WO2025059013A1 - Bacillus-based components for inhibiting or delaying the growth of enterococcus spp. in animals - Google Patents

Abstract

A method for Inhibiting or Delaying the Growth of Enterococcus spp. in animals using at least one Bacillus-based component is disclosed herein.

Description

BACILLUS-BASED COMPONENTS FOR INHIBITING OR DELAYING THE GROWTH OF ENTEROCOCCUS SPP. IN ANIMALS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/537,657, filed September 11, 2023, the disclosure of which is incorporated by reference herein in its entirety.

FIELD

The field relates to the use of Bacillus-based components for inhibiting or delaying the growth of Enterococcus spp. in animals.

BACKGROUND

Enterococcus is a large genus of lactic acid bacteria of the phylum Firmicutes.

Enterococci are Gram-positive cocci that often occur in pairs (diplococci) or short chains, and are difficult to distinguish from streptococci on physical characteristics alone. Enterococci are facultative anaerobic organisms, i.e., they are capable of cellular respiration in both oxygen-rich and oxygen-poor environments. Though they are not capable of forming spores, Enterococci are tolerant of a wide range of environmental conditions: extreme temperature (10-45°C), pH (4.5- 10.0), and high sodium chloride concentrations. Members of the genus Enterococcus were classified as group D Streptococcus until 1984, when genomic DNA analysis indicated a separate genus classification would be appropriate.

While Enterococcus species are known to be normal inhabitants of the intestine of birds and other vertebrates such as horses, cattle, pigs, dogs, cats, canaries, pigeons, turkeys and Muscovy ducks, some members of this genus are acknowledged to be opportunistic pathogens when they get outside the gut. This is the case with respect to E. avium, E. gallinarum and E. cecorum, wherein pathogenic E. cecorum in particular is important in terms of economic losses sustained by poultry farmers in broiler chicken production chains.

A part of the normal gut flora, disruptions or insult to normal gut function can result in Enterococcus spp. translocation to the spine of birds. Enterococcus spp. infections in the spine lead to vertebral and arthritic lesions, lameness and mortality in a condition known as cntcrococcal spondylothcsis or “kinky back,” as referred to by poultry producers. Spondylitis has been known in commercial production for many years and is typically seen in heavy, fastgrowing birds, especially males and broiler breeders (Aziz, T. & Barnes, H.J. (2009). Spondylitis is emerging in broilers. World Poultry, 25, 19).

While highly pathogenic and antibiotic-resistant strains of Enterococcus cecorum are known to cause economic losses to the broiler chicken industry (see, e.g, International Patent Application Publication No. WO 2018/112006, incorporated by reference herein), recent epidemiological surveys conducted in France between 1993 and 2020 (Souillard et al., 2022, Vet Microbiol. 2022 Jun;269: 109426) revealed that despite E. cecorum remaining the dominant pathogenic species encountered, a large variety of diseases and clinical symptoms are now attributed to Enterococcus species beyond E. cecorum. These species include E. faecalis, E. hirae, E. gallinarum, E. faecium, E. casseliflavus, E. durans, E. avium and E. columbae, and other Enterococcus species not yet identified.

Thus, routine farm hygiene procedures and antimicrobial therapy have proven insufficient to control outbreaks of pathogenic Enterococcus spp. and new, safe, and efficacious alternatives are needed to control these important emerging pathogens.

SUMMARY

Provided herein are methods for treating or preventing Enterococcus spp. infection in animals, for example, poultry.

Accordingly, in one aspect, provided herein are methods for inhibiting or delaying all or part of the growth of pathogenic Enteroccocus spp An an animal comprising administering an effective amount of at least one ZAzcF/u.s- based component selected from the group consisting of: a Bflcz7/u. -bascd direct fed microbial comprising one or more Bacillus bacterial strains, a supernatant obtained from a Bacillus culture or a combination thereof to an animal, wherein said pathogenic Enteroccocus spp. is selected from the group consisting of E. avium, E. casseliflavus, E. durans, E. faecalis, E. faecium, E. hirae, and E. mundtii. In some embodiments, the Bacillusbased direct fed microbial is selected from the group consisting of Bacillus velezensis, Bacillus amyloliquefaciens , Bacillus licheniformis, Bacillus pumilis and Baccillus subtilis. In some embodiments of any of the embodiments disclosed herein, the Bacillus-based direct fed microbial is selected from the group consisting of one or more of the following strains: Bacillus strain 2084 Accession No. NRR1 B-50013, Bacillus strain LSSAO1 (a.k.a. Bacillus strain BS8) Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507. In some embodiments of any of the embodiments disclosed herein, the animal is a monogastric animal. In some embodiments, the animal is a monogastric animal. In some embodiments of any of the embodiments disclosed herein, the animal is a multigastric animal. In some embodiments, the animal is a multigastric animal. In some embodiments of any of the embodiments disclosed herein, the monogastric animal is poultry. In some embodiments, the monogastric animal is poultry. In some embodiments of any of the embodiments disclosed herein, the at least one Bacillus -based component is administered directly to an animal through animal feed whether in the feed or on top of the feed or in a liquid. In some embodiments, the at least one ZAzd//u.s-bascd component is administered directly to an animal through a waterline. In some embodiments, the at least one Bacillus -based component is administered directly to an animal through animal feed whether in the feed or on top of the feed or in a liquid. In some embodiments, the at least one Bacillus -based component is administered directly to an animal through a waterline. In some embodiments, the at least one Bacillus -based component is administered to the animal in a form selected from the group consisting of a feedstuff, a feed additive composition, a premix or in a liquid. In some embodiments, the Bacillus-based component is administered to the animal in a form selected from the group consisting of a feedstuff, a feed additive composition, a premix or in in a liquid. In some embodiments of any of the embodiments disclosed herein, the method further comprises administering one or more enzymes selected from the group consisting of phytase, protease, amylase, xylanase, lipase, or glucoamylase to the animal. In some embodiments, the enzymes comprise a xylanase, an amylase, and a protease. In some embodiments, the method delays or inhibits the growth of pathogenic Enteroccocus spp.va an animal (such as one or more of E. avium, E. casseliflavus, E. durans, E. faecalis, E. faecium, E. hirae, and E. mundlii.) by about 5-100% or by about 10-70% (such as any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of any values falling in between these percentages), compared to animals that have not been administered an effective amount of at least one Bacillus-based component. Each of the aspects and embodiments described herein are capable of being used together, unless excluded cither explicitly or clearly from the context of the embodiment or aspect.

Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table depicting the Enterococcus species used in the Examples.

FIG. 2 is a bar graph depicting different classes Enterococcus species inhibition as a function of Bacillus strains tested.

FIG. 3 is a graph depicting the effect of the three tested Bacillus strains on the growth of multiple strains representing nine different Enterococcus species. Each dot represents a Bacillus strain while the horizontal bar is the average percentage of growth inhibition.

FIG. 4 is a graph depicting the delay of growth of Enterococcus mindtii 64247-EN01 when grown in presence of CFS of Bacillus strains 15AP4, 2084 and BS8 in combination with a ratio of 1 : 1 : 1.

FIG. 5 is a graph depicting the delay of growth of multiple isolates representing 8 species of Enterococcus grown in presence of Enviva® PRO CFS (horizontal bar represents the average).

FIG. 6 is a graph depicting how the difference in maximum optical density (OD) is determined using the growth of Enterococcus f aecium 62467-EN05 grown alone or in presence of the Bacillus CFS.

FIG. 7 is a graph depicting differences in the OD maximum comparing pathogen grown alone and in the presence of Enviva® PRO CFS for multiple strains representative of 8 different Enterococcus species associated with poultry diseases (horizontal bar represents the average).

FIG. 8 is a table depicting details of the 137 Enterococcus spp. isolates used in the study described in Example 6. 'U, university. ²C, clinical isolate (obtained from a diseased (symptomatic) bird); NP, non-pathogenic isolate (* = confirmed by in-embryo validation using the chicken embryo mortality assay; Huang et al. 2023); NC, non-clinical isolate obtained from a healthy bird. ³16 S, 16S ribosomal RNA sequencing; PCR, polymerase chain reaction; WGS, whole genome sequencing; ^T, type strain.

FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D are box plots of growth inhibition of 25 non- E. cecorum Enterococcus spp. isolates by cell-free supernatant (CFS) from three commercial strains of probiotic Bacillus strains (BS8 (FIG. 9A), 15AP4 (FIG. 9B), 2084 (FIG. 9C)) and by a 1:1:1 blend of the three probiotic strains (FIG. 9D) with results for the inhibition of 112 E. cecorum isolates shown on the far right of each image, for comparison. Isolates were a mixture of clinical and non-clinical isolates. Growth inhibition was measured at a time-point equivalent to the middle of the exponential growth phase of the PC. 70% inhibition is indicated by a horizontal dashed line.

FIG. 10 is a graph illustrating the growth kinetics of two E. avium (E-84-197 and 60268- EN05), measured as optical density, OD, seen during incubation in brain heart infusion medium (1% v/v) without or with a cell-free supernatant blend (10% v/v) from probiotic Bacillus strains BS8, 14AP4 and 2084 (added in the ratio 1:1:1) at 37°C.

DETAILED DESCRIPTION

All patents, patent applications, and publications cited are incorporated herein by reference in their entirety.

In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.

The articles “a”, “an”, and “the” preceding an element or component are intended to be nonrestrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore “a”, “an”, and “the” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term “comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of”. Similarly, the term “consisting essentially of” is intended to include embodiments encompassed by the term “consisting of’. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1- 2”, “1-2 & 4-5”, “1-3 & 5”, and the like.

As used herein in connection with a numerical value, the term “about” refers to a range of +/- 0.5 of the numerical value, unless the term is otherwise specifically defined in context. For instance, the phrase a “pH value of about 6” refers to pH values of from 5.5 to 6.5, unless the pH value is specifically defined otherwise.

It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

The terms "Enterococcus'' and “Enterococcus spp.” are used interchangeably and as used herein refers to a large genus of lactic acid bacteria of the phylum Firmicutes. Enterococci are Gram-positive cocci that often occur in pairs (diplococci) or short chains, and are difficult to distinguish from streptococci on physical characteristics alone. Enterococci are facultative anaerobic organisms, i.e., they are capable of cellular respiration in both oxygen-rich and oxygen-poor environments. Though they are not capable of forming spores, enterococci are tolerant of a wide range of environmental conditions: extreme temperature (10-45 °C), pH (4.5- 10.0), and high sodium chloride concentrations. Members of the genus Enterococcus were classified as group D Streptococcus until 1984, when genomic DNA analysis indicated a separate genus classification would be appropriate.

The Enterococcus spp. “Enterococcus cecorum” “Enterococcus gallinarum, ” “Enterococcus avium, ” “Enterococcus casseliflavus, ” “Enterococcus durans, ” “Enterococcus faecalis, ’’ “Enterococcus f aecium, ” “Enterococcus hirae, ” and “Enterococcus mundtii” are referenced interchangeably herein with the terms “E. cecorum” “E. gallinarum, ” “E. avium, ” “E. casseliflavus, ” “E. durans, ” “E. faecalis, ” “E. faecium, ” “E. hirae, ” and “E. mundtii,” respectively. Enterococcus spp. are bacterium of the intestinal tract of many domestic animals. The terms “animal” and “subject” are used interchangeably herein. An animal includes all non-ruminant (including humans) and ruminant animals. In a particular embodiment, the animal is a non-ruminant animal, such as a horse and a mono-gastric animal. Examples of monogastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, sows; poultry such as turkeys, ducks, chicken, broiler chicks, layers; fish such as salmon, trout, tilapia, catfish and carps; and crustaceans such as shrimps and prawns. In a further embodiment, the animal can be multigastric, such as a ruminant animal, including, but not limited to, cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and nilgai.

The term “ruminant” as used herein refers to a mammal that is able to acquire nutrients from plant-based food by fermenting it in a specialized stomach prior to digestion, principally, through microbial actions. The process typically requires the fermented ingesta (known as cud) to be regurgitated and chewed again. The process of rechewing the cud to further break down plant matter and stimulate digestion is called rumination. Roughly 150 species of ruminants include both domestic and wild species. Ruminating animals include, but are not limited to, cattle, cows, goats, sheep, giraffes, yaks, deer, elk, antelope, buffalo and the like.

The term “CFU” as used herein means “colony forming units” and is a measure of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell.

The term “direct-fed microbial” (“DFM”) as used herein is source of live (viable) naturally occurring microorganisms. A DFM can comprise one or more of such naturally occurring microorganisms such as bacterial strains. Categories of DFMs include spore-forming bacteria such Bacillus and Clostridium as well non-spore forming bacteria such as Lactic Acid Bacteria, Yeasts and Fungi. Thus, the term DFM encompasses one or more of the following: direct fed bacteria, direct fed yeast, direct fed yeast or fungi and combinations thereof.

Bacillus and Clostridium are unique, gram-positive rods that form spores. These spores are very stable and can withstand environmental conditions such as heat, moisture and a range of pH. These spores germinate into active vegetative cells when ingested by an animal and can be used in meal and pelleted diets. Lactic Acid Bacteria are gram-positive cocci that produce lactic acid which are antagonistic to pathogens. Since Lactic Acid Bacteria appear to be somewhat heat-sensitive, they are not used in pelleted diets as such and need to be protected (coated). Types of Lactic Acid Bacteria include Bifidobacterium, Lactobacillus and Enterococcus. The term “Bflc«7/i/.y-based direct-fed microbial” means a direct-fed microbial comprising one or more Bacillus bacterial species or strains. Non-limiting examples of Bacillus species include Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilis and Baccillus subtilis.

The term “Bacillus-based component” as used herein refers to (i) a Bacillus -based direct fed microbial comprising one or more Bacillus bacterial strains, (ii) a supernatant obtained from a Bacillus culture or (iii) a combination of (i) and (ii).

A "feed" and a "food", respectively, means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by a nonhuman animal and a human being, respectively.

As used herein, the term "food" is used in a broad sense - and covers food and food products for humans as well as food for non-human animals (i.e. a feed).

The term "feed" is used with reference to products that are fed to animals in the rearing of livestock. The terms “feed” and “animal feed” are used interchangeably. In a preferred embodiment, the food or feed is for consumption by non-ruminants and ruminants.

The term “probiotic” as used herein defines live microorganisms (including bacteria or yeasts for example) which, when for example ingested or locally applied in sufficient numbers, beneficially affects the host organism, i.e. by conferring one or more demonstrable health benefits on the host organism. Probiotics may improve the microbial balance in one or more mucosal surfaces. For example, the mucosal surface may be the intestine, the urinary tract, the respiratory tract or the skin. The term “probiotic” as used herein also encompasses live microorganisms that can stimulate the beneficial branches of the immune system and at the same time decrease the inflammatory reactions in a mucosal surface, for example the gut. Whilst there are no lower or upper limits for probiotic intake, it has been suggested that at least 10⁶- 10¹², for example, at least 1O⁶-1O¹⁰, for example 10⁸- 10⁹, efu as a daily dose will be effective to achieve the beneficial health effects in a subject.

The term “prebiotic” means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of beneficial bacteria.

The term “pathogen” as used herein means any causative agent of disease. Such causative agents can include, but are not limited to, bacterial, viral, fungal causative agents and the like. The terms “derived from” and “obtained from” refer to not only a protein produced or producible by a strain of the organism in question, but also a protein encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protein which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protein in question.

The term “effective amount” means a sufficient amount of the specified component.

As was noted above, Enteroccocus spp. are considered an emerging pathogen in poultry and can cause substantial losses in broiler and broiler breeder flocks. These pathogens have been increasingly recognized as a cause of enterococcal spondylitis, previously called enterococcal vertebral osteoarthritis (EVOA) in chickens. Disease outbreaks were diagnosed mostly in broiler chicken flocks raised under an intensive production system. Clinically affected birds suffered from locomotor problems due to compression of the spinal cord at the thoracic vertebrae resulting from Enteroccocus spp. - induced osteomyelitis and due to femoral head necrosis. Disease outbreaks can lead to high morbidity, mortality, culling, carcass condemnations, and may result in severe economic losses within a short time. Furthermore, isolates of Enteroccocus spp. have been demonstrating not only increased pathogenicity but also increased antimicrobial resistance.

Thus, the method described herein provides an alternative to the use of antibiotics since antimicrobial resistance is becoming a major global health threat.

In one embodiment, described herein is a method for inhibiting or delaying all or part of the growth of pathogenic Enteroccocus spp. in an animal which comprises administering an effective amount of at least one BaczV/i s -based component selected from the group consisting of: a Bac/Z/z/.s-bascd direct fed microbial comprising one or more Bacillus bacterial strains, a supernatant obtained from a Bacillus culture or a combination thereof to an animal.

The DFMs described herein comprise at least one viable microorganism such as a viable bacterial strain or a viable yeast or a viable fungus. In one embodiment, the DFM comprises at least one viable bacteria.

In one embodiment, the DFM may be a spore forming bacterial strain and hence the term DFM may be comprised of or contain spores, e.g. bacterial spores. Thus, the term “viable microorganism” as used herein may include microbial spores, such as endospores or conidia. Alternatively, the DFM in a feed additive composition described herein may not comprise of or may not contain microbial spores, c.g. cndosporcs or conidia.

The microorganism may be a naturally-occurring microorganism or it may be a transformed microorganism. Preferably, the microorganism is a combination of at least three suitable microorganisms, such as bacteria, that may be isolated.

A DFM as described herein may comprise microorganisms from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Camobacterium, Propionibacterium, Bifidobacterium, Clostridium, Paenibacillus and Megasphaera and combinations thereof.

Preferably, the DFM comprises one or more bacterial strains selected from the following Bacillus spp: Bacillus velezensis, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis.

The genus “Bacillus” , as used herein, includes all species within the genus “Bacillus”, as known to those of skill in the art, including but not limited to B. velezensis, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, B. pumilis and B. thuringiensis . It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as Bacillus stearothermophilus, which is now named “Geobacillus stearothermophilus” , or Bacillus polymyxa, which is now “Paenibacillus polymyxa”. The production of resistant endospores under stressful environmental conditions is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

In some embodiments, the DFM may be one or more of the bacterial strains found in Enviva® PRO which is commercially available from Danisco A/S. Enviva® PRO is a combination of Bacillus strain 2084 Accession No. NRR1 B-50013, Bacillus strain LSSAO1 (a.k.a. Bacillus strain BS8) Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507 (as taught in U.S. Patent No. 7,754,469 - incorporated herein by reference). In another aspect, the DFM may be further combined with the following Lactococcus spp: Lactococcus cremoris and Lactococcus lactis and combinations thereof.

The DFM may be further combined with the following Lactobacillus spp: Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus farciminis, Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus farciminis, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii and Lactobacillus j ens enii, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus ponds, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus zeae and combinations of any thereof.

With regard to the Lactobacillus spp. discussed above, it should be noted that, as recently as March 2020, Lactobacilli comprised 261 species that are extremely diverse phenotypically, ecologically, and genotypically. Given advances in whole genome sequencing and comparative genomics, the genus Lactobacillus was recently divided into 25 separate genera with strains belonging to previously designated Lactobacilli species being transferred to new species and/or genera (see Zheng et al., 2020, Int. J. Syst. Evol. Microbiol., 70:2782-2858; Pot et al., Trends in Food Science & Technology 94 (2019) 105-113; and Koutsoumanis et al., 2020, EFSA Journal, 18(7):6174 the disclosures of each of which are incorporated by reference herein). For purposes of the instant disclosure, the previous classification of Lactobacillus species will continue to be employed. However, in some embodiments Lactobacillus agilis is also classified as as Ligilactobacillus agilis. In other embodiments, Lactobacillus salivarius is also classified as Ligilactobacillus salivarius. In further embodiments, Lactobacillus reuteri is also classified as Limosilactobacillus reuteri.

In still another aspect, the DFM may be further combined with the following Bifidobacteria spp: Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacterium angulatum, and combinations of any thereof.

There can be mentioned bacteria of the following species: Bacillus velezensis, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pumilis, Pediococcus spp, Lactobacillus spp., Bifidobacterium spp., Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Bacillus subtilis, Propionibacterium thoenii, Lactobacillus farciminis, Lactobacillus rhamnosus, Megasphaera elsdenii, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillus salivarius ssp. Salivarius, Propionibacteria sp and combinations thereof.

The direct-fed microbial described herein comprising one or more bacterial strains may be of the same type (genus, species and strain) or may comprise a mixture of genera, species and/or strains. Preferably, direct-fed microbial described herein comprising one or more bacterial strains from the genus Bacillus.

Suitably the composition according to the present disclosure may be combined with one or more of the products or the microorganisms contained in those products disclosed in WO2012110778, and summarized as follows:

Bacillus strain 2084 Accession No. NRR1 B-50013, Bacillus strain LSSAO1 (a.k.a.

Bacillus strain BS8) Accession No. NRRL B-50104, and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507 (from Enviva® PRO®); Bacillus subtilis Strain C3102 (from Calsporin®); Bacillus licheniformis DSM28710 (B-ACT(R) from Huvepharma); Bacillus subtilis Strain PB6 (from Clostat®); Bacillus pumilis (8G-134); Bacillus subtilis Strain C3102 (from Gallipro® & GalliproMax®); Bacillus licheniformis (from Gallipro®Tect®); Lactobacillus salivarius, L. reuteri, Bifidobacterium animalis and Pediococcus acidilactici (from Poultry Star®); Lactobacillus, Bifidobacterium and/or Bacillus subtilis strain QST 713 (from Proflora®); Bacillus subtilis (QST 713) (from Baymix® Grobig BS (Bayer)); Bacillus amyloliquefaciens CECT-5940 (from Ecobiol® & Ecobiol® Plus); Bacillus subtilis and Bacillus licheniformis (from BioPlus2B®); Bacillus strain (from CSI®); Saccharomyces cerevisiae (from Yea-Sacc®); Pediococcus aciclilactici, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius (from Biomin C5®); Lactobacillus farciminis (from Biacton®); Lactococcus lactis DSM 1103(from Probios -pioneer PDFM®); Lactobacillus rhamnosus and Lactobacillus farciminis (from Sorbiflore®); Bacillus subtilis (from Animavit®); Saccharomyces cerevisiae (from Levucell SB 20®); Saccharomyces cerevisiae (from Levucell SC 0 & SCIO® ME); Pediococcus acidilacti (from Bactocell); Saccharomyces cerevisiae (from ActiSaf® (formerly BioSaf®)); Saccharomyces cerevisiae NCYC Sc47 (from Actisaf® SC47); Clostridium butyricum (from Miya-Gold®); Saccharomyces cerevisiae NCYC R-625 (from InteSwine®); Saccharomyces cerevisia (from BioSprint®); Lactobacillus rhamnosus (from Provita®); Bacillus subtilis and Aspergillus oryzae (from PepSoyGen-C®); Bacillus cereus (from Toyocerin®); Bacillus cereus var. toyoi NCIMB 40112/CNCM 1-1012 (from TOYOCERIN®), Lactobacillus plantarum (from LactoPlan®) or other DFMs such as Bacillus licheniformis and Bacillus subtilis (from BioPlus® YC) and Bacillus subtilis (from GalliPro®).

It is also possible to combine the DFM described herein with a yeast from the genera and species: Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia angusta, Pichia anomala, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces pastorianus (synonym of Saccharomyces carlsbergensis) and filamentus fungi from the genus Aspergillus.

Preferably, the DFM described herein comprises microorganisms which are generally recognized as safe (GRAS) and, in one embodiment, are GRAS-approved and/or Qualified Presumption of Safety by the European Food Safety Authority (EFSA)

In some embodiments, it is important that the DFM be heat tolerant, i.e., is thermotolerant. This is particularly the case when the feed is pelleted. Thus, the DFM may be a thermotolerant microorganism, such as a thermotolerant bacteria, e.g., spore-forming bacteria including for example Bacillus spp. Bacilli are able to form stable endospores when conditions for growth are unfavorable and are very resistant to heat, pH, moisture and disinfectants. If the bacterium/DFM is not a spore-former then it should be protected to survive feed processing as is described hereinbelow.

The Bacillus-based DFM as described herein described herein may inhibit or delay all or part of the growth of Enterococcus spp., e.g., E. cecorum, E. avium, E. gallinarum, E. casseliflavus, E. durans, E. faecalis, E. faecium, E. hirae, and/or E. mundtii. In other words, a Bacillus -based DFM as described herein is antipathogcnic. The term “antipathogcnic” as used herein means the DFM counters an effect (negative effect) of a pathogen, in this case, pathogenic Enterococcus spp., e.g.. E. cecorum, E. avium, E. gallinarum, E. casseliflavus, E. durans, E. faecalis, E. faecium, E. hirae, and/or E. mundtii.

For example, the following assay “DFM ASSAY” may be used to determine the suitability of a microorganism to be a DFM or in this embodiment, a Bacillus-based DFM as described herein. Such DFM can be run as follows:

The fully grown culture of a Bacillus strain was centrifuged and filter-sterilized (0.2 pm) so as to obtain sterile cell free supernatant (CFS). Each well of a 96- well microtiter plate is filled with 180 pl of a pathogen/BHI (or appropriate growth media) suspension (1%). The positive control wells are filled with extra 20 pl of the same broth media whereas the tested wells are filled with 20 pl of the tested CFSs. The negative controls contain the broth media only or broth media added with 20pl of CFS. The 96-well microtiter plate is then incubated aerobically at 37°C for 14 hours in a Flex station machine to record absorbance, with data transferred directly to a computer for analysis so as to generate kinetics growth curve. Measurements were taken every 15 minutes. Results are given as % of inhibition comparing the control group at an OD capturing the middle of the exponential growth phase (pathogen alone) and respective time point for the treatment group (pathogen incubated with Bacillus CFS). Delay in growth is calculated as the difference in time to reach an OD at the middle of the exponential growth phase between control and CFS -supplemented wells. All assays are conducted in biological duplicate (including 4 technical replicates). Means separation was conducted using Tukey’s HSD in JMP 11; differences were considered significant at P<0.05.

Antipathogcnic DFMs include one or more of the following bacteria and are described in W02013029013:

Bacillus subtilis strain 3BP5 Accession No. NRRL B-5O51O,

Bacillus subtilis strain 918 ATCC Accession No. NRRL B-50508, and

Bacillus subtilis strain 1013 ATCC Accession No. NRRL B-50509.

A Bacillus-based component as described herein may be prepared as culture(s) and carrier(s) (where used) and can be added to a ribbon or paddle mixer and mixed for about 15 minutes, although the timing can be increased or decreased. The components are blended such that a uniform mixture of the cultures and carriers result. The final product is, in one embodiment, a dry, flowable powder. Accordingly, a Bacillus-based component can comprise a: a Bacillus-based direct fed microbial comprising one or more Bacillus bacterial strains, a supernatant obtained from a Bacillus culture or a combination. Such a Bacillus-based component can then be added to animal feed or a feed premix. It can be added to the top of the animal feed (“top feeding”) or it can be added to a liquid such as the animal’s drinking water.

Inclusion of the individual strains in the Bacillus-based DFM as described herein can be in proportions varying from 1% to 99% and, in one embodiment, from 25% to 75%.

Suitable dosages of the Bacillus-based component as described herein in animal feed may range from about IxlO³ CFU/g feed to about IxlO¹⁰ CFU/g feed, suitably between about IxlO⁴ CFU/g feed to about IxlO⁸ CFU/g feed, suitably between about 7.5xl0⁴ CFU/g feed to about IxlO⁷ CFU/g feed.

A person of ordinary skill in the art will readily be aware of specific species and/or strains of microorganisms from within the genera described herein which are used in the food and/or agricultural industries and which are generally considered suitable for animal consumption. Animal feeds may include plant material such as com, wheat, sorghum, soybean, canola, sunflower or mixtures of any of these plant materials or plant protein sources for poultry, pigs, ruminants, aquaculture and pets.

The terms “animal feed”, “feed”, and “feedstuff” are used interchangeably and can comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as com gluten meal, Distillers Dried Grains with Solubles (DDGS) (particularly com based Distillers Dried Grains with Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; and/or e) minerals and vitamins.

When used as, or in the preparation of, a feed, such as functional feed, a Bacillus -based component as described herein may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient. For example, there could be mentioned at least one component selected from the group consisting of a protein, a peptide, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben and propyl paraben.

In a preferred embodiment, a Bacillus-based component as described herein may be admixed with a feed component to form a feedstuff. The term "feed component" as used herein means all or part of the feedstuff. Part of the feedstuff may mean one constituent of the feedstuff or more than one constituent of the feedstuff, e.g. 2 or 3 or 4 or more. In one embodiment the term "feed component" encompasses a premix or premix constituents. Preferably, the feed may be a fodder, or a premix thereof, a compound feed, or a premix thereof. A feed additive composition comprising a Bacillus-based component as described herein may be admixed with a compound feed or to a premix of a compound feed or to a fodder, a fodder component, or a premix of a fodder.

The term fodder as used herein means any food which is provided to an animal (rather than the animal having to forage for it themselves). Fodder encompasses plants that have been cut.

The term fodder includes hay, straw, silage, compressed and pelleted feeds, oils and mixed rations, and also sprouted grains and legumes.

Fodder may be obtained from one or more of the plants selected from: alfalfa (lucerne), barley, birdsfoot trefoil, brassicas, Chau moellier, kale, rapeseed (canola), rutabaga (swede), turnip, clover, alsike clover, red clover, subterranean clover, white clover, grass, false oat grass, fescue, Bermuda grass, brome, heath grass, meadow grasses (from naturally mixed grassland swards, orchard grass, rye grass, Timothy-grass, corn (maize), millet, oats, sorghum, soybeans, trees (pollard tree shoots for tree -hay), wheat, and legumes.

The term “compound feed” means a commercial feed in the form of a meal, a pellet, nuts, cake or a crumble. Compound feeds may be blended from various raw materials and additives. These blends are formulated according to the specific requirements of the target animal. Compound feeds can be complete feeds that provide all the daily required nutrients, concentrates that provide a part of the ration (protein, energy) or supplements that only provide additional micronutrients, such as minerals and vitamins.

The main ingredients used in compound feed are the feed grains, which include com, soybeans, sorghum, oats, and barley.

Suitably a premix as referred to herein may be a composition composed of microingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients. Premixes are usually compositions suitable for blending into commercial rations.

Any feedstuff described herein may comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as com gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins.

Furthermore, such feedstuff may contain at least 30%, at least 40%, at least 50% or at least 60% by weight com and soybean meal or corn and full fat soy, or wheat meal or sunflower meal.

In addition, or in the alternative, a feedstuff may comprise at least one high fibre feed material and/or at least one by-product of the at least one high fibre feed material to provide a high fibre feedstuff. Examples of high fibre feed materials include: wheat, barley, rye, oats, by products from cereals, such as com gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp. Some protein sources may also be regarded as high fibre: protein obtained from sources such as sunflower, lupin, fava beans and cotton.

As described herein, feed may be one or more of the following: a compound feed and premix, including pellets, nuts or (cattle) cake; a crop or crop residue: corn, soybeans, sorghum, oats, barley, corn stover, copra, straw, chaff, sugar beet waste; fish meal; freshly cut grass and other forage plants; meat and bone meal; molasses; oil cake and press cake; oligosaccharides; conserved forage plants: hay and silage; seaweed; seeds and grains, either whole or prepared by crushing, milling etc.; sprouted grains and legumes; yeast extract.

The term feed as used herein also encompasses in some embodiments pet food. A pet food is plant or animal material intended for consumption by pets, such as dog food or cat food. Pet food, such as dog and cat food, may be either in a dry form, such as kibble for dogs, or wet canned form. Cat food may contain the amino acid taurine.

The term feed may also encompass in some embodiments fish food. A fish food normally contains macro nutrients, trace elements and vitamins necessary to keep captive fish in good health. Fish food may be in the form of a flake, pellet or tablet. Pelleted forms, some of which sink rapidly, are often used for larger fish or bottom feeding species. Some fish foods also contain additives, such as beta carotene or sex hormones, to artificially enhance the color of ornamental fish.

Also encompassed within the term “feed” is bird food including food that is used both in birdfeeders and to feed pet birds. Typically, bird food comprises of a variety of seeds, but may also encompass suet (beef or mutton fat).

As used herein the term "contacted" refers to the indirect or direct application of the feed additive composition to the product (e.g. the feed). Examples of the application methods which may be used, include, but are not limited to, treating the product in a material comprising the feed additive composition, direct application by mixing the feed additive composition with the product, spraying the feed additive composition onto the product surface or dipping the product into a preparation of the feed additive composition.

The Bacillus-based component may be, in one embodiment, admixed with the product (e.g. feedstuff). Alternatively, it may be included in the emulsion or raw ingredients of a feedstuff.

For some applications, it is important that it is made available on or to the surface of a product to be affected/treated.

The Bacillus-based component may be applied to intersperse, coat and/or impregnate a product (e.g. feedstuff or raw ingredients of a feedstuff) with a controlled amount of a Bacillusbased component.

The DFM comprising at least one bacterial strain can be added in suitable concentrations, for example, in concentrations in the final feed product which offer a daily dose of between about 2xl0³ CFU/g of feed to about 2x10¹¹ CFU/g of feed, suitably between about 2xl0⁶ to about IxlO¹⁰, suitably between about 3.75xl0⁷ CFU/g of feed to about IxlO¹⁰ CFU/g of feed.

Preferably, the Bacillus-based component will be thermally stable to heat treatment up to about 70 °C; up to about 85°C; or up to about 95°C. The heat treatment may be performed from about 30 seconds up to several minutes. The term “thermally stable” means that at least about 50% of Bacillus-based component that was present/active before heating to the specified temperature are still present/active after it cools to room temperature. In a particularly preferred embodiment the Bacillus-based component is homogenized to produce a powder.

Alternatively, the Bacillus-based component is formulated to granules as described in W02007/044968 (referred to as TPT granules) incorporated herein by reference.

In another preferred embodiment when the feed additive composition is formulated into granules, the granules comprise a hydrated barrier salt coated over the protein core. The advantage of such salt coating is improved thermo-tolerance, improved storage stability and protection against other feed additives otherwise having adverse effect on the at least one protease and/or DFM comprising one or more bacterial strains. Preferably, the salt used for the salt coating has a water activity greater than 0.25 or constant humidity greater than 60% at 20°C. Preferably, the salt coating comprises a Na2SO4.

Feed containing the Bacillus-based component may be produced using a feed pelleting process. Optionally, the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets. The mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection. The mixture is heated in the conditioner up to a specified temperature, such as from 60-100°C, typical temperatures would be 70°C, 80°C, 85°C, 90°C or 95°C. The residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minutes 2 minutes., 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.

With regard to the granule at least one coating may comprise a moisture hydrating material that constitutes at least 55% w/w of the granule; and/or at least one coating may comprise two coatings. The two coatings may be a moisture hydrating coating and a moisture barrier coating. In some embodiments, the moisture hydrating coating may be between 25% and 60% w/w of the granule and the moisture barrier coating may be between 2% and 15% w/w of the granule. The moisture hydrating coating may be selected from inorganic salts, sucrose, starch, and maltodextrin and the moisture barrier coating may be selected from polymers, gums, whey and starch.

The granule may be produced using a feed pelleting process and the feed pretreatment process may be conducted between 70°C and 95 °C for up to several minutes, such as between 85°C and 95°C.

The Bacillus-based component may be formulated to a granule for animal feed comprising: a core; an active agent, the active agent of the granule retaining at least 80% activity after storage and after a steam-heated pelleting process where the granule is an ingredient; a moisture barrier coating; and a moisture hydrating coating that is at least 25% w/w of the granule, the granule having a water activity of less than 0.5 prior to the steam-heated pelleting process.

The granule may have a moisture barrier coating selected from polymers and gums and the moisture hydrating material may be an inorganic salt. The moisture hydrating coating may be between 25% and 45% w/w of the granule and the moisture barrier coating may be between 2% and 10% w/w of the granule.

A granule may be produced using a steam-heated pelleting process which may be conducted between 85°C and 95°C for up to several minutes.

Alternatively, the composition is in a liquid formulation suitable for consumption. In one embodiment, such liquid consumption contains one or more of the following: a buffer, salt, sorbitol and/or glycerol. In an additional embodiment, the composition containing the Bacillusbased component as described herein is formulated for delivery via a waterline. Information pertaining to formulations for waterline delivery of direct fed microbials can be found, for example, in International Patent Application Publication No. WO 2023/055850, incorporated by reference herein in its entirety.

Also, the feed additive composition may be formulated by applying, e.g. spraying, the Bacillus -based component onto a carrier substrate, such as ground wheat for example.

In one embodiment, such feed additive composition comprising a Bacillus -based component as described herein may be formulated as a premix. By way of example only the premix may comprise one or more feed components, such as one or more minerals and/or one or more vitamins. Alternatively, the composition is in a liquid formulation suitable for consumption. In one embodiment, such liquid consumption contains one or more of the following: a buffer, salt, sorbitol and/or glycerol.

Also, the feed additive composition may be formulated by applying, e.g., spraying, the Bacillus -based component onto a carrier substrate, such as ground wheat for example.

In one embodiment such Bacillus-based component as described herein may be formulated as a premix. By way of example only the premix may comprise one or more feed components, such as one or more minerals and/or one or more vitamins.

It will be understood that Bacillus-based component as disclosed herein is suitable for addition to any appropriate feed material.

As used herein, the term feed material refers to the basic feed material to be consumed by an animal. It will be further understood that this may comprise, for example, at least one or more unprocessed grains, and/or processed plant and/or animal material such as soybean meal or bone meal.

It will be understood by the skilled person that different animals require different feedstuffs, and even the same animal may require different feedstuffs, depending upon the purpose for which the animal is reared.

Preferably, the feedstuff may comprise feed materials comprising maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/ or any of the by-products, as well as protein rich components like soybean mean, rape seed meal, canola meal, cotton seed meal, sunflower seed mean, animal-by-product meals and mixtures thereof. In another embodiment, the feedstuff may comprise animal fats and/or vegetable oils.

Optionally, the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins. Preferably, the feedstuff is a com soybean meal mix.

In another aspect, there is provided a method for producing a feedstuff. Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and/or enzyme may be added, as discussed further below. Production of feedstuff may also involve an additional step that includes extrusion or expansion prior to pelleting, in particular, by suitable techniques that may include at least the use of steam.

The feedstuff may be a feedstuff for a monogastric animal, such as poultry (for example, broiler, layer, broiler breeders, turkey, duck, geese, water fowl), swine (all age categories), a pet (for example dogs, cats) or fish. In one embodiment, the feedstuff is for poultry.

The Bacillus-based component described herein can further include supplemental enzymes that can additionally be used as additives to animal feed, particularly poultry and swine feeds, as a means to improve nutrient utilization and performance characteristics. In one embodiment, the disclosure relates to administration of a composition comprising the Bacillus -based component described herein and one or more exogenous feed enzymes. The exogenous feed enzymes can include, but are not limited to, xylanase, amylase, phytase, beta- glucanase, glucoamylase, lipase, and protease.

Xylanase is the name given to a class of enzymes that degrade the linear polysaccharide -1,4-xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell walls. Xylanases, e.g., endo-P-xylanases (EC 3.2.1.8) hydrolyze the xylan backbone chain. In one embodiment, provided herein are compositions comprising any of Bacillus-based component described herein and one or more xylanase. In one embodiment, the xylanase may be any commercially available xylanase. Suitably the xylanase may be an endo- 1 ,4-P-d- xylanase (classified as EC 3.2.1.8) or a l,4p-xylosidase (classified as EC 3.2.1.37). In one embodiment, the disclosure relates to a Bacillus-based component described herein in combination with an endoxylanase, e.g. an endo-l,4-P-d-xylanase, and another enzyme. All E.C. enzyme classifications referred to herein relate to the classifications provided in Enzyme Nomenclature — Recommendations (1992) of the nomenclature committee of the International Union of Biochemistry and Molecular Biology — ISBN 0-12-226164-3, which is incorporated herein. In another embodiment, the xylanase may be a xylanase from Bacillus, Trichodermna, Therinomyces, Aspergillus and Penicillium. In still another embodiment, the xylanase may be the xylanase in Axtra XAP® or Avizyme 1502®, both commercially available products from Danisco A/S. In one embodiment, the xylanase may be a mixture of two or more xylanases. In still another embodiment, the xylanase is an endo-l,4-P-xylanase or a 1,4-P-xylosidase. In yet another embodiment, the xylanase is from an organism selected from the group consisting of: Bacillus, Trichoderma, Thermomyces, Aspergillus, Penicillium, and Humicola. The term protease as used herein is synonymous with peptidase or proteinase. The protease may be a subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.X.X). In one embodiment, the protease is a subtilisin. Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are also suitable. The protease may be a serine protease or a metalloprotease, e.g., an alkaline microbial protease or a trypsin-like protease. In one embodiment, provided herein are compositions comprising any of the oxygen- tolerant M. elsdenii strain compositions disclosed herein and one or more protease. In a further embodiment, the composition further comprises one or more yeast strains and/or yeast extract

In one embodiment, the disclosure relates to administration of a composition comprising a £>acz7/zz.s-bascd component described herein and xylanase. The composition can comprise 10- 50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500- 550, 550-600, 600-650, 650-700, 700-750, and greater than 750 xylanase units/g of composition. In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, and greater than 8000 xylanase units/g composition. It will be understood that one xylanase unit (XU) is the amount of enzyme that releases 0.5 pmol of reducing sugar equivalents (as xylose by the Dinitro salicylic acid (DNS) assay-reducing sugar method) from an oat-spelt-xylan substrate per min at pH 5.3 and 50° C. (Bailey, et al., Journal of Biotechnology , Volume 23, (3), May 1992, 257-270).

Amylase is a class of enzymes capable of hydrolysing starch to shorter-chain oligosaccharides, such as maltose. The glucose moiety can then be more easily transferred from maltose to a monoglyceride or glycosylmonoglyceride than from the original starch molecule. The term amylase includes a-amylases (E.C. 3.2.1.1), G4-forming amylases (E.C. 3.2.1.60), 0- amylases (E.C. 3.2.1.2) and y-amylases (E.C. 3.2.1.3). Amylases may be of bacterial or fungal origin, or chemically modified or protein engineered mutants. In one embodiment, provided herein are compositions comprising a Bacz7/us-based component described herein and one or more amylase for administration to an animal. In one embodiment, the amylase may be a mixture of two or more amylases. In another embodiment, the amylase may be an amylase, e.g. an a-amylase, from Bacillus licheniformis and an amylase, e.g. an a-amylase, from Bacillus amyloliquefaciens. In one embodiment, the a-amylase may be the a-amylase in Axtra XAP® or Avizyme 1502®, both commercially available products from Danisco A/S. In yet another embodiment, the amylase may be a pepsin resistant a-amylasc, such as a pepsin resistant Trichoderma (such as Trichoderma reesei) alpha amylase. A suitably pepsin resistant a- amylase is taught in UK application number 101 1513.7 (which is incorporated herein by reference) and PCT/IB2011/053018 (which is incorporated herein by reference).

In one embodiment, the disclosure relates to administration of a composition comprising a Bacillus-based component described herein and an amylase. The composition can additionally comprise a Bacillus-based component described herein, xylanase and amylase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 amylase units/g composition. In another embodiment, the composition comprises 500- 1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500- 5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, 8000-8500, 8500- 9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000- 15000 and greater than 15000 amylase units/g composition. It will be understood that one amylase unit (AU) is the amount of enzyme that releases 1 mmol of glucosidic linkages from a water insoluble cross-linked starch polymer substrate per min at pH 6.5 and 37° C. (this may be referred to herein as the assay for determining 1 AU).

The term protease as used herein is synonymous with peptidase or proteinase. The protease may be a subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.X.X). In one embodiment, the protease is a subtilisin. Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are also suitable. The protease may be a serine protease or a metalloprotease, e.g., an alkaline microbial protease or a trypsin-like protease. In one embodiment, provided herein are compositions comprising any of the Bacillusbased components described herein and one or more protease. In another embodiment, the compositions can comprise any of the Bacillus-based components described herein and one or more of a protease, amylase, and/or xylanase. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus sp., e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841), subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin), and Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583). Examples of useful proteases also include but arc not limited to the variants described in WO 92/19729 and WO 98/20115, incorporated by reference herein. In one embodiment, the protease is selected from the group consisting of subtilisin, a bacillolysin, an alkine serine protease, a keratinase, and a Nocardiopsis protease.

In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200- 250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700- 750, and greater than 750 protease units/g composition. In another embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000 protease units/g composition. [0099] It will be understood that one protease unit (PU) is the amount of enzyme that liberates from the substrate (0.6% casein solution) one microgram of phenolic compound (expressed as tyrosine equivalents) in one minute at pH 7.5 (40 mM Na2PC>4/lactic acid buffer) and 40° C. This may be referred to as the assay for determining 1 PU.

In yet further embodiments, the compositions can comprise any of the Bocri/t/. -bascd components described herein and one or more phytase. The phytase for use in the present invention may be classified a 6-phytase (classified as E.C. 3.1.3.26) or a 3-phytase (classified as E.C. 3.1.3.8). In one embodiment the phytase is a Citrobacter phytase derived from e.g. Citrobacter freundii, preferably C. freundii NCIMB 41247 and variants thereof e.g. as disclosed in W02006/038062 (incorporated herein by reference) and W02006/038128 (incorporated herein by reference), Citrobacter braakii YH-15 as disclosed in WO 2004/085638, Citrobacter braakii ATCC 51113 as disclosed in W02006/037328 (incorporated herein by reference), as well as variants thereof e.g. as disclosed in W02007/112739 (incorporated herein by reference) and WO2011/117396 (incorporated herein by reference), Citrobacter amalonaticus, preferably Citrobacter amalonaticus ATCC 25405 or Citrobacter amalonaticus ATCC 25407 as disclosed in W02006037327 (incorporated herein by reference), Citrobacter gillenii, preferably Citrobacter gillenii DSM 13694 as disclosed in W02006037327 (incorporated herein by reference), or Citrobacter intermedins, Citrobacter koseri, Citrobacter murliniae, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii, Citrobacter youngae, Citrobacter species polypeptides or variants thereof. In some embodiments, the phytase is an E. coli phytase marketed under the name Phyzymc XP™ Danisco A/S. Alternatively, the phytase may be a Buttiauxella phytase, e.g. a Buttiauxella agrestis phytase, for example, the phytase enzymes taught in WO 2006/043178, WO 2008/097619, WO2009/129489, W02008/092901, WO2009/129489, or WO2010/122532. The phytase can also be one of those disclosed in W02020/106796, all of which are incorporated herein by reference. In one embodiment, the phytase may be a phytase from Hajma, e.g. from Hafnia alvei, such as the phytase enzyme(s) taught in US2008263688, which reference is incorporated herein by reference. In one embodiment, the phytase may be a phytase from Aspegillus, e.g. from Apergillus orzyae. In one embodiment, the phytase may be a phytase from Penicillium, e.g. from Penicillium funiculo sum.

In one embodiment, the phytase is present in the Bacillus -based composition in range of about 200 FTU/kg to about 1000 FTU/kg feed, more preferably about 300 FTU/kg feed to about 750 FTU/kg feed, more preferably about 400 FTU/kg feed to about 500 FTU/kg feed. In one embodiment, the phytase is present at more than about 200 FTU/kg feed, suitably more than about 300 FTU/kg feed, suitably more than about 400 FTU/kg feed. In one embodiment, the phytase is present at less than about 1000 FTU/kg feed, suitably less than about 750 FTU/kg feed. In one embodiment, the phytase is present in the Bacillus-based composition in range of about 40 FTU/g to about 40,000 FTU/g composition, such as about 80 FTU/g composition to about 20,000 FTU/g composition, and such as about 100 FTU/g composition to about 10,000 FTU/g composition, and such as about 200 FTU/g composition to about 10,000 FTU/g composition. In one embodiment, the phytase is present in the composition at more than about 40 FTU/g composition, suitably more than about 60 FTU/g composition, suitably more than about 100 FTU/g composition, suitably more than about 150 FTU/g composition, suitably more than about 200 FTU/g composition. In one embodiment, the phytase is present in the composition at less than about 40,000 FTU/g composition, suitably less than about 20,000 FTU/g composition, suitably less than about 15,000 FTU/g composition, suitably less than about 10,000 FTU/g composition. It will be understood that as used herein 1 FTU (phytase unit) is defined as the amount of enzyme required to release 1 pmol of inorganic orthophosphate from a substrate in one minute under the reaction conditions defined in the ISO 2009 phytase assay — A standard assay for determining phytase activity and 1 FTU can be found at International Standard ISO/DTS 30024: 1-17, 2009. In one embodiment, the enzyme is classified using the E.C. classification above, and the E.C. classification designates an enzyme having that activity when tested in the assay taught herein for determining 1 FTU.

The Bacillus-based component described herein may be placed on top of the animal feed, i.e., top fed. Alternatively, the Bacillus-based component described herein may be added to a liquid such as in the drinking water of the animal.

As used herein the term "contacted" refers to the indirect or direct application of a Bacillus-based component as described herein to a product (e.g. the feed).

Examples of application methods which may be used, include, but are not limited to, treating the product in a material comprising the Bacillus-based component, direct application by mixing a feed additive composition Bacillus-based component as described herein with the product, spraying such feed additive composition onto the product surface, dipping the product into a preparation of the feed additive composition or delivering the Bacillus-based component as described herein in a liquid formulation via waterline. In one embodiment a feed additive composition Bacillus-based component as described herein is admixed with the product (e.g. feedstuff). Alternatively, the feed additive composition may be included in the emulsion or raw ingredients of a feedstuff. This allows the composition to impart a performance benefit.

A method of preparing the Bacillus-based component as described herein may also comprise the further step of pelleting the powder. The powder may be mixed with other components known in the art. The powder, or mixture comprising the powder, may be forced through a die and the resulting strands are cut into suitable pellets of variable length.

Optionally, the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets. The mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection. The mixture is heated in the conditioner up to a specified temperature, such as from 60-100°C, typical temperatures would be 70°C, 80°C, 85°C, 90°C or 95°C. The residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.

It will be understood by the skilled person that different animals require different feedstuffs, and even the same animal may require different feedstuffs, depending upon the purpose for which the animal is reared. Optionally, the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins. In some embodiments, the feedstuff is a corn soybean meal mix.

Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and enzyme may be added. Production of feedstuff may also involve an additional step that includes extrusion or expansion prior to pelleting, in particular by suitable techniques that may include at least the use of steam.

As was noted above, the Bacillus-based component and/or a feedstuff comprising the same may be used in any suitable form. It may be used in the form of solid or liquid preparations or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, pellets, tablets, dusts, and granules which may be wettable, spray-dried or freeze-dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous- organic solutions, suspensions and emulsions.

In some applications, the feed additive compositions may be mixed with feed or administered in the drinking water.

A Bacillus-based component, comprising admixing a Bacillus-based component as described herein with a feed acceptable carrier, diluent or excipient, and (optionally) packaging.

The feedstuff and/or Bacillus-based component may be combined with at least one mineral and/or at least one vitamin. The compositions thus derived may be referred to herein as a premix. The feedstuff may comprise at least 0.0001 % by weight of Bacillus-based component. Suitably, the feedstuff may comprise at least 0.0005%; at least 0.0010%; at least 0.0020%; at least 0.0025%; at least 0.0050%; at least 0.0100%; at least 0.020%; at least 0.100% at least 0.200%; at least 0.250%; at least 0.500% by weight of the Bacillus-based component.

Preferably, a food or Bacillus-based component may further comprise at least one physiologically acceptable carrier. The physiologically acceptable carrier is, in one embodiment, selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na^SOa, Talc, PVA and mixtures thereof. In a further embodiment, the food or feed may further comprise a metal ion chelator. The metal ion chelator may be selected from EDTA or citric acid.

In one embodiment a Bacillus-based component as described herein (whether or not encapsulated) can be formulated with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, NaiSCU, Talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate and mixtures thereof.

In some embodiments, a Bacillus-based component as described herein, will be in a physiologically acceptable carrier. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates. Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

EXAMPLES

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used with this disclosure.

The disclosure is further defined in the following Examples. It should be understood that the Examples, while indicating certain embodiments, is given by way of illustration only. From the above discussion and the Examples, one skilled in the art can ascertain essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt to various uses and conditions.

Example 1

Enterococcus spp. and Bacillus strains

Thirty Enterococcus spp. isolates were collected from culture collections in North America, as summarized in FIG. 1. During collection, emphasis was placed on sourcing Enterococcus spp. strains isolated from poultry outbreaks, allowing confidence that the tested strains were virulent and capable of causing disease.

The inhibitory potential of 3 to 4 Bacillus strains was tested in total. These included both DuPont proprietary DFM strains and one Bacillus isolated from a competitor DFM product, as summarized in Table 1. All tested Bacillus strains are commercialized for use in poultry production. Enviva® PRO, which is commercially available from Danisco A/S is a combination of Bacillus strain 2084 Accession No. NRR1 B-50013, Bacillus strain LSSAO1 (a.k.a. Bacillus strain BS8) Accession No. NRRL B-50104, and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507 (as taught in U.S. Pat. No. 7,754,469 - incorporated herein by reference).

Table 1: DFM Bacillus strains tested in this study

The non-DuPont Bacillus products were purchased, and isolated in triplicate from 3 separate production batches. All strains were identified to ensure that the strains recovered matched the strain claims on the product label. CFS from single Bacillus strains were tested against several isolates representative of each species. Number of representatives per species is listed in Table 2 below. Table 3: Representative isolates tested per species

Example 2 Production of Cell Free Supernatants (CFS) and Enterococcus Species Growth Inhibition Assay

An inoculating loop is used to inoculate a 30 ml shaker tube with 10 ml tryptic soy broth (TSB) from a frozen Bacillus stock. The tube is incubated in a 32°C incubator for 24 hours and shaken at 130 to grow the Bacillus.

The optical density (OD) was checked on a spectrometer (wavelength 600 nm, absorbance 0) after incubating flasks for 18 hours. 2 ml of sterile TSB was pipetted into a cuvette to create a blank control. A lOx dilution of Bacillus was created by pipetting 1.8 ml sterile TSB and 0.2 ml of 18 hr growth into each cuvette. Cuvettes were covered and inverted to ensure thorough mixing. The absorbance of the Bacillus dilutions was determined to be between 0.25 and 0.3 (samples with absorbance readings below 0.25 were re-incubated until absorbance reached acceptable levels).

The Bacillus growth was transferred from each flask into sterile 250 ml centrifuge bottles and centrifuged at 10,000 rpm for 10 minutes. After centrifuging, the supernatants of each Bacillus type were transferred to a Nalgene bottle top filter and pumped into 50 ml conical tubes.

This procedure was followed for all Bacillus strains. Cell Free Supernatant (CFS) was then frozen at -80 °C until required.

Enterococcus spp. strains were inoculated from deep frozen stock cultures in a BHI (brain and heart infusion) broth and a BHI agar plate (to check purity) and incubated overnight at 37 °C. All strains were subcultured at least twice before inclusion in the assay to ensure adaptation to the growth medium.

All assays were performed in biological duplicate (including 2 technical replicates) for each Bacillus-based DFM identified in Table 1 above. CFS from single Bacillus strain as well as the blend (BS8, 15AP4, 2084 at the ratio 1:1:1) were tested.

Twenty mL of BHI broth was incubated for 1 hour prior to assaying, to avoid thermic shock for the Enterococcus spp. cells.

In a 96-well UV treated microliter plate with flat-bottom wells, the medium (BHI broth) and the CFS and target microorganism were added as follows:

• Positive control: 200pl medium + 2pl bacterium (1%)

• Negative control : 200pl medium

• CFS assay well: 180pl medium + 20|al CFS + 2pl bacterium (1%)

• Negative CFS well: 180 1 medium + 20pl CFS

Plates were incubated for 18 hours at 37°C in a TECAN Spark Multimode readers to record absorbance, with data transferred directly to a computer for analysis. Measurements were taken every 15 minutes.

Results are given as % of inhibition comparing control at OD capture at the middle of the exponential growth phase of the Enterococcus spp. alone and compared with the corresponding OD of the treated {Enterococcus spp. incubated with Bacillus CFS).

Classes of inhibition was determined as follows:

• Strong inhibitory potential: % of inhibition >= 60

• Moderate inhibitory potential: % of inhibition is <60 and >=40

• Non-significant inhibitory potential: % of inhibition <40 and >=25

• No inhibitory potential

• Promotion of the growth of the pathogen <0

Inhibition showing a negative value means that the CFS promotes the growth of the pathogen. Inhibition over 100% indicates that the pathogen isolate has been lysed by the Bacillus strains, rather than simply inhibited. Example 3

Bacillus -Based Components for Inhibiting or Delaying the Growth of One Representative Strain of 9 Different Enterococuss spp. Encountered in Poultry Diseases

Average inhibition by each of the 3 Enviva® PRO strains (Bacillus-based component) varied according to the Enterococcus spp. tested. Table 3 shows the average growth inhibition of 9 Enterococcus spp. strains by Enviva® PRO strains (alone or in combination) and comparison with another Bacillus CFS obtained from a commercial product available in poultry market (Bacillus licheniformis DSM28710, B-ACT®, Huvepharma).

Table 3. Average inhibition of 9 Enterococcus spp. strains collected from poultry production by

4 Bacillus DFM strains (Bacillus-based component)

As shown in Table 3, Enviva® PRO strains Bacillus 15AP4, BS8 and 2084 (alone or in combination) demonstrate strong (>=60%) and consistent inhibitory potential against 7 out the 9 Enterococcus species tested. These species are E. cecorum; E. avium, E. durans, E. faecium, E. gallinarum, E. hirae and E. mundtii.

Additionally, FIG. 2 shows the different classes of inhibition as a function of Bacillus strains tested and for the different Enterococcus species tested. Thus, it was shown that both Bacillus 2084 and BS8 exhibited a strong inhibition against 88.88% of the tested Enterococcus spp. This percentage reached 77.77 % for the remaining strain constitutive of Enviva® PRO, specifically the strain 15AP4. At least 100% of the tested Enterococcus spp. are either strongly or moderately inhibited by the 3 Bacillus strains 15AP4, 2084 and BS8.

These percentages of inhibition differed significantly from those recorded for the Bacillus probiotic strain B. licheniformis DSM 28710. The highest percentage of inhibition recorded for the CFS obtained from DSM28710 was 44%, shown for the strain representative of the Enterococcus casseliflavus. Interestingly, this CFS promoted the growth (-18% in average) of 4 different isolates representative of the species Enterococcus cecorum. The growth of the 7 other Enterococcus species was considered non-affected by the B. licheniformis DSM28710 CFS as recorded percent inhibition ranged from between 0 and 20% (FIG. 2).

Example 4

Zlm/ZZu.s- Based Components for Inhibiting or Delaying Growth of Multiple Representative Strains of 9 different Enterococuss spp.

This assay was performed to evaluate the consistency of inhibition among the species. Therefore, multiple strains belonging to the same species were included in the assay. The CFS from the 3 Bacillus (15AP4, 2084 and BS8) were tested as a combined product (1:1:1 ratio). Depicted results show the average of 2 biological replicates (including 4 technical replicates).

FIG. 3 shows the percent inhibition recorded for the tested CFS (blend of the 3 Bacillus CFS at the ratio of 1:1:1 for BS8, 2084 and 15AP4) against multiple strains of Enterococcus belonging to 9 different species. The average percentage of inhibition against E. durans (n=3), E.faecium (n=4), E. hirae (n=4), E. cecorum (n=4) were all >= 60%, meaning that the CFS of the combined Enviva® PRO strains were considered strongly inhibitory. The average percent inhibition for the remaining Enterococcus species ranged between 40 and 59%, thus considered as moderately inhibitory.

FIG. 4 shows the antimicrobial activity of the CFSs of Bacillus strains 15AP4, BS8 and 2084 (Bacillus -based component in combination ratio 1:1:1) against Enterococcus mundtii 64247-EN01 expressed as percent inhibition at the exact end-point when the control pathogen curve reaches the middle of the exponential growth phase OD (here = 0.5) ) as well as how the delay of growth was determined.

FIG. 5 shows that the growth of the Enterococcus isolates belonging to species E. avium, E. casseliflavus and E.faecalis were delayed by 45, 41 and 40 minutes on average, respectively, compared to the pathogen alone. Additionally, the species E. durans and E. hirae experienced growth inhibition wherein growth was delayed by 70 and 65 minutes (on average), respectively.

The remaining Enterococcus species showed that growth was highly impacted by the presence of Enviva® PRO CFS, since the middle of their exponential growth phase was reached more than 2 hours in average compared to the control pathogen.

Previous work has shown that broilers develop sepsis between weeks 1-3 in production as is seen in both outright inhibition and delays in Enterococcus spp. hitting the exponential growth phase. Supplementation of poultry feed with the Bacillus- seA DFM(s) described herein may delay gut colonization, adherence and subsequent invasion, meaning that birds make it to slaughter with reduced incidence of clinical symptoms.

These data are quite compelling in comparison to the inhibition ranges of other pathogens that have been tested. The effect also seems quite consistent across the range of Enterococcus spp. isolates tested, despite a natural variation in Bacillus efficacy.

The in vitro results presented herein demonstrate that supplementation of poultry feed with a Bacillus-based component described herein may be very effective in inhibiting or delaying all or part of the growth of the emerging pathogen, Enterococcus species as well as inhibiting or delaying the growth of Enterococcus spp. in animals.

Example 5

Bacillus -Based Components for Limiting Maximum Growth of Enterococuss spp. in Animals

The Bacillus CFS also prevented the pathogen from reaching maximum growth as reflected by the max OD captured at the end of the exponential growth phase. This was another parameter used to assess the preventive effect of the Bacillus strain on the pathogen.

FIG. 6 illustrates how this parameter is calculated for each strain using as an example the strain E. f aecium 62467-EN05 where maximum growth is limited by the presence of the Bacillus CFS (here Bacillus CFS reduces maximum growth by 1.085 - 0.75= 0.33 point of O.D.). Thus, this figure illustrates that the Enviva® PRO CFS reduces by the max OD reached by the pathogen. Furthermore, FIG. 7 shows the difference in OD captured due to the limiting effect of the Bacillus CFS for all treated Enterococcus strains.

Example 6

Bacillus Strains Inhibit Enterococuss spp.

Materials and Methods

Isolates belonged to nine Enterococuss species (E. avium, E. casseliflavus, E. cecorum, E. durans, E.faecalis, E. faecium, E. gallinarum, E. hirae and E. mundtii), had been collected during 2007 to 2023 and were procured from a mixture of IFF customers and collaborating institutions in five different countries (Belgium, Israel, Finland, Poland and USA). They originated from a mixture of healthy and symptomatic poultry (broilers, breeders, eggs, laying hens, turkeys or unknown) from varied biological sites (gut, joint, spine, organ, litter or unknown). Summary details of the origin and diversity of the isolates are presented in FIG. 8.

The species identification of each isolate was confirmed by peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry, 16S ribosomal RNA sequencing (16S), whole genome sequencing (WGS), or PCR (FIG. 8.). Isolates were supplied in frozen vials in culture media and aliquots containing 30% glycerol were stored at -80°C. Isolates were cultured in Brain Heart Infusion medium (BHI; Biokar Diagnostics, Beauvais, France) at 37 °C in anaerobic conditions in the laboratory, until further use.

Results

The percentage growth inhibition of other (non-E. cecorum) Enterococcus spp. isolates by CFS from each of Enviva® PRO strains Bacillus 15AP4, BS8 and 2084 and by the 1:1:1 blend of these strains is shown in FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D, respectively. Notwithstanding the smaller sample sizes of the non-E. cecorum species relative to E. cecorum (n = 1-5 vs. n = 112), inhibition of the other Enterococcus spp. isolates was generally at a lower level than of E. cecorum (median inhibition 10-70% vs. > 70%; FIG. 9A - FIG. 9D). The inhibitory response was also more variable for the non-E. cecorum isolates, both across and within species. Nevertheless, isolates of several species, including E. avium, E. hirae and E. mundtii were consistently inhibited by all three Bacillus strains (median inhibition 50-90%). In addition, when tested as a blend, the Bacillus strains had a consistent growth inhibitory effect against all of the Enterococcus spp. isolates (to varying degrees), regardless of species, and inhibition of isolates of three non-E. cecorum species (E. avium, E. hirae and E. mundtii ) was relatively strong (median inhibition >50%; FIG. 9D). All of the tested isolates of these species were clinical isolates. An example of the effect of the blend of the three Bacillus strains on the growth kinetics of three clinical E. casseliflavus isolates is presented in FIG. 10. It can be seen that the probiotic blend inhibited and delayed the onset of the growth phase of the two isolates (61037-EC02 and 64246-EN01).

Previous studies of growth inhibition of poultry-relevant Enterococcus spp. isolates by probiotic bacteria are scarce, especially those that have evaluated the inhibition of non-E. cecorum species of Enterococcus, for which no previously published studies could be identified. In this Example, CFS from all of the tested probiotic Bacillus strains (BS8, 15AP4 and 2084), as well as the 1 : 1 :1 blend of these strains, inhibited the growth of the E. cecorum isolates, on average by >70%, with relatively low variability around the mean values (< 6% SEM).

Without being bound to theory, this suggests that despite the apparent high variability in animal and geographic origins as well as biological sampling origin, they were all highly and relatively consistently sensitive to the growth inhibitory effects of the probiotic Bacillus strains. A strong and consistent inhibitory effect against E. cecorum isolates in vitro makes these three probiotic strains a potential candidate for in vivo testing because, according to 27 years of data collected by the French epidemiological surveillance network (RNOEA), E. cecorum is the dominant Enterococcus spp. causing locomotor syndromes (77.9%), septicemia (53.4%) and other enterococcal diseases (36.2%) (Souillard et al., 2022). In contrast to the strong inhibition of E. cecorum by the Bacillus strains CFS, there was no growth inhibitory effect of CFS from the comparator strain (B. licheniformis DSM 28710).

Furthermore, the results appeared to indicate a degree of interaction among the CFS from the three Bacillus strains that together inhibited the majority of the non-E. cecorum Enterococcus spp. isolates - albeit it at a lower level then for the E. cecorum isolates (10 to 70% inhibition, on average, compared with >70%, on average, for the E. cecorum isolates) - whereas when applied individually they did not. No comparable studies are believed to exist that have tested interactions of probiotic strains against non-E. cecorum or E. gallinarum spp. of Enterococcus.

In conclusion, the results confirm and extend previous findings on the inhibition of E. cecorum isolates by probiotic Bacillus strains using a larger, more diverse, sample (n = 112 isolates). E. cecorum growth was consistently reduced by CFS from Bacillus strains BS8, 15AP4 and 2084, and a blend of the three, on average by >70%. In addition, presented for the first time is the first report of an interaction effect of the probiotic Bacillus blend in inhibiting the growth of isolates of other species of Enterococcus, some of which are also significant causative agents of Enterococcal disease in poultry.

Claims

CLAIMS What is claimed is:

1. A method for inhibiting or delaying all or part of the growth of pathogenic Enteroccocus spp.m' an animal comprising administering an effective amount of at least one Bacillus-based component selected from the group consisting of: a Bacillus-based direct fed microbial comprising one or more Bacillus bacterial strains, a supernatant obtained from a Bacillus culture, and a combination thereof to an animal, wherein said pathogenic Enteroccocus spp. is selected from the group consisting of E. avium, E. casseliflavus, E. durans, E. faecalis, E. faecium, E. hirae, and E. mundtii.

2. The method of claim 1, wherein the Bacillus-based direct fed microbial is selected from the group consisting of Bacillus velezensis, Bacillus amyloliquefaciens , Bacillus licheniformis, Bacillus pumilis and Baccillus subtilis.

3. The method of claim 1 or 2, wherein the Bacillus-based direct fed microbial is selected from the group consisting of one or more of the following strains: Bacillus strain 2084 Accession No. NRR1 B-50013, Bacillus strain LSSAO1 Accession No. NRRL B-50104 and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507.

4. The method of claim 1 or 2, wherein the animal is a monogastric animal.

5. The method of claim 3, wherein the animal is a monogastric animal.

6. The method of claim 1 or 2, wherein the animal is a multigastric animal.

7. The method of claim 3, wherein the animal is a multigastric animal.

8. The method of claim 1 or 2, wherein the monogastric animal is poultry.

9. The method of claim 3, wherein the monogastric animal is poultry.

10. The method of claim 1 or 2, wherein the at least one Bacillus -based component is administered directly to an animal through animal feed whether in the feed or on top of the feed or in a liquid.

11. The method of claim 10, wherein the at least one Bacillus-based component is administered directly to an animal through a waterline.

12. The method of claim 3, wherein the at least one Bacillus -based component is administered directly to an animal through animal feed whether in the feed or on top of the feed or in a liquid.

13. The method of claim 12, wherein the at least one Bacillus -based component is administered directly to an animal through a waterline.

14. The method of claim 10, wherein the at least one Bacillus-based component is administered to the animal in a form selected from the group consisting of a feedstuff, a feed additive composition, a premix or in a liquid.

15. The method of claim 12, wherein the Bacillus -based component is administered to the animal in a form selected from the group consisting of a feedstuff, a feed additive composition, a premix or in in a liquid.

16. The method of any one of claims 1-15, further comprising administering one or more enzymes selected from the group consisting of phytase, protease, amylase, xylanase, lipase, or glucoamylase to the animal.

17. The method of claim 16, wherein the enzymes comprise a xylanase, an amylase, and a protease.

18. The method of any one of claims 1-17, wherein the method delays or inhibits the growth of pathogenic Enteroccocus spp.in an animal by about 5-100% (such as any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of any values falling in between these percentages), compared to animals that have not been administered an effective amount of at least one Bacillus-based component.