EA043066B1 - COMPOSITION FOR THE PREVENTION OR CONTROL OF BACTERIAL COLONIZATION OR INFECTION IN THE INTESTINE OF POULTRY - Google Patents

Description

The present invention provides a strain of Bacillus subtilis selected from the group consisting of a) a strain deposited as DSM32324, b) a strain deposited as DSM32325, and c) a mutant strain according to (a) or (b), having sensitivity to ampicillin, vancomycin , gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol and has inhibitory activity against Escherichia coli and Clostridium perfringens.

Additionally, the invention relates to Bacillus compositions containing at least one strain of Bacillus subtilis according to the invention, preferably Bacillus subtilis strain DSM32324 and/or Bacillus subtilis strain DSM32325, as a probiotic (Direct Fed Microbial, DFM), premix, animal feed additive or feed for animals.

The present invention provides a method for improving one or more performance measures of an animal selected from the group consisting of 1) increased weight gain (WG), 2) reduced feed conversion ratio (FCR), 3) decreased necrotizing enteritis lesion score, 4) reduced incidence of necrotizing enteritis, 5) reduced mortality from necrotizing enteritis, 6) increased European Production Efficiency Factor (EPEF), and 7) reduced mortality by feeding an animal with a strain or composition of the present invention.

Prior Art

The cessation of the production of antibiotic-based growth promoters in the European Union in 2006 led to an increased need for cost-effective nutritional supplements with high efficacy and sensitivity to inhibitors of importance to humans and veterinary medicine.

Bacillus-based probiotic feed supplements are known to have positive effects on the health and productivity of pigs and poultry. These products are important for the food industry because the spores are heat resistant and can survive the granulation process at temperatures up to 90-95°C. The U.S. Food and Drug Administration (FDA) considers endospore-producing bacteria Bacillus subtilis and Bacillus licheniformis as safe (GRAS) and the Association of American Feed Control Officials (AAFCO) ) considers them acceptable for inclusion in animal diets or water.

WO 2013/153159 describes a method for selecting a Bacillus strain having antibiotic sensitivity, inhibitory activity against E. coli and Clostridium perfringens, and a high degree of sporulation.

Many of the isolates tested exhibited undesirable antibiotic resistance in excess of European Food Safety Authority (EFSA) breakpoints and were discarded for safety reasons. Several isolates showed inhibition of Clostridium perfringens, while only a few isolates inhibited E. coli. The best inhibition of pathogens was demonstrated mainly by strains of the species B. amyloliquefaciens.

In table. 7 in WO 2016/060934 shows the activity of 10 strains of Bacillus against E. coli. Five of the six strains of B. amyloliquefaciens showed activity against E. coli, while only one of the two strains of B. subtilis, a strain isolated from Kemin's Clostat product, showed activity against E. coli. Interestingly, this strain was subsequently found to be B. amyloliquefaciens, for comparison see WO 2016/118840 (line 23 on page 46).

WO 2016/118840 describes various strains of Bacillus for improving the health and productivity of farm animals, in particular two strains of B. amyloliquefaciens and two strains of B. subtilis (see Table 3.1). Only two of these strains, the B. subtilis strain deposited as DSM29870 and the B. amyloliquefaciens strain deposited as DSM29869, were found to be susceptible to all eight antibiotics tested. Results for ampicillin were not provided.

The B. subtilis strain deposited as DSM29870 was found to inhibit the growth of E. coli strains ATCC10535 and ATC25922 in vitro (Example 4). Example 7 shows the results of three Clostridium perfringens challenge experiments. No significant difference was found between the group treated with food DSM29870 (T4) and the group treated with food bacitracin (T3) in all parameters measured in these experiments. The results for BWG (live weight gain), FCR and mortality of birds with lesions from necrotic enteritis were intermediate between the uninfected untreated group (T1) and the infected untreated group (T2).

However, there is still a need for probiotic strains that can be used to improve the health and productivity of farm animals.

Brief summary of the invention

The present invention provides a strain of Bacillus subtilis selected from the group consisting of a) a strain deposited as DSM32324, b) a strain deposited as DSM32325, and c) a mutant strain according to (a) or (b), having sensitivity to ampicillin, vancomycin , gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol, and having inhibitory activity against E. coli and Clostridium perfringens.

The term bacterial strain refers to a bacterium that remains genetically unchanged when grown or propagated. Many such identical bacteria are included when reference is made to a strain.

Compositions containing at least one Bacillus subtilis strain of the present invention, such as, for example, a probiotic (DFM), animal feed additive or premix or animal feed, can be fed to animals.

At least one strain of Bacillus subtilis of the present invention can be added to the feed during production, after production by the supplier or employee who feeds the animal, immediately prior to the provision of food to the animal. The bacteria Bacillus subtilis used in the methods and compositions described herein are particularly suitable because they are able to survive (as spores) under high temperature and pressure conditions during the dry kibble production process.

Necrotizing enteritis caused by Clostridium perfringens has become a major economic problem in modern poultry production. The aim of some of the in vivo studies described in the examples was to investigate the effect of feed additives containing the Bacillus subtilis strain of the present invention on the pathogenesis of necrotic enteritis (NE) in caged broiler studies. Other in vivo studies in poultry have focused on performance with or without Clostridium perfringens challenge.

Separate cage/floor pen studies have been performed to evaluate the effect of Bacillus subtilis DSM32324 and DSM32325 on the development of subclinical necrotizing enteritis. Two different independent research bases were used, one located in Europe (Example 3, DSM32324) and the other in the USA (Example 4, DSM32325), which means that slightly different evaluation parameters were used to evaluate the effect of these two strains.

For Bacillus subtilis DSM3234, performance indicators such as feed conversion ratio (FCR) and average weight gain (AWG) measured on day 21, day 35 and day 42 showed a significant increase for all control points when considering Bacillus subtilis DSM3234 as a dietary supplement according to compared to an untreated infected control group. Surprisingly, the Bacillus treated group showed no significant difference from the uninfected, untreated control group, even though the latter group was not challenged and considered healthy (Table 7).

In an in vivo experiment with provocation of induced subclinical enteritis Bacillus subtilis DSM3234 significantly reduced the degree of damage from necrotizing enteritis in chickens and reduced mortality from necrotizing enteritis (Table 8).

Bacillus subtilis DSM32325 reduced the incidence of necrotizing enteritis in chickens compared to infected untreated controls in a statistically significant manner when data for days 25 and 26 were combined. Surprisingly, the incidence of necrotizing enteritis was even lower in the Bacillus treated group than in the amoxicillin treated control group (Table 1). 9).

In addition, Bacillus subtilis DSM32325 reduced the severity of necrotizing enteritis (mean score) compared to the infected untreated control in a statistically significant manner when day 25 and day 26 were combined. Unexpectedly, the mean score was even lower in the Bacillus treated group than in the amoxicillin treated group. (Table 10).

Two strains of B. subtilis were also evaluated in two feeding performance experiments, in Example 5 (DSM32324 and DSM32325) and Example 6 (DSM32324).

Example 5 shows the results of an experiment on 1800 male Ross 308 broilers, in which it was found that during the entire fattening period (age 0-42 days), broilers supplemented with strains DSM32324 or DSM32325 Bacilli grew significantly more than control animals. The feed conversion ratio (FCR) and EPEF (European Production Efficiency Factor) for all broilers supplemented with DSM19489, DSM32324 and DSM32325 improved significantly compared to control animals.

Example 6 presents the results of an experiment on 1300 Ross 308 male broilers per treatment group, showing that DSM19489 tended to decrease mortality (p=0.096) and Bacillus subtilis DSM32324 to a marked and significant decrease in mortality, especially in the final period, which also served to assess the statistical significance of mortality for the entire experiment.

In Example 7, the effect of three selected Bacilli strains (DSM32324, DSM32325 and DSM25840) on productivity and observed intestinal digestibility was examined and concluded that all three strains showed unexpectedly good and significantly improved results.

Birds supplemented with DSM32324 showed higher daily weight gain (Table 16), daily food intake and initial feed conversion rate (data not shown), and higher protein digestibility at day 42 (Table 17) compared to birds 2 043066 who did not receive the supplement.

Birds supplemented with DSM25840 exhibited higher daily weight gain (Table 16) and daily food intake during the initial period (data not shown), higher body weight on day 42 (Table 16), and higher digestibility of ash, protein and energy on day 42 (Table 17) compared to non-supplemented birds.

Birds supplemented with DSM32325 exhibited higher daily weight gain (Table 16) and higher daily food intake during the initial period (data not shown) and lower Ca and phosphorus absorption at Day 42 (Table 17) compared to birds not receiving the supplement.

In conclusion, these studies have shown that the Bacillus subtilis strains deposited as DSM32324 and DSM32325 show an action to reduce necrotizing enteritis in in vivo challenge tests and a positive effect on poultry performance indicators. The important findings were a significant increase in weight gain (WG), a significant decrease in the feed conversion ratio (FCR), a significant decrease in mortality, and a significant increase in the European Production Efficiency Factor (EPEF).

Example 8 shows that the Bacillus composition of the invention, EPB5, containing DSM32324, DSM25840 and DSM32325 in a ratio of 8:3:5, improved the performance of broilers compared to birds fed the corresponding diet without the addition of a probiotic. Positive responses were shown in relation to the feed conversion factor and average weight gain. Surprisingly, the EBP5 treated groups showed a significant improvement in some performance measures compared to the Bacillus single strain groups, again showing significant differences in performance compared to the untreated infected control group. In addition, both individual strains of Bacillus subtilis DSM32234, Bacillus subtilis DSM32235 and Bacillus amyloliquefaciens DSM25840 and the combination of EBP5 significantly reduced the necrotizing enteritis lesion score in chickens and reduced mortality from necrotizing enteritis in an in vivo challenge test.

Example 9 shows that the Bacillus composition of the invention improved the performance of turkeys aged from 1 to 147 days (feeding period of 147 days) at dose levels from 250 to 2000 mg/kg compared to birds fed the corresponding diet without the addition of a probiotic. Positive responses were shown for body weight gain, feed conversion rate and dry matter content of excrement.

Example 10 shows that the Bacillus composition of the invention can be combined with a vaccine, such as a live Salmonella Typhimurium vaccine, without affecting the initial colonization of the Salmonella vaccine and its subsequent ability to protect against Salmonella Heidelberg challenge in broiler chickens. This study shows that there may even be an additive effect in the combined action of this vaccine and the Bacillus composition (Table 20).

Definitions

Generally, the terms and expressions used herein have the meanings known in the art, which can be found by reference to standard texts, journal articles, and from the context known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the present invention.

As used here, the singular forms are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Animal food: The term animal food refers to any compound, preparation or mixture suitable for or intended for consumption by animals. The pet food for a monogastric animal contains concentrates as well as, for example, vitamins, minerals, enzymes, amino acids and/or other feed ingredients (such as in a premix). Animal feed may further comprise forage. Examples of poultry feed are given in Examples 3-7.

Composition: The term composition refers to a composition containing a carrier and at least one bacterial strain as described herein. The compositions described herein may be a probiotic (Direct Fed Microbial, DFM), an animal feed supplement, or a premix, or an animal feed.

Concentrate: The term concentrate means a feed with high concentrations of protein and energy substrates such as fishmeal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by grinding, milling, etc., e.g. from corn, oats , rye, barley, wheat), oilseed meal (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola seed, peanut or peanut), coconut meal, material derived from yeast, and vinasse (such as wet grain vinasse (WDS) and dry grain vinasse with soluble ingredients (DDGS)).

Control of C. perfringens infection and/or necrotic enteritis: The expression control of C. perfringens infection and/or necrotizing enteritis means a method and/or composition that partially or completely inhibits C. perfringens infections and/or necrotizing enteritis in an animal.

- 3 043066

Accordingly, the expression control of C. perfringens infection and/or necrotizing enteritis means that C. perfringens infections and/or necrotizing enteritis are suppressed or completely eliminated.

Probiotic (Direct Fed Microbial): The term probiotic or DFM means live microorganisms, including spores, which, when administered in adequate amounts, confer a host benefit such as improved digestion or health.

Enzymatic activity under anaerobic conditions: The expression enzymatic activity under anaerobic conditions means the activity of enzymes produced by a strain of Bacillus during growth under anaerobic conditions. Example 4 of WO 2013/153159 describes a method for testing Bacillus strains for the production of xylanase, cellulase and protease enzymes.

European Production Efficiency Factor (EPEF): The European Production Efficiency Factor is a way of comparing the performance of live poultry flocks. The single-number format facilitates comparisons of productivity within and between farms, and can be used to evaluate environmental, climate, and housing variables. EPEF is calculated as [(viability (%) x body weight (kg)/(Age of wasting (days) x FCR)] x 100 where viability is the percentage of live birds at the end of the study, body weight is the average weight gain at the end of the study, the age of attrition is the age of the birds at the end of the study and the FCR is the feed conversion rate at the end of the study.

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 performance of the animal. The true effective dosage in absolute numbers depends on factors including: the health status of the animal in question, the presence of other ingredients. The effective amount, effective concentration, or effective dosage of the bacterial strains can be determined by routine techniques known to those skilled in the art. An example of an effective amount for poultry is given in Examples 3-7.

Feed Conversion Rate (FCR): FCR is a measure of an animal's efficiency in converting feed mass into an increase in desired production. For animals that are bred for meat, such as pigs, poultry, cattle, sheep, and fish, the output is the mass achieved by the animal. Specifically, FCR is the mass of food eaten divided by products over a given period. FCR can be determined as described in Example 7. An improvement in FCR means a decrease in the FCR value.

Animal Feeding: The expressions fed to an animal or fed to an animal mean that the composition of the present invention is orally administered to the animal in an effective amount. Oral administration can be repeated, for example, one or more times a day for a certain period of time, such as several days, one week, several weeks, one month or several months. Feeding poultry may, for example, be carried out as described in Examples 3-7. Accordingly, the terms fed or fed means any type of oral administration, such as administration with animal food or drinking water, or in certain circumstances through a gastric tube or aerosol spray.

Forage: The term forage as defined here also includes roughage. Forage is fresh plant material such as forage hay and silage, grass and other forage plants, seaweed, germinated grains and legumes, or any combination thereof. Examples of fodder plants are alfalfa (Alfalfa, lucerne), horned bird, plants of the genus cabbage (e.g. collard greens, rapeseed (canola), swede (rutabaga, swede), turnip), clover (e.g. Swedish clover, red clover, clover subterranean, creeping clover), grasses (e.g., bermuda grass, brome, high ryegrass, fescue, trident, bluegrass, hedgehog, chaff, timothy grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat, and vegetables such as beets. Additionally, forage includes plant residues from grain production (such as corn straw, wheat straw, barley, oats, rye and other cereals), vegetable residues such as beet tops, oil mill wastes such as stalks and leaves of soybeans, rapeseed and others. leguminous plants, and fractions from grain refining for animal or human consumption or fuel production or other industries.

Inhibitory activity against Clostridium perfringens: The expression 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 conducting the test described in Example 1.

E. coli inhibitory activity: The expression E. coli inhibitory activity means that the growth of E. coli is inhibited and/or that some or all of the E. coli are killed. This can be determined by conducting the test described in Example 1.

Isolated: The term isolated means that the bacterial strains described herein are in a form or environment that does not occur naturally, ie. the strain is at least partially separated from one, or more than one, or all of the natural components with which it is associated in nature.

Pellet: The terms pellet and/or granulation refer to solid round, spherical and/or cylindrical tablets or pellets and processes for making such solid forms, in particular feed pellets and solid extruded animal food. As used herein, the terms extrusion or extrusion are terms well known in the art and refer to the process of forcing a composition, as described herein, through a pressurized orifice.

Poultry: The term poultry refers to domesticated birds that people keep for the eggs they produce and/or their meat and/or their feathers. Poultry includes producers, broilers and laying hens. Poultry includes members of the superorder Galloanserae (birds), specifically the order Galliformes (which includes chickens, guinea fowls, quails and turkeys) and the family Anatidae, the 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 chickens (including laying hens, broilers and chickens), ducks, geese, pigeons, turkeys and quails.

Prevent C. perfringens infections and/or necrotizing enteritis: Prevent C. perfringens infections and/or necrotizing enteritis means a method and/or composition that prevents the development of C. perfringens infection and/or necrotizing enteritis in an animal.

Reducing Sugar: A reducing sugar is any sugar that either has a reactive aldehyde group or has the ability to form one, allowing the sugar to act as a reducing agent. Reducing ends are formed as a result of enzymatic cleavage of the glycosidic bond between polymeric hydrocarbons. Reducing sugars include glucose, glyceraldehyde, and galactose, as well as disaccharides such as lactose and maltose, and can be measured by the Nelson-Somogyi (NS) method or using dinitrosalicylic acid (DNS). DNS is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which strongly absorbs light at 540 nm. This study simulates the situation when food is swallowed by an animal and digested in the digestive tract. The ability of various strains of Bacillus to degrade non-starch polysaccharides (NSPs) to reducing sugars was examined in Example 2.

Roughage: The term roughage refers to high-fiber dry plant material such as fibre, bran, seed and grain hulls and plant residues (such as dry livestock feed, copra, straw, chaff, beet sugar by-products).

Antibiotic-susceptible: The term antibiotic-susceptible means a phenotypic property of a bacterial strain such that the growth of said bacterial strain is inhibited under conditions in which the bacterial strain would otherwise grow. In this context, antibiotic susceptibility is tested according to the CLSI (Clinical and Laboratory Standards Institute) guidelines (M07-A8 and M45-A2). A Bacillus strain is considered susceptible if growth is determined only at the threshold concentration defined in EFSA Journal 2012; 10(6):2740 for vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol, or at a lower concentration. For ampicillin, there is no threshold given by the EFSA for Bacillus; a cut-off level of 4 mg/l was chosen for the strain that was considered susceptible.

Silage: The term silage refers to a fermented, high-moisture prepared feed that can be fed to herbivores such as horses and ruminants such as camels, llamas, cattle, and sheep, or used as feedstock for the production of biofuels for anaerobic fermenters. It is fermented and stored in a process called ensilage, ensiling, or silaging, and is usually obtained from grass or grains (e.g. corn, sorghum, oats, rye, timothy, forage grasses) or legumes such as clover (clovers/trefoils), alfalfa, peas, using the whole green plant (not just corn). Silage can be obtained from many field crops and specific terms may be used depending on the type (oatlage for oats, haylage for alfalfa). Silage is produced either by placing cut green plants in a silo store by stacking them in a large pile covered with plastic film, or by wrapping large rolls in plastic film.

Spore: The terms spore and endospore are used interchangeably and are used in their usual meaning, which is well known and understood by one of skill in the art. As used here, the term spore refers to a microorganism in its dormant, protected, non-reproductive state.

Stable: The term stable is a term known in the art, and in a preferred aspect, the term stable is intended to refer to the ability of a microorganism to remain in viable form prior to administration to an animal to improve the health of the animal.

Pig: The terms pig or hogs mean domesticated pigs that people keep for food, such as their meat. The term pig includes members of the genus Sus such as Sus scrofa domesticus or Sus domesticus and includes piglets, weaners, youngsters, finishers, hocks pigs, polts pigs, gilts, sows and gestating sows.

Plant protein: The term plant protein refers to any compound, preparation, or mixture that includes at least one protein derived from or derived from a plant, including modified proteins and protein derivatives.

Vegetable proteins can be obtained from vegetable protein sources such as legumes and cereals, for example from plant material of the Fabaceae (Leguminosae) families, for example soybean, lupine, pea or bean; Cruciferaceae, Chenopodiaceae, eg beetroot, sugar beet, spinach or quinoa quinoa; and Roaseae. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oats, maize (corn), rice and sorghum.

Weight Gain: Weight gain of an animal is the increase in weight of an animal over a period of time. An example of determining the average weight gain is given in Example 3, and an example of determining the daily weight gain is given in Example 7.

Detailed description of the invention

The present invention provides a strain of Bacillus subtilis selected from the group consisting of a strain deposited as DSM32324; a strain deposited as DSM32325 and a mutant strain DSM32324 or DSM32325 having sensitivity to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol; and having inhibitory activity against E. coli and Clostridium perfringens.

The bacterial strains described here are isolated, i. are in a form or environment that does not occur naturally.

The terms mutant bacterium or mutant strain refer to a natural (spontaneous, natural) mutant bacterium or bacterium with induced mutations, in the genome (DNA) of which there is one or more mutations that are absent in the DNA of the parent strain. An induced mutant is a bacterium where the mutation has been induced by a human-assisted treatment, such as any conventionally used mutagenic treatment, including treatment with chemical mutagens, such as chemical mutagens selected from:

(1) a mutagen that binds to or is incorporated into DNA, such as a base analog such as 2-aminopurine, or an intercalating agent such as ICR-191, (2) a mutagen that reacts with DNA, including alkylating agents, such as nitrosoguanidine or hydroxylamine or ethanemethylsulfonate (EMS) or K-methyl-K'-nitro-K-nitroguanidine (NTG), UV or gamma radiation, etc. In contrast, a spontaneous mutant or a natural mutant has not been subjected to human mutagenic effects.

A mutant may be subjected to several mutagenic treatments (one treatment should be understood as one mutagenesis step followed by a screening/selection step), but in the present invention it is preferred that no more than 20, or no more than 10, or no more than 5 treatments be carried out ( or screening/selection steps). In the mutant preferred in the present invention, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or even less than 0.0001% of the nucleotides in the bacterial genome are changed to other nucleotides, or deleted, according to compared to the parent strain.

Mutant bacteria as described above are non-GMO (Genetically Modified Organism), i.e. not modified by recombinant DNA technology. As an alternative to the above preferred method for producing a mutant by random mutagenesis, it is also possible to obtain such a mutant by site-directed mutagenesis, for example by using suitably designed cloning techniques.

When the proposed mutant is a spontaneous mutant, the strain is subjected to a selection step without any prior mutagenic treatment.

In one embodiment, the Bacillus subtilis strain of the invention has at least 98% (e.g., at least 98.5%, e.g., at least 99%, e.g., at least 99.5%, e.g., at least 99.6%, e.g. at least 99.7%, eg at least 99.8%, eg at least 99.9%) sequence identity with the DSM32324 nucleotide sequence.

In one embodiment, the Bacillus subtilis strain of the present invention has at least 98% (e.g., at least 98.5%, e.g., at least 99%, e.g., at least 99.5%, e.g., at least 99.6% , eg at least 99.7%, eg at least 99.8%, eg at least 99.9%) sequence identity with the amino acid sequence of DSM32324.

In one embodiment, the Bacillus subtilis strain of the present invention has at least 98% (e.g., at least 98.5%, e.g., at least 99%, e.g., at least

- 6 043066

99.5%, such as at least 99.6%, such as at least 99.7%, such as at least

99.8%, such as at least 99.9%) sequence identity with the nucleotide sequence of DSM32325.

In one embodiment, the Bacillus subtilis strain of the present invention has at least 98% (e.g., at least 98.5%, e.g., at least 99%, e.g., at least 99.5%, e.g., at least 99.6% , eg at least 99.7%, eg at least 99.8%, eg at least 99.9%) sequence identity with the amino acid sequence of DSM32325.

A Bacillus strain is considered to have E. coli inhibitory activity if the zone of inhibition is at least 0.5 mm (low inhibition). Preferably, the zone of inhibition is at least 0.5 to 2 mm (medium), more preferably greater than 2 mm (high). The zone of inhibition may differ for different strains of E. coli. For a strain to be considered as exhibiting E. coli inhibitory activity according to the present invention, it must provide a zone of inhibition of at least 0.5 mm for all E. coli strains tested. Preferably the zone of inhibition for two, three, four strains of E. coli, or even more preferably the zone of inhibition for all five strains of E. coli, is at least 0.5 to 2 mm. Even more preferably, the zone of inhibition for two, three, four strains of E. coli, or even more preferably, the zone of inhibition for all five strains of E. coli is greater than 2 mm.

A Bacillus strain is considered to have inhibitory activity against Clostridium perfringens if the zone of inhibition is at least 0.5 mm (low inhibition). Preferably, the zone of inhibition is at least 0.5 to 2 mm (medium), more preferably greater than 2 mm (high). The zone of inhibition may differ for different strains of Clostridium perfringens. For a strain to be considered as exhibiting inhibitory activity against Clostridium perfringens according to the present invention, it must provide a zone of inhibition of at least 0.5 mm for all strains of Clostridium perfringens tested. Preferably, the zone of inhibition for two, three, four strains of Clostridium perfringens, or even more preferably, the zone of inhibition for all five strains of Clostridium perfringens is at least 0.5 to 2 mm. Even more preferably, the zone of inhibition for two, three, four strains of Clostridium perfringens, or even more preferably, the zone of inhibition for all five strains of Clostridium perfringens is greater than 2 mm.

Preferably, the Bacillus strain should also have the ability to increase the amount of reducing sugars resulting from the breakdown of non-starch polysaccharides (NSPs). The ability of various strains of Bacillus to cleave NSP to reducing sugars was studied in Example 2, and the results are presented in table. 4. Strains having the ability to increase available sugars to at least 500 kJ/kg feed when tested as described in this example are considered preferred.

Based on the detailed descriptions of the studies, one skilled in the art can repeat these studies to determine if a particular strain of Bacillus meets parameters such as antibiotic susceptibility, inhibitory activity, and ability to cleave NSP. Thus, a person skilled in the art can constantly obtain strains with certain properties. Preferably, one skilled in the art can also include a vegetative cell sensitivity test at pH 4 and a bile resistance test to ensure that the strains are able to survive sufficiently in the gastrointestinal tract, for example as described in WO 2013/153159. Obviously, these studies can be performed in any order, and some strains may be excluded during the study if they do not meet the criteria.

Additionally, the invention provides a Bacillus composition containing at least one strain of Bacillus subtilis according to the invention. In one embodiment, the Bacillus composition contains one strain of Bacillus subtilis according to the invention. In another embodiment, the Bacillus composition contains two strains of Bacillus subtilis according to the invention, for example a combination of Bacillus subtilis DSM32324 and Bacillus subtilis DSM32325.

Bacillus compositions of the present invention may contain a combination of at least one Bacillus subtilis strain of the invention and at least one other Bacillus strain. The Bacillus composition may contain at least two strains, such as at least three, such as at least four, such as at least five Bacillus strains, at least one of which is a Bacillus subtilis strain of the present invention.

Bacillus strains can be used in any combination in a Bacillus composition. For example, a Bacillus composition may comprise at least one Bacillus subtilis strain of the invention and/or at least one Bacillus licheniformis strain and/or at least one Bacillus amyloliquiefaciens strain, such as two Bacillus subtilis strains of the invention and at least one strain of Bacillus amyloliquiefaciens. The composition may contain Bacillus subtilis DSM32324, and/or Bacillus

- 7 043066 subtilis DSM32325 in combination with Bacillus amyloliquiefaciens DSM25840 and/or Bacillus licheniformis DSM17236 and/or Bacillus subtilis DSM19489. Any other possible combinations of the Bacillus strains of the present invention with other Bacillus strains can also be made. As a specific example, the Bacillus composition contains Bacillus subtilis DSM32324, Bacillus subtilis DSM32325 and Bacillus amyloliquiefaciens DSM25840. In another specific example, the Bacillus composition contains Bacillus subtilis DSM32324, Bacillus subtilis DSM32325 and Bacillus licheniformis DSM17236. In another additional specific example, the Bacillus composition contains Bacillus subtilis DSM32324, Bacillus subtilis DSM32325 and Bacillus subtilis DSM19489.

If more than one strain is used, it is understood that the proportion of each strain in the composition is from 1 to 99%, in particular from 20 to 80%, for example from 30 to 70%, more specifically 20, 33, 40 or 50% of the total amount of bacterial isolates, calculated in CFU/g of composition. Individual strains may be present in nearly equal amounts or not in equal amounts.

In a preferred embodiment, the present invention provides a Bacillus composition containing DSM32324, DSM25840 and DSM32325 in a ratio of 8:3:5.

Suitable Bacillus strain or strains are provided in a form suitable for commercial use as known to those skilled in the art. Accordingly, in one embodiment, the Bacillus strain or strains are in dried (eg, spray dried) or frozen form in the composition. The composition may be provided in any suitable form, in particular in the form of a liquid such as a gel, suspension, powder or pellet.

In a preferred embodiment, the Bacillus composition contains from 105 to 10 ¹² cfu/g, in particular from 5x105 to 10 ¹² cfu/g, more preferably from 106 to 10 ¹² cfu/g and most preferably from 10 ⁷ to 10 ¹² cfu/g, in in particular from 10 ⁸ to 10 ¹¹ cfu/g, for example from 109 to 10 ¹⁰ cfu/g, of each of the bacterial strains in the composition. The Bacillus composition contains at least 5x104 cfu of each strain per 1 g of the composition, which distinguishes the composition of the present invention from, for example, animal feed with natural strains.

The term CFU/g refers to the weight (in grams) of the composition, including carriers such as calcium carbonate, anti-caking agents such as aluminum silicates and kieselguhr (diatomaceous earth), and other components present in the composition.

Compositions of the present invention contain at least one strain of Bacillus according to the invention and at least one carrier and/or other component which makes the composition suitable for feeding an animal or as an additive to drinking water.

As used here, the term premix refers to a strain of Bacillus added to a carrier to form a premix, which is then added to animal feed at the desired input percentage.

Alternatively, at least one Bacillus strain of the invention may be prepared in admixture with animal feed ingredients, as discussed in detail below. Such combinations may be in the form of pellets which are extruded by standard pelletizing processes.

The invention also provides a method for producing an animal feed, animal feed additive or premix containing the addition of at least one Bacillus strain of the invention to an animal feed or suitable components thereof.

Bacillus bacteria exist in the form of spores and vegetative cells, which can divide to form more vegetative cells. When reference is made here to Bacillus, this refers to both spores and vegetative cells, unless the context indicates otherwise.

In the Bacillus composition of the present invention, the Bacillus strain or strains are preferably provided in the form of spores. The main function of sporulation is usually to ensure the survival of the bacterium during periods of environmental stress. Therefore, they are resistant to UV and gamma radiation, desiccation, lysozyme, temperature, starvation and chemical disinfectants. The spore envelope is impermeable to many toxic molecules and may also contain enzymes that are involved in germination. The core has normal cellular structures such as DNA and ribosomes, but the spore is metabolically inactive.

The vegetative form of the bacteria produces effectors that can suppress bacterial pathogens or exert other beneficial effects in the animal's gastrointestinal tract. Thus, reactivation and germination of spores after administration to an animal are important.

From literary sources it is known that bile has a certain negative effect on survival and germination and development into vegetative cells in the gastrointestinal tract of animals. Therefore, probiotic bacteria in general must be able to survive and proliferate in the intestines of animals due to low pH tolerance and bile salt tolerance in order to be useful as Bacillus probiotic compositions for addition to animal feed. The examples provide useful in vitro tests in this regard. The low pH sensitivity test (simulating the environment of the stomach) focuses on the resistance of vegetative cells to pH 4.

- 8 043066

It is well known that spores are resistant at pH 2-3 and that vegetative cells die at pH 2. However, pH in the stomach can take values up to 4, especially under feeding conditions. This can lead to the germination of spores, and therefore it is relevant to test the sensitivity of vegetative cells to pH 4. The strains chosen should preferably be resistant to pH 4.

Bacillus subtilis strains deposited as DSM32324 and DSM32325 were tested for vegetative cell sensitivity to pH 4 and bile resistance in order to ensure that these strains are suitable.

The Bacillus composition may contain one or two different strains of B. subtilis and/or one or two strains of B. licheniformis and/or one or two strains of Bacillus amyloliquiefaciens, where each strain is independently selected to fulfill a particular role and/or function. The combination of these strains can be combined with animal feed, such as poultry feed, and ultimately used to improve the health and productivity of farm animals (eg, livestock and/or poultry). For example, the strains and/or combination strains can reduce the number of pathogens in the gut of poultry and increase the weight gain of industrial poultry.

In a further embodiment, the Bacillus composition of the invention may be combined with a vaccine, such as a live Salmonella Typhimurium vaccine, to suppress Salmonella infection and/or increase FCR.

In one aspect of the present invention, there is provided an animal feed, animal feed supplement or premix comprising at least one Bacillus subtilis strain of the present invention and further comprising one or more concentrate, vitamin, mineral, enzyme, amino acid and/or other feed ingredient. In one embodiment, the pet food, animal feed supplement, or premix comprises Bacillus subtilis strain DSM32324. In another embodiment, the pet food, animal feed supplement, or premix comprises Bacillus subtilis strain DSM32325. The animal feed, animal feed additive or premix may contain both Bacillus subtilis DSM32324 and Bacillus subtilis DSM32325.

In a specific embodiment, the pet food contains forage. Typically, forage contains a source of vegetable protein. In a specific embodiment, the plant protein source is material from one or more plants of the Fabaceae family. In another specific embodiment, the plant protein source is material from one or more plants of the Chenopodiaceae family. Other examples of vegetable protein sources are rapeseed and cabbage. In another specific embodiment, soy protein is the preferred plant protein source. As an example, the forage contains 0-80% corn and/or 0-80% sorghum and/or 0-70% wheat and/or 0-70% barley and/or 0-30% oats and/or 0-40% soy flour and/or 0-10% fish meal and/or 0-20% whey.

In one embodiment, the forage and at least one Bacillus subtilis strain of the invention are mixed with the concentrate. In another embodiment, the forage and at least one strain of Bacillus subtilis according to the invention are mixed with the premix. In a further embodiment, the feed and at least one Bacillus subtilis strain of the invention are mixed with vitamins and/or minerals. In a further embodiment, the forage and at least one Bacillus subtilis strain of the invention are mixed with one or more enzymes. In a further embodiment, the feed and at least one strain of Bacillus subtilis of the invention are mixed with other feed ingredients such as colorants, stabilizers, growth enhancers and aromatics/flavors, polyunsaturated fatty acids (PUFAs); substances generating reactive oxygen species, antimicrobial peptides, antifungal polypeptides and amino acids.

In a particular embodiment, the pet food consists of or contains milk (eg from pig, cow, goat, sheep) for example for feeding piglets. In another specific embodiment, the pet food consists of or contains a milk replacer, for example, for feeding piglets.

In another embodiment, the pet food 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 pet food may further include one or more minerals, such as one or more micronutrients and/or one or more macronutrients. Usually, fat-soluble and water-soluble vitamins and micronutrients form part of a so-called premix intended to be added to the feed, while macronutrients are usually added to the feed separately. Non-limiting examples of fat soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3. Non-limiting examples of water soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and pantothenate, such as Ca-D-pantothenate. Non-limiting examples of trace elements include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, and zinc. Non-limiting examples of macronutrients include calcium, magnesium, potassium, and sodium.

- 9 043066

The animal feed, animal feed additive or premix according to the invention may also contain at least one enzyme selected from the group consisting 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 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); an 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.

The animal feed, animal feed additive or premix according to the invention may additionally contain an additive of one or more amino acids. Examples of amino acids that are used in animal feed formulations are lysine, alanine, beta-alanine, threonine, methionine, and tryptophan. The animal feed, animal feed additive or premix of the invention may further comprise colorants, stabilizers, growth aids and aromatics/flavorings, polyunsaturated fatty acids (PUFAs); substances generating reactive oxygen species, antimicrobial peptides and antifungal polypeptides. Examples of dyes are carotenoids such as beta-carotene, astaxanthin and lutein. Examples of aromatic compounds/flavorants are creosol, anethole, deca-, undeca- and/or dodeca-lactones, ionones, iron, gingerol, piperidine, propylidene-phthalide, butylidene-phthalide, capsaicin and tannin. Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid. Examples of substances that generate reactive oxygen species are chemical compounds such as perborate, persulfate or percarbonate, and enzymes such as oxidase, oxygenase or synthetase.

In one embodiment, the pet food, animal feed supplement, or premix contains one or more coccidiostats.

Animal feed, animal feed additive or premix additionally contain a carrier. The carrier may contain one or more of the following compounds: water, glycerin, 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.

In one embodiment, one or more than one bacterial strain is stable when subjected to the pressure that is applied/achieved during the granulation extrusion process. In a certain embodiment, one or more than one bacterial strain is stable at a pressure in the range from 1 bar (1x105 Pa) to 40 bar (4x106 Pa).

In a certain embodiment, one or more than one bacterial strain is stable at high temperatures. In particular, bacterial strains are stable when subjected to the temperatures reached during the extrusion process for granulation. In another specific embodiment, one or more than one bacterial strain is stable in the temperature range from 70 to 120°C.

In one embodiment, the pet food, animal feed supplement, or premix further comprises one or more additional microorganism. In a particular embodiment, the pet food, animal feed additive, or premix 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 of them.

In a particular embodiment, the pet food, animal feed additive, or premix further comprises a bacterium from one or more of the following species: Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, 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.

In a specific embodiment, the pet food, animal feed supplement, or premix further comprises one or more types of yeast. One or more types of yeast may be selected from the group consisting of Saccharomycetaceae, Saccharomyces (in particular S. cerevisiae and/or S. boulardii), Kluyveromyces (in particular K. marxianus and K. lactis), Candida (in notably C. utilis, also called Torula yeast), Pichia (particularly P. pastoris), Torulaspora (particularly T. delbrueckii), Phaffia and Basidiomycota yeasts.

The animal ration may, for example, be in the form of a mash (non-granulated) or a kibble. Typically, ground food products are mixed and sufficient amounts of essential vitamins and minerals are added according to the specification for the species in question. Bacterial cultures and possibly enzymes can be added in the form of solid or liquid formulations. For example, for a stirrer, the culture composition in solid or liquid form may be added before or during the mixing step of the ingredients. For the gran

- 10 043066 lylated food (liquid or solid) the Bacillus composition can also be added before or during the mixing stage of the food ingredients. Typically, the liquid Bacillus composition of the invention contains the bacterial strain(s), optionally with a polyol such as glycerol, ethylene glycol or propylene glycol, and is added after the granulation step, for example by spraying the liquid composition onto pellets. The bacteria can also be included in the animal feed supplement or premix.

The composition of the present invention can be used to prevent or control bacterial colonization or infection, for example E. coli and/or Clostridium, in particular Clostridium difficile, Clostridium novyi, Clostridium perfringens or Clostridium septicum.

In another aspect of the present invention, a method is provided for preventing or controlling bacterial colonization or infection, for example E. coli and/or Clostridium, in particular Clostridium difficile, Clostridium novyi, Clostridium perfringens or Clostridium septicum, comprising administering an effective amount of a strain of the present invention or a composition of the present invention to an animal in need of such administration.

In another aspect of the present invention, there is provided a method of feeding an animal comprising administering a Bacillus composition of the invention to an animal, in particular a monogastric animal.

Monogastric animals include poultry such as broilers, producers, layers, turkeys, ostriches, quails, ducks and geese, herbivores such as horses and ruminants such as camels, llamas, cattle and sheep, calves, pigs piglets, weaners, youngsters, fattening piglets, hocks pigs, polts pigs, gilts, sows, farrowing sows, rodents such as rabbits, pets such as cats and dogs, and fish (including salmon, trout, tilapia, but not limited to catfish and carps, and crustaceans (including but not limited to small crustaceans and shrimp) Pigs and/or poultry are preferred monogastric animals.

In another aspect of the present invention, the use of at least one strain of Bacillus subtilis according to the invention or an animal feed, animal feed additive or premix containing at least one strain of Bacillus subtilis according to the present invention is provided to improve the productivity of an animal, in particular an animal with a monogastric .

As shown in the examples, administration of the Bacillus subtilis strain of the invention improves the gastrointestinal health of the animal, eg, prevents or controls enteritis, and provides improved animal performance in treated animals compared to controls. Animal performance indicators include, but are not limited to, weight gain (WG), feed conversion ratio (FCR), mortality reduction, and increase in European Production Efficiency Factor (EPEF).

Additionally, the invention provides a method of increasing the digestibility of animal feed, in particular the digestibility of proteins, comprising feeding the animal a strain of the present invention or a composition of the present invention.

Accordingly, the invention relates to the use of a strain of the present invention or a composition of the present invention for improving one or more animal performance indicators selected from the group consisting of:

1) increased weight gain (WG),

2) reduced feed conversion ratio (FCR),

3) reduced degree of damage from necrotic enteritis,

4) reduced frequency of necrotic enteritis,

6) reduced mortality from necrotizing enteritis,

7) increased European production efficiency factor (EPEF) and

8) reduced mortality.

In a preferred embodiment of the invention, the productivity of an animal is determined by the animal's body weight gain and/or by the feed conversion rate. By improved animal performance is meant that there is increased body weight gain and/or reduced feed conversion rate and/or improved nutrient uptake or feed energy and/or metabolizable energy and/or increased feed efficiency as a result of feed use. animal feed, animal feed additive or premix of the present invention in animal feed compared to animal feed that does not contain said animal feed, animal feed additive or premix. Preferably, improved animal performance is understood to mean that there is an increased body weight gain and/or a reduced feed conversion rate.

The expression increased weight gain refers to an animal having an increased body weight that is fed with a food containing a food composition compared to an animal that is fed with food without said food composition according to the invention. In particular, the weight gain (WG) of an animal is the increase in the weight of the animal over a period of time.

- 11 043066

In one embodiment, the improvement in body weight gain is 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 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%.

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: 1.8 to 2.0%, 2.0 to 2.2%, 2.2 to 2.4%, from 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%, from 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 .

By the terms reduced feed conversion ratio or improved feed conversion ratio is meant that the use of a feed additive composition in a feed results in less feed required to feed an animal to increase the animal's weight by a certain amount compared to the amount of feed that is required to increase the animal's weight by a certain amount. the same value when the feed does not contain said feed additive composition.

In one embodiment, improving the feed conversion ratio (FCR) results in an FCR of -2.5% or less than -2.5%, such as less than -2.6%, such as less than -2.7%, such as less than -2.8%, in particular less than -2.9%, in particular less than -3.0%. In a preferred embodiment, the improvement in FCR results in an FCR of -5 to -2%, in particular an FCR of -4 to -2%, in particular an FCR of -3.5 to -2.5%. In a certain embodiment, the improvement in FCR results in an FCR within a range selected from the group consisting of: -5 to -4.5%, -4.5 to -4%, -4 to -3.8%, from -3.8 to -3.6%, -3.6 to -3.4%, -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 term nutrient digestibility as used herein refers to the proportion of a nutrient that is absorbed from the gastrointestinal tract or a specific segment of the gastrointestinal tract, such as the small intestine. Nutrient absorption can be measured as the difference between what is administered to the subject and what is excreted in the subject's faeces, or between what is administered to the subject and what remains in the digestion site of a particular segment of the gastrointestinal tract, such as the ileum. Nutrient uptake, as used herein, can be measured as the difference between nutrient intake and nutrient excretion by total excretion collection over a period of time; or using an inert marker that is not digestible by the animal and allows the researcher to calculate the amount of the nutrient that has been absorbed throughout the entire gastrointestinal tract or in a segment of the gastrointestinal tract. Such an inert marker may be titanium dioxide, chromium oxide, or an acid-insoluble ash. Digestibility can be expressed as the percentage of a nutrient in the feed, or as the number of mass units of digestible nutrient per mass unit of nutrient in the feed. The term nutrient digestibility, as used herein, encompasses starch digestibility, fat digestibility, protein digestibility, mineral digestibility, and amino acid digestibility.

In another embodiment of the present invention, a method is provided for improving one or more performance measures of an animal selected from the group consisting of:

1) increased weight gain (WG),

2) reduced feed conversion ratio (FCR),

3) reduced degree of damage from necrotic enteritis,

4) reduced frequency of necrotic enteritis,

5) reduced mortality from necrotizing enteritis,

6) increased European production efficiency factor (EPEF) and

7) reduced mortality, including feeding the animal with the strain of the present invention or the composition of the present invention.

The composition of the present invention can also be used in a flexible food composition (FFF) when the animal is fed a feed having reduced metabolizable energy and the composition of the invention, whereby an acceptable animal production and/or feed conversion ratio is obtained despite the reduced metabolizable energy of the feed. The reduced metabolizable energy may be in the range of 97 to 99% of the metabolizable energy of the standard feed for the animal in question, in particular 97 to 98% or 98 to 99%.

Deposit and expert solution

The Bacillus licheniformis DSM17236 strain was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on April 7, 2005. Chr. Hansen

- 12 043066

A/S, Denmark. The deposit was made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus subtilis DSM19489 strain was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on June 27, 2007. Chr. Hansen A/S, Denmark. The deposit was made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Bacillus mojavensis strain DSM25839 was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on April 3, 2012. Chr. Hansen A/S, Denmark. The deposit was made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Bacillus amyloliquefaciens strain DSM25840 was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on April 3, 2012. Chr. Hansen A/S, Denmark. The deposit was made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus subtilis DSM25841 strain was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on April 3, 2012. Chr. Hansen A/S, Denmark. The deposit was made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus amyloliquefaciens strain DSM27032 was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on March 21, 2013. Chr. Hansen A/S, Denmark. The deposit was made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus subtilis DSM32324 strain was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on June 8, 2016. Chr. Hansen A/S, Denmark. The deposit was made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus subtilis DSM32325 strain was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on June 8, 2016. Chr. Hansen A/S, Denmark. The deposit was made in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

For all of the aforementioned deposited microorganisms, the following additional guidelines apply:

with respect to the respective Patent Offices of the respective designated States, the inventors request that a sample of the deposited microorganisms as defined above be made available only to an examiner designated by the requesting party until the date of grant of the patent or the date on which the application is rejected or withdrawn or considered withdrawn.

In vitro examples

Example 1

Screening for pathogen inhibition and antibiotic susceptibility.

Materials:

Veal Infusion Broth (VIB) (Difco, 234420).

Veal infusion broth agar (VIB) (VIB + 1.5% bacteriological agar (Agar no. 1), Oxoid LP0011).

Mueller-Hinton medium 2 with standardized cation content (Fluka).

T3 agar plates (per 1 L: 3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium dihydrogen phosphate and 0.005 g MnCl ₂ [pH 6.8] and 15 g agar).

Luria-Bertani medium (Laura-Bertani, LB) (g/l: Bactotryptone 10 (Difco 0123), Yeast extract 5 (Oxoid L21), NaCl 10 (Merck nr. 106404).

Brain Heart Extract (BHI) Agar (Oxoid CM375).

Bile salts (bile extract, porcine; Sigma B8631).

Bioassay dishes (Nunc 240845).

Petri dishes (Procudan 140096, Petri dish with separators).

Peptone Saline (0.9% Sodium Chloride, 1% Peptone) FKP.

ISO-SENSITEST medium (Oxoid CM0473).

- 13 043066

Microtiter plates (MTP) NUNC, Denmark.

Rectangular petri dishes (Omni tray)/single well plates N 242811 Thermo

Scientific/NUNC Denmark.

RNase-free/DNase-free 96-well deep-well (DW) microtiter plates (Thermo Fisher Science).

Ampicillin (Sigma, A9518-5G).

Vancomycin (Sigma, V1764-250MG).

Gentamicin (Sigma, G1264-50MG).

Kanamycin (Sigma, K1377-1G).

Streptomycin (Sigma, S6501-5G).

Erythromycin (Sigma E-5389).

Clindamycin (Sigma, C2569-10MG).

Tetracycline (Sigma T-7660).

Chloramphenicol (Sigma, C0378-5G).

Escherichia coli O101 H-, K99 F5 (State Serum Institute, Copenhagen, Denmark).

Escherichia coli O147:K89 F4 H19 (State Serum Institute, Copenhagen, Denmark).

Escherichia coli O149:k91,k88a,c,h10 NCTC10650, (National Collection of Type Cultures, England).

E. coli ATCC11775 (American type culture collection).

E. coli Cp6salp3 (Copenhagen Veterinary University).

Clostridium perfringens type A, DSM756, Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen.

Clostridium perfringens type C, NCTC3180, National Collection of Type Cultures (England).

Clostridium perfringens CCUG2036 (Culture Collection, University of Gothenburg, Sweden).

Clostridium perfringens CCUG2037 (Culture Collection, University of Gothenburg, Sweden).

Clostridium perfringens CCUG44727 (Culture Collection, University of Gothenburg, Sweden).

All pathogenic strains mentioned above were maintained in LB medium with 20% glycerol in BHI at -80°C.

Bacillus cultures:

Bacillus strains isolated from faeces, soil, food sources and obtained from strain bank collections were maintained in VIB with 20% glycerol in MTP master plates at -80°C.

Identification of isolates of spore-forming aerobic bacteria by the sequence of 16S and gyrB ribosomal RNA was carried out (Wang et al., 2007), screening for antibiotic sensitivity 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 tolerance and low pH sensitivity, enzymatic activity, media growth, heat tolerance and sporulation as described in WO 2013/153159.

Antibiotic susceptibility by measuring MIC.

Bacillus strains were analyzed for antibiotic susceptibility by measuring the minimum inhibitory concentration (MIC) for several antibiotics. The method used was the broth microdilution method as set forth in CLSI (Clinical and Laboratory Standards Institute M07-A8 and M45-A2).

An overnight culture suspension of the strain to be tested was inoculated into ISO-SENSITEST medium (Oxoid CM0473) in microtiter plates at a concentration of approximately 105 cfu/ml (colony forming units/ml) in two-fold serial dilutions of the antibiotic to be tested (total volume 100 μl/well) and incubated under aerobic conditions for 20-24 hours at 37°C. The results were recorded after 20 hours of incubation as the lowest antibiotic concentration that inhibited visible growth. The test was performed twice in two independent biological replicates.

Only strains of Bacillus that were susceptible to inhibitors according to the EFSA Guidance were included in the screening for inhibition of pathogenic E. coli and Clostridium perfringens.

Screening of Bacillus strains for inhibition of pathogenic E. coli.

Bacillus strains were added in a volume of 50 μl from MTP master plates to 700 μl VIB in DW (deep well) plates and incubated at 37° C. and 175 rpm overnight. E. coli strains were grown in LB at 30° C. overnight. 2 ml of E. coli overnight culture was mixed with 200 ml of liquid VIB agar at 50° C. and poured into each biological sample dish. The cups were dried in a sterile laminar flow cabinet. Overnight cultures of Bacillus, 2 μl each, were dropped onto the surface of VIB agar mixed with E. coli in biological sample dishes and incubated at 37° C. for 1 day.

The radii of the lightened zones of inhibition around Bacillus were measured and recorded as 3 means strong, ie. more than 2 mm, 2 means medium, i.e. from 0.5 to 2 mm, 1 means weak, i.e. less than 0.5 mm, and 0 means no inhibition.

Inhibition of Clostridium perfringens according to the agar drop test.

VIB agar was poured into biological sample dishes (200 ml per dish) and completely dried in

- 14 043066 sterile laminar flow cabinet. Overnight cultures of Bacillus, 2 μl each, were dropped onto the surface of VTB agar plates and incubated at 37° C. overnight. Strains of Clostridium perfringens were grown under anaerobic conditions on BHI agar at 37°C overnight. An overnight culture of Clostridium perfringens was added in a volume of 2 ml to 200 ml of BHI liquid agar, mixed and carefully layered onto biological sample dishes with Bacillus drops. Cups were incubated under anaerobic conditions at 37°C for 1 day.

The radii of the lightened zones of inhibition around Bacillus were measured and recorded as 3 means strong, ie. more than 2 mm, 2 means medium, i.e. from 0.5 to 2 mm, 1 means weak, i.e. less than 0.5 mm, and 0 means no inhibition.

All data were collected again on different days.

Results.

Table 1

E. coli inhibition results by selected Bacillus strains

inhibition E. coli inhibition DSM number if available E. coli O101 F5 E. coli O147:K89 F4 E. coli O149:k91,k88a E. coli ATCC 11775 E. coli Cp6salp3 Bacillus licheniformis 0 0 0 0 0 Bacillus licheniformis 17236 0 0 0 0 0 Bacillus subtilis 19489 0 0 0 0 0 Bacillus subtilis 0 0 1 1 1 Bacillus subtilis 32324 3 3 3 3 3 Bacillus subtilis 0 0 0 0 0 Bacillus subtilis 0 0 0 0 0 Bacillus subtilis 32325 2 2 3 2 2 Bacillus subtilis 25841 2 2 2 2 2 Bacillus amyloliquefaciens 25840 1 1 2 1 1 Bacillus amyloliquefaciens 27032 2 3 3 2 2

- 15 043066

table 2

Clostridium Inhibition Results by Selected Bacillus Strains

inhibition inhibition DSM number if available DSM756 NCTC3180 CCUG2036 CCUG2037 CCUG44727 Bacillus licheniformis 1 2 1 0 1 Bacillus licheniformis 17236 1 1 0 0 0 Bacillus subtilis 19489 0 0 0 1 0 Bacillus subtilis 0 2 0 0 1 Bacillus subtilis 32324 3 3 3 3 3 Bacillus subtilis 1 0 1 2 1 Bacillus subtilis 1 0 0 1 0 Bacillus subtilis 32325 3 3 3 2 3 Bacillus subtilis 25841 2 3 2 2 3 Bacillus amyloliquefaciens 25840 2 3 3 2 3 Bacillus amyloliquefaciens 27032 3 3 3 2 3

The results from the table. 1 and 2 for E. coli and Clostridium inhibition show that the Bacillus licheniformis strains tested and many of the Bacillus subtilis strains tested did not show any E. coli inhibition and showed weak Clostridium inhibition. However, two B. subtilis strains, DSM32324 and DSM32325, showed impressive results in both E. coli and Clostridium inhibition.

Example 2

Measurement of the amount of reducing sugars in feed incubated with a bacillus composition.

The purpose of the present experiment was to investigate the ability of different strains of Bacillus to degrade NSP in commercially available poultry starter feed and to increase the amount of available sugars.

Table 3

The composition of the complex feed used in the study

The complex feed based on wheat-corn-soybean (Table 3) was autoclaved at 121°C for 15 min for sterilization. The food sample was then diluted 20-fold with sodium phosphate buffer to maintain a pH of approximately 6-6.5 throughout the experiment. Bacillus products were obtained by inoculation of a 2% overnight culture of Bacillus strains grown in veal infusion broth (VIB) (Difco, 234420). A sample was taken for analysis for reducing sugar (DNS) (T=0). After incubation at 37°C for 24 h, the sample was taken for the determination of CFU. Another sample was centrifuged and the supernatant used for DNS determination.

The reducing sugar was analyzed with 3,5-dinitrosalicylic acid (DNS) as follows.

Na-acetate buffer (100 mM, pH 6) was mixed with sterile filtered Bacillus sample supernatant and incubated at 40°C for 10 min. The DNS reagent was added to the test tube, mixed, and incubated in a boiling water bath for 5 min. After cooling, the absorbance was measured at 540 nm using a spectrophotometer.

A standard curve was generated using concentrated glucose solution to report results in terms of reducing sugar or in units of enzyme activity (the amount of enzyme required to release 1 μmol of reducing glucose equivalent in 1 ml per unit of time).

The results are presented in table. 4.

Results.

Table 4

Sample kJ/kg feed DSM number if available Control 214 Bacillus aryabhattai 237 Bacillus licheniformis 17236 340 Bacillus licheniformis 15326 541 Bacillus subtilis 19489 403 Bacillus subtilis 25841 458 Bacillus subtilis 32325 642 Bacillus subtilis 32324 723 Bacillus amyloliquefaciens 16734 515 Bacillus amyloliquefaciens 27032 515 Bacillus amyloliquefaciens 14623 517 Bacillus amyloliquefaciens 15509 853 Bacillus amyloliquefaciens 25840 1142 Bacillus mojavensis 25839 939

In table. 4 shows the results for several different strains.

Bacillus and all strains of Bacillus tested were shown to provide more nutrition to the animal by providing more reducing sugars than the control, but there is also significant variation between individual strains.

Based on E. coli and Clostridium perfringens inhibition results, combined with results on the ability to provide increased amounts of reducing sugars, the two most productive strains, ie. B. subtilis strains DSM32324 and DSM32325 were selected for in vivo studies.

Examples in vivo

Example 3

Efficacy of Bacillus subtilis DSM32324 strain against Clostridium perfringens infection in in vivo studies.

- 17 043066

Table 5

diet

Ingredient name % (w/w) starter grower finisher Corn, grain 58.509 64.054 69.218 Soy flour, husked, solvent 35,550 29.771 24.511 vegetable fat 2,100 2.585 2.748 Calcium hydrogen phosphate 1.734 1,780 1.693 Calcium carbonate 1.150 0.910 0.873 Salt, (NaCl) 0.386 0.390 0.393

Methionine MHA, L-lysine, trace elements, vitamin premix and L-threonine were included as recommended by the breeder.

A diet based on unmedicated corn/soy flour was used (Table 5). 1.2x10 ⁶ cfu/g of Bacillus subtilis DSM32324 was added to the feed of one of the groups. Food and water were freely available throughout the study. All the food was on the paddock. Starter food was fed from day 0 to day 21. On day 21, unconsumed starter food was weighed and discarded. Grower food was fed up to day 35 and unconsumed grower food was weighed and discarded. Finishers were also fed until day 42, at which time unconsumed finishers were weighed and discarded.

Table 6

Treatment Clostridium inoculation perfringens СР-6 Pens/Trt birds/Trt No processing Not infected 8 320 medicine No processing Days 19, 20 and 21 8 320 medicine DSM32324 Days 19, 20 and 21 8 320

The experiment started with 40 male Ross 308 broiler chickens per pen. Treatments were carried out in repetitions in eight blocks, randomized within blocks of six pens each.

On days 19, 20, and 21, birds in all pens, except for the undrug-treated uninfected treatment group 1, were challenged with C. perfringens CP-6 broth culture (Knap I., et al., 2010). This strain is a field isolate of C. perfringens known to cause NE, obtained from commercial broiler operations and used as the infection organism in the present study. Fresh inoculum was used every day. Each pen received the same amount of inoculum, corresponding to approximately 1 x 10 ⁸ to 1 x 10 ⁹ CFU of C. perfringens CP-6. The inoculum was introduced by mixing it with the feed at the base of the tubular feeders.

On day 21, three birds from each pen were selected, sacrificed, the group weighed, and examined for the extent of necrotizing enteritis lesions present. Rated on a scale from 0 to 3, where 0 means normal and 3 means the most severe damage. Scored as follows: 0 for normal bowel, 1 for slight mucus and loss of tone, 2 for severe necrotizing enteritis, and 3 for extremely severe necrotizing enteritis with blood in the lumen.

All birds were weighed on days 31, 35, and 42 to evaluate the contribution of necrotizing enteritis to performance indicators: average live weight, average weight gain (AWG), feed intake, feed conversion ratio (FCR), necrotizing enteritis injury score, and mortality ( total and from NE) were calculated for all pens.

Statistical data analysis was performed using SAS Stat Version 9.2 using ANOVA analysis with a fully randomized design to establish differences between treatment groups. The pen was treated as a statistical experimental unit with diet as a fixed effect. Results were recorded as rms means and were considered different at P<0.05.

- 18 043066

Results.

Table 7

Day 21

AWG

Treatment FCR (kg) No drug treatment, non-infected 1.447v 0.562a Without drug treatment, infected 1.652a 0.5016 DSM32324 infected 1.5756 0.567a Day 3 5 AWG Treatment FCR (kg) No drug treatment, non-infected 1.5696 1,735ab Without drug treatment, infected 1.634a 1.6796 DSM32324 infected 1.5936 1.756a Day 42 AWG Treatment FCR (kg) No drug treatment, non-infected 1.6276 2.272a Without drug treatment, infected 1.731a 2.1556 DSM32324 infected 1.6456 2.303a

Letters appended to results represent treatment groups that are statistically significantly different from untreated controls (P < 0.05) or from each other.

Table 8

Score Mortality from

Damage treatment from NE NE %

No drug treatment means, uninfected 0.056 0.06 Without drug treatment infected 0.58a 4.3a DSM32324 infected 0.186 0.76

Letters appended to results represent treatment groups statistically significantly different from untreated controls (P<0.05) or from each other.

Conclusion.

For performance measures such as feed conversion ratio (FCR) and average weight gain (AWG) measured on day 21, day 35 and day 42, a significant improvement was observed for all control points when considering Bacillus subtilis DSM3234 as a feed additive compared to untreated infected control group. Surprisingly, the Bacillus treated group did not show any significant difference from the uninfected, untreated control group, even though the latter group was not infected and was considered healthy (Table 7).

With respect to subclinical enteritis induced in an in vivo challenge study, the results show that Bacillus subtilis DSM3234 reduced the necrotizing enteritis lesion score in chickens and significantly reduced necrotizing enteritis mortality (Table 8).

Example 4

Efficacy of Bacillus subtilis DSM32325 strain against Clostridium perfringens infection in in vivo studies.

A study was conducted to evaluate the effect of Bacillus subtilis strain DSM32325 on the incidence of necrotizing enteritis and the scoring of necrotizing enteritis lesions in chickens.

Ross 308 male broilers were divided into three groups of 48 birds per group:

infected control without treatment;

a group of infected treated with an antibiotic 20 mg amoxicillin/kg body weight; and a group of infected treated with Bacillus subtilis DSM32325 1.2x10 ⁶ cfu/g feed.

All animals were fed ad libitum during all phases and all animals were vaccinated by oprah.

- 19043066 on arrival at the study site (day 1) for infectious bronchitis and Newcastle disease. Up to day 9 birds were fed Kip 1-3 starter feed purchased from an industrial feed mill (Cibus, Kaaistraat 49; 8800 Roeselare, Belgium). The quantitative composition of the starter feed was the same for all animals, except for the inclusion of the appropriate product in each group. From day 9 to day 26 the birds received a high protein grower diet and fishmeal included at 40%. Teler2 grower food was purchased from an industrial feed mill (Cibus, Kaaistraat 49; 8800 Roeselare, Belgium). The composition of the grower food for the different treatment groups was exactly the same except for the inclusion of the appropriate product in each group. The foods were given a group code and then the treatment premix was mixed with the amount of food intended for each batch of food.

On days 19, 20, 21 and 22, approximately 109 cfu of Clostridium perfringens strain 56 (Timbermont et al. 2009) was orally administered three times daily to all birds as described in Timbermont et al. 2009. Lesions from necrotizing enteritis were assessed on days 25 and 26 (scores 0 to 6) (Johnson & Reid 1970). NE frequency and NE scores were analyzed using a logistic model and a linear regression model, respectively. Statistical significance was assessed at P<0.05.

Results.

The proportion of birds positive for macroscopic lesions from necrotizing enteritis (lesion grade >2) on each sampling day is shown in Table 1. 9. A linear regression model was built for each day to analyze differences between treatment groups and IUC (infected untreated control). An additional model was built on a combination of data on day 25 and day 26. In this model, days 25 and 26 and the group were added as a fixed effect.

Table 9

Day 25 Day 26 Days 25 and 26 Group %NE %NE %NE P < 0.05 Infected Raw control 50 23 36.5 Ref. Amoxicillin 44 14 29 Bacillus subtilis DSM32325 25 7 16 **

**Noted numbers represent treatment groups statistically significantly different from untreated control IUC (P<0.05).

Ref. - reference value.

Mean NE scores by group and day were calculated in a similar way.

Table 10

Day 25 Day 26 Days 25 and 26 Group Average score Average score Average score P < 0.05 Infected raw 1.75 1.15 1,450 Ref. control Amoxicillin 1.56 1.07 1.315 Bacillus subtilis DSM32325 1.25 1.00 1.125 ** **Numbers marked represent treatment groups, statistically significant

different from untreated control IUC (P<0.05).

The results show that Bacillus subtilis DSM32325 reduced the incidence of necrotizing enteritis in chickens compared to infected untreated controls in a statistically significant manner when data from days 25 and 26 were combined. Surprisingly, it was found that the incidence of necrotizing enteritis was even less in the Bacillus treated group than in the amoxicillin-treated group (Table 9).

Further, Bacillus subtilis DSM32325 reduced the severity of necrotizing enteritis (mean score) compared to the infected untreated control in a statistically significant manner when data from days 25 and 26 were combined. Surprisingly, it was found that the mean necrotizing enteritis score was even lower in the Bacillus treated group. than in the group treated with amoxicillin (Table 10).

Example 5

Feeding efficiency experiment.

Animals:

Ross 308 day old male broilers were randomly assigned to 72-level pens, each containing 25 chicks per pen, such that each treatment was repeated 12 times.

- 20 043066

The experimental groups were the negative control (NC), Bacillus subtilis

DSM19489, Bacillus subtilis DSM32324 and Bacillus subtilis DSM32325.

Diet:

A three-phase mash (Days 1-14, Days 15-28 and Days 29-42) free of any antibiotic compounds, inhibitors, performance enhancers, other probiotics, enzymes or acidifiers was provided ad libitum. The basis of the diet was corn, wheat, barley, rye and soy flour.

Table 11

The composition of the composition of the main diet

INGREDIENTS, % Starter day 0-14 Grower day 15-28 Finisher day 29-42 Corn 19.647 19.574 20.207 Soy flour 44% CP 30.147 24.190 20.210 Wheat 15,000 15,000 15,000 Full fat soy 12,000 15,000 15,000 Barley 10,000 10,000 10,000 Rye 5,000 7.5000 10,000 Soybean oil 4.141 4,833 - Animal fat (lard) - - 5.905 Calcium carbonate 1.139 1.067 1.045 calcium dihydrogen phosphate 1.546 0.462 1.286 Salt 0.327 0.302 0.303 Bicarbonate of soda 0.100 0.100 0.100 DL-Methionine 0.295 0.326 0.289 L-Lysine HO 0.186 0.168 0.176 L-Threonine 0.071 0.077 0.078 Vit&Min Premix 0.400 0.400 0.400

Products tested:

The test products Bacillus subtilis DSM19489, Bacillus subtilis DSM32324 and Bacillus subtilis DSM32325 were given with food during the 42 days of the experiment at a dosage of 1.2x10 ⁶ CFU/g of food.

Observations:

Average daily gain (ADG), body weight (BW), food intake as average daily food intake (ADF1) and feeding efficiency were measured; feed conversion ratio (FCR) at 1, 14, 28, 35 and 42 days of age. General health, medical treatment and mortality were assessed daily. The European Production Efficiency Factor (EPEF) was calculated:

[(viability, % x increase in BW, ^/(Duration of study in days x FCR)] x 100.

Statistical analysis and interpretation:

Analysis of variance was the main statistical method used. Data were analyzed as a fully randomized design by GLM SPSS v. 19.0 followed by Tukey's mean test. P<0.05 was considered as a statistically significant difference, while 0.05<P<0.10 was considered as a trend close to significant.

Results.

The health of the animals was considered normal throughout the study and no adverse events were observed. There were 23 deaths/culls (1.27%) between days 0 and 14, 19 deaths/culls (1.07%) between 14 and 28 days, and 16 deaths/culls (0.91%) between 28 to 42 days, and they were not related to processing. An overall mortality/cull rate of 58/1800 birds (3.22%) on day 42 was considered normal.

-21 043066

Table 12

Treatment Whole fattening phase, day 0-42 ADG, g/day ADFI, g/day FCR EPEF 1 Negative control 68.6 ^b 116.2 1.69 ^b 386 ^b 2 DSM19489 (B. subtilis) 70.4 ^ab 116.1 1.65 ^a 420 ^a 3 DSM32324 (B. subtilis) 71.9 ^a 116.6 1.62 ^a 424 ^a 5 DSM32325 (B. subtilis) 70.9 ^a 115.9 1.64 ^a 416 ^a SEM (n=12) 0.53 0.79 0.006 5.3 P (Probability) 0.0002 0.7429 < 0.0001 < 0.0001

The productivity of the animals was in accordance with the conditions of the experiment (male broilers were fed with a mash and grown in pens for floor keeping). At 28 days of age, DMS32324 treated broilers were 3.77% heavier than control birds (P<0.05). At 35 and 42 days of age, broilers supplemented with Bacillus strains DSM32324 or DSM32325 were significantly heavier than controls, with the DSM19489 group showing intermediate weights. During the start period (age 0 to 14 days), no significant differences were observed between treatment groups in terms of growth, feed intake or feed yield. During the grower period (age 15 to 28 days), chickens treated with DSM32324 grew significantly faster than control broilers. Feeding performance of all broilers supplemented with probiotics was significantly improved compared to control animals. No significant differences were observed between treatment groups in terms of growth, food intake or feed yield during the last week of the experiment (age 35 to 42 days).

For the overall fattening period (age 0-42 days), broilers supplemented with Bacillus strains DSM32324 or DSM32325 grew significantly faster than controls. The feed conversion ratio (FCR) and EPEF of all broilers supplemented with probiotics were significantly improved compared to control animals.

Example 6

Feeding efficiency experiment.

The objective of this study was to evaluate the supplement Bacillus subtilis DSM32324 and the commercial product Bacillus subtilis DSM19489 in broiler diets. The aim was to evaluate the effect of these products on performance in a wheat-based diet containing coccidiostats and feed enzymes.

For each treatment, 1300 day old chicks (males) ROSS 308 were taken, divided into 10 pens of 130 chicks. The chicks were fed ad libitum three-phase feed in granular form throughout all phases and drinking water was provided ad libitum in drip drinkers. The composition of the diet is shown in the table below. The feed was provided by Mezinarodni testovani drubeze, s.p., feed mill Lysa nad Labem.

Probiotic nutritional supplements were distributed as follows:

T1: Control group, no additives;

T2: Bacillus subtilis DSM19489 1.2x10 ⁶ cfu/g feed;

T3: Bacillus subtilis DSM32324 1.2x10 ⁶ cfu/g feed.

Table 13

The composition of the diet

Components Starter Diet grower diet Finisher ration Days 1-13 Day 14-28 Days 29-42 Wheat 40,000 51,880 56.450 Corn 19.460 10,000 10,000 Extra, soy flour 32,800 29,500 24,600 Soybean oil 4,000 5,000 5,800 L-lysine HO 0.170 0.220 0.200 DL-Methionine 0.060 0.100 0.120

- 22 043066

L-threonine 0.060 0.080 0.060 Limestone 1,500 1,500 1,400 Salt 0.250 0.250 0.240 dihydrogen phosphate calcium 1,000 0.770 0.500 Bicarbonate of soda 0.200 0.200 0.130 AMV BRI Plus 0.500 - - AMV BR2 Plus - 0.500 - AMV BR3 Plus - - 0.500

Body weight was measured on days 1 (all birds from each pen were weighed together), 13 and 28 (each bird was weighed individually, no fasting period) and 42 (each bird was weighed individually after 12 hours of fasting).

Food intake per 1 kg of live weight was recorded per pen on days 13, 28 and 42 and the feed conversion coefficient was calculated.

Statistical analysis:

Live weight performance at days 13, 28, and 42 and mortality were statistically assessed using a one-way ANOVA of the main treatment effect by Dunnett's test (all supplemented versus unsupplemented control (T1) diets).

Results.

The coefficient of variation (CV) for feed yield and feed yield ratio averaged 2.7%. The CV for final body weight and body weight gain was 3.7%, and the mean within-pen treatment variation in final BW (as a measure of herd uniformity) ranged from 10.3 to 15.3%.

Flock mortality was low (3.4% overall for non-supplemented birds) and unexpectedly low for the first 13 days.

Table 14

Treatment Mortality for the period Days 1-13 Day 14-28 Days 29-42 Days 1-42 Number birds G Number birds g Number of birds g Number birds g % Without additives 2 514 18 14421 24 45212 44 60147 3.38 DSM19489 4 1459 10 7074 14 25049 28 33582 2.15 DSM32324 3 1048 14 11295 7 11473 24 23816 1.85

Conclusion.

DSM19489 tended to reduce mortality (p=0.096), and Bacillus subtilis DSM32324 caused a marked and significant reduction in mortality, especially in the final period, which also served as a criterion for the statistical significance of mortality for the entire experiment.

Example 7

Effect of different strains of bacillus on growth, digestibility and gut health in broilers.

The birds were housed in 15 animals per pen measuring 1.2 m by 0.8 m from day 1 to the end of the experiment (day 42). The floor in each pen was covered with a layer of wood shavings about 5 cm thick. One commercial feeder tray with a feed tank was suspended inside the pen, and four drip drinkers were installed on the side of the pen.

Birds were fed a suboptimal diet including rye as a source of non-starch proteins and no feed enzymes. Up to a day, 22 birds were fed starter food. From day 22 to day 42, birds were fed grower food. The quantitative composition of the feed was the same for all animals, except for the inclusion of the appropriate strain in each group. A detailed description of the composition of the feed is given in table. 15. Test strains DSM32324, DSM25840 and DSM32325 were mixed with feed at a ratio of 1.2x10 ⁶ cfu/g feed.

-23 043066

Table 15

The composition of the composition of the main diet

INGREDIENTS, % Starter, day 0-21 Finisher, day 22-42 Corn 41.6 31.6 Soy flour 47.3% CP 26.0 24.0 Rapeseed flour 32.5 CP 4.0 5.0 Fishmeal 70.0CP 2.0 - Wheat 15.0 20.0 Rye 5.0 10.0 Soybean oil 1.0 1.0 Animal fat (lard) 1.5 5.0 Calcium carbonate 1.359 1, Yu calcium dihydrogen phosphate 1.2 0.85 Salt 0.18 0.21 Bicarbonate of soda 0.27 0.23 DL-Methionine 0.195 0.195 L-Lysine HC1 0.15 0.21 L-Threonine 0.045 0.065 L-valine 0.015 0.035 Vit&Min Premix 0.500 0.500

By the start of the experiment (Day 1), 960 animals were housed in 64 pens (i.e., 15 animals per pen). As mentioned above, starter food was given to all birds from day 1 to day 22. From day 22 to day 42, grower food was given to all birds.

On day 12, day 22 and day 42, tissue samples were taken from the duodenum, small intestine and ileum from 1 bird per pen.

On Day 1, Day 12, Day 22, Day 33, and Day 42, birds and feed were weighed to analyze the effects of probiotics on growth performance (weight gain, food intake, and feed yield) during various periods.

On day 22 and day 42, 6 birds per pen were euthanized. The contents of the ileum and caecum from the same pen were pooled. Additional samples of the contents of the ileum and caecum were frozen and stored at -80°C.

On days 19-22 and days 40-42, 3 faecal samples per pen were collected daily. Fecal samples from the same pen were pooled.

Health indicators were recorded daily. From day 1 until the end of the study on day 42, general health observations were made by experienced staff and recorded daily. If the animals showed signs of illness, general health observations were made at least twice a day. Mortality was recorded daily.

Body weight (BW) was measured per pen on day 1. BW weight of animals was measured individually on day 12, day 22, day 33 and day 42. Daily weight gain (DWG) was calculated per pen for the periods day 1 - day 12, day 1 - day 22 and day 1 - day 42. Daily weight gain (DWG) was calculated per animal for the periods day 12 - day 22, day 22 - day 33, day 33 - day 42 and day 22 - day 42.

The difference in BW between the start and end of each study period was the weight gain (WG) for that period. Daily weight gain (DWG) was calculated as WG divided by the number of days in the corresponding period. The DWG of dead animals was included when the average DWG for each group was calculated, considering the date of death of the bird as the end of the study period for that bird.

Daily feed intake (FC) and feed conversion ratio (FCR) were calculated at pen level for periods day 1 - day 12, day 12 - day 22, day 1 - day 22, day 22 - day 33, day 33 - day 42, day 22 - day 42 and day 1 - day 42.

Feed provided to animals (Feed IN) was weighed on day 1, day 12, day 22, and day 33. When on any following day the pen ran out of feed and more feed needed to be provided, the added feed was also weighed and recorded as Feed IN . The remaining feed in each pen (Feed OUT) was weighed on day 12, day 22, day 33 and day 42. The difference in feed weight at the start and end of each study period was calculated to determine feed intake (FC) per pen. The difference between the feed weight at the beginning and at the end of each study period (Feed IN - Feed OUT) was the FC of the respective pen for that period.

The average daily FC per bird was calculated as the FC divided by the number of days in the respective period multiplied by the number of animals that consumed food during that period in the respective pen.

The content of dry matter, total protein, total fat, Ca, P, energy and titanium dioxide was determined in the combined contents of the ileum and caecum collected on day 22 and day 42, and in the feed. Titanium oxide was added to the feed (0.3%) as an inert marker. Observed ileal digestibility was calculated as described in Waititu et al., 2014.

Samples were analyzed at the Department of Animal Sciences, Subdivision Animal Nutrition of Wageningen University under the supervision of Leon de Jonge. The following methods were used:

Dry matter: drying for 3 hours at 103°C based on ISO 6496 (1999).

Ash: heating for 3 hours at 550°C based on ISO 5984 (2002).

Protein: Kjeldahl method based on ISO 5983 (2005).

Fat: Extraction with petroleum ether after acid treatment based on ISO 6492 (1999).

Calcium: Absorption spectroscopy based on ISO 6869 (2000).

Phosphorus: Spectrometric determination based on ISO 6869 (2000).

Energy: bomb calorimeter method based on ISO 9831 (1998).

Titanium: Spectrometric determination based on Kjeldahl resolution followed by peroxide staining and absorption measurement at 408 nm.

The content of dry matter, total protein, total fat, Ca, energy and titanium dioxide was determined in the combined faeces collected on day 19 - day 22 and day 40 - day 42, and feed. Titanium oxide was added to the feed (0.3%) as an inert marker. The total apparent tract retention was calculated as described in Waititu et al., 2014.

Data were analyzed with RStudio (Version 0.99.467, RStudio, Inc.). All data except body weight and daily weight gain at the bird level were analyzed using linear regression models with the treatment group as a fixed effect (main package lm procedure). Body weight and daily weight gain at bird level were analyzed using linear mixed regression models with treatment group as fixed effect and pen as random effect to correct for bird clustering within the pen (nlme package lme procedure) (Protocol Deviation n° 1). Statistical significance was assessed at P<0.05.

Results.

Eight birds died during the study (0.8%).

In table. 16 shows the mean body weight by study day and treatment group. Differences were analyzed in linear mixed regression models with treatment as an unconditional fixed effect and a pen as a random effect to correct for clustering of birds within the pen.

Table 16

Average body weight

Group name Day 12 Day 22 Day 33 Day 42 Average Meaning Average Meaning Average Meaning Average Meaning NC 266 Ref. 687 Ref. 1569 Ref. 2390 Ref. DSM32324 272 0.220 730 0.010 1613 0.208 2459 0.176 DSM25840 286 <0.001 759 <0.001 1672 0.003 2525 0.009 DSM32325 283 0.001 744 0.001 1632 0.070 2476 0.087

In table. 17 shows the mean apparent ileal digestibility (AID) by nutrient and treatment group. Differences were analyzed by linear regression models with processing as an unconditional fixed effect (the lm procedure of the main package).

Table 17

Mean apparent ileal digestibility (AID)

Name groups Dry matter Ash Protein Fat Sa Phosphorus Energy Average Meaning Average Meaning Average Meaning Average Meaning Average Meaning Average Meaning Average Meaning NC 57.6 Ref. 38.2 Ref. 74.1 Ref. 61.6 Ref. 36.6 Ref. 53.8 Ref. 65.0 Ref. DSM32324 58.8 0.233 38.7 0.697 76.3 0.050 59.5 0.515 36.3 0.900 53.6 0.879 66.5 0.160 DSM25840 59.5 0.075 40.8 0.039 76.3 0.050 64.0 0.458 33.4 0.190 54.0 0.842 67.2 0.043 DSM32325 57.4 0.862 37.5 0.568 74.6 0.639 63.1 0.640 31.3 0.031 51.0 0.039 65.4 0.677

- 25 043066

Conclusion.

The aim of this study was to evaluate the effect of different strains

Bacillus in relation to growth and digestibility in broilers.

The strains tested showed significant effects on productivity and apparent digestibility in the ileum:

birds supplemented with DSM32324 showed higher daily weight gain (Table 16), daily food intake and feed conversion ratio at the start (data not shown) and higher protein digestibility on day 42 (Table 17) compared with birds not receiving the supplement;

birds supplemented with DSM25840 had higher daily weight gain (Table 16) and daily food intake at start (data not shown), higher body weight on day 42 (Table 16) and greater digestibility of ash, protein and energy on day 42 (Table 17) compared to non-supplemented birds;

birds supplemented with DSM32325 showed higher daily weight gain (Table 16) and daily food intake during the start period (data not shown) and lower Ca and phosphorus absorption on day 42 (Table 17) compared to birds not treated with additive.

In conclusion, all three strains showed unexpectedly good and significantly improved results.

Example 8

Comparative efficacy of four different dietary probiotics for the control of Clostridium perfringens necrotic enteritis in broiler chickens.

The aim of this study was to evaluate the effect of DSM32324, DSM25840, and DSM32325, as well as an 8:3:5 composition of all of them, EPB5, on the performance of Cobb 500 broilers infected with NE, and compare the effect of each strain with the effect of the composition .

On the day of hatching, Cobb 500 male chicks were obtained from the Cobb Vantress Hatchery, Cleveland, GA. 2250 chickens were brought to the study. All birds were vaccinated by spraying with coccidia vaccine at the dosage recommended in the instructions for the day of hatching.

Standard floor pen procedures were used throughout the experiment. The pens were checked daily for mortality. Bird weights (kg) per pen were recorded at baseline and on day of treatment (DOT) 21, 35 and 42.

Broiler diets were fed in the form of crumbs (starter feed) or pellets (grower and finisher). The quantitative composition of the feed was the same for all animals, except for the inclusion of Bacillus strains or composition for treatment groups.

The composition of the diet:

Table 18

Ingredient name % (w/w) starter grower finisher Corn, grain 58.509 64.054 69.218 Soy flour, husked, solvent 35,550 29.771 24.511 vegetable fat 2,100 2.585 2.748 Calcium hydrogen phosphate 1.734 1,780 1.693 Calcium carbonate 1.150 0.910 0.873 Salt, (NaCl) 0.386 0.390 0.393

Methionine MHA, L-lysine, trace elements, vitamin premix and L-threonine were included in accordance with the breeder's recommendations.

All feed was counted on the corral. Starter food was provided and fed from treatment day 0 to 21. On treatment day 21, unconsumed starter was weighed and discarded. Grower food was provided and fed until treatment day 35. On treatment day 35, unconsumed grower was weighed and discarded. Finisher food was provided and fed until treatment day 42. On treatment day 42, unconsumed finisher was weighed and discarded.

The experiment included 45 pens with 50 male broiler chicks per pen at the start of the study. Treatments were repeated in nine blocks, randomized within blocks of five pens each.

- 26 043066

Table 19

Treatment Description Target feed (cfu/g feed) Clostridium perfringens Pens/Trt T1 Control without probiotic 0 Day of processing 19, 20 and 21 9 T2 Bacillus subtilis DSM32324 8X 10 ⁵ Day of processing 19, 20 and 21 9 TK Bacillus subtilis DSM32325 5 HU ⁵ Day of processing 19, 20 and 21 9 T4 Bacillus amyloliquefaciens DSM25840 ЗХ10 ⁵ Day of processing 19, 20 and 21 9 T5 EBP5 ^1.6X106 Day of processing 19, 20 and 21 9

On days 19, 20, and 21, the birds in all pens were infected with a broth culture of C. perfringens. A field isolate of C. perfringens known to cause NE was used as the challenge organism. Fresh inoculum was used every day. Titration levels were approximately 10 ^8-9 cfu/pen. Each pen received the same amount of inoculum. The inoculum was introduced by mixing with the feed at the base of the tubular feeders.

On day 21, five birds from each pen were selected, sacrificed, the group weighed and examined for the presence of lesions caused by necrotizing enteritis. The assessment was carried out on a scale from 0 to 3 points, where 0 means the norm and 3 means the most severe damage. Scored as follows: 0 for normal bowel, 1 for slight mucus and loss of tone, 2 for severe necrotizing enteritis, and 3 for extremely severe necrotizing enteritis with blood in the lumen.

No concomitant drug therapy was used during this study. The corral was used as a statistical unit. Mean values were calculated for body weight, weight gain, feed intake, feed conversion ratio (FCR), NE injury score, and mortality (total and NE). Raw data statistical analysis (ANOVA) was performed using a randomized block design. Taki's HSD test (p<0.05) was used to separate means when F ANOVA values are significant (p<0.05).

The various indices in the rows show significance levels at P<0.05.

Results.

Table 20

Day 21 Treatment Feed intake FCR AWG (kg) 1. No drug treatment means 44.70a 1.903a 0.4306 2. Bacillus subtilis DSM32324 43.48a 1.694b 0.476a 3. Bacillus subtilis DSM32325 44.92a 1.649b 0.509a 4. Bacillus amyloliquefaciens DSM25840 42.32a 1.7866 0.4366 5.EBP5 43.14a 1.646b 0.488a

Table 21

Day 35 Treatment Feed intake FCR AWG (kg) 1. No drug treatment means 143.17a 1.909a 1.6066 2. Bacillus subtilis DSM32324 141.29a 1.8026 1.667a 3. Bacillus subtilis DSM32325 145.73a 1.8166 1.695a 4. Bacillus amyloliquefaciens DSM25840 140.13a 1.8016 1,641ab 5.EBP5 142.33a 1.7746 1.689a

- 27 043066

Table 22

Day 42 Treatment Feed intake FCR AWG (kg) Mortality rate 1. No drug treatment means 199.77a 1.957a 2.2376 4.2a 2. Bacillus subtilis DSM32324 197.67a 1.8736b 2.291ab 3.1a 3. Bacillus subtilis DSM32325 203.61a 1.8966 2.322a 2.7a 4. Bacillus amyloliquefaciens 3.3a DSM25840 196.86a 1.8986 2.2446 5.EBP5 199.87a 1.841b 2.342a 2.7a

Table 23

Damage Mortality

Machining from NE from NE %

1. No drug treatment means 1.0a 4.2a 2. Bacillus subtilis DSM32234 0.5b 0.46 3. Bacillus subtilis DSM32235 0.5b 0.96 4. Bacillus amyloliquefaciens DSM25840 0.76 1.66 5.EBP5 0.5b 0.46 Discussion and conclusion

For performance measures such as feed conversion ratio (FCR) and average weight gain (AWG) measured on day 21, day 35 and day 42, a significant improvement was observed for all control points when considering single-strain probiotic feed supplements and EBP5, in particular compared to an untreated infected control group.

Surprisingly, the EBP5 treated groups showed a significant improvement in some performance measures compared to the Bacillus single strain treatment groups, which on the other hand showed significant differences in performance measures with the undrug-treated infected control group.

With respect to subclinical enteritis induced in an in vivo challenge experiment, the results show that both Bacillus subtilis DSM32234, Bacillus subtilis DSM32235 and Bacillus amyloliquefaciens DSM25840 single strains and the combination of EBP5 reduced the scoring of lesions from necrotizing enteritis in chickens and significantly reduced mortality from necrotic enteritis.

Example 9

Composition of bacillus with three strains of bacillus in male turkeys.

The experiment involved 300 day old healthy male turkey poults (Kartzfehn Premium) who were randomly assigned to 60 pens of 5 birds per pen (10 repetitions per treatment group).

The diet consisted of an unmedicated commercial turkey diet having the composition of ingredients as described in Table 24 for turkeys aged from day 01 to day 63 and in Table 24. 25 for turkeys aged from day 64 to day 147. The quantitative composition of the feed was the same for all animals, except for the inclusion of the Bacillus composition for the treatment groups.

A composition of Bacillus EUR5 containing 1.6x10 ⁹ CFU/g DSM32324, 0.6x10 ⁹ CFU/g DSM25840 and ΙΟχΙΟ ⁹ CFU/g DSM32325, i.e. in a ratio of 8:3:5, mixed with feed.

-28043066

Table 24

Composition ingredients in diets P1, P2 and P3 for turkeys

feeding phase Р1 (age from day 01 to day 14) Р2 (age from day 15 to day 35) RH (age from day 36 to day 63) Ingredients Soy flour (total protein: 49%) g/kg 470.00 442.80 369.00 Corn g/kg 352.40 389.40 455.70 Wheat g/kg 79.30 79.30 79.30 Soybean Oil Dihydrogen Phosphate g/kg 35.00 35.00 41.00 calcium g/kg 25.00 17.90 17.90 Limestone g/kg 20.20 17.80 18.80 Premix g/kg 12.00 12.00 12.00 Methionine g/kg 2.50 2.20 2.20 Lysine g/kg 1.60 1.60 2.10 Limestone g/kg 2.00;1.90;1.75; 1.50;1.00;0 2.00;1.90;1.75; 1.50;1.00;0 2.00;1.90;1.75; 1.50;1.00;0 ERV5 g/kg 0;0.1;0.25;0.50; 1.00;2.00 0;0.1;0.25;0.50; 1.00;2.00 0;0.1;0.25;0.50; 1.00;2.00

* Content per 1 kg of Premix: 600000 ME Vit. A (acetate); 120000 ME Vit. D3; 6000 mg Vit. E (α-tocopherol acetate); 200 mg Vit. K3 (MSB); 250 mg Vit. B1 (mononitrate); 420 mg Vit. B2 (crystal riboflavin); 300 mg Vit. B6 (pyridoxine-HCl); 1500 mcg Vit. AT 12; 3000 mg niacin (niacinamide); 12500 mcg biotin (industrial, feed); 100 mg folic acid (crystal, industrial, feed); 1000 mg pantothenic acid (Ca d-pantothenate); 60,000 mg choline (chloride); 5000 mg iron (iron carbonate); 5000 mg zinc (zinc sulfate); 6000 mg manganese (manganese oxide); 1000 mg copper (copper oxide); 45 mg iodine (calcium iodate); 20 mg selenium (sodium selenite); 140 g sodium (NaCl); 55 g magnesium (magnesium sulfate); carrier: calcium carbonate (calcium min. 38%); Monteban G100: 5833 mg.

Table 25

Composition ingredients in diets P4, P5 and P6 for turkeys

feeding phase P4 (age from day 64 to day 91) P5 (age from day 92 to day 119) P6 (age from day 120 to day 147) Ingredients Corn g/kg 524.60 606.80 658.00 Soy flour (total protein: 49%) g/kg 303.00 225.00 173.00 Wheat g/kg 79.30 79.30 79.30 Soybean Oil Dihydrogen Phosphate g/kg g/kg 37.80 35.00 38.00 calcium 19.00 16.00 14.90 Limestone g/kg 18.70 17.00 15.30 Premix * g/kg 12.00 12.00 12.00 L-Lysine g/kg 2.00 2.80 3.50 DL-Methionine g/kg 1.60 1.50 1.50 L-Threonine g/kg 0.50 0.30 L-Tryptophan g/kg 0.10 0.20 Limestone g/kg 2.00; 1.90; 1.75; 1.50;1.00;0 2.00; 1.90; 1.75; 1.50;1.00;0 2.00; 1.90; 1.75; 1.50;1.00;0 Probiotic g/kg 0;0.1;0.25;0.50; 1.00;2.00 0;0.1;0.25;0.50; 1.00;2.00 0;0.1;0.25;0.50; 1.00;2.00

* Content per 1 kg of Premix: 600000 ME Vit. A (acetate); 120000 ME Vit. D3; 6000 mg Vit. E (α-tocopherol acetate); 200 mg Vit. K3 (MSB); 250 mg Vit. B1 (mononitrate); 420 mg Vit. B2 (crystal riboflavin); 300 mg Vit. B6 (pyridoxine-HCl); 1500 mcg Vit. AT 12; 3000 mg niacin (niacinamide); 12500 mcg biotin (industrial, feed); 100 mg folic acid (crystal, industrial, feed); 1000 mg pantothenic acid (Ca d-pantothenate); 60,000 mg choline (chloride); 5000 mg iron (iron carbonate); 5000 mg zinc (zinc sulfate); 6000 mg manganese (manganese oxide); 1000 mg copper (copper oxide); 45 mg iodine (calcium iodate); 20 mg selenium (sodium selenite); 140 g sodium (NaCl); 55 g magnesium (magnesium sulfate); carrier: calcium carbonate (calcium min. 38%); Monteban G100: 5833 mg

- 29 043066

Results.

Table 26

The effect of EUR5 on the productivity of male turkeys during the entire fattening period (P1-P6)

Processing groups T1 T2 tz T4 T5 Tb p-value Total Birds p° 50 50 50 50 50 50 Replays p° 10 10 10 10 10 10 EUR5 mg/kg 100 250 500 1000 2000 R 1 - R 6 (age from day 01 to day 147) Birds n ^and 48 49 48 48 49 49 Initial body weight G 61.2 ± 1.0 61.2±0.8 61.3 ± 1.0 61.2 ± 1.0 61.3±0.8 61.2 ± 1.0 1,000 Final body weight G 24169.3 ± 321.6 ^a 24522.9 ± 335.0 ^ab 24721.4 ± 252.2 ^bv 24917.7± ^251.2V 25333.3 ± 311.2 ^G 25759.3 ± 282.2 ^D <0.001 Weight gain G 24108.1 ± 320.9 ^a 24461.7 ± 334.6 ^ab 24660.2 ± 252.0 ^bv 24856.5 ± 251.1 ⁱⁿ 25272.0 ± 310.9 ^g 25698.1 ± 282.4 ^D <0.001 Weight gain/day g/day 164.0 ± 2.2 ^a 166.4 ± 2.3 ^a0 167.8 ± 1.7 ^0v 169.1± ^1.7V 171.9 ±2,G 174.8 ± 1.9 ^D <0.001 food intake G 51538.2± 735.3 ^ab 51309.4 ± 408.7 ^ab 51294.0 ± 377.0 ^ab 50941.6 ± 598.1 ^a6 51049.7 ± 404.7 ^a 51737.8 ± 581.9 ⁶ 0.016 Food intake/day g/day 350.6 ± 5.0 ^ab 349.0 ± 2.8 ^a 348.9 ± 2.6 ^ab 346.5 ± 4.1 ^ab 347.3 ± 2.8 ^a 352.0 ± 4.0 ^b 0.016 Feed yield 2.138±0.024' 2.098 ± 0.035 ⁱⁿ 2.080 ± 0.026 ^bU 2.050 ± 0.025 ^ab 2.020 ± 0.032 ^a 2.013 ±0.025 ^a <0.001

^ab Different row indices show significance levels at P<0.05.

Body weight gain.

The initial body weight of the poults was approximately 61.2 g and was almost the same in all treatment groups. The total body weight gain on the EBP5-free turkey diet (control group) was 24.11 kg; in relation to the 147-day feeding period, daily weight gain reached an average of 164 g. Total body weight gain showed a significant improvement when EBP5 was added at a dose of 250 or more (250 mg / kg: + 2.3%; 500 mg / kg : +3.1%; 1,000 mg/kg: +4.8%; 2000 mg/kg: +6.6%) compared with control, while in turkeys that received a diet containing EBP5 at a dose of level of 100 mg/kg, no significant changes were found (+1.0%) compared with the control.

The feed yield coefficient.

The total feed conversion ratio (feed:growth) of turkeys fed a diet without EBP5 reached 2.138, which indicates an excellent level of productivity and even exceeds the reference figures given by the breeder (2.540). Due to the positive effect on body weight gain in turkeys fed a diet containing EBP5, higher dose levels were associated with significantly reduced total feed conversion ratios (250 mg/kg: 2.080; 500 mg/kg: 2.050; 1000 mg/kg: 2.020; 2000 mg/kg: 2.013) compared to control.

Conclusion.

The overall mortality (including culling) was 2.7%, indicating the excellent health of the herd.

This experiment showed a significant improvement in performance in birds fed a diet containing 250 to 2000 mg/kg of the multi-strain probiotic EBP5 based on Bacillus. Body weight gain at 147 days of age was significantly increased by EBP5 up to 6.6% (2000 mg/kg) compared to control.

In turkey diets containing Bacillus-based EBP5 multi-strain probiotic, higher dose levels were associated with significantly improved total feed conversion rates compared to control, up to 5.8% (2000 mg/kg).

Noteworthy is the fact that at dose levels of 250 to 2000 mg/kg, the average total dry matter content of the faeces was significantly increased compared to the control.

Example 10

Combination of a bacillus formulation with three bacillus strains and a live attenuated salmonella vaccine in chickens.

120 day old Ross x Ross broiler chicks, not segregated by sex, were allocated to three different isolation rooms, each containing 40 broiler chicks at the start of the study. Each pen contained approximately 4 inches of fresh pine shavings, one tubular feeder and one bell type drinker for ad libitum feeding and drinking.

All birds were vaccinated against coccidia on the first day, and did not use any concomitant drug therapy. The pens were checked daily for mortality.

The starter diet (crumb) and grower diet (kibble) compositions from day 22 until the end of the study consisted of unmedicated industrial feed for

- 30 043066 broilers. The quantitative composition of the feed was the same for all animals except for the inclusion of the Bacillus composition for the treatment group. A Bacillus composition containing DSM32324,

DSM25840 and DSM32325 at a ratio of 8:3:5, mixed with feed at a ratio of 1.6x10 ⁶ CFU/g of feed.

AviPro® Megan® Vac 1, a live Salmonella Typhimurium vaccine manufactured by Lohmann Animal Health, Maine, USA, hereinafter referred to as Megan Vac, was coarsely sprayed onto day 1 chicks for treatment groups T2 and T3 at a single dose per bird in a volume of 0.25 ml for a chicken.

Table 27

Processing groups (40 birds in each group)

Group Megan Vac Composition of Bacillus in the diet T1 No No T2 Yes No TK Yes 1.6 x 10 ⁶ cfu/gram feed

On day 3, four caecal appendages and four spleens per treatment group were weighed and harvested to confirm vaccine colonization. All remaining birds were dosed orally (by gavage) with 3x10 ⁷ cfu of Salmonella Heidelberg (Alali et al., 2013) on day 4.

On day 40, just prior to slaughter, ten birds per treatment group were taken from each individual pen, sacrificed, and cecum removed under sterile conditions. Samples of the processes of the caecum were tested for Salmonella.

Results.

Salmonella prevalence results in caecal and liver/spleen specimens collected from four birds on Day 3 (see Table 28) support vaccine colonization.

Table 28 Salmonella Prevalence in Caecal and Liver/Spleen Samples

sample type Treatment no. Number of positive (%) Outgrowth of the caecum T1 4 0 T2 4 0 TK 4 0 Liver/spleen T1 4 0 T2 4 3 TK 4 4

Putative Salmonella isolates were confirmed with a specific Poly-O Salmonella antiserum (MiraVista, Indianapolis, IN).

Megan Vac alone or with the Bacillus formulation had numerically the lowest amount of Salmonella in the appendages of the cecum at day 40 compared to untreated birds (data not shown).

As follows from Table. 29, the FCR of birds fed Megan Vac plus Bacillus (T3) formulation was lower than that of untreated birds (T1) and birds fed Megan Vac alone (T2).

Table 29

Productivity per day 40

Treatment Food consumption (kg/pen) Weight gain (kg/live bird) FCR T1 95.86 1.86 1.47 T2 110.32 2.16 1.50 TK 106.70 2.15 1.38

Conclusion.

The aim was to evaluate the effect of a Bacillus based probiotic on colonization with a live Salmonella Typhimurium vaccine and the subsequent ability of a Bacillus composition to protect against Salmonella Heidelberg challenge in broiler chickens.

Day 3 samples showed that the Bacillus formulation had no effect on initial colonization with the Salmonella vaccine.

Also in this study it is shown that there may be an additive effect in the combined action of the vaccine and the composition of Bacillus.

Claims (7)

1. Composition for the prevention or control of bacterial colonization or infection in the intestines of poultry, containing at least one strain of Bacillus subtilis selected from the group consisting of: a) Bacillus subtilis strain deposited as DSM32324, and b) Bacillus subtilis strain deposited as DSM32325. 2. The composition according to claim 1, containing the Bacillus subtilis DSM32324 strain. 3. Composition according to claim 1 or 2, containing the strain Bacillus subtilis DSM32325. 4. Composition according to any one of claims 1 to 3, containing Bacillus subtilis DSM32324 and Bacillus subtilis DSM32325. 5. Composition according to any one of claims 1 to 4, wherein the Bacillus strain or strains are in the form of spores. 6. The composition according to any one of claims 1 to 5, which is a probiotic, premix, animal feed additive or animal feed. 7. A composition according to any one of claims 1 to 6, wherein the bacterial colonization or infection is a bacterial colonization or infection with E. coli and/or Clostridium perfringens.