AU2024208979A1

AU2024208979A1 - Probiotic strains capable of inhibiting fusobacterium necrophorum and improving gut barrier integrity

Info

Publication number: AU2024208979A1
Application number: AU2024208979A
Authority: AU
Inventors: Erik Juncker BOLL; Lena Catrine CAPERN; Bruno Ieda CAPPELLOZZA; Giuseppe COPANI; Jennifer SCHUTZ; Line SKJOET-RASMUSSEN
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2023-01-18
Filing date: 2024-01-16
Publication date: 2025-07-24
Also published as: MX2025008288A; EP4658288A1; WO2024153628A1

Abstract

The present disclosure generally relates to compositions comprising Lactobacillus animalis with inhibitory effect against negative effects transferred by pathogens on plants upon ingestion by the animal and/or inhibitory effect on gastrointestinal pathogenic infectionits gut, to a process for its preparation, to the use of Lactobacillus animalis to preventing, controlling, combating and/or inhibiting Fusobacterium necrophorum, to ameliorating the damaging effect of deoxynivalenol on gut barrier integrity and function, and to a kit.

Description

PROBIOTIC STRAINS CAPABLE OF INHIBITING FUSOBACTERIUM

NECROPHORUM AND IMPROVING GUT BARRIER INTEGRITY

FIELD OF THE DISCLOSURE

The present disclosure generally relates to compositions comprising Lactobacillus animalis with inhibitory effect against negative effects transferred by pathogens on plants upon ingestion by the animal and/or inhibitory effect on gastrointestinal pathogenic infection, to a process for its preparation, to the use of Lactobacillus animalis to controlling, combating and/or inhibiting Fusobacterium necrophorum, to ameliorating the damaging effect of deoxynivalenol on gut barrier integrity and function, and to a kit.

BACKGROUND

Beef serves as a major protein source in human nutrition. Over the last 25 years, global beef production has increased exponentially. With the global demand for meat and milk projected to double by 2050, beef production is expected to increase to meet future demands. This change in production scale has resulted in intensification in production systems. As a consequence, the need for better animal health, disease control, and "health management" has led to the increased use of antibiotics in the beef cattle production system. This increased use of antibiotics (especially for metaphylactic use and as growth promoters) has led to an increase in the microbial population that is resistant to antibiotics and in the Antimicrobial Resistance (AMR) gene pool within the beef cattle production system. As such, novel strategies to reduce antimicrobial use while improving animal health and efficiency are critical. One such alternative strategy is the use of probiotic strains of bacteria to help improve animal health replacing antibiotics. In the current disclosure, several isolated species of bacteria were identified and tested for their ability to control or inhibit the growth of F. necrophorum, a pathogen known to be involved in liver abscesses occurrence in cattle.

Liver abscess is an important metabolic disease that is affecting ruminants and especially beef cattle due to their its specific diet containing a greater amount of grains, minerals and vitamins, and thus richer in energy and/or protein, but low in fiber. The common feedstuffs used in beef cattle diets include corn, soybean meal, oats, wheat, molasses, etc. The losses related to liver abscess are different and wide, ranging from liver discards due to contamination, to decrease in body weight and performance, poor feed efficiency, low carcass yield as well as other problems related to the slaughterhouses. In 2010, Brown and Lawrence estimated an annual economic loss of $7,007,797 to the beef industry due to liver abscessation in cattle.

This disclosure demonstrates the potential of using probiotics as an alternative to antibiotics to control pathogens involved in the formation of liver abscesses, i.e. the occurrence of liver abscesses in ruminants, like F. necrophorum. Fusobacterium necrophorum is one of the etiological agents associated with liver abscesses in ruminants. The disclosure also demonstrates the probiotic L. animalis strain's ability to support gut barrier integrity and function under stressful conditions. The disclosure also demonstrates a reduction in number of liver abscesses observed in beef cattle after administration of the composition comprising probiotic strain(s) of the disclosure to beef cattle during the finishing period in the feedlot.

The composition comprising probiotic strain(s) of the present disclosure has the added benefit of being able to ameliorate the damaging effect of deoxynivalenol (DON) on gut barrier integrity. The mycotoxin DON is produced by various Fusarium fungi known to frequently infect various grains in the field or during storage. It is one of the most common mycotoxin contaminants of cereal-based food and animal feed. This fact, along with the stability of DON through food processing and its known cytotoxic effects has made it a major public health concern as a food contaminant for humans and livestock. Upon ingestion, DON causes impaired integrity of the intestinal barrier. DON-induced increased epithelial permeability to toxins, pathogenic bacteria and viruses then leads to gastrointestinal inflammation. DON exerts both acute and chronic toxic effects in humans and livestock with symptoms including diarrhea, vomiting, abdominal pain, and weight loss. At high dosages, DON ingestion may lead to tissue damage and ultimately death, whereas chronic exposure to low dosages caused reduced weight gain and altered nutritional efficiency. From an economical perspective, the mitigation of the toxic effects of DON and other mycotoxins in livestock was recently estimated to be $466 million.

The present disclosure demonstrates the ability of L. animalis when it is combined with other probiotic bacterial strains to ameliorate the damaging effect on the gut barrier integrity caused by exposure to DON.

Bartenschlager et al. discuss the use of probiotics as additives to cattle feed and present work on identifying new probiotic strains useful as feed additives for cattle. A Bacillus pumilus and a Bacillus licheniformis strains are identified as being particularly useful as Direct Feed Microbial as they among other things inhibit F. necrophorum. However, in vivo trials show no difference in liver scores between the control and the test group.

Amachawadi et al. (2016) is a review of the incidences of liver abscess in Holstein cows and discuss the bacteriology of the same and the development of a vaccine approach to control liver abscesses in cattle.

Thus, there is still a need for novel ways of improving the health of ruminants by minimizing pathogenic pressure on the animal in an easy and safe way. It would be advantageous to obtain these benefits without having to use antibiotics, but relying solely on a biological solution using probiotic strains. The inventors of the present disclosure have proceeded with extensive screening and research in order to identify probiotic strains capable of inhibiting pathogens that pose a significant problem to the ruminant stock, and at the same time being able to improve gut barrier integrity, thereby solving the problem of providing safe, biological solutions to the field of ruminants.

SUMMARY OF THE DISCLOSURE

The present disclosure provides a method of inhibiting a gastrointestinal pathogenic Fusobacterium necrophorum infection in an animal, the method comprising, administering to said animal a composition that inhibits the growth of said pathogenic bacterium, said composition comprising Lactobacillus animalis.

The isolated bacterial species of the present disclosure were screened for their ability to inhibit in vitro F. necrophorum using agar diffusion assays and supernatant assays to characterize and compare their abilities. The results revealed the inhibition of E necrophorum by probiotic bacteria of the genus Enterococcus, Bacillus, and Lactobacillus. Of the tested Lactobacillus strains the best Lactobacillus strain was identified and its ability to improve gut barrier integrity was examined. A combination of probiotic bacteria was examined for its ability to improve gut barrier integrity in the absence of stressful conditions and after exposure to deoxynivalenol (DON) mycotoxin.

Within the disclosure is also an animal feed, animal feed additive, or premix for use in inhibiting a gastrointestinal pathogenic Fusobacterium necrophorum infection in an animal, comprising Lactobacillus animalis and further comprises one or more additional components selected from concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s), and other feed ingredient(s).

The composition of the disclosure and the animal feed, animal feed additive or premix may further comprise additional probiotic strains selected from the group consisting of the following: Enterococcus faecium, Propionibacterium freudenreichii, Bacillus subtilis, Bacillus altitudinis, Bacillus pumilus and Bacillus licheniformis.

In one embodiment of the present disclosure, the Lactobacillus animalis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 33570.

The disclosure describes and demonstrates the effect of probiotic bacteria on the inhibition of F. necrophorum and of a Lactobacillus animalis strain with superior inhibitory effect. Further described is the additional counteracting effect on DON- induced impaired intestinal epithelial barrier integrity when a combination of probiotic bacteria including L. animalis is used.

While not wishing to be bound by theory, it is believed that having a probiotic solution to limit the severity of liver abscess or controlling directly one of the major etiological agents associated with liver abscesses, leads to a lower infection rate with F. necrophorum and thus limiting the prevalence and/or severity of liver abscess, will be a great advantage for the cattle industry. With the additional effect of ameliorating the damaging effects of DON exposure on the gut barrier integrity, the suggested probiotic solution has the potential to solve simultaneously two major challenges involving gastrointestinal infection in the cattle industry.

The disclsoure is as described in the claims.

DEFINITIONS

In general, the terms and phrases used herein have their art- recognized meaning, which can be found by reference to standard textbooks, journal references, and context known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

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

Composition: As used herein the term "composition" refers to a composition comprising a carrier and at least one bacterial strain as described herein.

Effective amount/concentration/dosage: As used herein the terms "effective amount", "effective concentration", or "effective dosage" are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the overall health of the animal and confer benefits similar to the ones demonstrated in the examples.

The actual effective dosage in absolute numbers depends on factors including the state of health of the animal in question, and other ingredients present. The "effective amount", "effective concentration", or "effective dosage" of the bacterial strains may be determined by routine assays known to those skilled in the art.

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

By "inhibit" or other forms of the word, such as "inhibiting" or "inhibition," may in certain instances refer to the lowering of an event or characteristic (e.g., microorganism growth or survival). It is understood that this is typically in relation to some standard or expected value, in other words, it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, "inhibits the population of bacteria" in certain instances may refer to lowering the amount of bacteria relative to a standard or a control. The term "probiotic" in certain instances may refer to one or more live microorganisms that confer beneficial effects on a host organism. Benefits derived from the establishment of probiotic microorganisms within the digestive tract include reduction of pathogen load, improved microbial fermentation patterns, improved nutrient absorption, improved immune function, aided digestion and relief of symptoms of irritable bowel disease and colitis.

The term "pathogen" in certain instances may refer to any microorganism that produces a harmful effect and/or disease state in a human or animal host.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1

Figure 1 discloses an example of inhibition zones when Fusobacterium necrophorum is subjected to Lactobacillus animalis in an in vitro agar diffusion assay.

FIGURE 2

Figure 2 discloses the inhibitory potential of several L. animalis strains against F. necrophorum which was tested in two different agars (Muller Hinton agar and Wilkins Chalgren agar).

FIGURE 3

Figure 3 discloses the result of TEER assay without challenge. Intestinal epithelial Caco-2 cell monolayers seeded and differentiated for three weeks in transwells were exposed on the apical side to 1.7xl0⁷ CFU pr. transwell of L. animalis or Bovamine® Defend Plus (BDP) normalized to 1.7xl0⁷ CFU pr. transwell of L. animalis. TEER was measured for a total of 24 hrs. Data is expressed as means of percent of relative TEER (A) or area under the curve (AUC, baseline of 100% relative TEER) (B) ± SD for triplicate samples. ** (p<0.01); **** (p<0.0001) indicate significant differences from the "Bovamine® Defend Plus" group. One-way ANOVA followed by Dunnetts's multiple comparisons.

FIGURE 4

Figure 4 discloses the result of TEER assay with DON challenge. Caco-2 cell monolayers seeded and differentiated for three weeks in transwells were exposed on the apical side to Bovamine® Defend Plus (BDP, 1.7xl0⁷ CFU of L. animalis; IxlO⁸ total CFU pr. transwell) and FITC-dextran 20 kDa (FD20, 400pg pr. transwell), and four hours later challenged on both the apical and basolateral side with 50pM DON. TEER was measured for a total of 24 hrs (A), after which the amount of FD20 translocated to the basolateral compartment was quantified (B). Data is expressed as means of percent of relative TEER or percent of translocated FD20 ± SD for triplicate samples. ** (p<0.01); *** (p<0.001) indicate significant differences from the "DON only" group. One-way ANOVA followed by Dunnetts's multiple comparisons.

DETAILED DESCRIPTION

Liver abscesses in cattle

The current hypothesis of liver abscess formation, i.e. liver abscess occurrence in cattle is described as a dysbiosis of the microbiome due to increased availability of fermentable carbohydrates in high-grain diets leading to an increase in the abundance of Streptococcus bovis (S. bovis) in the rumen. S. bovis is a lactic acid producing organism commonly present within the rumen. As such, when fermentable carbohydrates are abundant, S. bovis increases in abundance and changes its metabolism to homolactic fermentation, resulting in increased lactic acid production. This shift enables the ruminal epithelium to become compromised, as a result of lactic acidosis to occur. Thus, the ruminal pH drops from ~6.5 to <5. This condition leads to ruminitis, a condition which dehydrates and impairs the integrity of the epithelial layer of the ruminal wall, allowing for Fusobacterium necrophorum, the major pathogen involved in liver abscess formation, to enter into the liver via the portal blood flow (Bartenschlager et al). Dysbiosis may also occur in the lower GIT of ruminants, leading to the occurrence of the hyperpermeable syndrome which, ultimately may lead to leakage of F. necrophorum and other pathogens in the blood circulation and the occurrence of liver abscess (Pinnell et al., 2023).

Currently, liver abscesses are being treated by a prophylactic management with tylosin, an antibiotic from the macrolide family. Thus, the overuse of antibiotic classes, that are also used to treat human disease, can lead to antibiotic resistant pathogens. These pathogens can resist antibiotics currently used in the medical industry and thus, prophylactic use of antibiotics in the beef industry has come under great scrutiny. Additionally, the use of tylosin in the feed is not approved for use in dairy cows, leaving this group of cattle without a treatment for liver abscesses and, therefore, more susceptible to their negative effects on health and performance of the dairy cow herd.

Although tylosin is widely used in the feedlot industry, there is considerable interest in evaluating antibiotic alternatives, such as probiotics, essential oils, and vaccines, to control liver abscesses.

Fusobacterium necrophorum and liver abscesses in cattle

Liver abscesses are almost always polymicrobial infections with Gram-negative anaerobe bacteria constituting the predominant flora. Almost all studies have concluded that F. necrophorum, a ruminal bacterium, is the primary causative agent.

Fusobacterium necrophorum, a Gram-negative, rod-shaped, and aerotolerant anaerobe, is a normal inhabitant of the rumen of cattle. The organism is in ruminal contents and adherent to the ruminal wall. Its role in ruminal fermentation is to metabolize lactic acid and degrade feed and epithelial proteins. The ruminal concentration is higher in grain-fed than forage-fed cattle. From the rumen, the organism gains entry into the portal circulation and is trapped in the liver to cause abscesses. The organism is an opportunistic pathogen and a primary causative agent of liver abscesses, an economically important disease of grain-fed cattle. Liver abscesses are often secondary to ruminal acidosis and rumenitis in grain-fed cattle. Two subspecies of F. necrophorum, subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B), are recognized that can be differentiated based on morphological, biochemical, biological and molecular characteristics. The subsp. necrophorum is more virulent and is isolated more frequently from infections than the subsp. funduliforme. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis), either specific or non-specific infections, in a variety of animals. Of these, bovine liver abscesses and foot rot are of significant concern to the cattle industry. Liver abscesses arise with the organisms (including F. necrophorum) that inhabit the rumen gaining entry into the portal circulation, and are often secondary to ruminal acidosis and rumenitis complex in grain-fed cattle where impaired integrity of the epithelial layer of the ruminal wall can be observed. Foot rot is the major cause of lameness in dairy and beef cattle. The pathogenic mechanism of F. necrophorum is complex and not well defined. Several toxins or secreted products, such as leukotoxin, endotoxin, hemolysin, hemagglutinin, proteases, and adhesin, etc., have been implicated as virulence factors. The major virulence factor appears to be leukotoxin, a secreted protein of high molecular weight, active specifically against leukocytes from ruminants. Moreover, F. necrophorum may reach the liver also following the breakdown of the integrity of the lower GIT. T. pyogenes is the second most commonly isolated bacterium from abscesses of liver in feedlot cattle. T. pyogenes is commonly associated with Fusobacterium necrophorum and Porphyromonas levii in necrotic infections of the laminae of the feet.

The present disclosure provides a probiotic L. animalis strain which demonstrates the ability to inhibit F. necrophorum and thus has the potential to solve challenges in the cattle industry of high relevance by controlling, combatting and/or inhibiting F. necrophorum infection and hence limiting the negative effects of same. By being able to limit the amount of F. necrophorum in the rumen of the cattle, it is possible to limit the burden of the pathogen on the liver if impaired integrity of the epithelial layer of the ruminal wall should arise. To be able to control pathogens, such as F. necrophorum, is of high relevance for the livestock industry.

The present disclosure also demonstrates that the L. animalis strain of the disclosure has the ability to inhibit T. pyogenes when provided in combination with P. freundenreichii, B. subtilis, and B. licheniformis. As T. pyogenes also is a major pathogen involved in liver abscesses in cattle, it is believed that this ability provides an additional benefit. While not wishing to be bound by theory it is believed that this makes the L. animalis strain of the disclosure highly relevant in the fight against liver abscesses in cattle, either alone or in combination with other probiotic strains.

In one embodiment of the present disclosure a method of inhibiting a gastrointestinal pathogenic Fusobacterium necrophorum infection in an animal, the method comprising, administering to said animal a composition that inhibits growth of said pathogenic bacterium, said composition comprising Lactobacillus animalis is provided.

Fusarium spp. and deoxynivalenol

Among the plant diseases, fungal pathogens are the biggest global threat causing huge losses in agriculture and food/feed production. Fungal pathogens, Fusarium graminearum, Fusarium culmorum, and other Fusarium spp. fungi, cause fusarium head blight (FHB), which is a devastating crop disease resulting in billions of dollars in economic losses worldwide annually. The Fusarium spp. spores are often present in soils, can remain there for several seasons and reinfect the plants. The symptoms associated with FHB include discoloration or yellowing of glumes and spikelets after flowering. In relation to the cattle industry, the most devasting problem with these fungi is that they contaminate seeds of the crops infected with the fungi mainly with two types of mycotoxins; trichothecene deoxynivalenol and zearalenone, which are produced during infection. Both mycotoxins have hazardous effects on human and animal health.

Various strategies have been implemented to inhibit and control phytopathogens, such as Fusarium graminearum and Fusarium culmorum, including application of chemical fungicides, crop rotation, seed treatment etc. Even though the correct usage of fungicide at an early heading date can reduce FHB occurrence by 50-60%, the application of fungicides is challenging due to the overlapping of different developmental stages within the crop. To note, FHB disease generally develops late in the season or also during storage of the crops/seeds indicating that early application of fungicides might only be partially effective. Furthermore, prolonged use of chemically synthesized fungicides reduces microbial biodiversity in soil, increases pathogen resistance, and generally degrades the soil quality.

Hence, fully controlling the Fusarium fungi is at the moment not possible and some degree of infection is always present and must be expected to pose a risk for animals fed a high grain diet. The mycotoxin deoxynivalenol (DON), produced by various Fusarium fungi, often infects various grains in the field and/or during storage. It is one of the most common mycotoxin contaminants of cereal-based food and animal feed. This fact, along with the stability of DON through food processing and its known cytotoxic effects has made it a major public health concern as a food contaminant for humans and livestock. Upon ingestion, DON causes impaired integrity of the intestinal barrier. DON-induced increased epithelial permeability to toxins, pathogenic bacteria, and viruses then leads to gastrointestinal inflammation. DON exerts both acute and chronic toxic effects in humans and livestock with symptoms including diarrhea, vomiting, abdominal pain, and weight loss. At high dosages, DON ingestion may lead to tissue damage and ultimately death, whereas chronic exposure to low dosages causes reduced weight gain and altered nutritional efficiency. From an economical perspective, the mitigation of the toxic effects of DON and other mycotoxins in livestock was recently estimated to be $466 million.

High performing cattle, especially beef cattle, are often fed a high grain diet, making the Fusarium associated mycotoxin DON a concern in livestock management due to potential excessive ingestion of DON.

The present disclosure provides a probiotic L. animalis strain which demonstrates the ability to ameliorate the damaging effects of DON exposure on the gut barrier integrity when administered in combination with other probiotic bacteria. This has the potential to solve challenges in the livestock industry of high relevance.

In one embodiment of the disclosure a method of ameliorating the damaging effects of DON exposure on the gut barrier integrity is provided.

Probiotic bacteria and Lactobacillus spp

Probiotics have proven to be a substantial substitute for antibiotics used in the animal diet and thus have gained popularity. Probiotics are live and non-pathogenic microbes commercially utilized as modulators of gut microflora, hence exerting advantageous effects on the health and productivity of animals. The Lactobacillus genus includes one of the most prevalently administered probiotic bacteria. Lactobacillus is a genus of more than 25 species of gram-positive, catalase-negative, non-sporulating, rodshaped organisms. Lactobacillus species are generally anaerobic, non-motile, and do not reduce nitrate.

The most common method used today to control pathogenic bacterial populations in livestock is through the use of antimicrobial compounds. While these are effective for short-term treatments, prolonged application of antimicrobial compounds leads to the evolution of antibiotic resistance in pathogenic organisms. The widespread occurrence of antibiotic-resistant microorganisms is well known, some of the most common being methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Bacteria are remarkably adaptable to deleterious environments with their abilities to rapidly reproduce and modify their genetic content. Thus, it is inevitable that after prolonged application of any method that disrupts or kills bacteria, a population that is recalcitrant to its effects will eventually arise.

The application of probiotic bacteria that are meant to inhibit or reduce the numbers of pathogenic bacteria within a gastrointestinal system have been utilized for some years in the animal feed industry. Significantly better animal performance and pathogen reductions can be seen in treated animals and positive effects on weight gain, feed conversion ratio and diarhea scores have been reported. Several probiotic products also exists on the market, however no product exists that targets F. necrophorum specifically.

In one embodiment of the present disclosure it is contemplated that the L. animalis is combined with other probiotic strains in a composition and/or animal feed, animal feed additive or premix. There are numerous advantages for the inclusion of multiple strains of microorganisms in a microbial product. The potential advantages, whether working independently or concurrently, allow for a superior microbial product and enhanced benefits for the host.

A benefit of a multiple strain-containing composition is the ability to target more than one pathogen population. Microbial pathogens are very diverse and require different methods to reduce or eliminate their populations. Thus, a composition containing different microorganisms that can affect different pathogenic populations will result in an overall healthier system. Different microorganisms positively influence the gastrointestinal system through different mechanisms. One strain may reduce pathogen populations, while another may have an immunostimulative effect, and another may produce micronutrients essential for the host. Interestingly, multiple strains may also provide synergistic effects upon the host or pathogen inhibition abilities. One strain alone may not be able to reduce certain populations, but the combination of two different strains working through different mechanisms can reduce pathogen populations.

Additionally, the use of multiple beneficial microorganisms can help overcome bacteriophages that infect and kill bacteria. Bacteriophages are very common in gastrointestinal systems and have profound effects upon the microbial community. Bacteriophages require specific sites on a cell to bind and infect. Thus, including multiple microorganisms in a product, the greater the likelihood that at least some populations from the product will evade bacteriophage attack and elicit beneficial effects upon the microbial community and host.

Certain embodiments of the present disclosure concern a method of inhibiting or reducing a population of pathogenic bacteria in or on an animal. In such embodiments, the population of pathogenic bacteria may be on the skin of the animal, in the blood of the animal or in one or more organs of the animal. In specific embodiments, the disclosure concerns a method of inhibiting or reducing a population of pathogenic bacteria in the gastrointestinal tract of the animal.

In embodiments concerning the inhibition or reduction of a population of pathogenic bacteria, the method comprises providing to the animal a composition comprising at least one probiotic microorganism.

In one embociments of the disclosure, the probiotic microorganism is L. animalis.

In the embodiments of the disclosure, at least one additional probiotic microorganism is comprised in the composition. This probiotic microorganism may be any probiotic organism or any strain of a probiotic microorganism. In specific embodiments the composition comprises Enterococcus faecium, Bacillus licheniformis, Bacillus paralicheniformis, Bacillus subtilis, Lactococcus lactis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium thermophilum, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus alactosus, Lactobacillus alimentarius, Lactobacillus amylophilus, Lactobacillus amylovorans, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus batatas, Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus buchnerii, Lactobacillus bulgaricus, Lactobacillus catenaforme, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus confusus, Lactobacillus coprophilus, Lactobacillus coryniformis, Lactobacillus corynoides, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus desidiosus, Lactobacillus divergens, Lactobacillus enterii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus frigidus, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus gasseri, Lactobacillus halotolerans, Lactobacillus helveticus, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus hordniae, Lactobacillus inulinus, Lactobacillus jensenii, Lactobacillus jugurti, Lactobacillus kandleri, Lactobacillus kefir, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus minor, Lactobacillus minutus, Lactobacillus mobilis, Lactobacillus murinus, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pseudoplantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rogosae, Lactobacillus tolerans, Lactobacillus torquens, Lactobacillus ruminis, Lactobacillus sake, Lactobacillus salivarius, Lactobacillus sanfrancisco, Lactobacillus sharpeae, Lactobacillus sobrius, Lactobacillus trichodes, Lactobacillus vaccinostercus, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus xylosus, Lactobacillus yamanashiensis,

Lactobacillus zeae, Pediococcus acidlactici, Pediococcus pentosaceus, Streptococcus cremoris, Streptococcus diacetylactis, Streptococcus faecium, Streptococcus intermedius, Streptococcus lactis, Streptococcus thermophilus, Propionibacterium freudenreichii, Propionibacterium acidipropionici, Propionibacterium jensenii, Propionibacterium thoenii, Propionibacterium cyclohexanicum, Propionibacterium granulosum, Propionibacterium microaerophilum, Propionibacterium propionicum, Propionibacterium acnes, Propionibacterium australiense, Propionibacterium avidum or a combination thereof. In other embodiments, the composition comprises one or more different strains of the aforementioned species of probiotic microorganisms.

In one embodiment the composition could contain any number of microorganisms and or microbial components and/or metabolites. Examples of bacterial species that could be used for the probiotic mixture include but are not limited to the group consisting of: Enterococcus faecium, Bacillus licheniformis, Bacillus subtilis, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus animalis, Lactobacillus brevis, Lactobacillus buchnerii, Lactobacillus bulgaricus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus hilgardii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Pediococcus acidlactici, Pediococcus pentosaceus, Propionibacterium freudenreichii, Propionibacterium acidipropionici, or a combination thereof. In other embodiments, the composition comprises one or more different strains of the aforementioned species of probiotic microorganisms.

In one embodiment of the present disclosure, the composition comprises a mixture of L. animalis together with at least one probiotic strain selected form the group consisting of Propionibacterium freudenreichii, Bacillus subtilis and Bacillus licheniformis.

In one embodiment of the present disclosure the composition comprises a combination of Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis and

Bacillus licheniformis. In one embodiment of the present disclosure the composition comprises a combination of the Lactobacillus animalis strain deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen as DSM 33570, with any one of the following strains: the Propionibacterium freudenreichii strain deposited as DSM 34127, the Bacillus licheniformis strain deposited as DSM 17236, the Bacillus subtilis strain deposited as DSM 32324 and/or the Bacillus subtilis strain deposited as DSM 17231.

In embodiments of the present disclosure wherein an administration of probiotic microorganisms is contemplated, the number of microorganisms per administration may be any amount capable of providing some inhibition or reduction of a population of pathogenic bacteria. In specific embodiments, the number of microorganisms per administration is between Ix lO⁴ and IxlO¹⁰ CFU/g. In one embodiment the number of microorganisms in an administration is at approximately IxlO⁹ CFU/g.

In one embodiment of the present disclosure, the probiotic microorganisms are provided in an animal feed, animal feed additive, or premix in an amount to provide a total of between 1 x 10⁶ to 1 x 10¹¹ CFU/animal/day when the animal feed, animal feed additive or premix is fed to the animal. In one embodiment the animal feed, animal feed additive or premix comprises probiotic microorganisms in an amount to provide a total of between 1 x 10⁸ and 1 x 10¹¹ CFU/animal/day. In one embodiment the number of microorganisms in an administration of animal feed, animal feed additive or premix is at approximately Ix lO⁹ CFU/animal/day.

In one embodiment, the administration is oral administration. In embodiments wherein the administration is oral administration, the composition comprising one or more probiotic bacteria may be mixed with or incorporated in animal feed, animal feed additive or premix, or mixed with animal drinking water. In such embodiments, the composition or compositions may be formulated as a liquid formulation for administration, or as a freeze-dried formulation, or as a gel formulation or as a spore formulation. Formulations

In certain aspects of the disclosure contemplate a carrier formulation for the probiotic microorganisms. In certain aspects of the disclosure, the carrier may be any number of different percentages (weight per weight, weight per volume, or volume per volume) of the final product. The carrier can comprise any amount of about 99.9%, about 95%, about 90%, about 80%, about 70%, about 60% about 50%, about 40%, about 30%, and so on. The remaining composition can also include other carriers such as lactose, glucose, sucrose, salt, cellulose, etc. In specific aspects of the disclosure, the carrier may be 50% or more of the total product.

In certain aspects of the disclosure, other chemicals or materials mainly used for the reduction or absorption of moisture may also be included. These may include, but are not limited to: calcium stearate, sodium aluminosilicate, silica, calcium carbonate, zeolite, bicarbonates, sodium sulfate, silicon dioxide, or ascorbic acid.

In certain aspects of the disclosure, other chemicals or materials mainly used for the reduction or absorption of oxygen may also be included. These may include, but are not limited to, iron oxides, ascorbic acid, sodium sulfide, and silica materials.

Antioxidants included in a preservation matrix may be provided to retard oxidative damage to the microbial cells during the preservation and storage process. A particularly preferred antioxidant is sodium ascorbate. An antioxidant typically comprises from about 0.1% to about 1.0% by weight of the preservation matrix and preferably comprises about 0.5% by weight of the preservation matrix. In one embodiment, a preservation matrix of the present disclosure comprises about 0.5% sodium ascorbate by weight of the preservation matrix.

Examples of suitable saccharide carrier components are sucrose, fructose, maltose, dextrose, lactose and maltodextrin. An example of a suitable polyol is glycerol. Examples of suitable amino acids are glutamic acid, aspartic acid, and the salts thereof. An example of a suitable peptide carrier is peptone. An example of a milk- derived compound is, in addition to the abovementioned maltodextrin, also sweet whey powder. Suitable organic carboxylic acids are, for example, citric acid, malic acid and L-ascorbic acid. Examples of suitable mineral carriers are montmorillonite and palygorskite.

Freeze-drying for preparing dry microorganism cultures according to the disclosure can be carried out.

Another, drying process contemplated for use in the present disclosure is spraydrying. Those methods that can be used according to the disclosure are essentially all spray-drying techniques known in the art. The material to be sprayed can, for example, be dried concurrently or countercurrently; spraying can be carried out by means of a single-component or multiple-component nozzle or by means of an atomizer wheel.

The drying process according to the disclosure may be carried out in such a manner that a very low residual moisture content is present in the dry material.

Instead of the above-described physical post-drying processes, it is also conceivable to add specific desiccants to the dry material obtained from the spray-drying. Examples of suitable desiccants are inorganic salts, such as calcium chloride and sodium carbonate, organic polymers, such as the product obtainable under the trade name Kollidion 90 F, and silicon-dioxide-containing desiccants, such as silica gel, zeolites and desiccants which are obtainable under the trade name Tixosil 38, Sipernat 22 S or Aerosil 200.

Some bacteria can survive environmental stresses through the formation of spores. This complex developmental process is often initiated in response to nutrient deprivation. It allows the bacterium to produce a dormant and highly resistant cell. Spores can survive environmental assaults that would normally kill other bacteria. Some stresses that endospores can withstand include exposure to high temperatures, high ultraviolet irradiation, desiccation, chemical damage, and enzymatic destruction. The extraordinary resistance properties of endospores make them of particular importance because they are not readily killed by many antimicrobial treatments. Common bacteria that form spores include species from the Bacillus and Clostridium genera. Spores formed by these bacteria remain in their dormant state until the spores are exposed to conditions favorable for growth. The inclusion of spores in a probiotic composition is appealing because of their ability to withstand processing methods and can have extended shelf life viabilities. Additionally, bacterial spores can require less processing because they do not require additional steps for preservation (such as freeze drying, spray drying, freezing, etc.), as is required for many other probiotic organisms.

In one embodiment of the disclosure Bacillus spp. probiotic bacteria are added to the composition together with the L. animalis strain, such as Bacillus subtilis, Bacillus altitudinis and/or Bacillus licheniformis.

Uses of Formulated Bacterial Products

The methods and formulations of the present disclosure can be adjusted to provide beneficial effects to many types of animals, including ruminal fermenters, i.e. ruminants. In one preferred embodiment, the product is fed to ruminal fermenters to inhibit a gastrointestinal pathogenis caused by F. necrophorum infection and/or to ameliorate the damaging effects of DON exposure on the gut barrer integrity, thereby improving animal health and animal productivity. Ruminal fermentors that might benefit from the present disclosure include but are not limited to: cattle, sheep, goats, camels, llama, bison, buffalo, deer, wildebeest, antelope, and any other pre-gastric fermentor.

The various embodiments of the disclosure include the application of a combination of probiotic microorganisms to the animal feed, animal feed additive or premix. The different microorganisms can be of different species, or they may be of the same species but constitute different strains within said species. The product may contain multiple species and multiple strains. For example, two, three, four, five, six, and so on different microorganisms and/or strains can be applied. The application of multiple types of different microorganisms and/or different strains lead to additive or more preferably super additive or more preferably synergistic effects in maintaining or improving animal health or decreasing or eliminating the presence of pathogenic bacteria.

The amount of microorganism administered to the animal feed can be any amount sufficient to achieve the desired increase in animal health. This amount can be anywhere from 1 to 10¹³ CFU per kg of animal feed. For example, amounts of about 10⁴ CFU/gram feed, about 5xl0⁴ CFU/gram feed, about 10⁹ CFU/gram feed, about 5xl0⁹ CFU/gram feed, or ranges between 1 to 10¹³ CFU per kg of animal feed can be used. In some embodiments, the dried biological may be administered to an animal through a variety of means including, but not limited to, being distributed in an aqueous solution and subsequently being applied to animal feed, water source, or directly fed to the animal, or through direct application of the product onto animal feed or direct administration or consumption by the animal.

In certain embodiments, the compositions and methods of the present disclosure involve two or more probiotic bacteria. These compositions would be provided in a combined amount effective to achieve the desired effect, for example, the killing or growth inhibition of a pathogenic microorganism. This process may involve administering different strains or species microorganisms at the same time. In certain embodiments the different strains or species may be combined into a single formulation for administration. In other embodiments, the different strains or species may be each in a single formulation for administration. Still in other embodiments, some microorganism strains or species may be combined into a single formulation and others may be combined into a different formulation. When more than one formulation is used, the formulations may be administered to the animal at the same time or subsequent to each other.

In such instances where administration is subsequent to each other, it is contemplated that one may administer both formulations within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for administration significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It is further contemplated that other administrations may be used with three or more different formulations of microorganisms.

In one embodiment, the composition of the present disclosure is designed for continual or periodic administration to ruminants throughout the feeding period in order to reduce the incidence and severity of intestional infections associated with F. necrophorum and/or ameliorate the damaging effects of DON exposure on the gut barrier integrity.

The following listed aspects are further comprised by present disclosure:

ASPECTS

1. A method of inhibiting a gastrointestinal pathogenic Fusobacterium necrophorum infection in an animal, the method comprising, administering to said animal a composition that inhibits growth of said pathogenic bacterium, said composition comprising Lactobacillus animalis. The method of aspect 1, wherein said composition is administered to said animal in an amount to provide a total amount of said probiotic strain of between 1 x 10⁸ and 1 x 10¹¹ CFU/animal/day. The method according to any of the preceding aspects, wherein said composition further comprises at least one additional probiotic strain selected from the group consisting of the following species: Enterococcus faecium, Propionibacterium freudenreichii, Bacillus subtil is and Bacillus licheniformis. The method according to any of the preceding aspects, wherein the Lactobacillus animalis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 33570. The method according to any of the preceding aspects, wherein the Enterococcus faecium is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 22502. The method according to any of the preceding aspects, wherein the Propionibacterium freudenreichii is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 34127. The method according to any of the preceding aspects, wherein the Bacillus subtilis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 32324 or the strain deposited with accession no. DSM 17231. The method according to any of the preceding aspects, wherein the Bacillus licheniformis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 17236. The method according to any of the preceding aspects, wherein said composition comprises a combination of Lactobacillus animalis,

Propionibacterium freudenreichii, Bacillus subtilis and Bacillus licheniformis. 10. The method according to any of the preceding aspects, wherein said composition comprises Lactobacillus animalis strain DSM 33570, Propionibacterium freudenreichii strain DSM 34127, Bacillus licheniformis strain DSM 17236 and Bacillus subtilis strain DSM 32324.

11. The method according to any of the preceding aspects, wherein said composition further ameliorates the damaging effects of deoxynivalenol (DON) exposure on gut barrier integrity.

12. An animal feed, animal feed additive or premix for use in inhibiting a gastrointestinal pathogenic Fusobacterium necrophorum infection in an animal, comprising Lactobacillus animalis and further comprises one or more additional components selected from concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s), and other feed ingredient(s).

13. The animal feed, animal feed additive or premix according to aspect 12, wherein said Lactobacillus animalis are present in said animal feed, animal feed additive or premix in an amount to provide a total of between 1 x 10⁸ to 1 x 10¹¹ CFU/animal/day.

14. The animal feed, animal feed additive or premix according any of aspects 12 to 13, further comprising additional probiotic strains selected from the group consisting of the following species: Enterococcus faecium, Propionibacterium freudenreichii, Bacillus subtilis, and Bacillus licheniformis.

15. The animal feed, animal feed additive or premix according to aspect 14, wherein the Lactobacillus animalis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 33570.

16. The animal feed, animal feed additive or premix according to aspect 14, wherein the Enterococcus faecium is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM

22502. he animal feed, animal feed additive or premix according to aspect 14, wherein the Propionibacterium freudenreichii is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 34127. he animal feed, animal feed additive or premix according to aspect 14, wherein the Bacillus subtilis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 32324 or the strain deposited with accession no. DSM 17231. he animal feed, animal feed additive or premix according to aspect 14, wherein the Bacillus licheniformis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 17236. he animal feed, animal feed additive or premix according to any of aspects 12 to 19, wherein said composition comprises a combination of Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis and Bacillus licheniformis. he animal feed, animal feed additive or premix according to aspect 20, wherein said composition comprises Lactobacillus animalis strain DSM 33570, Propionibacterium freudenreichii strain DSM 34127, Bacillus licheniformis strain DSM 17236 and Bacillus subtilis strain DSM 32324. he animal feed, animal feed additive or premix according to any of aspects 12 to 21, wherein said animal feed, animal feed additive or premix further ameliorates the damaging effects of deoxynivalenol (DON) exposure on gut barrier integrity. 1

23. A composition for treating or inhibiting a pathogenic infection by a Fusobacterium necrophorum in an animal, said composition comprising Lactobacillus animalis.

24. The composition of aspect 23, wherein composition comprises said Lactobacillus animalis in an amount of between IxlO⁴ and Ix lO¹⁰ CFU/g.

25. The composition according to any of aspects 23 to 24, further comprising additional probiotic strains selected from the group consisting of the following species: Enterococcus faecium, Propionibacterium freudenreichii, Bacillus subtilis, and Bacillus licheniformis.

26. The composition according to any of aspects 23 to 24, wherein the Lactobacillus animalis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 33570.

27. The compostion according to aspect 25, wherein the Enterococcus faecium is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 22502.

28. The composition according to aspect 25, wherein the Propionibacterium freudenreichii is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 34127.

29. The composition according to aspect 25, wherein the Bacillus subtilis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 32324 or the strain deposited with accession no. DSM 17231.

30. The composition according to aspect 25, wherein the Bacillus licheniformis is the strain deposited at Deutsche Sammlung von Mikroorganismen und

Zellkulturen with accession No. DSM 17236. 31. The composition according to any of aspects 23 to 30, wherein said composition comprises a combination of Lactobacillus animalis,

Propionibacterium freudenreichii, Bacillus subtil is and Bacillus licheniformis.

32. The composition according to any of aspects 23 to 31, wherein said composition further ameliorates the damaging effects of deoxynivalenol (DON) exposure on the gut barrier function.

33. The composition according to any of aspects 23 to 31 for use in the reduction in the number of liver abscesses observed in an animal receiving the composition.

34. Process for preparing a composition, as defined in any one of claims 24 to 32, comprising mixing, in desired ratios, effective amounts of the Lactobacillus animalis for feeding, together with concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s), and other feed ingredient(s).

35. Kit, comprising the composition, as defined in any one of claims 24 to 32, or obtainable from a process as defined in claim 33, and instructions for use.

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

EXAMPLES

Trials have been conducted to evaluate the inhibitory effects of the Lactobacillus animalis strain against Fusobacterium necrophorum, comparing the abilities of different L. animalis strains in different agar media, and evaluating the ability of one of the L. animalis' to reverse the effects of DON on gut barrier integrity when combined with additional probiotic strains. Trials in beef cattle were also performed to evaluate the beneficial effect of the L. animalis strain in vivo when applying it as part of a combination of probiotic strains resulting in a significant reduction in the total number of liver abscesses observed. Lastly, the inhibitory effects of the Lactobacillus animalis strain in combination with P. freundenreichii, B. subtilis, B. licheniformis against Trueperella pyrogenes were also evaluated in vitro.

EXAMPLE 1

Efficacy ofL. animalis in inhibiting in vitro Fusobacterium necrophorum in an agar diffusion assay.

Method:

Day 1 :

Fusobacterium necrophorum inoculated on Fastidious Anaerobe agar (FAA) agar and incubated at 37°C anaerobically for 24h.

Overnight cultures (in BHI broth) of probiotic strain (Lactobacillus animalis - DSM 33570).

Day 2:

Test strain material: Lactobacillus animalis - DSM 33570 overnight culture in BHI.

Agar preparation: Wilkins Chalgren agar melted and cooled to 50°C.

Fusobacterium necrophorum preparation: Using a cotton swap, a 0,5 McFarland suspension are made in Maximum Recovery Diluent (MRD).

35ml melted agar and 10pl pathogen suspension are mixed in a 50ml falcon tube and cast in Omnitray plates, immediately appling the Nunc™lmmuno TSP. Plates left to solidify for 20 mins before removing Nunc™lmmuno TSP lid. Plates dry with normal lid on for additional 20 mins. 10pl of the L. animalis overnight culture applied to selected wells in the agar (n=2). After anaerobically incubation at 37°C for 48h, the inhibition zone diameter is measured from full growth to full growth, with a lower limit of 3,5mm (width of well)

Results:

Clear inhibition zones, measuring on average 8,5mm in diameter, visible for Lactobacillus animalis - DSM 33570 spots (see figure 1).

EXAMPLE 2

Effect of various probiotic strains to inhibit in vitro Fusobacterium necrophorum - Supernatant method

Method:

Generation of supernatants:

Probiotic sample strains and pathogen strains were inoculated in BHI broth and incubated according to strain growth conditions. After incubation, samples were centrifuged and the supernatant were filtered (0,22pm) in two aliquots. One aliquot was pH adjusted to approximately pH 7 and the other aliquot had no further treatment. The supernatants were stored at 5°C until the assay was performed.

Supernatant assay setup:

Day one: Pathogenic strain, F. necrophorum ATCC 25286 were inoculated on Brucella agar and incubated anaerobically at 37°C for 24h..

Day two (pathogen suspension preparation) : A lxlO⁶ and lxlO⁷ CFU/ml suspension of F. necrophorum in MRD was prepared. Control A: BHI (unspent media) and Control B: Supernatant from F. necrophorum (spent media).

Assay setup in 96 well microtiter plate:

130pl BHI/Supernatant added to wells. 15pl pathogen suspension added to test wells.

Plate incubated anaerobically at 37°C and read at OD 600nM after 30 and 48 hours of incubation. Results are reported as % reduction of OD 600nM compared to control and as OD 600nM readings plotted in graphs.

Results:

EXAMPLE 3

Ability of different L. animalis strains to inhibit F. necrophorum in different agarmedia. Cell free supernatants are routinely used to preliminary screen for antimicrobial activity of bacteria by means of the agar well diffusion method, but the supernatant may also include other molecules (such as medium components and/or intracellular compounds) accidentally released during cell free supernatant preparation, which may interfere with the assay. Reproducibility of bacteriocin activity against the same testmicroorganisms is an important factor to be considered.

Azevedo et al. (2018) showed that the selection of the agar-media is crucial for the bioassay response. In the work by Azevedo et al., growth inhibition by means of the agar well diffusion assays was carried out on different agar-media showing a strong dependence on the agar-media used, indicating that the inhibitory effects could also depend on the diffusion of exudates that are included in the cell-free supernatant. The assays in that work were performed on Mueller Hinton {Enterococcus sp. and Listeria sp.) and MacConkey {E. coli) agar-media.

A further investigation of the ability of different L. animalis strains to inhibit F. necrophorum was investigated. To ensure that the results of the present disclosure were not influenced by the agar-media, the same assay was performed on both media types.

Day one:

The pathogenic strain, F. necrophorum ATCC 25286 was inoculated on Brucella agar and incubated anaerobically at 37°C for 24h.

Four different Lactobacillus animalis strains were inoculated in BHI broth and incubated anaerobically at 37°C for 24h. The four Lactobacillus animalis strains were NCIMB702937, NCIMB702941, CCUG31155, and L. animalis strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM

33570. Day two:

Agar preparation: Wilkins Chalgren and Muller Hinton agar was prepared according to the manufacturer and cooled to 50°C.

Pathogen strain preparation: A 0.5 McFarland suspension made in MRD using a cotton swap.

Plate preparation: 35 mL melted agar and 10 pl pathogen suspension mixed in a 50 mL falcon tube and cast in Omnitray plates, immediately applying the Nunc™Immuno TSP. Plates are left to solidify for 20 minutes before removing the Nunc™Immuno TSP lid. Plates dry with the normal lid on for additional 20 minutes.

Sample application: 10 pL of the L. animalis overnight cultures were applied to selected wells in the agar (n=2).

Incubation & results: After anaerobically incubation at 37°C for 48h, the inhibition zone diameter is measured from full growth to full growth, with a lower limit of 3.5mm (width of the well) and an upper limit of 16mm (distance between wells).

Not all L. animalis strains showed the ability to inhibit F. necrophorum. The inhibitory potential was tested in two different agar types as shown in Figure 2.

One strain was not able to inhibit F. necrophorum in either agar type (strain NCIMB702941) and one strain was only able to inhibit F. necrophorum in one agar type (strain NCIMB702937). Two of the strain were able to inhibit F. necrophorum in both media types with the strain PTA-6750 being the most efficifient showing the biggest inhibition zones (see figure 2).

The fact is that not only do some strains show no inhibitory effect towards F. necrophorum on either agar type, but some strains have a different pattern, showing an inhibitory effect in one of two agar types. This tells us that different inhibitory mechanisms that are in play for the strains and not a general ability within the species. EXAMPLE 4

L. animalis alone and in combination with other probiotic strains increases transepithelial electrical resistance (TEER).

Epithelial and endothelial cells form barriers in the body. The strength and integrity of these barriers can be assessed via measurements of the electrical resistance across the cell layer in vitro, called TEER.

TEER is a well-established method of evaluating and monitoring epithelial tissue in a non-destructive assay. In particular, the confluence of the monolayer is quickly determined. The confluence can be tracked and monitored in real-time as the TEER measurement will rise as the gaps in the monolayer close.

TEER is often used with epithelial and endothelial cells in a monolayer as a strong indicator of cell barrier integrity and permeability.

Method:

Human cancer-derived epithelial intestinal Caco-2 cell monolayers were seeded on 1.12- cm² transwells (0.4 pm pore size, Corning) at 5xl0⁴ cells pr. transwell. Culture medium (Dulbecco's Modified Eagle Medium supplemented with non-essential amino acids, penicillin-streptomycin-amphotericin B and 10% fetal bovine serum) was changed every 3-4 days. After 20-22 days, upon reaching a confluent, polarized and differentiated state, the cells were equilibrated overnight in antibiotic-free cell culture medium in a CellZscope2 system. On the day of the experiment, pure L. animalis bulk material or Bovamine® Defend Plus product (containing a combination of Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis and Bacillus licheniformis') was added to the apical compartments of the cell monolayers at a concentration normalized to 1.7xl0⁷ CFU of L. animalis pr. transwell. Hourly TEER measurements were carried out for a total of 24 hours. TEER results are calculated as relative to baseline values before addition of the bacteria to the epithelial cells. FD20 translocation results are calculated as relative to the apically added amount.

Results:

As shown in Figure 3, exposure of Caco-2 cell monolayers to L. animalis increases transepithelial electrical resistance (TEER) compared to unstimulated Caco-2 cells. Combining L. animalis with other probiotic bacteria is contemplated to increase efficiency. When the Caco-2 cell monolayers are exposed to a combination of L. animalis and other probiotic bacteria (Propionibacterium freudenreichii, Bacillus subtilis and Bacillus licheniformis a combination also commercially known as Bovamine® Defend Plus.), a significantly higher increase in TEER is observed compared to L. animalis by itself.

EXAMPLE 5 - BOVAMINE DEFEND® Plus probiotic product ameliorates the damaging effect of deoxynivalenol on gut barrier integrity

The mycotoxin deoxynivalenol (DON) is produced by various Fusarium fungi known to frequently infect various grains in the field or during storage (Eskola et al., 2020; Sobrova et al., 2010). It is one the most common mycotoxin contaminants of cerealbased food and animal feed (Recharla et al., 2022). Upon ingestion, DON causes impaired integrity of the intestinal barrier, its first site of exposure. DON-induced increased epithelial permeability to toxins, pathogenic bacteria and viruses then leads to gastrointestinal inflammation (Pinton & Oswald, 2014).

The TEER experiment described in Example 4 was performed again under similar conditions but with the addition that after four hours of start of the experiment, deoxynivalenol (DON) was added to the apical and basolateral compartments. Start of the experiment is considered the time point where the BOVAMINE DEFEND® Plus probiotic product (containing a combination of Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis and Bacillus licheniformis) is added to the apical compartments of the cell monolayers at a concentration normalized to 1.7xl0⁷ CFU of L. animalis pr. transwell along with 400pg of fluorescein isothiocyanate dextran-20 kDa (FD20). Hourly TEER measurements were carried out for a total of 24 hours. At the end of the experiment the amount of FD20 translocated to the basolateral compartment was quantified by measuring fluorescence (490nm emission/520nm excitation). TEER results are calculated as relative to baseline values before addition of the bacteria to the epithelial cells. FD20 translocation results are calculated as relative to the apically added amount.

As shown in Figure 4, DON causes a profound TEER decrease and subsequent increase in apical-to-basolateral FD20 translocation across the Caco-2 cell monolayers. However, when applied four hours prior to the DON-challenge, the BOVAMINE DEFEND® Plus probiotic product ameliorates the DON-induced TEER decrease and significantly decreases the DON-induced FD20 translocation.

Conclusions:

L. animalis supports gut barrier integrity in vitro, as assessed by meassuring TEER across Caco-2 cell monolayers. This effect is even more pronounced when L. animalis is applied as part of the BOVAMINE DEFEND® Plus probiotic product (in combination with P. freundenreichii, B. subtilis, B. licheniformis'). The BOVAMINE DEFEND® Plus probiotic product furthermore protects the intestinal barrier from the damaging effect of the mycotoxin deoxynivalenol by counteracting DON-induced TEER decrease and FD20 translocation. Thus, L. animalis shows a potential for supporting gut barrier integrity in vivo, and when applying it as part of a combination of probiotic strains an even greater beneficial effect on gut barrier support may be observed. EXAMPLE 6 - Administration of BOVAMINE DEFEND® Plus probiotic product to beef cattle reduces the number of observed liver abscesses, i.e. the occurrence of liver abscesses

An in vivo trial was performed to evaluate the effect of feeding the composition of the present disclosure comprising L. animalis in combination with P. freundenreichii, B. subtilis, B. licheniformis as part of the BOVAMINE DEFEND® Plus probiotic product to beef cattle. The study was performed at a research facility in Kansas, United States . The in vivo trial was conducted over a 6 month period. Several parameters relevant for the farmer was investigated including feed efficiency, average daily gain, cost of gain, mortality and number of liver abscesses observed in the slaughtered animal.

1,625 crossbred feedlot steers were assigned to 1 of 24 feedlot pens with 55-85 steers per pen. The initial body weight (BW) was determined to be 370 ± 15.9 kg and the average initial age of the steers were 12 months. The study period had a duration of 133 days in average. The steers were twice a day offered basal diet and were divided into two groups. One group receiving no probiotic supplementation (Control, n=812) and one group receiving a probiotic supplementation according to the present disclosure comprising L. animalis in combination with P. freundenreichii, B. subtilis, B. licheniformis as part of the BOVAMINE DEFEND® Plus probiotic product (Lactobacillus animalis (DSM 33570), Propionibacterium freudenreichii (DSM 34127), Bacillus licheniformis (DSM 17236), and Bacillus subtilis (DSM 32324) on a lactose carrier having 1.2¹¹ CFU/g) included directly into the feed at 50 mg/head per day (BDP, n = 812).

The diet is described in the below table A and table B:

Table A

Table B

Based on the in vitro results described in Example 1-5 the number and degree of liver abnormalities were investigated. Any liver abnormalities observed were ranked in three groups: A-, A or A+. A category denoted "Others" were also included and covers signs of contamination, flukes, telangiectasis, heart failure and cirrhosis.

Conclusions:

It was found that when L. animalis is applied as part of the BOVAMINE DEFEND® Plus probiotic product (in combination with P. freundenreichii, B. subtilis, B. licheniformis) the total number of liver abscesses observed is significantly reduced by 4.4%-units (14.81 vs. 10.41; P < 0.01). Furthermore the probiotic composition administered improved the feed efficience by 5% (7.08 vs. 6.71; P < 0.01) and the average daily gain tended to be improved (1.48 vs. 1.55; P < 0.06). This resulted in the cost of gain which h also tended to be improved (159 vs. 151; P < 0.06; dollars per 45kg of live weight gained throughout the study period). In addition and importantly the mortality was descreased by 1.49%-units (2.23% vs. 0.74%; P < 0.01).

Thus, the beneficial effect of L. animalis described in Examples 1-5 is further supported and the potential for supporting gut barrier integrity in vivo is confirmed and when applying it as part of a combination of probiotic strains an even greater beneficial effect may be observed resulting in a significant reduction in the total number of liver abscesses observed.

EXAMPLE 7 - Efficacy ofL. animalis in combination with P. freundenreichii, B. subtilis, B. licheniformis in inhibiting in vitro Trueperella pyrogenes in an agar diffusion assay.

Method:

Day 1 :

Trueperella pyrogenes inoculated on Tryptic Soy Agar with 5% Shepp blood (TSA-SB) and incubated at 37°C anaerobically for 24h.

Overnight cultures (in BHI broth) of probiotic strains (Lactobacillus animalis - DSM 33570 in combination with P. freundenreichii, B. subtilis, B. licheniformis').

Day 2: Test strain material: Lactobacillus animalis - DSM 33570 in combination with P. freundenreichii, B. subtilis, B. licheniformis overnight culture in BHI. Agar preparation: Wilkins Chalgren agar melted and cooled to 50°C. Trueperella pyrogenes preparation: Using a cotton swap, a 0,5 McFarland suspension are made in Maximum Recovery Diluent (MRD).

35ml melted agar and 10pl pathogen suspension are mixed in a 50ml falcon tube and cast in Omnitray plates, immediately appling the Nunc™lmmuno TSP. Plates left to solidify for 20 mins before removing Nunc™lmmuno TSP lid. Plates dry with normal lid on for additional 20 mins. 10 .l of the L. animalis in combination with P. freundenreichii, B. subtilis, B. licheniformis overnight culture applied to selected wells in the agar (n=3). After anaerobically incubation at 37°C for 48h, the inhibition zone diameter is measured from full growth to full growth, with a lower limit of 3,5mm (width of well) and an upper limit of 16mm (distance between wells).

Results:

L. animalis in combination with P. freundenreichii, B. subtilis and B. licheniformis showed a high inhibitory potential towards T. pyrogenes, with clear inhibition zone diameters exceeding the upper limit of measurement of 16mm.

The present invention has been described with reference to various embodiments, aspects, examples, or the like. It is not intended that these elements be read in isolation from one another. Thus, the present disclosure provides for the combination of two or more of the embodiments, aspects, examples, or the like.

All embodiments described herein are intended to be within the scope of the invention disclosed. These and other embodiments of the present invention will become readily apparent to those skilled in the art from the following detailed description of the preferred embodiments having reference to the whole description, the invention not being limited to any particular preferred embodiment(s) disclosed. TAXONOMY

Lactobacillus animalis is now known as Ligilactobacillus animalis, as described in Zheng et al., Int. J. Syst. Evol. Microbiol. DOI 10.1099/ijsem.0.004107. The two different names are used interchangeably herein.

DEPOSIT AND EXPERT SOLUTION

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

Table 1: Deposits made at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germanybeing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtil is and Bacillus licheniformis REFERENCES

Amachawadi, R.G., Nagaraja, T. G. (2016), Liver abscesses in cattle: A review of incidence in Holsteins and of bacteriology and vaccine approaches to control in feedlot cattle. J Anim Sci. Apr;94(4): 1620-32. doi: 10.2527/jas.2015-0261.

Azevedo, P. (2018), Importance of the agar-media in the evaluation of bacteriocin activity against the same test-microorganisms. Braz. J. Pharm. Sci. 54(1). http://doi.org/10.1590/s2175-97902018000117533

Bartenslager, A. (2020), Investigating microbiomes and developing direct-fed microbials to improve cattle health, University of Nebraska - Lincoln. Theses and Dissertations in Animal Science. 194

Eskola, M., Kos, G., Elliott, C. T., Hajslova, J., Mayar, S., & Krska, R. (2020). Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited 'FAO estimate' of 25%. Critical Reviews in Food Science and Nutrition, 60(16), 2773- 2789. https://doi.org/10.1080/10408398.2019.1658570

Pinnell, L. J., Young, J. D., Thompson, T. W., Wolfe, C. A., Bryant, T. C., Nair, M. N., ... & Morley, P. S. (2023). Establishing the link between microbial communities in bovine liver abscesses and the gastrointestinal tract. Animal Microbiome, 5(1), 58.

Pinton, P., & Oswald, I. P. (2014). Effect of Deoxynivalenol and Other Type B Trichothecenes on the Intestine: A Review, https://doi.org/10.3390/toxins60

Recharla, N., Park, S., Kim, M., Kim, B., & Jeong, J. Y. (2022). Protective effects of biological feed additives on gut microbiota and the health of pigs exposed to deoxynivalenol : a review. J Anim Sci Technol, 64(4), 640-653.

Sobrova, P., Adam, V., Vasatkova, A., Beklova, M., Zeman, L., & Kizek, R. (2010). Deoxynivalenol and its toxicity. Interdisciplinary Toxicology, 3(3), 94-99. https://doi.org/10.2478/vl0102-010-0019-x

Claims

1. A method of inhibiting a gastrointestinal pathogenic Fusobacterium necrophorum infection in an animal, the method comprising, administering to said animal a composition that inhibits growth of said pathogenic bacterium, said composition comprising Lactobacillus animalis.

2. The method of claim 1, wherein said composition is administered to said animal in an amount to provide a total amount of said probiotic strain of between 1 x 10⁸ and 1 x 10¹¹ CFU/animal/day.

3. The method according to any of the preceding claims, wherein said composition further comprises at least one additional probiotic strain selected from the group consisting of the following species: Enterococcus faecium, Propionibacterium freudenreichii, Bacillus subtil is and Bacillus licheniformis.

4. The method according to any of the preceding claims, wherein the Lactobacillus animalis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 33570.

5. The method according to any of the preceding claims, wherein said composition comprises a combination of Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtil is and Bacillus licheniformis.

6. The method according to any of the preceding claims, wherein said composition further ameliorates the damaging effects of deoxynivalenol (DON) exposure on gut barrier integrity.

7. An animal feed, animal feed additive or premix for use in inhibiting a gastrointestinal pathogenic Fusobacterium necrophorum infection in an animal, comprising Lactobacillus animalis and further comprises one or more additional components selected from concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s), and other feed ingredient(s).

8. The animal feed, animal feed additive or premix for use according to claim 7, wherein said Lactobacillus animalis are present in said animal feed, animal feed additive or premix in an amount to provide a total of between 1 x 10⁸ to 1 x 10¹¹ CFU/animal/day.

9. The animal feed, animal feed additive or premix for use according any of claims 7 to 8, further comprising additional probiotic strains selected from the group consisting of the following species: Enterococcus faecium, Propionibacterium freudenreichii, Bacillus subtilis, and Bacillus licheniformis.

10. The animal feed, animal feed additive or premix for use according to claims 7 to 9, wherein the Lactobacillus animalis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 33570.

11. The animal feed, animal feed additive or premix for use according to any of claims 7 to 10, wherein said composition comprises a combination of Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus subtilis and Bacillus licheniformis.

12. The animal feed, animal feed additive or premix for use according to any of claims 7 to 11, wherein said animal feed, animal feed additive or premix further ameliorates the damaging effects of deoxynivalenol (DON) exposure on gut barrier integrity and function.

13. A composition for treating or inhibiting a pathogenic infection by a Fusobacterium necrophorum in an animal, said composition comprising Lactobacillus animalis.

14. The composition according to claim 13, further comprising additional probiotic strains selected from the group consisting of the following species:

Enterococcus faecium, Propionibacterium freudenreichii, Bacillus subtilis, and

Bacillus licheniformis.

15. The composition according to any of claims 13 to 14, wherein the Lactobacillus animalis is the strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen with accession No. DSM 33570.

16. The composition according to any of claims 13 to 15, wherein said composition further ameliorates the damaging effects of deoxynivalenol (DON) exposure on the gut barrier function.

17. Process for preparing a composition, as defined in any one of claims 13 to 16, comprising mixing, in desired ratios, effective amounts of the Lactobacillus animalis for feeding, together with concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s), and other feed ingredient(s).

18. Kit, comprising the composition, as defined in any one of claims 13 to 16, or obtainable from a process as defined in claim 17, and instructions for use.