WO2022067382A1 - Compositions and methods for maintaining and improving mammary gland health - Google Patents

Abstract

Disclosed herein are methods for treating a mammalian teat, udder or breast, or protecting a teat, udder or breast from infection, comprising applying to the teat, udder or breast of a mammal a hygiene-maintaining composition comprising at least one Lactobacillus species selected from L. paracasei, L. casei and L. buchneri. Also disclosed are methods for maintaining or improving mammary gland health and for protecting against mastitis in a mammalian teat, breast or udder. The present invention also describes teat conditioner compositions comprising at least one Lactobacillus species selected from L. paracasei, L. casei and L. buchneri.

Description

COMPOSITIONS AND METHODS FOR MAINTAINING AND IMPROVING MAMMARY GLAND HEALTH

Field of the Art

[001] The present disclosure relates generally to teat conditioner compositions and to methods for maintaining and improving the health of mammary glands, in particular teats and udders of dairy animals. Also provided are methods for preventing and reducing the incidence of mastitis.

Background

[002] Mastitis is the most prevalent production problem of animal welfare, production and economic loss facing the dairy industry worldwide. The prevalence risk of mastitis is high and is influenced by animal (e.g. parity, stage of lactation), farm (e.g. herd-size, geographical location), and nutritional factors in the herd. Teat canal and the integrity of teat-end tissue play pivotal roles against the introduction of mastitis-associated pathogens into the udder. Teat-end hyperkeratosis is the teat canal response to the forces imposed by milking. Milking machine and animal level factors can lead to severe teat-end hyperkeratosis, increase the roughness of the teat end, and increase the risk of intramammary infections by mastitis-causing pathogens in the herd.

[003] Somatic cell count in milk is considered a biomarker of mammary gland inflammation and is often used as an indicator of intramammary infections. When a lactating animal is infected, the somatic cell count increases in milk from the animal. An elevated somatic cell count causes significant problems in milk production. Milk with a high somatic cell count may need to be discarded, or partitioned from the remainder of a batch and blended with low somatic cell count milk to make it allowable for human consumption. With the automation of milking operations milk with a high somatic cell count from one animal may contaminate an entire milking run. Furthermore, even relatively low levels of intramammary infection can significantly reduce the volume and quality of milk produced by an animal. A relative reduction in somatic cell count while holding constant other animal and herd-level risk factors, reflects a lower risk of exposure to intramammary infections. Reduced milk somatic cell count is regarded as a good indicator of effective mastitis management practices in a herd.

[004] Avoiding intramammary infection is also an important factor in maintaining good health of a female mammal. This can be achieved with the use of teat conditioners and/or teat disinfectants.

[005] Teat disinfection is a common mastitis prevention practice that has proven to be an effective tool in the control of mastitis, associated with a lower incidence of new intramammary infections, a reduction in bulk milk somatic cell count and fewer teat skin abnormalities. Commercially available teat disinfectants are typically chemical-based formulations, comprising for example, chlorine and iodine, such as an iodophor, chlorhexidine acetate and chlorhexidine gluconate, raising concerns about the risk of chemical residues in milk.

[006] There remains a clear need for the development of improved and/or less harmful or environmentally damaging compositions and methods for treating teats to maintain or improve mammary gland health and prevent or reduce the incidence of mastitis.

Summary of the Disclosure

[007] A first aspect of the present invention provides a method for treating a mammalian teat, udder or breast, or protecting a teat, udder or breast from infection, comprising applying to the teat, udder or breast of a mammal at least one Lactobacillus species selected from L. paracasei, L. casei an L. buchneri.

[008] The method may comprise applying two of L. paracasei, L. casei and L. buchneri. In an exemplary embodiment, the method comprises applying L. paracasei, L. casei and L. buchneri.

[009] In a particular embodiment, the method comprises applying a hygiene -maintaining amount of a composition comprising the at least one Lactobacillus species, the two Lactobacillus species or the L. paracasei, L. casei and L. buchneri.

[0010] In an embodiment, the hygiene -maintaining amount comprises at least about 10⁶ CFU/ml of each of the at least one Lactobacillus species, at least about 10⁶ CFU/ml of each of the two Lactobacillus species, or at least about 10⁶ CFU/ml of each of L. paracasei, L. casei and L. buchneri.

[0011] In an embodiment the composition is applied as a teat spray or teat dip.

[0012] The composition may further comprise one or more emollients. [0013] The composition may further comprise one or more surfactants.

[0014] Typically the mammal is a human or a dairy animal. In an exemplary embodiment, the dairy animal is a cow. In particular embodiments, the mammal is lactating. The application may be performed pre-milking and/or post-milking.

[0015] A second aspect of the present invention provides a method for maintaining or improving mammary gland health in a mammal, comprising administering to the mammal at least one Lactobacillus species selected from L. paracasei, L. casei and L. buchneri.

[0016] The method may comprise administering to the mammal two of L. paracasei, L. casei and L. buchneri. In an exemplary embodiment, the method comprises administering L. paracasei, L. casei and /.. buchneri.

[0017] Typically the Lactobacillus species are administered topically to a teat, breast or udder of the mammal. In an exemplary embodiment, the topical administration comprises administering a composition comprising the one or more Lactobacillus species, the two Lactobacillus species or the /.. paracasei, L. casei and /.. buchneri, optionally as a teat spray or a teat dip.

[0018] The composition may further comprise one or more emollients.

[0019] The composition may further comprise one or more surfactants.

[0020] Typically the mammal is a human or a dairy animal. In an exemplary embodiment, the dairy animal is a cow. In particular embodiments, the mammal is lactating. The application may be performed pre-milking and/or post-milking.

[0021] Accordingly, in an embodiment the method of the second aspect is a method for maintaining or improving teat health or udder health in a dairy animal or a herd of dairy animals.

[0022] Assessing or determining mammary gland health may comprise determining or measuring somatic cell count in milk produced by the mammal.

[0023] A third aspect of the present invention provides a method for protecting against mastitis in a mammalian teat, breast or udder, comprising applying to the teat, breast or udder at least one Lactobacillus species selected from /.. paracasei, L. casei and /.. buchneri. [0024] The method may comprise applying two of L. paracasei, L. case! and L. buchneri. In an exemplary embodiment, the method comprises applying L. paracasei, L. casei and L. buchneri.

[0025] In a particular embodiment, the method comprises applying a composition comprising the at least one Lactobacillus species, the two Lactobacillus species or the L. paracasei, L. casei and A. buchneri.

[0026] In an embodiment the composition is applied as a teat spray or teat dip.

[0027] The composition may further comprise one or more emollients.

[0028] The composition may further comprise one or more surfactants.

[0029] The composition may further comprise xanthan gum.

[0030] Typically the mammal is a human or a dairy animal. In an exemplary embodiment, the dairy animal is a cow. In particular embodiments, the mammal is lactating. In an exemplary embodiment, the application is performed post-milking.

[0031] Assessing or determining protection against mastitis may comprise determining or measuring somatic cell count in milk produced by the mammal.

[0032] A further aspect of the present invention provides a method for protecting against mastitis, or reducing the incidence of mastitis, in a herd of dairy animals, comprising administering to at least one animal of the herd at least one Lactobacillus species selected from L. paracasei, L. casei and A. buchneri.

[0033] The method may be employed as defined in a hereinbefore described aspect.

[0034] Another aspect of the present invention provides a method for decreasing somatic cell count in the milk from a lactating mammal, comprising administering to the mammal at least one Lactobacillus species selected from L. paracasei, L. casei and L. buchneri.

[0035] The method may be employed as defined in a hereinbefore described aspect. [0036] Another aspect of the present invention provides a teat conditioner composition comprising at least one Lactobacillus species selected from L. paracasei, L. casei and L. buchneri.

[0037] The teat conditioner composition may comprise two of L. paracasei, L. casei and L. buchneri. In an exemplary embodiment, the teat conditioner composition comprises L. paracasei, L. casei and /.. buchneri.

[0038] Typically the teat conditioner composition is formulated for topical application to the teats, breast or udder of a mammal. In an exemplary embodiment, the mammal is a dairy animal.

[0039] In an exemplary embodiment, the teat conditioner composition comprises at least about 10⁶ CFU/ml of each of the at least one Lactobacillus species, at least about 10⁶ CFU/ml of each of the two Lactobacillus species, or at least about 10⁶ CFU/ml of each of L. paracasei, L. casei and A. buchneri.

[0040] The teat conditioner composition may further comprise one or more emollients.

[0041] The teat conditioner composition may further comprise one or more surfactants.

[0042] The teat conditioner composition may further comprise xanthan gum.

Brief Description of the Drawings

[0043] Aspects and embodiments of the present invention are described herein, by way of non-limiting example only, with reference to the following drawings.

[0044] Figure 1. Schemata of the study design (A), and the observed average teat end scores (B) and average somatic cell count (C) with 95% confidence interval as observed in the current study. Iodine-based (control) group - black solid line and solid squares. Lactobacillus- based (treatment) group - black dashed line, white triangles. Treatment periods were between study days 1-7, 23-31, and 40-48. Washout periods were between study days 14 -18, and 31-34. Detailed Description

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, typical methods and materials are described.

[0046] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0047] In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0048] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0049] As used herein, the terms "effective amount" and “hygiene -maintaining amount” refer to an amount of a composition applied to a teat, breast or udder, or otherwise administered to a mammal, that is sufficient to effect one or more beneficial or desired outcomes, for example, in terms of maintaining mammary gland health, protecting against mastitis, and/or reducing somatic cell counts in milk. An “effective amount” or “hygiene -maintaining amount” can be provided in one or more administrations. The exact amount required will vary depending on factors such as the identity and number of individual Lactobacillus species employed, the mammal being treated, the nature of the composition to be applied, the form in which the composition is applied and the means by which it is applied. Thus, it is not possible to specify an exact “effective amount” or “hygiene-maintaining amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. [0050] The term "optionally" is used herein to mean that the subsequently described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiments in which the event or circumstance occurs as well as embodiments in which it does not.

[0051] As described herein, the present inventors have identified a Lactobacillus -containing composition that has protective effects on teat ends, that can reduce somatic cell counts in milk and that can improve teat end sphincter functions and udder health. As exemplified herein, a composition comprising L. paracasei, L. casei and L. buchneri applied as a post-milking teat spray was compared in a cross-over clinical trial in a single herd with a commercial iodine-based post-milking teat disinfectant product. There were fewer abnormal teat end hyperkeratosis events and a clear trend to a lower mean somatic cell count in the animals administered the Lactobacillus composition. There was a relatively increased number of chapped teat ends in the animals administered the commercial iodine product, despite the known skin conditioning properties of this product.

[0052] Embodiments of the present invention therefore relate to compositions comprising one, two or three of L. paracasei, L. casei and L. buchneri and to uses of such compositions as described hereinbelow. Such compositions contain live Lactobacilli . As such, compositions of the present invention can offer advantages over existing compositions and products that do not contain living organisms (for example, lactic acid-based, iodine-based and other chemical-based products). For example, without wishing to be bound by theory, the Lactobacilli produce antibacterial substances, including bacteriocins. In addition to the bactericidal effect of such substances, use of a composition of the present invention may provide a longer lasting effect, contrasting to the one-time high dose exposure associated with commercial chemical-based teat disinfectants; a high initial dose which may then taper off to below the threshold of efficacy. In addition, /z7c/o/?«c/7/z/.s-containing compositions of the invention benefit from the absence of harmful residues, making them GRAS (“generally regarded as safe”) compositions suitable for human use.

[0053] One aspect of the present invention provides a method for protecting or treating a mammalian teat, udder or breast, comprising applying to the teat, udder or breast of a mammal at least one Lactobacillus species selected from L. paracasei, L. casei and L. buchneri, typically a composition comprising at least one Lactobacillus species selected from L. paracasei, L. casei and Z. buchneri.

[0054] By "protecting or treating” it is meant that the Lactobacilli or composition comprising same once applied maintains or improves the health or condition of the teat, udder or breast, in particular the health or condition of the skin of the teat, udder or breast, and/or protects the teat, udder or breast from infection and from mastitis.

[0055] Another aspect of the invention provides a method for maintaining or improving mammary gland health in a mammal, comprising administering to the mammal at least one Lactobacillus species selected from L. paracasei, L. casei and L. buchneri, typically a composition comprising at least one Lactobacillus species selected from L. paracasei, L. casei and Z. buchneri.

[0056] The term "improving" and grammatical equivalents thereof as used herein in relation to mammary gland, teat, breast or udder health, means improvement in one or more manifestations or symptoms of, or associated with, mammary gland, teat, breast or udder health, or in one or one or more parameters associated with mammary gland, teat, breast or udder health resulting from administration or application in accordance with the present invention, beyond that seen in the absence of said administration or application. Such improvement may be observable, detectable or measurable. Thus, the improvement may be quantifiable or qualitative in nature.

[0057] In exemplary embodiments as described herein, mammary gland, teat, breast or udder health may be determined, for example, by determination of somatic cell counts in milk produced by the mammal, by teat condition scoring using assessments well known in the art such as determination of teat end hyperkeratosis, and/or by observation or detection of skin dryness, chapping or cracking on or around the teat or on the breast or udder. Those skilled in the art will readily appreciate that the described measures and parameters associated with mammary gland, teat, breast and udder health are provided as examples only and the scope of the invention is not to be limited by reference to these specific measures and parameters. Any measure or parameter known to those skilled in the art for assessing and monitoring mammary gland, teat, breast and udder health may be employed in accordance with the invention and is contemplated within the scope of the invention. [0058] Another aspect of the invention provides a method for protecting against mastitis, or reducing the incidence of mastitis, in a herd of dairy animals, comprising administering to at least one animal of the herd at least one Lactobacillus species selected from L. paracasei, L. casei and L. buchneri, typically a composition comprising at least one Lactobacillus species selected from A. paracasei, L. casei and A. buchneri.

[0059] The term “protecting against” and grammatical equivalents thereof, as used herein in relation to mastitis refers to any and all applications which prevents, hinders, or retards mastitis or an infection associated with or that results in mastitis, or which otherwise delays the onset of mastitis or an infection associated with or that results in mastitis.

[0060] Another aspect of the invention provides a method for decreasing somatic cell count in the milk from a lactating mammal, comprising administering to the mammal at least one Lactobacillus species selected from A. paracasei, L. casei and A. buchneri, typically a composition comprising at least one Lactobacillus species selected from A. paracasei, A. casei and A. buchneri.

[0061] The methods of the present invention typically comprise topically administering, or applying, a composition to a teat, breast or udder of a female mammal.

[0062] In accordance with the present invention the mammal may be any milk-producing mammal. Exemplary mammals include, but are not limited to, dairy animals (such as dairy cattle, sheep, goats, llamas, pigs etc) kept for the purpose of producing milk, performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals and humans. In exemplary embodiments, the mammal is a human or dairy animal. In an exemplary embodiment the dairy animal is a cow.

[0063] Typically, methods of the present invention may be employed (i.e. compositions may be administered or applied) while the mammal is lactating. The method may be employed (i.e. a composition administered or applied) either before the lactating mammal delivers milk (premilking) or after the lactating mammal delivers milk (post-milking). In exemplary embodiments, application of the present invention to dairy animals may be post-milking. Those skilled in the art will appreciate that in relation to dairy animals, the terms pre-milking and post-milking refer to before or after the animals have been milked. Administration or application in accordance with the invention may occur, for example, within minutes or hours of milking. For example, a post-milking application may take place within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes of milking.

[0064] In exemplary embodiments it is contemplated that the Lactobacilli, or compositions comprising same, may be administered to a mammal daily or less than daily, for example every second day or every third day for the duration of treatment required to achieve the desired outcome. Administration may be continuous, for example on a daily basis or every second day, or may be intermittent with spacing between administrations determined by those skilled in the art.

[0065] The compositions of the present invention may comprise or consist of one of L. paracasei, L. casei and L. buchneri. Alternatively, the compositions may comprise or consist of L. paracasei and L. casei. Alternatively, the compositions may comprise or consist of L. paracasei and L. buchneri. Alternatively, the compositions may comprise or consist of L. casei and L. buchneri. Alternatively, the compositions may comprise or consist of L. paracasei, L. casei and L. buchneri. The compositions may comprise two or more strains of any or all of the Lactobacillus species described herein.

[0066] In an exemplary embodiment, the L. paracasei may comprise the strain deposited with the National Measurement Institute, Australia on 14 December 2012 under Accession Number V12/022849 (strain designated ‘T9’). In an exemplary embodiment, the L. casei may comprise the strain deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number VI I/022948 (strain designated L. zeae). In an exemplary embodiment, the L. buchneri may comprise the strain deposited with National Measurement Institute, Australia on 27 October 2011 under Accession Number VI I/022946. These deposited strains have been previously described and disclosed, for example in WO 2013/063658 and WO 2014/172758, the disclosures of which are incorporated herein in their entirety.

[0067] A composition for use in accordance with the present invention may comprise or consist of the strain deposited with the National Measurement Institute, Australia under Accession Number V 12/022849, or a variant thereof. A composition for use in accordance with the present invention may comprise or consist of the strain deposited with National Measurement Institute, Australia under Accession Number VI 1/022948, or a variant thereof. A composition for use in accordance with the present invention may comprise or consist of the strain deposited with National Measurement Institute, Australia under Accession Number VI 1/022946.

[0068] A composition for use in accordance with the present invention may comprise or consist of the strain deposited with the National Measurement Institute, Australia under Accession Number V12/022849, or a variant thereof, and the strain deposited with National Measurement Institute, Australia under Accession Number V 11/022948, or a variant thereof. A composition for use in accordance with the present invention may comprise or consist of the strain deposited with the National Measurement Institute, Australia under Accession Number V12/022849, or a variant thereof, and the strain deposited with National Measurement Institute, Australia under Accession Number V 11/022946, or a variant thereof. A composition for use in accordance with the present invention may comprise or consist of the strain deposited with the National Measurement Institute, Australia under Accession Number VI I/022948, or a variant thereof, and the strain deposited with National Measurement Institute, Australia under Accession Number VI 1/022946, or a variant thereof.

[0069] A composition for use in accordance with the present invention may comprise or consist of the strain deposited with the National Measurement Institute, Australia under Accession Number V12/022849, or a variant thereof, the strain deposited with National Measurement Institute, Australia under Accession Number VI I/022948, or a variant thereof, and the strain deposited with National Measurement Institute, Australia under Accession Number VI 1/022946, or a variant thereof.

[0070] As used herein, the term "variant" refers to both naturally occurring and specifically developed variants or mutants of the microbial strains disclosed and exemplified herein. Variants may or may not have the same identifying biological characteristics of the specific strains exemplified herein, provided they share similar advantageous properties in terms of treating or preventing infections caused by, or treating or preventing diseases caused by or associated with, microbial pathogens. Illustrative examples of suitable methods for preparing variants of the microbial strains exemplified herein include, but are not limited to, gene integration techniques such as those mediated by insertional elements or transposons or by homologous recombination, other recombinant DNA techniques for modifying, inserting, deleting, activating or silencing genes, intraspecific protoplast fusion, mutagenesis by irradiation with ultraviolet light or X-rays, or by treatment with a chemical mutagen such as nitrosoguanidine, methylmethane sulfonate, nitrogen mustard and the like, and bacteriophage- mediated transduction. Suitable and applicable methods are well known in the art and are described, for example, in J. H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); and J. Sambrook, D. Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), inter alia.

[0071] Also encompassed by the term “variant” as used herein are microbial strains phylogenetically closely related to strains disclosed herein and strains possessing substantial sequence identity with the strains disclosed herein at one or more phylogenetically informative markers such as rRNA genes, elongation and initiation factor genes, RNA polymerase subunit genes, DNA gyrase genes, heat shock protein genes and recA genes. For example, the 16S rRNA genes of a “variant” strain as contemplated herein may share about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a strain disclosed herein.

[0072] The concentrations of individual Lactobacillus species to be employed in accordance with the present invention may depend on a variety of factors including the identity and number of individual species employed, the form in which a composition is applied, the means by which it is applied, and the general health of the mammal to which it is applied.. For any given case, appropriate concentrations may be determined by one of ordinary skill in the art using only routine experimentation. By way of example only, the concentration of each species present in the composition or otherwise to be administered may be from about 1 x 10² cfu/ml to about 1 x 10¹⁰ cfu/ml, and may be about 1 x 10³ cfu/ml, about 2.5 x 10³ cfu/ml, about 5 x 10³ cfu/ml, 1 x 10⁴ cfu/ml, about 2.5 x 10⁴ cfu/ml, about 5 x 10⁴ cfu/ml, 1 x 10⁵ cfu/ml, about 2.5 x 10⁵ cfu/ml, about 5 x 10⁵ cfu/ml, 1 x 10⁶ cfu/ml, about 2.5 x 10⁶ cfu/ml, about 5 x 10⁶ cfu/ml, 1 x 10⁷ cfu/ml, about 2.5 x 10⁷ cfu/ml, about 5 x 10⁷ cfu/ml, 1 x 10⁸ cfu/ml, about 2.5 x 10⁸ cfu/ml, about 5 x 10⁸ cfu/ml, 1 x 10⁹ cfu/ml, about 2.5 x 10⁹ cfu/ml, or about 5 x 10⁹ cfu/ml. In particular exemplary embodiments the final concentration of each Lactobacillus species is about 1 x 10⁶ cfu/ml.

[0073] The Lactobacilli may be cultured according to any suitable method known to the skilled addressee and may be prepared for addition to a composition for administration by, for example, freeze-drying, spray-drying or lyophilisation. Thus, in embodiments of the present invention the Lactobacilli may be in a dried form (such as lyophilized or sporulated form) in a suitable carrier medium, for example a FOS medium or other soluble fiber, sugar, nutrient or base material for the composition. The Lactobacilli may be encapsulated in, for example, a suitable polymeric matrix to improve long-term stability and storage of the compositions. In one example, encapsulation may comprise alginate beads, although those skilled in the art will appreciate that any suitable encapsulation material or matrix may be used. Encapsulation may be achieved using methods and techniques known to those skilled in the art.

[0074] Compositions disclosed herein may optionally further comprise one or more additional microbial organisms which may, for example, act in synergy or concert with, or otherwise cooperate with the Lactobacilli disclosed herein. Examples of suitable additional microbial organisms include additional Lactobacillus species such as L. parafarraginis, L. rapi, L. bulagaricus, L. leveticus, L. reutreri, L. salivarius, and Streptococcus species such as .S'. thermophilus . Those skilled in the art will appreciate that this list is merely exemplary only, and is not limited by reference to the specific examples here provided.

[0075] The present invention provides compositions and methods for application which maintain hygiene and help prevent mammary infections. In accordance with the present invention Lactobacilli and compositions comprising Lactobacilli are typically applied topically to the teats, breast or udder of subject mammals. In particular embodiments, in accordance with the invention compositions are typically applied to the skin of the teat, in particular to skin surrounding the teat orifice, for example as a liquid, emulsion, foam, gel, cream or paste. The compositions may be topically applied in any manner. For example, the composition can be applied by dipping the teat of a mammal into the composition, or by wiping, brushing, or spraying the composition onto the skin of the teat. Thus, in particular embodiments, the composition may be applied in the form of a teat dip or teat spray.

[0076] A teat dip may be applied to the teat directly from a cup or similar container. In an exemplary embodiment, the container may comprise an applicator suitable for applying the composition to the skin of the teat of a dairy animal. Such an applicator can comprise, for instance, a cup portion of a size and shape that will allow the teat to be inserted into the cup and contact the composition. [0077] A teat spray may be an aerosol or pump spray. If it is desired that the composition is applied as a spray, the spray may be an aerosol using a propellant such as dimethyl ether, which can also function as a carrier. The propellant component, which is preferably water or alcohol soluble, may be present in a range of about 10% to 50%, for example about 30%. A composition can also be applied by non-aerosol spray methods such as trigger or pump sprays.

[0078] The compositions, especially liquid compositions, can have any suitable viscosity. In some instances, however, it may be desired to use a thicker composition so that the composition is retained on the skin of the teat for a longer period of time without dripping. Thus, the composition can be formulated so as to have a higher viscosity, such as about 50 cP or more, about 100 cp or more, about 200 cp or more, about 500 cP ormore, about 1000 cP or more, about 2500 cp or more, or even about 5000 cp or more. The viscosity of the composition will typically be less than about 10,000 cp. Viscosity refers to the kinematic viscosity measured at standard temperature and pressure (25°C and 1 atm).

[0079] Compositions to be applied in accordance with the invention may further comprise one or more emollients. Suitable emollients include, for example, glycerol, sorbitol, lanolin, linoleic acid, propylene glycol, glycerin, D-panthenol, poly ethylene glycol (PEG) (e.g. molecular weight 200-10,000) and esters thereof, acyl lactylates, polyquatemium compounds, glycerol laurate, glycerol cocoate, PEG-7 glycerol cocoate, stearic acid, hydrolyzed silk peptide, silk protein, aloe vera gel, guar hydroxypropyltrimonium chloride, alkyl poly glucoside/glyceryl luarate, shea butter, coco butter, and combinations or mixtures of any two or more of said emollients. In exemplary embodiments, the one or more emollients may comprise glycerol, sorbitol, lanolin, linoleic acid, or a combination or mixture of two or more thereof. In some embodiments, emollients may be present in an amount of between about 1% to about 40% by weight of the total weight of the composition, for example, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% or about 40% by weight of the total weight of the composition.

[0080] Compositions to be applied in accordance with the invention may further comprise one or more surfactants. Suitable surfactants include, for example, anionic, cationic, nonionic, zwitterionic and amphoteric surfactants, and can be high foaming, low foaming, moderate foaming, or non-foaming type surfactants. Anionic surfactants include, for example, sodium stearate, linear alkyl benzene sulfonic acid, linear alkyl benzene sulfonate, alkyl sulfomethyl ester, a-olefin sulfonate, alcohol ether sulfate, alkyl sulfate, alkylsulfo- and dialkylsulfo succinate, and salts thereof. Nonionic surfactants include, for example, polysorbates such as polysorbate 20 and polysorbate 80, alkyl polyglucoside, alkyl ethoxylated alcohol, alkyl propoxylated alcohol, ethoxylatedpropoxylated alcohol, alkylphenol ethoxylates, sorbitan, sorbitan ester, alkanol amide, and polyethoxylated polyoxypropylene block copolymers (poloxamers). Amphoteric surfactants include, for example, alkyl betaines and alkyl amphoacetates such as cocoamidopropyl betaine, coco betaine, sodium cocoamphoacetate, sodium lauroamphoacetate and sodium cocoamphodiacetate. The surfactant component may comprise a combination or mixture of any two or more of said surfactants. In exemplary embodiments, the one or more surfactants may comprise an alkyl betaine, polysorbate, sodium stearate, or a combination or mixture of two or more thereof. In some embodiments, surfactants may be present in an amount of between about 1% to about 40% by weight of the total weight of the composition, for example, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% or about 40% by weight of the total weight of the composition.

[0081] Compositions to be applied in accordance with the invention may further comprise a barrier or film-forming agent, or a thickener. Suitable barrier and film-forming agents and thickeners include, for example, polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA or PVOH), polyacrylate, polyacrylamide, latex, carbomer, glycerol, hemicelluloses (e.g., arabinoxylanes and glucomannanes), plant gum materials (e.g., guar gum, gum arabic, and johannistree gums), cellulose and derivatives thereof (e.g., methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, methylhydroxypropylcellulose (HPMC) and ethylhydroxyethylcellulose), starch and starch derivatives (e.g., hydroxyethyl starch or cross linked starch), microbial, seaweed and other polysaccharides (e.g., xanthan gum, sodium alginate, carrageenan, curdlan, pullulan, dextran and molasses), dextran sulfate, whey, collagen, pectin, gelatin, chitosan, chitosan derivatives, and polysulfonic acids and their salts. Clays and modified clays (e.g., bentonite or laponite), colloidal alumina or silica, and fatty acids or salts thereof can also be used as thickeners, cothickeners, or stability agents for thickeners. In some embodiments, barrier and film -forming agents and thickeners may be present in an amount of between about 1% to about 40% by weight of the total weight of the composition, for example, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% or about 40% by weight of the total weight of the composition.

[0082] Compositions employed in accordance with the present invention may include additional additives, such as pigments or dyes to act as an indicator whether a particular animal has been treated. For example, a dye such as methylene blue or FD & C Blue Dye #3 may be included in a range of, for example, from about 0.01% to 1.0%, of the total weight of the composition.

[0083] Methods of the present invention may further comprise administering to the mammal one or more antimicrobial agents. Administration or exposure to a composition disclosed herein and an antimicrobial agent may be at the same time or at different times, i. e. simultaneous or sequential. Antimicrobial agents may be co-formulated with Lactobacillus species used in the present invention. Compositions disclosed herein may therefore comprise one or more antimicrobial agents. In instances where the Lactobacilli and antimicrobial agents are formulated in different compositions, they can be administered or delivered by the same or different routes or means.

[0084] As used herein the term "antimicrobial agent" refers to any agent that, alone or in combination with another agent, is capable of killing or inhibiting the growth of one or more species of microorganism. Suitable antimicrobial agents include, but are not limited to, antibiotics, detergents, surfactants, agents that induce oxidative stress, bacteriocins and antimicrobial enzymes (e.g. lipases, pronases and lyases) and various other proteolytic enzymes and nucleases, peptides and phage. Reference to an antimicrobial agent includes reference to both natural and synthetic antimicrobial agents.

[0085] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

[0086] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. [0087] The present disclosure will now be described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples

[0088] The following examples are illustrative of the invention and should not be construed as limiting in any way the general nature of the disclosure of the description throughout this specification.

Example 1 - Efficacy of a Lactobacillus-based teat spray on udder health in lactating dairy cows

Study design and animal management

[0089] The inventors conducted a positive-controlled, randomised 2x3 cross-over study involving two experimental groups (LA CT treated, positive control [PC] treated) and three treatment periods (Figure 1 A). The study was conducted using the year-round calving University of Queensland-Gatton 230 milking cow research dairy herd. The herd is managed as two groups of milking cows typical of Queensland dairies: a combined fresh and early lactation group (up to 100 days in milk (DIM); fed a total mixed ration; n = 90); and a second group comprising mid- and late lactation cows (> 100 DIM; fed pasture and a mixed ration; n = 140). Grazing pasture consisted of a mixture of temperate and tropical plant species. Pasture supplemented with a silage-based mixed ration was sufficient to meet the maintenance and production requirements of a cow producing 25 L of milk per day. The experimental procedures were approved by the University of Queensland Animal Ethics Committee prior to the start of the study.

[0090] The sample size required to evaluate changes in udder health (i.e. somatic cell count (SCC)), 13 cows, was based on the a priori assumptions for SCC of an alpha of 95%, a power of 90%, a common standard deviation of 20,000 cells/mE, a difference of 40,000 cells/mE and a correlation between group means of 0.1. For teat end hyperkeratosis score (TES) comparison, it was assumed that TES improvements (lower scores) would be associated with a decreased risk of mastitis in this herd. Sample size calculations determined that fifty cows would allow detection (with 95% confidence, power of 90%, pooled variance one teat end score, 1: 1 ratio treatment to control sample size) of a difference of one teat end score (one to five scoring scale, see below) between the mean TES before and after LACT teat spray treatment. Therefore, the study sample size was 50 cows divided equally into the two experimental groups.

[0091] Fifty apparently healthy lactating dairy cows of mixed age, stage of lactation and breed, were randomly selected (simple random sampling without replacement) from a pool of 60 eligible cows (median TES of 2 or less, apparently normal quarter milk, SCC less than 300,000 for at least the past 8 weeks, no history of systemic disease or clinical mastitis in the eight weeks preceding the study start date). Enrolled cows were managed, fed and milked as per the routine farm practices with the exception that the study animals were maintained as separate groups from the main herd.

[0092] Cows were milked twice daily at 04:00 hr and 15:00 hr in a double 14 rapid exit high line parallel parlour (GEA Westfalia™, Victoria, Australia). The automatic cup removal (ACR) system was set to detach milking clusters when milk flow decreased to 0.2 L/min. Milk-line vacuum pressure was checked during the milking of each turn of cows in the parlour. The vacuum pressure was maintained between 45 and 48 kPa by a variable frequency vacuum pump. To ensure a consistent milking routine, all milking staff received ongoing training in milking machine operation and milking protocols. Based on study farm animal health records, the estimated incidence risk of clinical mastitis in the source herd was, on average, 22% (95% confidence interval [CI] = 17 - 33%).

Experimental design and procedures

[0093] The Lactobacillus based teat spray evaluated was a liquid formulation suitable for application as a post-milking teat spray. The formulation contained a mixture of three Lactobacillus species (Lactobacillus paracasei, L. buchneri, L. casei,' minimum 10⁶ cfii/mL of each strain) in saline (0.9%NaCl). This preparation was stored at 4°C until applied to the teats (without an emollient) using a hip mounted, hand operated mobile teat sprayer (HipSpray™, Ambic Equipment Ltd., Davies Way, Brisbane, Australia) set to deliver 10 mL per teat as per manufacturer instructions. The positive control treatment (PC) was a commercial, iodine-based, post-milking teat disinfectant (Dairy Power Mastidyne™ Iodophor 20g/L available iodine, 2% free iodine, ECOLAB, Sydney, Australia) supplied as a 2-part concentrate and automatically mixed with potable water on an as needed basis for application to the teats using a spray wand. The product as applied consisted of three parts Dairy Power Mastidyne™ Iodophor teat sanitiser, eight parts cool potable water, and one-part Dairy Power Glysoft™ udder emollient 10% solution (ECOLAB, Sydney, Australia) as per the manufacturer’s instructions. The inline sprayer gun (AMBIC™, Davisway/DASCO, Victoria, Australia) was set to pump a volume of 10 mL per teat covering the entire teat as per manufacturer’s instructions.

[0094] Cows were assigned to groups using simple random sampling without replacement. Simple random sampling was also used to assign a specific teat treatment (either LACT or PC) to one ofthe two 25-cow study groups forthe first and second 2-week long experimental periods. The third 2-week long experimental period was a replica of the second period. The two groups of animals were milked separately with an abbreviated milking machine cleaning cycle run before and between the milkings. To minimize the risk of residual effect of the two treatments, each treatment period was separated by a minimum washout period of 48 hours during which there were no applications of either treatments. This minimum washout period was based on an absence of detectable (c/PCR) biological residues of the LACT organisms at 36 hours posttreatment.

[0095] Composite milk samples (50 mL) were collected bi-weekly from each cow at the Monday and Friday morning milkings, preserved with Acticide L-Bronopol, and SCC determined by the Australian Herd Recording Services (Kenilworth, Queensland, Australia) using an automated cell counter (Fossomatic 5000, Foss Electric). Teat ends were evaluated and scored weekly.

[0096] The TES for each cow were determined by a single individual during a single milking session using the one to five TES scale adapted from Mein et al. (2001): score 1 = normal with no apparent ring present at the teat end; score 2 = smooth and slightly rough ring; score 3 = rough ring; score 4 = very rough ring; score 5 = open lesions or scabs. The scorer was blinded to the treatment allocations to study animals. The TES were averaged (median) at the cow level. The median TES were used in subsequent analyses.

Statistical analyses

[0097] Summary statistics generated for continuous or categorical variables (as appropriate) included: mean, median, standard deviation (SD), first and third quartiles (1^st and 3^rd Q), minimum, maximum, counts and percentage, as appropriate. Chi-squared test was used to assess the homogeneity of TES count distribution between the treatment groups. The statistical analysis was conducted in R (R Development Core Team, 2019).

[0098] The association between the LACT and PC groups and TES was assessed using a multivariate, mixed effects, ordered logistic regression model. The analysis was performed at the quarter-level to allow the model to account for the repeated measurements in TES and clustering of teats within cows (the sampling unit, i.e. the udder quarters nested within the cow, i.e. the experimental unit). Square root transformation was applied to the study day to maintain the linearity of the modelled log odds across the study days and reduce the risk of violating the proportional odds assumption (see below). Statistical significance was declared at an alpha of 0.05 or less. The association between LACT and PC and individual cow SCC was quantified using a multivariate mixed-effects linear regression model with cow fitted as a random effect. Model building followed forward selection procedure. First order interaction terms were tested and were retained of interaction terms was significant at a likelihood ratio test P value of 0.05 or less. Models specification followed that described by St-Pierre (2007) and took the following generic forms:

Somatic cell counts - animal level data [1]

Teat end scores - quarter data [2] ytjmk = Intercept + Tretamenti + Period] + ^Time + Quarter_m + S_mik + £ij_mk

[0099] Where ytjk and ytjmk denote the response observed at the cow and quarter levels, respectively for model [1] and [2], in period/ of treatment,, and Si* and St_mk are the random error term forthe A ¹¹ cow (or Quarter_m nested within cow, respectively) in the z^th treatment group. The outcome variable (SCC) was log transformed to stabilise the SCC variance and restore normality of the data. Study day was modelled as a polynomial (of the 4^th order) variable with the order of the polynomial determined using the Akaike Information Criterion (AIC). The residuals (eij_k and Eij_mk) of the random effect term were assumed to be normally distributed with a mean of zero, a variance of a², and an autoregressive correlation structure of the first order. Overall model fit was based on AIC, Bayesian information criterion (BIC) and visual assessment of Pearson ’s residuals against fitted values, Q-Q standardised residuals against standardised normal quantiles violated the normality assumption (Bates and Maechler, 2010). The proportional odds assumption was checked visually by examining the vertical consistency of distances between any two of the orders TES scores (at the logit scale) within explanatory variable in the model. The overall effect of LACT and PC over the three treatment periods was further explored using least-squares means (LSM) prediction from the final mixed-effects linear model. (LSM means predictions were averaged predictions across all covariates in the model fixed at the reference levels.) Statistical significance was declared at an alpha of 0.05 or less. Because SCC were log transformed, interpretation of the coefficient represents a unit change in log SCC. The antilog of each coefficient is interpreted as follows: for a continuous explanatory variable, the antilog of the coefficient represent a change in average (geometric mean) SCC for each unit change in the continuous variable. For a categorical variable, the antilog of the coefficient is the ratio of the means for each level of the categorical variable compared with the reference category. All analysis using ordinal (Christensen, 2019), emmenas (Lenth, 2020), visreg (Breheny, 2017), nlme and lme4 (Bates, 2007; Bates and Maechler, 2010) statistical packages in R.

Results

[00100] The teat end scores of the cows in the LACT and PC groups followed a similar curvilinear trend (Figures 1A, B). The TES values were highest during the second of the three 2-week treatment periods. Overall, the LACT group was associated with fewer TES 4 (13%) and 5 (1%) and more TES 1 (7%), (Chi-squared = 29.042, df = 4, P < 0.01) compared to the PC group (15%, 2% and 2% respectively, Figure IB). In parallel with the TES results, the SCC associated with both treatments followed a similar trend (Figure 1C).

[00101] The results from the multivariable model for TES are shown in Table 1. Holding the covariates at their reference, on average, the odds of a shift from low to high TES tended to be lower for cows in the LACT group compared to TES for cows in the PC group (OR = 0.74, 95% CI 0.54 - 1.01, P = 0.06; Table 1). The TES value of individual cows at baseline influenced the odds of observing a high TES during the study. Irrespective of treatment assignment, for each unit TES score above score 1 at baseline, the odds of TES changing from a low to a high score during the study increased approximately three-fold (OR = 3.46, 95% 2.07 - 5.78, P < 0.01; Table 1). Table 1. Coefficient (standard errors) and odd ratios (95% confidence interval) from final multivariate mixed-effects ordered logistic regression model fitted on cows teat end scores (TESf) for the study animals.

Variable Coefficient (SE) Odd Ratio (95% CI) P-value

Teat end scores at baseline 1.24 (0.26) 3.46 (2.07 - 5.78) <0.01

Daily milk production (L; centred^) -0.01 (0.02) 0.99 (0.95 - 1.03) 0.59

Time (Day; square root) -0.01 (0.01) 0.99 (0.98 - 1.01) 0.15

Treatment Group

PC Reference 1

LACT -0.30 (0.16) 0.74 (0.54 - 1.01) 0.06

Treatment Period

Period 1 Reference 1

Period 2 2.03 (0.18) 7.61 (5.36 - 10.80) <0.01

Period 3 1.95 (0.29) 7.01 (3.98 - 12.39) <0.01

Udder Quarter

Fore quarters Reference 1

Hind quarters -1.32 (0.19) 0.27 (0.18 - 0.39) <0.01

Random effect Variance (SE) 95% Confidence Interval

Cow 0.33 (0.18) 0.11 - 0.94

Quarter 0.60 (0.20) 0.31 - 1.15 f Teat end scores (scale 1 to 5; one is a normal teat end with no ring apparent; 5 is a severely abnormal teat end, rough, raised and obvious ring at teat end)

{centred on the mean.

Key: SE Standard Error; CI Confidence Interval, L litres. PC iodine-based positive control, LACT lactobacillus based product. Model fitted using mixed-effect linear regression procedure in R. Model fitted with quarter nested with cow and animal fitted as random effect. Robust standard error estimation was used to adjust for clustering within cow. Final model AIC = 2700.561, Wald Chi-squared = 237.20 P <0.001, Log psuedolikelihood = - 1337.2804.

[00102] The results from the multivariable model for SCC are shown in Table 2. After controlling for the effect of TES at baseline, milk production, and holding the remaining covariates at their reference, on average, there was a tendency for SCC in the LACT group to be 9% lower (antilog of coefficient = 0.91, 95% CI 0.80 - 1.03, P = 0.13) compared to the PC group (Table 2). Across all three treatment periods, the average SCC for cows in the PC group was 14% higher (1.14, 95% CI = 0.97 - 1.33, P = 0.07; (Table 3) compared to the LACT treated

COWS. Table 2. Coefficient (standard errors) and antilog of the estimated coefficients (95% confidence interval) from final multivariate mixed-effects linear regression model fitted on individual cows somatic cell count (SCC; 000’s cells/mL) for the study animals.

tcentred around the mean.

$ The order of the fitted polynomial was assessed using model AIC.

Key: Se Standard Error; CI Confidence Interval, PC iodine-based positive control, LACT lactobacillus based product. Model fitted using mixed-effect linear regression procedure in R. Model fitted with cow fitted as random effects. Final model AIC = 628.929, Loglikelihood = -303.4645.

Table 3. Least-square means predictions (marginal means; at the log and antilog scales) and mean ratios obtained over the grid of predictors settings from linear mixed-effects model shown in Table 2.

Predicted marginal effect

fvalues are in 000’s cells/mL’ . $ Bonferroni adjusted for multiple comparisons Key: SE Standard Error; CI Confidence Interval.

[00103] The present study evaluated the effect of a probiotic -based, post-milking teat skin spray on the health of the mammary glands using the proxy parameters of SCC and TES. There were fewer abnormal TES in the LACT group. The odds of a TES shifting (throughout the study) from a lower to a higher score was lower (OR = 0.74, 95% CI 0.54 - 1.01, P = 0.06) for cows in the LACT group. When the effect of the explanatory variables was controlled, there was a clear trend to a lower mean SCC in the LACT group (antilog of coefficient = 0.91, 95% CI 0.80 - 1.03, P = 0.13) compared to the PC group cows. There was a relatively increased number of chapped teat ends in the control group cows. This occurred despite the skin conditioning properties of this commercial product relative to the product under study in the LACT group. In contrast to treatment with the commercial product, cows in the LACT group showed a strong propensity towards a lower risk of an increase in TES. This suggests the Lactobacillus -based product has a protective effect on teat ends. References

Bates, D. (2007). Ime4: Linear mixed-effects models using S4 classes. R package version 0.99875-9.

Bates, D., and Maechler, M. (2010). Ime4: Linear mixed-effects models using S4 classes.

Breheny, P.B., W. (2017). Visualization of regression models using visreg. R

56 -71.

Christensen, R.H.B. (2019). Ordinal - Regression models for ordinal data. R package version 2019.12-10. htlps://CRAN.R-project.org/package^::::ordinal.

Lenth, R. (2020). "emmeans: Estimated marginal means, aka least-squares means. R package version 1.4.8. https://CRAN.R-proiect.org/package^:::emmeans".

Mein, G.A., Neijenhuis, F., Morgan, W.F., Reinemann, D.J., Hillerton, J.E., Baines, J.R., et al. (2001). "Evaluation of bovine teat condition in commercial dairy herds: 1. Non-infectious factors", in: Proc. 2nd International Symposium on Mastitis and Milk Quality.

R Development Core Team (2019). "R: A Language and Environment for Statistical Computing". (Vienna, Austria: R Foundation for Statistical Computing).

St-Pierre, N.R. (2007). Design and analysis of pen studies in the animal sciences. J Dairy Sci 90, E87-E99. doi: 10.3168/jds.2006-612.

Claims

26 Claims

1. A method for treating a mammalian teat, udder or breast, or protecting a teat, udder or breast from infection, comprising applying to the teat, udder or breast of a mammal a hygienemaintaining composition comprising at least one Lactobacillus species selected from L. paracasei, L. casei an L. buchneri.

2. A method according to claim 1, wherein the composition comprises two of A. paracasei, L. casei and L. buchneri.

3. A method according to claim 1 , wherein the composition comprises L. paracasei, L. casei and A. buchneri.

4. A method for maintaining or improving mammary gland health in a mammal, comprising administering to the mammal a composition comprising at least one Lactobacillus species selected from A. paracasei, L. casei and A. buchneri.

5. A method according to claim 4, wherein the composition comprises two of A. paracasei, A. casei and A. buchneri.

6. A method according to claim 4, wherein the composition comprises A. paracasei, A. casei and A. buchneri.

7. A method according to any one of claims 4 to 6, wherein the Lactobacillus species are administered topically to a teat, breast or udder of the mammal.

8. A method according to any one of claims 4 to 7, wherein the method is for maintaining or improving teat health or udder health in a dairy animal or a herd of dairy animals.

9. A method according to any one of claims 4 to 8, wherein assessing or determining mammary gland health comprises determining or measuring somatic cell count in milk produced by the mammal.

10. A method for protecting against mastitis in a mammalian teat, breast or udder, comprising applying to the teat, breast or udder a composition comprising at least one Lactobacillus species selected from A. paracasei, A. casei and A. buchneri.

11. A method according to claim 10, wherein the composition comprises two of A. paracasei, A. casei and A. buchneri.

12. A method according to claim 10, wherein the composition comprises L. paracasei, L. casei and Z. buchneri.

13. A method according to any one of claims 10 to 12, wherein assessing or determining protection against mastitis comprises determining or measuring somatic cell count in milk produced by the mammal.

14. A method according to any one of claims 1 to 13, wherein the mammal is lactating.

15. A method for decreasing somatic cell count in the milk from a lactating mammal, comprising administering to the mammal a composition comprising at least one Lactobacillus species selected from Z. paracasei, L. casei and Z. buchneri.

16. A method according to claim 15, wherein the composition comprises two ofZ. paracasei,

L. casei and Z. buchneri.

17. A method according to claim 15, wherein the composition comprises L. paracasei, L. casei and Z. buchneri.

18. A method according to any one of claims 1 to 17, wherein the mammal is a dairy animal.

19. A method according to claim 18, wherein the dairy animal is a cow.

20. A method according to claim 18, wherein the composition is applied post-milking.

21. A method according to any one of claims 1 to 17, wherein the mammal is a human.

22. A method according to any one of claims 1 to 21, wherein the composition is applied to a teat, breast or udder as a teat spray, dip, cream or gel.

23. A method according to any one of claims 1 to 22, wherein the composition further comprises one or more emollients.

24. A method according to any one of claims 1 to 23, wherein the composition further comprises one or more surfactants.

25. A method according to any one of claims 1 to 24, wherein the composition further comprises xanthan gum.

26. A teat conditioner composition comprising at least one Lactobacillus species selected from Z. paracasei, L. casei and Z. buchneri.

27. A teat conditioner composition according to claim 26, comprising two of Z. paracasei,

Z. casei and Z. buchneri.

28. A teat conditioner composition according to claim 26, comprising L. paracasei, L. case! and /.. buchneri.

29. A teat conditioner composition according to any one of claims 26 to 28, formulated for topical application to the teats, breast or udder of a mammal.

30. A teat conditioner composition according to any one of claims 26 to 29, comprising at least about 10⁶ CFU/ml of each of the at least one Lactobacillus species, at least about 10⁶ CFU/ml of each of the two Lactobacillus species, or at least about 10⁶ CFU/ml of each of L. paracasei, L. casei an L. buchneri.

31. A teat conditioner composition according to any one of claims 26 to 30, further comprising one or more emollients.

32. A teat conditioner composition according to any one of claims 26 to 31, further comprising one or more surfactants.

33. A teat conditioner composition according to any one of claims 26 to 32, further comprising xanthan gum.