Microbiome modulation for mammary health and reduction of mastitis

Abstract

The present disclosure provides a composition and methods for preventing, reducing, or eliminating a mastitis infection in a bovine animal. The composition includes an inhibitory composition in an amount effective to inhibit the growth of infectious mastitis pathogens in a mammary system of the bovine animal, where the inhibitory composition includes a microbial composition or an extract of one or more microbes of a mammary system-associated microbiota of healthy bovines, including Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp., Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali, or any combinations thereof.

Description

MICROBIOME MODULATION FOR MAMMARY HEALTH AND REDUCTION OF MASTITIS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 510,037, filed June 23, 2023, which is incorporated by reference herein in its entirety. FIELD

[0002] The present disclosure is directed to the modulation of the mammary system microbiome for maintaining mammary health and for the prevention, reduction, or elimination of mastitis in bovine animals. BACKGROUND

[0003] Mastitis is one of the most frequent and costly health problems for organic and conventional dairy producers worldwide. Mastitis is an inflammation of any portion of the mammary system of the cow that is mainly caused by infectious pathogens, including bacteria, archaea, fungi or algae, and can be classified as subclinical mastitis or clinical mastitis. Mastitis causes deleterious impacts on dairy herd productivity, longevity, and profitability as a result of discarded milk, decreased milk production during the remainder of lactation and penalties for increased milk somatic cell count, decreased reproductive performance, increased costs associated with treatments and labor, higher culling and replacement rate, and an increased rate of death to affected animals.

[0004] The incidence of new mammary infections in the cow is high during the first week of the drying off period and reaches a peak during the transition period. the drying off period defines the period of time when lactation ceases before calving, which can typically last from 40 days to 70 days, and the transition period defines the period from 14 days before to 14 days after calving. The incidence of mastitis remains low during mid to late lactation. In a healthy cow, a keratin plug maintains a seal on the teat canal during the dry period and prevents entry of infectious mastitis pathogens into the teat canal. However, as the keratin plug disappears prior to calving, the cow becomes more susceptible to entry of infectious mastitis pathogens into the teat canal. Furthermore, some cows fail to create an effective keratin plug at all. These failures accompanied with natural suppression of the immune system of the cow during the transition period increases theirsusceptibility to mastitis. Thus, management of pathogens during the drying off and transition periods is of critical importance.

[0005] Mastitis is the main reason for antimicrobial use in conventional dairy herds, and there is no effective solution for mastitis in organic dairies reared without antimicrobials. The effectiveness of antimicrobial therapies in conventional herds is insufficient, having efficacy of between 30-60%. Available strategies in organic dairy systems are even more limited and less effective. For at least these reasons, and the growing concerns over imprudent use of antimicrobials and development of antimicrobial resistance in infectious mastitis pathogens with zoonotic potential, identifying alternative solutions for its prevention and treatment is an important priority both for the dairy industry and from public health perspective. SUMMARY

[0006] The present disclosure provides a composition for preventing, reducing, or eliminating a mastitis infection in a bovine anima. The composition can include an inhibitory composition in an amount effective to inhibit the growth of infectious mastitis pathogens in a mammary system of the bovine animal, where the inhibitory composition includes a microbial composition or an extract of one or more microbes of a mammary system-associated microbiota of healthy bovines. The mammary system-associated microbiota can include: Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali, or any combinations thereof.

[0007] In an aspect, the infectious mastitis pathogens include one or more strains of bacteria, archaea, fungi, or algae.

[0008] In an aspect, the infectious mastitis pathogens include one or more of: Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcusagalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof.

[0009] In an aspect, the mastitis pathogens suitable for targeting with the inhibitory compositions herein can include at least one or more microbes including Escherichia coli, Corynebacterium bovis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, and Trueperella pyogenes, or any combinations thereof.

[0010] In an aspect, the mastitis infection includes a clinical mastitis infection or a subclinical mastitis infection.

[0011] In an aspect, the inhibitory composition includes a teat dip, a wipe, a cream, a lotion, a balm, a powder, a spray, a gel, or an ointment suitable for external application to the mammary system.

[0012] In an aspect, the inhibitory composition includes an injectable composition suitable for injection into at least one teat canal.

[0013] The present disclosure provides a method for preventing, reducing, or eliminating a mastitis infection in bovine animals. The method can include injecting an inhibitory composition into an intramammary environment via a teat canal or applying an inhibitory composition to at least a portion of an exterior surface of a mammary system of a bovine animal, where the inhibitory composition is applied in an amount effective to inhibit the growth of infectious mastitis pathogens. The inhibitory composition includes a microbial composition or an extract of one or more of microbes of a mammary system-associated microbiota of healthy bovines, where the mammary system-associated microbiota includes Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali , or any combinations thereof.

[0014] In an aspect, the infectious mastitis pathogens include one or more strains of bacteria, archaea, fungi, or algae.

[0015] In an aspect, the infectious mastitis pathogens include one or more of Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof.

[0016] In an aspect, the mammary system includes an udder and one or more teats.

[0017] In an aspect, the inhibitory composition is injected or applied once daily.

[0018] In an aspect, the inhibitory composition is injected or applied at least once daily to eliminate mastitis pathogens.

[0019] In an aspect, the inhibitory composition is injected or applied at least once daily until a subclinical mastitis infection has cleared.

[0020] In an aspect, the inhibitory composition is injected or applied one time per 24 hours, two times per 24 hours, three times per 24 hours, four times per 24 hours, five times per 24 hours, or six times per 24 hours.

[0021] In an aspect, the inhibitory composition is injected or applied once daily for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, or for an entire lactation period to prevent mastitis pathogens.

[0022] In an aspect, the inhibitory composition is injected or applied to at least a portion of the exterior surface of the mammary system of the bovine animal until a mastitis infection is cleared.

[0023] In an aspect, the mastitis infection includes clinical mastitis or subclinical mastitis.

[0024] In an aspect, the inhibitory composition is injected or applied to at least a portion of the exterior surface of the mammary system of the bovine animal at any time.

[0025] In an aspect, the inhibitory composition is injected or applied daily to at least a portion of the exterior surface of the mammary system of the bovine animal.

[0026] In an aspect, the inhibitory composition is injected or applied to at least a portion of the exterior surface of a sub-clinically infected mammary system of the bovine animal at any time during a subclinical infection.

[0027] In an aspect, the inhibitory composition is injected or applied daily to at least a portion of the exterior surface of an infected mammary system of the bovine animal during an infection.

[0028] In an aspect, the inhibitory composition is injected or applied to at least a portion of an exterior surface of the mammary system of the bovine animal at any time during a period of oneto three weeks prior to calving or at any time during a period of from one to three weeks following calving.

[0029] In an aspect, the inhibitory composition is injected or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal at any time during a period of one to three weeks prior to calving or at any time during a period of from one to three weeks following calving.

[0030] In an aspect, the inhibitory composition is injected or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal on a first day of In an aspect, the inhibitory composition is injected or applied to at least a portion of the exterior surface of the mammary system of the bovine animal at any time during a drying off period.

[0031] In an aspect, the inhibitory composition is injected or applied daily to at least a portion of the exterior surface of the mammary system of the bovine animal a drying off period.

[0032] In an aspect, the inhibitory composition is injected or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal at any time during a drying off period, at any time in one to three weeks leading up to the drying off period, or at any time in one to three weeks following the drying off period.

[0033] In an aspect, the inhibitory composition is injected or applied to an exterior surface of the mammary system during a subclinical mastitis infection.

[0034] In an aspect, the inhibitory composition is injected or applied to an exterior surface of at least one quarter of an udder of the bovine animal during a subclinical mastitis infection.

[0035] In an aspect, the inhibitory composition is injected or applied to an exterior surface of all four teats or all four quarters of an udder of the bovine animal during a clinical mastitis infection.

[0036] In an aspect, the inhibitory composition is injected or applied to an exterior surface of all four teats or all four quarters of an udder of the bovine animal during a subclinical mastitis infection.

[0037] In the disclosure herein, a method for inhibition of infectious mastitis pathogens in the mammary system of bovine animals is provided The method can include injecting an inhibitory composition into an intramammary environment via a teat canal or applying an inhibitory composition to at least a portion of an exterior surface of a mammary system of a bovine animal, where the inhibitory composition is applied in an amount effective to inhibit the growth of infectious mastitis pathogens. The inhibitory composition suitable for the methods herein includes a microbial composition or an extract of one or more of microbes of a mammary system-associated microbiota, where the one or more microbes of the mammary system-associated microbiotainclude Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp., Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali, or any combinations thereof; and where the infectious mastitis pathogens include one or more of Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof. BRIEF DESCRIPTION OF THE FIGURES

[0038] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0039] FIG.1 is a schematic representation of a phylogenetic tree summarizing antimicrobial and anti-virulence activities of milk isolates against mastitis pathogens in accordance with various aspects herein.

[0040] FIG.2 is a photograph showing the effects of various inhibitory compositions herein against S. dysgalactiae in accordance with various aspects herein.

[0041] FIG.3 is a photograph showing the effects of various inhibitory compositions herein against S. uberis in accordance with various aspects herein.

[0042] FIG.4 is a photograph showing the effects of various inhibitory compositions herein against S. aureus in accordance with various aspects herein.

[0043] FIG.5 is a photograph showing the effects of various inhibitory compositions herein against E. coli in accordance with various aspects herein.

[0044] FIG.6 is a photograph showing the effects of various inhibitory compositions herein against A. baumannii in accordance with various aspects herein.DETAILED DESCRIPTION

[0045] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0046] The balanced microbiota of the mammary system can play an important role in maintaining udder health and mastitis susceptibility. The microbiota in the udder can be classified as healthy when there is high diversity of bacteria and resistance to colonization by opportunistic or pathogenic microbes. Development of clinical mastitis and inflammatory responses in the udder can be associated with decreased diversity of the microbiota and disruption of the proper balance between the proportion of bacterial species that do not cause disease against opportunistic and pathogenic microbes. It is understood that a clinical mastitis infection can be identified in a cow through visible changes in the milk, teats, or udders, or combinations thereof. Changes in milk color and consistency, such as caused by fibrin clots present in the milk, milk quality as measured by protein content, and decreased milk production volume can be observed. The condition of clinical mastitis further can include swelling, reddening, or hardening of the udder or teats; heat or pain at the udder or teats; systemic symptoms including fever, aches, and chills; and in severe cases, death. Subclinical mastitis can also occur in a cow in response to pathogenic microbes and is often asymptomatic. A subclinical mastitis infection can be identified as a decrease in milk production volume and quality, or by sampling the milk for somatic cell counts past an industry defined threshold indicative of infection. Infectious mastitis pathogens can be transmitted from cow to cow, or they can be spread through the animals’ habitat.

[0047] The present disclosure provides a culturomics approach combined with metagenomics and culture-enriched metagenomics to characterize the beneficial members of microbiota of the mammary system that are present in milk of healthy dairy cows. The disclosure further describes the inhibitory effects of the beneficial members of the microbiota mammary system against common mastitis pathogens. The disclosure provides novel formulations based on these defined microbial communities or their metabolites when applied to the mammary system at various life stages to maintain the proper balance and diversity of the mammary system microbiota and to suppress the growth of infectious mastitis pathogens. The formulations disclosed herein can be applied topically to an exterior surface of the mammary system or injected into intramammary environment via the teat canal to promote mammary microbiota and / or to suppress mastitispathogens and prevent mastitis infection. The formulations disclosed herein further can be applied as a prophylaxis to improve the natural balance of mammary system microbiota or following mastitis infection and antimicrobial therapy to help with recovery of the mammary system microbiota following those perturbations. The formulations disclosed herein further can be administered as an intramammary application within the teat or udder.

[0048] As used herein, the term “mammary system” can include the udder and one or more teats, where the udder can include four quarters and each quarter can include at least one teat. The term mammary system further can be used to describe the internal components of the mammary system including the teat canal; and any tissues, cells, and fluids contained within the udder and the teats.

[0049] As used herein, the terms “microbiota,” “microbiome,” and “microbial community” can be used interchangeably and can refer to the collection of microorganisms (e.g., bacteria, archaea, viruses, algae, and fungi) present in a defined environment. The microorganisms can interact with each other and / or a host species, and can include commensal, opportunistic, or pathogenic species. The microbiome can include the genes and genomes of the microbes as well as the products produced by the microbiota in the defined environment. Bovine Mammary-Associated Microbiota Present in Healthy Animals

[0050] The mammary system-associated microbiota present in healthy bovine animals can include a microbial community that can inhibit colonization of one or more types of infectious mastitis pathogens. Such microbial communities can include one or more microbes in balanced concentrations so that no single species is present in a high enough concentration to outcompete other species within the community and can result in any given species playing a beneficial or commensal role to a pathogenic role. For example, in some cases commensal microbial species can become highly abundant in a microbial community to the point where they trigger immune response and can cause inflammation of the mammary system also known as mastitis.

[0051] For the disclosure herein, a mammary system-associated microbiota present in healthy bovine animals was identified using a culturomics approach that included techniques such as culturing various mammary gland isolates, using matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry fingerprint, 16S ribonucleic acid (RNA) gene sequencing for the identification of unique bacterial species, and whole genome sequencing of select isolates to identify the entire genomic content of those strains. It will be appreciated that many similartechniques can be used to identify microbial species within a microbial community, including additional techniques such as shotgun metagenomic sequencing.

[0052] The microbes identified within the mammary system-associated microbiota of healthy bovines herein include, at least, Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali. It will be appreciated that the classification of various microbes can change as taxonomy continues to be refined. If any of the microbes in this list are reclassified at a later date, they are to be considered to fall within the scope of this disclosure.

[0053] In various aspects, herein, the inhibitory compositions can include one or more of the microbes identified as part of the mammary system-associated microbiota. The inhibitory composition can include a composition containing live microbes of the mammary system- associated microbiota. The inhibitory composition can include lyophilized microbes of the mammary system-associated microbiota. The inhibitory composition can include extracts of one or more microbes. In various aspects, the extracts of one or more microbes of the mammary system-associated microbiota can include whole-cell extracts or supernatant extracts. Infectious Mastitis Pathogens

[0054] The inhibitory compositions herein are suitable for inhibiting the growth of various infectious mastitis pathogens in bovine animals, including on or more strains of bacteria, archaea, fungi, or algae. The mastitis pathogens can invade the teats or udders of bovine animals and can lead to either of a clinical mastitis infection or a subclinical mastitis infection. By way of example, the mastitis pathogens suitable for targeting with the inhibitory compositions herein can include at least one or more pathogens including Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae,Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof. In an aspect, the mastitis pathogens suitable for targeting with the inhibitory compositions herein can include at least one or more microbes including Escherichia coli, Corynebacterium bovis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, and Trueperella pyogenes. It will be appreciated that the classification of various microbes can change as taxonomy continues to be refined. If any of the microbes herein are reclassified at a later date, they are to be considered to fall within the scope of this disclosure. Inhibitory Compositions

[0055] The present disclosure provides compositions for preventing, reducing, or eliminating a mastitis infection in bovine animals. The composition can include an inhibitory composition in an amount effective to inhibit the growth of infectious mastitis pathogens in the mammary system of the bovine animal. The inhibitory composition can include a microbial composition or an extract of one or more microbes (i.e. a microbial extract) of a mammary system-associated microbiota of healthy bovines. The microbial composition or an extract can be created from one or more of Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali., or any combinations thereof.

[0056] The inhibitory compositions can inhibit the growth of one or more infectious mastitis pathogens including, but not to be limited to, Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof. In an aspect, the mastitis pathogens suitable for targeting with the inhibitory compositions herein can include at least one or more microbes including Escherichia coli, Corynebacteriumbovis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, and Trueperella pyogenes, or any combinations thereof.

[0057] The inhibitory composition can include live microbes of the mammary system- associated microbiota in healthy bovine animals. The inhibitory composition can include lyophilized microbes of the mammary system-associated microbiota. The inhibitory composition can include extracts of one or more microbes, such as whole-cell extracts or supernatant extracts. The microbial extracts can include supernatants where cellular components have been removed by centrifugation or filtration, or both. In an aspect, the microbial extracts in supernatant form can be concentrated to remove excess water. In an aspect, the inhibitory composition can include multiple microbes of the mammary system-associated microbiota, where each of the multiple microbes is cultured under microbe-specific conditions, lyophilized, and then each lyophilized microbe is mixed to form the inhibitory composition. In another aspect, the inhibitory composition can include multiple microbial extracts from the microbes of the mammary system-associated microbiota, where each of the multiple microbes is cultured under microbe-specific conditions, the extracts are separated from cellular components, and then each extract is mixed to form the inhibitory composition.

[0058] The inhibitory compositions can be formulated for application to the bovine animal on any of the four quarters of the udder, or on any of the four teats of the animal, or any combination of application to the teats and udder. The inhibitory compositions can include multiple delivery forms for external topical application, including but not to be limited to, a teat dip, a wipe soaked or embedded with the composition, a cream, a lotion, a balm, a powder, a spray, a gel, or an ointment. The inhibitory compositions further can be formulated for injection into the intramammary environment via a teat canal. The inhibitory composition can be applied to one or more teats as a pre-dip composition, a post-dip composition, or by injecting the inhibitory composition into the udder via one or more teat canals, or any combinations thereof

[0059] Inhibitory compositions formulated for external topical application can include various components, including one or more of an emollient, a surfactant, preservatives, thickeners, pH adjusters, or stabilizers. Such ingredients can include water, petrolatum, bees wax, cocoa butter, shea butter, glycerin, paraffin, mineral oil, vegetable oils, ceramides, propylene glycol, polyethylene glycol, short or long chain fatty acids, disodium or tetrasodium EDTA, lanolin, aloe vera, essential oils, vitamin E, xanthan gum, citric acid, or sodium hydroxide.Methods for Preventing, Reducing, or Eliminating Mastitis

[0060] The present disclosure provides for multiple methods of preventing, reducing, or eliminating mastitis in bovine animals using the inhibitory compositions described elsewhere herein. The methods can include applying an inhibitory composition to at least a portion of an exterior surface of a mammary system of a bovine animal and / or injected to the intramammary environment via a teat canal, where the inhibitory composition is applied in an amount effective to optimize the balance of mammary system microbiota and / or inhibit the growth of infectious mastitis pathogens. The inhibitory composition can include a microbial composition or an extract of one or more microbes of a mammary system-associated microbiota of healthy bovines. Suitable microbes for use in the inhibitory compositions used in the methods herein are described in more detail elsewhere herein. In an aspect, the inhibitory composition can be applied to any portion of an exterior surface of the mammary system and / or injected into intramammary environment including one or more teats or to one or more quarters of an udder of the bovine animal, or any combinations thereof, as a prophylaxis or following mastitis and antimicrobial therapy to achieve optimum balance of mammary microbiota and suppress mastitis pathogens and / or prevent from their regrowth. In an aspect, optimum mammary microbiota balance can be achieved, and a mastitis infection can be considered to be cleared when there are no longer visible signs of clinical mastitis or subclinical mastitis and / or when milk production volume and quality returns to a normal production volume and quality relative to a similar bovine animal without a mastitis infection. The inhibitory compositions can be applied to at least a portion of an exterior surface of the mammary system that is not infected when a separate portion of the mammary system is infected with a clinical mastitis infection. In some aspects, the inhibitory compositions can be applied during the routine course of milking, such as when sanitizing the udder and teats post milking.

[0061] The methods herein are suitable for preventing, reducing, or eliminating mastitis in bovine animals using by inhibiting the growth of various infectious pathogens such as bacteria, archaea, fungi, or algae, including, but not to be limited to one or more of Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof. In an aspect, the mastitis pathogens suitable for targeting with the inhibitory compositions herein can include at least one or moremicrobes including Escherichia coli, Corynebacterium bovis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, and Trueperella pyogenes. In an aspect, methods herein are suitable for optimizing the balance of mammary system microbiota by in bovine animals using by inhibiting the growth of various infectious bacteria, archaea, fungi, or algae as described herein.

[0062] The methods herein for preventing, reducing, or eliminating mastitis in bovine animals involve applying the inhibitory composition to at least a portion of the mammary system of a cow. It will be appreciated that the normal mammary system of most cows includes an udder having four quarters, where each quarter further includes one teat. It is within the scope of the methods of the present disclosure to apply the inhibitory compositions to a bovine animal with mammary anatomy that deviates from the normal population of cows, such as a cow with five udders, or three udders or two udders, and any other deviations that may arise as compared to normal mammary system anatomy. Accordingly, the inhibitory compositions can be injected into intramammary environment via a teat canal and / or applied to at least a portion of an exterior surface of one or more of the teats, at least a portion of the udder, or at least a portion of an exterior surface of one or more of the four quarters of the udder of a bovine animal. The inhibitory compositions can be injected into intramammary environment via a teat canal and / or applied to an exterior surface of one, two, three, or four teats, or any combinations thereof, to prevent, reduce, or eliminate mastitis pathogens in bovine animals. The inhibitory compositions further can be injected into intramammary environment via a teat canal and / or applied to an exterior surface of one, two, three, or four quarters of the udder, or to the entire udder of the bovine animal. The inhibitory compositions can be applied to any combination of teats and udder quarters.

[0063] The inhibitory compositions can be applied to a bovine animal at any life stage, including at the drying off period, during the transition period, or during early-, mid- or late lactation at any time. . As used herein, the terms “drying off period” and “dry off” are used interchangeably unless otherwise noted and can refer to the period of time when lactation ceases before calving and can typically last from 40 days to 70 days.

[0064] The inhibitory compositions can be injected into intramammary environment via a teat canal and / or applied to an exterior surface of at least one quarter of an udder during a subclinical mastitis infection. The inhibitory compositions can be injected into intramammary environment via a teat canal and / or applied to an exterior surface of all four quarters of an udder of the bovine animal during a subclinical mastitis infection. In an aspect, the inhibitory composition can beapplied to an exterior surface of one or more teats and one or more quarters of the udder, or any combination thereof, during a subclinical mastitis infection.

[0065] Numerous scenarios of application of the inhibitory compositions to promote an optimum balance of mammary system microbiota to prevent infection and reduce or eliminate a mastitis infection can be suitable for the methods herein. The inhibitory compositions can be prophylactically injected via teat canal and / or applied to at least a portion of the mammary system when not infected or can be injected via teat canal and / or applied to at least a portion of the mammary system at regularly scheduled time periods during a subclinical mastitis infection. In the methods herein, the inhibitory composition can be applied to at least a portion of the exterior surface of the mammary system of the bovine animal at any time. In the methods herein, the inhibitory composition can be applied daily to at least a portion of the exterior surface of the mammary system of the bovine animal. In the methods herein, the inhibitory composition can be injected into mammary environment via teat canal and / or applied to at least a portion of the exterior surface of a sub-clinically infected mammary system of the bovine animal at any time during a subclinical infection. In the methods herein, the inhibitory composition can be injected into mammary environment via teat canal and / or applied daily to at least a portion of the exterior surface of a sub-clinically infected mammary system of the bovine animal.

[0066] The inhibitory compositions can be injected into mammary environment via teat canal and / or applied to the mammary system to at least a portion of the mammary system at the day of parturition, at any time in the one to three weeks leading up to parturition or at any time in the one to three weeks following parturition. It will be appreciated that the term “parturition” is understood to refer to the birth of a calf and can also be referred to as “calving.” In the methods herein, the inhibitory composition can be injected into mammary environment via teat canal and / or applied to at least a portion of an exterior surface of the mammary system of the bovine animal at any time during a period of one to three weeks prior to calving or at any time during a period of from one to three weeks following calving. In the methods herein, the inhibitory composition can be injected into mammary environment via teat canal and / or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal at any time during a period of one to three weeks prior to calving or at any time during a period of from one to three weeks following calving. In the methods herein, the inhibitory composition can be injected into mammary environment via teat canal and / or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal on the first day of parturition.

[0067] The inhibitory compositions can be injected into mammary environment via teat canal and / or applied to the mammary system to at least a portion of the mammary system at the time of drying off, at any time in the one to three weeks leading up to the drying off period, or at any time in the one to three weeks following the drying off period. In the methods herein, the inhibitory composition can be injected into mammary environment via teat canal and / or applied to at least a portion of the exterior surface of the mammary system of the bovine animal at any time during the drying off period, at any time in the one to three weeks leading up to the drying off period, or at any time in the one to three weeks following the drying off period. In the methods herein, the inhibitory composition can be applied daily to at least a portion of the exterior surface of the mammary system of the bovine animal during the drying off period, daily during the one to three weeks leading up to the drying off period, or daily during the one to three weeks following the drying off period. During the drying off period, the inhibitory compositions can be applied to the exterior surface of the mammary system at any time as needed to prevent infection and reduce or eliminate a mastitis infection.

[0068] The inhibitory compositions can be applied in the various methods herein at least once daily to prevent mastitis, can be applied at least once daily to reduce the incidence of mastitis, or can be applied at least once daily following a mastitis infection and antimicrobial therapy to optimize the microbiome of the mammary system and clear pathogen. In an aspect, the inhibitory composition can be applied one time per 24 hours, two times per 24 hours, three times per 24 hours, four times per 24 hours, five times per 24 hours, or six times per 24 hours. In an aspect, the inhibitory composition can be applied once daily for at least one week, at least two weeks, at least three weeks, at least four weeks, or at least five weeks, or for the entire lactation period to prevent mastitis pathogens. As the incidence of mastitis infections begins to subside in the herd, application can be reduced to from once daily to two to four times weekly. In an aspect, in high health herds with low incidence of mastitis, the inhibitory compositions can be applied once weekly for at least one week, at least two weeks, or at least three weeks or during the entire lactation period to reduce mastitis infections or the possibility of mastitis infections. In an aspect, the inhibitory compositions can be applied once weekly for at least one week during the entire period of dry off, as a prophylactic measure to prevent mastitis infection during this period. In an aspect, the inhibitory compositions can be applied once weekly for at least one week, at least two weeks, or at least three weeks prior to or after parturition to prevent mastitis infection following the birth of a calf.

[0069] The inhibitory compositions further can be injected into at least one, two, three, or four teat canals to prevent, reduce, or eliminate mastitis in a bovine animals. The inhibitory compositions herein can be formulated to be injected into the teat canal where it can act to prevent, reduce, or eliminate a mastitis infection in the teats and / or udder. It will be appreciated that any combination of exterior topical application and injection into a teat canal can be performed to prevent, reduce, or eliminate a mastitis infection in a bovine animal.

[0070] It will be appreciated that one teat of one udder quarter of a cow can have an active mastitis infection while the remaining teats and udder quarters of the same cow are unaffected. It will further be appreciated that one, two, three, or four teats and their respective udder quarters can have an active mastitis infection while those remaining are unaffected. For those teats and udder quarters with a clinical mastitis infection, a higher dose (e.g., 2X) of the inhibitory composition can be applied during following completion of antimicrobial therapy, For those teats and udder quarters that are not affected or are experiencing subclinical mastitis, the inhibitory composition can be administered during healthy or subclinical mastitis to prevent the spread of infectious pathogens that cause mastitis. The inhibitory compositions can be injected into a teat canal with a subclinical mastitis infection and can additionally be injected into one or more of the teat canals without a mastitis infection. In the methods herein, the inhibitory composition can be applied to an exterior surface of at least one quarter of an udder of the bovine animal during a subclinical mastitis infection or following antimicrobial therapy after a clinical mastitis infection. In the methods herein, the inhibitory composition can be applied to an exterior surface of at least one quarter of an udder of the bovine animal during a subclinical mastitis infection, even if only a fraction of the udder is infected. In the methods herein, the inhibitory composition can be applied to an exterior surface of all four teats or all four quarters of an udder of the bovine animal during a subclinical mastitis infection, even if only a fraction of the udder is infected. Methods for Inhibiting Pathogenic Microbes

[0071] The methods herein can inhibit of infectious mastitis pathogens in the mammary system of bovine animals. The method can include applying an inhibitory composition to at least a portion of an exterior surface of a mammary system of a bovine animal, and / or injecting into intramammary environment via teat canal, where the inhibitory composition is applied in an amount effective to inhibit the growth of infectious mastitis pathogens, where the inhibitory composition includes a microbial composition or an extract of one or more microbes of a mammary system-associated microbiota as described elsewhere herein. The infectious mastitispathogens subject to inhibition by the inhibitory compositions herein include one or more of. Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof. In an aspect, the mastitis pathogens suitable for targeting with the inhibitory compositions herein can include at least one or more microbes including Escherichia coli, Corynebacterium bovis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, and Trueperella pyogenes.

[0072] The methods herein for inhibiting infectious mastitis pathogens in the mammary system of bovine animals involve applying the inhibitory composition to at least a portion of the mammary system of a cow. As discussed above, it will be appreciated that the normal mammary system of most cows includes an udder having four quarters, where each quarter further includes one teat. It is within the scope of the methods of the present disclosure to apply the inhibitory compositions to a bovine animal with mammary anatomy that deviates from the normal population of cows, such as a cow with five udders, or three udders or two udders, and any other deviations that may arise as compared to normal mammary system anatomy. Accordingly, the inhibitory compositions can be injected into intramammary environment via teat canal and / or applied to at least a portion of an exterior surface of one or more of the teats, at least a portion of the udder, or at least a portion of an exterior surface of one or more of the four quarters of the udder of a bovine animal. The inhibitory compositions can be injected into intramammary environment via teat canal and / or applied to an exterior surface of one, two, three, or four teats, or any combinations thereof, to prevent, reduce, or eliminate mastitis in a bovine animals. The inhibitory compositions further can be injected into intramammary environment via teat canal and / or applied to an exterior surface of one, two, three, or four quarters of the udder, or to the entire udder of the bovine animal. The inhibitory compositions can be applied to any combination of teats and udder quarters.

[0073] The inhibitory compositions can be applied to a bovine animal to inhibit infectious mastitis pathogens in the mammary system of bovine animals at any life stage, including at the drying off period, the week prior to calving, two to three weeks following calving, or any time during a subclinical mastitis infection, or any time following antimicrobial therapy of mammary system.

[0074] The inhibitory compositions can be injected into intramammary environment via teat canal and / or applied to an exterior surface of at least one quarter of an udder during a subclinical mastitis infection. The inhibitory compositions can be injected into intramammary environment via teat canal and / or applied to an exterior surface of all four quarters of an udder of the bovine animal during a clinical mastitis infection. In an aspect, the inhibitory composition can be injected into intramammary environment via teat canal and / or applied to an exterior surface of one or more teats and one or more quarters of the udder, or any combination thereof, during a clinical or subclinical mastitis infection.

[0075] Various application scenarios are described in reference to methods for preventing, reducing, or eliminating a mastitis infection in bovine animals, and are applicable to these methods for inhibiting mastitis pathogens as well. Animals

[0076] The animals suitable for preventing, reducing, or eliminating mastitis herein can include bovine animals. Bovine animal, or cattle (Bos taurus or Bos indicus) suitable for topical application of the inhibitory compositions herein can include, but are not to be limited to, dairy and beef cattle breeds such as Anatolian Black, Andalusian Black, Angus (e.g., black or red), Aubrac, Aussie Red, Ayrshire, Belgian (e.g., blue or red), Belted Galloway, Brahman, Brangus, Braunvieh, Brown Swiss, Butana, Canadienne, Caracu, Charolais, Chianina, Corriente, Criollo, Danish Red, Darkensberger, Dexter, Dutch Belted, Fleckvieh, Gelbvieh, German Angler, Gir, Guernsey, Hereford, Holstein, Illawarra, Jersey, Kankrej, Kerry, Kenana, Limousin, Maine-Anjou, Milking Devon, Milking Shorthorn, Mongolian, Montbeliarde, N’Dama, Normande, Norwegian Red, Piedmontese, Red Sindhi, Red and White (a.k.a., Red and White Holstein), Santa Gertruids, Sahiwal, Salers, Scottish Highland, Swedish Red, Simmental, Tarentaise, Tharparkar, Texas Longhorn, Viking Red, Wagyu, and Watusi, and any crosses thereof. EXAMPLES

[0077] Various aspects of the present disclosure can be better understood by reference to the following Examples which are offered by way of illustration. The present disclosure is not limited to the Examples given herein. Example 1: Investigation of Raw Colostrum and Raw Milk from Bovines on Inhibition of Infectious Mastitis Pathogens

[0078] This example analyzed the effects of raw colostrum and raw milk on the inhibition of infectious mastitis pathogens. The experiments focused on the effects of raw colostrum and raw milk specifically on Staphylococcus aureus and Escherichia coli.

[0079] Samples of colostrum, milk from a healthy cow, or milk from mastitic cows were obtained. Briefly, pre-milking teat disinfection was performed using 0.5% iodine pre-dip solution, teats were thoroughly dried using individual paper towels and scrubbed for 15 seconds using cotton pads moistened in 70% alcohol. Composite colostrum and milk samples (~ 20 mL from two quarters of each cow combined into a 50 mL sterile containers) were collected and placed on ice in the milking parlor and transferred over ice to the laboratory for further processing.

[0080] Five aliquots of 5 mL or milk or colostrum were prepared and processed independently according to one of the following conditions: 1.) storage at 4 °C; 2.) heating to 75 °C for five minutes and cooled to 4 °C; 3.) addition of 750 µL of saponin, incubation at room temperature for 10 minutes followed by cooling to 4 °C; 4.) centrifugation at max speed for 10 minutes, followed by filter sterilization using a 0.2 µm filter; and 5.) centrifugation at max speed for 10 minutes, followed by filter sterilization using a 0.2 µm filter, heating at 80 °C for 5 minutes, followed by cooling to 4 °C.

[0081] The mastitis pathogens to be tested were prepared as follows: pick a single colony from a growth plate containing the pathogen of interest and culture in 5 mL of brain heart infusion broth (BHI) broth overnight. Transfer 200 µL of culture to a fresh 5 mL of BHI broth and further dilute to 1 / 100 or an O.D. at 600 nm of approximately 0.1.

[0082] For each milk or colostrum sample, an “original plate” was prepared by co-incubating 150 µL of the 1 / 100 diluted pathogen with 150 µL of each of the milk treatments at two different concentrations (D1 & D2), using transparent sterile 96-well plates with lids. Plates were covered by breathable covers and incubated at 37 °C in a CO2incubator. At 12h-16h after incubation, the plate was removed from the incubator and 50 µL of each well was transferred to a new sterile 96- well plate, and 1 / 10 serial dilutions were made using sterile BHI in a “dilution plate.” Using a multichannel pipette, a spot assay was performed by spotting 5 µL from each well on a BHI agar plate followed by incubation at 37 °C for 24h-48h and followed by colony counting. After 6h- 12hof incubation, the spot assay plates were used for cfu counting.

[0083] Results from 8 samples (2 colostrum and 6 milk) showed inhibitory effects against both S. aureus and E. coli. In general, the highest growth rate for both pathogens were observed by co-incubating with filter-sterilized heat-inactivated milk samples whereas co-incubation withuntreated, raw samples in general resulted in the lowest growth rate of both pathogens (data not shown).

[0084] Overall, inhibitory effects of raw, saponin treated (lysis of immune cells), and pasteurized milk and colostrum samples (inactivation of bacteria and proteins by heat) on the growth rate of pathogens were inconsistent both among samples and between pathogens. However, when comparing the inhibitory effects of heat treated vs. non-heat treated filter- sterilized samples, for vast majority of samples heat treatment decreased the inhibitory effect of filter-sterilized milk on the growth of pathogens; suggesting potential antimicrobial activities for bioactive proteins of the milk. However, the inhibitory effects observed was minimal (less than 1 log decrease in CFU of pathogens), reflecting the low concentrations of these bioactive molecules. Example 2: Characterization of Cultured Microbial Communities at Species Level Using Shallow Shotgun Metagenomics

[0085] This example analyzed the high-resolution characterization of cultured microbial communities at species level using shallow shotgun metagenomics. Shallow shotgun metagenomics was used to determine accurate taxonomic classifications of all bacterial species recovered from each milk / colostrum sample. For this purpose, a single metagenomic DNA pool was created for each sample by combining bacterial cell pellets cultured on all different media types, culture conditions (anaerobic and aerobic), and across all time-points into a single tube followed by DNA extraction as previously described. The resulting metagenomic DNA pools (n=16) were subjected to shotgun library preparation as previously described13. In brief, miniaturized Illumina short-read libraries were prepared using the NEBNext Ultra II FS DNA Library Prep Kit. Individually barcoded libraries were subjected to dual size-selection using the ProNex® Size-Selective Purification System to enrich for insert sizes of 800-1000 bp. Final libraries were sequenced on an Illumina HiSeq2500 platform in rapid run mode, paired-end 2x250nt, at the McMaster Genomics Facility (Hamilton, ON, Canada). Adaptor sequences were trimmed from the resulting sequences using Trimmomatic. The resulting high-quality paired-end reads were used for species-level and strain-level taxonomic classification using Kraken2 and MetaPhlAnv.3 bioinformatic packages, respectively.

[0086] The shallow shotgun sequencing was performed on the pool of all bacterial colonies recovered from different culturing media / conditions of each sample. Taxonomic classification of the resulting metagenomics reads identified a total of 73 bacterial species, belonging to 28 genera and spanning 3 major bacterial phyla Actinobacteria, Firmicutes, and Proteobacteria. The mostdiverse bacterial genera identified were Bacillus (11 species), Corynebacterium (10 species), and Staphylococcus (18 species). Among these, non-aureus staphylococci species (NAS), in particular S. saprophyticus, S. haemolyticus, S. chromogenes, and S. epidermidis were the most prevalent isolates detected across all samples. In addition, one species of fungi (Candida parapsilosis) was also detected in 4 samples.

[0087] In combination with data provided by 16S rRNA gene sequencing, shallow metagenomics also enabled identification of frozen culture stocks containing the highest diversity of bacterial genera of interest (e.g. Corynebacterium, Bacillus, Bifidobacterium) for re-culturing, isolation, and screening for potential antimicrobial activities against mastitis pathogens. Example 3: Identification and Isolation of Individual Bacterial Species with Inhibitory Properties Against Infectious Mastitis Pathogens

[0088] This example analyzed the ability to isolate individual commensal bacteria and creation of a strain bank to screen against mastitis pathogens.

[0089] In order to identify strains from the milk and colostrum samples collected from Examples 1-2, individual bacterial isolates that were cultured on different media and under different culturing conditions were transferred on large agar plate in 96-well plate format, allowing high-throughput replication, storage, and characterization with the use of 96-well pin replicators. Following initial transfer of colonies from the original mixed plates to the 96-well format, the latter plates were incubated at 37 °C (24h for aerobic plates and 72h for anaerobic plates). For each plate, sterile pin replicators were used for: a) making duplicate backup plates; b) making frozen stock in deep 96-well plates containing BHI broth and 20% glycerin; and c) transferring colonies onto 96-well PCR plates containing lysis buffer for high-throughput DNA extraction followed by shallow 16S rRNA gene sequencing and taxonomic classification of individual or probably mixed colonies.

[0090] Screening the collection of commensal bacterial isolates for antimicrobial and anti- virulence activity against a panel of infectious mastitis pathogens was performed. In order to screen potential inhibitory activities such as antimicrobial and antiviral activities of the pilot strain bank against common infectious mastitis pathogens, a panel of infectious mastitis pathogens including 4 different strains of each of the following pathogens: Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, and one strain of pathogenic Enterococcus faecium was used. For the first round of screening, one strain of each of the following groups: S. agalactiae, S. aureus, E. coli, and E. faecium was chosen.

[0091] Co-culture of selected pathogens with commensals was performed as follows. Pathogens were grown overnight in BHI broth, diluted to OD600 of approximately 0.1, and then 150 µL of the dilution was spread over large BHI3+RF agar plates to create pathogen lawns. At the same time, 96-well pin replicators were used to stamp fresh cultures of milk isolates over the pathogen lawn. For aerobes, the co-culture plate was incubated at 37 °C and inhibitory activities against each pathogen (i.e. potential zone of inhibition around milk isolates) was monitored at 24h and 48h (photos were taken at each time point and potential hits were recorded accordingly). For milk isolates showing activity against each pathogen, that isolate was streaked twice for purity, the original pathogen inhibitory assay was repeated to confirm reproducibility, and if positive, all other strains of that pathogen were also screened against that milk isolate to evaluate the spectrum of inhibitory activity. For isolates with confirmed activity against mastitis pathogens, individual frozen stocks were created in duplicate, and isolate was characterized by Sanger 16S rRNA gene sequencing. The inhibitory activities of the milk strain isolates in exemplary co-culturing experiments are presented in Table 1.muicerutluC-oCnisnegohtaPtsniagAsetalosIniartSfoseitivitcAyroetaii+ + +tbi ca + + +hlnaIg.syd + +1E.LS + + + + +BATan aan sailsiim lium iuis mcmcsis s rereuc iuul irel ul ireml mr umpu upsisillisismuna tctacaetcao qlc eoecaoaorvcoavsofis s ssn ssnca nei rldiel beoo eo rueo ll nullullullerullerbietrtot n sibsen sissrni ani aniicehiciciconiconvesrsoIeArunAmanAmaaBci laBaBaBosaBosrBobl oryroryrorCeaoCexoCex+++++++ +++ ++ + ++ + + ++ +aciatcitmss s s yulylsuiilulc sic u ucucmsuilaaro o anro onsul su ull suretlaco ni cocoiv ai s paacrbiioa csl palli scc ii c amansiy c llca al all ia gtxsiy caisoc aicx bioc erni ll oet rorl oro ali mii mi xi b n o s biilaell ic e ni es joy hcetmi eanu tn ceet dnuekc caabrc daigrc daiagteebraci uetneubiab repuimpo tr istsi ae sieeagCok Eqa Eaf Esp Fat Lla LlaMinMul Pap Pis Poc TrcWoj

[0092] Screening the ability of milk isolates to inhibit the expression of accessory gene regulator (agr) locus of S. aureus was also performed on milk samples. The milk samples were cultured in broth media in 96-well format to reach to the stationary phase, supernatant of individual cultures were then filter sterilized using 0.22 µm filters and used to screen for ability to inhibit the expression of agr locus of an engineered S. aureus strain containing a lux reporter system. For isolates showing inhibitory activities, the assay was repeated in duplicate to make sure of reproducibility of results, and isolates were characterized by Sanger 16S rRNA gene sequencing.

[0093] In parallel with culture-enriched sequencing and creation of mixed bacterial communities, individual isolates were also picked from different culture conditions of each sample and arrayed on 96-well plate formats for IDSeq characterization (i.e. partial 16S rRNA sequencing). Overall, approximately 1000 isolates were picked from the total of 16 milk and colostrum samples resulting in identification of 353 unique ASVs, belonging to 164 distinct species (79 Firmicutes sp., 49 Actinobacteria sp., 20 Bacteroidetes sp., 16 Proteobacteria sp., and 2 species of Verrucomicrobiota). Among the classified ASVs, those belonging to Staphylococcus (98 unique ASVs), Bacillus (27 unique ASVs), and Corynebacterium (31 unique ASVs) accounted for more than 44% of the entire culture collection; implying the high prevalence of these bacterial genera in the mammary gland ecosystem.

[0094] Antimicrobial and anti-virulence activities of milk isolates: Isolates of the culture collection were further screened for their potential antimicrobial activities against common mastitis pathogens. The initial round of screening was performed by coculturing individual isolates (either aerobically or anaerobically, according to the original culture condition of each isolate) with one strain of each of the following mastitis pathogens: S. disgalactiae, S. uberis, S. aureus, and E. coli. Not surprisingly, several non-aureus staphylococci isolates (NAS), including S. chromogenes, S. felis, S. haemolyticus, and S. edaphicus showed inhibitory activities against S. aureus. Other bacterial isolates showing activity against S. aureus included Bacillus zhangzhouensis, Aerococcus viridans, Escherichia fergusonii, Cutibacterium acnes, Cellulosimicrobium funkei, Brachybacterium sacelli, and several species of Corynebacterium. The only isolates showing activity against E. coli were A. viridans and C. acnes. With respect to streptococci pathogens, a wide range of NAS species, B. zhangzhouensis, A. viridans, B. sacelli, Phycicoccus badiiscoriae, and Corynebacterium showed inhibited the growth of S. disgalactiae and S. uberis.

[0095] Co-incubation of the S. aureus agr reporter strain with filter-sterilized supernatants of all milk isolates identified several species capable of inhibiting the expression of agr locus,including: all the identified NAS species, Bacillus sp., A. viridans, Paraclostridium benzoelyticum, C. acnes, and B. sacelli.

[0096] The initial screening assay was performed using a high throughput approach to enable screening a large number of isolates against different mastitis pathogens. However, this approach has several limitations: a) isolates of the culture collection were not further purified following the standard microbiology technique (i.e. 3 rounds of streaking and purification), b) lack of high- resolution taxonomic classification using full-length sanger 16S rRNA sequencing, and c) screening assay was not performed in duplicate / triplicate, therefore lacking evidence of reproducibility.

[0097] To address the abovementioned limitations of the high-throughput assay, all the isolates showing activity through the initial assay were re-streaked twice on individual agar plates for purity, and then tested against different strains of the target pathogen(s) for which they showed activity in the initial assay. This was followed by full length Sanger 16S rRNA gene sequencing of the isolates showing activity against different strains of the same pathogen (i.e. isolates with potential broad-spectrum antimicrobial activity). This confirmatory assay identified 3 Bacillus strains (B. pumilis and B. zhangzhouensis) with broad spectrum inhibitory activity against S. disgalactiae, S. uberis, and S. aureus. Two strains of Aerococcus (A. urinaeequi and A. viridans) with strong inhibitory activity against all mastitis pathogens tested were also identified. A C. acnes strain that inhibited the growth of S. aureus and E. coli was further identified. Data are presented in FIG. 1, which shows a schematic representation of a phylogenetic tree summarizing antimicrobial and anti-virulence activities of milk isolates against various mastitis pathogens. It will be appreciated that while the isolated strains of Aerococcus urinaeequi showed strong antimicrobial effects against the mastitis pathogens, it was confirmed to present a safety risk and thus not pursued as a component of the inhibitory composition presented herein.

[0098] With respect to the inhibition of agr expression in S. aureus, all the initial hits were re- cultured in broth media and filter-sterilized supernatants were used for confirmatory assay (in duplicate). Microbes including B. pumilus, A. viridans, Clostridium disporicum, Paraclostridium benzoelyticum, Blautia glucerasea, Brevibacterium epidermidis, and two strains of Corynebacterium that inhibited the expression of agr locus in S. aureus were further identified as shown in FIG.1. Without wishing to be bound by any particular theory, it is understood that an increased expression of the agr locus can be an indication of increased virulence of a given microbe, and that inhibition of the agr locus, such as by the beneficial microbes as described herein, can indicate a decrease in virulence. Decreasing virulence by inhibiting the agr locus can be aviable mechanism for suppressing virulence of a pathogen while not exerting evolutionary pressures that can result in antimicrobial resistance when inhibiting growth of a given pathogen. Example 4: Antimicrobial Effects of Various Crude Extracts of Microbes from the Mammary System-Associated Microbiota

[0099] This example analyzed the inhibitory effects of various crude extracts of microbial isolates on several infectious mastitis pathogens.

[0100] Briefly, selected isolates were cultured on BHI3 medium using a cotton swab to obtain a dense lawn. Cultures were incubated at 37 °C for either 2 days (aerobic isolates) or 4 days (anaerobic isolates). After the cultures reached the desired lawn density, a metal spatula was used to cut fermented agar into small pieces that were transferred to a 50-mL Falcon tube. Approximately 15-20 mL of 100% methanol was added to each tube, followed by shaking on a spinning wheel at room temperature to let the extraction process continue for 24h. Each mixture of bacterial agar and methanol was passed through a coffee filter and collected into 16mm x 100mm glass tubes. The resulting methanol extracts were concentrated and evaporated on a Genevac Centrifugal Evaporator (SP-Scientifics, NY, US). The resulting dry powders from the methanol crude extracts were resuspended using 750 µL of 100% dimethyl sulfoxide (DMSO) as a universal solvent. The DMSO suspensions were further centrifuged for 2-3 minutes at approximately 8000 rpm, to allow collection of the supernatant containing microbial crude extracts. The crude extracts were used to screen indirect metabolite-driven inhibitory activities of milk isolates against mastitis pathogens using a disc-diffusion assay.

[0101] Antimicrobial disc diffusion assays were performed by growing pathogens overnight in BHI broth, diluted to OD600of approximately 0.1, and then 150 µL of the dilution was spread over large BHI3 agar plates. Sterile susceptibility blank discs (BD BBLTMSensi-DiscTM, Becton Dickinson, NJ, US) were placed on the surface of the agar plate and 2 × 15 µL of the crude extracts from each milk isolate were pipetted on the discs. Plates were then incubated at 37 °C for 24h to allow for the growth of pathogens to a visible lawn, after which potential antimicrobial activities of the extracts were evaluated by measuring inhibitory zones around the discs. For each assay, a disc containing 2 × 15 µL of 100% DMSO were also included as control for potential inhibitory effect of the solvent on the target pathogen.

[0102] Results are summarized in Table 2, where a positive value indicates inhibition by the microbial crude extract and a negative value indicates no significant inhibition by the microbial crude extract.TABLE 2. Antimicrobial Effects of Various Individual Microbial Crude Extracts Reported by Disc Diffusion Assay

[0103] Crude extracts were further combined to determine the antimicrobial effects of various combinations on infectious mastitis pathogens. Combinations of crude extracts and the results showing antimicrobial effects against various pathogens are shown in Table 3. Antimicrobial activity by the microbial crude extracts was recorded as follows: a double positive value indicates additive inhibition, a single positive value indicates individual inhibition, and a negative value indicates no significant inhibition. Combinations of crude extracts that showed additive inhibitory activity had increased antimicrobial activity against the target pathogens compared to the same crude extracts individually. Combinations of crude extracts that showed individual inhibitory activity showed approximately equal antimicrobial activity against the target pathogens compared to the same crude extracts individually. Combinations of crude extracts that showed negativeinhibitory activity showed no antimicrobial activity or weaker antimicrobial activity against the target pathogens compared to the same crude extracts individually. TABLE 3. Antimicrobial Effects of Various Combinations of Microbial Crude Extracts Reported by Disc Diffusion AssayExample 5: Antimicrobial Effects of Various Combinations of Crude Extracts of Microbes from the Mammary System-Associated Microbiota of Healthy Bovines Against Infectious Mastitis Pathogens

[0104] This example analyzed the inhibitory effects of various fractionated extracts of microbial isolates on several infectious mastitis pathogens.

[0105] Briefly, selected isolates were cultured on BHI3 medium using a cotton swab to obtain a dense lawn. Cultures were incubated at 37 °C for either 2 days (aerobic isolates) or 4 days (anaerobic isolates). After the cultures reached the desired lawn density, a metal spatula was used to cut fermented agar into small pieces that were transferred to a 50-mL Falcon tube. Approximately 15-20 mL of 100% methanol was added to each tube, followed by shaking on a spinning wheel at room temperature to let the extraction process continue for 24h. Each mixture of bacterial agar and methanol was passed through a coffee filter and collected into 16mm x 100mm glass tubes. The resulting methanol extracts were concentrated and evaporated on a Genevac Centrifugal Evaporator (SP-Scientifics, NY, US). The resulting dry powders from the methanol crude extracts were resuspended using 750 µL of 100% dimethyl sulfoxide (DMSO) as a universal solvent. The DMSO suspensions were further centrifuged for 2-3 minutes at approximately 8000 rpm, to allow collection of the supernatant containing microbial crude extracts. The crude extracts were subjected to partial purification and fractionation using reversed phase flash chromatography (CombiFlash RF 200; Teledyne ISCO, NE, US). All fractionations were performed using 15g C18 Gold columns (RediSep Rf Gold C18; Teledyne ISCO, NE, US) with 2.5 mL of each crude extract as input. The resulting fractions (24 original fractions per crude extract) were evaporated using a Genevac and resuspended using 750 µL of 100% DMSO. These fractions were used to screen indirect metabolite-driven inhibitory activities of milk isolates against mastitis pathogens using a disc-diffusion assay

[0106] Antimicrobial disc diffusion assays were performed by growing pathogens overnight in BHI broth, diluted to OD600 of approximately 0.1, and then 150 µL of the dilution was spread over large BHI3 agar plates. Sterile susceptibility blank discs (BD BBLTMSensi-DiscTM, Becton Dickinson, NJ, US) were placed on the surface of the agar plate and 2 × 15 µL of the crude extract or CombiFlash chromatography fractions from each milk isolate were pipetted on the discs. Plates were then incubated at 37 °C for 24h to allow for the growth of pathogens to a visible lawn, after which potential antimicrobial activities of extracts were evaluated by measuring inhibitory zones around the discs. For each assay, a disc containing 2 × 15 µL of 100% DMSO were also included as control for potential inhibitory effect of the solvent on the target pathogen.

[0107] An exemplary disc diffusion assay showing the inhibitory effects of CombiFlash chromatography fractions for each of Paraclostridium bifermentans_A; Lacrimispora sp.; and Brevibacillus borstelensis individually and in combination is shown in FIGS.2-6. Referring now to FIG.2, the target pathogen, S. dysgalactiae was plated as described above. Discs were placed on the surface of the agar plate and 2 × 15 µL of the CombiFlash chromatography fractions were pipetted onto the discs (a disc containing 2 × 15 µL of 100% DMSO were also included as control) alone or in combination of all three. Plates were assayed to assess the potential antimicrobial activities of the fractionated extracts by measuring inhibitory zones around the discs. The disc diffusion assays were repeated using the Paraclostridium bifermentans_A; Lacrimispora sp.; and Brevibacillus borstelensis fractions against S. uberis (FIG.3), S. aureus (FIG.4), E. coli (FIG.5), and A. baumannii (FIG.6).

[0108] Results of all of the crude extracts tested are summarized in Table 4, where a positive value indicates inhibition by the fractionated microbial extract and a negative value indicates no significant inhibition by the microbial crude extract. Antimicrobial activity by the fractionated microbial extracts was recorded as follows: a double positive value indicates additive inhibition, a single positive value indicates individual inhibition, and a negative value indicates no significant inhibition. Combinations of fractionated extracts that showed additive inhibitory activity had increased antimicrobial activity against the target pathogens compared to the same crude extracts individually. Combinations of fractionated extracts that showed individual inhibitory activity showed approximately equal antimicrobial activity against the target pathogens compared to the same crude extracts individually. Combinations of crude fractionated extracts that showed negative inhibitory activity showed no antimicrobial activity or weaker antimicrobial activity against the target pathogens compared to the same fractionated extracts individually.TABLE 4: Antimicrobial Effects of Various Combinations of CombiFlash Fractionated Microbial Extracts Reported by Disc Diffusion Assay

[0109] It should be understood that the definitions described herein apply to all aspects as described unless otherwise stated.

[0110] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference is to be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0111] Values expressed in a range format are to be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1 % to about 5 %” or “about 0.1 % to 5 %” is to be interpreted to include not just about 0.1 % to about 5 %, but also the individual values (e.g., 1 %, 2 %, 3 %, and 4 %) and the sub-ranges (e.g., 0.1 % to 0.5 %, 1.1 % to 2.2 %, 3.3 % to 4.4 %) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0112] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis (% w / w or w / w %) is also referred to as weight percent (wt. %) or percent by weight (% wt.) herein. ADDITIONAL EXAMPLES

[0113] The following is a non-comprehensive list of additional examples of the invention.

[0114] Example 1. A composition for preventing, reducing, or eliminating a mastitis infection in a bovine animal comprising: an inhibitory composition in an amount effective to inhibit the growth of infectious mastitis pathogens in a mammary system of the bovine animal; wherein the inhibitory composition comprises a microbial composition or an extract of one or more microbes of a mammary system-associated microbiota of healthy bovines; and wherein the mammary system-associated microbiota comprises: Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis,Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali, or any combinations thereof.

[0115] Example 2. The composition of example 1, wherein the infectious mastitis pathogens comprise one or more strains of bacteria, archaea, fungi, or algae.

[0116] Example 3. The composition of examples 1 or 2, wherein infectious mastitis pathogens comprise one or more of: Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof.

[0117] Example 4. The composition of any of examples 1-3, wherein the mastitis infection comprises a clinical mastitis infection or a subclinical mastitis infection.

[0118] Example 5. The composition of any of examples 1-4, wherein the inhibitory composition comprises a teat dip, a wipe, a cream, a lotion, a balm, a powder, a spray, a gel, or an ointment suitable for external application to the mammary system.

[0119] Example 6. The composition of any of examples 1-5, wherein the inhibitory composition comprises an injectable composition suitable for injection into at least one teat canal.

[0120] Example 7. A method for preventing, reducing, or eliminating a mastitis infection in bovine animals comprising: injecting an inhibitory composition into an intramammary environment via a teat canal or applying an inhibitory composition to at least a portion of an exterior surface of a mammary system of a bovine animal, where the inhibitory composition is applied in an amount effective to inhibit the growth of infectious mastitis pathogens; wherein the inhibitory composition comprises a microbial composition or an extract of one or more of microbes of a mammary system-associated microbiota of healthy bovines; wherein the mammary system-associated microbiota comprises Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis,Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali , or any combinations thereof.

[0121] Example 8. The method of example 7, wherein the infectious mastitis pathogens comprise one or more strains of bacteria, archaea, fungi, or algae.

[0122] Example 9. The method of examples 7 or 8, wherein infectious mastitis pathogens comprise one or more of Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof.

[0123] Example 10. The method of any one of examples 7-9, wherein the mammary system comprises an udder and one or more teats.

[0124] Example 11. The method of any one of examples 7-10, wherein the inhibitory composition is injected or applied once daily.

[0125] Example 12. The method of any one of examples 7-10, wherein the inhibitory composition is injected or applied at least once daily to eliminate mastitis pathogens.

[0126] Example 13. The method of any one of examples 7-10, wherein the inhibitory composition is injected or applied at least once daily until a subclinical mastitis infection has cleared.

[0127] Example 14. The method of any one of examples 7-10, wherein the inhibitory composition is injected or applied one time per 24 hours, two times per 24 hours, three times per 24 hours, four times per 24 hours, five times per 24 hours, or six times per 24 hours.

[0128] Example 15. The method of any one of examples 7-10, wherein the inhibitory composition is injected or applied once daily for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, or for an entire lactation period to prevent mastitis pathogens.

[0129] Example 16. The method of any one of examples 7-15, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of the mammary system of the bovine animal until a mastitis infection is cleared.

[0130] Example 17. The method of any one of examples 7-16, wherein the mastitis infection comprises clinical mastitis or subclinical mastitis.

[0131] Example 18. The method of any one of examples 7-17, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of the mammary system of the bovine animal at any time.

[0132] Example 19. The method of any one of examples 7-18, wherein the inhibitory composition is injected or applied daily to at least a portion of the exterior surface of the mammary system of the bovine animal.

[0133] Example 20. The method of any one of examples 7-19, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of a sub-clinically infected mammary system of the bovine animal at any time during a subclinical infection.

[0134] Example 21. The method of any one of examples 7-20, wherein the inhibitory composition is injected or applied daily to at least a portion of the exterior surface of an infected mammary system of the bovine animal during an infection.

[0135] Example 22. The method of any one of examples 7-21, wherein the inhibitory composition is injected or applied to at least a portion of an exterior surface of the mammary system of the bovine animal at any time during a period of one to three weeks prior to calving or at any time during a period of from one to three weeks following calving.

[0136] Example 23. The method of any one of examples 7-22, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal at any time during a period of one to three weeks prior to calving or at any time during a period of from one to three weeks following calving.

[0137] Example 24. The method of any one of examples 7-23, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal on a first day of parturition.

[0138] Example 25. The method of any one of examples 7-24, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of the mammary system of the bovine animal at any time during a drying off period.

[0139] Example 26. The method of any one of examples 7-25, wherein the inhibitory composition is injected or applied daily to at least a portion of the exterior surface of the mammary system of the bovine animal a drying off period.

[0140] Example 27. The method of any one of examples 7-26, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal at any time during a drying off period, at any time in one to three weeks leading up to the drying off period, or at any time in one to three weeks following the drying off period.

[0141] Example 28. The method of any one of examples 7-27, wherein the inhibitory composition is injected or applied to an exterior surface of the mammary system during a subclinical mastitis infection.

[0142] Example 29. The method of any one of examples 7-28, wherein the inhibitory composition is injected or applied to an exterior surface of at least one quarter of an udder of the bovine animal during a subclinical mastitis infection.

[0143] Example 30. The method of any one of examples 7-29, wherein the inhibitory composition is injected or applied to an exterior surface of all four teats or all four quarters of an udder of the bovine animal during a clinical mastitis infection.

[0144] Example 31. The method of any one of examples 7-30, wherein the inhibitory composition is injected or applied to an exterior surface of all four teats or all four quarters of an udder of the bovine animal during a subclinical mastitis infection.

[0145] Example 32. A method for inhibition of infectious mastitis pathogens in the mammary system of bovine animals comprising: injecting an inhibitory composition into an intramammary environment via a teat canal or applying an inhibitory composition to at least a portion of an exterior surface of a mammary system of a bovine animal, where the inhibitory composition is applied in an amount effective to inhibit the growth of infectious mastitis pathogens; wherein the inhibitory composition comprises a microbial composition or an extract of one or more of microbes of a mammary system-associated microbiota; wherein the one or more microbes of the mammary system-associated microbiota comprise Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis,Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali, or any combinations thereof; and wherein the infectious mastitis pathogens comprise one or more of Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof.

Claims

CLAIMS What is claimed is:

1. A composition for preventing, reducing, or eliminating a mastitis infection in a bovine animal comprising: an inhibitory composition in an amount effective to inhibit the growth of infectious mastitis pathogens in a mammary system of the bovine animal; wherein the inhibitory composition comprises a microbial composition or an extract of one or more microbes of a mammary system-associated microbiota of healthy bovines; and wherein the mammary system-associated microbiota comprises: Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali, or any combinations thereof.

2. The composition of claim 1, wherein the infectious mastitis pathogens comprise one or more strains of bacteria, archaea, fungi, or algae.

3. The composition of claims 1 or 2, wherein infectious mastitis pathogens comprise one or more of: Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof.

4. The composition of any of claims 1-3, wherein the mastitis infection comprises a clinical mastitis infection or a subclinical mastitis infection.

5. The composition of any of claims 1-4, wherein the inhibitory composition comprises a teat dip, a wipe, a cream, a lotion, a balm, a powder, a spray, a gel, or an ointment suitable for external application to the mammary system.

6. The composition of any of claims 1-5, wherein the inhibitory composition comprises an injectable composition suitable for injection into at least one teat canal.

7. A method for preventing, reducing, or eliminating a mastitis infection in bovine animals comprising: injecting an inhibitory composition into an intramammary environment via a teat canal or applying an inhibitory composition to at least a portion of an exterior surface of a mammary system of a bovine animal, where the inhibitory composition is applied in an amount effective to inhibit the growth of infectious mastitis pathogens; wherein the inhibitory composition comprises a microbial composition or an extract of one or more of microbes of a mammary system-associated microbiota of healthy bovines; wherein the mammary system-associated microbiota comprises Anaerocolumna aminovalerica, Bacillus glycinifermentans, Bacillus licheniformis, Bacillus pumilis, Bacillus sonorensis, Bacillus_AY sp002271785, Bacillus butanolivorans, Brevibacillus borstelensis, Brevibacterium epidermis, Clostridium aerotolerans, Corynebacterium sp.(some but not all species), Corynebacterium xerosis, Cutibacterium acnes, Cytobacillus kochii, Dietzia sp., Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus_A pseudoavium, Facklamia sp., Facklamia tabacinasalis, Hungatella, Lacrimispora sp., Lacrimispora algidixylanolytica, Metabacillus niabensis, Micrococcus luteus, Paenibacillus pabuli, Paraclostridium bifermentans, Peribacillus simplex, Populibacterium corticicola, Timonella sp., Tissierella creatinini, and Weissella jogaejeotgali , or any combinations thereof.

8. The method of claim 7, wherein the infectious mastitis pathogens comprise one or more strains of bacteria, archaea, fungi, or algae.

9. The method of claims 7 or 8, wherein infectious mastitis pathogens comprise one or more of Acinetobacter baumannii, Corynebacterium bovis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycoplasma sp., Prototheca zopfii, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus chromogens, Staphylococcus hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Trueperella (formerly Arcanobacterium) pyogenes, or any combinations thereof.

10. The method of any one of claims 7-9, wherein the mammary system comprises an udder and one or more teats.

11. The method of any one of claims 7-10, wherein the inhibitory composition is injected or applied once daily.

12. The method of any one of claims 7-10, wherein the inhibitory composition is injected or applied once daily for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, or for an entire lactation period to prevent mastitis pathogens.

13. The method of any one of claims 7-12, wherein the mastitis infection comprises clinical mastitis or subclinical mastitis.

14. The method of any one of claims 7-13, wherein the inhibitory composition is injected or applied daily to at least a portion of the exterior surface of the mammary system of the bovine animal.

15. The method of any one of claims 7-14, wherein the inhibitory composition is injected or applied to at least a portion of an exterior surface of the mammary system of the bovine animal at any time during a period of one to three weeks prior to calving or at any time during a period of from one to three weeks following calving.

16. The method of any one of claims 7-15, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal on a first day of parturition.

17. The method of any one of claims 7-16, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of the mammary system of the bovine animal at any time during a drying off period.

18. The method of any one of claims 7-17, wherein the inhibitory composition is injected or applied to at least a portion of the exterior surface of an infected mammary system of the bovine animal at any time in one to three weeks leading up to the drying off period or at any time in one to three weeks following the drying off period.

19. The method of any one of claims 7-18, wherein the inhibitory composition is injected or applied to an exterior surface of at least one quarter of an udder of the bovine animal during a subclinical mastitis infection.

20. The method of any one of claims 7-19, wherein the inhibitory composition is injected or applied to an exterior surface of all four teats or all four quarters of an udder of the bovine animal during a clinical or subclinical mastitis infection.

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