Probiotic bacterial strain for improving the health performance of an animal

Abstract

The present disclosure generally relates to compositions and methods for reducing the development and / or severity of respiratory disease using novel combinations of probiotic bacterial strains, thereby improving the health performance of animals. In addition, the present disclosure relates to compositions and methods for inhibiting the growth of pathogenic bacteria using novel combinations of probiotic bacterial strains.

Description

Technical Field

[0001] This disclosure generally relates to novel combinations of probiotic strains, such as compositions and methods for improving animal health by using or maintaining respiratory health in animals through reducing the incidence and / or severity of respiratory diseases. Background Technology

[0002] One of the most common respiratory diseases in livestock is pneumonia, defined as an inflammation of the lungs. Bovine respiratory disease (BRD) is the single largest health problem facing the fattening industry, causing enormous economic and production losses. The disease accounts for approximately 75% of morbidity and 50% to 70% of mortality in fattening farms. It is estimated that the disease costs the U.S. fattening industry $800 million to $900 million annually due to reduced animal production and increased veterinary treatment costs.

[0003] Furthermore, BRD leads to loss of live productivity, which ultimately affects the quality and yield of carcasses obtained from animals. In the United States, as the number of BRD treatments increased from 0 to ≥3, the total value of calves decreased linearly, indicating losses in productive performance and carcass traits, as well as additional pharmacological costs affecting overall profitability. In a comprehensive economic assessment of the economic aspects of BRD conducted in Australia, Blakebrough-Hall et al. ( J. Anim.Sci. According to a report in the Journal of Animal Science, Vol. 98, No. 2, 2020, as the number of treatments for BRD increased, the production performance and carcass traits of fattening farms declined linearly, and the value of slaughtered animals also decreased.

[0004] Among the pathogens involved in BRD syndrome, Pasteurella multocida (…) Pasteurella multocida ), hemolytic Mansonia ( Mannheimia haemolytica ), Sleeping histophilia ( Histophilus somni ) and bovine mycoplasma ( Mycoplasma bovis ) is considered the main pathogen causing bovine pneumonia (Czuprynski et al.) Anim.Health Res. Rev. [Animal Health Research Review] 5:277-282, 2004); Bringhenti et al., ( J. Dairy Sci.[Journal of Dairy Science] 104:10291–10309, 2021). A noteworthy and interesting point is that these bacteria are thought to be ubiquitous in the upper respiratory tract of cattle, meaning that animals themselves carry most (if not all) of these microbes, which coexist harmoniously with the animals but may later cause BRD. The most common approach to treating bovine pneumonia involves the use of broad-spectrum antibiotics after assessing the animal's health parameters (e.g., nasal discharge, appetite, respiratory rate, and rectal temperature). However, the use of antibiotics does not guarantee a full recovery of the herd's health and productivity, as demonstrated by others (Wilson et al., (J. Dairy Sci. [Journal of Dairy Science] 104:10291–10309, 2017); Blakebrough-Hall et al., ibid.).

[0005] Globally, livestock production systems (regardless of species) are seeking alternatives that can minimize antibiotic use. These alternatives include natural microbial solutions that can support the overall health of animals and thus reduce the number of sick animals requiring antibiotic treatment.

[0006] Although extensive research has been conducted on the determinants of bovine respiratory diseases, only a few conclusive findings have been made, and bovine respiratory diseases will remain a very costly condition until effective treatments or preventative measures are identified.

[0007] Therefore, there is still a need for probiotic strains and specific mixtures of probiotic strains that can be used to improve or maintain the health of production animals, especially those with health benefits for respiratory diseases. Summary of the Invention

[0008] This disclosure provides a composition comprising a novel combination of probiotic strains that have proven effects on animal health and production performance by maintaining or improving respiratory health.

[0009] Respiratory infections can occur due to a combination of one or more factors, including animal stress, exposure to high levels of infectious agents, and / or impaired animal immune function. Examples disclosed herein demonstrate the effects of the compositions disclosed on the health performance of animals, similarly related to respiratory diseases.

[0010] Compositions containing novel combinations of probiotic strains disclosed herein can be fed to animals, for example, as direct feed microorganisms (DFM), as animal feed additives, in premix form, or directly incorporated into animal feed.

[0011] The novel combination of probiotic strains disclosed herein can be added to feed by the producer by mixing the strains with the feed to be fed to the animals during production, immediately after production by the supplier, or before feeding to the animals.

[0012] Typical treatment for animals exhibiting clinical signs primarily consists of antibiotics (especially penicillin) and vaccination. In addition, the industry generally recognizes good livestock management as a preventative measure to minimize the risk of disease outbreaks. This management includes maintaining livestock in good overall health and minimizing stress in production facilities. This is a costly procedure to implement, and despite various efforts, complete prevention cannot be achieved.

[0013] The purpose of the in vitro studies described in the examples is to investigate the inhibitory effect of strains present in the novel probiotic combination according to this disclosure on hemolytic Mansicae, a major cause of respiratory diseases in ruminants. The purpose of the in vivo studies described in the examples is to investigate the subsequent effects of feed additives containing the novel probiotic combination according to this disclosure on the incidence and / or severity of respiratory diseases in ruminants, in a production performance trial of dairy calves and a challenge study of weaned offspring in sows and nursery pens.

[0014] 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 containing the probiotic strains disclosed herein.

[0015] These results demonstrate that the novel combination of the aforementioned probiotic strains improves animal health, thus providing a cost-effective and easily administered alternative to traditional treatments involving antibiotics and vaccination against respiratory pathogens. While not wishing to be bound by theory, it is believed that daily supplementation with the composition disclosed herein will maintain or improve the respiratory health of animals receiving the composition.

[0016] definition Generally, the terms and phrases used herein have their generally accepted meanings in the art, which can be discovered by referring to standard textbooks, journal articles, and context known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of this disclosure.

[0017] As used in this article, the singular forms “a / an” and “the” are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0018] As used herein, the term “and / or” is intended to mean both combined (“and”) and exclusive (“or”) use, i.e., “A and / or B” is intended to mean “A alone, or B alone, or A and B together”.

[0019] Animal feed: As used herein, the term "animal feed" means any compound, preparation, or mixture suitable for or intended for use by animals. Animal feed for monogastric animals typically contains concentrates along with vitamins, minerals, enzymes, direct fed microbial, amino acids, and / or other feed ingredients (as in premixes), while animal feed for ruminants typically contains forage (including roughage and silage) and may further contain concentrates along with vitamins, minerals, enzymes, direct fed microbial, amino acids, and / or other feed ingredients (as in premixes). Animal feed components may consist of or contain milk (e.g., from dairy cows, goats, sheep), for example, for feeding young animals. Examples of ingredients suitable for animal feed and animal feed formulations are described and illustrated in the "Compositions, Forms of Administration, and Formulations" section.

[0020] 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 can be used in direct feed microorganisms (DFM), animal feed additives, premixes, or total mixed diets (TMR). The composition can be provided in any suitable form, such as in liquid or solid form.

[0021] Concentrates: As used herein, the term “concentrate” means feed with high protein and energy concentrations, such as fishmeal, molasses, oligosaccharides, seeds and grains (e.g., whole or prepared from corn, oats, rye, barley, sorghum, wheat, or by crushing, milling, etc.), oilseed filter cake (e.g., from cottonseed, safflower, sunflower, soybean (e.g., soybean meal), rapeseed / canola, peanut, or ground peanut), palm kernel cake, yeast-derived materials, and distillers' grains (e.g., wet distillers' grains (WDS) and dry distillers' grains with solubles (DDGS)).

[0022] Inhibition of the growth of hemolytic Manslaughter: As used herein, the term "inhibition of the growth of hemolytic Manslaughter" refers to a method and / or composition that partially or completely inhibits hemolytic Manslaughter infection in an animal. Therefore, the term "inhibition of the growth of hemolytic Manslaughter" means that hemolytic Manslaughter infection is reduced or completely eliminated, and the overall health of the animal is improved.

[0023] Direct feeding microorganisms: As used herein, the term “direct feeding microorganisms” or “DFM” refers to live microorganisms, including spores, that, when applied in appropriate amounts, impart beneficial effects to the animals receiving them, such as improved digestion or health.

[0024] Effective amount / effective concentration / effective dose: As used herein, the terms “effective amount,” “effective concentration,” or “effective dose” are defined as the amount, concentration, or dose of one or more bacterial strains sufficient to improve the health and production performance (feed digestibility or efficiency) of an animal, as shown in the examples.

[0025] The actual effective dose, in absolute terms, depends on a variety of factors, including the health status of the animal in question and other components present. The “effective amount,” “effective concentration,” or “effective dose” of a bacterial strain can be determined by routine assays known to those skilled in the art, taking into account the guidance provided herein.

[0026] Feeding animals: As used herein, the terms “feeding animals” or “administered to animals” mean administering an effective amount of the composition disclosed herein orally to an animal. This oral administration may be repeated, for example, once or more daily for a specified period of time (e.g., days, weeks, weeks, months, or months). Therefore, the terms “feeding” or “feeding” mean any type of oral administration, such as via animal feed or via drinking water, or in some cases via oral gavage or aerosol spray.

[0027] Isolated: As used herein, the term “isolated” means that the bacterial strain described herein is in a form or environment not present in nature, that is, the strain is isolated at least in part from one or more or all naturally occurring components associated with it in nature.

[0028] Prevention and / or minimization of respiratory diseases: As used herein, the term "prevention and / or minimization of respiratory diseases" refers to methods and / or compositions for preventing and / or minimizing the incidence of respiratory diseases in animals. Those skilled in the art can readily assess whether an animal has developed a respiratory disease. An example of such an assessment method is described in the examples disclosed herein.

[0029] Maintaining or improving respiratory health may alternatively or otherwise be intended to prevent or reduce the occurrence of respiratory infections in animal populations or farms; and / or reduce the likelihood of respiratory infection outbreaks; or reduce the prevalence of respiratory infections in animal populations; and / or reduce or prevent respiratory infections caused by pathogens in animals; or reduce the severity of respiratory infections; or reduce the pathogen load in animals.

[0030] Weight gain: As used herein, weight gain in an animal is the increase in the animal's live body weight over a specified period of time. The term "average daily gain (ADG)" is also used to describe weight gain. As used herein, the terms "average daily gain" and "ADG" have the same meaning and are used interchangeably. The term "average daily gain" refers to the body weight gained by an animal in one day, that is, in 24 hours.

[0031] Overall impression: As used herein, the term “overall impression” is a measure of the overall impression of a given animal on a given date and is assessed using methods known in the art.

[0032] Dry matter intake (DMI): As used herein, the terms “dry matter intake” and “DMI” have the same meaning and are used interchangeably. The term “dry matter intake” refers to the amount of feed consumed by an animal (excluding water content).

[0033] Feed efficiency (FE): As used herein, the terms “feed efficiency” and “FE” have the same meaning and are used interchangeably. “Increased feed efficiency” means that the use of the feed additive composition disclosed herein results in a greater weight gain per kg of feed consumed compared to the weight gain observed in animals when the feed does not contain the feed additive composition disclosed herein. Attached Figure Description

[0034] Figure 1 The inhibition zone (in mm) achieved after incubating hemolytic Mansonia with either a control or a probiotic (Bacillus licheniformis DSM 17236) is shown.

[0035] Figure 2 This shows the University of Wisconsin's scoring system for calf pneumonia.

[0036] Figure 3 This shows the proportion of pre-weaning dairy calves requiring pharmacological treatment for pneumonia who were fed either the control (CON) or probiotics (PRO) (i.e., calves scoring 2 or 3 using the University of Wisconsin-Madison scoring system for calf pneumonia). The observed treatment effects ( P = 0.05).

[0037] Figure 4This is an overview of the study in Example 3. Twenty-four pigs were divided into three groups (n = 8) and fed either a diet supplemented with probiotics (PRO group) or a diet supplemented with a placebo (positive and negative controls) for 14 days prior to intranasal challenge with swIAV (H1N2) at DPI 0. Only the probiotic group and the positive control group were challenged with swIAV. The negative control group underwent a simulated infection. As shown in the figure, serum, nasal swabs for immunological and viral load assays, and fecal samples were collected during the study. All pigs were euthanized at DPI 7. DPI = Days post-inoculation.

[0038] Figure 5 The extent of gross lung pathology was shown. Pigs were fed a diet containing or without PRO for 14 days, followed by intranasal challenge with swIAV for 7 days, after which they were euthanized and their gross lung lesions were assessed. These groups were the negative control group (CON; no challenge, no probiotics), the positive control group (CHL; challenged, no probiotics), and the probiotic group (PRO; challenged and probiotics). Gross lung lesions were calculated as the percentage of lungs with red atelectasis at 7 days post-infection (DPI 7). Differences between the two infection groups were analyzed by the Mann-Whitney t-test, indicating the median for each treatment group. A p-value ≤ 0.05 was considered statistically significant. Furthermore, if P ≤ 0.10, then a trend is considered to exist.

[0039] Figure 6 The study shows the level of lung lesions in two groups of pigs infected with influenza A virus (a positive control group and a group fed with the probiotic Bacillus subtilis strain).

[0040] Figure 7 The C-reactive protein (CRP) levels in three groups of pigs are shown: a negative control group of pigs not infected with influenza A virus, a positive control group of pigs infected with influenza A virus, and a group of pigs infected with influenza A virus and fed with the probiotic Bacillus subtilis strain.

[0041] Figure 8 The changes in serum amyloid A (SAA) and SAA3, inflammatory markers, in ileal tissue were shown in two groups of pigs infected with influenza A virus (control group and group fed with the probiotic Bacillus subtilis strain).

[0042] Figure 9 The figure shows the average total number of lung lobes with lesions per pig in the control group and pigs treated with Bacillus subtilis during H1N2 influenza challenge in nursery pigs. Detailed Implementation

[0043] This disclosure relates to compositions comprising at least one strain of Bacillus, and the use of such compositions and methods for maintaining or improving respiratory health, the compositions being used, for example, to maintain or improve the respiratory health of animals by reducing the incidence and / or severity of respiratory diseases in animals receiving the compositions.

[0044] As stated above, bovine respiratory diseases (BRD) are the single largest health problem facing the fattening industry, resulting in significant economic and production losses. Treatment of respiratory diseases is difficult and inadequate, relying primarily on antibiotic administration and careful management of livestock production facilities. The ability to reduce the incidence and / or severity of respiratory diseases in animals would be of immense value to producers, and improvements in animal welfare and feed quality through improved respiratory health are highly desirable. Therefore, this disclosure enables these beneficial effects by allowing easy administration of the compositions disclosed herein without the need for the administration of antibiotics and similar substances to animals.

[0045] This disclosure includes a method for maintaining or improving the respiratory health of an animal, the method comprising administering to the animal a composition comprising at least one of two different probiotic strains of the genus Bacillus, and the composition providing improved respiratory health compared to a similar animal not administered the composition.

[0046] This disclosure includes a method for inhibiting the incidence and / or severity of respiratory diseases in animals, the method comprising administering to the animals a composition that inhibits the development of clinical signs of a disease associated with the respiratory disease, the composition comprising at least one of two different probiotic strains.

[0047] Respiratory diseases Respiratory infections (such as pneumonia) can occur due to a combination of one or more factors, including animal stress, exposure to high levels of infectious agents, and / or impaired animal immune function. The initial problem in many pneumonia cases is thought to be a sudden change in the normal nasal bacterial flora, leading to a sudden and dramatic increase in one or more bacterial species. Bacterial proliferation is often caused by the collapse of host defense mechanisms due to stress (e.g., transport, concurrent disease) or cellular damage (e.g., viral infection, virulence). These bacteria are inhaled in large quantities into the lungs and may overwhelm normal defense mechanisms, colonizing, proliferating locally, and triggering inflammation.

[0048] Furthermore, stress is often a precursor to viral and / or bacterial respiratory infections, especially in animal populations that have recently been stressed due to transport, handling, and mixing. Pneumonia can also be caused by direct infection with viruses, bacteria, and fungi, as well as by toxins acquired through bloodstream routes, inhalation or aspiration of food or gastric contents.

[0049] Pneumonia is a major cause of loss in the cattle industry worldwide, and hemolytic Mansonia is a key respiratory pathogen in this disease. Hemolytic Mansonia belongs to the Pasteurellaceae family and is an inactive, non-spore-forming, facultative anaerobic, oxidase-positive, and fermentative Gram-negative bacillus or cocci.

[0050] In one embodiment of this disclosure, the respiratory disease to be prevented and / or minimized is pneumonia.

[0051] Bovine respiratory disease (BRD) is the most common and economically damaging disease affecting beef cattle worldwide. It is a complex bacterial or viral infection that causes potentially fatal pneumonia in calves. The infection is usually the sum of three interdependent factors: stress, underlying viral infection, and new bacterial infection. Diagnosis of this disease is complex because there are multiple possible causes. One approach to diagnosing BRD is to follow the University of Wisconsin scoring system, as illustrated in the example.

[0052] The disease can manifest at any point in an animal's life, but it is most common in calves within four weeks of weaning, when they are sorted and often sold to different farms; BRD is commonly known as "shipping fever." It is unclear whether stress itself, group mixing, or transport conditions are the primary triggers, and while studies have identified general stressors such as transport and cold weather conditions, there is still no conclusive evidence for more specific factors (e.g., distance, transport pattern, temperature, or temperature fluctuations). In this context, stress can be triggered by many different events and conditions. Removing calves from their mothers is a known stressor, as is isolating them. High (or relatively high) pathogen loads are also known stressors, such as those present on production farms.

[0053] Therefore, BRD is a "multifactorial syndrome" that depends on many different causes. The pathological condition usually occurs when the pathogenic organism colonizes the body through a secondary infection following a primary bacterial or viral infection, which may occur after stress (e.g., due to handling or transport).

[0054] The most commonly associated bacterial agents with BRD are *Mannella hemolyticus*, *Pasteurella multocida*, *Histomostomyces hirsutus*, 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 compositions that inhibit one or more of these bacterial agents (e.g., *Mannella hemolyticus*) would be advantageous in reducing the incidence and / or severity of BRD. One method for evaluating inhibitory activity against *Mannella hemolyticus* can be seen in the examples disclosed herein.

[0055] The most prominent sign of pneumonia caused by BRD is depression, manifested as drooping ears, a blank stare, and solitary behavior. In addition, most cows will develop a fever above 104°F (40°C). Other symptoms include coughing, decreased appetite, and difficulty breathing.

[0056] Because of the numerous possible viral / bacterial precursors to BRD, there are many treatment options surrounding the following three main aggravating factors of the disease: viral, bacterial, and stress.

[0057] Vaccinations exist for several biological precursors of BRD, but the large number of possible precursors complicates the process of selecting a vaccination regimen. Furthermore, vaccines are not entirely effective in preventing disease, but only in alleviating it. Many questions regarding vaccine effectiveness relate to misuse, such as failure to administer the vaccine dose at the proper time or failure to administer the vaccine before transport (because stress during animal handling and treatment can reduce vaccine efficacy and immune protection against respiratory pathogens).

[0058] In the absence of vaccination (often because calves are purchased unvaccinated), antibiotics can help prevent the bacterial factors that cause disease. The phase-out of antibiotic use in the EU and the US has led to increased demand for cost-effective alternatives to combat pathogens that have significant impacts on both humans and veterinary medicine.

[0059] Stress often acts as a final precursor to BRD. The disease that constitutes BRD can persist in a herd for a long time before symptoms appear, but a weakened immune system due to stress may stop controlling the disease. There are many causes of stress, and one of the main sources is transportation and mixing of cattle, but weather may also be another possible factor.

[0060] In one embodiment of this disclosure, the respiratory disease with reduced incidence and / or severity is a bovine respiratory disease.

[0061] Probiotic bacterial strains The probiotic strains disclosed herein are isolated, i.e., exist in forms or environments not found in nature, and have surprising effects when used in compositions as described herein.

[0062] In one embodiment of this disclosure, the probiotic strain in the composition is Bacillus paralicheniformis (B. paralicheniformis). Bacillus paralicheniformis ) and optionally Bacillus subtilis. In one embodiment of this disclosure, the probiotic strain in the composition is Bacillus subtilis and optionally Bacillus licheniformis. In one embodiment of this disclosure, the composition further comprises other probiotic strains, such as Lactobacillus animalis ( Lactobacillus animalis ) and / or Propionibacterium fischeri ( Propionibacterium freudenreichii ).

[0063] In one embodiment of this disclosure, the composition comprising a probiotic strain is used to provide a total of 1 × 10 8 With 1 × 10 11 Between CFU / animal / day, for example, in 1 × 10 8 With 1 × 10 10 Between CFU / animal / day, for example, in 1×10 9 With 1 × 10 10 Between CFU / animal / day, for example, in 5 × 10 9 With 5 × 10 10 The amount of CFU / animal / day is administered to the animal.

[0064] In a preferred embodiment of this disclosure, the composition comprising a probiotic strain is used to provide a total of 6 × 10 9 With 8 × 10 9 CFU / animal / day, for example, approximately 6 × 10 9 CFU / animal / day, e.g., approximately 7 × 10 9 CFU / animal / day, e.g., approximately 8 × 10 9 The animal was given a dose of CFU / animal / day.

[0065] The term "CFU / animal / day" refers to the amount of probiotic strain administered to each animal per day. Therefore, this term excludes carriers (e.g., calcium carbonate), anti-caking agents (e.g., aluminum silicate and diatomaceous earth), and other components that may optionally be present in the composition.

[0066] The compositions disclosed herein comprise at least one species of the probiotic strain disclosed herein and at least one carrier and / or other components that make the compositions suitable for feeding animals or as an additive to drinking water or milk / milk substitutes.

[0067] In one embodiment of this disclosure, the composition comprises more than one probiotic strain, with equal portions of Bacillus subtilis strain and Bacillus licheniformis strain present.

[0068] In another embodiment of this disclosure, the composition comprises more than one probiotic strain, with unequal portions of Bacillus subtilis strain and Bacillus licheniformis strain.

[0069] In one embodiment of this disclosure, the composition comprises Bacillus subtilis deposited as DSM 32324 or Bacillus subtilis deposited as DSM 17231.

[0070] In one embodiment of this disclosure, the Bacillus subtilis strain present in the composition is a strain deposited as DSM17231.

[0071] In one embodiment of this disclosure, the Bacillus licheniformis strain present in the composition is a strain deposited as DSM17236.

[0072] In a preferred embodiment disclosed herein, the composition comprises a Bacillus subtilis strain deposited as DSM 17231 and a Bacillus licheniformis strain deposited as DSM 17236.

[0073] In one embodiment of this disclosure, the composition comprises a Bacillus subtilis strain preserved as DSM 32324.

[0074] In one embodiment, the composition comprises a Bacillus subtilis strain deposited as DSM 32324 and a Bacillus licheniformis strain deposited as DSM 17236.

[0075] In one embodiment of this disclosure, the composition comprises two different probiotic strains, namely Bacillus subtilis preserved as DSM17231 and Bacillus licheniformis preserved as DSM 17236, optionally in a ratio of 1:1 in terms of colony forming units (CFU).

[0076] In one embodiment of this disclosure, the composition comprises two different probiotic strains: Bacillus subtilis preserved as DSM17231 and Bacillus licheniformis preserved as DSM 17236, optionally in a 1:1 ratio of colony-forming units (CFU). In such an embodiment, the composition does not contain any other microbial components, but may contain other components, such as one or more carriers and / or one or more feed ingredients.

[0077] In one embodiment, the microbial component of the compositions, methods, or uses disclosed herein consists of a Bacillus subtilis strain deposited as DSM 32324 and a Bacillus licheniformis strain deposited as DSM 17236.

[0078] The probiotic strains of the compositions disclosed herein can be provided in the form of spores and / or bacterial cells. In one embodiment of this disclosure, Bacillus strains (Bacillus subtilis and Bacillus licheniformis) are provided in the form of spores.

[0079] In one embodiment of this disclosure, the composition comprises a certain amount of Bacillus subtilis strain DSM32324 or DSM 17231 to provide at 0.15 × 10 8 With 0.15 × 10 11 Bacillus subtilis strains DSM 32324 or DSM 17231 with a CFU / day ratio, preferably provided at 1 × 10⁻⁶ CFU / day. 9 With 1 × 10 10 Bacillus subtilis strain DSM 32324 or DSM 17231 with a CFU / day. In one embodiment, the composition disclosed herein comprises a certain amount of Bacillus subtilis strain DSM 32324 or DSM 17231 to provide approximately 1 × 10⁻⁶ CFU / day. 9 Bacillus subtilis strains DSM 32324 or DSM 17231, with a CFU / day.

[0080] In one embodiment of this disclosure, the composition comprises an amount of Bacillus licheniformis strain DSM17236 to provide a concentration of 0.15 × 10⁻⁶. 8 With 0.15 × 10 11 Bacillus licheniformis strain DSM 17236, preferably provided at 1 × 10⁻⁶ CFU / day. 9 With 1 × 10 10 CFU / day of Bacillus licheniformis strain DSM 17236. In one embodiment, the composition disclosed herein comprises a certain amount of Bacillus licheniformis strain DSM 17236 to provide approximately 1 × 10 9Bacillus licheniformis strain DSM 17236, CFU / day.

[0081] In one embodiment of this disclosure, the composition comprises a certain amount of probiotic strains to provide approximately 1 × 10⁻⁶ bacteria. 9 CFU / day of Bacillus subtilis preserved as DSM 17231 and approximately 1 × 10⁻⁶ CFU / day 9 Bacillus licheniformis preserved as DSM 17236 CFU / day.

[0082] In one embodiment of this disclosure, the microbial component of the composition consists of a certain amount of probiotic strains to provide approximately 1 × 10⁻⁶. 9 CFU / day of Bacillus subtilis preserved as DSM 17231 and approximately 1 × 10⁻⁶ CFU / day 9 CFU / day of Bacillus licheniformis preserved under DSM 17236. In such embodiments, the composition does not contain any other microbial components, but may contain other components, such as one or more carriers and / or one or more feed ingredients.

[0083] Composition application form and formulation The probiotic strains present in the compositions disclosed herein are provided in commercially relevant forms known to those skilled in the art. Therefore, in the embodiments, the probiotic strains of the composition are present in a dried (e.g., spray-dried or freeze-dried) or frozen form. Different probiotic strains present in the compositions disclosed herein may be present in different forms in the composition; for example, some may be spray-dried and some may be freeze-dried. The composition may be provided in any suitable form, such as in a liquid (e.g., gel, slurry, etc.) or in a solid (e.g., powder or pellet) form.

[0084] For compositions in premix form, the probiotic strains disclosed herein can be added to a carrier to prepare a mineral-vitamin mixture (premix), which can then be added to animal feed at the desired inclusion rate.

[0085] Alternatively, for compositions in the form of animal feed, the probiotic strains disclosed herein can be formulated together with animal feed ingredients, as shown below. Such combinations of the disclosed compositions and animal feed ingredients may optionally be in the form of pellets extruded by a standard pelleting process.

[0086] This disclosure also provides a method for producing animal feed, animal feed additives or premixes, the method comprising adding the probiotic strains disclosed herein to the animal feed or related components thereof.

[0087] The compositions of the present invention can be added to silage: “Silage” refers to a fermented, high-moisture stored feed that 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 known as ensilage, ensiling, or silaging, and is typically made from whole green plants (not just cereals) of grass or cereal crops (such as corn, sorghum, oats, rye, timothy, or other forage plants) or legume crops (such as clover, trefoils, alfalfa, and peas). Silage can be made from many field crops, and specific terms may be used depending on the type (oatlage for oats, haylage for alfalfa). Silage is made by placing cut green vegetation in a silage pit, by piling it in a large heap covered with plastic sheets, or by wrapping large bales in plastic film.

[0088] In one embodiment of this disclosure, the composition comprising a probiotic strain is used to provide a total of 1 × 10 5 With 1 × 10 11 Between CFU / g feed, for example at 1 × 10 6 With 1 × 10 10 Between CFU / g feed, for example at 1 × 10 7 With 1 × 10 9 Between CFU / g feed, for example at 5 × 10 6 With 5 × 10 10 The animals were given an amount between CFU / g of feed.

[0089] In one embodiment, this disclosure provides an animal feed, animal feed additive, or premix that contains the probiotic strain disclosed herein and further contains one or more concentrates, vitamins, minerals, enzymes, amino acids, and / or other feed ingredients.

[0090] In one embodiment, the animal feed, animal feed additive, or premix comprises the composition disclosed herein (the composition comprises Bacillus subtilis deposited as DSM 32324 or DSM 17231 and optionally Bacillus licheniformis deposited as DSM17236), and further comprises one or more concentrates, vitamins, minerals, enzymes, amino acids, and / or other feed ingredients.

[0091] In a preferred embodiment, the animal feed, animal feed additive, or premix comprises the composition disclosed herein (the composition comprises Bacillus subtilis deposited as DSM 17231 and Bacillus licheniformis deposited as DSM 17236), and further comprises one or more concentrates, vitamins, minerals, enzymes, amino acids, and / or other feed ingredients.

[0092] In a preferred embodiment, the animal feed, animal feed additive, or premix comprises the composition disclosed herein (comprising Bacillus subtilis deposited as DSM 17231 and Bacillus licheniformis deposited as DSM 17236), and further comprises one or more concentrates, vitamins, minerals, enzymes, amino acids, and / or other feed ingredients.

[0093] In a specific embodiment, the animal feed is a total mixed ration (TMR) comprising 0% to 80% forage sources (e.g., hay, silage, or forage). Typically, forage includes the edible parts of plants (in addition to isolated grains) that can provide feed for the animals or can be harvested for animal feeding. As an example, a TMR may comprise 0% to 80% corn; and / or 0% to 80% sorghum; and / or 0% to 70% wheat; and / or 0% to 70% barley; and / or 0% to 30% oats; and / or 0% to 40% soybean meal.

[0094] In one embodiment, the composition disclosed herein containing probiotic strains is mixed with forage, concentrates, and optionally other feed components to obtain TMR.

[0095] In another embodiment, the composition of this disclosure containing probiotic strains is mixed with concentrates, vitamins, and / or minerals to obtain a premix. This premix can be mixed with a final diet to obtain a TMR. In another embodiment, the TMR and the composition of this disclosure containing probiotic strains are mixed with one or more enzymes. In another embodiment, the composition of this disclosure containing probiotic strains is mixed with one or more other feed ingredients, such as colorants, stabilizers, growth-improving additives and flavor compounds / flavorings, saturated or polyunsaturated fatty acids (PUFAs), essential oils, antioxidants, antimicrobial peptides, antifungal peptides, and amino acids.

[0096] In a particular embodiment, the animal feed component consists of or contains milk (e.g., from dairy cows, goats, or sheep), for example, for feeding calves. In another particular embodiment, the animal feed component consists of or contains milk substitutes, for example, for feeding calves. In one embodiment, the composition of this disclosure containing probiotic strains is mixed with water, milk, or a milk replacer for feeding calves, piglets, or other weaned animals.

[0097] In another embodiment, the animal feed component may contain 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 component may optionally contain one or more minerals, such as one or more trace minerals and / or one or more macro minerals. Typically, fat-soluble vitamins and water-soluble vitamins, along with trace minerals, form part of a so-called premix intended to be added to the feed, while macro minerals are typically added separately to the feed. Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3. Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and pantothenate, such as Ca-D-pantothenate. Non-limiting examples of trace 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.

[0098] The animal feeds, animal feed additives, or premixes disclosed herein may also contain at least one enzyme selected from the group consisting of: phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); α-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 α-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); cellulase (EC 3.2.1.4); and β-glucanase (EC 3.1.1.17). 3.2.1.6), or any mixture thereof.

[0099] The animal feeds, animal feed additives, or premixes disclosed herein may further contain one or more added amino acids. Examples of amino acids used in animal feeds are rumen-protected or non-rumen-protected lysine, alanine, β-alanine, threonine, methionine, and tryptophan. The animal feeds, animal feed additives, or premixes disclosed herein may further contain colorants, stabilizers, growth-improving additives, and flavor compounds / flavorings, polyunsaturated fatty acids (PUFAs), essential oils, antioxidants, antimicrobial peptides, and antifungal peptides. Examples of colorants are carotenoids, such as β-carotene, astaxanthin, and lutein. Examples of flavor compounds / flavorings are methoxycresol, anethole, decadecyl, undecyl, and / or dodecadecyl lactones, ionone, irisone, gingerol, piperidine, propylidene phthalide, butylidene phthalide, capsaicin, and tannins. Examples of saturated fatty acids are C16 and C18, such as palmitic acid and oleic acid, while polyunsaturated fatty acids are C18, C20, and C22 polyunsaturated fatty acids, such as linoleic acid, linolenic acid, arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, and γ-linolenic acid. Examples of reactive oxygen species (ROS) generators are chemicals such as perborates, persulfates, or percarbonates; and enzymes such as oxidases, oxygenases, or synthases.

[0100] In one embodiment, the animal feed, animal feed additive, or premix contains one or more anticoccidial agents.

[0101] In one embodiment, the animal feed, animal feed additive, or premix further comprises a carrier. The carrier may comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1,2-propanediol or 1,3-propanediol, 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.

[0102] In embodiments, the animal feed, animal feed additive, or premix further comprises one or more additional microorganisms. In specific embodiments, the animal feed, animal feed additive, or premix further comprises bacteria from one or more genera including Lactobacillus. Lactobacillus Lactococcus spp. Lactococcus Streptococcus ( Streptococcus ), Bacillus spp. ( Bacillus ), Pediococcus ( Pediococcus ), Enterococcus spp. Enterococcus Leuconostoc ( ) Leuconostoc ), Clostridium carnivorum ( Carnobacterium ), Propionibacterium spp. PropionibacteriumBifidobacterium spp. Bifidobacterium Clostridium ( Clostridium ) and the genus *Macrococcus* ( Megasphaera (or any combination thereof).

[0103] In one embodiment, the animal feed, animal feed additive, or premix further comprises bacteria from one or more strains of Bacillus amyloliquefaciens (BAM). Bacillus amyloliquefaciens ), Bacillus subtilis, Bacillus pumilus ( Bacillus pumilus ), Polymyxin Bacillus ( Bacillus polymyxa ), Bacillus licheniformis, Bacillus megaterium ( Bacillus megaterium Bacillus coagulans ( Bacillus coagulans ), Bacillus circularis ( Bacillus circulans ), Bacillus simplex ( Bacillus simplex Mojave Bacillus ( Bacillus mojavensis ), Bacillus safortiformis ( Bacillus safensis ), Bacillus simplex, Bacillus atrophus ( Bacillus atrophaeus ), Methyltrophic Bacillus ( Bacillus methylotrophicus ), Bacillus sicca ( Bacillus siamensis ), Bacillus cereus ( Bacillus vallismortis ), Bacillus tekiria ( Bacillus tequilensis (or any combination thereof).

[0104] In one embodiment, the animal feed, animal feed additive, or premix contains strains of Bacillus subtilis, Bacillus licheniformis, Lactobacillus animalis, and Propionibacterium fischeri.

[0105] In one embodiment, the animal feed, animal feed additive, or premix further comprises one or more types of yeast. The one or more types of yeast may be selected from the group consisting of: Saccharidae (…). Saccharomycetaceae ), Yeast ( Saccharomyces (such as brewer's yeast) S. cerevisiae ) and / or Bula yeast ( S. boulardii Kluyveromyces ( )), Kluyveromyces ( Kluyveromyces (such as Max Kluyveromycin) K. marxianus ) and Kluyveromycin ( K. lactis )), Candida genus ( Candida (such as Candida utilis) C. utilis ), also known as the genus *Saccharomyces* ( Torula ) yeast), Pichia pastoris ( Pichia (such as Pichia pastoris) P. pastoris ), and the genus *Cytomyces* ( Torulaspora(e.g., Delphireco spores) T. delbrueckii ), Phaefoetida ( Phaffia Yeasts and Basidiomycetes ( Basidiomycota ).

[0106] The compositions disclosed herein may additionally comprise cryoprotectants, lyophilization protectants, antioxidants, nutrients, fillers, flavorings, or mixtures thereof. The compositions may be in freeze-dried or lyophilized form. Preferably, the compositions comprise one or more of cryoprotectants, lyophilization protectants, antioxidants, and / or nutrients; more preferably, they comprise cryoprotectants, lyophilization protectants, and / or antioxidants; and most preferably, they comprise either a cryoprotectant or a lyophilization protectant, or both. The uses of protectants such as cryoprotectants and lyophilization protectants are known to those skilled in the art. Suitable cryoprotectants or lyophilization protectants include monosaccharides, disaccharides, trisaccharides, and polysaccharides (e.g., glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch, and gum arabic), polyols (e.g., erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol), amino acids (e.g., proline, glutamic acid), complex substances (e.g., skim milk, peptone, gelatin, yeast extract), and inorganic compounds (e.g., sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and its salts, gallate, cysteine, sorbitol, mannitol, and maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, and vitamins (e.g., B vitamins, vitamin C). The composition may optionally contain additional substances, including fillers (e.g., lactose, maltodextrin), and / or flavoring agents.

[0107] Animal diets can be formulated as, for example, powdered (non-pelletized) or pelleted feed. Typically, the milled feed is mixed and supplemented with adequate amounts of essential vitamins and minerals according to the instructions for the type discussed. Bacterial cultures and, optionally, enzymes can be added as solid or liquid formulations. For example, for powdered feed, solid or liquid culture formulations can be added before or during the ingredient mixing step. For pelleted feed, the (liquid or solid) composition of this disclosure containing probiotic strains can be added to the pelleted food after the pelleting step. Typically, the liquid composition of this disclosure contains probiotic strains, optionally accompanied by polyols (e.g., glycerol, ethylene glycol, or propylene glycol), and is added after the pelleting step, for example, by spraying the liquid formulation onto pellets.

[0108] Another aspect of this disclosure relates to a method for feeding animals, the method comprising administering to an animal (e.g., a monogastric animal or a ruminant) a composition comprising a probiotic strain of the present disclosure. The term "animal" means all animals other than humans. Examples of animals are non-ruminants and ruminants. Ruminants include, for example, animals such as sheep, goats, cattle (e.g., beef cattle, dairy cattle, and young calves), deer, yaks, camels, llamas, and kangaroos. Non-ruminants include monogastric animals such as 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 broilers and laying hens); horses (including but not limited to hot-blooded horses, cold-blooded horses, and warm-blooded horses); and young calves.

[0109] Ruminants include dairy cows, cattle, sheep, deer, and goats. Monogastric animals include poultry, pigs, and pets (cats and dogs).

[0110] Ruminants have digestive systems that are fundamentally different from those of monogastric animals, and therefore it cannot be concluded that compositions designed for monogastric animals and proven effective in monogastric animals will also be suitable for and effective in ruminants. Therefore, providing compositions that are effective in both monogastric and ruminant animals is not straightforward.

[0111] An animal's digestive system is involved in the mechanical and chemical digestion of food, the absorption of nutrients, and the elimination of indigestible materials from the body. The main difference between a monogastric digestive system and a ruminant digestive system is that digestion in a monogastric system primarily occurs in the stomach, while digestion in a ruminant system is foregut fermentation. A monogastric digestive system consists of a single stomach, while a ruminant digestive system consists of four stomachs (reticulum, rumen, omasum, and abomasum). Monogastric digestion is primarily found in omnivores and carnivores, while ruminants are herbivores.

[0112] A monogastric digestive system is an organ system that helps digest both animal and plant materials. This digestive system is called monogastric because it consists of a single stomach. Humans, horses, pigs, poultry, dogs, cats, birds, and rabbits have monogastric digestive systems. Digestion begins when feed enters the mouth. Both chemical and mechanical digestion begin in the mouth. Saliva contains enzymes to digest carbohydrates. The esophagus is the passageway that carries feed to the stomach. Various enzymes are secreted into the stomach cavity to digest proteins in the feed. Animals with monogastric digestive systems primarily feed on animal tissues. Their diet is easily digestible; therefore, a single stomach is sufficient for this purpose. The small intestine primarily absorbs nutrients from digested feed. The large intestine absorbs water from poorly digestible materials.

[0113] Ruminants are mammals that primarily digest plant-based foods through bacterial action, first fermenting / degrading them in the first compartment of the stomach, then ruminating on the semi-digested clump (now called "ruminant food (cud)") and chewing it again. This process of further chewing ruminant food to break down plant matter and stimulate digestion is called "rumination." Examples of ruminants include cattle, dairy cows, beef cattle, young calves, goats, sheep, lambs, deer, yaks, camels, and llamas. The ruminant digestive system refers to the organ system in which the digestion of plant material occurs. Cattle, sheep, deer, and goats are examples of animals with a ruminant digestive system. Ruminants lack teeth in the front of their upper jaw, instead possessing a hard pad of skin called a dentate pad. In addition to the basic anatomy of the animal's digestive system, the ruminant digestive system consists of four stomachs: the rumen, reticulum, omasum, and abomasum. The first three stomachs (rumen, reticulum, and omasum) are involved in the breakdown of plant fibers and the digestion of non-fibrous compounds. The microbiome is involved in this process. The microbiome breaks down cellulose and starch, for example, through fermentation, producing volatile fatty acids such as acetic acid, butyric acid, and propionic acid. These volatile fatty acids are used as an energy source by ruminants. Digestive enzymes are secreted in the fourth stomach, called the abomasum. Therefore, in ruminants, fermentation occurs before feed digestion. This process is thus called foregut fermentation. Furthermore, ruminants chew these partially digested or regurgitated foods by returning them from the first stomach. The small and large intestines of ruminants resemble a monogastric digestive system. However, ruminants also contain a large cecum for further digestion of fibers.

[0114] In a preferred embodiment of this disclosure, the composition is used in a method for maintaining or improving the respiratory health of ruminants or reducing the incidence and / or severity of respiratory diseases.

[0115] In another embodiment of this disclosure, the composition of this disclosure is used in a method for maintaining or improving the respiratory health of a monogastric animal or reducing the incidence and / or severity of respiratory diseases.

[0116] As demonstrated in the examples, the compositions disclosed herein inhibit pathogens associated with respiratory diseases and affect the development of lung lesions, and administration of the compositions disclosed herein containing the probiotic strains described herein reduces the incidence and / or severity of respiratory diseases in animals compared to controls.

[0117] "Reducing the incidence and / or severity of respiratory diseases" means that the use of the compositions disclosed herein in animal feeding will result in an improvement in the respiratory health of animals to which the compositions disclosed herein are administered, thereby reducing the number of observed lung lesions and / or the number of signs of respiratory diseases, as determined by, for example, the University of Wisconsin scoring system (see Example 1).

[0118] In one embodiment, the reduction in the number of lung lesions results in a reduction of 3.0% or more, for example, more than 3.5%, for example, more than 4.0%, for example, more than 4.5%, for example, more than 5.0%, for example, more than 5.5%.

[0119] In one embodiment, the reduction in the number of lung lesions results in a reduction between 3.0% and 5.5%, for example, between 3.0% and 5.0%, for example, between 3.0% and 4.5%, for example, between 3.0% and 4.0%, for example, between 3.0% and 3.5%.

[0120] In one embodiment, a reduction in the number of signs of respiratory disease, as determined by, for example, the University of Wisconsin scoring system, results in a reduction of 10% or more, such as more than 15%, more than 20%, more than 30%, more than 40%, or 50%.

[0121] In one embodiment, a reduction in the number of signs of respiratory disease, as determined by, for example, the University of Wisconsin scoring system, results in a reduction between 10% and 50%, such as between 10% and 40%, such as between 10% and 30%, such as between 10% and 20%.

[0122] Taxonomy Bacillus licheniformis is now known as Bacillus paralicheniformis, as described in Dunlap et al., 2015 (International Journal of Systematic and Evolutionary Microbiology, 65, 3487–3492). These two different names are used interchangeably in this article.

[0123] Preservation and expert solutions The applicant requests that, prior to the patent grant date, only the following deposited microbial samples be provided to experts, subject to the current regulations of the industrial property offices of the contracting parties to the Budapest Treaty.

[0124] Table 1: The applicant has made the following deposits with depositary institutions that have obtained international depositary status, in accordance with the Budapest Convention on the international recognition of the preservation of microorganisms for patent proceedings: Leibniz Institute DSMZ - German Microbiology and Cell Culture Cultural Relics Preservation Center 7B Inhoffenstr., 38124, Braunschweig, Germany.

[0125] The invention is further defined by the following numbered paragraphs:

[0126] 1. A composition comprising at least one strain of Bacillus subtilis and optionally at least one strain of Bacillus licheniformis, the composition being used to reduce the incidence and / or severity of respiratory diseases in animals.

[0127] 2. A composition comprising at least one strain of Bacillus subtilis and optionally at least one strain of Bacillus licheniformis, the composition being used for the prevention of respiratory diseases in animals.

[0128] 3. A composition comprising at least one Bacillus subtilis strain and optionally at least one Bacillus licheniformis strain, the composition being used for maintaining or improving the respiratory health of animals.

[0129] 4. The composition as described in any one of paragraphs 1-3, wherein the Bacillus subtilis strain is selected from strains deposited as DSM17231 and DSM 32324.

[0130] 5. The composition as described in any one of paragraphs 1-3, wherein the Bacillus licheniformis strain is a strain deposited as DSM17236.

[0131] 6. The composition according to any of the preceding paragraphs, wherein the composition comprises strains preserved as DSM 17231 and DSM17236.

[0132] 7. The composition of any one of paragraphs 1-3, wherein the microbial component of the composition comprises Bacillus subtilis deposited as DSM17231 and Bacillus licheniformis deposited as DSM 17236.

[0133] 8. The composition according to any of the preceding paragraphs, wherein the respiratory disease is selected from the group consisting of pneumonia and bovine respiratory diseases.

[0134] 9. The composition according to any of the preceding paragraphs, wherein the animal is a ruminant and the respiratory disease is a bovine respiratory disease.

[0135] 10. A method for producing animal feed, animal feed additives or premixes, the method comprising adding the composition as described in any one of paragraphs 1 to 9 to the animal feed, animal feed additives or premixes or related components thereof.

[0136] 11. The method as described in paragraph 10, wherein the animal feed, animal feed additive or premix is ​​used for animals selected from the group consisting of: poultry, herbivores, ruminants, pigs, rodents, pets, fish and crustaceans, preferably wherein the animal is a ruminant, such as a dairy cow.

[0137] 12. An animal feed, animal feed additive or premix comprising a composition as described in any one of paragraphs 1 to 9, and further comprising one or more concentrates, vitamins, minerals, enzymes, amino acids and / or other feed ingredients.

[0138] 13. Animal feed, animal feed additive or premix as described in paragraph 12, wherein the composition comprises Bacillus subtilis preserved as DSM 17231 and Bacillus licheniformis preserved as DSM 17236.

[0139] 14. A method for reducing the incidence and / or severity of respiratory diseases in animals or for preventing respiratory diseases in animals, the method comprising administering to the animal a composition for reducing the incidence and / or severity of clinical signs of a disease associated with the respiratory disease, the composition comprising at least one Bacillus subtilis strain and optionally at least one Bacillus licheniformis strain, wherein the composition is in animal feed, animal feed additives, or premixes to provide a concentration of 1 x 10 8 With 1 x 10 11 The total amount of the probiotic strain administered to the animal is between CFU / animal / day.

[0140] 15. The method as described in paragraph 14, wherein the animal feed, animal feed additive, or premix is ​​the animal feed, animal feed additive, or premix as described in paragraph 12 or 13.

[0141] 16. The method as described in paragraph 14 or 15, further comprising evaluating the effect of the application of the composition on reducing pathogens.

[0142] 17. The method described in paragraph 16, wherein the pathogen to be reduced is hemolytic Mansonia.

[0143] 18. A method for 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 an animal a composition as described in any one of paragraphs 1 to 9, or an animal feed, animal feed additive or premix as described in paragraphs 12 or 13.

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

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

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

[0147] 22. The method as described in paragraph 21, wherein the microbial component of the composition comprises a Bacillus subtilis strain preserved as DSM 17231 and a Bacillus licheniformis strain preserved as DSM 17236.

[0148] 23. The use of a composition comprising at least one strain of Bacillus subtilis and optionally at least one strain of Bacillus licheniformis for the prevention of respiratory diseases in animals or for reducing the incidence and / or severity of respiratory diseases in animals.

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

[0150] 25. Uses as described in paragraph 23 or 24, wherein the animal is a ruminant.

[0151] 26. The use as described in any one of paragraphs 23-25, wherein the Bacillus subtilis strain is selected from strains deposited as DSM17231 and as DSM 32324.

[0152] 27. The use as described in any one of paragraphs 23-25, wherein the Bacillus licheniformis strain is a strain deposited as DSM17236.

[0153] 28. Use as described in any one of paragraphs 23-27, wherein the composition comprises strains preserved as DSM 17231 and DSM 17236.

[0154] 29. The use as described in any one of paragraphs 23-25, wherein the microbial component of the composition comprises a Bacillus subtilis strain deposited as DSM17231 and a Bacillus licheniformis strain deposited as DSM 17236.

[0155] 30. The use as described in any one of paragraphs 23-29, wherein the respiratory disease is selected from the group consisting of pneumonia and bovine respiratory diseases.

[0156] 31. The use as described in any one of paragraphs 23-30, wherein the animal is a ruminant and the respiratory disease is a bovine respiratory disease.

[0157] 32. Use as described in any one of paragraphs 23-31, for the prevention or reduction of the incidence and / or severity of respiratory diseases caused by hemolytic Mansicae.

[0158] 33. The use of a composition comprising a Bacillus subtilis strain deposited as DSM 17231 and a Bacillus licheniformis strain deposited as DSM 17236, or an animal feed, animal feed additive or premix comprising said composition for the purpose of maintaining or improving the respiratory health of an animal, wherein the animal is a ruminant.

[0159] 34. A method for maintaining or improving the respiratory health of an animal, wherein the animal is a ruminant, the method comprising administering to the animal a composition comprising a Bacillus subtilis strain preserved as DSM 17231 and a Bacillus licheniformis strain preserved as DSM 17236, or an animal feed, animal feed additive or premix comprising said composition.

[0160] Example Example 1 Bacillus licheniformis DSM 17236 was inoculated into brain and heart broth and incubated aerobically at 37°C for 48 h.

[0161] Hemolytic Mansonia solani strains were inoculated onto tryptone soy agar and incubated aerobically at 37°C for 24 h. On the day of assay, the hemolytic Mansonia solani culture was resuspended in maximum recovery dilution medium using a swab with 0.5 μL McFarland suspension. Following this step, 35 mL of molten LB agar and 10 μL of the pathogen suspension were mixed in a 50 mL Falcon tube and poured into Omnitray plates, immediately covered with Nunc™ Immuno TSP (Thermo Fisher Scientific, Waltham, MA). The plates were then allowed to solidify for 20 min, after which the caps were removed. The plates were then allowed to dry for another 20 min with the caps on. Finally, 5 µL of the overnight strain culture was applied to selected wells in the agar.

[0162] Three copies of the analysis sample were prepared. After aerobic incubation at 30°C for 48 h, the inhibition zone from complete growth to complete growth was measured using Photoshop, with a lower limit of 3.5 mm (width of each well in the plate). The inhibition zone (in mm) was measured 48 h after adding Bacillus licheniformis to the agar plate.

[0163] In this example, the addition of Bacillus licheniformis DSM 17236 to hemolytic Mansonia produced a 10.4 mm inhibition zone (see [link to original text]). Figure 1 These results support the efficacy of probiotic strains in directly inhibiting this important pathogen associated with bovine respiratory diseases.

[0164] Example 2 The role of probiotic supplementation in the health and production performance of pre-weaning milk calves In vivo studies were conducted to evaluate the effects of feeding a combination of probiotic strains, Bacillus subtilis (DSM 17231) and Bacillus licheniformis (DSM 17236), on the health and production performance of pre-weaning Gyr × Holstein dairy calves. The experiment was conducted at a commercial dairy farm in Passos, Minas Gerais, Brazil.

[0165] In this study, 60 newborn calves were included after ingesting colostrum (approximately 24 hours after birth) and were designated as: (1) whole milk without probiotics ( n = 30) and (2) The probiotic combination was added at a dose of 1 g / head / day of whole milk (3.2 × 10 9 CFU / head / day; n = 30). Treatment is provided daily for 77 days.

[0166] The milk feeding program was as follows: from day 1 to day 42 of the study, 6 L / calf / day was given twice daily; from day 43 to weaning (day 77), 3 L / calf / day was given once daily. From day 1 of birth to weaning, a concentrated feed containing cornmeal (40.0%), soybean meal (35.2%), dried distillers grains (17.1%), premix (6.8%), and urea (0.9%) was provided, as shown in Table 2 below. Table 2: Daily feed amount

[0167] Throughout the experiment, assess the animals for any signs of adverse health events, including evaluating pneumonia according to the Calf Health Scorer Scale from the University of Wisconsin (https: / / www.vetmed.wisc.edu / fapm / svm-dairy-apps / calf-health-scorer-chs / ), such as... Figure 2 As shown. The calves in this study were subjected to different stress triggers, such as early removal from their mothers, solitary placement, and exposure to high pathogen loads.

[0168] In this example, by feeding a combination of probiotic strains, the incidence of pneumonia decreased from 46.7 (n = 14 calves) to 23.0 (n = 7 calves). P = 0.05; Figure 3 ).

[0169] Although the mean age of onset (P = 0.76) and the number of days of pharmacological intervention were similar (P = 0.76), P = 1.00) or the cost of pharmacological intervention per calf ( P No statistical significance was observed in terms of (= 0.55), but the total cost of pharmacological treatment for CON was numerically higher than that for PRO (277.2 USD vs. 102.9 USD).

[0170] Furthermore, during the study period described in this example, calves fed with the combination of probiotic strains had a heavier weaning weight (81.7 kg vs. 88.0 kg; P = 0.04) and showed an increasing trend in ADG (0.618 kg / day vs. 0.682 kg / day) and FE (0.353 kg gain / kg feed vs. 390 kg gain / kg feed). P ≤ 0.09).

[0171] Example 3 Preparation of viral inoculum Viral isolate A / sw / Denmark / 12687 / 03 (H1N2) (lineage 1C.2) was multiplied and passaged three times in Madin-Darby canine kidney (MDCK) cells and stored in aliquots at -80°C until use. Viral titers in MDCK cells were determined using a half-maximal tissue culture infectious dose (TCID50) assay, followed by dilution in Eagle's minimum essential medium (MEM) (Gibco) to obtain 1.58 x 10⁻⁶ saturates. 5 TCID50 / ml.

[0172] Experimental Design A total of 24 crossbred Duroc x Landrace-Yorkshire pigs (females and castrated males, 7 weeks old, initial weight 12 kg) were used. All pigs were tested for swIAV virus and antibodies at approximately four weeks of age, confirming negative results for both swIAV and IAV-specific antibodies. After stratification based on sex and weight, the pigs were assigned to one of three groups: 1) unchallenged and not fed probiotics (CON; n = 8), 2) challenged but not fed probiotics (CHL; n = 8), and 3) challenged and fed 6.95 × 10⁻⁶ probiotics. 6 Pigs were fed CFU / g of Bacillus subtilis DSM 32324 (PRO; n = 8). All groups received the same diet (the CON and CHL groups did not contain Bacillus subtilis DSM 32324), and all pigs had free access to feed and water throughout the study.

[0173] Two weeks after being fed a diet containing or without Bacillus subtilis DSM 32324, all animals from CHL and PRO were administered 3 mL of H1N2 swIAV suspension (1.58 x 10⁻⁶). 5 Animals from CON were challenged intranasally with TCID50 / mL, and aerosol exposure was performed in the right nostril using a MAD Nasal™ intranasal mucosal device to administer simulated culture medium (MEM, Gibbsco). Clinical signs of disease were monitored daily in pigs, and body weight and temperature were measured on day 14 before infection (-14), day of infection (0), and day 7 post-infection (DPI 7).

[0174] Sampling procedure All pigs were euthanized at DPI 7. At the time of euthanasia, pigs were anesthetized by intramuscular injection of 0.1 ml / kg Zoletil 50 VET (teletamine 25 mg / ml, zoprazepam 25 mg / ml) and euthanized by intracardiac injection of 20% sodium pentobarbital (KELA, 100 mg / kg). Lung and ileum tissue samples were isolated and immediately stored in RNAlater (Invitrogen) at room temperature until transferred to -20°C the following day. During necropsy, schematic diagrams of lung lesions were drawn, and photographs of the dorsal and ventral sides of the lungs were taken. The area of ​​observed red atelectasis was measured by a pathologist in a blinded state using Adobe Acrobat Reader, combining the lung photographs and schematic diagrams. A biopsy of the right middle lobe of the lung was performed for histopathological examination and fixed in formalin (BiopSafe) until embedded in paraffin. The biopsy was then cut into 2-3 µm sections and stained with hematoxylin and eosin (H&E) to examine for histopathology and influenza virus, thereby quantifying swIAV positive cells.

[0175] DSM 32324 can reduce swIAV-induced gross lung symptoms. Influenza challenge induced mild infection in pigs, but without clinical symptoms. No differences in microscopic changes in lung tissue were observed between the CHL and PRO groups, nor were there any differences in the number of IAV-positive cells. However, gross lung lesions were observed in all influenza-challenged pigs (CHL and PRO groups), but not in the CON group. When comparing the total area of ​​lesions, pigs from the PRO group tended to have ( ) compared to the CHL group. P = 0.065) even lower gross lung lesions.

[0176] Example 4 In H1N2 influenza challenge in nursery pigs, supplementing sows and weaned offspring with Bacillus-based probiotics has shown effectiveness. Helps support live animal production performance and manage gross lung lesions A series of recent studies have shown that providing effective probiotics to sows and their offspring allows pigs to respond more effectively to viral challenge. Sixteen pregnant sows (parity 1 to 5) assigned by gestation pen were exposed to one of two treatments: control (CON), in which sows were fed a standard gestation and lactation diet without probiotics; or Bacillus spp. (BAC), in which sows were fed a standard diet from 5 weeks before farrowing until lactation, with daily feed application to achieve 1.65 x 10 6 CFU of Bacillus composition / g complete feed. The Bacillus composition used consisted of Bacillus licheniformis DSM17236 and Bacillus subtilis DSM 17231. Ninety-six nursery piglets weaned from CON and BAC sows were assigned to the nursery portion of this study at day 21 (48 piglets per CON or BAC treatment). Throughout the 3-week nursery period of this study, CON piglets selected for nursery received a probiotic-free diet, and BAC piglets selected for nursery received 1.10 x 10 g of complete feed. 6 CFU / g complete feed. Pigs were divided into 8 pens with 6 pigs per pen by treatment. Individual pig weight and pen feed intake were recorded on days 0, 7, 14, and 21. After a 14-day post-weaning feed acclimation period, all pigs were challenged with H1N2 influenza virus via intratracheal gavage (Haesebrouck et al.). Vet.Microbiol.[Veterinary Microbiology] 11:239-249, 1986). Clinical signs, including rectal temperature, respiratory score, abdominal score, and behavioral score, were collected daily from the day before challenge (day 13) until the end of the study. On day 21, after body weight collection, all pigs were euthanized, lungs were removed, and the percentage of each of the seven lobes (right anterior, left anterior, right middle, left middle, accessory, right posterior, and left posterior) with gross (visible) pulmonary lesions was scored (Halbur et al., J. Vet. Diagn. Invest. [Veterinary Diagnostic Survey Journal] 8:11-20, 1996). BAC treatment improved F:G ratio before challenge (P<0.01, 1.177 vs. 1.309), after challenge (P<0.01, 1.418 vs. 1.633), and throughout growth (P<0.001, 1.287 vs. 1.464). Furthermore, in the BAC-treated group, the percentage of pigs with gross pulmonary lesions in the left anterior, right anterior, accessory, left posterior, and right posterior lobes was reduced. Additionally, in the BAC-treated group, the total number of diseased lobes per pig was reduced (P<0.001, 4.1 vs. 5.3). These data indicate that when pigs are exposed to H1N2 influenza challenge, administration of a Bacillus-based probiotic composition containing Bacillus licheniformis DSM 17236 and Bacillus subtilis DSM 17231 to sows and subsequently to their weaned offspring is associated with improved feed efficiency and reduced lung lesions.

[0177] Example 5 Changes in lung pathology and inflammation during influenza A virus infection induced by Bacillus subtilis DSM 32324 in pigs Materials and Methods Prior to intranasal inoculation with the H1N2 strain of swine influenza A (1 C.2 lineage), pigs were fed a diet containing or without the probiotic strain Bacillus subtilis DSM 32324 (2.6E+06 CFU / g feed) for 14 days. Throughout the study, fecal samples, blood samples, and nasal swabs were collected to examine viral shedding and immune gene expression. Seven days after infection, pigs were euthanized, and lung and ileum tissues were collected for gene expression analysis and pathological examination.

[0178] result The results are shown in Figure 6 , Figure 7 and Figure 8 middle. Figure 6The total area of ​​lesions in the two infection groups was compared, showing a significant trend toward a decrease in the amount of gross lung lesions in infected pigs fed with Bacillus subtilis DSM 32324 (Bacillus subtilis group) compared to infected pigs fed a diet not supplemented with Bacillus subtilis DSM 32324 (positive control group) (P = 0.065).

[0179] Figure 7 The CRP levels are shown on days 0, 1, 4, and 7 post-infection. It can be seen that on day 4 post-infection (DPI 4), the CRP level in the positive control group was significantly increased due to infection compared to the negative control group (P = 0.03), with the Bacillus subtilis group showing a moderate level (P = 0.12).

[0180] Figure 8 Serum amyloid A (SAA) levels in pigs are shown. A decreasing trend in SAA levels was observed in the Bacillus subtilis group compared to the positive control group (P = 0.06). Similar results were observed for SAA3, which was significantly decreased in the Bacillus subtilis group (P = 0.04).

[0181] These findings suggest that administration of Bacillus subtilis DSM 32324 has the potential to reduce lung lesions, which may be attributed to overall suppression of the immune system, as indicated by decreased serum C-reactive protein (CRP) levels, reduced interferon-stimulated gene (ISG) expression, and localized reductions in the inflammatory marker serum amyloid A (SAA) in ileal tissue. Notably, the immunomodulatory effects of Bacillus subtilis DSM 32324 appear to be independent of the gut microbiota, as neither influenza infection nor administration of Bacillus subtilis DSM 32324 altered the microbiota composition.

[0182] Example 6 In H1N2 influenza challenge in nursery pigs, probiotic supplementation based on Bacillus spp. was beneficial for sows and their weaned offspring. effect Materials and Methods Five weeks before farrowing, 16 pregnant sows were assigned to two treatment groups: CON (control) and BAC (Bacillus spp.). BAC sows received a probiotic diet supplemented with Bacillus subtilis DSM 17231 and Bacillus licheniformis DSM 17236 (1.65E+09 CFU / kg feed) during gestation and lactation, while CON sows received a standard commercial diet. On day 21, 96 sows weaned from CON and BAC sows were assigned to eight pens, six sows per pen (48 sows per treatment group). During the three-week nursery period, BAC sows were supplemented with a Bacillus spp.-based probiotic (1.10E+09 CFU / kg feed), while CON sows were fed a standard diet.

[0183] After a 14-day acclimatization period following weaning, all pigs were challenged with H1N2 influenza via intratracheal gavage. Production performance indicators were recorded weekly from day 13 (the day before challenge) until the end of the study, while rectal temperature and clinical severity were assessed daily. Viral load in nasal swabs collected on days 16, 18, and 21 was analyzed by qPCR. On day 21, all pigs were euthanized, and lungs were removed and scored based on the percentage of lung lobes with gross (visible) pulmonary lesions.

[0184] result The results are shown in Tables 3 and 4 below. Figure 9 middle.

[0185] Table 3: Improvement in feed conversion ratio (FCR) in pigs fed probiotics before and after viral challenge.

[0186] Table 4: Percentage of pigs with rectal temperature above 39.7℃

[0187] Compared with CON pigs, BAC pigs had improved feed conversion ratio (FCR) before and after challenge (P<0.01; Table 3). During the post-challenge period, the proportion of pigs with rectal temperatures above 39.7°C was lower in the BAC-treated group (P<0.01; Table 4), and their clinical severity scores (combined behavioral, abdominal, and respiratory scores) were also lower on the same day (P<0.05). On days 16 and 18, viral intensity was 1–2 ct lower in nasal swabs associated with BAC-fed pigs, and the average total number of lesions per pig was lower in the BAC-treated group (P<0.001). Figure 9 ).

[0188] The present invention has been described with reference to various embodiments, aspects, examples, etc. It is not intended that these elements be interpreted independently of each other. Therefore, this disclosure provides for combinations of two or more embodiments, aspects, examples, etc.

[0189] All embodiments described herein are intended to fall within the scope of the disclosed invention. These and other embodiments of the invention will be readily understood by those skilled in the art through the following detailed description of preferred embodiments throughout the specification. The invention is not limited to any one or more specific preferred embodiments disclosed.

Claims

1. A composition comprising at least one Bacillus subtilis ( B. subtilis ) strains and optionally at least one Bacillus licheniformis ( B. licheniformis ( ) strains, the composition being used for maintaining or improving respiratory health, for preventing respiratory diseases in animals, or for reducing the incidence and / or severity of respiratory diseases in animals.

2. The composition of claim 1, wherein the Bacillus subtilis strain is selected from strains preserved as DSM 17231 and DSM32324.

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

4. The composition according to any of the preceding claims, wherein the composition comprises the strains preserved as DSM 17231 and DSM17236.

5. The composition of claim 1, wherein the microbial component of the composition comprises the Bacillus subtilis strain preserved as DSM 17231 and the Bacillus licheniformis strain preserved as DSM 17236.

6. The composition according to any of the preceding claims, wherein the animal is a ruminant.

7. An animal feed, animal feed additive or premix comprising the composition as described in any one of claims 1 to 6, and further comprising one or more concentrates, vitamins, minerals, enzymes, amino acids and / or other feed ingredients.

8. A method for maintaining or improving the respiratory health of an animal, the method comprising administering to the animal a composition as described in any one of claims 1-6, or an animal feed, animal feed additive, or premix as described in 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. The use of a composition comprising at least one Bacillus subtilis strain and optionally at least one Bacillus licheniformis strain for maintaining or improving the respiratory health of animals.

12. The use as claimed in claim 11, wherein the animal is a ruminant.

13. The use as claimed in claim 11 or 12, wherein the Bacillus subtilis strain is selected from strains deposited as DSM 17231 and DSM 32324.

14. The use according to any one of claims 11-13, wherein the Bacillus licheniformis strain is a strain deposited as DSM17236.

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

16. The use as claimed in claim 15, wherein the microbial component of the composition comprises the Bacillus subtilis strain preserved as DSM 17231 and the Bacillus licheniformis strain preserved as DSM 17236.

17. The use as claimed in any one of claims 12-16, wherein the respiratory disease is selected from the group consisting of pneumonia and bovine respiratory diseases, for example, wherein the animal is a ruminant and the respiratory disease is a bovine respiratory disease.

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