PROBIOTIC STRAINS TO IMPROVE ANIMAL HEALTH PERFORMANCE

Abstract

In general, the invention relates to a component and method for reducing the development and / or severity of respiratory disease by using a novel combination of probiotic strains, thereby improving the health performance of animals. Furthermore, the invention relates to a component and method for inhibiting the growth of pathogens by using a novel combination of probiotic strains.

Description

[0001] PROBIOTIC STRAINS TO IMPROVE HEALTH PERFORMANCE OF ANIMALS

[0002] FIELD

[0003] The present disclosure generally relates to compositions and methods for improving or maintaining respiratory health of animals, e.g. by reducing the occurrence and / or severity of respiratory disease, using a novel combination of probiotic strains, thereby improving the health performance of the animals.

[0004] BACKGROUND

[0005] One of the most common respiratory diseases in livestock animals is pneumonia, which is defined as inflammation of the lungs. Bovine respiratory disease (BRD) is the single largest health issue faced by the feedlot industry, causing substantial economic and productive losses. The disease accounts for approximately 75% of morbidity and 50% to 70% of mortality in feedlots. It has been estimated to cost the American feedlot industry between $800 and $900 million annually due to decreased animal production and increased veterinary treatment costs.

[0006] Moreover, BRD leads to in vivo productive losses that ultimately impacts the quality and return on the carcass obtained from the animals. In the United States, total calf value was linearly decreased as the number of treatments for BRD increased from 0 to > 3, indicating losses in performance and carcass traits, and additional pharmacological costs that impact overall profitability of the operation. In a thorough evaluation of the economic aspects of BRD in Australia, Blakebrough-Hall et al. (J. Anim. Sci., Volume 98, Issue 2, 2020) reported that as the number of treatments for BRD increased, feedlot performance and carcass traits linearly decreased, with a decrease in the value of slaughtered animals as well.

[0007] Among the pathogens involved in the BRD complex, Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, and Mycoplasma bovis are recognized as the main ones causing pneumonia in cattle (Czuprynski et al. (Anim. Health Res. Rev. 5:277-282, 2004); Bringhenti et al., (J. Dairy Sci. 104:10291-10309, 2021)). An interesting note is that these bacteria are considered ubiquitous in the bovine upper respiratory tract, meaning that the animal itself hosts most, if not all, of these microorganisms in a harmonious state that can later take an opportunity and cause BRD. The most common approach when treating pneumonia in cattle includes the utilization of broad-spectrum antibiotics following the assessment of health parameters in the animals, such as nasal discharge, appetite, respiratory rate, and rectal temperature. Nonetheless, the utilization of antibiotics does not ensure full health and performance recovery to the herd, as demonstrated by others (Wilson et al., (J. Dairy Sci. 104:10291-10309, 2017); Blakebrough-Hall et al., supra). Across the globe, the livestock production system, regardless of the species, is looking for alternatives that could minimize antibiotic utilization. These alternatives include natural, microbial solutions that can support the overall health of the animals and, therefore, reduce the number of animals being sick and requiring antibiotic treatment.

[0008] Although much research has been done regarding the determinants of bovine respiratory disease, there are only a few conclusive findings and bovine respiratory disease will continue to be an extremely costly condition until an efficient treatment or prevention is identified.

[0009] Thus, there is still a need for probiotic strains and specific mixtures of probiotic strains which can be used to improve or maintain the health of production animals, especially with health effects on respiratory conditions.

[0010] SUMMARY

[0011] The present disclosure provides a composition comprising a novel combination of probiotic strains which have a proven effect on the health and performance of animals, by maintaining or improving respiratory health.

[0012] Respiratory disease infection may develop due to one or a combination of factors including stress of the animal, exposure to high levels of infectious agents and / or immunocompromised animals. The examples of the present disclosure demonstrate the effect of the composition of the disclosure on the health performance of animals also in relation to respiratory disease.

[0013] Compositions comprising the novel combination of probiotic strains of the disclosure may be fed to animals, e.g., as Direct Fed Microbial (DFM), as an animal feed additive, in a premix, or incorporated in the animal feed directly.

[0014] The novel combination of probiotic strains of the disclosure may be added to the feed during production, after production by the supplier, or by the producer mixing the strains with the feed to be fed to the animal, just prior to providing the feed to the animal.

[0015] Typical treatments for animals with clinical signs consist largely of antibiotics (especially penicillin) and vaccination. Besides this, there is a general awareness of good management of livestock as a preventive measurement to minimize the risk of outbreaks. This management includes keeping the livestock in general good health and minimizing stress in the production facility. This is all a costly procedure to undertake, and full prevention cannot be achieved despite all efforts being made.

[0016] The purpose of the in vitro studies described in the examples was to investigate the inhibition effect of the strains present in the novel combination of probiotics according to the disclosure on Mannheimia haemolytica, which is one of the major causative agents of respiratory disease in ruminants. The purpose of the in vivo studies described in the examples was to investigate the subsequent effect of feed additives comprising a novel combination of probiotics according to the disclosure on the occurrence and / or severity of respiratory disease in ruminants in a performance trial on dairy calves, and in a challenge study on sows and weaned progeny in a nursery.

[0017] This disclosure describes the benefits of a novel combination of probiotic strains (Bacillus licheniformis and Bacillus subtilis) in maintaining or improving respiratory health in animals receiving a composition comprising the probiotic strains of the present disclosure.

[0018] These results demonstrate that the aforementioned novel combination of probiotic strains improves the health of the animals, thereby providing a cost-effective and easy-to-administer alternative to the traditional treatment approach using antibiotics and vaccination against respiratory pathogens. While not wishing to be bound by theory, it is believed that a daily supplement of the composition of the present disclosure will maintain or improve respiratory health in the animals receiving the composition.

[0019] DEFINITIONS

[0020] 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 context of the present disclosure.

[0021] 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.

[0022] 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”.

[0023] Animal feed: As used herein the term "animal feed" refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a mono-gastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbials, amino acids and / or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and / or other feed ingredients (such as in a premix). The animal feed components may consist of or comprise milk (e.g., from cow, goat, sheep), e.g., for feeding of younger animals. Examples of ingredients suitable for animal feed and formulations of animal feed are described and exemplified in the section “Composition administration form and formulation”. Composition: As used herein the term "composition" refers to a composition comprising a carrier and at least one bacterial strain as described herein. The compositions described herein may be used in a Direct Fed Microbial (DFM), an animal feed additive, a premix or a Total Mixed Ration (TMR). The composition may be provided in any suitable form such as in the form of a liquid or solid.

[0024] Concentrate: As used herein the term "concentrate" means a feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, sorghum, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed / canola, peanut or groundnut), palm kernel cake, yeast-derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

[0025] Inhibit the growth of Mannheimia haemolytica: As used herein the term "inhibit the growth of M. haemolytica" means a method and / or composition that partly or completely inhibits M. haemolytica infections in an animal. Accordingly, the term "inhibit the growth of M. haemolytica" means the M. haemolytica infections are reduced or completely eliminated and the overall health of the animal is improved.

[0026] Direct Fed Microbial: As used herein the term "direct fed microbial" or “DFM” means live microorganisms including spores which, when administered in adequate amounts, confer a benefit, such as improved digestion or health, on the animal to which it is administered.

[0027] 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 health and performance (feed digestion or efficiency) of an animal, as illustrated in the examples.

[0028] 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 in view of the guidance provided herein.

[0029] Feeding an animal: As used herein the terms "feeding an animal" or "fed to an animal" means that the composition of the present disclosure is administered orally to the animal in an effective amount. The oral administration may be repeated, e.g., one or more times daily over a specified period of time, such as several days, one week, several weeks, one month or several months. Accordingly, the terms "feeding" or "fed" mean any type of oral administration, such as administration via an animal feed or via drinking water or, in certain circumstances, by oral gavage or aerosol spray. 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.

[0030] Preventing and / or minimizing respiratory disease: As used herein the term "preventing and / or minimizing respiratory disease" means a method and / or composition that prevents and / or minimizes occurrence of a respiratory disease in an animal. Whether an animal has developed a respiratory disease can easily be evaluated by a person skilled in the art. One example of such an evaluation method is described in the examples of the present disclosure.

[0031] Maintaining or improving respiratory health is alternatively or also intended to mean preventing or reducing the onset of respiratory infections in an animal population or farm; and / or reducing the likelihood of onset of respiratory infections; or reducing the prevalence of respiratory infections in an animal population; and / or reducing or preventing respiratory infections in an animal by a pathogen; or reducing the severity of a respiratory infection; or reducing the load of a pathogen in an animal.

[0032] Body Weight Gain: As used herein the Body Weight Gain of an animal is the increase of live weight of the animal over a specified period of time. Weight gain is also described using the term “Average Daily Gain (ADG)”. As used herein the term “Average Daily Gain” and “ADG” means the same and are used interchangeably. The term “Average Daily Gain” means the amount of weight gained by one animal within one day, i.e. within 24 hours.

[0033] General impression: As used herein the term “General impression” is a measure of the overall general impression of a given animal at a given day and is assessed by methodologies known in the field.

[0034] Dry Matter Intake (DMI): As used herein the term “Dry Matter Intake” and the term “DMI” mean the same and are used interchangeably. The term “Dry Matter Intake” means the amount of feed consumed by the animal, excluding water content.

[0035] Feed efficiency (FE): As used herein the term “Feed efficiency” and the term “FE” mean the same and are used interchangeably. By “increased feed efficiency” it is meant that the use of the feed additive composition of the disclosure in feed results in a greater body weight gain per kg of feed consumed compared to the amount of body weight gain observed in an animal when the feed does not comprise said feed additive composition of the disclosure.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1 shows the inhibitory zones achieved (in mm) after incubation of Mannheimia haemolytica with either control or probiotic (B. licheniformis DSM 17236). Figure 2 shows the University of Wisconsin scoring system for pneumonia in calves.

[0038] Figure 3 shows the proportion of pre-weaning dairy calves fed control (CON) or probiotic (PRO) that required pharmacological treatments for pneumonia (i.e. calves receiving a score of 2 or 3 using the University of Wisconsin scoring system for pneumonia in calves). A treatment effect was observed (P = 0.05).

[0039] Figure 4 is an overview of the study in Example 3. 24 pigs were allocated into three groups (n=8) and either fed a diet supplemented with probiotics (PRO group) or placebo (positive and negative control) 14 days before being challenged intranasally with swIAV (H1 N2) on DPI 0. Only the probiotic group and the positive control were challenged with swIAV. The negative control group was mock infected. Serum, nasal swabs for immunology and viral load and fecal samples were collected during the study as shown in the figure. All pigs were euthanized at DPI 7. DPI = days post inoculation.

[0040] Figure 5 shows the extent of gross lung pathology. Pigs were fed a diet with or without the PRO for 14 days before intranasal challenge with swIAV for 7 days, after which the pigs were euthanized, and their lungs were assessed for gross lesions. The groups are Negative control group (CON; not challenged, not fed probiotic), Positive control group (CHL; challenged, not fed probiotic), and probiotics group (PRO; challenged and fed probiotic). Gross lung lesions were calculated as the percentage of lung with red atelectasis 7 days post infection (DPI 7). Differences between the two infected groups were analyzed by Mann-Whitney t test and the median is indicated for each treatment group. Differences were considered significant if the P value was <0.05; * and considered a tendency if P<0.10.

[0041] Figure 6 shows levels of lung lesions in two groups of pigs infected with influenza virus A, a positive control group and a group fed a probiotic B. subtilis strain.

[0042] Figure 7 shows C-reactive protein (CRP) levels in three groups of pigs, a negative control group with pigs not infected with influenza virus A, a positive control group with pigs infected with influenza virus A, and a group with pigs infected with influenza A and fed a probiotic B. subtilis strain.

[0043] Figure 8 shows changes in inflammatory marker serum amyloid A (SAA) and SAA3 in ileum tissue in two groups of pigs infected with influenza virus A, a control group and a group fed a probiotic B. subtilis strain.

[0044] Figure 9 shows the average total lung lobes with lesions per pig in control and in Bacillus-treated pigs in a nursery H1 N2 influenza challenge. DETAILED DESCRIPTION OF THE INVENTION

[0045] This disclosure relates to a composition comprising at least one Bacillus strain for use in maintaining or improving respiratory health in an animal, e.g. by reducing the occurrence and / or severity of respiratory disease in an animal receiving said composition, as well as use of the composition and methods for maintaining or improving respiratory health.

[0046] As noted above, bovine respiratory disease (BRD) is the single largest health issue faced by the feedlot industry, causing substantial economic and productive losses. The treatment of respiratory disease is difficult and insufficient, relying mainly on the administration of antibiotics and on careful management of the livestock production facility. Being able to reduce the occurrence and / or severity of respiratory disease in an animal would be of high value for the producer and provide an improvement in animal welfare and food quality by improving respiratory health, which is highly desired. The present disclosure is thus capable of achieving these advantageous actions by an easy administration of the composition of the disclosure without administering antibiotics and like substances to the animals.

[0047] The present disclosure includes a method of maintaining or improving the respiratory health of an animal, comprising administering to said animal a composition comprising at least one of two different probiotic Bacillus strains and that provides improved respiratory health compared to a similar animal to which said composition has not been administered.

[0048] The present disclosure includes a method of inhibiting the occurrence and / or severity of development of respiratory disease in an animal comprising administering to said animal a composition that inhibits the development of clinical signs of disease associated with said respiratory disease, said composition comprising at least one of two different probiotic strains.

[0049] Respiratory disease

[0050] Respiratory disease infections such as pneumonia may develop as a result of one or a combination of factors including stress of the animal, exposure to high levels of infectious agent and / or immunocompromised animals. The initial problem in many cases of pneumonia is thought to be a sudden change in the normal nasal bacterial flora, which results in a sudden dramatic increase in one or more species of bacteria. Bacterial proliferation is usually caused by a breakdown of the host defenses as a result of stress (e.g., transportation, concurrent illness) or cellular insult (e.g., viral infection, toxicity). These bacteria are breathed into the lungs in large numbers and may overwhelm the normal defense mechanisms, localize, multiply, and initiate inflammation.

[0051] In addition, stress is often a precursor of viral and / or bacterial respiratory infections, particularly in groups of animals that have recently been congregated and stressed by travel, handling, and mixing. Pneumonia also can be caused by direct infection with viruses, bacteria, and fungi, as well as by toxins arriving hematogenously, by inhalation, or by aspiration of food or gastric contents.

[0052] Pneumonia is a leading cause of loss to the cattle industry throughout the world and Mannheimia haemolytica is a key respiratory pathogen in this disease. Mannheimia haemolytica belongs to the family Pasteurellaceae, being non-motile, non-spore-forming, facultatively anaerobic, oxidase-positive, and fermentative gram-negative rods or coccobacilli.

[0053] In one embodiment of the present disclosure, the respiratory disease being prevented and / or minimized is pneumonia.

[0054] Bovine respiratory disease (BRD) is the most common and costly disease affecting beef cattle in the world. It is a complex, bacterial or viral infection that causes pneumonia in calves which can be fatal. The infection is usually a sum of three codependent factors: stress, an underlying viral infection, and a new bacterial infection. The diagnosis of the disease is complex since there are multiple possible causes. One way of diagnosing BRD is to follow the University of Wisconsin scoring system as also shown in the examples.

[0055] The disease may manifest itself at any time point in the animals’ life, however most often in calves within four weeks of weaning, when calves are sorted and often sold to different farms; a common nickname for BRD is "shipping fever. It is not known whether the stress itself, co-mingling, or travel conditions are at most to blame, and while studies have identified general stressing factors like transport and cold weather conditions, there is still no conclusive evidence on more specific factors (e.g. distance, transport mode, temperature, or temperature volatility). Stress in this context may be triggered by many different events and conditions. The removal of calves from their mother is a known event for stress and so is individually housing. A high(er) pathogen load is also a known stressor, such as the pathogen load present in a production farm.

[0056] BRD is thus a "multi-factorial syndrome" that is dependent on a number of different causes. The pathological condition commonly arises where the causative organism becomes established by secondary infection, following a primary bacterial or viral infection, which may occur after stress, e.g., from handling or transport.

[0057] The bacterial agents most commonly linked with BRD are Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Viral agents include Bovine viral diarrhea (BVD), Infectious Bovine Rhinotracheitis (IBR), Bovine respiratory syncytial virus (BRSV), and Parainfluenza type-3 virus (PI-3). Without being bound by theory it is believed that providing a composition with inhibitory effect on one or more of these bacterial agents, such as M. haemolytica, will be an advantage in reducing the occurrence and / or severity of BRD. One way of evaluating the inhibitory effect against M. haemolytica can be seen in the examples of the present disclosure.

[0058] The biggest sign of the pneumonia that BRD causes is depression, shown as droopy ears, dull eyes, and social isolation. Additionally, most cows will have a fever above 104 °F (40 °C). Other symptoms include coughing, decreased appetite, and breathing difficulty.

[0059] Because of the number of possible viral / bacterial precursors to BRD, there are a number of treatment options circling around the three main aggravators of the disease: viruses, bacteria, and stress.

[0060] Vaccinations exist for several biological BRD precursors, but the multitude of possible precursors complicates the process of choosing a vaccine regime. Additionally, vaccines are not completely effective in stopping the disease, but are merely helpful in mitigation. Many of the problems with vaccine effectiveness rest with improper use, such as failing to time vaccine doses appropriately, or not administering them before shipping, as the stress during handling and processing of the animals reduces the efficacy of the vaccines and the immune protection against respiratory pathogens.

[0061] In the absence of vaccination (often because calves are bought unvaccinated), antibiotics can help to stop the bacterial factors of the disease. The phase-out of the use of antibiotics in the European Union and the USA has resulted in an increased need for cost-effective alternatives to handle pathogens of human and veterinary importance.

[0062] Stress often serves as the final precursor to BRD. The diseases that make up BRD can persist in a cattle herd for a long period of time before becoming symptomatic, but immune systems weakened by stress can stop controlling the disease. There are many causes of stress and one of the major sources of stress comes from the shipping process and from the co-mingling of cattle, but also the weather may be another possible factor.

[0063] In one embodiment of the present disclosure, the respiratory disease being reduced in occurrence and / or severity is bovine respiratory disease.

[0064] Probiotic bacterial strains

[0065] The probiotic strains of the present disclosure described herein are isolated, i.e., present in a form or environment which does not occur in nature, and have a surprising effect when used in a composition as described herein.

[0066] In one embodiment of the disclosure said probiotic strain(s) in said composition are Bacillus paralicheniformis and optionally Bacillus subtilis. In one embodiment of the disclosure said probiotic strain(s) in said composition are Bacillus subtilis and optionally Bacillus licheniformis. In one embodiment of the disclosure said composition further comprises other probiotic strains, such as Lactobacillus animalis and / or Propionibacterium freudenreichii.

[0067] In one embodiment of the disclosure said composition comprising probiotic strains is administered to said animal in an amount to provide a total of between 1 x 108and 1 x 1011CFU / animal / day, such as between 1 x 108and 1 x 1O10CFU / animal / day, such as between 1 x 109and 1 x 1O10CFU / animal / day, such as between 5 x 109and 5 x 1O10CFU / animal / day.

[0068] In a preferred embodiment of the disclosure, said composition comprising probiotic strains is administered to said animal in an amount to provide a total of between 6 x 109and 8 x 109CFU / animal / day, such as about 6 x 109CFU / animal / day, such as about 7 x 109CFU / animal / day, such as about 8 x 109CFU / animal / day.

[0069] The term “CFU / animal / day” relates to the amount of probiotic strains administered to each animal per day. This term thus excludes carriers such as calcium carbonate, anti-caking agents such as aluminum silicates and kieselguhr (diatomaceous earth) and other components which optionally may be present in the composition.

[0070] Compositions of the present disclosure include at least one species of probiotic strains of the disclosure and at least one carrier and / or other components that make the composition suitable for feeding an animal or as an additive for drinking water or milk / milk substitute.

[0071] In one embodiment of the disclosure, wherein said composition comprises more than one probiotic strain, equal parts of Bacillus subtilis strains and Bacillus licheniformis strains are present.

[0072] In another embodiment of the disclosure, wherein said composition comprises more than one probiotic strain, unequal parts of Bacillus subtilis strains and Bacillus licheniformis strains are present.

[0073] In one embodiment of the present disclosure the composition comprises the Bacillus subtilis deposited as DSM 32324 or the Bacillus subtilis deposited as DSM 17231 .

[0074] In one embodiment of the present disclosure the Bacillus subtilis strain present in said composition is the strain deposited as DSM 17231.

[0075] In one embodiment of the present disclosure the Bacillus licheniformis strain present in said composition is the strain deposited as DSM 17236.

[0076] In preferred embodiments of the present disclosure, the composition comprises the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236.

[0077] In one embodiment of the present disclosure the composition comprises the Bacillus subtilis strain deposited as DSM 32324. In one embodiment, the composition comprises the Bacillus subtilis strain deposited as DSM 32324 and the Bacillus licheniformis strain deposited as DSM 17236

[0078] In one embodiment of the present disclosure the composition comprises two different probiotic strains, namely the Bacillus subtilis deposited as DSM 17231 and the Bacillus licheniformis deposited as DSM 17236, optionally in a ratio in colony forming unit (CFU) of said probiotic strains of 1 :1.

[0079] In one embodiment of the present disclosure the composition consists of two different probiotic strains, namely the Bacillus subtilis deposited as DSM 17231 and the Bacillus licheniformis deposited as DSM 17236, optionally in a ratio in colony forming unit (CFU) of said probiotic strains of 1 :1. In such embodiments, the composition does not include any other microorganism components, but may include other components, such as one or more carriers and / or one or more feed ingredients.

[0080] In one embodiment, the microorganism component of the composition, method or use disclosed herein consists of the Bacillus subtilis strain deposited as DSM 32324 and the Bacillus licheniformis strain deposited as DSM 17236.

[0081] The probiotic strains of the composition of the present disclosure may be provided in the form of spores and / or bacterial cells. In one embodiment of the present disclosure the Bacillus strains (Bacillus subtilis and Bacillus licheniformis) are provided in the form of spores.

[0082] In one embodiment of the present disclosure the composition of the present disclosure comprises an amount of Bacillus subtilis strain DSM 32324 or DSM 17231 to provide between 0.15 x 108and 0.15 x 1011CFU / day of Bacillus subtilis strain DSM32324 or DSM 17231 , preferably between 1 x 109and 1 x 1O10CFU / day Bacillus subtilis strain DSM32324 or DSM 17231. In one embodiment the composition of the present disclosure comprises an amount of Bacillus subtilis strain DSM32324 or DSM 17231 to provide about 1 x 109CFU / day of Bacillus subtilis strain DSM32324 or DSM 17231.

[0083] In one embodiment of the present disclosure the composition of the present disclosure comprises an amount of Bacillus licheniformis strain DSM 17236 to provide between 0.15 x 108and 0.15 x 1011CFU / day of Bacillus licheniformis strain DSM 17236, preferably between 1 x io9and 1 x 1O10CFU / day Bacillus licheniformis strain DSM 17236. In a one embodiment the composition of the present disclosure comprises an amount of Bacillus licheniformis strain DSM 17236 to provide about 1 x 109CFU / day of Bacillus licheniformis strain DSM 17236.

[0084] In one embodiment of the present disclosure the composition comprises amounts of the probiotic strains to provide about 1 x 109CFU / day of the Bacillus subtilis deposited as DSM 17231 and about 1 x 109CFU / day of the Bacillus licheniformis deposited as DSM 17236. In one embodiment of the present disclosure the microorganism components of the composition consist of amounts of the probiotic strains to provide about 1 x 109CFU / day of the Bacillus subtilis deposited as DSM 17231 and about 1 x 109CFU / day of the Bacillus licheniformis deposited as DSM 17236. In such embodiments, the composition does not include any other microorganism components, but may include other components, such as one or more carriers and / or one or more feed ingredients.

[0085] Composition administration form and formulation

[0086] The probiotic strains present in the composition of the present disclosure are provided in a commercially relevant form known to the skilled person. Accordingly, in an embodiment, the probiotic strains of the composition are present in a dried (e.g., spray-dried or freeze-dried) or frozen form. The different probiotic strains present in the composition of the present disclosure may be present in different forms in the composition, e.g., some may be spray-dried and some may be freeze-dried. The composition may be provided in any suitable form such as in the form of a liquid e.g., a gel, a slurry, etc., or in the form of a solid, e.g., a powder or a pellet.

[0087] For compositions in the form of a premix, the probiotic strains of the disclosure may be added to a carrier to make a mineral-vitamin mixture (premix), which may then be added to an animal feed at a desired inclusion rate.

[0088] Alternatively, for compositions in the form of an animal feed, the probiotic strains of the disclosure may be formulated with animal feed ingredients, as illustrated below. Such combinations of the composition of the disclosure and animal feed ingredients optionally may be in the form of pellets that are extruded through standard pelleting processes.

[0089] The disclosure also provides a method for producing an animal feed, animal feed additive, or premix comprising adding the probiotic strains of the disclosure to an animal feed or relevant components thereof.

[0090] The composition of the invention may be added to silage: “Silage” means fermented, high- moisture stored fodder which can be fed to ruminants (cud-chewing animals such as cattle and sheep) or used as a biofuel feedstock for anaerobic digesters. It is fermented and stored in a process called ensilage, ensiling or silaging, and is usually made from grass or cereal crops (e.g. maize, sorghum, oats, rye, timothy grass or other forage grass plants) or legume crops like clovers / trefoils, alfalfa, vetches, using the entire green plant (not just the grain). Silage can be made from many field crops, and special terms may be used depending on the type (oatlage for oats, haylage for alfalfa). Silage is made either by placing cut green vegetation in a silo, by piling it in a large heap covered with plastic sheet, or by wrapping large bales in plastic film. In one embodiment of the disclosure said composition comprising probiotic strains is administered to said animal in an amount to provide a total of between 1 x 105and 1 x 1011CFU / g feed, such as between 1 x 106and 1 x 1O10CFU / g feed, such as between 1 x 107and 1 x 109CFU / g feed, such as between 5 x 106and 5 x 1O10CFU / g feed.

[0091] In one embodiment, the present disclosure provides an animal feed, animal feed additive, or premix comprising the probiotic strain(s) of the disclosure, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0092] In one embodiment the animal feed, animal feed additive, or premix comprises the composition of the disclosure comprising the Bacillus subtilis deposited as DSM 32324 or DSM 17231 and optionally the Bacillus licheniformis deposited as DSM 17236, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0093] In a preferred embodiment, the animal feed, animal feed additive, or premix comprises the composition of the disclosure comprising the Bacillus subtilis deposited as DSM 17231 and the Bacillus licheniformis deposited as DSM 17236, and further comprises one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0094] In a preferred embodiment the animal feed, animal feed additive or premix comprises the composition of the disclosure consisting of the Bacillus subtilis deposited as DSM 17231 and the Bacillus licheniformis deposited as DSM 17236, and further comprises one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0095] In a specific embodiment, the animal feed is a Total Mixed Ration (TMR) comprising 0 to 80% forage source, such as hay, silage, or pasture. Generally, forage comprises edible parts of a plant (other than separated grain) that can provide feed for animals or can be harvested for feeding animals. As an example, the TMR comprises 0-80% maize; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% barley; and / or 0-30% oats; and / or 0-40% soybean meal.

[0096] In one embodiment, the composition of the present disclosure comprising the probiotic strains is mixed with forage, concentrate, and optionally other feed components to obtain a TMR.

[0097] In a further embodiment, the composition of the present disclosure comprising the probiotic strains is mixed with concentrate, vitamins and / or minerals to obtain a premix. This premix can be mixed with a final diet to obtain a TMR. In a further embodiment, the TMR and the composition of the present disclosure comprising the probiotic strains is mixed with one or more enzymes. In a further embodiment, the composition of the present disclosure comprising the probiotic strains is mixed with other feed ingredients, such as one or more of coloring agents, stabilizers, growth improving additives and aroma compounds / flavorings, saturated or polyunsaturated fatty acids (PUFAs), essential oils, antioxidants, anti-microbial peptides, anti-fungal polypeptides and amino acids. In a particular embodiment, the animal feed components consist of or comprise milk (e.g., from cow, goat, sheep), e.g., for feeding of calves. In another particular embodiment, the animal feed components consist of or comprise milk replacement, e.g., for feeding of calves. In one embodiment the composition of the present disclosure comprising the probiotic strains is mixed with water, milk or milk replacer for feeding of calves, piglets or other weaning animals.

[0098] In another embodiment, the animal feed components may include one or more vitamins, such as one or more fat-soluble vitamins and / or one or more water-soluble vitamins. In another embodiment, the animal feed components may optionally include one or more minerals, such as one or more trace minerals and / or one or more macro minerals. Usually fat- and water-soluble vitamins, as well as trace minerals, form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed. Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3. Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and pantothenate, e.g., Ca-D-pantothenate. Nonlimiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, and zinc. Non-limiting examples of macro minerals include calcium, magnesium, potassium, and sodium.

[0099] The animal feed, animal feed additive or premix of the disclosure may also comprise at least one enzyme selected from the group comprising of phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1 .32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alphaamylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); cellulase (EC 3.2.1.4); and beta-glucanase (EC 3.2.1.6), or any mixture thereof.

[0100] The animal feed, animal feed additive or premix of the disclosure may further comprise one or more added amino acids. Examples of amino acids which are used in animal feed are rumen- protected or not rumen-protected lysine, alanine, beta-alanine, threonine, methionine and tryptophan. The animal feed, animal feed additive or premix of the disclosure may further comprise coloring agents, stabilizers, growth improving additives and aroma compounds / flavorings, polyunsaturated fatty acids (PUFAs), essential oils, antioxidants, antimicrobial peptides and anti-fungal polypeptides. Examples of coloring agents are carotenoids such as beta-carotene, astaxanthin, and lutein. Examples of aroma compounds / flavorings are creosol, anethol, deca-, undeca- and / or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phthalide, butylidene phthalide, capsaicin and tannin. Examples of saturated fatty acids are C16 and C18, such as palmitic and oleic acids, and polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as linoleic, linolenic, arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid. Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a synthetase.

[0101] In one embodiment the animal feed, animal feed additive or premix comprises one or more coccidiostats.

[0102] In one embodiment the animal feed, animal feed additive or premix further comprises a carrier. The carrier can comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, whey, whey permeate, wheat flour, wheat bran, corn gluten meal, starch and cellulose.

[0103] In an embodiment, the animal feed, animal feed additive or premix further comprises one or more additional microorganisms. In a particular embodiment, the animal feed, animal feed additive or premix further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera or any combination thereof.

[0104] In one embodiment, the animal feed, animal feed additive or premix further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus simplex, Bacillus mojavensis, Bacillus safensis, Bacillus simplex, Bacillus atrophaeus, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, Bacillus tequilensis or any combination thereof.

[0105] In one embodiment, the animal feed, animal feed additive or premix comprises a Bacillus subtilis, a Bacillus licheniformis, a Lactobacillus animalis and a Propionibacterium freudenreichii strain.

[0106] In one embodiment, the animal feed, animal feed additive or premix further comprises one or more types of yeast. The one or more type of yeast can be selected from the group consisting of Saccharomycetaceae, Saccharomyces (such as S. cerevisiae and / or S. boulardii), Kluyveromyces (such as K. marxianus and K. lactis), Candida (such as C. utilis, also called Torula yeast), Pichia (such as P. pastoris), Torulaspora (such as T. delbrueckii), Phaffia yeasts and Basidiomycota.

[0107] The composition of the present disclosure may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition may be in frozen or freeze-dried form. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as cryoprotectants and lyoprotectants known to a skilled person in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and / or flavorants.

[0108] Animal diets can, e.g., be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feedstuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. The bacteria cultures and optionally enzymes can be added as solid or liquid formulations. For example, for mash feed a solid or liquid culture formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) composition of the disclosure comprising the probiotic strain(s) may be added to the pelleted food after the pelleting step. Typically, a liquid composition of the disclosure comprises the probiotic strain(s) optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets.

[0109] Another aspect of the disclosure relates to a method for feeding an animal comprising administering the composition of the disclosure comprising the probiotic strains to an animal, such as a monogastric animal or a ruminant. The term “animal” refers to all animals except humans. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, cattle, e.g. beef cattle, cows, and young calves, deer, yaks, camels, llamas and kangaroos. Non-ruminant animals include mono-gastric animals, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chickens (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods), and young calves.

[0110] Ruminants include cows, cattle, sheep, deer, and goats. Monogastric animals include poultry, swine and pets (cats and dogs).

[0111] Ruminants have a fundamentally different digestive system than monogastric animals, and it therefore cannot be concluded that a composition designed for a monogastric animal and with proven effect in the same would also be suitable for and have an effect in a ruminant. Hence, providing a composition effective in both monogastric animals and ruminants is not straightforward.

[0112] The digestive system of animals is involved in the mechanical and chemical digestion of food, absorption of nutrients, and elimination of indigestible materials from the body. The main difference between monogastric and ruminant digestive system is that digestion in the monogastric digestive system mainly occurs in the stomach, whereas digestion in the ruminant digestive system is a foregut fermenter type digestion. The monogastric digestive system is composed of a single stomach while the ruminant digestive system is composed of four stomachs (reticulum, rumen, omasum, and abomasum). Monogastric digestive systems mainly occur in omnivores and carnivores, while ruminants are herbivores.

[0113] The monogastric digestive system refers to the organ system which helps the digestion of both animal and plant materials. It is called monogastric since this digestive system is composed of a single stomach. Humans, horses, swine, fowl, dogs, cats, birds, and rabbit-like animals have a monogastric digestive system. The digestion begins with the entering of feed into the mouth. Both chemical and mechanical digestion start at the mouth. Saliva contains enzymes to digest carbohydrates. The esophagus is the passage that leads feed to the stomach. Various enzymes are secreted into the lumen of the stomach to digest proteins in the feed. Animals with a monogastric digestive system mainly take animal tissues as food. Their diet is easy to digest. Thus, a single stomach is enough for the purpose. The small intestine mainly absorbs the nutrients from the digested feed. The large intestine absorbs water from the indigestible materials.

[0114] Ruminants are mammals that digest plant-based food by initially fermenting / degrading it within the animal's first compartment of the stomach, principally through bacterial actions, then regurgitating the semi-digested mass, now known as cud, and chewing it again. The process of re-chewing the cud to further break down plant matter and stimulate digestion is called "ruminating". Examples of ruminants are cattle, cow, beef cattle, young calf, goat, sheep, lamb, deer, yank, camel and llama. The ruminant digestive system refers to the organ system in which the digestion of plant materials occurs. Cattle, sheep, deer, and goats are examples of animals having a ruminant digestive system. The top jaw of ruminant animals lacks teeth in the front, but instead, a hard pad of skin is present, which is called the dental pad. Other than the basic anatomy of an animal digestive system, the ruminant digestive system is composed of four stomachs. They are rumen, reticulum, omasum, and abomasum. The first three stomachs, the rumen, reticulum, and omasum, are involved in the breaking down of plant fibers and the digestion of non-fibrous compounds. The population of microflora is involved in this process. It breaks down cellulose and starch, for example, through fermentation, producing volatile fatty acids such as acetate, butyrate, and propionate. These volatile fatty acids are utilized by the ruminant as an energy source. Digestive enzymes are secreted in the fourth stomach called the abomasum. Therefore, fermentation occurs before the digestion of the feed in ruminant animals. Hence, this process is called the foregut fermentation. Furthermore, ruminant animals chew the partly digested food or cud by returning them from the first stomach. The small intestine and large intestine of ruminants are similar to the monogastric digestive system. However, ruminants comprise a large caecum for further digestion of the fibers.

[0115] In preferred embodiments of the present disclosure, the composition of the present disclosure is used in a method of maintaining or improving respiratory health or reducing the occurrence and / or severity of respiratory disease in a ruminant.

[0116] In another embodiment of the present disclosure, the composition of the present disclosure is used in a method of maintaining or improving respiratory health or reducing the occurrence and / or severity of respiratory disease in a monogastric animal.

[0117] As evidenced in the examples, the composition of the disclosure inhibits relevant pathogens for respiratory disease, and affects the development of lung lesions, and administration of the composition of the disclosure comprising the probiotic strains described herein reduces the occurrence and / or severity of respiratory disease in animals as compared to controls.

[0118] By “reducing the occurrence and / or severity of respiratory disease” it is meant that the use of the composition of the disclosure in the feeding of animals will lead to an improvement in respiratory health among the animals being administered the composition of the disclosure, resulting in a reduced number of observed lung lesions, and / or reduced number of signs of respiratory disease as determined by e.g. the University of Wisconsin scoring system (see Example 1).

[0119] In one embodiment, the reduced number of lung lesions results in a reduction of 3.0% or more than 3.0%, such as more than 3.5%, such as more than 4.0%, such as more than 4.5%, such as more than 5.0%, such as more than 5.5%.

[0120] In one embodiment, the reduced number of lung lesions results in a reduction of between 3.0% and 5.5%, such as between 3.0% and 5.0%, such as between 3.0% and 4.5%, such as between 3.0% and 4.0%, such as between 3.0% and 3.5%.

[0121] In one embodiment, the reduced number of signs of respiratory disease as determined by e.g. the University of Wisconsin scoring system results in a reduction of 10% or more than 10%, such as more than 15%, such as more than 20%, such as more than 30%, such as more than 40%, such as 50%.

[0122] In one embodiment, the reduced number of signs of respiratory disease as determined by e.g. the University of Wisconsin scoring system results in a reduction of between 10% and 50%, such as between 10% and 40%, such as between 10% and 30%, such as between 10% and 20%. TAXONOMY

[0123] Bacillus licheniformis is now known as Bacillus paralicheniformis, as described in Dunlap et al. 2015 (International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3487-3492). The two different names are used interchangeably herein.

[0124] DEPOSIT AND EXPERT SOLUTION

[0125] 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.

[0126] Table 1 : The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0127] The invention is further defined by the following numbered paragraphs:

[0128] 1. A composition comprising at least one B. subtilis strain and optionally at least one B. licheniformis strain for use in reducing the occurrence and / or severity of respiratory disease in an animal.

[0129] 2. A composition comprising at least one B. subtilis strain and optionally at least one B. licheniformis strain for use in preventing respiratory disease in an animal.

[0130] 3. A composition comprising at least one B. subtilis strain and optionally at least one B. licheniformis strain for use in maintaining or improving respiratory health in an animal.

[0131] 4. The composition of any of paragraphs 1-3, wherein said B. subtilis strain is selected from the strains deposited as DSM 17231 and DSM 32324. 5. The composition of any of paragraphs 1-3, wherein said B. licheniformis strain is the strain deposited as DSM 17236.

[0132] 6. The composition according to any preceding paragraph, wherein said composition comprises the strains deposited as DSM 17231 and DSM 17236.

[0133] 7. The composition of any of paragraphs 1-3, wherein the microorganism component of said composition consists of the Bacillus subtilis deposited as DSM 17231 and the Bacillus licheniformis deposited as DSM 17236.

[0134] 8. The composition according to any preceding paragraph, wherein said respiratory disease is selected from the group consisting of pneumonia and bovine respiratory disease.

[0135] 9. The composition according to any preceding paragraph, wherein said animal is a ruminant and said respiratory disease is bovine respiratory disease.

[0136] 10. A method of producing an animal feed, animal feed additive, or premix, comprising adding the composition of any of paragraphs 1 to 9 to an animal feed, animal feed additive, or premix or relevant components thereof.

[0137] 11. The method according to paragraph 10, wherein the animal feed, animal feed additive or premix is for an animal selected from the group consisting of poultry, herbivores, ruminants, swine, rodents, pets, fish and crustaceans, preferably wherein the animal is a ruminant such as a cow.

[0138] 12. An animal feed, animal feed additive, or premix comprising the composition of any of paragraphs 1 to 9, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

[0139] 13. The animal feed, animal feed additive, or premix of paragraph 12, wherein said composition comprises the Bacillus subtilis deposited as DSM 17231 and the Bacillus licheniformis deposited as DSM 17236.

[0140] 14. A method of reducing the occurrence and / or severity of a respiratory disease in an animal, or for preventing respiratory disease in an animal, the method comprising administering to said animal a composition that reduces the occurrence and / or severity of clinical signs of disease associated with said respiratory disease, said composition comprising at least one B. subtilis strain and optionally at least one B. licheniformis strain, wherein said composition is administered in an animal feed, animal feed additive, or premix to said animal in an amount to provide a total amount of said probiotic strains of between 1 x 108and 1 x 1011CFU / animal / day.

[0141] 15. The method of paragraph 14, wherein said animal feed, animal feed additive, or premix is an animal feed, animal feed additive, or premix according to paragraph 12 or 13. 16. The method of paragraph 14 or 15, further comprising assessing the effects of pathogen reduction by said administration of said composition.

[0142] 17. The method of paragraph 16, wherein the pathogen to be reduced is Mannheimia haemolytica.

[0143] 18. A method of maintaining or improving respiratory health, reducing the number of observed lung lesions, and / or reducing the number of signs of respiratory disease as determined by the University of Wisconsin scoring system, the method comprising feeding a composition according to any of paragraphs 1 to 9 or an animal feed, animal feed additive, or premix according to paragraph 12 or 13 to an animal.

[0144] 19. The method of paragraph 18, which is a method of maintaining or improving respiratory health in an animal.

[0145] 20. The method of paragraph 18, wherein the animal is a ruminant.

[0146] 21. The method of paragraph 19 or 20, comprising administering to said animal a composition comprising the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236, or an animal feed, animal feed additive or premix comprising said composition.

[0147] 22. The method of paragraph 21, wherein the microorganism component of said composition consists of the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236.

[0148] 23. Use of a composition comprising at least one B. subtilis strain and optionally at least one B. licheniformis strain for preventing respiratory disease in an animal or for reducing the occurrence and / or severity of respiratory disease in an animal.

[0149] 24. Use of a composition comprising at least one B. subtilis strain and optionally at least one B. licheniformis strain for maintaining or improving respiratory health in an animal.

[0150] 25. Use according to paragraph 23 or 24, wherein the animal is a ruminant.

[0151] 26. Use according to any of paragraphs 23-25, wherein said B. subtilis strain is selected from the strains deposited as DSM 17231 as DSM 32324.

[0152] 27. Use according to any of paragraphs 23-25, wherein said B. licheniformis strain is the strain deposited as DSM 17236.

[0153] 28. Use according to any of paragraphs 23-27, wherein said composition comprises the strains deposited as DSM 17231 and DSM 17236. 29. Use according to any of paragraphs 23-25, wherein the microorganism component of said composition consists of the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236.

[0154] 30. Use according to any of paragraphs 23-29, wherein said respiratory disease is selected from the group consisting of pneumonia and bovine respiratory disease.

[0155] 31. Use according to any of paragraphs 23-30, wherein said animal is a ruminant and said respiratory disease is bovine respiratory disease.

[0156] 32. Use according to any of paragraphs 23-31 , for preventing or for reducing the occurrence and / or severity respiratory disease caused by Mannheimia haemolytica.

[0157] 33. Use of a composition comprising the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236, or an animal feed, animal feed additive or premix comprising said composition, for maintaining or improving respiratory health of an animal, wherein the animal is a ruminant.

[0158] 34. A method of maintaining or improving respiratory health in an animal, wherein the animal is a ruminant, comprising administering to said animal a composition comprising the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236, or an animal feed, animal feed additive or premix comprising said composition.

[0159] EXAMPLES

[0160] EXAMPLE 1

[0161] Bacillus licheniformis DSM 17236 was inoculated in brain heart infusion broth and incubated aerobically for 48h at 37C°.

[0162] Mannheimia haemolytica strain was inoculated on trypticase soy agar medium and incubated at 37°C aerobically for 24h. On the day of the assay, the culture of M. haemolytica was suspended by using a cotton swab with a 0.5 McFarland suspension in a maximum recovery diluent medium. Following this step, a suspension of 35 mL melted LB agar and 10 pL of pathogen was mixed in a 50 mL Falcon tube and the mixture was cast in Omnitray plates with an immediate application of the Nunc™lmmuno TSP (Thermo Fisher Scientific, Waltham, MA). Then, the plates were left to solidify for 20 min before lid removal. Thereafter, plates were allowed to dry with a normal lid on for an additional 20 min. Lastly, 5 pL of the strain overnight culture was applied to the selected wells in the agar.

[0163] Samples were analyzed in triplicate. After aerobic incubation at 30°C for 48h, the inhibition zone was measured via Photoshop from full growth to full growth, with a lower limit of 3.5 mm (width of each well in the plate). The inhibition zone was measured at 48 h post-B. licheniformis addition on the agar plate (in mm).

[0164] In the present example, adding B. licheniformis DSM 17236 with M. haemolytica yielded an inhibition zone of 10.4 mm (see Figure 1). These results support the efficacy of the probiotic strain in directly inhibiting this important pathogen associated with respiratory disease in cattle.

[0165] EXAMPLE 2

[0166] The effects of probiotics supplementation on the health and performance of pre-weaning dairy calves

[0167] An in vivo study was conducted to evaluate the effects of feeding a combination of the probiotic strains 8. subtilis (DSM 17231) and 8. licheniformis (DSM 17236) on the health and performance of pre-weaning dairy Gyr x Holstein dairy calves. The experiment was conducted at a commercial dairy farm located in Passos, MG, Brazil.

[0168] In this study, 60 newborn calves were enrolled in the study following the colostrum intake (approximately 24 hours post-birth) and assigned to: (1) whole milk without probiotic addition (n = 30) and (2) whole milk with the addition of 1 g / head per day of the probiotic combination (3.2 x 109CFU / head per day; n = 30). Treatments were offered daily for 77 days.

[0169] The milk feeding scheme was as follows: from day 1 to 42 of the study = 6 L / calf per day split in two feedings; from day 43 to weaning (day 77) = 3 L / calf per day offered once a day. A concentrate feed containing corn meal (40.0%), soybean meal (35.2%), dried distiller's grains (17.1 %), premix (6.8%), and urea (0.9%) was offered from day 1 of age through weaning as shown in Table 2 below:

[0170] Table 2: Feeding amount per day Throughout the experimental period, animals were evaluated for any signs of adverse health events, including pneumonia following the Calf Health Scoring Chart from University of Wisconsin (https: / / www.vetmed.wisc.edu / fapm / svm-dairy-apps / calf-health-scorer-chs / ) as shown in Figure 2. The calves in this study were subjected to different triggers of stress, such as early removal from the mother, individual housing and subjection to a higher load of pathogens.

[0171] In the present example, pneumonia occurrence decreased from 46.7 (n = 14 calves) to 23.0 (n = 7 calves) by feeding the combination of probiotic strains (P = 0.05; Figure 3).

[0172] Although no statistical significance was observed on the mean age of occurrence (P = 0.76), days of pharmacological intervention (P = 1.00), or cost of pharmacological intervention per calf (P = 0.55), the total expense of pharmacological treatment was numerically higher for CON vs. PRO (277.2 vs. 102.9 USD, respectively).

[0173] Moreover, calves that were fed the combination of probiotic strains were heavier at weaning (81.7 vs. 88.0 kg; P = 0.04) and tended (P < 0.09) to have a greater ADG (0.618 vs. 0.682 kg / day) and FE (0.353 vs. 390 kg gain / kg feed) during the study described in this example.

[0174] EXAMPLE 3

[0175] Preparation of virus inoculum

[0176] The virus isolate A / sw / Denmark / 12687 / 03 (H1 N2) (lineage 1C.2) was propagated and passaged three times in Madin-Darby canine kidney (MDCK) cells and stored in aliquots at -80°C until use. A tissue culture infectious dose 50% (TCID50) assay was used to determine the virus titers in MDCK cells followed by dilution in Eagle’s Minimum Essential Medium (MEM) (Gibco) to obtain a TCID50 / ml of 1.58 x 105.

[0177] Experimental design

[0178] A total of 24 cross-bred Duroc x Landrace-Yorkshire pigs (females and castrated males, 7 weeks old, initial body weight 12 kg) were used. All pigs were tested for swIAV virus and antibodies approx, four weeks of age and confirmed negative for swIAV and lAV-specific antibodies. After stratification based on sex and weight, pigs were allocated to one of three groups: 1) Not challenged and not fed probiotic (CON; n=8), 2) Challenged, but not fed probiotic (CHL; n=8), and 3) Challenged and fed B. subtilis DSM 32324 at 6.95 x 106CFU / g feed (PRO; n = 8). All groups received the same diet (the CON and CHL groups without B. subtilis DSM 32324) and all pigs had ad libitum access to feed and water throughout the study.

[0179] After two weeks of being fed the diets with or without B. subtilis DSM 32324, all animals from CHL and PRO were challenged intranasally with 3 mL of H1 N2 swIAV suspension (1.58 x 105TCID50 / mL) and the animals from CON were given mock media (MEM, Gibco) in the right nostril by aerosol exposure with MAD Nasal™ intranasal mucosal devices. The pigs were monitored daily for clinical signs of disease, and body weights and temperatures were measured at days -14, 0, and 7 days post-infection (DPI).

[0180] Sampling procedures

[0181] All pigs were sacrificed at DPI 7. At euthanasia, the pigs were anesthetized by intramuscular injection of 0.1 ml / kg Zoletil 50 VET (tiletamine 25 mg / ml, zolazepam 25 mg / ml) and euthanized by intracardiac injection of 20 % sodium pentobarbital (KELA, 100 mg / kg). Specimens of lung and ileum tissue were isolated and stored immediately in RNAIater (Invitrogen) and kept at room temperature until being transferred to -20°C the following day. At necropsy, the lung lesions were sketched and photos of both dorsal and ventral sides of the lungs were taken. The area of red atelectasis observed in the lung photos combined with the lung sketches was measured blinded by a pathologist using Adobe Acrobat Reader. Lung biopsies of lobus dexter medius were taken for histopathological examination and stabilized in formalin (BiopSafe) until being embedded in paraffin wax, sliced into 2-3 pm sections and stained with hematoxylin and eosin (H&E) to examine histopathology and influenza virus to quantify swIAV positive cells.

[0182] DSM 32324 may reduce swIA V-induced gross lung pathology

[0183] Influenza challenge caused a mild infection in the pigs with no clinical symptoms. No differences in microscopic changes were observed and no difference in number of lAV-positive cells was observed in the lung tissue between CHL and PRO groups. However, gross lung lesions were observed in all influenza-challenged pigs (CHL and PRO group), whilst no gross lesions were observed in the CON group. When comparing the total area with lesions, pigs from the PRO group tended to have (P=0.065) lower amount of gross lung lesions compared to the CHL group.

[0184] EXAMPLE 4

[0185] Bacillus-based probiotic supplementation to sows and weaned progeny help to support live performance and manage gross lung lesions in a nursery H1N2 Influenza challenge

[0186] A series of recent studies have indicated that providing an effective probiotic to dams and their progeny allows the pigs to respond more favorably to viral challenges. Sixteen pregnant dams (parity 1 to 5) allotted by gestation pen were exposed to one of two treatments: Control (CON) where dams consumed a standard, non-probiotic containing, gestation and lactation diet or Bacillus (BAC) where dams consumed standard diets with a daily top dress that was applied to achieve 1.65 x 106CFUs of Bacillus composition / gram of complete feed from 5 weeks prior to farrowing through lactation. The Bacillus composition used comprised Bacillus licheniformis DSM 17236 and B. subtilis DSM 17231 . Ninety-six nursery pigs weaned from CON and BAC sows were allotted (48 per treatment CON or BAC) to the nursery portion of this study at weaning on day 21. CON pigs selected for the nursery received non-probiotic containing feed and BAC pigs selected for the nursery received 1.10 x 106CFUs Bacillus / gram of complete feed for the entire 3-week nursery period of the study. Pigs were allotted to 8 pens with 6 pigs per pen by treatment. Individual pig weights and pen feed intakes were recorded on days 0, 7, 14, and 21. Following 14-days of feed acclimatization post-weaning, all pigs received via intratracheal gavage, an H1 N2 Influenza viral challenge (Haesebrouck et al., Vet. Microbiol. 11 :239-249, 1986). Clinical signs, including rectal temperatures, respiratory score, abdominal score, and demeanor score were collected daily beginning one day prior to challenge (day 13) through the end of the study. On day 21 , following weight collection, all pigs were euthanized, lungs were removed, and each of 7 lung lobes (right and left cranial, right and left middle, accessory, and right and left caudal) were scored on percentage of lobe bearing gross (visible) lung lesions (Halbur et al., J. Vet. Diagn. Invest. 8:11-20, 1996). BAC treatment resulted in improved F:G during the pre-challenge (P < 0.01 , 1.177 v. 1.309), post-challenge (P < 0.01 , 1.418 v. 1.633), and entire growth (P < 0.001 , 1.287 v. 1.464) periods. Also, in the BAC treatment group, the percentage of pigs with gross lung lesions in the left and right cranial, accessory, and left and right caudal lung lobes was reduced. Additionally, total lung lobes per pig with lesions (P < 0.001 4.1 v. 5.3) were reduced in pigs in the BAC treatment group. These data indicate that application of a Bacillus-based probiotic composition comprising Bacillus licheniformis DSM 17236 and B. subtilis DSM 17231 to sows and subsequently their weaned progeny is associated with improved feed efficiency and reduced lung lesions when pigs are exposed to an H1 N2 Influenza challenge.

[0187] EXAMPLE 5

[0188] Bacillus subtilis DSM 32324 induces changes in lung pathology and inflammation during influenza A virus infection in pigs

[0189] Materials and methods

[0190] Pigs were fed a diet either with or without the probiotic strain B. subtilis DSM 32324 (2.6E+06 CFU / g feed) for 14 days before being intranasally inoculated with a swine influenza A H1 N2 strain (1 C.2 lineage). Throughout the study, fecal samples, blood samples and nasal swabs were collected to examine viral shedding and immune gene expression. After seven days of infection, the pigs were euthanized, and lung and ileum tissues were collected for gene expression analysis and pathological examination.

[0191] Results

[0192] The results are shown in Figures 6, 7 and 8. Figure 6 compares the total area with lesions of the two infected groups, showing was a clear tendency (P=0.065) for a lower amount of gross lung lesions in infected pigs fed B. subtilis DSM 32324 (B. subtilis group), compared to the infected group fed a diet not supplemented with B. subtilis DSM 32324 (Positive control group).

[0193] Figure 7 shows CRP levels at 0, 1 , 4 and 7 days post infection. It is seen that at 4 days postinfection (DPI 4), the CRP levels were significantly increased by infection in the positive control group compared to the negative control group (P=0.03), with the 8. subtilis group being intermediate (P=0.12).

[0194] Figure 8 shows serum amyloid A (SAA) levels in the pigs. There was a tendency (P=0.06) for reduced SAA in the 8. subtilis group compared to the positive control group. Similar results were observed for SAA3, which was significantly reduced in the 8. subtilis group (P=0.04).

[0195] These findings indicates that administration of B. subtilis DSM 32324 has the potential to reduce lung lesions, possibly attributable to a general suppression of the immune system as indicated by reduced C-reactive protein (CRP) levels in serum, decreased expression of interferon-stimulated genes (ISGs), and localized reduction of the inflammatory marker serum amyloid A (SAA) in ileum tissue. Notably, the immune-modulatory effects of B. subtilis DSM 32324 appeared to be unrelated to the gastrointestinal microbiota, as the microbiota composition remained unaltered by both the influenza infection and the administration of 8. subtilis DSM 32324.

[0196] EXAMPLE 6

[0197] Effect of Bacillus-based probiotic supplementation to sows and their weaned offspring in a nursery h 1n2 influenza challenge

[0198] Materials and methods

[0199] Five weeks prior to farrowing, 16 gestating sows were allotted to two treatment groups: CON (control) and BAC (Bacillus). Gestation and lactation diets of the BAC sows were supplemented with a probiotic consisting of 8. subtilis DSM 17231 and B. licheniformis DSM 17236 (1.65E+09 CFU / kg feed) while the CON sows received standard commercial diets. On day 21 , 96 pigs weaned from CON and BAC sows were allotted to eight pens with six pigs / pen (48 pigs / treatment group). During the 3-week nursery period, BAC pigs were supplemented with the Bacillus-based probiotic (1.10E+09 CFU / kg feed) and CON pigs were fed a standard diet.

[0200] Following 14 days of acclimatization post-weaning, all pigs were challenged with H1 N2 influenza via intratracheal gavage. Performance metrics were recorded weekly while rectal temperatures and clinical severity were assessed daily beginning one day prior to challenge (day 13) through study termination. Nasal swabs collected on days 16, 18 and 21 were analyzed for viral loads via qPCR. On day 21 , all pigs were euthanized, and the lungs were removed and scored on percentage of lobe bearing gross (visible) lung lesions. Results

[0201] The results are shown in Tables 3 and 4 below and in Figure 9.

[0202] Table 3: Improvement in feed conversion ratio (FCR) pre- and post-challenge in probiotic-fed pigs

[0203] Table 4: Percent of pigs with rectal temperature above 39.7°C

[0204] Compared with the CON pigs, the BAC pigs had an improved feed conversion ratio (FCR) during the pre- and post-challenge periods (P<0.01 ; Table 3). The proportion of pigs with rectal temperatures above 39.7°C was lower in the BAC treatment group during the post-challenge period (P<0.01 ; Table 4), as were clinical severity scores for the same days (combined demeanor, abdominal, and respiratory score) (P<0.05). Viral intensity was 1-2 ct lower in nasal swabs associated with the BAC fed pigs on days 16 and 18, and the average total lung lobes with lesions per pig was lower in the BAC treatment (P<0.001 ; Figure 9).

[0205] 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.

[0206] 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.

Claims

CLAIMS1. A composition comprising at least one B. subtilis strain and optionally at least one B. licheniformis strain for use in maintaining or improving respiratory health, for use in preventing respiratory disease in an animal, or for use in reducing the occurrence and / or severity of respiratory disease in an animal.

2. The composition of claim 1 , wherein said B. subtilis strain is selected from the strains deposited as DSM 17231 and DSM 32324.

3. The composition of claim 1 or 2, wherein said B. licheniformis strain is the strain deposited as DSM 17236.

4. The composition according to any preceding claim, wherein said composition comprises the strains deposited as DSM 17231 and DSM 17236.

5. The composition of claim 1 , wherein the microorganism component of said composition consists of the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236.

6. The composition according to any preceding claim, wherein said animal is a ruminant.

7. An animal feed, animal feed additive, or premix comprising the composition of any of claims 1 to 6, and further comprising one or more concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and / or other feed ingredient(s).

8. A method of maintaining or improving the respiratory health of an animal, comprising administering to said animal a composition according to any of claims 1-6 or an animal feed, animal feed additive or premix according to claim 7.

9. The method of claim 8, wherein the animal is a ruminant.

10. The method of claim 8 or 9, wherein the composition comprises the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236.11 . Use of a composition comprising at least one B. subtilis strain and optionally at least one B. licheniformis strain for maintaining or improving respiratory health in an animal.

12. Use according to claim 11 , wherein the animal is a ruminant.

13. Use according to claim 11 or 12, wherein said B. subtilis strain is selected from the strains deposited as DSM 17231 and DSM 32324.

14. Use according to any of claims 11-13, wherein said B. licheniformis strain is the strain deposited as DSM 17236.

15. Use according to any of claims 11-14, wherein said composition comprises the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236.

16. Use according to claim 15, wherein the microorganism component of said composition consists of the Bacillus subtilis strain deposited as DSM 17231 and the Bacillus licheniformis strain deposited as DSM 17236.

17. Use according to any of claims 12-16, wherein said respiratory disease is selected from the group consisting of pneumonia and bovine respiratory disease, e.g. wherein said animal is a ruminant and said respiratory disease is bovine respiratory disease.