Bacillus pumilus strains improving animal performance parameters

Abstract

The present disclosure generally relates to compositions comprising Bacillus pumilus with positive effects on animal performance parameters, to the use of Bacillus pumilus for the prevention, reduction or control of a bacterial colonization or infection, to a method of improving one or more animal performance parameters and to a kit. Particularly, the disclosure relates to Bacillus pumilus DSM 34300 and DSM 34299.

Description

[0001] BACILLUS PUMILUS STRAINS IMPROVING ANIMAL PERFORMANCE

[0002] PARAMETERS

[0003] FIELD

[0004] The present disclosure generally relates to compositions comprising Bacillus pumilus with positive effects on animal performance parameters, to the use of Bacillus pumilus for the prevention, reduction or control of bacterial colonization or infection, to a method of improving one or more animal performance parameters and to a kit. Particularly, the disclosure relates to Bacillus pumilus strains DSM 34300 and DSM 34299.

[0005] BACKGROUND

[0006] Ingestion of pathogens, especially of bacterial origin, but also including viruses and other disease-causing microorganisms, is a common problem in most livestock animals. Pathogens have been known to cause illness in animals, resulting in several deleterious effects, including weight loss, diarrhea, abdominal cramping, and renal failure. For animals that are immunosuppressed or malnourished, just the effects of diarrhea can be fatal. Therefore, the well-being of the animals is very important, and any illness could potentially affect the health and performance of the animal, thereby influencing the productivity and profitability of the operation. This is especially important in relation to production animals where the final body weight of the animal at slaughter and carcass weight are of high economic importance.

[0007] Pathogens are often transferred between animals and often animal-to-animal transfer cannot be prevented. The most common solution to this problem has been to provide antibiotics to the animals, but besides being costly, this solution can also result in the generation of antibiotic-resistant strains of bacteria, leading to a general agreement in the field that the excessive utilization of antibiotics should be avoided. This phase-out of antibiotics has resulted in an increased need for cost-effective feed additives with high efficiency to support health of the livestock herd.

[0008] The utilization of Bacillus-based probiotics in the diets of livestock animals has yielded beneficial health and production effects. More specifically, strains of Bacillus spp. have gained a greater importance in the feed industry because spores are heat stable and can survive the pelletizing process of animal feed. The phase-out of antibiotic growth promoters in the European Union in 2006 has resulted in an increased need for cost-effective feed additives with high efficacy and susceptibility to inhibitorys of human and veterinary importance.

[0009] Bacillus- based probiotic feed additives are known for their positive effects on health and production in livestock animals. These products are relevant for the feed industry because spores are heat stable and can survive the pelletizing process at temperatures up to 90-95°C. The endospore-forming bacteria Bacillus subtilis and Bacillus licheniformis are Generally Regarded as Safe (GRAS) by the U.S. Food and Drug Administration (FDA) and acceptable for inclusion in an animal diet or water by the Association of American Feed Control Officials (AAFCO).

[0010] W02015 / 091770 describes a marine-derived B. pumilus strain and the use of this strain in weaning feed for pigs. Specifically mentioned is a rifampicin-resistant strain isolated from seaweed. No overall treatment effect was observed and no positive effect on mean piglet diarrhea scores were obtained.

[0011] W02016 / 060935 describes compositions comprising one or more bacteria for improving the in vitro true digestibility and / or neutral detergent fiber digestibility of an animal feed. Various Bacillus strains are mentioned, namely Bacillus pumilus, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus licheniformis. The Bacillus pumilus strain is described to have enzyme activity under aerobic conditions against casein and / or xylan. Effects of different deposited strains effect on in vitro true digestibility and / or neutral detergent fiber digestibility of an animal feed is investigated, however no reference is made to strain species of the deposited strains and thus at no point is B. pumilus particularly preferred or connected with any beneficial effect.

[0012] W02019 / 002476 describes an isolated B. pumilus strain (DSM 32539) which shows characteristics of being able to survive under simulated gastric passage and proved able to grow in the presence of 0.3% chicken bile and at pH 6. The isolated strain shows significantly increased xylanase activity compared to benchmark strains. The strain also demonstrates inhibition of different pathogens. No significant difference was observed on average daily weight gain, feed conversion ratio, feed intake as well as the diarrhea score when compared to the positive control group.

[0013] WO94 / 11492 describes Bacillus pumilus and Bacillus coagulans and mixtures thereof for use in direct-fed microbials for poultry feeding. No significant improvement on mortality, total body weight gain was observed in the trials performed.

[0014] Thus, there is still a need for probiotic strains which can be used for improving health and performance of production animals. The inventors of the present disclosure have proceeded with extensive screening and research in order to solve the object of providing probiotic strains which can be used for improving health and performance of production animals and have identified two novel Bacillus pumilus strains, which show effective pathogen inhibition and have proven improvement of health and performance of production animals of both monogastric and ruminant origin.

[0015] SUMMARY

[0016] The present disclosure provides a Bacillus pumilus strain selected from the group consisting of a) the strain deposited as DSM 34299, b) the strain deposited as DSM 34300, and c) a variant strain of DSM 34299 or DSM 34300 obtained by using the deposited strain as starting material and which has retained or further improved the properties of DSM 34299 or DSM 34300.

[0017] The Bacillus pumilus of the present disclosure has sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol; and has inhibitory activity against E. coli, S. carnosus (Staphylococcus carnosus) and Clostridium perfringens.

[0018] A bacterial strain refers to a bacterium that remains genetically unchanged when grown or multiplied. The multiplicity of such identical bacteria is included when reference is made to a strain.

[0019] The strains and compositions comprising the strains of the present disclosure have a proven effect on the health and growth performance of both monogastric animals and ruminants.

[0020] Compositions comprising the novel probiotic strains of the disclosure may be fed to an animal, e.g., as a Direct Fed Microbial (DFM), as an animal feed additive, in a premix, or incorporated in the animal feed directly.

[0021] At least one Bacillus pumilus strain according to the disclosure may be added to the feed during production, after production by the supplier or by the person feeding the animal, just prior to providing the feed to the animal. The Bacillus pumilus bacteria used in the methods and compositions described herein are particularly suitable because they are capable of surviving (as spores) the heat and pressure conditions of the process of producing a dry pelleted feed product.

[0022] The purpose of the studies described in the examples was to investigate the effect of DSM 34299 and DSM 34300 on pathogen growth and animal performance parameters. The disclosure describes and demonstrates the benefits of the Bacillus pumilus strains DSM 34299 and DSM 34300 in improving the health of the animals to which they have been administered by demonstrating pathogen inhibition in vitro and improvement of animal performance in vivo.

[0023] The clostridial diseases are caused by bacteria which belong to a group of organisms called Clostridium. These bacteria are important in farm animal production because they can cause significant disease problems. The Clostridium group of organisms produces very potent exotoxins (poisons), which are damaging to animals.

[0024] Necrotic enteritis (NE) caused by Clostridium perfringens has become a grave economic problem in modern poultry production. The purpose of some of the in vivo studies described in the examples was to investigate the effect of feed additives comprising a Bacillus subtilis strain according to the disclosure on the pathogenesis of NE in broiler cage studies. Other in vivo studies in poultry focused on performance with or without challenge with Clostridium perfringens.

[0025] Bovine enterotoxemia is a major cause of mortality in veal calves. Predominantly veal calves of beef cattle breeds are affected, and losses due to enterotoxemia may account for up to 20% of total mortality. Clostridium perfringens type A is considered to be the major causative agent.

[0026] Typical treatments for calves with milder clinical signs consist largely of antibiotics (especially penicillin) and the use of C. perfringens antitoxin products. Several injectable antitoxin preparations that contain specific antibodies directed against toxins produced by C. perfringens are currently available, however, there is no effective treatment available at the moment. At present, the prevention of clostridial diseases relies largely on vaccination, however, such vaccination is reported to only be partially effective in prevention. The whole herd should be vaccinated following the recommendations on the vaccine label. This is a costly procedure to undertake, and full prevention cannot be achieved.

[0027] The purpose of the in vitro studies described in the Examples was to investigate several B. pumilus strains for their ability to inhibit relevant pathogens, while at the same time being susceptible to relevant antibiotics and have other characteristics relevant to the utilization of the B. pumilus strain as an animal feed or feed additive. A further purpose of the in vivo studies described in the Examples was to investigate the subsequent effect of a composition comprising identified B. pumilus strains according to the disclosure on the growth and health performance of monogastric and ruminant animals.

[0028] This disclosure describes the benefits of two novel B. pumilus probiotic strains in inhibiting relevant animal pathogens and improving the growth and health parameters of animals. These benefits have been assessed in (i) an in vitro assay demonstrating the inhibitory effect of the novel combination of probiotic strains on growth of E. coli and Staphylococcus carnosus (Example 1), and (ii) an in vivo trial demonstrating the efficacy of Bacillus pumilus in controlling experimentally induced subclinical necrotic enteritis in broilers under floor pen conditions (Example 2), and (iii) an in vivo trial with small ruminant (sheep and lambs) demonstrating the effect on weight gain, milk yield, and milk quality of supplementing the diet with Bacillus pumilus (Example 3).

[0029] These results demonstrate that the aforementioned composition comprising novel B. pumilus strains improves the health and performance of production animals within both the monogastric and ruminant livestock population, thereby providing a cost-effective and easy-to-administer alternative to the traditional approach of treatment and minimization of pathogen infection and disease in a livestock herd using antibiotics and vaccination, while at the same time improving growth performance in the receiving animals.

[0030] DEFINITIONS

[0031] In general, the terms and phrases used herein have their art- recognized meaning, which can be found by reference to standard texts, journal references, and context known to those skilled in the art. The following definitions are provided to clarify their specific use in context of the disclosure.

[0032] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0033] As used herein, the term "and / or" is intended to mean the combined ("and") and the exclusive ("or") use, i.e. "A and / or B" is intended to mean "A alone, or B alone, or A and B together".

[0034] Animal feed: The term "animal feed" refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a monogastric animal comprises concentrates as well as for example vitamins, minerals, enzymes, amino acids and / or other feed ingredients (such as in a premix). The animal feed may further comprise forages and / or silages as a source of fiber. Examples of poultry and ruminant feed are given in the Examples.

[0035] Composition: The term "composition" refers to a composition comprising a carrier and at least one bacterial strain as described herein. The compositions described herein may be a Direct Fed Microbial (DFM), an animal feed additive or premix, or an animal feed.

[0036] Concentrate: The term "concentrate" means a feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds, and grains (either whole or prepared by crushing, milling, etc. from, e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed / canola, peanut or groundnut), palm kernel cake, yeast-derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

[0037] Control or prevent C. perfringens infections and / or necrotic enteritis: The term "control or prevent C. perfringens infections and / or necrotic enteritis" means a method and / or composition that partly or completely inhibits C. perfringens infections and / or necrotic enteritis in an animal. Accordingly, the term "control or prevent C. perfringens infections and / or necrotic enteritis" means the C. perfringens infections and / or the necrotic enteritis are reduced or completely eliminated.

[0038] Direct Fed Microbial: The term "direct fed microbial" or "DFM" means live microorganisms including spores which, when administered in adequate amounts, confer a benefit, such as improved digestion or health, on the host.

[0039] Effective amount / concentration / dosage: The terms "effective amount", "effective concentration", or "effective dosage" are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the digestion or yield of an animal. The actual effective dosage in absolute numbers depends on factors including: the state of health of the animal in question, other ingredients present. The "effective amount", "effective concentration", or "effective dosage" of the bacterial strains may be determined by routine assays known to those skilled in the art. An example of an effective amount for poultry and ruminants is given in the Examples.

[0040] Feeding an animal: The terms "feeding an animal" or "fed to an animal" means that the composition of the present disclosure is administered orally to the animal in an effective amount. The oral administration may be repeated, e.g. one or more times daily over a specified time period such as several days, one week, several weeks, one months or several months. Feeding of poultry can, e.g. be performed as described in Example 2. Feeding of ruminants can, e.g. be performed as described in Example 3. Accordingly, the terms "feeding" or "fed" mean any type of oral administration such as administration via an animal feed or via drinking water or, in certain circumstances, by oral gavage or aerosol spray.

[0041] Inhibitory activity against Clostridium perfringens-. The term "Inhibitory activity against Clostridium perfringens" means that the growth of Clostridium perfringens is inhibited and / or that some or all of the Clostridium perfringens are killed. This can be determined by the assay described in Example 1. Inhibitory activity against E. coir. The term "Inhibitory activity against E. coli" means that the growth of f. coli is inhibited and / or that some or all of the E. coli are killed. This can be determined by the assay described in Example 1.

[0042] Inhibitory activity against S. carnosus-. The term "Inhibitory activity against S. carnosus" means that the growth of S. carnosus (Staphylococcus carnosus) is inhibited and / or that some or all of the S. carnosus are killed. This can be determined by the assay described in Example 1.

[0043] Isolated: The term "isolated" means that the bacterial strains described herein are in a form or environment which does not occur in nature, i.e. the strain is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.

[0044] Sensitive to antibiotics: The term "sensitive to antibiotics" means the phenotypic property of a bacterial strain, that the growth of said bacterial strain is inhibited under conditions where the bacterial strain would otherwise grow. In this context, sensitivity to antibiotics is tested after the CLSI guidelines (M07-A8 and M45-A2). A strain of Bacillus is considered sensitive if growth is only detected at or below the breakpoint concentration specified in EFSA Journal 2012; 10(6):2740 for vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol. With regard to ampicillin, there is no breakpoint given by EFSA for Bacillus; the breakpoint 4 mg / L has been chosen for a strain to be considered sensitive.

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

[0046] Weight Gain: The Weight Gain of an animal is the increase of weight of the animal over a specified time period. One parameter to look at could be average Weight Gain or daily Weight Gain. One could also look at the final mean Body Weight of the animals (like done in Example 2) or the Average Daily Gain (ADG) and final Body weight as done in Example 3.

[0047] BRIEF DESCRIPTION OF THE FIGURES

[0048] Figure 1 shows the effect of feeding ewes with the composition of the disclosure comprising a B. pumilus strain on the birth weight of the lambs.

[0049] Figure 2 shows the interactive effect of feeding ewes with the composition of the disclosure comprising a B. pumilus strain on lambs' body weight over time after birth. Figure 3 shows the effect of feeding ewes with the composition of the disclosure comprising a B. pumilus strain on average daily gain (ADG) of the lambs.

[0050] Figure 4 shows the interactive effect of feeding ewes with the composition of the disclosure comprising a B. pumilus strain on corrected milk yield.

[0051] Figure 5 shows the interactive effect of feeding ewes with the composition of the disclosure comprising a B. pumilus strain on milk fat yield.

[0052] Figure 6 shows the interactive effect of feeding ewes with the composition of the disclosure comprising a B. pumilus strain on milk protein yield.

[0053] DETAILED DESCRIPTION

[0054] Necrotic enteris

[0055] Necrotic enteritis caused by Clostridium perfringens has become a grave economic problem in modern poultry production. It is an important concern to the poultry industry because of production losses, increased mortality, reduced welfare of birds, and increased risk of contamination of poultry products for human consumption. There is a general consensus that although clinical outbreaks of necrotic enteritis may cause high levels of mortality, the subclinical form of the disease is more important than the clinical form because it may persist in broiler flocks without overt clinical manifestation. Since the disease is undetected and birds remain untreated, subclinical necrosis causes the greatest economic losses in the poultry production industry. Thus, there remains a need for an animal feed or animal feed additive, which can prevent or minimize the development and impact of necrotic enteritis.

[0056] Predisposing factors of necrotic enteritis have been identified to be nutrition, stress in the flock, presence of poultry pathogenic C. perfringens strains, and coccidiosis. The latter, namely mucosal damage caused by coccidial pathogens, is the best-known predisposing factor for necrotic enteritis. In Example 2 the effect of the composition of the disclosure comprising a B. pumilus strain is investigated and a significant reduction in lesion score is observed.

[0057] The purpose of some of the in vivo studies described in the examples was to investigate the effect of the composition of the disclosure comprising a B. pumilus strain according to the disclosure on the pathogenesis of necrotic enteritis (NE) in broiler cage studies. Other in vivo studies in poultry focused on performance with or without challenge with Clostridium perfringens and Eimera oocysts.

[0058] Growth-promoting antibiotics have been banned from animal feed in many parts of the world and thus worldwide the use of antimicrobial growth promoters is being reduced because of concern for the spread of antimicrobial resistance. It is therefore of importance when choosing a Bacillus pumilus strain for use in animal feed or as an animal feed additive that the strain chosen er susceptible to antibiotics. The in vitro screening of B. pumilus strains for antibiotic susceptibility can be seen in Example 1.

[0059] Animal performance parameters

[0060] The disclosure provides a method of improving one or more animal performance parameters selected from the group consisting of i) increased body weight (BW), ii) increased birth weight, iii) increased Average Daily Gain (ADG), iv) lower coccidiosis lesion scoring, v) lower E. acervuline lesion scoring, vi) lower overall mortality, vii) increased milk yield, viii) increased milk fat content, and ix) increased milk protein content, by feeding a strain or a composition according to the disclosure to an animal.

[0061] In another aspect, the disclosure relates to the use of at least one B. pumilus strain of the disclosure or an animal feed, animal feed additive or premix comprising at least one B. pumilus strain of the disclosure to improve the performance of an animal, in particular a monogastric animal or a ruminant.

[0062] As evidenced in the examples, administration of a B. pumilus strain of the disclosure improves the gastrointestinal health of the animal, e.g. prevents or controls lesion scoring and provides improved animal performance parameters for the treated animals as compared to controls. Animal performance parameters include but are not limited to average daily gain (ADG), body weight (BW), decrease of mortality, and increase in milk yield and quality (increased milk fat and protein content).

[0063] Accordingly, the disclosure relates to the use of a strain according to the disclosure, or a composition according to the disclosure, for improving one or more animal performance parameters selected from the group consisting of: i) increased body weight (BW), ii) increased birth weight, iii) increased Average Daily Gain (ADG), iv) lower coccidiosis lesion scoring, v) lower E. acervuline lesion scoring, vi) lower overall mortality, vii) increased milk yield, viii) increased milk fat content, and ix) increased milk protein content In one embodiment of the disclosure, "animal performance" is determined by the body weight gain of the animal and / or by the average daily gain. By "improved animal performance" it is meant that there is increased body weight gain and / or average daily gain resulting from the use of animal feed, animal feed additive or premix of the present disclosure in animal feed in comparison to animal feed which does not comprise said animal feed, animal feed additive or premix. Preferably, by "improved animal performance" it means that there is increased body weight gain and / or average daily gain.

[0064] An "increased weight gain" refers to an animal having increased body weight on being fed feed comprising a feed composition compared with an animal being fed a feed without said feed composition of the disclosure. Specifically, the Weight Gain (WG) of an animal is the increase of weight of the animal over a specified time period. In one embodiment, the improvement in body weight gain is of at least 0.5%, such as at least 1%, such as at least 2%, such as at least 2.5%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least 10%.

[0065] In one embodiment, the improvement in weight gain results in a body weight gain of at least 0.5%, such as at least 0.8%, such as at least 1.2%, such as at least 1.5%, such as at least 1.8%, such as at least 2.0%, such as at least 2.5%, such as at least 3.0%, such as at least 4.0%, such as at least 5.0%, such as at least 6.0%, such as at least 7.0%. In a preferred embodiment, the improvement in weight gain results in a weight gain selected from the group consisting of from 1.8% to 2.0%, from 2.0% to 2.2%, from 2.2% to 2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from 2.8% to 3.0%, from 3.0% to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from 3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from 7% to 10%, or any combination thereof.

[0066] In another embodiment, "animal performance" is determined by a decrease in overall mortality. A "decrease in overall mortality" refers to a reduction in overall mortality of at least 0.5%, such as at least 1%, such as at least 2%, such as at least 2.5%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least 10%.

[0067] In another embodiment, "animal performance" is determined by a decrease in coccidiosis and / or E. acervuline lesion scoring. A "decrease in coccidiosis and / or E. acervuline lesion scoring" refers to a reduction in coccidiosis and / or E. acervuline lesion scoring of at least 0.5%, such as at least 1%, such as at least 2%, such as at least 2.5%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least In another embodiment, "animal performance" is determined by an increase in milk yield. An "increase in milk yield" refers to an increase in milk yield of at least 0.5%, such as at least 1%, such as at least 2%, such as at least 2.5%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least 10%.

[0068] In another embodiment, "animal performance" is determined by an increase in milk fat content and / or milk protein content. An "increase in milk fat content and / or milk protein content" refers to an increase in milk fat content and / or milk protein content of at least 0.5%, such as at least 1%, such as at least 2%, such as at least 2.5%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least 10%.

[0069] In another embodiment, the disclosure relates to a method of improving one or more animal performance parameters selected from the group consisting of: i) increased body weight (BW), ii) increased birth weight, iii) increased Average Daily Gain (ADG), iv) lower coccidiosis lesion scoring, v) lower E. acervuline lesion scoring, vi) lower overall mortality, vii) increased milk yield, viii) increased milk fat content, and ix) increased milk protein content the method comprising feeding a strain according to the disclosure or a composition according to the disclosure to an animal.

[0070] Probiotic bacterial strains

[0071] The probiotic strains of the present disclosure described herein are isolated, i.e., present in a form or environment that does not occur in nature, and have a surprising effect when used in a composition as described herein. The probiotic strains of the present disclosure are B. pumilus.

[0072] When choosing a B. pumilus strain for use in an animal feed or animal feed additive (including as a Direct Fed Microbial (DFM) or premix) there are some characteristics that are relevant to evaluate, because these characteristics will influence the performance and fitness of the strain for the intended application as a probiotic for animals. Such characteristics are the ability of the strain to grow on bile agar, its cellulase activity, its ability to form a biofilm, its protease activity, and its protein and fiber degradation ability. Finally, it is highly relevant to evaluate the ability of the B. pumilus strain to grow at different temperatures and for a probiotic intended for use in monogastric and ruminants, the relevant temperature to test growth at is 37°C (relevant body temperature of most monogastric animals and ruminants) and 41°C (relevant body temperature for poultry).

[0073] These characteristics can be evaluated as described in Example 1.

[0074] In one embodiment of the disclosure said composition comprising probiotic strain(s) is administered to said animal in an amount to provide a total of between 1 x 108to 1 x 1011CFU / animal / day, such as between 1 x 108to 1 x IO10CFU / animal / day, such as between 1 x 109to 1 x IO10CFU / animal / day, such as between 5 x 109to 5 x IO10CFU / animal / day.

[0075] In a preferred embodiment of the disclosure, said composition comprising probiotic strain(s) is administered to said animal in an amount to provide a total of between 2 x 109to 8 x 109CFU / animal / day, such as 2,5 x 109CFU / animal / day, such as 3 x 109CFU / animal / day, such as 4 x 109CFU / animal / day, such as 5 x 109CFU / animal / day, such as 6 x 109CFU / animal / day, such as 7 x 109CFU / animal / day, such as 8 x 109CFU / animal / day.

[0076] The term "CFU / animal / day" relates to the amount of probiotic strain(s) administered to each animal per day. This term thus excludes carriers such as calcium carbonate, anticaking agents such as aluminum silicates and kieselguhr (diatomaceous earth), and other components which optionally may be present in the composition.

[0077] Compositions of the present disclosure include at least one species of probiotic strain(s) of the disclosure and at least one carrier and / or other components that make the composition suitable for feeding an animal or as an additive for drinking water.

[0078] In one embodiment of the disclosure, wherein said composition comprises more than one probiotic strain, the Bacillus pumilus strains should account for at least half of the CFU / g of composition.

[0079] In another embodiment of the disclosure, wherein said composition comprises more than one probiotic strain, the Bacillus pumilus strains may account for less than half of the CFU / g of composition.

[0080] In another embodiment of the disclosure, wherein said composition comprises more than one probiotic strain, the composition may comprise more than one Bacillus pumilus strains present in either equal or unequal parts.

[0081] In one embodiment of the present disclosure the Bacillus pumilus strain present in said composition is the strain deposited as DSM 34300. In one embodiment of the present disclosure the Bacillus pumilus strain present in said composition is the strain deposited as DSM 34299.

[0082] In one embodiment of the present disclosure the composition comprises the Bacillus pumilus deposited as DSM 34300 or the Bacillus pumilus deposited as DSM 34299.

[0083] In one embodiment of the present disclosure the composition comprises two different probiotic strains, namely the Bacillus pumilus deposited as DSM 34300, and the Bacillus pumilus deposited as DSM 34299, optionally in a ratio in colony forming unit (CFU) of said probiotic strains of 1 : 1.

[0084] In one embodiment of the present disclosure the composition comprises two different probiotic strains consisting of the Bacillus pumilus deposited as DSM 34300, and the Bacillus pumilus deposited as DSM 34299, optionally in a ratio in colony forming unit (CFU) of said probiotic strains of 1: 1. In such embodiments, the composition does not include any other microorganism components, but may include other components, such as one or more carriers and / or one or more feed ingredients.

[0085] The probiotic strains of the composition of the present disclosure may be provided in the form of spores and / or bacterial cells. In one embodiment of the present disclosure the Bacillus strains are provided in the form of spores.

[0086] In one embodiment of the present disclosure the composition of the present disclosure comprises an amount of Bacillus pumilus strain DSM 34300 or DSM 34299 to provide between 1 x 108and 1 x 1011CFU / day of Bacillus pumilus strain DSM 34300 or DSM 34299, preferably between 1 x 109and 1 x 1010CFU / day Bacillus pumilus strain DSM 34300 or DSM 34299. In a preferred embodiment the composition of the present disclosure comprises an amount of Bacillus pumilus strain DSM 34300 or DSM 34299 to provide 3 x 109CFU / day of Bacillus pumilus strain DSM 34300 or DSM 34299.

[0087] In one embodiment of the present disclosure the microorganism components of the composition consist of amounts of the probiotic strains to provide 1 x 109CFU / day of the Bacillus pumilus deposited as DSM 34300 or 3 x 109CFU / day of the Bacillus pumilus deposited as DSM 34299. In such embodiments, the composition does not include any other microorganism components, but may include other components, such as one or more carriers and / or one or more feed ingredients.

[0088] In one embodiment of the present disclosure the composition of the present disclosure is present in an animal feed or animal feed additive and may in this embodiment comprises an amount of Bacillus pumilus strain DSM 34300 or DSM 34299 to provide between 1 x 106and 1 x 109CFU / g feed of Bacillus pumilus strain DSM 34300 or DSM 34299, such as between 1 x 106and 5 x 106CFU / g feed. Composition administration form and formulation

[0089] The probiotic strains present in the composition of the present disclosure are provided in a commercially relevant form known to the skilled person. Accordingly, in an embodiment, the probiotic strains of the composition are present in a dried (e.g., spray dried or freeze dried) or frozen form. The different probiotic strains present in the composition of the present disclosure may be present in different forms in the mixture, e.g. some may be spray dried and some may be freeze dried. The composition may be provided in any suitable form such as in the form of a liquid e.g., a gel, a slurry, etc., or a in the form of a solid, e.g., a powder or a pellet.

[0090] For compositions in the form of a premix, the probiotic strains of the disclosure may be added to a carrier to make a mineral-vitamin mixture (premix), which may then be added to an animal feed at a desired inclusion rate.

[0091] Alternatively, for compositions in the form of an animal feed, the probiotic strains of the disclosure may be formulated with animal feed ingredients, as illustrated below. Such combinations of the composition of the disclosure and animal feed ingredients optionally may be in the form of pellets that are extruded through standard pelleting processes.

[0092] The disclosure also provides a method for producing an animal feed, animal feed additive, or premix comprising adding the probiotic strains of the disclosure to an animal feed or relevant components thereof.

[0093] In one embodiment, the present disclosure provides an animal feed, animal feed additive, or premix comprising the probiotic strain(s) of the disclosure, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0094] In one embodiment the animal feed, animal feed additive, or premix comprises the composition of the disclosure comprising the Bacillus pumilus deposited as DSM 34300 and optionally the Bacillus pumilus deposited as DSM 34299, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0095] In one embodiment the animal feed, animal feed additive, or premix comprises the composition of the disclosure comprising the Bacillus pumilus deposited as DSM 34299 and optionally the Bacillus pumilus deposited as DSM 34300, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0096] In one embodiment the animal feed, animal feed additive, or premix comprises the composition of the disclosure comprising the Bacillus pumilus deposited as DSM 34300 and the Bacillus pumilus deposited as DSM 34299, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0097] In a preferred embodiment the animal feed, animal feed additive or premix comprises the composition of the disclosure consisting of the Bacillus pumilus deposited as DSM 34300 and / or the Bacillus pumilus deposited as DSM 34299, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0098] In a specific embodiment, the animal feed is a Total Mixed Ration (TMR) comprising 0 to 80% forage source, such as hay, silage or pasture. Generally, forage comprises edible parts of a plant (other than separated grain) that can provide feed for animals or can be harvested for feeding animals. As an example, the TMR comprises 0-80% maize; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% barley; and / or 0-30% oats; and / or 0-40% soybean meal.

[0099] In one embodiment, the composition of the present disclosure comprising the probiotic strains are mixed with forage, concentrate, and optionally other feed components to obtain a TMR.

[0100] In a further embodiment, the composition of the present disclosure comprising the probiotic strains is mixed with concentrate, vitamins and / or minerals to obtain a premix. This premix can be mixed with a final diet to obtain a TMR. In a further embodiment, the TMR and the composition of the present disclosure comprising the probiotic strains is mixed with one or more enzymes. In a further embodiment, the composition of the present disclosure comprising the probiotic strains are mixed with other feed ingredients, such as one or more of coloring agents, stabilizers, growth improving additives and aroma compounds / flavorings, saturated or polyunsaturated fatty acids (PUFAs), essential oils, antioxidants, anti-microbial peptides, anti-fungal polypeptides and amino acids.

[0101] In a particular embodiment, the animal feed components consist of or comprise milk (e.g., from cow, goat, sheep), e.g., for feeding of calves. In another particular embodiment, the animal feed components consist of or comprise milk replacement, e.g., for feeding of calves. In one embodiment the composition of the present disclosure comprising the probiotic strains is mixed with water, milk or milk replacer for feeding of calves, piglets or other weaning animals.

[0102] In another embodiment, the animal feed components may include one or more vitamins, such as one or more fat-soluble vitamins and / or one or more water-soluble vitamins. In another embodiment, the animal feed components may optionally include one or more minerals, such as one or more trace minerals and / or one or more macro minerals. Usually fat- and water-soluble vitamins, as well as trace minerals, form part of a so- called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed. Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3. Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin Bl, vitamin B2, vitamin B6, niacin, folic acid and pantothenate, e.g., Ca-D-pantothenate. Nonlimiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium and zinc. Non-limiting examples of macro minerals include calcium, magnesium, potassium and sodium.

[0103] The animal feed, animal feed additive or premix of the disclosure may also comprise at least one enzyme selected from the group comprising of 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 Al (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); cellulase (EC 3.2.1.4); and beta-glucanase (EC 3.2.1.6), or any mixture thereof.

[0104] The animal feed, animal feed additive or premix of the disclosure may further comprise one or more added amino acids. Examples of amino acids which are used in animal feed are rumen -protected or not rumen -protected lysine, alanine, beta-alanine, threonine, methionine and tryptophan. The animal feed, animal feed additive or premix of the disclosure may further comprise coloring agents, stabilizers, growth improving additives and aroma compounds / flavorings, polyunsaturated fatty acids (PUFAs), essential oils, antioxidants, anti-microbial peptides and anti-fungal polypeptides. Examples of coloring agents are carotenoids such as beta-carotene, astaxanthin, and lutein. Examples of aroma compounds / flavorings are creosol, anethol, deca-, undeca- and / or dodecalactones, ionones, irone, gingerol, piperidine, propylidene phthalide, butylidene phthalide, capsaicin and tannin. Examples of saturated fatty acids are C16 and C18, such as palmitic and oleic acids, and polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as linoleic, linolenic, arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid. Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a synthetase.

[0105] In one embodiment the animal feed, animal feed additive or premix comprises one or more coccidiostats.

[0106] In one embodiment the animal feed, animal feed additive or premix further comprises a carrier. The carrier can 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, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, whey, whey permeate, wheat flour, wheat bran, corn gluten meal, starch and cellulose.

[0107] In an embodiment, the animal feed, animal feed additive or premix further comprises one or more additional microorganisms. In a particular embodiment, the animal feed, animal feed additive or premix further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Lentilactobacillus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium, Weizmania and Megasphaera or any combination thereof.

[0108] In a particular embodiment, the animal feed, animal feed additive or premix further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, Bacillus paralicheniformis, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus simplex, Bacillus mojavensis, Bacillus safensis, Bacillus simplex, Bacillus atrophaeus, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, Bacillus tequilensis or any combination thereof.

[0109] In a particular embodiment, the animal feed, animal feed additive or premix further 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. I act is), Candida (such as C. utilis, also called Torula yeast), Pichia (such as P. pastoris), Torulaspora (such as T. delbrueckii), Phaffia yeasts and Basidiomycota.

[0110] The composition of the present disclosure may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition may be in frozen or freeze-dried form. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as cryoprotectants and lyoprotectants known to a skilled person in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and / or flavorants.

[0111] Animal diets can, e.g., be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feedstuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. The bacteria cultures and optionally enzymes can be added as solid or liquid formulations. For example, for mash feed a solid or liquid culture formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) composition of the disclosure comprising the probiotic strain(s) may be added to the pelleted food after the pelleting step. Typically, a liquid composition of the disclosure comprises the probiotic strain(s) 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.

[0112] Another aspect of the disclosure relates to a method for feeding an animal comprising administering the composition of the disclosure comprising the probiotic strains to an animal, such as a monogastric animal or a ruminant. In one embodiment of the disclosure the monogastric animal being fed is poultry. The term "poultry" means domesticated birds kept by humans for the eggs they produce and / or their meat and / or their feathers. Poultry includes breeders, broilers and layers. Poultry include members of the superorder Galloanserae (fowl), especially the order Galliformes (which includes chicken, Guineafowls, quails and turkeys) and the family Anatidae, in order Anseriformes, commonly known as "waterfowl" and including domestic ducks and domestic geese. Poultry also includes other birds that are killed for their meat, such as pigeons and ostriches. Examples of poultry include chicken (including layers, broilers and chicks), ducks, geese, pigeons, turkeys and quail.

[0113] In one embodiment of the present disclosure the animal being fed is a ruminant. Ruminants include cows, cattle, sheep, deer, and goats.

[0114] Ruminants have a fundamentally different digestive system than monogastric animals, and it therefore cannot be concluded that a composition designed for a monogastric animal and with proven effect in same would also be suitable for and have an effect in a ruminant. Hence, providing a composition effective in both monogastric animals and ruminants is not straight forward.

[0115] The digestive system of animals is involved in the mechanical and chemical digestion of food, absorption of nutrients, and elimination of indigestible materials from the body. The main difference between monogastric and ruminant digestive system is that digestion in the monogastric digestive system mainly occurs in the stomach, whereas digestion in the ruminant digestive system is a foregut fermenter type digestion. The monogastric digestive system is composed of a single stomach while the ruminant digestive system is composed of four stomachs (reticulum, rumen, omasum, and abomasum). Monogastric digestive systems mainly occur in omnivores and carnivores, while ruminants are herbivores.

[0116] The monogastric digestive system refers to the organ system which helps the digestion of both animal and plant materials. It is called monogastric since this digestive system is composed of a single stomach. Human, horse, swine, fowl, dog, bird and rabbit-like animals have a monogastric digestive system. The digestion begins with the entering of feed to the mouth. Both chemical and mechanical digestion starts at the mouth. Saliva contains enzymes to digest carbohydrates. The esophagus is the passage that leads feed to the stomach. Various enzymes are secreted into the lumen of the stomach to digest proteins in the feed. Animals with a monogastric digestive system mainly take animal tissues as food. Their diet is easy to digest. Thus, a single stomach is enough for the purpose. Small intestine mainly absorbs the nutrients from the digested feed. The large intestine absorbs water from the indigestible materials.

[0117] The ruminant digestive system refers to the organ system in which the digestion of plant materials occurs. Cows, cattle, sheep, deer, and goats are examples of the animals having a ruminant digestive system. The top jaw of ruminant animals lacks teeth in the front, but instead, a hard pad of skin is present, which is called the dental pad. Other than the basic anatomy of an animal digestive system, the ruminant digestive system is composed of four stomachs. They are rumen, reticulum, omasum, and abomasum. The first three stomachs, the rumen, reticulum, and omasum, are involved in the breaking down of plant fibers and digestion of non-fibrous compounds. The population of microflora is involved in this process. It breaks down cellulose and starch, for example, through fermentation, producing volatile fatty acids such as acetate, butyrate and propionate. These volatile fatty acids are utilized by the ruminant as an energy source. Digestive enzymes are secreted in the fourth stomach called the abomasum. Therefore, fermentation occurs before the digestion of the feed in ruminant animals. Hence, this process is called the foregut fermentation. Furthermore, ruminant animals chew the partly digested food or cud by returning them from the first stomach. Small intestine and large intestine of ruminants are similar to the monogastric digestive system. However, ruminants comprise a large caecum for further digestion of the fibers.

[0118] In one embodiment of the present disclosure, the composition of the present disclosure is used in a method of preventing and / or minimizing respiratory disease in a ruminant. In another embodiment of the present disclosure, the composition of the present disclosure is used in a method of preventing and / or minimizing respiratory disease in a monogastric animal.

[0119] As evidenced in the examples, administration of the composition of the disclosure comprising the probiotic strains described herein improves animal performance including increased body weight (BW), increased birth weight, increased Average Daily Gain (ADG), lower coccidiosis lesion scoring, lower E. acervuline lesion scoring, lower overall mortality, increased milk yield, increased milk fat content, and increased milk protein content in animals receiving the composition as compared to controls.

[0120] In the context of the present disclosure, a "variant thereof" is to be understood as a Bacillus pumilus with an alteration in the wild-type nucleotides of the genome of an organism (e.g. Bacillus pumilus DSM 34300) resulting in changes in the phenotype of said organism, wherein the alteration may be a deletion of one or more nucleotides, a substitution of one or more nucleotides, an insertion of one or more nucleotides, and / or a modification of one or more nucleotides. In the context of the present disclosure, a deletion is to be understood as a genetic mutation resulting in the removal of one or two nucleotides of wild-type nucleotide sequence of the genome of an organism; a insertion is to be understood as the addition of one or more nucleotides to the wild-type nucleotide sequence; a substitution (or point mutation) is to be understood as a genetic mutation where a nucleotide of wild-type nucleotide sequence is changed by another nucleotide; a frameshift is to be understood as a genetic mutation caused by a insertion or deletion of a number of nucleotides in a wild-type nucleotide sequence that is not divisible by three, therefore changing the reading frame and resulting in a completely different translation from the original reading frame; an introduction of a stop codon is to be understood as a point mutation in the DNA sequence resulting in a premature stop codon; a inhibition of substrate binding of the encoded protein is to be understood as any mutation in the nucleotide sequence that leads to a change in the protein sequence responsible for preventing binding of a substrate to its catalytic site of the protein. Furthermore, a knockout mutant is to be understood as genetic mutation resulting in the removal or deletion of a gene, such as an entire gene or an entire open reading frame from the genome of an organism.

[0121] Algorithms for aligning sequences and determining the degree of sequence identity between them are well known in the art. For the purpose of the present disclosure and as an example, one of these algorithms is based on aligning both sequences with the blastp as provided by the National Center for Biotechnology Information (NCBI) on https: / / blast.ncbi.nlm.nih.gov applying standard parameter settings (Matrix: BLOSUM62, Gap Costs: Existence: 11 Extension: !, Conditional compositional score matrix adjustment) and subsequent quantification of identical amino acid pairs in identical positions over the aligned amino acid sequences. A similar process may be carried out for aligning nucleotide sequences using, in this case, blastn as provided by the National Center for Biotechnology Information (NCBI) on https: / / blast.ncbi.nlm.nih.gov applying standard parameter.

[0122] In one embodiment the present disclosure relates to a variant of Bacillus pumilus DSM 34300, wherein the average nucleotide identity (ANI) of the Bacillus pumilus variant is at least 99%, such as e.g. at least 99.5%, such as e.g. at least 99.8%, such as e.g. at least 99.9% identical to the genome of the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 34300.

[0123] In one embodiment the present disclosure relates to a variant of Bacillus pumilus DSM 34299, wherein the average nucleotide identity (ANI) of the Bacillus pumilus variant is at least 99%, such as e.g. at least 99.5%, such as e.g. at least 99.8%, such as e.g. at least 99.9% identical to the genome of the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 34299.

[0124] The illustrative examples presented below serve to better describe the present disclosure. However, the formulations described merely refer to some means to some embodiments of the present disclosure and should not be taken as limiting the scope thereof.

[0125] EXAMPLES

[0126] Trials have been conducted to evaluate the beneficial effects of the Bacillus pumilus strains of the disclosure both under in vitro and under in vivo conditions in both mongastric animals and ruminants.

[0127] EXAMPLE 1

[0128] In vitro trial investigating antibiotic resistance and inhibition effect on selected pathogens and investigating other performance characteristica of probiotics for use in animal feed.

[0129] Materials:

[0130] Veal Infusion Broth (VIB) (Difco, 234420)

[0131] Veal Infusion Broth (VIB) agar (VIB + 1.5% Agar bacteriological (Agar no. 1), Oxoid LP0011) Muller Hinton Broth 2, Cation-adjusted (Fluka)

[0132] T3 agar plates (per liter: 3 g of tryptone, 2 g of tryptose, 1.5 g of yeast extract, 0.05 M sodium dihydrogen phosphate and 0.005 g of MnCI2 [pH 6.8], and 15 g agar) Laura-Bertani (LB) broth (g / L: Bacto tryptone 10 (Difco 0123), Yeast extract 5 (Oxoid L21), NaCI 10 (Merck nr. 106404))

[0133] Brain Heart Infusion (BHI) agar (Oxoid CM375)

[0134] Bile salts (Bile extract, porcine; Sigma B8631)

[0135] Bioassay dishes (Nunc 240845)

[0136] Petri dishes (Procudan 140096, petridish with ribs)

[0137] Physiological saline solution with peptone (0.9% sodium chloride, 1% peptone) FKP ISO-SENSITEST Broth (Oxoid CM0473)

[0138] Microtitre plates (MTP) NUNC, Denmark

[0139] Omni tray / single well plates N 242811 Thermo Scientific / NUNC Denmark

[0140] Deep well microtitre 96 well trays (DW) Rnase / DNase free (Thermo Fisher Science)

[0141] Ampicillin (Sigma, A9518-5G)

[0142] Vancomycin (Sigma, V1764-250MG)

[0143] Gentamicin (Sigma, G1264-50MG)

[0144] Kanamycin (Sigma, K1377-1G)

[0145] Streptomycin (Sigma, S6501-5G)

[0146] Erythromycin (Sigma E-5389)

[0147] Clindamycin (Sigma, C2569-10MG)

[0148] Tetracycline (Sigma T-7660)

[0149] Chloramphenicol (Sigma, C0378-5G)

[0150] Escherichia coli O101 H-, K99 F5 (State Serum Institute, Copenhagen, Denmark) Escherichia coli O147:K89 F4 H19 (State Serum Institute, Copenhagen, Denmark)

[0151] Escherichia coli O149:k91,k88a,c,hl0 NCTC10650, (National Collection of Type Cultures, England)

[0152] E. coli ATCC11775 (American type culture collection)

[0153] E. coli Cp6salp3 (Copenhagen Veterinary University)

[0154] Clostridium perfringens Type A, DSM756, Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen

[0155] Clostridium perfringens type C, NCTC3180, National Collection of Type Cultures (England)

[0156] Clostridium perfringens CCUG2036 (Culture Collection, University of Gothenburg, Sweden)

[0157] Clostridium perfringens CCUG2037 (Culture Collection, University of Gothenburg, Sweden)

[0158] Clostridium perfringens CCUG44727 (Culture Collection, University of Gothenburg, Sweden)

[0159] Staphylococcus carnosus DSM20501, Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen

[0160] All pathogen strains mentioned above were maintained in LB with 20% glycerol in BHI at -80°C

[0161] Bacillus Cultures:

[0162] 101 Bacillus pumilus strains isolated from different sources such as feces, soil, food sources and collected from strain bank collections were maintained in VIB with 20% glycerol in MTP master plates at -80°C.

[0163] Bacteria spore-forming aerobic isolates were subjected to identification by 16S ribosomal sequence and gyrB (Wang et al., 2007), screening for antibiotic susceptibility according to "Guidance on the assessment of bacterial susceptibility to inhibitorys of human and veterinary importance." EFSA Journal 2012; 10(6) :2740 as described below, and bile resistance and sensitivity to low pH, enzymatic activity, growth in different media, heat resistance and sporulation as described in WO2013 / 153159.

[0164] Antibiotic susceptibility measured by MIC

[0165] Bacillus pumilus strains were analyzed for antibiotic susceptibility by measuring the minimum inhibitory concentration (MIC) for a number of antibiotics. The method used was a broth microdilution method as outlined by the standard of CLSI (Clinical and Laboratory Standards Institute M07-A8 and M45-A2).

[0166] A suspension of an overnight culture of the strain to be tested was inoculated in ISO- SENSITEST Broth (Oxoid CM0473) in microtitre plates at an approximate concentration of 105CFU / ml (colony-forming units / ml) in two-fold serial dilutions of the antibiotic to be tested (total volume 100 pl / well) and incubated aerobically for 20-24 hours at 37°C. The results were recorded after 20 hours of incubation as the lowest concentration of the antibiotic to inhibit visible growth. The test was performed twice as two independent biological replicates. Screening of Bacillus strains for inhibition of pathogenic E. coli and S. carnosus

[0167] Pure colonies (grown at 37°C on TSA+SB agar) of E. coli and S. carnosus were suspended in sterile PepSalDiluent to obtain 0.5 McFarland, corresponding to 1 x 108CFU / ml approximately.

[0168] Standard inoculum (5. coli and S. carnosus ; 2, 5 and 10 pL) was added to LB agar (35 ml). The inoculated agar was poured into single-well plates, and 96-pin lids were attached. The agar was allowed to solidify for 20-30 min. The 96-pin lids were removed and the wells were dried for 30-60 min in a laf bench. Bacillus (5 pL overnight cultures grown at 37°C in BHI-broth, with shake, 150rpm) was added to the wells in triplicates. The plates were incubated at 30°C for 48 h. Each assay was carried out in duplicates.

[0169] Approximate concentration ofE. coli and S. carnosus in agar plates

[0170] 2 pL of 0.5 McFarland / 35 ml agar: (1 x 108CFU / ml) / (35ml / 0.002ml) = 6 x 103CFU / ml

[0171] 5 pL of 0.5 McFarland / 35 ml agar: (1 x 108CFU / ml) / (35ml / 0.005ml) = 1.5 x 104CFU / ml

[0172] 10 pL of 0.5 McFarland / 35 ml agar: (1 x 108CFU / ml) / (35ml / 0.01ml) = 3 x 104CFU / ml Radii of clarified inhibition zones around the Bacillus were measured and recorded as "3 = high" - more than 2 mm, "2= medium" - between 0.5 - 2 mm and "1= low" - less than 0.5 mm and 0= no inhibition.

[0173] All data were replicated on separate days.

[0174] Clostridium perfringens inhibition by agar spot test

[0175] VIB agar was poured into bioassay dishes (200 ml per dish) and dried thoroughly in a sterile bench. Overnight Bacillus pumilus cultures, 2 pl of each, were spotted onto the surface of the VIB agar dishes and incubated at 37°C overnight. Clostridium perfringens strains were grown anaerobically on BHI agar at 37°C overnight. Overnight culture of Clostridium perfringens was added in a volume of 2 ml to 200 ml liguid BHI agar, mixed and overlaid gently into the bioassay dishes with Bacillus spots. The dishes were incubated anaerobically at 37°C for 1 day.

[0176] Radii of clarified inhibition zones around the Bacillus were measured and recorded as "3 = high" - more than 2 mm, "2= medium" - between 0.5 - 2 mm and "1= low" - less than 0.5 mm and 0= no inhibition.

[0177] All data were replicated on separate days. Other fitness parameters

[0178] When choosing a Bacillus pumilus strain for use in an animal feed or animal feed additive (including as a Direct Fed Microbial (DFM) or premix) there are some characteristics that are relevant to evaluate, because these characteristics will influence the performance and fitness of the strain for the intended application as a probiotic for animals. Such characteristics are the ability of the strain to grow in the presence of bile, its cellulase activity, its ability to form biofilm, and its capabilities to degrade fibre, starch, protein and feed components. Finally, it is highly relevant to evaluate the ability of the B. pumilus strain to grow at different temperatures and for a probiotic intended for use in monogastric and ruminants, the relevant temperature to test growth at is 37°C (relevant body temperature of most monogastric animals and ruminants) and 41°C (relevant body temperature for poultry).

[0179] RESULTS The results of the different tests are summarized herein below in Tables 1-3.

[0180] Table 1

[0181] Results of selected Bacillus pumilus strains' susceptibility to selected antibiotics Table 2

[0182] Results of selected Bacillus pumilus strains and their performance and fitness Table 3

[0183] Results of selected Bacillus pumilus strains and their inhibition of E.coli and

[0184] Staphylococcus carnosus and their growth ability at different temperatures As can be seen from the above Tables 1-3, not all B. pumilus strains have the same abilities. Only B. pumilus strain #3 (deposited as DSM 34299) and #5 (deposited as DSM 34300) comprise all the needed abilities for a probiotic strain intended to be used in an animal feed or animal feed additive. These two strains are susceptible to antibiotics, are good at forming biofilm and can degrade protein and fiber. Furthermore, they are efficient in inhibiting E. coli and S. carnosus and can grow both at 37 and 41°C, making them useful for ruminants and monogastrics including poultry. The other B. pumilus strains tested failed in one or more of these parameters, making them less useful or not usefull as a probiotic component of an animal feed or animal feed additive.

[0185] EXAMPLE 2

[0186] In vivo trial with monogastric animals (poultry) - Efficacy of Bacillus pumilus in controlling experimentally induced subclinical necrotic enteritis in broilers under floorpen conditions.

[0187] Summary

[0188] Necrotic enteritis (NE) remains one of the most challenging diseases in poultry production despite constant advances in nutrition, stable management and genetics. Acute or clinical NE can cause high mortality in poultry, while sub-clinical NE reduces growth due to poor health. Among the two severity levels of NE, the sub-clinical form is the most common form compared to the acute or clinical form.

[0189] Since the ban of antimicrobial growth promotors that served successfully in control of NE, there has been a constant search for alternative methods for either prevention and / or treatment. The use of microbes and microbe-derived products might offer the preventive immunity needed in chicken production management. Microbes produce a plethora of molecules with antimicrobial propertiesand they can also have beneficial effects through interactions with their host.

[0190] Thus, the objective of this study was to evaluate the preventive potential of different Bacillus pumilus strains against experimentally induced necrotic enteritis (NE) in broilers. The efficacy of the treatments was assessed by individual intestinal lesion scoresand secondary by survey of mortality rate, and body weight (BW). Three different treatment groups were included: an infected untreated control (IUC) group and two treatment groups receiving different strains of Bacillus pumilus. Materials and methods

[0191] Experimental induction of NE is dependent on a combination of several predisposing factors prior to inoculation of a virulent Clostridium perfringens strain. In the current study, all animals were exposed to a high protein content grower feed with 30% fishmeal from D17 until the end of the study. A high protein content grower feed is standard in production of poultry, but it is also considered a predisposing factor that increases the risk of development of necrotic enteritis in the herd.

[0192] All birds were challenged with an overdose of a coccidiosis inoculum (Paracox-8) on D14 and D16via oral gavage.

[0193] An overview of the composition of the inoculum can be found in Table 4.

[0194] Table 4: Number of oocysts per mLinocula

[0195] The objective of this study was to evaluate the preventive properties of two different Bacillus pumilus strains against subclinical necrotic enteritis in broiler chickens. Finally, necrotic enteritis was induced by oral administration of approximately 109CFU Clostridium perfringens / bird on D18, D19, D20 & D21 to all groups. The strain was NetB-positive and alpha toxin producing.

[0196] Firstly, the administration of probiotics did not cause any adverse events within broiler chickens, in this specific experimental setup. There was no coccidiosis or treatment- related mortality and the clinical behavior of all animals was normal throughout the study period.

[0197] Induction of subclinical NE was successful since 72.6% of birds in the IUC demonstrated lesions due to NE and 1.9% NE-related mortality was recorded.

[0198] Results

[0199] The inclusion of the test products was measured by Chr. Hansen. In the IUC group the level of spore forming strains present were as expected, i.e. 6 x 103-7 x 103CFU / g feed. Both treatments groups (DSM 34300 and DSM 34299) had an inclusion rate as expected, i.e. 1 x 106- 4 x 106CFU / g feed. No major deviations in Bacillus counts were observed. Mortality

[0200] The study results revealed an overall mortality frequency above normal, even though that was not seen to be challenge-related.

[0201] Significant differences in overall mortality were observed for day 1 to 27 for DSM 34300: 1.9% versus 8.7% in the infected, untreated control group.

[0202] Non-significant difference, however numerical difference observed for DSM 34299: 4.9% versus 8.7% in the infected, untreated control group.

[0203] It is worth noting that any numerical reduction in mortality is a benefit for the producer even if not significant.

[0204] Table 5: Overview of percent total mortality and NE-related mortality during the study

[0205] Coccidiosis results

[0206] Coccidiosis lesion scores were studied as ordinal data with cumulative link models (procedure elm of the package ordinal). Treatment and day were added as fixed effect. All groups were compared to T01. Post-hoc pairwise comparisons were made without adjustment method. Groups with different letters (e.g. a and b) were significantly different (P < 0.05).

[0207] The Coccidiosis lesion scores results revealed a mean total Coccidiosis lesion score of 1.36 for the infected untreated control group (IUC), which was significant higher than both treatment groups.

[0208] The same pattern was seen on the Eimeria acervulina lesion score measurements, which could be expected as the two measurements are interconnected. Table 6a: Total coccidiosis lesion score Day 22 + D23

[0209] Table 6b: Eimeria acervulina lesion score for Day 22 + Day 23 The body weights were monitored and are presented in Table 7 below for the different groups on study day 22. This revealed an unexpected 8.4% increased body weight for group receiving DSM 34299. Such huge differences in body weight are rarely seen in poultry production.

[0210] Differences in body weights at D22 were studied with linear mixed regression models with pen as random effect and treatment as fixed effect. All treatments were compared to the IUC. Least square means and their standard error are also shown in the tables below. 95% confidence intervals (CI) of the differences with T01 were estimated. Post-hoc pairwise comparisons were made without adjustment method. Groups with different letters (e.g. a and b) were significantly different (P < 0.05).

[0211] Table 7: Body weights The objective of this study wasto evaluate the preventive properties oftwo Bacillus pumilus strains against subclinical necrotic enteritis in broiler chickens. As the part of the experimental model, the birds were subjected to inculation with a moderate number of coccidia I oocysts consisting of two species, Eimeria acervulina and E. max / ma with the provision of low quality grower feed with high inclusion offish meal protein (30% fish meal) from day 20 of the study.

[0212] Firstly, the administration of probiotics did not cause any adverse events within broiler chickens, in this specific experimental setup. There was no coccidiosis or treatment- related mortalityand the clinical behaviour of all animals was normal throughout the study period. The results demonstrates that the B. pumilus strain DSM 34300 significant lowered the mortality, while the B. pumilus strain DSM 34299 provided a numerical, but not significant reduction in mortality. Both the tested B. pumilus strains showed a significant reduction in coccidiosis lesion score and Eimeria acervulina lesion score. Both the tested B. pumilus strains also increased the body weight of the animals receiving the composition of the disclosure comprising either of the B. pumilus strains.

[0213] EXAMPLE 3

[0214] In vivo trial with small ruminant (sheep) - Effect on weight gain of supplementing diet with Bacillus pumilus

[0215] The experimental study was conducted in a dairy sheep farm in Sicily and lasted 120 days in total. On the same farm, 100 pregnant ewes were selected from a large flock for homogeneity of the state of pregnancy (assessed by ultrasound and examination by expert personnel). About 45 days before giving birth, ewes were randomly allotted to two treatments: the control group was fed with a conventional diet (CON), and the treated group received the same diet supplemented with Bacillus pumilus deposited as DSM 34299 (BP). All the animals always had free access to water, indoor spaces and outdoor paddocks.

[0216] During the pregnancy phase, the ewes were fed with 2 kg hay / sheep / day, distributed over two meals, and with 300 g / sheep of concentrate per day in a single meal. While during the lactation phase, the ewes received 500 g / sheep of concentrate and 2 kg hay / sheep / day distributed in two meals. The ingredients of pregnancy and lactation concentrates are shown in Table 8. Probiotic products were thoroughly mixed with the concentrate daily, just before feeding to sheep.

[0217] The lambs born were weighed on the same day. Lambs were then weighed every other week (14 days between weighings) until weaning. At the age of 45 days, lambs were weaned and ewes were milked twice a day. For 30 days, milk yield was weekly recorded for each sheep, and individual milk samples were collected for milk composition analysis by Milkoscan and Bactoscan (Foss Analytics, Hamburg, Germany).

[0218] Table 8. Ingredients of experimental concentrates.

[0219] Results

[0220] The birth weight of the lambs where the ewes had been administered B. pumilus deposited as DSM 34299 (BP) during pregnancy was significantly higher than the birth weight of the ewes that were not administered probiotics (CON). This can be seen in Figure 1.

[0221] The body weight of the lambs was followed from delivery until after weaning, i.e. from day 0 until day 45. 4 different measurements were made with approximately 1 week between each measurement. The results of the weighing can be seen in Figure 2. A significant difference between the body weight of the lambs born from ewes fed the pregnancy and lactation concentrate and the control lambs can be seen. The lambs in the BP group gain significantly more body weight than the CON lambs. As can be seen from Figure 2 the body weight of the BP lambs is higher at all measuring points and the difference between the two groups increases over time. Looking at the Average Daily Gain (ADG) supports this finding as a significant difference is found between the BP and CON group (see Figure 3).

[0222] EXAMPLE 4

[0223] In vivo trial with small ruminant (sheep) - Effect on milk quality and yield of supplementing diet with Bacillus pumilus

[0224] To investigate the effect of feeding a conventional diet supplemented with Bacillus pumilus deposited as DSM 34299 (BP) on the milk yield and milk quality the ewes were milked twice a day after the lambs were weaned at age 45 days. For 30 days, milk yield was weekly recorded for each sheep, and individual milk samples were collected for milk composition analysis by Milkoscan and Bactoscan (Foss Analytics, Hamburg, Germany).

[0225] Overall Conclusion

[0226] The strains DSM 34299 have the ability to improve animal performances (weight at birth of the lambs, growth of the lambs, milk yield, and quality).

[0227] DEPOSIT AND EXPERT SOLUTION

[0228] The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

[0229] Table 9: Deposits made at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

Claims

CLAIMS1. A Bacillus pumilus strain selected from the group consisting of: a. the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 34299, b. the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 34300, and c. a variant strain of DSM 34299 or DSM 34300 obtained by using the deposited strain as starting material and which has retained or further improved the properties of DSM 34299 or DSM 34300.

2. The Bacillus pumilus strain or variant thereof according to claim 1, wherein said B. pumilus strain has the properties of: a. sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol; and b. inhibitory activity against E. coli, S. carnosus and Clostridium perfringens.

3. A composition comprising at least one Bacillus pumilus strain of claim 1 or 2 and acceptable excipients and / or carriers.

4. The composition according to claim 3 which comprises the Bacillus pumilus strain deposited as DSM 34299.

5. The composition according to claim 3 or 4 which comprises the Bacillus pumilus strain deposited as DSM 34300.

6. The composition according to claim 3 which comprises the Bacillus pumilus strain deposited as DSM 34300 and the Bacillus pumilus strain deposited as DSM 34299.

7. The composition according to any of claims 3 to 6 which comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Lentilactobacillus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

8. The composition according to any one of claims 3 to 7, wherein the Bacillus pumilus strain or strains are present as spores.

9. The composition according to any of claims 3 to 8, wherein said composition comprises from 1.0 x 106CFU / gram to 1.0 x 109CFU / gram of said Bacillus pumilus.

10. A composition according to any one of claims 3 to 9 for use in the prevention, reduction or control of a bacterial colonization or infection.

11. The composition according to claim 9 for use in the prevention, reduction or control of a bacterial colonization or infection by Clostridium spp., Escherichia coli and / or Streptococcus carnosus.

12. A composition according to any one of claims 3 to 9 for use in the improvement of animal performance parameters, such as increase of body weight, increase in milk yield and / or increase in milk quality.

13. An animal feed, animal feed additive or premix comprising the composition according to any one of claims 3 to 9, wherein said composition is present in said animal feed, animal feed additive or premix in an amount to provide a total of between 1 x 108to 1 x 1011CFU / head / day of said composition, wherein said animal feed, animal feed additive or premix further comprises one or more additional components selected from concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s), and other feed ingredient(s).

14. A method for the prevention, reduction or control of a bacterial colonization or infection, the method comprising administering an effective amount of a strain according to claim 1 or 2 or a composition according to any one of claims 3 to 9 to an animal in need thereof.

15. The method according to claim 14 wherein colonization or infection by Clostridium spp., Escherichia coli and / or Streptococcus carnosus is prevented or controlled.

16. The method according to claim 14 or 15, wherein the animal is a monogastric animal, such as poultry and / or swine.

17. The method according to claim 14 or 15, wherein the animal is a ruminant, such as a cow, cattle, sheep, deer or goat.

18. A method of improving one or more animal performance parameters selected from the group consisting of: a. increased body weight (BW), b. increased birth weight, c. increased Average Daily Gain (ADG), d. lower coccidiosis lesion scoring, e. lower E. acervuline lesion scoring, f. lower overall mortality, g. increased milk yield, h. increased milk fat content, andi. increased milk protein content the method comprising feeding a strain according to claim 1 or 2 or a composition according to any of claims 3 to 9 to an animal.

19. A kit, comprising the composition as defined in any one of claims 3 to 9 and instructions for use.

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