or poultry and, at the same time, improve body weight gain and/or feed conversion rate (in both Clostridium perfringens challenged and non-challenged chickens).

BACILLUS STRAINS OF THE INVENTION

[0048] The invention relates to the following aspects in relation to Bacillus strains:

[0049] Aspect 1: Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and iii) the Bacillus strain enhances body weight gain and/or the feed conversion rate in chickens fed the Bacillus strain.

[0050] Aspect 2: Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and iii) the Bacillus strain is sensitive to at least seven , as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.

[0051] Aspect 3: Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain is sensitive to at least seven, such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline and iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.

[0052] Aspect 4: Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, iv) the Bacillus strain has antimicrobial activity against Clostridium perfringens and optionally v) i) the Bacillus strain has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyze one or more of the substrates selected from the group consisting of Amylose, Arabinane, Arabinoxylan, Casein and Xylan.

[0053] In a preferred embodiment, the Bacillus according to Aspects 1, 2, 3 or 4 has a high compatibility with monensin as being compatible with at least 2.3 μg/ml of monensin as determined in Example 12. It is further It is more preferred that the Bacillus strain is compatible with at least 2.4 μg/ml monensin as determined in Example 12 (such as at least 2.5 μg/ml monensin as determined in Example 12, such as at least 2.6 μg /ml monensin as determined in Example 12 or as at least 2.7 μg/ml monensin as determined in Example 12).

[0054] Sensitivity to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline can, for example, be determined as described in Example 5.

[0055] Enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinane, Arabinoxylan, Casein and Xylan can, for example, be determined as described in Example 6.

[0056] In one embodiment the Bacillus strain according to Aspects 1, 2 or 3 has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan , Casein and Xylan.

[0057] In one embodiment, the Bacillus strain according to Aspects 2, 3 or 4 improves one or more performance parameters in poultry selected from the list consisting of body weight gain, Production Efficiency Factor European and feed conversion rate in chickens fed with the Bacillus strain.

[0058] In one embodiment, the Bacillus strain according to Aspect 3 has antimicrobial activity against Clostridium perfringens. Antimicrobial activity against Clostridium perfringens can, for example, be determined as described in Example 2.

[0059] In one embodiment, the Bacillus strain according to Aspects 1 or 4 is sensitive to at least seven to eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.

[0060] In one embodiment, improving the power conversion rate (FCR) for Aspects 1, 2, 3 or 4 results in an FCR of -2.5% or less than -2.5%, such as less than -2.6%, as less than -2.7%, as less than -2.8%, as less than -2.9%, as less than -3.0%. In a preferred embodiment, the improvement of FCR results in an FCR of -5% to -2%, such as an FCR of -4% to -2%, such as an FCR of -3.5% to -2.5%. In a specific embodiment, the FCR enhancement for Aspects 1, 2, 3, or 4 results in an FCR in a range selected from the group consisting of -5% to -4.5%, from -4.5% to -4%, from -4% to - 3.8%, from -3.8% to -3.6%, from -3.6% to -3.4%, from -3.4% to - 3.2%, -3.2 to -3.0%, -3.0% to -2.8%, and -2.8 to -2.5%, or any combination of these ranges. The FCR can be determined as described in Example 8.

[0061] In one embodiment, the improvement in body weight gain for Aspects 1, 2, 3 or 4 results in a body weight gain of at least 0.5%, such as at least 0.8%, such as at least 1.5%, like at least 1.8%, like at least 2.0%, like at least 2.3%, like at least 3.5%, like at least 4.2%, like at least 5, 2%, like at least 6.5%, like at least 7.3%. In a preferred embodiment, the improvement in body weight gain for Aspects 1, 2, 3, or 4 results in a body weight gain selected from the group consisting of 1.8% to 2.0%, of 2. .0% to 2.2%, 2.2% to 2.4%, 2.4% to 2.6%, 2.6% to 2.8%, 2.8% to 3, 0%, 3.0% to 3.2%, 3.2% to 3.4%, 3.4% to 3.6%, 3.6% to 3.8%, 3, 8% to 4.0%, 4% to 5%, 5% to 7%, 7% to 10%, or any combination thereof. Body weight gain can be determined as described in Example 8.

[0062] In one embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4, wherein the Bacillus strain comprises 16S rDNA that is more than 98% (such as more than 98.5%, such as more than 99%, as in greater than 99.5%, as in greater than 99.5%) sequence identity with SEQ ID NO: 1.

[0063] In one embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4, wherein the Bacillus strain comprises 16S rDNA that is more than 98% (such as more than 98.5%, such as more than 99%, as in greater than 99.5%) sequence identity with SEQ ID NO: 2.

[0064] In one embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4 wherein the Bacillus strain comprises 16S rDNA that is more than 98% (such as more than 98.5%, such as more than 99%, as in greater than 99.5%) sequence identity with SEQ ID NO: 3.

[0065] In one embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4, wherein the Bacillus strain comprises 16S rDNA that is more than 98% (such as more than 98.5%, such as more than 99%, such as greater than 99.5%) sequence identity with SEQ ID NO: 4.

[0066] In one embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4 is a strain of Bacillus subtilis, a strain of Bacillus amyloliquefaciens or a strain of Bacillus licheniformis.

[0067] In one embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4 has an antimicrobial effect against E. coli. The effect against E. coli can, for example, be determined as described in Example 4.

[0068] In one embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4 is the Bacillus strain that has deposit accession number DSM 29869, or a strain that has all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. In another embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4 is the Bacillus strain having deposit accession number DSM 29870, or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. In yet another embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4 is the Bacillus strain having deposit accession number DSM 29871, or a strain having all the identifying characteristics of DSM 29871 of Bacillus or a mutant thereof that has antimicrobial activity against Clostridium perfringens. In a further embodiment, the Bacillus strain according to Aspects 1, 2, 3 or 4 is the Bacillus strain having the deposit accession number DSM 29872, or a strain having all the identifying characteristics of DSM 29872. of Bacillus or a mutant thereof that has antimicrobial activity against Clostridium perfringens.

[0069] The invention relates, in one embodiment, to a strain of Bacillus that has the deposit accession number DSM 29869 or a strain that has all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof. The invention relates, in another embodiment, to a strain of Bacillus having the deposit accession number DSM 29870 or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. The invention relates, in another embodiment, to a strain of Bacillus having the deposit accession number DSM 29871 or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof. The invention relates, in a further embodiment, to a strain of Bacillus having deposit accession number DSM 29872 or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.

[0070] The invention also relates to a biologically pure culture of the Bacillus strain according to aspects 1, 2, 3 or 4. In a further embodiment, the invention relates to a biologically pure culture of the Bacillus strain having deposit accession number DSM 29869 or a strain that has all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof. In a further embodiment, the invention relates to a biologically pure culture of the Bacillus strain having deposit accession number DSM 29870 or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. In a further embodiment, the invention relates to a biologically pure culture of the Bacillus strain having deposit accession number DSM 29871 or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof. In a further embodiment, the invention relates to a biologically pure culture of the Bacillus strain having deposit accession number DSM 29872 or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.

[0071] The invention also relates to a Bacillus strain isolated according to aspects 1, 2, 3 or 4. In a further embodiment, the invention also relates to an isolated Bacillus strain having deposit accession number DSM 29869 or an isolated strain that has all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof. In a further embodiment, the invention also relates to an isolated Bacillus strain having deposit accession number DSM 29870 or an isolated strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. In a further embodiment, the invention also relates to an isolated Bacillus strain having deposit accession number DSM 29871 or an isolated strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof. In a further embodiment, the invention also relates to an isolated Bacillus strain having deposit accession number DSM 29872 or an isolated strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.

COMPOSITIONS OF THE INVENTION

[0072] The invention relates to a composition comprising spores of one or more strains of Bacillus according to the invention.

[0073] More specifically, the invention relates to the following aspects in relation to compositions comprising Bacillus strains:

[0074] Aspect 5: A composition comprising spores of a Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and iii) the Bacillus strain Bacillus improves body weight gain and/or feed conversion rate in chickens fed the Bacillus strain.

[0075] Aspect 6: A composition comprising spores of a Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and iii) the Bacillus strain Bacillus is sensitive to at least seven, as well as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.

[0076] Aspect 7: A composition comprising spores of a Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain is sensitive to at least seven, such as eight of the selected antibiotics from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline and iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the strain of Bacillus.

[0077] Aspect 8: A composition comprising spores of a Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and optionally iii) the strain of Bacillus has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyze one or more of the substrates selected from the group consisting of Amylose, Arabinane, Arabinoxylan, Casein and Xylan.

[0078] In a preferred embodiment, the Bacillus strain (or strains) of the composition according to Aspects 5, 6, 7 or 8 has a high compatibility with monensin as being compatible with at least 2.3 μg/ml of monensin as determined in Example 12. It is further preferred that the Bacillus strain is compatible with at least 2.4 μg/ml monensin as determined in Example 12 (such as at least 2.5 μg/ml monensin as determined in Example 12 , as at least 2.6 μg/ml monensin as determined in Example 12 or as at least 2.7 μg/ml monensin as determined in Example 12).

[0079] Sensitivity to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline can, for example, be determined as described in Example 5.

[0080] The enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinane, Arabinoxylan, Casein and Xylan can, for example, be determined as described in Example 6.

[0081] In one embodiment, the Bacillus strain of the composition according to Aspects 5, 6 or 7 has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinane , Arabinoxylan, Casein and Xylan.

[0082] In one embodiment, the Bacillus strain of the composition according to Aspects 6, 7 or 8 improves one or more performance parameters in poultry selected from the list consisting of body weight gain, Efficacy Factor of European Production and feed conversion rate in chickens fed with the Bacillus strain.

[0083] In one embodiment, the Bacillus strain of the composition according to Aspect 8 has antimicrobial activity against Clostridium perfringens. Antimicrobial activity against Clostridium perfringens can, for example, be determined as described in Example 2.

[0084] In one embodiment, the Bacillus strain of the composition according to Aspects 5 or 8 is sensitive to at least seven, such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.

[0085] In one embodiment, improving the power conversion rate (FCR) for Aspects 5, 6, 7 or 8 results in an FCR of -2.5% or less than -2.5%, such as less than -2.6%, as less than -2.7%, as less than -2.8%, as less than -2.9%, as less than -3.0%. In a preferred embodiment, the improvement of FCR results in an FCR of -5% to -2%, such as an FCR of -4% to -2%, such as an FCR of -3.5% to -2.5%. In a specific embodiment, the FCR enhancement for Aspects 1, 2, 3, or 4 results in an FCR in a range selected from the group consisting of -5% to -4.5%, from -4.5% to -4%, from -4% to - 3.8%, from -3.8% to -3.6%, from -3.6% to -3.4%, from -3.4% to - 3.2%, -3.2 to -3.0%, -3.0% to -2.8%, and -2.8 to -2.5%, or any combination of these ranges. The FCR can be determined as described in Example 8.

[0086] In one embodiment, the improvement in body weight gain for Aspects 5, 6, 7 or 8 results in a body weight gain of at least 0.5%, such as at least 0.8%, such as at least 1.5%, like at least 1.8%, like at least 2.0%, like at least 2.3%, like at least 3.5%, like at least 4.2%, like at least 5, 2%, like at least 6.5%, like at least 7.3%. In a preferred embodiment, the improvement in body weight gain for Aspects 1, 2, 3, or 4 results in a body weight gain selected from the group consisting of 1.8% to 2.0%, of 2. .0% to 2.2%, 2.2% to 2.4%, 2.4% to 2.6%, 2.6% to 2.8%, 2.8% to 3, 0%, 3.0% to 3.2%, 3.2% to 3.4%, 3.4% to 3.6%, 3.6% to 3.8%, 3, 8% to 4.0%, 4% to 5%, 5% to 7%, 7% to 10%, or any combination thereof. Body weight gain can be determined as described in Example 8.

[0087] In one embodiment the Bacillus strain of the composition according to Aspects 5, 6, 7 or 8 comprises 16S rDNA that is more than 98% (such as more than 98.5%, such as more than 99%, such as more than 99.5%) sequence identity with SEQ ID NO: 1 and/or greater than 98% (such as greater than 98.5%, such as greater than 99%, such as greater than 99.5%) sequence identity with SEQ ID NO: 2 and/or greater than 98% (such as greater than 98.5%, such as greater than 99%, such as greater than 99.5%) sequence identity with SEQ ID NO: 3 and/or greater than 98% (as in greater than 98.5%, as in greater than 99%, as in greater than 99.5%) sequence identity with SEQ ID NO: 4.

[0088] In one embodiment, the Bacillus strain of the composition according to Aspects 5, 6, 7 or 8 is a strain of Bacillus subtilis, or a strain of Bacillus amyloliquefaciens.

[0089] In one embodiment, the Bacillus strain of the composition according to Aspects 5, 6, 7 or 8 has an antimicrobial effect against E. coli. The effect against E. coli can, for example, be determined as described in Example 4.

[0090] In one embodiment, the Bacillus strain of the composition according to Aspects 5, 6, 7 or 8 is the Bacillus strain having deposit accession number DSM 29869, or a strain having all the characteristics of identification of Bacillus DSM 29869 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. In another embodiment, the Bacillus strain of the composition according to Aspects 5, 6, 7 or 8 is the Bacillus strain having deposit accession number DSM 29870, or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. In yet another embodiment, the Bacillus strain of the composition according to Aspects 5, 6, 7 or 8 is the Bacillus strain having deposit accession number DSM 29871, or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. In a further embodiment, the Bacillus strain of the composition according to Aspects 5, 6, 7 or 8 is the Bacillus strain having deposit accession number DSM 29872, or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.

[0091] The invention relates, in one embodiment, to a composition comprising a Bacillus having the deposit accession number DSM 29869 or a strain having all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof. The invention relates, in another embodiment, to a composition comprising a Bacillus having the deposit accession number DSM 29870 or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. The invention relates, in another embodiment, to a composition comprising a Bacillus having the deposit accession number DSM 29871 or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof. The invention relates, in a further embodiment, to a composition comprising a Bacillus having deposit accession number DSM 29872 or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.

[0092] In an embodiment of Aspects 5, 6, 7 or 8, the bacillus spores of the composition are present as dry spores such as spray-dried spores. In an embodiment of Aspects 5, 6, 7 or 8, the bacillus spores of the composition are present as stable spores. The composition according to Aspects 5, 6, 7 or 8 may also be a liquid composition and/or comprise culture supernatant comprising one or more Bacillus strains of the invention.

[0093] In an embodiment of Aspects 5, 6, 7 or 8, the composition additionally comprises a carrier. The carrier may comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, sulfate potassium, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch, cellulose, farigel , cassava kernels, sodium aluminum silicate, colloidal amorphous silica, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000 and carbopol.

[0094] In a preferred embodiment of Aspects 5, 6, 7 or 8, the composition additionally comprises calcium carbonate and sodium aluminum silicate.

[0095] In a preferred embodiment of Aspects 5, 6, 7 or 8, the composition additionally comprises calcium carbonate, sodium aluminum silicate and sucrose.

[0096] In another preferred embodiment of Aspects 5, 6, 7 or 8, the composition further comprises one or more carriers such as one or more carriers selected from the group consisting of calcium carbonate, sodium sulfate, starch, farigel and cassava kernels.

[0097] In another additional embodiment of Aspects 5, 6, 7 or 8, the composition additionally comprises one or more flow agents such as sodium aluminum silicate and/or colloidal amorphous silica (for example, Sipernat 50S).

[0098] In another further embodiment of Aspects 5, 6, 7 or 8, the composition further comprises one or more binders such as one or more binders selected from the group consisting of sucrose, sorbitol, glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, dextrin, maltodextrin and carbopol.

[0099] In a preferred embodiment, the composition comprises Bacillus DSM 29869, calcium carbonate and sodium aluminum silicate.

[00100] In a preferred embodiment, the composition comprises Bacillus DSM 29869, calcium carbonate, sodium aluminum silicate and sucrose.

[00101] In a preferred embodiment, the composition comprises Bacillus DSM 29869 and one or more carriers such as one or more carriers selected from the group consisting of calcium carbonate, sodium sulfonate, starch, farigel and cassava kernels.

[00102] In a preferred embodiment, the composition comprises Bacillus DSM 29869 and one or more flow agents such as sodium aluminum silicate and/or colloidal amorphous silica (for example, Sipernat 50S).

[00103] In a preferred embodiment, the composition comprises Bacillus DSM 29869 and one or more binders such as one or more binders selected from the group consisting of sucrose, sorbitol, glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, dextrin, maltodextrin and carbopol.

[00104] In a preferred embodiment, the composition comprises Bacillus DSM 29870, calcium carbonate and sodium aluminum silicate.

[00105] In a preferred embodiment, the composition comprises Bacillus DSM 29870, calcium carbonate, sodium aluminum silicate and sucrose.

[00106] In a preferred embodiment, the composition comprises Bacillus DSM 29870 and one or more carriers such as one or more carriers selected from the group consisting of calcium carbonate, sodium sulfonate, starch, farigel and cassava kernels.

[00107] In a preferred embodiment, the composition comprises Bacillus DSM 29870 and one or more flow agents such as sodium aluminum silicate and/or colloidal amorphous silica (for example, Sipernat 50S).

[00108] In a preferred embodiment, the composition comprises Bacillus DSM 29870 and one or more binders such as one or more binders selected from the group consisting of sucrose, sorbitol, glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, dextrin, maltodextrin and carbopol.

[00109] In a preferred embodiment, the composition comprises Bacillus DSM 29871, calcium carbonate and sodium aluminum silicate.

[00110] In a preferred embodiment, the composition comprises Bacillus DSM 29871, calcium carbonate, sodium aluminum silicate and sucrose.

[00111] In a preferred embodiment, the composition comprises Bacillus DSM 29871 and one or more carriers such as one or more carriers selected from the group consisting of calcium carbonate, sodium sulfonate, starch, farigel and cassava kernels.

[00112] In a preferred embodiment, the composition comprises Bacillus DSM 29871 and one or more flow agents such as sodium aluminum silicate and/or colloidal amorphous silica (for example, Sipernat 50S).

[00113] In a preferred embodiment, the composition comprises Bacillus DSM 29871 and one or more binders such as one or more binders selected from the group consisting of sucrose, sorbitol, glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, dextrin, maltodextrin and carbopol.

[00114] In a preferred embodiment, the composition comprises Bacillus DSM 29872, calcium carbonate and sodium aluminum silicate.

[00115] In a preferred embodiment, the composition comprises Bacillus DSM 29872, calcium carbonate, sodium aluminum silicate and sucrose.

[00116] In a preferred embodiment, the composition comprises Bacillus DSM 29872 and one or more carriers such as one or more carriers selected from the group consisting of calcium carbonate, sodium sulfonate, starch, farigel and cassava kernels.

[00117] In a preferred embodiment, the composition comprises Bacillus DSM 29872 and one or more flow agents such as sodium aluminum silicate and/or colloidal amorphous silica (for example, Sipernat 50S).

[00118] In a preferred embodiment, the composition comprises Bacillus DSM 29872 and one or more binders such as one or more binders selected from the group consisting of sucrose, sorbitol, glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, dextrin, maltodextrin and carbopol.

[00119] In one embodiment, the composition comprises one or more coccidiostats in which the composition is, for example, a premix.

[00120] In a preferred embodiment of the composition according to Aspects 5, 6, 7 or 8, the composition comprises from 105 to 1018 CFU/g of isolated Bacillus spores.

[00121] In a further embodiment, the composition according to the invention comprises one or more bacterial strains such as at least two of the above strains up to and including all strains in the group consisting of DSM 29869, DSM 29870, DSM 29871 and DSM 29872 .

[00122] In one embodiment for any of the previously mentioned embodiments, the Bacillus spore exterminates/inhibits at least 40% (such as at least 45%, at least 50%, at least 60%, at least 70% or at least 80 %) of Clostridium perfringens after, for example, 24 hours, for example, determined as described in Example 2.

[00123] In another embodiment of the invention, the composition additionally comprises one or more additional microbes. In another embodiment of the invention, the composition additionally comprises one or more enzymes. In another embodiment of the invention, the composition additionally comprises one or more vitamins. In another embodiment of the invention, the composition additionally comprises one or more minerals. In another embodiment of the invention, the composition additionally comprises one or more amino acids. In another embodiment of the invention, the composition additionally comprises one or more feed ingredients.

[00124] In one embodiment for any of the previously mentioned embodiments, the composition also improves the health of the monogastric animal when provided as food to said animal. In another embodiment for any of the aforementioned embodiments, the composition can also increase the egg yield of poultry when said poultry are fed. In one embodiment for any of the aforementioned embodiments, the composition increases the yield of meat from the monogastric animal when provided as food to said animal.

[00125] In a preferred embodiment, the composition comprises one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1x104 and 1x1018 CFU/kg of composition, preferably between 1x107 and 1x1016 CFU/kg of composition, more preferably between 1x1010 and 1x1015 CFU/kg of composition and, most preferably, between 1x1011 and 1x1014 CFU/kg of composition.

[00126] In a preferred embodiment, the bacterial count of each of the bacterial strains in the composition is between 1x104 and 1x1018 CFU/kg of dry matter, preferably between 1x107 and 1x1016 CFU/kg of dry matter, more preferably between 1x1010 and 1x1015 CFU/kg of dry matter and with maximum preference between 1x1011 and 1x1014 CFU/kg of dry matter. In a more preferred embodiment, the bacterial count of each of the bacterial strains in the animal feed is between 1x108 and 1x1010 CFU/kg of dry matter.

[00127] In a preferred embodiment, the composition has a bacterial count of each Bacillus spore between 1x103 and 1x1013 CFU/animal/day, preferably between 1x105 and 1x1011 CFU/animal/day, more preferably between 1x106 and 1x1010 CFU/animal/day and, most preferably, between 1x107 and 1x109 CFU/animal/day.

[00128] In yet another embodiment of the invention, one or more bacterial strains are present in the composition in the form of a spore as a stable spore. In yet a further embodiment of the invention, the stable spore will germinate in the intestine and/or stomach of the monogastric animal.

[00129] In one embodiment, the one or more bacterial strains are stable when subjected to pressures applied/obtained during an extrusion process for pelletization. In a specific embodiment, the one or more bacterial strains are stable at pressures ranging from 0.1 MPa (1 bar) to 4 MPa (40 bars), particularly from 1 MPa (10 bars) to 4 MPa (40 bars), more particularly, from 1.5 MPa (15 bars) to 4 MPa (40 bars), even more particularly, from 2 MPa (20 bars) to 4 MPa (40 bars), even more particularly from 3.5 MPa (35 bars) ) to 3.7 MPa (37 bars), even more particularly 3.6 MPa (36 bars).

[00130] In a specific embodiment, the one or more bacterial strains are stable at high temperatures. In particular, bacterial strains are stable when subjected to the temperatures obtained during an extrusion process for pelletization. In an even more particular embodiment, the one or more bacterial strains are stable at temperatures ranging from 80 °C to 120 °C, particularly temperatures ranging from 90 °C to 120 °C, even more particularly, temperatures ranging from 95°C to 120°C.

[00131] In another aspect, the invention relates to a composition comprising a carrier and one or more bacterial cultures that have characteristics substantially identical to those of a strain selected from the group consisting of: the strain having the DSM deposit accession number 29869; the strain having deposit accession number DSM 29870; the strain having deposit accession number DSM 29871; and the strain having deposit accession number DSM 29872; or any combination thereof.

[00132] In one embodiment, the composition comprises a carrier and the strain having the deposit accession number DSM 29869, or a strain having all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof.

[00133] In one embodiment, the composition comprises a carrier and the strain having the deposit accession number DSM 29870, or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.

[00134] In one embodiment, the composition comprises a carrier and the strain having the deposit accession number DSM 29871, or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof.

[00135] In one embodiment, the composition comprises a carrier and the strain having the deposit accession number DSM 29872, or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.

[00136] In one embodiment, the composition additionally comprises one or more additional microbes. In a particular embodiment, the composition further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

[00137] In a specific embodiment, the composition additionally comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, Bacillus megaterium, Bacillus coagulans, Bacillus circulans or any combination thereof same.

[00138] In a specific embodiment, the composition additionally comprises one or more types of yeast. The one or more types of yeast can be selected from the group consisting of Saccharomycetaceae, Saccharomyces (such as S. cerevisiae and/or S. boulardii), Kluyveromyces (such as K. marxianus and K. lactis), Candida (such as C. utilis, also called Torula yeast), Pichia (such as P. pastoris), Torulaspora (such as T. delbrueckii), Phaffia yeasts, and Basidiomycota.

[00139] In an embodiment for any of the previously mentioned embodiments, the composition additionally comprises a carrier. The carrier may comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, sulfate potassium, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch, cellulose, farigel , cassava kernels, sodium aluminum silicate, colloidal amorphous silica, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000 and carbopol.

[00140] In another embodiment, the composition described herein may optionally include one or more enzymes. Enzymes can be classified based on the NC-IUBMB Enzyme Nomenclature manual, 1992, see also the ENZYME website: http://www.expasy.ch/enzyme/. ENZYME is a repository of information regarding enzyme nomenclature. It is based mainly on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and describes each type of characterized enzyme for which an EC number (Commission of Enzyme) was provided (Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305). This nomenclature of IUB-MB Enzymes is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural characteristics of these enzymes.

[00141] Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, xylanase, galactanase, mannanase, dextranase and alpha-galactosidase, into families based on amino acid sequence similarities was proposed a few years ago. They are currently classified into 90 different families: See the CAZy(ModO) website (Coutinho, P.M. & Henrissat, B. (1999) Carbohydrate-Active Enzymes server at URL: http://afmb.cnrs-mrs.fr/ ~cazy/CAZY/index.html (corresponding articles: Coutinho, P.M. & Henrissat, B. (1999) Carbohydrate-active enzymes: an integrated database approach. In "Recent Advances in Carbohydrate Bioengineering", edited by H.J. Gilbert, G. Davies , B. Henrissat and B. Svensson, The Royal Society of Chemistry, Cambridge, pages 3 to 12; Coutinho, P. M. & Henrissat, B. (1999) The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach. In "Genetics, Biochemistry and Ecology of Cellulose Degradation", edited by K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimura, Uni Publishers Co., Tokyo, pages 15 to 23 ).

[00142] Therefore, the composition of the invention may also comprise at least one other enzyme selected from the group comprising phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4), phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixture thereof.

[00143] In a specific embodiment, the composition of the invention comprises a phytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially available phytases include Phytase Bio-FeedTM (Novozymes), Ronozyme® P and HiPhosTM (DSM Nutritional Products), NatuphosTM (BASF), Finase® and Quantum® Blue (AB enzymes), a Phyzyme® XP (Verenium/DuPont ) and Axtra® PHY (DuPont). Other preferred phytases include those described, for example, in WO 98/28408, WO 00/43503, and WO 03/066847.

[00144] In a specific embodiment, the composition of the invention comprises a xylanase (EC 3.2.1.8). Examples of commercially available xylanases include Ronozyme® WX and G2 (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium) and Axtra® XB (Xylanase/beta-glucanase, DuPont).

[00145] In a specific embodiment, the composition of the invention comprises a protease (EC 3.4). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).

MANUFACTURING

[00146] The composition of the invention can, for example, be manufactured as a bulk composition (non-pelletized) or pelletized composition. Bacterial cultures and optionally enzymes can be added as solid and liquid formulations. For example, for bulk compounding, a solid or liquid culture formulation may be added before or during the ingredient mixing step. Typically, a liquid culture preparation comprises the culture of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets.

[00147] The enzyme can be added to the composition as a granule, which is optionally pelletized or extruded. The granule typically comprises a core particle and one or more coatings, which are typically salt and/or wax coatings. The core particle can be any one of a homogeneous mixture of an active compound optionally together with salts (for example, organic or inorganic zinc or calcium salt) or an inert particle with an active compound applied thereto. The active compound is the culture of the invention optionally combined with one or more enzymes. The inert particle may be water-soluble or water-insoluble, for example, starch, a sugar (such as sucrose or lactose), or a salt (such as NaCl, Na2SO4). The salt coating is typically at least 1 μm thick and can be a specific salt or a mixture of salts, such as Na2SO4, K2SO4, MgSO4 and/or sodium citrate. Other examples are those described, for example, in WO 2008/017659, WO 2006/034710, WO 97/05245, WO 98/54980, WO 98/55599, WO 00/70034 or polymer coating as described in WO 01 /00042.

PREFERRED MODALITIES

[00148] Preferred embodiments of the invention are described in the two sets of items below in the present document. SET OF ITEMS I: 1. A strain of Bacillus distinguished by the fact that: i) the strain of Bacillus is non-hemolytic, ii) the strain of Bacillus has antimicrobial activity against Clostridium perfringens, and iii) the strain of Bacillus enhances the gain of body weight and/or feed conversion rate in chickens fed with the Bacillus strain. 2. A strain of Bacillus distinguished by the fact that: i) the strain of Bacillus is non-hemolytic, ii) the strain of Bacillus has antimicrobial activity against Clostridium perfringens, and iii) the strain of Bacillus is sensitive to at least seven, as eight of antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline. 3. A strain of Bacillus distinguished by the fact that: i) the strain of Bacillus is non-hemolytic, ii) the strain of Bacillus is sensitive to at least seven, such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin , Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline and iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain. 4. A Bacillus strain distinguished by the fact that: i) the Bacillus strain is non-hemolytic, ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens, and optionally iii) the Bacillus strain has enzymatic activity under aerobic conditions and/ or anaerobic that hydrolyze one or more of the substrates selected from the group consisting of Amylose, Arabinane, Arabinoxylan, Casein and Xylan. 5. The Bacillus strain according to any one of items 1 to 3, wherein the Bacillus strain has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose , Arabinan, Arabinoxylan, Casein and Xylan (e.g., determined as described in Example 6). 6. The Bacillus strain according to any one of items 2 to 4, wherein the Bacillus strain improves one or more performance parameters in poultry selected from the list consisting of body weight gain, European Production Efficacy and feed conversion rate in poultry fed the Bacillus strain. 7. The Bacillus strain according to item 3, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens (e.g., determined as described in Example 2). 8. The Bacillus strain according to any one of items 1 and 4, wherein the Bacillus strain is sensitive to at least seven, such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin , Kanamycin, Streptomycin, Erythromycin and Tetracycline (e.g., determined as described in Example 5). 9. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain comprises 16S rDNA that is greater than 98% sequence identity with SEQ ID NO: 1 and/or greater than 98% identity of sequence with SEQ ID NO: 2 and/or greater than 98% sequence identity with SEQ ID NO: 3 and/or greater than 98% sequence identity with SEQ ID NO: 4. 10. The Bacillus strain, according to any of the preceding items, wherein the Bacillus strain is a Bacillus subtilis strain or a Bacillus amyloliquefaciens strain. 11. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain has an antimicrobial effect against E. coli (e.g., determined as described in Example 4). 12. The strain of Bacillus according to any of the preceding items, wherein the strain of Bacillus is the strain of Bacillus having deposit accession number DSM 29869 or a strain having all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. 13. The strain of Bacillus according to any of the preceding items, wherein the strain of Bacillus is the strain of Bacillus having deposit accession number DSM 29870 or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. 14. The strain of Bacillus according to any of the preceding items, wherein the strain of Bacillus is the strain of Bacillus having deposit accession number DSM 29871 or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. 15. The strain of Bacillus according to any of the preceding items, wherein the strain of Bacillus is the strain of Bacillus having deposit accession number DSM 29872 or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof that has antimicrobial activity against Clostridium perfringens. 16. A Bacillus that has deposit accession number DSM 29869 or a strain that has all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof. 17. A Bacillus that has deposit accession number DSM 29870 or a strain that has all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. 18. A Bacillus that has deposit accession number DSM 29871 or a strain that has all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof. 19. A Bacillus that has deposit accession number DSM 29872 or a strain that has all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof. 20. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain has a high compatibility with monensin as being compatible with at least 2.3 μg/ml monensin as determined in Example 12 (as per at least 2.4 μg/ml monensin, at least 2.5 μg/ml monensin, at least 2.6 μg/ml monensin, or at least 2.7 μg/ml monensin as determined in Example 12 ). 21. A composition comprising spores of the Bacillus strain according to any one of items 1 to 20. 22. The composition according to item 21, wherein the Bacillus spores of the composition are present as dry spores. 23. The composition according to any one of items 21 to 22, which further comprises a carrier. 24. The Composition according to item 23, wherein the carrier comprises one or more of the following compounds: water, glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium aluminum silicate , sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, flour wheat, wheat bran, corn gluten flour, starch, farigel, cassava kernels, sodium aluminum silicate, colloidal amorphous silica, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, carbopol and cellulose. 25. The composition according to any one of items 21 to 24, wherein the composition comprises from 105 to 1018 CFU/g of isolated Bacillus spores. 26. The Composition according to any one of items 21 to 25, which further comprises one or more components selected from the list consisting of: one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients. 27. The Composition according to any one of items 21 to 26, wherein the bacterial count of each Bacillus spore is between 1x104 and 1x1018 CFU/kg of composition, preferably between 1x107 and 1x1016 CFU/kg of composition, with more preferably, between 1x1010 and 1x1015 CFU/kg of composition and, most preferably, between 1x1011 and 1x1014 CFU/kg of composition. 28. The Composition according to any one of items 21 to 26, wherein the bacterial count of each Bacillus spore is between 1x104 and 1x1018 CFU/kg of composition, preferably between 1x106 and 1x1015 CFU/kg of composition, and, more preferably, between 1x107 and 1x1011 CFU/kg of composition. 29. A biologically pure culture of the Bacillus strain according to any one of items 1 to 20. 30. An isolated Bacillus strain according to any one of items 1 to 20. 31. An isolated Bacillus strain selected from from the group consisting of Bacillus strain DSM 29869, DSM 29870, DSM 29871 and DSM 29872, or a strain that has all the identifying characteristics of Bacillus DSM 29869, DSM 29870, DSM 29871 and DSM 29872 or a mutant thereof. 32. The strain of Bacillus isolated in accordance with item 31, wherein the identifying characteristics may be one or more (as all) of the characteristics selected from the group consisting of i) non-hemolytic, e.g., as determined in Example 1, ii) antimicrobial activity against Clostridium perfringens, for example, as determined in Example 2, iii) antimicrobial activity against E. coli, for example, as determined in Example 4, iv) sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin , Kanamycin, Streptomocin, Erythromycin and Tetracycline, and v) high compatibility with monensin as being compatible with at least 2.3 μg/ml of monensin as determined in Example 12 (as at least 2.4 μg/ml of monensin, as at least 2.5 μg/ml monensin, as at least 2.6 μg/ml monensin, or as at least 2.7 μg/ml monensin as determined in Example 12). SET OF ITEMS II: 1. A strain of Bacillus distinguished by the fact that: i) it has deposit accession number DSM 29870 or a strain that has all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof; ii) has deposit accession number DSM 29869 or a strain that has all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof; iii) has deposit accession number DSM 29871 or a strain that has all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof; or iv) has deposit accession number DSM 29872 or a strain that has all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof. 2. The Bacillus strain according to item 1, wherein the Bacillus strain is non-hemolytic. 3. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens (e.g., determined as described in Example 2). 4. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain enhances body weight gain and/or feed conversion rate in chickens fed the Bacillus strain. 5. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain comprises 16S rDNA that is greater than 98% sequence identity with SEQ ID NO: 1 and/or greater than 98% identity of sequence with SEQ ID NO: 2 and/or greater than 98% sequence identity with SEQ ID NO: 3 and/or greater than 98% sequence identity with SEQ ID NO: 4. 6. The Bacillus strain, according to any of the preceding items, wherein the Bacillus strain comprises 16S rDNA that is greater than 99% sequence identity with SEQ ID NO: 1 and/or greater than 99% sequence identity with SEQ ID NO: 2 and/or greater than 99% sequence identity with SEQ ID NO: 3 and/or greater than 99% sequence identity with SEQ ID NO: 4. 7. The Bacillus strain according to any of the items above, wherein the Bacillus strain is a Bacillus subtilis strain or a Bacillus amyloliquefaciens strain. 8. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain has an antimicrobial effect against E. coli (e.g., determined as described in Example 4). 9. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain is an isolated Bacillus strain. 10. A biologically pure culture of the Bacillus strain according to any one of items 1 to 9. 11. The Bacillus strain according to any one of the preceding items, wherein the Bacillus strain is sensitive to at least seven , as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline (e.g., determined as described in Example 5). 12. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinane , Arabinoxylan, Casein and Xylan (e.g., determined as described in Example 6). 13. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain improves one or more performance parameters in poultry selected from the list consisting of body weight gain, Efficacy Factor of European production and feed conversion rate in poultry fed the Bacillus strain. 14. The Bacillus strain according to any of the preceding items, wherein the Bacillus strain has a high compatibility with monensin as being compatible with at least 2.3 μg/ml monensin as determined in Example 12 (as per at least 2.4 μg/ml monensin, at least 2.5 μg/ml monensin, at least 2.6 μg/ml monensin, or at least 2.7 μg/ml monensin as determined in Example 12 ). 15. A composition comprising spores of the Bacillus strain according to any one of items 1 to 14. 16. The composition according to item 15, wherein the Bacillus spores of the composition are present as spores such as dried spores as spray-dried spores. 17. The Composition according to item 16, wherein the bacterial count of each Bacillus spore is between 1x104 and 1x1018 CFU/kg of composition, preferably between 1x106 and 1x1015 CFU/kg of composition, and, more preferably, between 1x107 and 1x1011 CFU/kg of composition. 18. The composition according to any one of items 15 to 17, which further comprises a carrier. 19. The Composition according to item 18, wherein the carrier comprises one or more of the following compounds: water, glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium aluminum silicate , sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, flour wheat, wheat bran, corn gluten flour, starch, farigel, cassava kernels, sodium aluminum silicate, colloidal amorphous silica, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, carbopol and cellulose. 20. The composition according to item 18 or 19, wherein the carrier comprises calcium carbonate. 21. The composition according to any one of items 15 to 20, wherein the composition comprises from 105 to 1018 CFU/g of isolated Bacillus spores. 22. The Composition according to any one of items 15 to 21, which further comprises one or more components selected from the list consisting of: one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients. 23. The Composition according to any one of items 15 to 22, wherein the bacterial count of each Bacillus spore is between 1x104 and 1x1018 CFU/kg of composition, preferably between 1x107 and 1x1016 CFU/kg of composition, with more preferably, between 1x1010 and 1x1015 CFU/kg of composition and, most preferably, between 1x1011 and 1x1014 CFU/kg of composition. 24. The Composition according to any one of items 15 to 23, wherein the bacterial count of each Bacillus spore is between 1x104 and 1x1018 CFU/kg of composition, preferably between 1x106 and 1x1015 CFU/kg of composition, and, more preferably, between 1x107 and 1x1011 CFU/kg of composition. 25. An isolated Bacillus strain selected from the group consisting of Bacillus strain DSM 29869, DSM 29870, DSM 29871 and DSM 29872, or a strain that has all the identifying characteristics of Bacillus DSM 29869, DSM 29870, DSM 29871 and DSM 29872 or a mutant thereof. 26. The Bacillus strain isolated in accordance with item 25, wherein the identifying characteristics may be one or more (as all) of the characteristics selected from the group consisting of i) non-hemolytic, e.g., as determined in Example 1, ii) antimicrobial activity against Clostridium perfringens, for example, as determined in Example 2, iii) antimicrobial activity against E. coli, for example, as determined in Example 4, iv) sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin , Kanamycin, Streptomocin, Erythromycin and Tetracycline, and v) high compatibility with monensin as being compatible with at least 2.3 μg/ml of monensin as determined in Example 12 (as at least 2.4 μg/ml of monensin, as at least 2.5 μg/ml monensin, as at least 2.6 μg/ml monensin, or as at least 2.7 μg/ml monensin as determined in Example 12). 27. The strain of Bacillus isolated in accordance with item 25 or 26 for use in the treatment of necrotic enteritis and/or a clostridium perfringens infection. 28. An isolated Bacillus strain selected from the group consisting of Bacillus strain DSM 29869, DSM 29871 and DSM 29872 or a strain having all the identifying characteristics of Bacillus DSM 29869, DSM 29871 and DSM 29872 or a mutant of even for the treatment of necrotic enteritis and/or a clostridium perfringens infection. 29. Use of the Bacillus strain or the Composition according to any of the preceding items, to improve one or more performance parameters of an animal selected from the group consisting of: improving feed conversion ratio, improving gain of body weight, improve the European Production Efficiency Factor, improve feeding efficiency and improve the health of, for example, poultry such as chickens. 30. Use, in accordance with item 29, in which the Bacillus strain is selected from the group consisting of the Bacillus strain DSM 29869, DSM 29871 and DSM 29872, or a strain that has all the identifying characteristics of Bacillus DSM 29869, DSM 29871 and DSM 29872 or a mutant thereof. 31. A method of improving one or more performance parameters of an animal selected from the group consisting of: improving feed conversion ratio, improving body weight gain, improving European Production Efficiency Factor, improving feeding efficacy and improving health, which comprises administering the Bacillus strain or the Composition according to any of the preceding items, to the animal. 32. The method, according to item 31, in which the Bacillus strain is selected from the group consisting of the Bacillus strain DSM 29869, DSM 29871 and DSM 29872, or a strain that has all the identifying characteristics of Bacillus DSM 29869, DSM 29871 and DSM 29872 or a mutant thereof. 33. A method for feeding an animal comprising administering the Bacillus strain or the Composition according to any of the preceding items, to said animal optionally together with other animal feed ingredients. 34.The method of feeding an animal in accordance with item 33, wherein the animal is selected from the group consisting of poultry and swine.

EXAMPLES EXAMPLE 1: Screening for Hemolysis-negative Bacillus strains

[00149] Hemolysis was tested in accordance with the Technical Manual on the assessment of the toxigenic potential of Bacillus species used in animal nutrition, EFSA Journal 2011; 9(11):2445.

[00150] Sheep blood agar plates were purchased ready for use (Becton Dickenson art 254053 or 254087). Alternatively, agar plates can be prepared by adding 5% defibrated sheep blood (obtained from Statens Serum Institute, Denmark) to TS-agar (Oxoid CM 131). The agar should be autoclaved at 121°C for 20 minutes and cooled to about 40°C before adding the blood immediately before pouring onto the plates.

[00151] Bacillus strains were extracted from preservation at -80 °C and applied by swabbing onto the TS agar plate, which was incubated at 30 °C overnight or until growth appeared. From a single colony, as little material as possible was used for application by swabbing into a line in % of an agar plate. The plate was incubated at 30 °C for 72 hours. The hemolysis/whitening zones of the Bacillus strains to be tested were compared with the positive and negative control. As a positive control, Bacillus subtilis ATCC21332 was used. As a negative control, Bacillus subtilis 168, (BGSC-1A1, Bacillus Genetic Stock Center) was used.

[00152] In a screening, of 599 independent isolates of Bacillus strains 223 (37%) were negative for hemolysis. In another screen, 21 of 65 independent Bacillus isolates (32%) were negative for hemolysis. Both screenings comprised, exclusively, strains that are based on the identification for sequencing of 16S rDNA that belongs to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus or Bacillus amyloliquefaciens or in close relation to these four species.

[00153] Non-hemolytic Bacillus strains therefore appear to be common and reasonably abundant in nature, but comprising only a minority of natural strains. Nonhemolytic strains seen as more abundant in Bacillus licheniformis, while Bacillus amyloliquefaciens strains appear hemolytic. All non-hemolytic strains were tested for growth inhibition of Clostridium perfringens. The following strains from the screening were all negative for hemolysis and from those selected for further study: DSM 29869, DSM 29870, DSM 29871 and DSM 29872. Clostat (Bacillus PB6) was determined to be hemolytic.

EXAMPLE 2: DETERMINATION OF GROWTH INHIBITION OF CLOSTRIDIUM PERFRINGENS

[00154] All non-hemolytic strains were tested for growth inhibition of Clostridium perfringens strains 23 and 48 (both are positive for netB) [Gholamiandekhordi et al., 2006, Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status , Vet. Microbiol. 113:143-152], were grown overnight in tryptic soy broth (BD part 211822) supplemented with 0.6% yeast extract (BD part 212750) at 35 °C under static anaerobic conditions. 250 μl of overnight Clostridium perfringens culture was added to 250 ml of tryptic soy agar supplemented with 0.6% yeast extract at 40°C and poured into rectangular petri dishes (Nunc part 267060). The inoculated agar was allowed to cool to room temperature after which an 8 mm diameter well was produced in the agar. The plates were stored in the absence of oxygen until use.

[00155] The Bacillus strain DSM 29869, DSM 29870, DSM 29871 or DSM 29872 was cultivated overnight in tryptic soy broth at 35 ° C under aerobic conditions. 1,000 μl of Bacillus culture was collected and fractionated into cell-free supernatant and cells via centrifugation. 20 μl of cell-free supernatant or 100x cells diluted in phosphate-buffered saline were added directly to the wells on agar plates inoculated with Clostridium perfringens. A control well contained 20 μl of phosphate-buffered saline. The plates were incubated for 18 hours at 35 °C under anaerobic conditions.

[00156] Inhibition of the Clostridium perfringens strain was noted by a zone of whitening around the well of interest. The phosphate buffered saline well was considered a negative control based on the absence of the brightening zone around the well.

[00157] Cell-free supernatant and 100x diluted cells from Bacillus strains DSM 29869, DSM 29870, DSM 29871 and DSM 29872 had the ability to consistently inhibit the growth of C. perfringens 23 and 48 strains in vitro. Inhibition was also seen with the "CloSTAT" competitor strain, both for supernatant and cells, but was not seen with the GalliproTect competitor strain. "CloSTAT" is a strain of Bacillus amyloliquefaciens that was isolated from the commercial DFM product CloSTAT, Kemin. "GalliproTect" is a strain of Bacillus licheniformis that was isolated from the commercial product Gallipro Tect, Chr Hansen.

EXAMPLE 3: IDENTIFICATION, CHARACTERIZATION AND DEPOSIT OF BIOLOGICAL MATERIAL

[00158] The following biological materials have been deposited under the terms of the Budapest Treaty at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH, Inhoffenstraβe 7 B, 38124 Braunschweig, Germany, and have been assigned the following accession numbers : TABLE 3.1: DEPOSIT OF BIOLOGICAL MATERIAL

[00159] The strains were deposited under conditions that ensure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposits represent a substantially pure culture of the deposited strain. Deposits are available as required by foreign patent laws in countries in which counterparties to this application or their descendants are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the present invention in derogation of patent rights granted by governmental action.

SEQUENCING OF 16S rDNA GENES

[00160] DNA was extracted from a culture of DSM 29869, DSM 29870, DSM 29871 and DSM 29872 using the QiaAmp DNA Blood Mini Kit (Qiagen art 51106). The kit was used as recommended for DNA extraction from gram-positive bacteria.

[00161] 16S rDNA was amplified in a total volume of 50 μl by mixing: 10 pmol of each of Primer 16S F and 16S R, 0.2 mM of each nucleotide, 2.5 units of Ampli taq, 1 x Ampli taq buffer, 5 μl of DNA template and using the following PCR program: 94°C 2 min (94°C 30 s, 52°C 30 s, 72°C 1 min)x35.72° C 10 min in a Perkin Elmer PCR machine. The PCR product was sequenced by the Novozymes DNA sequencing facility using primer 530R, 357F, 1390R and 1100F. TABLE 3.2: INITIATORS:

[00162] 1390-R Primer Degeneration: R is A or G. The 16S rDNA sequences of DSM 29869, DSM 29870, DSM 29871 and DSM 29872 are shown as SEQ ID NO: 1 to 4 in the sequence listing, respectively . The 16S rDNA sequences of DSM 29869, DSM 29870, DSM 29871 and DSM 29872 were analyzed by BLAST against the EMBL database and showed identity with 16S rDNA sequences of Bacillus subtilis (SEQ ID NO: 2 and SEQ ID NO: 3) and with Bacillus amyloliquefaciens (SEQ ID NO: 1 and SEQ ID NO: 4).

[00163] In order to study the phylogenetic affiliation of SEQ ID NO: 1 to SEQ ID NO: 4, the sequences were analyzed through a ClustalW alignment in MegAlign (DNASTAR) using SEQ ID NO: 5 to SEQ ID NO: 7 as performance test. These sequences are 16S rDNA sequences from Bacillus vallismortis strains extracted from AB021198 (SEQ ID NO: 5), Bacillus subtilis extracted from AJ276351 (SEQ ID NO: 6) and Bacillus amyloliquefaciens extracted from AB255669 (SEQ ID NO: 7).

[00164] The ClustalW alignment of SEQ ID NO: 1 to SEQ ID NO: 7 (Figure 1) shows 7 nucleotide positions in which 2 or more sequences have a nucleotide that deviates from each other. For numbering, the positions in SEQ ID NO: 6 are used. At position 152 SEQ ID NO: 2 it is identical to Bacillus amyloliquefaciens and Bacillus vallismortis, but different from the rest, which are identical to each other. At position 174 SEQ ID NO: 1 and SEQ ID NO: 4 are identical to Bacillus amyloliquefaciens, but different from the rest that are identical to each other. At position 257 SEQ ID NO: 4 it is identical to Bacillus amyloliquefaciens, but different from the rest, which are identical to each other. At position 437, 444 and 455 SEQ ID NO: 2 and SEQ ID NO: 3 are identical to Bacillus subtilis, but different from the rest that are identical to each other. At position 1223 SEQ ID NO: 1 it is identical to Bacillus amyloliquefaciens, but different from the rest, which are identical to each other. Variation in 16S rDNA genes supports the species affiliation seen in the BLAST report.

DESCRIPTION OF BIOLOGICAL MATERIAL

[00165] Bacillus amyloliquefaciens DSM 29869 was isolated by Novozymes (Novo Nordisk) from an environmental sample collected in Jamaica in 1990. The strain was identified as Bacillus amyloliquefaciens based on 16S rDNA sequencing.

[00166] Bacillus subtilis DSM 29870 was isolated by Novozymes (Novo Nordisk) from an environmental sample collected in Jamaica in 1990. The strain was identified as Bacillus subtilis based on 16S rDNA sequencing.

[00167] Bacillus subtilis DSM 29871 was isolated for Novozymes by a Danish high school student at Br0nderslev Gymnasium from a privately owned soil sample in Denmark in 2008. The strain was identified as Bacillus subtilis based on 16S rDNA sequencing .

[00168] Bacillus amyloliquefaciens DSM 29872 was isolated from a US environmental sample in 2008. The strain was identified as Bacillus amyloliquefaciens based on 16S rDNA sequencing.

EXAMPLE 4: DETERMINATION OF GROWTH INHIBITION OF ESCHERICHIA COLI

[00169] Strains of Escherichia coli, ATCC10536 or ATCC25922, were grown overnight in tryptic soy broth (BD part 211822) supplemented with 0.6% yeast extract (BD part 212750) at 35 ° C under static anaerobic conditions. 100 μl of the overnight Escherichia coli culture was added to 250 ml of tryptic soy agar supplemented with 0.6% yeast extract at 40°C and poured into rectangular petri dishes (Nunc part 267060). The inoculated agar was then allowed to cool to room temperature after which an 8 mm diameter well was produced in the agar.

[00170] The Bacillus strain DSM 29869, DSM 29870, DSM 29871 or DSM 29872 was cultivated overnight in tryptic soy broth at 35 ° C under aerobic conditions. 1,000 μl of Bacillus subtilis culture was collected and fractionated into cell-free supernatant and cells via centrifugation. 20 μl of cell-free supernatant or 100x cells diluted in phosphate buffer saline were added directly to the wells on the agar plates inoculated with Escherichia coli. A control well contained 20 μl of phosphate-buffered saline. The plates were incubated for 18 hours at 30 °C under aerobic conditions.

[00171] Inhibition of the Escherichia coli strain was noted by a zone of whitening around the well of interest. The phosphate buffered saline well was considered a negative control based on the absence of the brightening zone around the well.

[00172] Cell-free supernatant and 100x diluted cells from Bacillus strains DSM 29869, DSM 29870, DSM 29871 and DSM 29872 had the ability to consistently inhibit the growth of E. coli strains ATCC10535 and ATCC25922 in vitro. Inhibition was also seen using the CloSTAT competitor strain for both supernatant and cells.

EXAMPLE 5: SENSITIVITY TO ANTIBIOTICS

[00173] The minimum inhibitory concentrations (MIC) of eight antibiotics against Bacillus strains DSM 29869, DSM 29870, DSM 29871 and DSM 29872 were determined using broth microdilution essentially as described in the CLSI guidelines (M07-A9 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Only modification was that the volumes were changed; 90 μl of Mueller Hinton Broth 2 with bacteria was added to 10 μl of antibiotic dilutions, bacteria cfus and antibiotic concentration were changed so that the final concentrations and final cfus were compatible with the guideline. Plates were incubated for 20 to 24 h instead of 16 to 20 h. The control strain recommended in the CLSI standard Staphylococcus aureus ATCC 29213 was used as the control strain. STRAINS: Bacillus amyloliquefaciens DSM 29869 Bacillus subtilis DSM 29870 Bacillus subtilis DSM 29871 Bacillus amyloliquefaciens DSM 29872 S. aureus ATCC 29213 ANTIBIOTICS: Chloramphenicol (Sigma C1919, 10 mg/ml, solubilized in 96% ethanol) Clindamycin (Sigma PHR1159, 10 mg/ ml, solubilized in water) Erythromycin (ABBOTICIN from Amdipharm 40501, 50 mg/ml, solubilized in 96% ethanol) Gentamicin (Biomedicals inc., 190057, 10 mg/ml, solubilized in water) Kanamycin (Sigma, CAS no. 25389 -94-0, 50 mg/ml, solubilized in water) Streptomycin (Sigma, S1277, 10 mg/ml, solubilized in water) Tetracycline (Sigma, T3383, 10 mg/ml, solubilized in water) Vancomycin (Sigma, V- 8138, 10 mg/ml, solubilized in water) Mueller Hinton Broth 2 (Sigma/Fluka 90922) 0.9% NaCl (Sigma/RdH 31434/Merck 106404) Tryptic soy agar plates (Oxoid CM 131) Microtiter plates : Costar plate, polypropylene, round bottom, Corning 3879

PREPARATION OF BACTERIA:

[00174] Some colonies of Bacillus spp. (<1 day old) were inoculated into Mueller Hinton Broth 2 (MHB) and incubated for about 4 hours at 37°C. OD600 (BioPhotometer plus, Eppendorf) was measured and adjusted to 0.25 (equivalent to McFarland 0.5) in MHB. For the control strain, direct colony suspension was used. A few colonies of S. aureus ATCC 29213 (<1 day old) were suspended in MHB and OD600 (BioPhotometer plus, Eppendorf) was measured and adjusted to 0.10 to 0.12 (McFarland equivalent 0.5) in MHB. Bacterial suspensions were diluted 200x in MHB.

PREPARATION OF TEST PLATES:

[00175] The antibiotics were diluted to a concentration of 640 μg/ml in MHB. A two-fold dilution series was prepared in MHB to a concentration of 0.625 μg/ml. 10 μl of each dilution and each antibiotic was pipetted into a microtiter plate. Later, when the antibiotics were mixed with the bacteria suspension, the samples were diluted 10x (10 μl of sample in a total volume of 100 μl). This resulted in the final test range of 0.06 to 64 μg/ml.

[00176] If the plates were not used promptly, the plates would be stored in the freezer at -20 °C until use.

[00177] 90 μl of the bacterial suspensions was added to the assay plates. The assay plates were incubated in a plastic bag with a damp cloth at 37 °C for 20 to 24 h. The MIC was determined as the lowest concentration of antibiotic that completely inhibited bacterial growth as detected by the naked eye.

CFU ESTIMATION:

[00178] A 10-fold dilution series in 0.9% NaCl was produced for 10-3 of the cultures inoculated into the microtiter plate. 2x 100 ul of the 10-3 dilution was placed on two TSA plates. The plates were incubated overnight at 37 °C. The number of CFU/ml was counted.

[00179] Three biological replicates of the assay were performed for Bacillus subtilis DSM 29871, Bacillus amyloliquefaciens DSM 29869 and Bacillus subtilis DSM 29870 (MIC 1-3), while four biological replicates were performed for Bacillus amyloliquefaciens DSM 29872 (MIC 2-5).

RESULTS:

[00180] The MIC values obtained for B. subtilis DSM 29870 showed that the breakpoint values were equal to or below the breakpoint values given in the EFSA guidelines (EFSA journal 2012; 10(6):2740) . For B. subtilis DSM 29869, MIC values obtained in two of three biological replicates showed MIC values at or below the breakpoint. At MIC 1, the value for Tetracycline was 16. However, due to the analytical variance of the method MIC results from a dilution above the breakpoint is generally accepted and the strain can be considered as sensitive. Therefore, according to EFSA, these two strains are considered sensitive to all 8 antibiotics included in the test (Tables 5.2 and 5.3).

[00181] For strains of B. subtilis DSM 29871 and B. amyloliquefaciens DSM 29872, MIC values showed that the two strains were sensitive to seven of the eight antibiotics (Tables 5.1 and 5.4). An increased tolerance was observed towards Streptomycin and, according to EFSA, the strains were classified as resistant to Streptomycin.

[00182] As a control S. aureus ATCC 29213 was tested in parallel and obtained MIC values within the ranges given by the CLSI standard (M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014) (Table 5.5).

[00183] The amount of bacteria inoculated into the assay plates was measured (CFU/ml). In general, the CFU/ml was very close to the target value of 5*105 CFU/ml. However, the CFU/ml for Bacillus strains may be associated with some uncertainty since bacteria tend to aggregate and, once aggregated, will only result in one colony forming unit (Tables 5.1 and 5.2). Table 5.1: MIC RESULTS FOR B. SUBTILIS DSM 29871 EFSA Journal 2012; 10(6):2740 nr not registered TABLE 5.2: MIC RESULTS FOR B. AMYLOLIQUEFACIENS DSM 29869 * EFSA Journal 2012; 10(6):2740 Table 5.3: MIC RESULTS FOR B. SUBTILIS DSM 29870 * EFSA Journal 2012; 10(6):2740 Table 5.4: MIC RESULTS FOR B. AMYLOLIQUEFACIENS DSM 29872 * EFSA Journal 2012; 10(6):2740 Table 5.5: MIC RESULTS FOR S. AUREUS ATCC 29213 * M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014

EXAMPLE 6: ENZYME ACTIVITY PREPARATION OF PRECULTURES FROM ONE DAY TO NIGHT

[00184] Overnight cultivation was performed in a 96-deep well plate with 1.25 ml of LB broth (Difco; BD #244610) per well. Each strain was tested in duplicate and inoculated with a single colony from a Standard Methods Agar plate (SMA plates, Smith River Biologicals #11-00450). Prior to incubation, a single sterile Aeraseal breathable gasket (Fisher #50-212-463) was placed on top of the plate and the plate was incubated with shaking at 35°C overnight under aerobic conditions.

DETERMINATION OF ANAEROBIC GROWTH

[00185] SMA plates were swabbed from overnight cultures using a 10 μl inoculation cycle. Up to four strains were swabbed per plate, in separate quadrants.

[00186] The plates were incubated at 39 °C in an AnaeroJar (Thermo Scientific Oxoid, Fisher no. OXAG0025A) together with an AnaeroGen sachet (Thermo Scientific Oxoid, Fisher no. OXAN0025A) to maintain anaerobic conditions (~0.1% of oxygen). The work was carried out under aseptic conditions in a biological cabinet, using sterile materials.

DETERMINATION OF ENZYME ACTIVITY WITH THE USE OF AZCL SUBSTRATE AGAR PLATES

[00187] Preparation of media with AZCL substrates which may be AZCL cellulose subjected to autoclaving (AZCL-HE cellulose, I-AZCEL, Megazyme), arabinoxylan (AZCL-Arabinoxylan, wheat, I-AZWAX, Megazyme), arabinan (AZCL-Arabinano, debranched, I-AZDAR, Megazyme):

[00188] In two (2) 500 ml beakers, 300 ml of 0.1x and 1x LB agar were prepared for each beaker, with magnetic stir bars added to each. While stirring, 100 ml of LB condition from the beakers was added to three (3) 250 ml Wheaton bottles thus producing 3 bottles with 0.1x LB and 3 bottles with 1x LB. AZCL cellulose, arabinan and arabinoxylan were then added separately to each of a 1x and a 0.1x bottle with shaking. A total of 0.05 grams of substrate was added per 100 ml of media. If more than 100 ml of media is being used, the amount of substrate added is adjusted using the ratio 0.05 g/100 ml. The media were autoclaved to sterilize. Note: Only these three AZCL substrates can be subjected to autoclaving without obvious dye release indicating substrate instability. After autoclaving, media are kept at >50°C until ready for culture plates. The pH was checked and, if necessary, adjusted to pH 7 using 10% HCl or 2N NaOH, maintaining sterility.

PREPARATION OF MEDIA WITH AZCL SUBSTRATES THAT CANNOT BE SUBJECTED TO AUTOCLAVAGE

[00189] These substrates were: AZCL amylose (I-AZAMY, Megazyme), AZCL casein (I-AZCAS, Megazyme), AZCL xylan (Xylan, birchwood (I-AZXBW, Megazyme).

[00190] In two (2) 1,000 ml Wheaton bottles, 400 ml of 0.1x LB and 1x LB was prepared and a magnetic stir bar was added to each. The media were autoclaved to sterilize before adding the AZCL reagent. After autoclaving, the media were kept at 50 to 55 °C until ready to add the AZCL substrate and culture plates. The pH was checked and, if necessary, adjusted to pH 7 using 10% HCl or 2N NaOH, maintaining sterility.

PLACEMENT OF AZCL SUBSTRATE MEDIA AGAR PLATES

[00191] The work was carried out under aseptic conditions in a biological cabinet, using sterile materials. These steps were performed quickly so that the agar did not solidify before transfer to the 96-well plate.

[00192] For substrates that could be autoclaved (AZCL cellulose, AZCL arabinoxylan, AZCL arabinan): The autoclaved and heated (~50 °C) liquid agar media were magnetically stirred in order to suspend the substrate of insoluble AZCL. An aliquot was transferred to a sterile solution basin (Periodic mixing of the material in the solution basin was necessary to keep the substrate suspended). A repeating multichannel pipette (e.g., 1,250-μl Matrix pipette along with corresponding sterile 1,250-μl pipette tips) was used to dispense 180 μl into each well of a sterile 96-well plate. A set of spikes would typically last up to six columns per plate. Once the plate was filled, a sterile lid was added and allowed to solidify. One plate was produced for each substrate (AZCL cellulose, AZCL arabinoxylan, AZCL arabinan) and condition (0.1x and 1x LB).

[00193] For substrates that could not be subjected to autoclaving (AZCL amylose, AZCL casein, AZCL xylan): In a sterile 250 ml beaker with a magnetic stir bar, 100 ml of 0.1x or 1X media was added Warm sterile LB (no added AZCL substrate). While stirring, 0.05 mg of AZCL substrate is added, and stirring is continued until resuspended. It may help to add the substrate slowly at first. Once the substrate was resuspended, it was poured into the solution basin and added to the plates as described above. One plate was produced for each substrate (AZCL cellulose, AZCL arabinoxylan, AZCL arabinan) and condition (0.1x and 1x LB).

INOCULATION OF 96-WELL PLATES, INCUBATION AND EVALUATION

[00194] The work was carried out under aseptic conditions in a biological cabinet, using sterile materials. AZCL substrate agar plates were inoculated by applying 2 μl of the overnight culture that was dispensed on top of the agar. The added liquid should be visible on top of the agar. When moving to the next plate, it must be ensured that there are no drops left on the end of the pipette tips to ensure that each well has been inoculated. Repeat for all AZCL substrate plates. Control enzyme dilutions were prepared, each at 1/100, by diluting in sterile phosphate dilution buffer: 10 μl enzyme preparation + 990 μl buffer. Control enzyme wells were then inoculated with 10 μl of the appropriate enzyme dilution for each substrate. TABLE 6.1: ENZYMES AND RESPECTIVE SUBSTRATES Enzyme Substrate TABLE 6.2: ENZYME PRODUCTION UNDER AEROBIC CONDITIONS. TABLE 6.3: ENZYME PRODUCTION UNDER ANAEROBIC CONDITIONS, 24 HOURS TABLE 6.4: ENZYME PRODUCTION UNDER ANAEROBIC CONDITIONS, 48 HOURS

Example 7: Efficacy of strain DSM 29870 under Clostridium perfringens challenge conditions MATERIAL AND METHODS

[00195] Three independent battery studies were conducted to evaluate the influence of the DSM 29870 strain on the development of induced necrotic enteritis.

[00196] In each experiment, a total of 256 one-day-old male Cobb x Cobb broilers were allocated to Petersime battery cages (8 birds/cage). Cages were used in a completely randomized, factorial design with 8 cages per treatment (i.e., 64 animals/treatment).

[00197] An unmedicated corn/soybean-based commercial broiler starter feed was formulated (Table 7.1). Food and water were available ad libitum throughout the tests. TABLE 7.1: COMPOSITION OF THE BASAL4 EXPERIMENTAL DIET (START D1-D28) 1Vitamin mixture provided below (per kg of diet): thiamine mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin),12.0 μg; pyridoxine*HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1,892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. 2Trace mineral mix provided below (per kg diet): manganese (MnSO4^H2O), 60 mg; iron (FeSO^lhO), 30 mg; zinc (ZnO), 50 mg; copper (CuSO.i^IhO), 5 mg; iodine (ethylene diamine dihydroiodine), 0.15 mg; selenium (NaSe03), 0.3 mg. 3 Met= methionine; Cys= cysteine. 4The basal feed will not contain any probiotic/prebiotic additives, NSPases, coccidiostats or antibiotic growth promoter.

[00198] The 4 experimental groups consisted of (Table 7.2): T1, uninfected and untreated animals; T2, untreated animals infected with C. perfringens; T3, animals infected by C. perfringens fed the basal diet containing bacitracin (included at 50 ppm, from d1 to d28); T4, animals infected by C. perfringens fed the basal diet containing the DSM 29870 strain (included in 1.109 CFU/kg of feed in test 1 and 5.108 CFU/kg of feed in tests 2 and 3, from d1 to d28). TABLE 7.2: EXPERIMENTAL TREATMENTS

[00199] On day 14, all birds were orally inoculated with a coccidial inoculum containing approximately 5,000 Eimeria maxima oocysts per bird.

[00200] Once a day on d19, d20 and d21, all birds except T1 were administered (via esophageal tube) with 1 ml of a fresh broth culture of C. perfringens.

[00201] On d21, three birds from each cage were selected, sacrificed, weighed and examined for the degree of presence of necrotic enteritis lesions. Scoring was based on a score of 0 to 3, with 0 being normal and 3 being the most severe.

[00202] Live body weight and feed were recorded at the beginning (d1) and end of the experiment (d28), based on the cage, to calculate performance: - Body Weight Gain (BWG) = (Live Body Weight) d28 — (Live Body Weight)di - Feed Conversion Ratio (FCR) = ratio between feed consumed and weight gained

[00203] Mortality was recorded daily and the FCR was adjusted accordingly.

[00204] Necrotic enteritis injury scores, total mortality and % of dead birds with necrotic enteritis injuries were also calculated

[00205] Data (n = 32) were subjected to an ANOVA, with a randomized complete block design using the XLSTAT ANOVA procedure (Addinsoft 1995-2014) to establish differences between diets. The pen was considered as the experimental unit. The model included diets (n=4) and block as a fixed effect. Results are reported as least squares means. LS means were assumed to be different at P < 0.05.

EXPERIMENTAL RESULTS

[00206] The results obtained in performance, necrotic enteritis injury scores, mortality and % of dead birds with necrotic enteritis injury parameters are shown in Tables 7.3. TABLE 7.3: EFFECT OF DIETARY COMPLEMENTATION OF DSM 29870 STRAIN ON THE NEGATIVE IMPACT ON PRODUCTION PARAMETERS DUE TO NECROTICAL ENTERITIS INDUCED IN THREE INDEPENDENT TESTS. 1 Uninfected, untreated group fed basal diet. 2 Infected, untreated group fed basal diet. 3 Infected group fed the basal diet supplemented with bacitracin (50 ppm). 4 Infected group fed the basal diet supplemented with DSM 29870 (1.109 CFU/kg of feed). 5 Means of 8 replicates of 8 birds per cage for each group. The raw data (n=32) were analyzed using analysis of variance with block (n=8) and treatments (n=4) according to a fixed effect. 6 Means of 8 replicates of 3 birds per cage for each group. The raw data (n=32) were analyzed using analysis of variance with block (n=8) and treatments (n=4) according to a fixed effect. a,b,c Values with different superscripts are significantly (P<0.05) different from each other. 1 Uninfected, untreated group fed basal diet. 2 Infected, untreated group fed basal diet. 3 Infected group fed the basal diet supplemented with bacitracin (50 ppm). 4 Infected group fed the basal diet supplemented with DSM 29870 (5.108 CFU/kg of feed). 7 Means of 8 replicates of 8 birds per cage for each group. The raw data (n=32) were analyzed using analysis of variance with block (n=8) and treatments (n=4) according to a fixed effect. 8 Means of 8 replicates of 3 birds per cage for each group. The raw data (n=32) were analyzed using analysis of variance with block (n=8) and treatments (n=4) according to a fixed effect. a,b,c Values with different superscripts are significantly (P<0.05) different from each other. 9 Uninfected, untreated group fed basal diet. 10 Infected, untreated group fed basal diet. 11 Infected group fed the basal diet supplemented with bacitracin (50 ppm). 12 Infected group fed the basal diet supplemented with DSM 29870 (5.108 CFU/kg of feed). 13 Means of 8 replicates of 8 birds per cage for each group. The raw data (n=32) were analyzed using analysis of variance with block (n=8) and treatments (n=4) according to a fixed effect. 6 Means of 8 replicates of 3 birds per cage for each group. The raw data (n=32) were analyzed using analysis of variance with block (n=8) and treatments (n=4) according to a fixed effect. a,b,c Values with different superscripts are significantly (P<0.05) different from each other.

[00207] Data obtained from the three challenge studies showed that the DSM 29870 strain can nullify the negative impact of induced necrotic enteritis on production parameters with a significant effect across all challenge tests on FCR and mortality due to necrotic enteritis, in relation to the positive control group (uninfected, untreated animals).

[00208] Furthermore, there was no significant difference between the group fed with DSM 29870 and the group fed with bacitracin in relation to all parameters measured in these tests.

[00209] The challenge studies described above showed that administration of the DSM 29870 strain could prevent necrotic enteritis in broiler chickens.

EXAMPLE 8: FIRST PERFORMANCE TEST WITH BROILER (521) MATERIAL AND METHODS

[00210] A total of 2,000 one-day-old male Cobb 500 broilers were weighed individually (body weight 42 g ±1 g) and allocated to pens on the floor (50 birds/pen). Pens were used in a completely randomized, factorial design with 8 pens per treatment (i.e., 400 animals/treatment). The 5 experimental treatments consisted of (Table 8.1): T1, negative control basal diets; T2, basal diets containing the DSM 29870 strain (included in 5,108 CFU/kg of feed, from d1 to d35); T3, basal diets containing the DSM 29871 strain (included in 5,108 CFU/kg of feed, from d1 to d35); T4 basal diets containing strain DSM 29872 (included at 5,108 CFU/kg of feed, from d1 to d35); T5, basal diet containing a commercialized probiotic, GalliPro Max (included in the commercial dose, 8,108 CFU/kg of feed, from d1 to d35).

[00211] The basal diets consisted of a 3-phase feeding program: initial phase (from day 1 to day 12), fattening phase (from day 13 to day 28) and final phase (from day 29 to day 35). All phases were formulated to meet or exceed animal requirements and agree with commercial US corn and soy meal based broiler diets. The dietary and gross material composition of these diets is given in Table 8.2. All experimental diets include phytase (Ronozyme P, 250 FYT/kg) and do not contain any coccidiostat, NSPase or growth-promoting substance. Food and water were available ad libitum throughout the test. TABLE 8.1: EXPERIMENTAL TREATMENTS TABLE 8.2: COMPOSITION OF BASIC EXPERIMENTAL DIETS 1Vitamin Blend will provide the following (per kg of diet): Thiamine mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin),12.0 μg; pyridoxine^HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1,892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. Trace mineral mix provided the following (per kg of diet): manganese (MnSOsHiO), 60 mg; iron (FeSOsVHiO), 30 mg; zinc (ZnO), 50 mg; copper (CuSO^HiO), 5 mg; iodine (ethylene diamine dihydroiodine), 0.15 mg; selenium (NaSe03), 0.3 mg.

[00212] Live body weight and feed were recorded at the beginning (d1) and end of the experiment (d35), based on the pen, to calculate performance: - Feed Admission (FI) = (Remaining Feed)d35 - (Feed Delivered)d1 - Body Weight Gain (BWG) = (Live Body Weight)d35 - (Live Body Weight)d1 - Feed Conversion Ratio (FCR) = ratio between feed consumed and weight gained

[00213] Mortality was recorded daily and the FCR was adjusted accordingly.

[00214] Data (n = 40) were subjected to an ANOVA, with a randomized complete block design using the XLSTAT ANOVA procedure (Addinsoft 1995-2014) to establish differences between diets. The pen was considered as the experimental unit. The model included diets (n=5) and block as a fixed effect. Results are reported as least squares means. LS means were assumed to be different at P < 0.05.

[00215] Similar calculations were performed in examples 9 and 10.

SIMILAR CALCULATIONS WERE PERFORMED IN EXAMPLES 3 AND 4. EXPERIMENTAL RESULTS

[00216] The results obtained in the performance parameters for the entire experimental period are shown in Table 8.3. TABLE 8.3: EFFECT OF DIETARY COMPLEMENTATION OF DSM 29870, DSM 29871 AND DSM 29872 STRAINS ON BROIL CHICKEN PERFORMANCE 1 Group fed with basal diet. 2 Group supplemented with DSM 29870/29871/29872 fed basal diet including strain DSM 29870/29871/29872 at 5,108 CFU/kg of feed. 3 Group supplemented with GalliPro Max fed the basal diet including GalliPro Max at 8,108 CFU/kg of feed. 4 Means of 8 replicates of 50 birds per pen for each group. The raw data (n=24) were analyzed using analysis of variance with block (n=8) and treatments (n=3) according to a fixed effect. a,b,c Values with different superscripts are significantly (P<0.05) different from each other.

[00217] Feed additives did not significantly affect the intake of animal feed compared to the control. Body weight was slightly increased with the use of NZB strains while GalliPro Max tended to increase this parameter.

[00218] The strains of DSM 29870, DSM 29871 and DSM 29872 also significantly improved the feed conversion ratio by 1.6%, 1.5% and 1.9%, respectively, with no significant differences between the groups fed with strains from NZB. GalliPro Max had only a slight and non-significant effect on the power conversion ratio.

[00219] No probiotic tested in this document had a significant effect on the level of mortality.

Example 9: SECOND PERFORMANCE TEST WITH BROILER (522) MATERIAL AND METHODS

[00220] A total of 900 one-day-old male Ross 708 broilers were weighed individually (body weight 47.1 ±1.1 g) and allocated to pens on the floor (15 birds/pen). Pens were used in a completely randomized, factorial design with 12 pens per treatment (i.e., 180 animals/treatment). The 5 experimental treatments consisted of (Table 9.1): T1, negative control basal diets; T2, basal diets containing the DSM 29870 strain (included in 5,108 CFU/kg of feed, from d1 to d35); T3, basal diets containing the DSM 29871 strain (included in 5,108 CFU/kg of feed, from d1 to d35); T4 basal diets containing strain DSM 29872 (included at 5,108 CFU/kg of feed, from d1 to d35); T5, basal diet containing a commercialized probiotic, GalliPro Max (included in the commercial dose, 8,108 CFU/kg of feed, from d1 to d35).

[00221] The basal diets consisted of a 3-phase feeding program: initial phase (from day 1 to day 14), fattening phase (from day 15 to day 21) and final phase (from day 22 to day 35). All phases were formulated to meet or exceed animal requirements and agree with commercial US corn and soy meal based broiler diets. The dietary and gross material composition of these diets is given in Table 9.2. All experimental diets included vitamin and mineral premixes, but did not contain any coccidiostat, NSPase, phytase or growth-promoting substance. Food and water were available ad libitum throughout the test. TABLE 9.1: EXPERIMENTAL TREATMENTS TABLE 9.2: COMPOSITION OF BASIC EXPERIMENTAL DIETS 1Vitamin mix will provide the following (per kg of diet): vitamin A, 13,227,513 IU; vitamin D3, 3,968,254 IU; vitamin E, 66 138 IU; vitamin B12, 40 mg; biotin, 254 mg; menadione, 3,968 mg; thiamine, 3,968 mg; riboflavin, 13,228 mg; d-pantothenic acid, 22,046 mg; pyridoxine, 7,937 mg; niacin, 110 229 mg; folic acid, 2,205 mg; Selenium Premix: selenium, 600 ppm + calcium, 36% 2Trace mineral mix provided below (per kg diet): calcium, Min: 15.7% and Max: 18.7%; manganese (Mn), 6.0%; zinc (Zn), 6.0%; iron (Fe), 4.0%; copper (Cu), 5,000 ppm; iodine (I), 1,250 ppm; cobalt (Co), 500 ppm

EXPERIMENTAL RESULTS

[00222] The results obtained in the performance parameters for the entire experimental period are shown in Table 9.3. TABLE 9.3: EFFECT OF DIETARY COMPLEMENTATION OF DSM 29870, DSM 29871 AND DSM 29872 STRAINS ON BROIL CHICKEN PERFORMANCE 1 Group fed with basal diet 2 Group supplemented with DSM 29870/29871/29872 fed with basal diet including strain DSM 29870/29871/29872 at 5.108 CFU/kg of feed. 3 Group supplemented with GalliPro Max fed the basal diet including GalliPro Max at 8,108 CFU/kg of feed. 4 Means of 12 replicates of 15 birds per pen for each group. The raw data (n=36) were analyzed using analysis of variance with block (n=12) and treatments (n=3) according to a fixed effect. a,b,c Values with different superscripts are significantly (P<0.05) different from each other

[00223] Also, in this experiment, the administration of strains DSM 29870, DSM 29871, DSM 29872 and GalliPro Max did not significantly reduce feed intake, but improved body weight gain by 2.3%, 1.7%, 2 .6% and 1.5%, respectively. Therefore, the relative difference to control was significant for DSM 29870 and DSM 29872.

[00224] Strains DSM 29870 and DSM 29872 significantly improved the feed conversion ratio by 3.6% and 4.2%, respectively. DSM 29871 and GalliPro Max also improved (slightly for DSM 29871) the power conversion ratio in this experiment, but not significantly.

[00225] The DSM 29870 strain allowed a decrease in the level of mortality in a numerically greater manner than GalliPro Max.

EXAMPLE 10: THIRD PERFORMANCE TEST WITH BROILER (051) MATERIAL AND METHODS

[00226] A total of 1080 one-day-old male Ross PM3 broilers were weighed individually (body weight 42 ± 3.5 g) and allocated to pens on the floor (18 birds/pen). Pens were used in a completely randomized, factorial design with 12 pens per treatment (i.e., 216 animals/treatment). The 3 experimental treatments consisted of (Table 10.1): T1, negative control basal diets; T2, basal diets containing the DSM 29870 strain (included in 5,108 CFU/kg of feed, from d1 to d35); T3, basal diets containing the DSM 29871 strain (included in 5,108 CFU/kg of feed, from d1 to d35); T4 basal diets containing strain DSM 29872 (included at 5,108 CFU/kg of feed, from d1 to d35); T5, basal diet containing a commercialized probiotic, GalliPro Max (included in the commercial dose, 8,108 CFU/kg of feed, from d1 to d35).

[00227] The basal diets consisted of a 3-phase feeding program: initial phase (from day 1 to day 21) and fattening phase (from day 22 to day 35). All phases have been formulated to meet or exceed animal requirements and comply with commercial EU corn and soy meal based broiler diets. The dietary and gross material composition of these diets is given in Table 10.2. All experimental diets included vitamin and mineral premixes, but did not contain any coccidiostat, NSPase, phytase or growth-promoting substance. Food and water were available ad libitum throughout the test. TABLE 10.1: EXPERIMENTAL TREATMENTS Table 10.2: COMPOSITION OF BASIC EXPERIMENTAL DIETS 1Premix will provide the following (per kg of diet): vitamin A, 12,000 IU; vitamin D3, 3 000 IU;, vitamin E = 300 IU;, vitamin K3, 3 mg; vitamin B, 2 mg; vitamin B2, 8 mg; vitamin B6, 3 mg; vitamin B12, 0.02 mg; folic acid, 1 mg; biotin, 0.2 mg; calcium pantothenate, 15 mg; nicotinic acid, 40 mg; manganese (Mn), 80 mg; zinc (Zn), 60 mg; iodine (I), 1 mg, iron (Fe), 80 mg; copper (Cu), 15 mg; cobalt (Co), 0.4 mg; selenium (Se), 0.2 mg; magnesium (Mg), 5 mg; Ethoxyquin, 0.5 mg; BHA, 0.5 mg, citric acid, 5 mg; phosphoric acid, 5 mg.

EXPERIMENTAL RESULTS

[00228] The results obtained in the performance parameters for the entire experimental period are shown in Table 10.3. Table 10.3: EFFECT OF DIETARY COMPLEMENTATION OF DSM 29870, DSM 29871 AND DSM 29872 STRAINS ON BROIL CHICKEN PERFORMANCE 1 Group fed with basal diet. 2 Group supplemented with DSM 29870/29871/29872 fed basal diet including strain DSM 29870/29871/29872 at 5,108 CFU/kg of feed. 3 Group supplemented with GalliPro Max fed the basal diet including GalliPro Max at 8,108 CFU/kg of feed. 4 Means of 12 replicates of 18 birds per pen for each group. The raw data (n=36) were analyzed using analysis of variance with block (n=12) and treatments (n=3) according to a fixed effect. a,b,c Values with different superscripts are significantly (P<0.05) different from each other.

[00229] In this experiment, administration of strains DSM 29870, DSM 29871, DSM 29872 (such as GalliPro Max) had no significant effect on feed intake, but led to significant improvement in body weight gain by 6.5%, 7. 3% and 5.2%, respectively. GalliPro Max also improved body weight gain (+4.2%), but not significantly.

[00230] All NZB strains significantly improved the feed conversion ratio by 4.0%, 5.3% and 4.8% (DSM 29870, DSM 29871 and DSM 29872, respectively) as GalliPro Max reaching 3 .4% improvement.

[00231] Regarding the level of mortality, there were no significant differences between the treatments.

EXAMPLE 11: SYNTHESIS AND META-ANALYSIS

[00232] The results of the three performance experiments described above are summarized in Table 11.1. TABLE 11.1: RESULTS OF INDIVIDUAL STUDIES a,b,c Values with different superscripts are significantly (P<0.05) different from each other.

[00233] These tests which have a similar experimental design, all data obtained were pooled and combined in a meta-analysis (after being tested for homogeneity). This meta-analysis involved 3,980 broiler chickens in 160 replicates (32 replicates and 796 animals for each experimental group). The results are shown in Table 11.

[00234] Data (n = 160) were subjected to a mixed-mode ANOVA, using the SAS GLIMMIX procedure (SAS Institute, 2002 to 2012) to establish differences between diets. The pen was considered as the experimental unit. The model included diets (n=5) as a fixed effect and experiment (n=3) as a random effect. Results are reported as least squares means. LS means were assumed to be different at P < 0.05. CHICKEN 1 Group fed with basal diet 2 Group supplemented with DSM 29870/29871/29872 fed with basal diet including strain DSM 29870/29871/29872 at 5.108 CFU/kg of feed. 3 Group supplemented with GalliPro Max fed the basal diet including GalliPro Max at 8,108 CFU/kg of feed. 4 Means of 32 observations for each experimental group (796 animals/group). a,b,c Values with different superscripts are significantly (P<0.05) different from each other.

[00235] This meta-analysis showed that the use of the DSM 29870, DSM 29871 or DSM 29872 strain in a corn/soy meal-based diet significantly improved, on average, body weight gain (+2.9%, +2.6% and +3.0%, respectively) and the feed conversion ratio (- 2.4%, -2.2% and -2.7%, respectively) with no effect on feed intake.

CONCLUSION

[00236] In the working example above, the positive effects of the DSM 29870 strain were demonstrated on the performances of broiler chickens (i.e., body weight gain and feed conversion ratio) fed a diet based on corn meal/ soy.

[00237] All data obtained from the experiments described in the examples above also showed that the effect of the DSM 29870 strain on the feed conversion ratio is due to its effect on body weight. These effects may be associated with the effect on health or the enhancement of metabolism.

[00238] The DSM 29870 strain showed positive and significant effects on body weight gain and feed conversion ratio in 66% and 100% of the above working examples, respectively.

[00239] GalliPro Max showed no significant effect on body weight gain in any of the above working examples and showed a significant and positive effect on feed conversion ratio in one third of the experiments.

[00240] In the experiments described in the examples above, the better effect of the DSM 29870 strain compared to GalliPro Max on the performance of broiler chickens was demonstrated. The effect observed using DSM 29870 was, on average, twice that observed for GalliPro Max.

EXAMPLE 12: DETERMINATION OF COMPATIBILITY WITH MONENSIN

[00241] Monensin compatibility of Bacillus strains DSM 29869, DSM 29870, DSM 29871 and DSM 29872 was determined using a modified broth microdilution similar to the method described in Example 5. Briefly, a single colony of Bacillus spp. (from tryptic soy agar plates overnight) was inoculated into Mueller Hinton Broth (MHB) and cultured overnight. Sterile media were inoculated with the culture overnight and allowed to grow for 4 hours to test bacteria in the logarithmic growth phase. The cultures were then diluted again 1:200 in fresh MHB and 90 μl of this inoculated broth was added to monensin diluted at the indicated concentrations. Prior art strains were also tested for comparison: NN019785, NN062266 (NRRL B-50013), NN062267 (NRRL B-50104), NN062278 (PTA-6507), NN062319 (FERM BP-1096), NN062440, NN062441 (DSM 17236 ), NN062439. Strains: Bacillus amyloliquefaciens DSM 29869 -50104) Bacillus subtilis NN062278 (PTA-6507) Bacillus amyloliquefaciens NN062319 (FERM BP-1096) Bacillus subtilis NN062440 Bacillus licheniformis NN062441 (DSM 17236) Bacillus amyloliquefaciens NN062439 MATERIALS: Monensin sodium salt (Sigma, CAS no. 22373-78-0, solubilized in 96% ethanol) Mueller Hinton Broth (Becton , Dickinson and Company, 275730) Tryptic soy agar (Becton, Dickinson and Company, 236920) Microtiter plates: Costar plate, polypropylene, flat bottom, Corning 3628 Borosilicate glass tubes: Kimbale, 16 x 125 mm, 73500-16125 Adhesive Gas Permeable Seals: Thermo Scientific, AB-0718

PREPARATION OF BACTERIA:

[00242] Bacillus spp. It was grown overnight on tryptic soy agar plates (40 g/l) at 37 °C. Mueller Hinton broth (21 g/l) was dissolved in water and autoclaved in glass tubes containing 5 ml of broth each. A single colony of Bacillus spp. (from overnight plates) was inoculated into Mueller Hinton Broth (MHB) and incubated overnight at 37 °C, shaking at 200 rpm. A 5 ml glass tube of fresh sterile media was then inoculated with 25 ml of overnight culture and allowed to grow for 4 hours at 37°C. The cultures were then diluted again 1:200 in fresh MHB. 90 μl of this inoculated broth was then added to the antibiotic diluted at the indicated concentrations.

PREPARATION OF TEST PLATES:

[00243] Monensin was diluted in 96% ethanol to a concentration of 800 μg/ml. This solution was then diluted 10-fold in sterile phosphate buffer to a concentration of 80 μg/ml. A two-fold dilution series was prepared in MHB to a concentration of 2.5 μg/ml. 10 μl of each dilution and each antibiotic was pipetted into a microtiter plate. Later, when the antibiotics were mixed with the bacteria suspension, the samples were diluted 10x (10 μl of sample in a total volume of 100 μl). This resulted in the final test range of 0.25 to 8 μg/ml.

[00244] 90 μl of the bacterial suspensions was added to the assay plates. The assay plates were then covered with a permeable adhesive glass seal and incubated overnight at 37°C shaking at 200 rpm. The maximum compatible concentration was determined similar to an MIC analysis as the above concentration that inhibited 80% of bacteria as detected by the naked eye.

RESULTS:

[00245] A potential challenge of applying Bacillus spp. in feed is the common use of antibiotics as growth promoters in feed. Therefore, it is necessary to determine strain compatibility with commonly used feed antibiotics in order to identify any personal conflicts with use as a direct feed microorganism. Therefore, the monensin compatibility of Bacillus strains DSM 29869, DSM 29870, DSM 29871 and DSM 29872 was determined together with the prior art strains. Bacillus strains DSM 29869, DSM 29870, DSM 29871 and DSM 29872 indicated a higher level of compatibility with monensin than most prior art strains included herein: NN019785, NN062266 (NRRL B-50013), NN062267 (NRRL B-50104), NN062278 (PTA-6507), NN062319 (FERM BP-1096), NN062440, NN062441 (DSM 17236), NN062439. TABLE 12.1: MONENSIN COMPATIBILITY RESULTS

Claims (14)

1. Composition comprising a strain of Bacillus, characterized by the fact that said strain: i) has deposit accession number DSM 29870; ii) has deposit access number DSM 29869; iii) has deposit access number DSM 29871; or iv) has deposit accession number DSM 29872, wherein the composition additionally comprises a carrier; wherein the carrier comprises a compound selected from the group consisting of glycerol, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, sodium aluminum silicate, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate , potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch, farigel , cassava cores, sodium aluminum silicate, colloidal amorphous silica, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, carbopol. and cellulose; wherein the Bacillus strain improves one or more performance parameters of an animal selected from the group consisting of: improving the feed conversion rate, improving body weight gain, improving the European Production Efficiency Factor, improving the efficiency of feeding and improving the health of, for example, birds such as chickens; wherein the Bacillus strain is sensitive to at least seven, such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline; wherein the Bacillus strain has a high compatibility with monensin; and in which the Bacillus strain is non-hemolytic. 2. Composition according to claim 1, characterized by the fact that the Bacillus strain is compatible with at least 2.3 μg/ml of monensin, such as at least 2.4 μg/ml of monensin, such as at least 2, 5 μg/ml monensin, such as at least 2.6 μg/ml monensin or at least 2.7 μg/ml monensin. 3. Composition according to claim 1 or 2, characterized by the fact that the Bacillus strain has antimicrobial activity against Clostridium perfringens. 4. Composition according to any one of claims 1 to 3, characterized by the fact that the Bacillus strain improves body weight gain and/or the feed conversion rate in chickens fed with the Bacillus strain. 5. Composition according to any one of claims 1 to 4, characterized by the fact that the Bacillus strain comprises 16S rDNA having SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4. 6. Composition according to any of the preceding claims, characterized by the fact that the Bacillus strain is a strain of Bacillus subtilis or a strain of Bacillus amyloliquefaciens. 7. Composition according to any of the previous claims, characterized by the fact that the Bacillus strain has an antimicrobial effect against E. coli. 8. Composition according to any of the preceding claims, characterized by the fact that the Bacillus strain is an isolated Bacillus strain. 9. Composition according to any one of claims 1 to 8, characterized in that it comprises a pure culture of the Bacillus strain. 10. Composition according to any one of the preceding claims, characterized by the fact that the Bacillus spores of the composition are present as spores such as dry spores. 11. Composition according to any one of the preceding claims, characterized by the fact that the bacterial count of each Bacillus spore is between 1x104 and 1x1018 CFU/kg of composition, preferably between 1x106 and 1x1015 CFU/kg of composition and, more preferably, between 1x107 and 1x1011 CFU/kg of composition. 12. Composition according to any one of claims 1 to 11, characterized in that the carrier comprises calcium carbonate. 13. Use of the composition as defined in any one of claims 1 to 12, characterized by the fact that it is to improve one or more performance parameters of an animal, selected from the group consisting of: increasing the feed conversion rate, increasing weight gain, increase the European Production Efficiency Factor, increase feeding efficiency and increase the health of, for example, poultry such as chickens. 14. Use according to claim 13, characterized in that the Bacillus strain is selected from the group consisting of: DSM 29869, DSM 29871 and DSM 29872.