RU2789148C2 - Components based on microorganism of bacillus genus for inhibition or deceleration of enterococcus spp. growth in animals - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: invention relates to a method for inhibition or deceleration of growth of representatives of pathogenic Enterococcus cecorum in poultry. The proposed method includes administration to an animal of effective amount of at least one of microorganism strains for eating, selected from a group of 2084 Bacillus strain under access No. NRRl B-50013 and 15A-P4 Bacillus strain under access No. ATCC 15 PTA-6507.

EFFECT: invention provides effective inhibition or deceleration of growth of representatives of pathogenic Enterococcus cecorum in poultry.

3 cl, 7 dwg, 7 tbl, 3 ex

Description

FIELD OF TECHNOLOGY

The technical field to which the present invention relates relates to the use of components based on Bacillus to inhibit or slow the growth of Enterococcus spp. in animals.

BACKGROUND OF THE INVENTION

Enterococcusis a large genus of lactic acid bacteria belonging to the phylum Firmicutes.Enterococciare Gram-positive cocci that often occur as pairs (diplococci) or short chains and are difficult to distinguish from streptococci by physical characteristics alone.Enterococci are facultative anaerobic organisms, i.e. they are capable of cellular respiration in both oxygen-rich and oxygen-poor environments. Although they cannot spore, Enterococci are tolerant of a wide range of environmental conditions: extreme temperatures (10–45°C), pH (4.5–10.0) and high concentrations of sodium chloride. Representatives of the genusEnterococcus until 1984 were classified asStreptococcus group D when analysis of genomic DNA showed that it would be appropriate to classify them in a separate genus. It is known that some speciesEnterococcus are opportunistic pathogens if they enter the intestines. This takes place in relationE. avium, E. gallinarum AndE. cecorum, andE. cecorum is important from the point of view of economic losses incurred by poultry farmers in the mass production of broiler chickens.

Enterococcus cecorum is a natural inhabitant of the intestines of birds and other vertebrates such as horses, cattle, pigs, dogs, cats, canaries, pigeons, turkeys and musky ducks. It is considered an emerging pathogen in poultry and other bird species. Necrosis of the femoral head and spondylitis have been described as the main pathological changes in infected chickens.

Disturbances or damage to the normal function of the intestine can lead to the transfer of Enterococcus cecorum , which is part of the normal intestinal flora, into the spine of birds. Spinal infections caused by Enterococcus cecorum result in spinal and joint lesions, lameness, and death in a condition known as enterococcal spondylolisthesis or "ruffled back syndrome".

Spondylitis, referred to by poultry owners as "ruffled back syndrome", has been known in the industry for many years and is commonly seen in heavy, fast growing birds, especially males and broilers (Aziz, T. & Barnes, H.J. (2009). Spondylitis is emerging in broilers, World Poultry, 25, 19).

Highly pathogenic and antibiotic resistant strains of Enterococcus cecorum continue to cause economic damage to commercial broiler chicken production. Thus, standard hygiene procedures and antimicrobial therapy on farms have been shown to be insufficient to control outbreaks of pathogenic Enterococcus spp., especially Enteroccocus cecorum .

Therefore, a safe and effective alternative is needed to control this important new pathogen.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, a method is disclosed for inhibiting or slowing the growth of all or part of pathogenic Enteroccocus spp. in an animal, which involves administering to the animal an effective amount of at least one component based on a microorganism of the genus Bacillus , selected from the group consisting of a microbial preparation for ingestion based on a microorganism of the genus Bacillus , containing one or more bacterial strains of the genus Bacillus , the supernatant obtained from a culture of Bacillus , or combinations thereof.

In a second embodiment, the Bacillus edible microbial preparation is selected from the group consisting of Bacillus amyloliquefaciens , Bacillus licheniformis , Bacillus pumilis , and Bacillus subtilis .

In a third embodiment, the Bacillus edible microbial preparation is selected from one or more of the following strains: Bacillus strain 2084 Accession No. NRRl B-50013, Bacillus strain LSSAO1 Accession No. NRRL B-50104, and Bacillus strain 15A-P4 under ATCC accession number PTA–6507.

In a fourth embodiment, the animal may be a monogastric animal, preferably a poultry.

In a fifth embodiment, the animal may be a multi-gastric animal.

In a sixth embodiment, at least one component based on a microorganism of the genus Bacillus can be administered directly to the animal with the animal food, either as contained in the food, or added on top of the food, or in the form of a liquid, such as water.

In a seventh embodiment, the Bacillus microorganism component may be administered to the animal in a form selected from the group consisting of a feed product, a feed additive composition, a premix, or in a liquid such as water.

BRIEF DESCRIPTION OF GRAPHICS

The figure 1 shows the average value of inhibition against strains of E. cecorum collected in the American and European poultry industry, using strains of Enviva ^® PRO.

Figure 2 shows the growth kinetics of E. cecorum strain G84-68 with/without BS8 Bacillus .

Figure 3 shows the growth kinetics of E. cecorum strain D45-08 with/without BS8 Bacillus .

Figure 4 shows the growth inhibition profile of Enterococcus cecorum strain 11976-2 incubated with or without cell-free supernatant (CFS) Bacillus BS8, 15AP4 Bacillus or 2084 Bacillus .

Figure 5 shows the antimicrobial activity of B. amyloliquefaciens subsp. plantarum CFS 15AP4, BS8 and 2084 against Enterococcus gallinarum VTT E–97776T, expressed as % inhibition at an endpoint exactly corresponding to the achievement of the control pathogen OD curve of 0.4.

Figure 6 shows the growth profiles of Enterococcus avium E 84197 incubated with or without Bacillus strain 2084 CFS.

Figure 7 shows the antimicrobial activity of CFS DSM7 ^T Bacillus amyloliquefaciens , DSM10 ^T B. subtilis and DSM13 ^T B. licheniformis against 10 clinical isolates of Enterococcus cecorum , expressed as % inhibition at an endpoint exactly corresponding to the achievement of the control pathogen OD curve of 0.4 .

DETAILED DESCRIPTION OF THE INVENTION

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

A number of terms and abbreviations are used in this disclosure. Unless expressly stated otherwise, the following definitions apply.

The singular forms of the element or component are intended to be non-limiting as to the number of examples (ie, occurrences) of the element or component. Therefore, the singular should be understood to include one or at least one, and the singular form of an element or component also includes the plural, except where the singular is expressly intended.

The term "comprising" means the presence of the stated features, integers, steps, or components as set forth in the claims, but in such a way that it does not exclude the presence or addition of one or more features, integers, steps, components, or groups thereof. The term "comprising" is meant to include the embodiments encompassed by the terms "substantially consisting of" and "consisting of". Similarly, the term "substantially consisting of" is intended to include the embodiments encompassed by the term "consisting of".

All ranges, if any, are inclusive and combinable. For example, when referring to the range "1 to 5", said range should be considered to include the ranges "1 to 4", "1 to 3", "1-2", "1-2 and 4-5", "1 -3 and 5", etc.

As used herein in relation to a numerical value, the term "approximately" refers to the range of +/- 0.5 of the numerical value, unless the specified term is otherwise specifically defined in the context. For example, the phrase "pH value of about 6" refers to pH values from 5.5 to 6.5, unless a different pH value is specifically defined.

Each maximum numerical limit disclosed herein is intended to include every lower numerical limit, as if such lower numerical limits would be expressly given herein. Each minimum numerical limit disclosed herein will include each upper numerical limit as if such upper numerical limits would be explicitly given herein. Each numerical range disclosed herein will include each narrower numerical range that falls within such broader numerical range as if all such narrower numerical ranges were expressly given herein.

Terms "Enterococcus" And "Enterococcus spp." are used interchangeably, and in the context of this document they refer to a large genus of lactic acid bacteria belonging to the phylum Firmicutes. Enterococci are Gram-positive cocci that often occur in pairs (diplococci) or short chains and are difficult to distinguish from streptococci Enterococci are facultative anaerobic organisms, i.e. they are capable of cellular respiration in both oxygen-rich and oxygen-poor environments. Although they cannot spore, Enterococci are tolerant of a wide range of environmental conditions: extreme temperatures (10–45°C), pH (4.5–10.0) and high concentrations of sodium chloride. Representatives of the genusEnterococcus until 1984 were classified asStreptococcus group D when analysis of genomic DNA showed that it would be appropriate to classify them in a separate genus.

The terms " Enterococcus cecorum " and " E. cecorum " are used interchangeably in this document. E. cecorum is a species of the genus Enterococcus and is a bacterium in the intestinal tract of many domestic animals.

The terms "animal" and "subject" are used interchangeably herein. Animal includes all non-ruminants (including humans) and ruminants. In a particular embodiment, the animal is a non-ruminant animal such as a horse and a monogastric animal. Examples of monogastric animals include, but are not limited to, pigs and domestic pigs such as piglets, nursery pigs, sows; poultry such as turkeys, ducks, chickens, broiler chickens, laying hens; fish such as salmon, trout, tilapia, catfish and carps; and crustaceans such as small shrimp and large shrimp. In a further embodiment, the animal may be a multi-chambered animal such as a ruminant, including but not limited to cattle, calves, goats, sheep, giraffes, bison, elk, wapiti, yaks, water buffaloes, deer, camels, alpacas , llamas, antelope, pronghorn and nilgai.

The term "ruminant" as used herein refers to a mammal that is able to obtain nutrients from food of plant origin by fermenting it in a specialized stomach before digestion, mainly through the action of microorganisms. The process typically requires burping and re-chewing the fermented contents of the rumen (known as cud). The process of re-chewing the cud to further refine the plant material and promote digestion is called cud chewing. Approximately 150 ruminant species include both domesticated and wild species. Ruminants include, without limitation, cattle, cows, goats, sheep, giraffes, yaks, deer, elks, antelopes, buffaloes, and the like.

As used herein, the term "CFU" stands for "colony forming units" and is a measure of viable cells, where a colony is a population of cells derived from a single progenitor cell.

As used herein, the term "food microbial"("DFM") is a source of live (viable) naturally occurring microorganisms. The DFM may contain one or more of these naturally occurring microorganisms, such as bacterial strains. DFM categories include spore-forming bacteria such as Bacillus and Clostridium , as well as non-spore-forming bacteria such as lactic acid bacteria, yeasts, and fungi. Thus, the term DFM encompasses one or more of the following: edible bacteria, edible yeast, edible yeast or fungi, and combinations thereof.

Bacillus and Clostridium are unique gram-positive rods that produce spores. These spores are very stable and can withstand environmental conditions such as heat, moisture and a certain pH range. These spores germinate into active vegetative cells when ingested by animals and can be used in ground and pelleted diets. Lactic acid bacteria are Gram-positive lactic acid-producing cocci that antagonize pathogenic organisms. Because lactic acid bacteria appear to be heat sensitive to some extent, they are not used as such in pelleted diets and must be protected (coated). Types of lactic acid bacteria include Bifidobacterium , Lactobacillus , and Enterococcus.

The term "microbial preparation for ingestion based on a microorganism of the genus Bacillus " means a microbial preparation for ingestion containing one or more bacterial strains of the genus Bacillus .

As used herein, the term “ Bacillus microorganism component” refers to (i) a Bacillus microorganism microbial food preparation containing one or more bacterial strains of the Bacillus genus, (ii) a supernatant obtained from a culture of Bacillus , or (iii) combinations of (i) and (ii).

"Feed" and "food" respectively means any natural or artificial diet, food or the like, or components of such food, intended or suitable for consumption, ingestion, digestion by animals other than humans and humans, respectively.

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

The term "feed" is used in relation to products that are fed to animals in the rearing of farm animals. The terms "food" and "pet food" are used interchangeably. In a preferred embodiment, the food or feed is suitable for consumption by non-ruminants and ruminants.

As used herein, the term "probiotic" refers to live microorganisms (including, for example, bacteria or yeast) that, for example, when ingested or applied topically in sufficient amounts, benefit the host organism, i.e., provide one or several clear health benefits to the host. Probiotics may improve the microbiota balance on one or more mucosal surfaces. For example, the mucosal surface may be in the intestine, urinary tract, respiratory tract, or on the skin. As used herein, the term "probiotic" also encompasses live microorganisms that can stimulate important branches of the immune system while at the same time reducing inflammatory responses on mucosal surfaces such as the intestines. Although there are no upper or lower limits for probiotic intake, it has been suggested that at least 10 ⁶ -10 ¹² , preferably at least 10 ⁶ -10 ¹⁰ , preferably 10 ⁸ -10 ⁹ cfu as daily dose will be effective to achieve beneficial effects on the health of the subject.

The term "prebiotic" means a non-digestible food ingredient that benefits the host by selectively stimulating the growth and/or activity of one or a limited number of beneficial bacterial species.

Used in this document, the term "pathogen" means any causative agent of the disease. Such pathogens may include, without limitation, bacterial, viral, fungal pathogens, and the like.

The terms "derived from" and "derived from" refer not only to a protein produced or capable of being produced by a strain of the organism in question, but also to a protein encoded by a DNA sequence isolated from such a strain and produced in a host organism containing such a DNA sequence. In addition, this term refers to a protein that is encoded by a DNA sequence of synthetic origin and/or a DNA sequence derived from cDNA, and which has the distinctive characteristics of the protein in question.

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

As noted above, Enteroccocus cecorum is considered a new pathogen in the poultry industry and can cause significant losses in broiler and broiler breeders. E. cecorum is increasingly recognized as the causative agent of enterococcal spondylitis, formerly called enterococcal spinal osteoarthritis (EVOA) in chickens. Outbreaks of the disease were diagnosed mainly in broiler flocks raised under intensive production systems. Clinically affected birds suffered from motor problems due to compression of the spinal cord in the thoracic vertebrae from E. cecorum -induced osteomyelitis and due to necrosis of the femoral head. Disease outbreaks can lead to high disease prevalence, mortality, culling, carcass rejection and can result in severe economic losses in a short period of time. Moreover, E. cecorum isolates not only showed increased pathogenicity, but also increased resistance to antimicrobial agents.

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

In one embodiment, this document describes a method of inhibiting or slowing the growth of all or part of the pathogenic Enteroccocus spp. in an animal, which involves administering to the animal an effective amount of at least one component based on a microorganism of the genus Bacillus , selected from the group consisting of a microbial preparation for ingestion based on a microorganism of the genus Bacillus , containing one or more bacterial strains of the genus Bacillus , the supernatant obtained from a culture of Bacillus , or combinations thereof.

The DFMs described herein contain at least one viable microorganism, such as a viable bacterial strain, or a viable yeast, or a viable fungus. Preferably the DFM contains at least one viable bacterium.

In one embodiment, the DFM may be a spore-forming bacterial strain, and therefore the term DFM may imply that the DFM is composed of or contains spores, such as bacterial spores. Thus, as used herein, the term "viable microorganism" may include microbial spores such as endospores or conidia. Alternatively, the DFM in the feed additive composition described herein may not or may not contain microbial spores, such as endospores 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, which can be isolated.

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

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

As used herein, the genus " Bacillus " includes all species within the genus " Bacillus " known to those skilled in the art, including, but not limited to, 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 clear that the genus Bacillus continues to undergo taxonomic reorganization. Thus, the genus is intended to include species that have been reclassified, including, but not limited to, organisms such as Bacillus stearothermophilus , now called " Geobacillus stearothermophilus ", or Bacillus polymyxa , now called " Paenibacillus polymyxa ". The production of resistant endospores under environmental stress is considered a 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 .

Preferably, 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. NRRl B-50013, Bacillus Strain LSSAO1, Accession No. NRRL B-50104, and Bacillus Strain 15A-P4, Accession No. ATCC PTA-6507 (as disclosed in US Pat. No. 7,754,469 B, incorporated to this document by reference).

In another aspect, DFM can be further combined with the following Lactococcus spp: Lactococcus cremoris and Lactococcus lactis and combinations thereof.

DFM можно дополнительно комбинировать со следующими 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 и Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii , 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 pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus zeae и комбинациями любых of them.

In yet another aspect, DFM can 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 any combinations thereof.

Bacteria of the following species may be mentioned: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pumilis, Enterococcus, Enterococcus spp., and Pediococcus spp, Lactobacillus spp. Bifidobacterium spp. animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillus salivarius ssp. Salivarius, Propionibacteria sp and combinations thereof.

Described herein, a microbial preparation for ingestion containing one or more bacterial strains may contain a single type (genus, species, and strain) or may contain a mixture of genera, species, and/or strains. Preferably, the microbial preparation for ingestion described herein comprises one or more bacterial strains from the genus Bacillus .

Where appropriate, the composition of the present disclosure may be combined with one or more of the products or microorganisms contained in such products as disclosed in WO2012110778 and summarized as follows:

Bacillus subtilis strain 2084, Accession No. NRRl B-50013, Bacillus subtilis strain LSSAO1, Accession No. NRRL B-50104, and Bacillus subtilis strain 15A-P4, Accession No. ATCC PTA-6507 (from Enviva ^® PRO ^® (formerly known as Avicorr ^® ) ; Bacillus subtilis strain C3102 (from Calsporin ^® ); Bacillus subtilis strain PB6 (from Clostat ^® ); Bacillus pumilis (8G-134); NCIMB 10415 Enterococcus (SF68) (from Cylactin ^® ); Bacillus subtilis strain C3102 (from Gallipro ^® and ^{GalliproMax®} ); Bacillus licheniformis (from ^{Gallipro® Tect®} ); Enterococcus faeciul, Lactobacillus salivarius, L. reuteri, Bifidobacterium animalis and Pediococcus acidilactici (from Poultry ^Star® ); Lactobacillus, Bifidobacterium and/or Enterococcus from ^Protexin® ); Bacillus subtilis strain QST 713 (from ^Proflora® ); CECT-5940 Bacillus amyloliquefaciens (from Ecobiol ^® and Ecobiol ^® Plus); SF68 Enterococcus faecium (from ^Fortiflora® ); Bacillus subtilis and Bacillus licheniformis (from BioPlus2B ^® ); lactic acid bacteria 7 Enterococcus faecium (from Lactiferm ^® ); Bacillus strain (from ^CSI® ); Saccharomyces cerevisiae (from Yea- ^Sacc® ); Enterococcus (from Biomin IMB52 ^® ); Pediococcus acidilactici, Enterococcus, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius (from Biomin ^C5® ); Lactobacillus farciminis (from ^Biacton® ); Enterococcus (from Oralin ^E1707® ); Enterococcus (2 strains), DSM 1103 Lactococcus lactis (from Probios-pioneer PDFM ^® ); Lactobacillus rhamnosus and Lactobacillus farciminis (from ^Sorbiflore® ); Bacillus subtilis (from ^Animavit® ); Enterococcus (from ^Bonvital® ); Saccharomyces cerevisiae (from Levucell SB ^20® ); Saccharomyces cerevisiae (from Levucell SC 0 and ^SC10® ME); Pediococcus acidilacti (from Bactocell); Saccharomyces cerevisiae (from ActiSaf ^® (Wound BioSaf ^® )); NCYC Sc47 Saccharomyces cerevisiae (from ^Actisaf® SC47); Clostridium butyricum (from Miya– ^Gold® ); Enterococcus (from Fecinor and Fecinor Plus®); NCYC R-625 Saccharomyces cerevisiae (from InteSwine®); Saccharomyces cerevisia (from ^BioSprint® ); Enterococcus and Lactobacillus rhamnosus (from Provita ^® ); Bacillus subtilis and Aspergillus oryzae (from PepSoyGen–C ^® ); Bacillus cereus (from ^Toyocerin® ); NCIMB 40112/CNCM I–1012 Bacillus cereus var. toyoi (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 yeast from the genera and species : Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia angusta, Pichia anomala, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces pastorianus (syn. Saccharomyces carlsbergensis) and filamentous mushrooms from the genus Aspergillus.

Preferably, the DFM described herein contains microorganisms that are generally recognized as safe (GRAS) and are preferably approved for GRAS status and/or have been awarded Qualified Presumption of Safety by the European Food Safety Authority (EFSA).

In some embodiments, it is important that the DFM is heat resistant, i.e. was heat resistant. This is especially true if the feed is granulated. Thus, the DFM may be a heat-resistant microorganism such as heat-resistant bacteria, such as spore-forming bacteria, including, for example,bacillusspp. Bacilli are able to form stable endospores under unfavorable growth conditions and are extremely resistant to high temperatures, pH, humidity and disinfectants. If the bacterium/DFM does not have the ability to form spores, then it must be protected to survive the feed handling as described below.

DFM based on Bacillus , which is described in this document, can inhibit or slow down the growth of all or part of Enterococcus spp., such as E. cecorum . In other words, Bacillus DFM as described herein is anti-pathogenic. As used herein, the term "anti-pathogenic" means that DFM counteracts the effect (negative effect) of a pathogen, in this case pathogenic Enterococcus spp., eg E. cecorum .

For example, the following assay, referred to as "DFM Assay", can be used to determine the suitability of a microorganism to act as a DFM or, in this embodiment, a Bacillus.beta. DFM as described herein. Such a DFM analysis can be carried out as follows.

A fully grown culture of the Bacillus strain was centrifuged and sterilized by filtration (0.2 μm) to obtain a sterile cell-free supernatant (CFS). Each well of a 96-well microtiter plate is filled with 180 µl of a suspension (1%) of the pathogen/BHI (or appropriate growth medium). Positive control wells are filled with an additional 20 µl of the same broth growth medium, while test wells are filled with 20 µl of test CFS. Negative controls contain broth media only or media supplemented with 20 µl of CFS. The 96-well microtiter plate is then aerobically incubated at 37°C for 14 hours on a stationary Flex absorbance recorder, with the data transferred directly to a computer for analysis to generate a growth kinetics curve. Measurements were taken every 15 minutes. The results are presented as % inhibition comparing control at OD=0.4 (pathogen only) and treated sample (pathogen incubated with Bacillus CFS). Growth retardation is calculated as the difference in time to reach an OD of 0.4 between control wells and CFS-supplemented wells. All analyzes are carried out in duplicate. Separation of means was performed using Tukey's HSD in JMP 11; differences were considered significant at P<0.05.

Antipathogenic DFMs include one or more of the following bacteria and are described in WO2013029013:

strain 3BP5 Bacillus subtilis accession number NRRL B-50510,

strain 918 Bacillus subtilis accession number ATCC NRRL B-50508 and

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

The microorganism component of the genus Bacillus as described herein may be obtained as culture(s) and carrier(s) (if applicable) and may be added to a ribbon or paddle mixer and mixed for approximately 15 minutes, however time may be increased or reduce. The components are mixed in such a way that the result is a homogeneous mixture of cultures and carriers. The end product is preferably a dry free flowing powder. Accordingly, the Bacillus microorganism component may comprise a Bacillus microbial food preparation containing one or more bacterial strains of the Bacillus genus, a supernatant obtained from a Bacillus culture, or a combination. Such a component based on a microorganism of the genus Bacillus can then be added to the animal feed or feed premix. It can be added on top of the pet food ("backfill"), or it can be added to a liquid such as the animal's drinking water.

The incorporation of individual strains into the Bacillus DFM described herein can be made in proportions ranging from 1% to 99% and preferably from 25% to 75%.

Suitable doses of the Bacillus microorganism component described herein in pet food can range from about 1x103 cfu/g feed to about 1x1010 cfu/g feed, suitably from about 1x104 cfu/g feed up to about 1x108 cfu/g feed, suitably from about 7.5x104 cfu/g feed to about 1x107 cfu/g feed.

One of ordinary skill in the art will have no difficulty knowing the specific species and/or strains of microorganisms from the genera described herein that are used in the food and/or agricultural industries and that are generally considered suitable for animal consumption. Animal feeds may include plant material such as corn, wheat, sorghum, soybeans, canola, sunflower, or mixtures of any of these plant materials, or sources of plant proteins for poultry, pigs, ruminants, aquaculture, and pets.

The terms "animal feed", "feed" and "feed" are used interchangeably and may include one or more feed materials selected from the group consisting of a) cereals such as small grains (e.g. wheat, barley, rye, oats and combinations thereof) and/or coarse grains such as maize or sorghum; b) grain processing wastes such as corn gluten meal, dried vinasse with solubles (DDGS) (in particular dried vinasse with solubles based on corn (cDDGS)), wheat bran, wheat semolina, wheat fine bran, rice bran , rice husk, oat husk, coconut kernel and citrus pulp; c) protein derived from sources such as soybeans, sunflowers, peanuts, lupins, peas, fava beans, cotton, canola, fishmeal, dry blood plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats derived from vegetable and animal sources, and/or e) minerals and vitamins.

In the case of use as a feed, or in the preparation of a feed such as a functional feed, the Bacillus microorganism component described herein may be used in combination with one or more of a food acceptable carrier, food acceptable diluent, food acceptable a food-grade excipient, a food-acceptable excipient, a food-active ingredient. For example, at least one component selected from the group consisting of protein, peptide, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride can be mentioned. , 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 metabisulphite, methylparaben and propylparaben.

In a preferred embodiment, the Bacillus microorganism component described herein may be mixed with a feed component to form a feed product. As used herein, the term "feed component" means all or part of a food product. A food portion may mean one food constituent or more than one food constituent, for example 2 or 3 or 4 or more. In one embodiment, the term "feed component" encompasses the premix or premix constituents. Preferably, the feed may be forage or a premix thereof, compound feed or a premix thereof. A feed additive composition containing a component based on a microorganism of the genus Bacillus described herein can be mixed with compound feed or compound feed premix or forage, forage component or forage premix.

The term forage, as used herein, means any food that is provided to an animal (as opposed to food that the animal obtains on its own). Forage covers plants that have been cut.

The term forage includes hay, straw, silage, pressed and pelleted feed, oils and mixed rations, as well as sprouted grains and legumes.

The nutritional substance can be obtained from one or more of the plants selected from: alfalfa (alfalfa), barley, lollipop, representatives of the genus cabbage, cabbage variety Chau moellier, fodder cabbage, rapeseed (canola), turnip (rutabaga), turnip , clover, hybrid clover, red clover, underground clover, white clover, grass, high French ryegrass, fescue, Bermuda grass, bonfire, trident, bluegrass species (from pasture grasses mixed in nature), cocksfoot, perennial ryegrass, meadow timothy , corn (maize), millet, oats, sorghum, soybeans, trees (sprouts of cut trees for hay from trees), wheat and legumes.

The term "compound feed" means industrial feed in the form of flour, granules, nuts, cake or crumbs. Compound feeds can be mixed using various raw materials and additives. These mixtures are formulated according to the specific requirements of the target animal.

Compound feeds can be complete feeds that provide all the daily essential nutrients, concentrates that provide part of the diet (protein, energy), or supplements that provide only additional micronutrients such as minerals and vitamins.

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

Suitably, a premix, as referred to herein, may be a composition consisting of micro-ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other necessary ingredients. Premixes are usually compositions suitable for mixing with industrial rations.

Any feed product described herein may contain one or more feed materials selected from the group consisting of a) cereals such as small grains (e.g. wheat, barley, rye, oats and combinations thereof) and/or coarse grains such like maize or sorghum; b) grain processing residues such as corn gluten meal, dried vinasse with solubles (DDGS), wheat bran, wheat semolina, wheat fine bran, rice bran, rice husk, oat husk, coconut kernel and citrus pulp; c) protein derived from sources such as soybeans, sunflowers, peanuts, lupins, peas, fava beans, cotton, canola, fishmeal, dry plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats derived from vegetable and animal sources; e) minerals and vitamins.

Moreover, such feed product may contain at least 30%, at least 40%, at least 50%, or at least 60% by weight of corn and soybean meal, or corn and full fat soybean, or wheat flour, or feed sunflower seed cake flour.

Additionally or alternatively, the food product may contain at least one high fiber food material and/or at least one processed product of at least one high fiber food material to produce a high fiber food product. Examples of high fiber feed materials include wheat, barley, rye, oats, cereal by-products such as corn gluten meal, dried vinasse with solubles (DDGS), wheat bran, wheat semolina, wheat fine bran, rice bran, rice husk, oat husk, coconut kernel and citrus pulp. Some sources of protein can also be considered high-fiber sources: protein derived from sources such as sunflowers, lupins, fava beans, and cottonseed.

As described herein, the feed may be one or more of the following: compound feed and premix, including pellets, nuts or cake (for cattle); crop or crop residues: corn, soybeans, sorghum, oats, barley, corn on the cob, copra, straw, chaff, sugar beet waste; fish flour; freshly cut grass and other fodder plants; meat and bone meal; molasses; oil cake and pressed cake; oligosaccharides; canned fodder plants: hay and silage; seaweed; seeds and grains, whole or prepared by crushing, grinding, etc.; sprouted grains and legumes; yeast extract.

As used herein, the term food also encompasses, in some embodiments, pet food. Pet food is a material of plant or animal origin 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 dog food pellets, or in a wet, canned form. Cat food may contain the amino acid taurine.

The term food may also encompass, in some embodiments, fish food. Fish food normally contains the macronutrients, micronutrients and vitamins needed to maintain the health of captive fish. The fish food may be in the form of a flake, granule or tablet. Granular forms, some of which sink quickly in water, are often used for larger or bottom-feeding fish. Some fish foods also contain additives such as beta-carotene or sex hormones to artificially enhance the coloration of ornamental fish.

The term "food" also encompasses bird food, including food used in both bird feeders and poultry feed. Bird food usually contains various seeds, but may also include lard (beef or mutton fat).

As used herein, the term "contacted" refers to the indirect or direct application of a feed additive composition to a product (eg, feed). Examples of uses that can be used include, without limitation, treating the product in a material containing the feed additive composition, direct application by mixing the feed additive composition with the product, spraying the feed additive composition onto the surface of the product, or dipping the product into a feed additive formulation formulation.

The component based on the microorganism of the genus Bacillus can preferably be mixed with a product (eg a feed product). Alternatively, it can be included in the emulsion or raw ingredients of the feed product.

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

The Bacillus microorganism component can be used to embed, coat and/or impregnate a product (eg, feed product or feed raw ingredients) with a controlled amount of the Bacillus microorganism component.

DF M containing at least one bacterial strain can be added at suitable concentrations, such as concentrations in the final feed product that provide a daily dose of from about 2x103 cfu/g feed to about 2x1011 cfu/g feed, at a suitable about 2x106 to about 1x1010, suitably from about 3.75x107 cfu/g feed to about 1x1010 cfu/g feed.

Preferably, the Bacillus microorganism component will be heat-stable up to about 70°C; up to about 85°C or up to about 95°C. The heat treatment can be carried out for from about 30 seconds to several minutes. The term "heat-stable" means that at least about 50% of the Bacillus microorganism component that was present/active before being heated to a certain temperature is still present/active after it is cooled to room temperature. In a particularly preferred embodiment, the Bacillus microorganism component is homogenized to obtain a powder.

Alternatively, the Bacillus microorganism component is formulated as granules as described in WO2007/044968 (referred to as TPT granules), incorporated herein by reference.

In another preferred embodiment, when the feed additive composition is formulated as granules, these granules contain a hydrated barrier salt that coats the protein core. Such a salt coating has the advantage of improved thermal stability, improved storage stability and protection from other feed additives otherwise adversely affecting at least one protease and/or DFM containing one or more bacterial strains. Preferably, the salt used for salt coating has a water activity of greater than 0.25 or a permanent moisture content of greater than 60% at 20°C. Preferably the salt coating contains Na ₂ SO ₄ .

Feed containing a component based on a microorganism of the genus Bacillus can be obtained using a feed pelleting method. Optionally, the granulation step may include steaming or a conditioning step prior to forming the granules. The mixture containing the powder can be placed in a conditioning device such as a steam injection mixer. The mixture is heated in a conditioning device to a certain temperature, such as 60-100°C, with temperatures typically being 70°C, 80°C, 85°C, 90°C or 95°C. The residence time can be varied from seconds to minutes and even hours. For example, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.

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

The pellet can be produced by a feed pelleting method, and the feed pretreatment method can be carried out at a temperature of 70°C to 95°C for several minutes, for example at a temperature of 85°C to 95°C.

The component based on the microorganism of the genus Bacillus can be formulated as an animal feed pellet containing: a core; an active agent, wherein the active agent of the granule retains at least 80% activity after storage and after the steam heated granulation process, where the granule is an ingredient; a moisture barrier coating; and a moisturizing hydrating coating that makes up at least 25% w/w of the granule, wherein the granule has a water activity of less than 0.5 prior to the steam heated granulation process.

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

The granule can be produced by a steam heated granulation process, which can be carried out at 85°C-95°C for several minutes.

Alternatively, the composition is in the form of a liquid formulation suitable for consumption, preferably such a consumable liquid contains one or more of the following: buffer, salt, sorbitol and/or glycerin.

In addition, the composition of the feed additive can be formulated by applying, for example spraying, a component based on a microorganism of the genus Bacillus onto a carrier substrate, such as, for example, ground wheat.

In one embodiment, such a feed additive composition containing the Bacillus microorganism component described herein may be formulated as a premix. By way of example only, the premix may contain one or more feed components such as one or more minerals and/or one or more vitamins.

Alternatively, the composition is in the form of a liquid formulation suitable for consumption, preferably such a consumable liquid contains one or more of the following: buffer, salt, sorbitol and/or glycerin.

In addition, the composition of the feed additive can be formulated by applying, for example spraying, a component based on a microorganism of the genusbacillus onto a carrier substrate such as, for example, ground wheat.

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

It will be appreciated that the Bacillus microorganism component disclosed herein is suitable for addition to any appropriate feed material.

As used herein, the term feed material means the main feed material for consumption by an animal. It will further be understood that it may contain, for example, at least one or more varieties of raw cereals, and/or processed plant material, and/or animal material, such as soybean meal or bone meal.

One skilled in the art will appreciate that different animals require different foods, and even the same animal may require different foods depending on the purpose for which the animal is raised.

Preferably, the feed product may contain feed materials including maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum and/or any processed products, as well as protein-rich components such as soy flour, rapeseed flour, flour canola, cottonseed meal, sunflower seed meal, animal derived meal, and mixtures thereof. More preferably, the food product may contain animal fats and/or vegetable oils.

Optionally, the food product may also contain additional minerals, such as, for example, calcium, and/or additional vitamins. Preferably, the feed product is a mixture of corn and soybean meal.

In another aspect, a method for producing a food product is provided. The feed product is usually obtained in feed mills, in which the raw materials are first crushed to a suitable particle size and then mixed with the appropriate additives. The food product can then be made into a slurry or granules; with the latter, as a rule, a method is provided in which the temperature is raised to a predetermined level, and then the feed is passed through a matrix to obtain granules of a specific size. The granules are allowed to cool. Subsequently, liquid additives such as fat and enzyme can be added. The preparation of the food product may also include an additional step which includes extrusion or swelling prior to granulation, in particular by suitable techniques which may include at least the use of steam.

The feed product may be feed for a monogastric animal such as poultry (e.g. broiler, laying hen, broiler breeders, turkey, duck, geese, waterfowl), pig (all age categories), pet (e.g. dogs , cats) or fish, preferably the feed product is intended for poultry.

The component based on the microorganism of the genus Bacillus , described in this document, can be placed on top of animal food, ie fall asleep. Alternatively, the Bacillus.beta . microorganism component described herein can be added to a liquid, such as an animal's drinking water.

As used herein, the term "contacted" refers to the indirect or direct application of a component based on a microorganism of the genusbacillusdescribed in this document in relation to the product (for example, stern).

Examples of application methods that can be used include, without limitation, treatment of the product in a material containing a Bacillus microorganism component, direct application by mixing a feed additive composition containing a Bacillus microorganism component, as described herein, with the product, application spraying such a feed additive composition onto the surface of the product or immersing the product in a preparation based on the feed additive composition. In one embodiment, a feed additive composition containing a component based on a microorganism of the genus Bacillus , as described herein, is preferably mixed with a product (eg, a feed product). Alternatively, the feed additive composition may be included in the emulsion or raw ingredients of the feed product. This improves the performance of the composition.

The process for producing the Bacillus microorganism component described herein may also include an additional step of powder granulation. The powder can be mixed with other components known in the art. The powder or mixture containing the powder can be forced through a mold and the resulting filaments cut into suitable granules of various lengths.

Optionally, the granulation step may include steaming or a conditioning step prior to forming the granules. The mixture containing the powder can be placed in a conditioning device such as a steam injection mixer. The mixture is heated in a conditioning unit to a predetermined temperature, eg 60-100ºC, typical temperatures would be 70ºC, 80ºC, 85ºC, 90ºC or 95ºC. The residence time can vary from seconds to minutes and even hours. For example, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.

One skilled in the art will appreciate that different animals require different foods, and even the same animal may require different foods depending on the purpose for which the animal is raised.

Optionally, the food product may also contain additional minerals, such as, for example, calcium, and/or additional vitamins. In some embodiments, the food product is a mixture of corn and soy flour.

The feed product is usually obtained in feed mills, in which the raw materials are first crushed to a suitable particle size and then mixed with the appropriate additives. The food product can then be made into a slurry or granules; with the latter, as a rule, a method is provided in which the temperature is raised to a predetermined level, and then the feed is passed through a matrix to obtain granules of a specific size. The granules are allowed to cool. Subsequently, liquid additives such as fat and enzyme can be added. The preparation of the food product may also include an additional step which includes extrusion or swelling prior to granulation, in particular by suitable techniques which may include at least the use of steam.

As noted above, the component based on the microorganism of the genus Bacillus and/or the feed product containing it can be used in any suitable form. It can be used in the form of solid or liquid preparations or their alternatives. Examples of solid preparations include powders, pastes, boluses, capsules, dragees, tablets, powders and granules, which can be wetted, spray dried or lyophilized. Examples of liquid preparations include, without limitation, aqueous, organic or aqueous-organic solutions, suspensions, and emulsions.

In some applications, the feed additive composition may be mixed with feed or introduced into drinking water.

The Bacillus microorganism component comprises mixing the Bacillus microorganism component described herein with a feed-acceptable carrier, diluent or excipient and optionally packaging.

The food product and/or component based on a microorganism of the genus Bacillus can be combined with at least one mineral and/or at least one vitamin. The compositions thus obtained may be referred to herein as a premix. The feed product may contain at least 0.0001% by weight of a component based on a microorganism of the genus Bacillus . Suitably, the feed product may contain 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 a component based on a microorganism of the genus Bacillus .

Preferably, the food product or component based on a microorganism of the genus Bacillus may further comprise at least one physiologically acceptable carrier. The physiologically acceptable carrier is preferably selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat component, sucrose, starch, Na[2]SO[4], talc, PVA and mixtures thereof. In a further embodiment, the food or feed may further comprise a metal ion chelator. The metal ion chelator can be selected from EDTA or citric acid.

In one embodiment, the Bacillus microorganism component described herein (either encapsulated or unencapsulated) may be formulated with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate) , cyclodextrin, wheat or a component of wheat, sucrose, starch, Na ₂ SO ₄ , talc, PVA, sorbitol, benzoate, sorbate, glycerin, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulphite, formate and mixtures thereof.

In some embodiments, the Bacillus microorganism component described herein will be in a physiologically acceptable carrier. Suitable carriers can be large slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive viral particles. Pharmaceutically acceptable salts may be used, for example mineral acid salts such as hydrochlorides, hydrobromides, phosphates and sulfates, or organic acid salts 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, adjuvants such as wetting or emulsifying agents or pH buffering agents may be present in such compositions. Such carriers make it possible to formulate pharmaceutical compositions in the form of tablets, pills, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by the patient. Once compiled, they can be administered directly to the subject. The subjects to be treated may be animals.

Non-limiting examples of compositions and methods disclosed herein include:

1. A method for inhibiting or slowing down the growth of all or part of representatives of pathogenic Enteroccocus spp. in an animal, which involves administering to the animal an effective amount of at least one component based on a microorganism of the genus Bacillus , selected from the group consisting of a microbial preparation for ingestion based on a microorganism of the genus Bacillus , containing one or more bacterial strains of the genus Bacillus , the supernatant obtained from a culture of Bacillus , or combinations thereof.

2. The method according to Embodiment 1, wherein the Bacillus microbial edible preparation is selected from the group consisting of Bacillus amyloliquefaciens , Bacillus licheniformis , Bacillus pumilis , and Bacillus subtilis .

3. The method according to embodiment 1 or 2, wherein the microbial preparation for ingestion based on a microorganism of the genus Bacillus is selected from one or more of the following strains: Bacillus strain 2084 accession number NRRl B-50013, Bacillus strain LSSAO1 accession number NRRL B –50104 and Bacillus strain 15A–P4 Accession No. ATCC PTA–6507.

4. The method according to embodiment 1, 2 or 3, wherein the animal is a monogastric animal.

5. The method according to embodiment 1, 2 or 3, wherein the animal is a multi-gastric animal.

6. The method according to embodiment 1, 2, 3 or 4, wherein the monogastric animal is poultry.

7. The method according to any of the above embodiments 1-6, wherein at least one component based on a microorganism of the genus Bacillus is administered directly to the animal with the animal feed, either as contained in the feed, or added on top of the feed, or in the form of a liquid, like water.

8. The method of any one of embodiments 1-7, wherein at least one Bacillus microorganism component is administered to the animal in a form selected from the group consisting of a feed product, a feed additive composition, a premix, or a liquid such as water.

EXAMPLES

Unless otherwise defined herein, all technical and scientific terms used in this document have the same meaning as generally understood by a person skilled in the art to which the present invention 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, NY (1991) provide a general a dictionary for many of the terms used in this disclosure.

The present invention is further defined in the following examples. It should be understood that the examples, while showing certain embodiments, are provided for purposes of illustration only. From the foregoing discussion and examples, one skilled in the art will be able to ascertain the essential features of the present invention, and without deviating from its spirit and scope, will be able to make various changes and modifications to adapt it to various applications and conditions.

Example 1

Strains Enterococcus cecorum And bacillus

Fifty-two strains of Enterococcus cecorum were collected from culture collections in North America and Europe, which are summarized in Table 1 below. that the strains tested were virulent and capable of causing disease.

Table 1 Enterococcus cecorum strains used in this study

Internal designation
strain Geographic origin biological origin Year
allocation Source 11915–3 North Carolina, USA spinal abscess 2013 DuPont Internal Collection 11937–2 North Carolina, USA spinal abscess 2013 DuPont Internal Collection 11944–2 North Carolina, USA spinal abscess 2013 DuPont Internal Collection 12108–2 Delaware, USA spinal abscess 2013 DuPont Internal Collection 11976–5 North Carolina, USA Caecal smear 2013 DuPont Internal Collection 12123–1 Delaware, USA gastrointestinal tract 2013 DuPont Internal Collection 12476–5 Delaware, USA spinal abscess 2013 DuPont Internal Collection 11914–3 North Carolina, USA spinal abscess 2013 DuPont Internal Collection 12147–1** North Carolina, USA spinal abscess 2013 DuPont Internal Collection 14191–2 Minnesota, USA spinal abscess 2014 DuPont Internal Collection 14201–1 Minnesota, USA spinal abscess 2014 DuPont Internal Collection 11920–1 North Carolina, USA spinal abscess 2013 DuPont Internal Collection 12161–6 North Carolina, USA gastrointestinal tract 2013 DuPont Internal Collection 14194–5 Minnesota, USA spinal abscess 2014 DuPont Internal Collection 11955–1 North Carolina, USA spinal abscess 2013 DuPont Internal Collection 14192–2 Minnesota, USA spinal abscess 2014 DuPont Internal Collection 11976–2 North Carolina, USA Caecal smear 2013 DuPont Internal Collection 11952–1 North Carolina, USA spinal abscess 2013 DuPont Internal Collection 12163–2 North Carolina, USA gastrointestinal tract 2013 DuPont Internal Collection 14202–4 Minnesota, USA spinal abscess 2014 DuPont Internal Collection 11960–1 North Carolina, USA spinal abscess 2013 DuPont Internal Collection 12696M-1** North Carolina, USA spinal abscess 2013 DuPont Internal Collection 12418–1 Minnesota, USA gastrointestinal tract 2013 DuPont Internal Collection 11957–3** North Carolina, USA spinal abscess 2013 DuPont Internal Collection 12435–3 Minnesota, USA blind gut 2013 DuPont Internal Collection 11951–1** North Carolina, USA spinal abscess 2013 DuPont Internal Collection 12119–2 Delaware, USA gastrointestinal tract 2013 DuPont Internal Collection 904971 Pennsylvania, USA yolk sac 2 Pennsylvania State University 903797 Pennsylvania, USA chicken knee joint Pennsylvania State University 900118 Pennsylvania, USA Liver Pennsylvania State University 843323 Pennsylvania, USA chicken bone Pennsylvania State University 842805 Pennsylvania, USA chicken pericardium Pennsylvania State University 841976 Pennsylvania, USA dead chicken bone 1 Pennsylvania State University 840394 Pennsylvania, USA chicken bone 2 Pennsylvania State University 840387 Pennsylvania, USA chicken knee joint Pennsylvania State University 910525 Pennsylvania, USA spinal abscess Pennsylvania State University 209247 Pennsylvania, USA Bone marrow Pennsylvania State University E.58.33 Belgium femoral head 2014 PoulPharm E.53.14 Belgium femur 2014 PoulPharm E.59.56 ** Belgium femoral head 2014 PoulPharm F.68,19 ** Belgium joint 2015 PoulPharm C.34,19 ** Belgium Bone marrow 2013 PoulPharm D.42.11 Belgium joint 2013 PoulPharm D.44,11 ** Belgium Bone marrow 2014 PoulPharm D.45.08 Belgium joint 2014 PoulPharm G.84.68 Belgium Bone marrow 2015 PoulPharm J.5.76 Belgium hock 2015 PoulPharm G.77.23 Belgium bone marrow from the femur 2015 PoulPharm G.75.17 ** Belgium articular surface 2015 PoulPharm G.80.72 Belgium femoral head 2015 PoulPharm G.79.39 Belgium Bone marrow 2015 PoulPharm J.6.81 Belgium bone marrow from the femur 2015 PoulPharm

** - strain used when testing strains not from the DuPont collection

The inhibitory potential of a total of 11 Bacillus strains was tested. These included both DuPont proprietary DFM strains and Bacillus isolated from competitor DFM products, summarized in Table 2. All Bacillus strains tested are commercially available for use in the poultry industry. ^Enviva® PRO, which is commercially available from Danisco A/S, is a combination of Bacillus strain 2084, Accession No. NRRl B-50013, Bacillus strain LSSAO1, Accession No. NRRL B-50104, and Bacillus strain 15A-P4, Accession No. ATCC PTA- 6507 (as disclosed in US 7754469 B, incorporated herein by reference).

Table 2 DFM Bacillus strains tested in this study

Internal designation
strain Commercial designation
strain Type (declared on the label) Commercial
the product's name Manufacturing company BS8 NRRL B-50104 Bacillus amyloliquefaciens Enviva® PRO DuPont 2084 NRRL B-50013 Bacillus amyloliquefaciens Enviva® PRO DuPont 15A-P4 PTA-6507 Bacillus amyloliquefaciens Enviva® PRO DuPont CS1 C-3102
DSM15544 Bacillus subtilis Calsporin Calpis 2B1 DSM5750 Bacillus subtilis BioPlus 2B Chr Hansen 2B2 DSM5749 Bacillus licheniformis BioPlus 2B Chr Hansen #11/1 DSM17299 Bacillus subtilis Gallipro Chr Hansen #12/1 DSM17236 Bacillus licheniformis Gallipro Tect Chr Hansen #10/4 Bacillus subtilis Sporulin Novus #10B/1 Bacillus subtilis Sporulin Novus #10B/4 Bacillus subtilis Sporulin Novus

All non-DuPont Bacillus products were purchased and isolated in triplicate from 3 separate serial lots. All strains were identified to ensure that the isolated strains matched the strains declared on the product label. Non-DuPont products were tested on a subset of 9 isolates from the complete Enterococcus cecorum collection as indicated in Table 2 above.

Example 2

Preparation of cell-free supernatants (CFS) and growth of strains Enterococcus cecorum , Enterococcus avium And Enterococcus gallinarum

The inoculation loop was used to inoculate a 30 ml shaker tube with 10 ml tryptic soy broth (TSB) with frozen Bacillus stock culture. The tube was incubated in an incubator at 32 ^° C for 24 hours and shaken at 130 to grow Bacillus .

After the flasks were incubated for 18 hours, the optical density (OD) was checked on a spectrometer (wavelength 600 nm, absorbance 0). To create a blank control, 2 ml of sterile TSB was pipetted into the cuvette. A 10-fold dilution of Bacillus was created by pipetting into each cuvette 1.8 ml of sterile TSB and 0.2 ml of an 18-hour culture. The cuvettes were closed and inverted to ensure thorough mixing. Absorbance dilutions of Bacillus α were found 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 culture from each flask was transferred to sterile 250 ml centrifuge vials and centrifuged at 10,000 rpm. within 10 minutes. After centrifugation, the supernatants of each Bacillus type were transferred to a vial-mounted Nalgene bottle filter and pumped into 50 ml conical tubes.

This procedure was carried out for all Bacillus strains. The cell-free supernatant (CFS) was then frozen at -80 ^° C. until needed for use.

E. cecorum strains selected from deep-frozen stock cultures were inoculated into BHI broth (brain and heart infusions) and BHI agar plate (to check purity) and incubated overnight at 37 ^° C. the strains were subcultured at least twice to ensure adaptation to the growth medium.

All analyzes were performed in duplicate for each Bacillus DFM listed in Table 2 above.

To avoid thermal shock to Enterococcus cecorum cells, 20 ml of BHI broth was incubated for 1 hour before analysis.

Medium (BHI broth) and CFS, as well as the target microorganism, were added to a UV-treated 96-well flat-bottomed microtiter plate as follows:

positive control: 200 µl medium + 2 µl bacteria (1%);

negative control: 200 µl of medium;

CFS assay well: 180 µl medium + 20 µl CFS + 2 µl bacteria (1%);

negative well with CFS: 180 µl medium + 20 µl CFS.

The plates were incubated for 14 hours at 37 ^° C. in a stationary Flex absorbance recorder, with the data transferred directly to a computer for analysis. Measurements were taken every 15 minutes.

Results are reported as % inhibition comparing control at OD=0.4 ( E. cecorum only) and treated sample ( E. cecorum incubated with CFS Bacillus ). Among 42 individual strains, the mean inhibition of each of the 3 strains of Enviva® PRO ( Bacillus based component) was >85% as shown in Figure 1. It was determined that >50% inhibition was considered acceptable and ≥75% inhibition was considered excellent. Inhibition greater than 100% indicated that the pathogen isolate was not inhibited, but lysed by Bacillus activity.

Table 3 shows the mean growth inhibition of 51 Enterococcus cecorum strains by Enviva® PRO strains. All strains were able to significantly inhibit the growth of Enterococcus cecorum compared to E. cecorum incubated without CFS Bacillus . The most effective strain was 15AP4 (a component based on a microorganism of the genus Bacillus ) with an average inhibition value of 88.79%, although the differences between strains were not statistically significant.

Table 3. Mean value of inhibition of Enterococcus cecorum strains collected in American and European poultry industry by 3 strains of Bacillus DFM (a component based on a microorganism of the genus Bacillus )

European strains American strains All strains Average value
Ingibiro
value, % SEM Average value
inhibitiro
value, % SEM Average value
Ingibi
ration, % SEM 15AP4 88.27 2.887 89.03 3.086 88.79 2.30 BS2084 89.87 2.887 88.08 2.955 88.60 2.233 BS8 86.80 2.887 84.24 2.955 84.99 2.233 P-value 0.7552 0.4898 0.4054

Table 4 shows the mean growth inhibition of a subgroup of 9 Enterococcus cecorum strains by 12 Bacillus strains. The most effective strain was BS2084 (a component based on a microorganism of the genus Bacillus ) with an average inhibition value of 85.33%. The least effective Bacillus strain was 2B2 with an average inhibition value of -80.17%, indicating that Enterococcus cecorum grew faster in the presence of CFS Bacillus (a component based on a microorganism of the genus Bacillus ) than in its absence. There were significant differences in efficacy among Bacillus strains.

Table 4 Mean Growth Inhibition of 11 Enterococcus cecorum strains by 11 DFM Bacillus strains

15AP4 BS2084 BS8 CS1 #11/1 #12/1 #10/4 #10B/1 #10B/4 2B1 2B2 11951–1 73.90 89.09 73.49 -23.50 47.98 31.67 36.26 - - 37.57 31.80 11957–3 102.15 102.39 99.83 97.67 -6.57 25.89 -11.83 52.68 -6.57 -55.86 -2.80 11960–1 93.12 88.99 74.47 83.30 -54.90 -5.51 32.99 94.25 51.11 -83.02 -50.94 12696M–1 32.17 49.62 67.50 99.12 -0.10 15.12 -15.19 100.36 73.90 -17.63 -6.77 C.34,19 89.79 77.34 75.19 99.35 -6.28 5.25 -25.09 94.81 53.57 -67.24 -40.41 D.42.11 98.69 99.40 99.30 86.33 -67.20 -35.45 -29.53 50.36 25.55 -128.06 -54.36 E.59.56 50.60 84.74 71.20 86.33 -16.02 -6.04 -8.02 82.07 57.95 -146.62 -87.93 F.68,19 89.38 83.21 78.16 79.76 -4.68 -9.61 -13.67 66.94 23.08 -121.79 -41.97 G.75,17 100.99 93.22 97.43 79.76 -81.97 -9.07 -77.68 69.90 32.96 -138.92 -35.45 Average value ^{1 81.20a 85.33a 81.84a 76.46a} -21.04 ^{s 1.36bc -12.41bc 76.46a 38.94ab -80.17d -32.09cd} Standard err. 8.176 5.168 4.365 12.771 13.391 6,883 11.287 6,861 8.948 20.559 11.624

¹ P-value=P<0.0001; values a, b, c in the series without common superscripts are characterized by significant differences at P<0.05

REFERENCE: Verslyppe B, De Smet W, De Baets B, De Vos P, Dawyndt P 2014. StrainInfo introduces electronic passports for microorganisms. Syst Appl Microbiol. 37(1):42–50.

The data in Table 5 demonstrates the antimicrobial activity of CFS 15AP4, BS8 and 2084 B. amyloliquefaciens subsp. plantarum (a component based on a microorganism of the genus Bacillus ) against VTT E–97776T Enterococcus gallinarum .

Table 5. In vitro growth retardation of Enterococcus gallinarum VTT E-97776T culture induced by a Bacillus- based component of CFS 15AP4, BS8 and 2084 Bacillus amyloliquefaciens subsp . plantarum .

Figures 1 and 2 show the mean inhibition for E. cecorum strains collected from American and European poultry, respectively.

Figure 3 shows the growth kinetics of E. cecorum with/without Enviva® PRO strains. Some E. cecorum were not completely inhibited by Bacillus supernatants, ie the Bacillus based component. At the end of the 14-hour incubation period, pathogen concentrations were similar for control and treated plates, but the lag phase increased and exponential growth slowed down. Considering the time of passage through the intestines of poultry, this result would mean that (despite the absence of general inhibition) the component based on the microorganism of the genus Bacillus can prevent the proliferation of the pathogen during its passage through the intestines, reducing the risk of adhesion and movement out of it. Other strains both retarded and inhibited growth over a 14 hour period, as shown in Figure 4.

Figure 5 shows the antimicrobial activity of CFS 15AP4, BS8, and 2084 B. amyloliquefaciens subsp. plantarum (a component based on a microorganism of the genus Bacillus ) against VTT E–97776T Enterococcus gallinarum , which is expressed as % inhibition at an end point exactly corresponding to the achievement of the control pathogen OD curve 0.4.

Figure 6 shows the growth profiles of Enterococcus avium E 84197 incubated with or without CFS of Bacillus strain 2084 (a component based on a microorganism of the genus Bacillus ).

Figure 7 shows the antimicrobial activity of CFS strain DSM7 ^T Bacillus amyloliquefaciens , DSM10 ^T B. subtilis and DSM13 ^T B. licheniformis against 10 clinical isolates of Enterococcus cecorum isolated from the poultry system in the USA and in Belgium, which is expressed as % inhibition at the end point , exactly corresponding to the achievement of the curve of the control pathogen OD 0.4 (black bars correspond to the average value of % inhibition).

Previous work has shown that broilers develop sepsis in the range of 1–3 weeks during production, as evidenced by both direct inhibition and delay in reaching the exponential growth phase of E. cecorum . Supplementation of poultry feed with Bacillus- based DFM(s) described herein can delay intestinal colonization, adhesion and subsequent invasion, which means a reduction in the incidence of clinical symptoms in birds reaching slaughter age.

These data are quite convincing when compared to the inhibition ranges of other tested pathogens. The effect also appeared to be fairly consistent across the range of E. cecorum isolates tested, despite the natural variation in Bacillus potency.

The in vitro results presented herein demonstrate that the addition of the Bacillus.beta. component described herein to poultry feed can be very effective in inhibiting or slowing the growth of all or part of the novel E. cecorum pathogen as well as inhibition or retardation of the growth of Enterococcus spp. in animals (as shown in figure 5).

Example 3

Components based on a microorganism of the genus bacillus to inhibit or

growth retardation Enterococus spp. in animals

The type strain in the International Code of Nomenclature for Bacteria is defined as the "name-bearing type of a species" and is the "starting point" against which all other strains are compared to determine if they belong to that species. The inclusion of type strains of each of the bacterial species (see Table 6 below) included in this study allowed comparison of the inhibitory potential of strains characterized as "probiotic" with strains belonging to the same species but not characterized as "probiotic" . In this case, the hypothesis was that the probiotic properties are unique to particular strains and these properties cannot be transferred to any other strains belonging to the same species. Only those strains that have been carefully characterized and evaluated can be labeled as "probiotic". The type strains included in this experiment were representative of three different Bacillus species related to the strains tested herein. Their origins and a non-exhaustive list of equivalent strains available in other culture collections are presented in Table 6.

Table 6 Type strains used in this study, their origin and non-exhaustive list of equivalents in other culture collections (adapted from Verslyppe et al., 2014; http://www.straininfo.net/)

Types of bacteria Bacillus amyloliquefaciens Bacillus subtilis Bacillus licheniformis Type strains used in this study B. amyloliquefaciens DSM7T B. subtilis DSM10T B. licheniformis DSM13T Origin of the type strains used in this study DSMZ, Germany DSMZ, Germany DSMZ, Germany American Type Culture Collection ATCC USA ATCC ^23350T ATCC ^6051T ATCC ^14580T Bioresource Collection and Research Center, BCRC, Taiwan BCRC ^11601T BCRC ^10255T BCRC ^11702T VTT Culture Collection, Finland VTT E– ^80124T VTT E– ^97009T VTT E– ^95571T NBRC Biological Resource Center, Japan NBRC ^15535T NBRC ^12210T NBRC ^13719T ; NBRC ^16412T NBRC ^12200T NCIMB National Collections of Industrial Food and Marine Bacteria, UK NCIMB ^12077T NCIMB ^3610T NCIMB ^50016T ; NCIMB ^9375T NRRL Agricultural Research Service Crop Collection, USA NRRL B- ^14393T NRRL B-4219 ^T NRRL NRS- ^1264T Belgian coordinated collections of microorganisms LMG/Collection of bacteria LMG LMG 12234 ^T LMG ^7135T LMG ^12363T ; LMG ^12407T ; LMG ^6933T CCUG Culture Collection, University of Gothenburg, Sweden CUCG 163B ^T CCUG ^7422T Collection of the Institute. Pasteur CIP, France C.I.P. 52.65 ^T ; C.I.P. ^5265T C.I.P. ^52.71T Korean Type Culture Collection KCTC, Korea (Republic of) KCTC ^3555T KCTC ^1753T

The antimicrobial activity of these three type strains was evaluated against a set of 10 Enterococcus cecorum isolates (Table 7) as previously described.

Table 7. Characteristics of the clinical isolates of Enterococcus cecorum used in this study, obtained from poultry production.

Geographic Market Item Strain number Isolate origin biological origin Date of allocation Collection NAM ¹ clinical disease 12147–1 Mountainair, North Carolina spinal abscess (23.05.2013) WAU_US NAM clinical disease 12696M–1 Mountainair, North Carolina spinal abscess 09.12.13 WAU_US NAM clinical disease 11951–1 Mountainair, North Carolina Spinal abscess smear (10.05.13) WAU_US NAM clinical disease 11957–3 Mountainair, North Carolina Spinal abscess smear (10.05.13) WAU_US EU ² clinical disease C.34,19 BE ³ broiler bone marrow 08/05/2013 Poulpharm_BE EU clinical disease D.42.11 BE Joint, out of production 11/28/2013 Poulpharm_BE EU clinical disease E.59.56 BE femoral head, manufacturer 09/10/2014 Poulpharm_BE EU clinical disease F.68,19 BE Joint, out of production 01/12/2015 Poulpharm_BE EU clinical disease G.75,17 BE articular surface, broiler production 04.05.2015 Poulpharm_BE

¹⁼ North America

²⁼ Europe

³ = Belgium

The percentage growth inhibition of ten pathogenic Enterococcus cecorum isolates by each of the CFSs derived from 3 different type strains is shown in Figure 7. In contrast to the 3 strains of B. amyloliquefaciens from Enviva Pro (15AP4; BS8 and 2084) (see Table 4 above) the B. amyloliquefaciens type strain DSM7T showed no antimicrobial activity against the tested pathogens (FIG. 7). In this experiment, CFS from the type strain (DSM7 ^T ) of B. amyloliquefaciens promoted pathogen growth and was unable to inhibit any of the tested pathogens. These results confirm the strain specificity of the antimicrobial property. The assignment of probiotic status to a particular strain is the result of a scientific screening approach, the purpose of which is to identify a given strain, or strain among thousands of strains, that is unique and therefore may provide benefits to the host as well as economic value.

Thus, it was surprising to find that B. licheniformis DSM13 ^T showed greater antimicrobial activity (68.58% on average) against pathogenic isolates of E. cecorum compared to commercial probiotic strains of B. licheniformis and #10/4 with indicators constituting 1.36% and -12.41%, respectively (Table 4). The B. subtilis type DSM10 ^T strain exhibited an inhibition percentage of 26% on average, which was nevertheless higher than the average percentage exhibited by the commercial B. subtilis probiotic strain #11/1 (–21%) (Fig. .7 and table 4).

Claims (3)

1. A method of inhibiting or slowing down the growth of representatives of pathogenic Enterococcus cecorum in poultry, which includes administering to the animal an effective amount of at least one of the strains of a microorganism for ingestion selected from the group of Bacillus strain 2084 under accession number NRRl B-50013 and strain 15A- P4 Bacillus accession no. ATCC 15 PTA-6507. 2. The method of claim 1, wherein the at least one edible microorganism is administered directly to the animal with the animal food, either as contained in the food, added on top of the food, or in the form of a liquid. 3. The method of claim 1, wherein at least one edible microorganism is administered to the animal in a form selected from the group consisting of a feed product, a feed additive composition, a premix, or a liquid.

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