WO2015000022A1 - Treatment and prevention of mastitis - Google Patents

Abstract

The present invention relates to mastitis and to the identification of factors that play a role in the aetiology and progression of mastitis. In particular, an association between mastitis and toll-like receptors has been identified. The present invention therefore provides, amongst other things, methods for the treatment and prevention of mastitis, improving lactation in a mastitis affected subject, and/or treating and/or preventing mammary tissue damage in a mastitis affected subject, by inhibiting the activity of at least one toll-like receptor in the subject.

Description

TREATMENT AND PREVENTION OF MASTITIS

PRIORITY CLAIM

[0001] This international patent application claims priority to Australian provisional patent application 2013902497 filed on 5 July 2013, the contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to mastitis and to the identification of factors that play a role in the aetiology and progression of mastitis. In particular, an association between mastitis and toll-like receptors has been identified. The present invention therefore provides, amongst other things, methods for the treatment and prevention of mastitis by inhibiting the activity of at least one toll-like receptor in an affected subject.

BACKGROUND OF THE INVENTION

[0003] Mastitis is a common condition the affects the mammary gland of lactating mammals. In humans, mastitis has been shown to affect between 17% and 27% of lactating mothers primarily in the first three-months post-partum, whilst in dairy cattle mastitis has been estimated to cost between 90 and 135 dollars per cow per year primarily due to a loss of milk production and milk quality in mastitis affected animals.

[0004] Mastitis has a diverse range of manifestations and symptoms. It can cause localised pain, fever, engorgement of the mammary glands as a whole or localised engorgement (focal engorgement) caused by a blocked mammary duct, tissue damage including damage to mammary ducts and alveoli, changes in the functioning of the mammary gland that ultimately lead to a decrease in milk production and change in composition, the development of breast abscesses, and in some severe cases mastitis has led to death of the affected subject.

[0005] Approximately 80 years ago, bacterial infections were proposed as a causative agent of mastitis. Over the following 40 years improved hygiene standards and the widespread administration of antibiotics, primarily in the dairy industry, significantly reduced the incidence of mastitis. This led to the view that bacterial infections were the cause of mastitis. As a consequence of this, antibiotics are commonly employed to prevent and treat mastitis in both humans and dairy cattle. However, despite the wide spread use of antibiotics, the rates of mastitis remain problematic. [0006] Many cases of mastitis present without evidence of an active bacterial infection. Furthermore, bacterial counts in non-mastitis affected subjects are often comparable to those of mastitis affected subjects and recent studies have shown that mastitis affected women administered antibiotics do not shown improved response rates compared to mastitis affected women not administered antibiotics. This brings into doubt the role of bacterial infections as the causative agent in mastitis. As a result, the presence of a bacterial infection is not identified as a hallmark diagnostic or causative feature of mastitis. Presently, the World Health Organisation (WHO) identifies milk stasis as the primary cause of mastitis, which may or may not be further accompanied by an active bacterial infection.

[0007] Despite the identified role of milk stasis in mastitis, the mechanism by which it causes the development of mastitis is not understood. Consequently, there is a need for new methods and techniques for the treatment and prevention of mastitis that do not solely target bacterial infection of the mammary gland.

[0008] Unfortunately, there have not been suitable animal models to sufficiently study the factors that may be involved in the development and progression of mastitis over a prolonged period of time, and therefore there has not been the ability to sufficiently study methods and techniques for the prevention and treatment of mastitis. In this regard, animals such as cattle are not considered suitable for the study of mastitis development and progression due to their diverse genetic backgrounds, their long gestational period, and critically, the inability to easily genetically manipulate cattle so as to isolate contributory factors in disease development.

[0009] Consequently, murine models are typically used for the study of mastitis. However, currently used murine models fail to truly replicate mastitis, and do not permit the study of the physiological outcomes of the disease. For example, mouse models currently utilised to study mastitis rely on the perfusion into an entire mammary gland, typically via the teat canal, of an inflammatory stimulus such as lipopolysaccharide (LPS). This results in the entirety of the mammary gland receiving LPS. As a result, this model necessitates that the pups cannot be housed with the lactating mother due to the risk of exposure to LPS. Therefore, these experiments are either performed on pre-partum mice (typically at gestational day 15 or greater - i.e. approximately 5 days prior to delivery), or postpartum mice where the pups are removed. However, neither of these models sufficiently replicates mastitis and therefore neither of these models make it possible or feasible to actually study mastitis. [0010] In the case where the inflammatory stimulus is administered pre-partum the mammary gland has not sufficiently formed such that it is analogous to a functioning postpartum mammary gland. While in models whereby the stimulus is administered postpartum, the necessary removal of the pups, or the fact that pups will not feed on the treated gland, means that the mammary gland will rapidly go into involution and the architecture of the mammary gland will rapidly change leading to significant cellular apoptosis and loss of alveoli and ducts. Thereby, when using these models it is impossible to distinguish between long-term changes in the gland as a result of the inflammatory stimulus and changes in the gland due to natural involution of the gland as a result of the weaning of the pups. In any event, in currently known models of mastitis, the administration of an inflammatory stimulus only represents an acute inflammatory event and is typically resolved in a short period of time (24 to 48 hours). In comparison, when an animal suffers from mastitis it is usually typified by a chronic inflammatory stimulus (such as milk stasis) in a localised area of the mammary gland (as opposed to the entirety of the mammary gland). At the same time the lactating mother usually continues to feed the infant from the mastitis affected mammary gland.

[0011] Given the clear difference between the current animal models used for studying mastitis-like disease and how mastitis actually develops and progresses, there is a need for the development of new methods and techniques for the treatment and prevention of mastitis which are derived from utilisation of an animal model that more accurately replicates the induction and progression of mastitis.

[0012] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

[0013] In addressing the aforementioned issues, the inventors have developed an in vivo mouse model of mastitis that more accurately resembles the development and progression of the disease by maintaining a functional mammary gland and creating a localised area of chronic inflammatory stimulus, thereby simulating ductal blockage and consequential milk stasis. The inventors have demonstrated that the early local and systemic immune response in this model is consistent with human studies of the disease. However, unlike previous studies in mouse models, which only look at the first 24 to 48 hours, the current model has permitted the inventors to track progression of the disease over a prolonged period of time (up to 10 days). Accordingly, for the first time, this model has permitted the inventors to investigate factors playing a role in mastitis, and in particular the role that toll-like receptors play in establishment, progression and resolution of the disease. Critically, this model, unlike previous models, has permitted the inventors to develop methods for improving the function of a mastitis affected mammary gland, such as improving lactation and preventing mammary tissue damage.

[0014] Accordingly, in a first aspect the present invention provides a method of treating and/or preventing mastitis in a subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject.

[0015] In one embodiment, the method includes administering to the subject an agent which inhibits the activity of the at least one toll-like receptor.

[0016] In some embodiments, inhibiting the activity of at least one toll-like receptor includes inhibiting the signalling of the at least one toll-like receptor. In one embodiment, inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R- associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-KB transcription factor, TIR-domain-containing adapter-inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factors-activated kinase (TAK1 ), TAK- binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

[0017] In some embodiments, the toll-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9. In one embodiment, the toll-like receptor is tolllike receptor 4.

[0018] In some embodiments, the method includes the further step of administering to the subject at least one antimicrobial agent. In one embodiment, the at least one antimicrobial agent is an antibacterial agent.

[0019] In some embodiments, the method includes the further step of administering to the subject at least one anti-inflammatory agent. In one embodiment, the at least one antiinflammatory agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

[0020] In some embodiments, the subject is non-human. For example, the subject may be bovine. In some embodiments the subject is a human.

[0021] Through use of the in vivo mouse model of mastitis, the inventors have established that toll-like receptor deficient mice exhibit better disease resolution as evidenced by functional lactation.

[0022] Accordingly, in a second aspect the present invention provides a method of improving lactation in a mastitis affected subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject.

[0023] In one embodiment of the second aspect of the invention, the method includes administering to the subject an agent which inhibits the activity of the at least one toll-like receptor.

[0024] In some embodiments of the second aspect of the invention, inhibiting the activity of at least one toll-like receptor includes inhibiting the signalling of the at least one toll-like receptor. In one embodiment, inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R-associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-κΒ transcription factor, TIR-domain-containing adapter- inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factor-3-activated kinase (TAK1 ), TAK-binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

[0025] In some embodiments of the second aspect of the invention, milk production in the subject is increased. [0026] In some embodiments of the second aspect of the invention, the toll-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9. In one embodiment, the toll-like receptor is toll-like receptor 4.

[0027] In some embodiments of the second aspect of the invention, the method includes the further step of administering to the subject at least one antimicrobial agent. In one embodiment, the at least one antimicrobial agent is an antibacterial agent.

[0028] In some embodiments of the second aspect of the invention, the method includes the further step of administering to the subject at least one anti-inflammatory agent. In one embodiment, the at least one anti-inflammatory agent is is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

[0029] In some embodiments of the second aspect of the invention, the subject is non- human. For example, the subject may be bovine. In some embodiments the subject is a human.

[0030] It is known that damage to mammary tissue, including damage to mammary ducts and alveoli, is a feature of severe mastitis. Accordingly, in a third aspect the present invention provides a method of treating and/or preventing mammary tissue damage in a mastitis affected subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject.

[0031] In one embodiment of the third aspect of the invention, the method includes administering to the subject an agent which inhibits the activity of the at least one toll-like receptor.

[0032] In some embodiments of the third aspect of the invention, inhibiting the activity of at least one toll-like receptor includes inhibiting the signalling of the at least one toll-like receptor. In one embodiment, inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R-associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-κΒ transcription factor, TIR-domain-containing adapter- inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factor-3-activated kinase (TAK1 ), TAK-binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

[0033] In some embodiments of the third aspect of the invention, the toll-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9. In one embodiment, the toll-like receptor is toll-like receptor 4.

[0034] In some embodiments of the third aspect of the invention, the method includes the further step of administering to the subject at least one antimicrobial agent. In one embodiment, the at least one antimicrobial agent is an antibacterial agent.

[0035] In some embodiments of the third aspect of the invention, the method includes the further step of administering to the subject at least one anti-inflammatory agent. In one embodiment, the at least one anti-inflammatory agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

[0036] In some embodiments of the third aspect of the invention, the subject is non-human. For example, the subject may be bovine. In some embodiments the subject is a human.

[0037] In a fourth aspect, the present invention provides use of an agent that inhibits the activity of at least one toll-like receptor for the treatment and/or prevention of mastitis in a subject, for improving lactation in a mastitis affected subject, and/or for treating and/or preventing mammary tissue damage in a mastitis affected subject. [0038] In a fifth aspect, the present invention provides use of an agent that inhibits the activity of at least one toli-Iike receptor in the manufacture of a medicament for the treatment and/or prevention of mastitis in a subject, for improving lactation in a mastitis affected subject, and/or for treating and/or preventing mammary tissue damage in a mastitis affected subject.

[0039] In a sixth aspect, the present invention provides a pharmaceutical composition when used for treating and/or preventing mastitis in a subject, improving lactation in a mastitis affected subject, and/or treating and/or preventing mammary tissue damage in a mastitis affected subject, the composition including an effective amount of an agent that inhibits the activity of at least one toil-like receptor in the subject.

[0040] In some embodiments of the sixth aspect of the invention, the agent inhibits the signalling of the at least one toll-like receptor in the subject. In one embodiment, inhibiting the signaliing of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1R- associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-actlvated protein kinase (MARK), an Ι Β kinase (IKK), a NF-KB transcription factor, TIR-domain-containing adapter-inducing interferon-β (TRIP), interferon regulatory factor (!RF) 3, iRF7, transforming growth factors-activated kinase (TAK1 ), TAK- binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

[0041] In some embodiments of the sixth aspect of the invention, milk production in the subject is increased,

[0042] In some embodiments of the sixth aspect of the invention, the toil-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toli-Iike receptor 4, toli-Iike receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9. In one embodiment, the toil-tike receptor is toli-Iike receptor 4.

[0043] In some embodiments of the sixth aspect of the invention, the composition further includes at feast one antimicrobial agent, in one embodiment, the at least one antimicrobial agent is an antibacterial agent.

[0044] In some embodiments of the sixth aspect of the invention, the composition further includes at least one anti-infiammatory agent. In one embodiment, the at least one antiinflammatory agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

[0045] In some embodiments of the sixth aspect of the invention, the composition is adapted to be administered orally, to be administered topically, to be administered as an injection, or is adapted for mucosal administration.

[0046] In some embodiments of the sixth aspect of the invention, the subject is non-human. For example, the subject may be bovine. In some embodiments the subject is a human.

[0047] In a seventh aspect, the present invention provides a combination product including:

(i) an agent that inhibits the activity of at least one toll-like receptor; and

(ii) at least one antimicrobial agent

the agent and the at least one antimicrobial agent provided in a form for co-administration to a subject or in a form for separate administration to a subject.

[0048] In some embodiments of the seventh aspect of the invention, the agent inhibits the signalling of the at least one toll-like receptor in the subject. In one embodiment, inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R- associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-KB transcription factor, TIR-domain-containing adapter-inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factor- -activated kinase (TAK1 ), TAK- binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

[0049] In some embodiments of the seventh aspect of the invention, the toll-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9. In one embodiment, the toll-like receptor is toll-like receptor 4. [0050] In some embodiments of the seventh aspect of the invention, the at least one antimicrobial agent is an antibacterial agent.

[0051] In some embodiments of the seventh aspect of the invention, the combination product further includes at least one anti-inflammatory agent. In one embodiment, the at least one anti-inflammatory agent is selected from the group consisting of a non-steroidal antiinflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

[0052] In some embodiments of the seventh aspect of the invention, the combination product is used for the treatment and/or prevention of mastitis in a subject, for improving lactation in a mastitis affected subject, and/or for treating and/or preventing mammary tissue damage in a mastitis affected subject. In one embodiment, milk production in the subject is increased.

[0053] In some embodiments of the seventh aspect of the invention, the subject is non- human. For example, the subject may be bovine. In some embodiments the subject is a human.

BRIEF DESCRIPTION OF THE FIGURES

[0054] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description, taken in conjunction with the accompanying drawings.

[0055] FIGURE 1 - Picomicrographs of H&E stained mammary gland tissue administered LPS-containing matrigel and quantification of affected areas as a percentage of total mammary gland.

[0056] FIGURE 2 - Picomicrographs of anti-RB6 antibody stained mammary glands illustrating neutrophil recruitment into the mammary tissue administered LPS-containing matrigel and quantification of neutrophils recruitment into the tissue. [0057] FIGURE 3 - Picomicrographs of anti-F4/80 antibody stained mammary glands illustrating macrophage recruitment into the mammary tissue administered LPS-containing matrigel and quantification of macrophage recruitment into the tissue.

[0058] FIGURE 4 - Picomicrographs of anti-RB6 antibody stained mammary glands illustrating neutrophil recruitment into the mammary tissue of TLR4-/- and wild-type mice administered LPS-containing matrigel and quantification of neutrophils recruitment into the tissue.

[0059] FIGURE 5 - Picomicrographs of anti-F4/80 antibody stained mammary glands illustrating macrophage recruitment into the mammary tissue of TLR4-/- and wild-type mice administered LPS-containing matrigel and quantification of macrophage recruitment into the tissue.

[0060] FIGURE 6 - Graphs showing altered abundance of serum cytokines and chemokines in TLR4-/- compared to wild-type mice following administration of LPS-containing matrigel.

[0061] FIGURE 7 - Picomicrographs of H&E stained mammary glands showing involution of alveoli and intracellular localisation of phosphorylated-STAT5 (p-STAT5) in TLR4-/- and wild- type mice and quantification of the percentage of glandular area following administration of LPS-containing matrigel and nuclear localisation of p-STAT5.

[0062] FIGURE 8 - Graphs showing the increase in CXCL1 production from macrophages exposed to fresh or static bovine and human milk and the role of TLR4 and TLR2 signalling in the induction of CXCL1 .

DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention is predicated in part on the development of an in vivo mouse model of mastitis that resembles the clinical manifestation observed in humans with the disease. Using this mouse model, the inventors have found that toll-like receptor deficient mice exhibit better mastitis disease resolution than wild-type counterparts.

[0064] Accordingly, in a first aspect the present invention provides a method of treating and/or preventing mastitis in a subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject. [0065] Mastitis in general is characterised by an array of symptoms including; swelling (oedema) of the mammary tissue; redness and localised heat as a result of increased blood flow to the tissue; pain; systemic fever; systemic changes in cytokine and chemokine concentrations; changes in milk characteristics, including increased leukocyte (somatic cell) counts (SCC) in the milk, decreased viscosity of milk, decreased milk protein (primarily decrease casein), clotting of milk, a decline in potassium concentration, a decline in calcium concentration, a decline in lactoferrin concentration; a decreased volume of milk produced by the mammary gland; development of one or more abscesses in the mammary gland; and pathological damage to the mammary tissue, including the mammary ducts and milk producing alveoli.

[0066] The causative aetiology of mastitis is currently defined by the World Health Organisation (WHO) as milk stasis which may, or may not, be followed by a bacterial infection. However as the causative aetiology of mastitis is not readily diagnosed the disease is indicated by the presence of one or more symptoms.

[0067] In view of this, the term "mastitis" as used throughout the specification refers to the presence of one or more of the symptoms of mastitis, including those listed above. In one embodiment, relevant clinical symptoms may include, but are not limited to, systemic fever, systemic changes in cytokine and chemokine concentrations, changes in milk characteristics, a decreased volume of milk produced by the mammary gland, development of one or more abscesses in the mammary gland, and pathological damage to the mammary tissue, including the mammary ducts and milk producing alveoli.

[0068] Toll-like receptors (also referred to herein as "TLRs") are type I integral membrane glycoproteins that consist of eleven currently identified members of the toll-like receptor family (TLR 1 to TLR 1 1 ), with TLR 1 1 being exclusively expressed in mice. In general, tolllike receptors are comprised of an extracellular domain, a transmembrane domain and an intracellular (or cytoplasmic) domain. The transmembrane and intracellular domains of the toll-like receptors share considerable homology amongst the members. The extracellular region of the toll-like receptors contains leucine-rich repeat (LRR) motifs with some degree of variation that accounts for varying specificities for the ligand/agonist of each receptor. In addition to variation of cognate ligands, toll-like receptors exhibit different patterns of expression, as well as differing cell origins. In spite of their difference, many of the toll-like receptors binding to shared ligands/agonists and are expressed on the same cell types. To this end there is a degree of redundancy within the family of receptors with one of the member of the family being able to compensate, in some circumstances, for the loss or deficiency of another.

[0069] As used throughout the specification the term "toll-like receptor" refers to any one of the members of the toll-like receptor family of proteins. Gene and protein sequences for known members of the TLRs in humans can be derived from the HUGO Gene Nomenclature Committee (HGNC)(http://www.genenames.org/). The HGNC reference numbers for the members of toll-like receptor family in humans are listed in Table 1 below, together with GenBank Accession numbers and GenelDs.

TABLE 1

[0070] Gene and protein sequences for members of the TLRs in bovine have the GenBank Accession numbers listed in Table 2. The GenelD for each bovine TLR is also provided.

TABLE 2

GenBank GenBank

Toll-like Receptor

Accession Number Accession Number GenelD (TLR)

(nucleotide) (amino acid)

TLR 1 NM_001046504.1 NP_001039969.1 574090

TLR 2 NM_174197.2 NP_776622.1 281534

TLR 3 NM_001008664.1 NP_001008664.1 281535

TLR 4 NM_174198.6 NP_776623.5 281536

TLR 5 NM_001040501 .1 NP_001035591.1 444870 TLR 6 NM_001001 159.1 NP_001001 159.1 407237

TLR 7 NM_001033761 .1 NP_001028933.1 493686

TLR 8 NM_001033937.1 NP_001029109.1 532262

TLR 9 NM_183081.1 NP_898904.1 282602

TLR 10 NM_001076918.2 NP_001070386.1 539791

[0071] It is to be made clear that reference herein to a toll-like receptor, includes a reference to its naturally-occurring variants. In this regard, a "variant" of a toll-like receptor may exhibit a nucleic acid or an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to a native toll-like receptor. In some embodiments, a variant of a toll-like receptor is expected to retain native biological activity or a substantial equivalent thereof. For example, the human TLR 4 gene encodes three protein variants as represented by GenBank Accession Numbers NP_612564.1 (variant 1 - isoform a), NP_003257.1 (variant 2 - isoform c) and NP_612567.1 (variant 3 - isoform d). The nucleotide sequences encoding these TLR 4 variants are represented by GenBank Accession Numbers NM_138554.4 (variant 1 ), NM_003266.3 (variant 2) and NM_138557.2 (variant 3). Furthermore, the human TLR 10 gene encodes five protein variants as represented by GenBank Accession Numbers NP_1 12218.2 (variant 1 - isoform a), NP_001017388.1 (variant 2 - isoform a), NP_001 182035.1 (variant 3 - isoform a), NP_001 182036.1 (variant 4 - isoform a), and NP_001 182037.1 (variant 5 - isoform b). The nucleotide sequences encoding these TLR 10 variants are represented by GenBank Accession Numbers NM_030956.3 (variant 1 ), NM_001017388.2 (variant 2), NM_001 195106.1 (variant 3), NM_001 195107.1 (variant 4), and NM 001 195108.1 (variant 5).

[0072] In some embodiments of the present invention, the toll-like receptor may be selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9. In one embodiment, the toll-like receptor is toll-like receptor 4.

[0073] The term "inhibiting the activity" of a toll-like receptor as used throughout the specification is taken to mean a decrease in the expression of, the level of, or the function of (including the signalling of), one or more of the toll like receptors when compared to an untreated, or non-inhibited, toll-like receptor. This includes, but is not limited to the following: removing a gene that encodes for one or more of the toll-like receptors; altering/mutating a gene, or selecting for an animal that has an altered/mutated gene, that encodes for one or more of the toll-like receptors in a manner that decreases the activity of the toll-like receptor; decreasing the rate of, or preventing, the transcription of a gene encoding for one or more of the toll-like receptors; decreasing the rate of, or preventing, the translation of a nucleic acid that encodes for one or more of the toll-like receptors; decreasing the quantity of one or more toll-like receptors on the surface of a cell; decreasing the rate of transportation of one or more of the toll-like receptors to the surface of a cell; increasing the rate of internalisation from the surface of the cell of one or more of the toll-like receptors; increasing the rate of proteolysis of one or more or the toll-like receptors; antagonising one or more of the toll-like receptors; decreasing, or preventing, the interaction of one or more of the toll-like receptors with a co-factor/co-receptor required for induction of signalling by the receptor, which includes altering the expression, level and/or function of the co-factor/co-receptor in the cell or on the surface of the cell; decreasing the rate of, or preventing dimerization of, the toll-like receptors; decreasing or preventing signalling of one or more of the toll-like receptors; and inhibiting the down-stream signalling pathway of one or more of the toll-like receptors which includes altering the level of, or function of, any one or more of the downstream signalling factors in a manner that decreases or prevents signalling of one or more of the toll-like receptors.

[0074] Reference herein to "decrease" with respect to the expression of, or the level of, a toll-like receptor, whether at the transcriptional (mRNA) or translational (protein) stage is intended to mean, for example, at least a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1 .1 -fold, 1 .2-fold, 1 .3-fold, 1 .4-fold, 1 .5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 2.1 -fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3- fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 5- fold, 10-fold, 20 fold, 50-fold, or 100-fold reduction in the expression or level of a toll-like receptor mRNA or protein compared to an untreated, or non-inhibited, toll-like receptor.

[0075] Reference herein to "decrease" with respect to the function of a toll-like receptor is intended to mean a reduction in, or the elimination of, the function of the receptor in the affected subject, including a reduction or elimination in the signalling of the toll-like receptor. In effect, the activity of a toll-like receptor in the affected subject is to be reduced to a level commensurate to an untreated, or non-inhibited, toll-like receptor. In some embodiments, the activity of a toll-like receptor may be reduced by at least 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1 .1 -fold, 1 .2-fold, 1 .3-fold, 1.4-fold, 1 .5-fold, 1 .6-fold, 1.7- fold, 1.8-fold, 1.9-fold, 2-fold, 2.1 -fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1 -fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8- fold, 3.9-fold, 4-fold, 5-fold, 10-fold, 20 fold, 50-fold, or 100-fold in the affected subject. [0076] Methods which can be used to measure the extent of reduction in expression or level of a toll-like receptor in the subject would be known in the art. With respect to measuring a decrease in the transcription of a toll-like receptor gene into mRNA, levels of mRNA may be measured by techniques which include, but are not limited to, Northern blotting, RNA in situ hybridisation, reverse-transcriptase PCR (RT-PCR), real-time (quantitative) RT-PCR, microarrays, or "tag based" technologies such as SAGE (serial analysis of gene expression). Microarrays and SAGE may be used to simultaneously quantitate the expression of more than one gene. Primers or probes may be designed based on a nucleotide sequence of a toll-like receptor gene or transcripts thereof. Methodology similar to that disclosed in Paik ei al., 2004 {NEJM, 351 (27): 2817-2826), or Anderson et al., 2010 {Journal of Molecular Diagnostics, 12(5): 566-575) may be used to measure the extent of expression of a toll-like receptor gene. Many methods are also disclosed in standard molecular biology text books such as Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory Press, 2001.

[0077] With respect to RT-PCR, the first step is typically the isolation of total RNA from a sample obtained from the subject under investigation. Typical samples in this instance would include a biopsy sample (and corresponding normal tissue if possible) from affected mammary tissue or a milk sample taken from the affected subject. RNA can also be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin- fixed) tissue samples previously obtained from the subject. Messenger RNA (mRNA) may be subsequently purified from the total RNA sample. The total RNA sample (or purified mRNA) is then reverse transcribed into cDNA using a suitable reverse transcriptase. The reverse transcription step is typically primed using oligo-dT primers, random hexamers, or primers specific for a toll-like receptor gene under investigation, depending on the RNA template. The cDNA derived from the reverse transcription reaction then serves as a template for a typical PCR reaction. In this regard, two oligonucleotide PCR primers specific for a toll-like receptor gene are used to generate a PCR product. A third oligonucleotide, or probe, designed to detect a nucleotide sequence located between the other two PCR primers is also used in the PCR reaction. The probe is non-extendible by the Taq DNA polymerase enzyme used in the PCR reaction, and is labelled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together, as they are on the probe. During the PCR amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template- dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is freed from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0078] In real-time RT-PCR the amount of product formed, and the timing at which the product is formed, in the PCR reaction correlates with the amount of starting template. RT- PCR product will accumulate quicker in a sample having an increased level of mRNA compared to a standard or "normal" sample. Real-time RT-PCR measures either the fluorescence of DNA intercalating dyes such as Sybr Green into the synthesized PCR product, or can measure PCR product accumulation through a dual-labelled fluorigenic probe (i.e., TaqMan probe). The progression of the RT-PCR reaction can be monitored using PCR machines such as the Applied Biosystems' Prism 7000 or the Roche LightCycler which measure product accumulation in real-time. Real-time RT-PCR is compatible both with quantitative competitive PCR and with quantitative comparative PCR. The former uses an internal competitor for the target sequence for normalization, while the latter uses a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.

[0079] The production and application of microarrays for measuring the extent of decrease or reduction in expression of a toll-like receptor gene at the transcriptional level are well known in the art. In general, in a microarray, a nucleotide sequence (for example an oligonucleotide, a cDNA, or genomic DNA) representing a portion or all of a toll-like receptor gene occupies a known location on a substrate. A nucleic acid target sample (for example total RNA or mRNA) obtained from a subject of interest is then hybridized to the microarray and the amount of target nucleic acid hybridized to each probe on the array is quantified and compared to the hybridisation which occurs to a standard or "normal" sample (e.g. to an untreated, or non-inhibited, toll-like receptor sample). One exemplary quantifying method is to use confocal microscope and fluorescent labels. The Affymetrix GeneChip™ Array system (Affymetrix, Santa Clara, Calif.) and the Atlas™ Human cDNA Expression Array system are particularly suitable for quantifying the hybridization; however, it will be apparent to those of skill in the art that any similar systems or other effectively equivalent detection methods can also be used. Fluorescently labelled cDNA probes may also represent a toll-like receptor nucleic acid target sample. Such probes can be generated through incorporation of fluorescent nucleotides during reverse transcription of total RNA or mRNA extracted from a sample of the subject to be tested. Labelled cDNA probes applied to the microarray will hybridize with specificity to the equivalent spot of DNA on the array. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance in the sample compared to the abundance observed in a standard or "normal" sample. With dual colour fluorescence, separately labelled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization using microarray analysis affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels.

[0080] Methods which can be used to measure a decrease in the expression of or the level of a toll-like receptor at the translational level (protein level) would be known in the art. For example, the level of toll-like receptor protein may be measured by techniques which include, but are not limited to, antibody-based testing (including Western blotting, immunoblotting, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation and dissociation-enhanced lanthanide fluoro immuno assay (DELFIA)), proteomics techniques, surface plasmon resonance (SPR), versatile fibre-based SPR, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemistry, immunofluorescence, matrix-assisted laser desorption/ionization mass spectrometry (MALDI- MS), as described in WO 2009/004576 (including surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS), especially surface-enhanced affinity capture (SEAC), protein microarrays, surface-enhanced need desorption (SEND) or surface- enhanced photo label attachment and release (SEPAR)), matrix-assisted laser desorption/ionization time-of-f light (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry.

[0081] With respect to antibody-based testing methods such as immunohistochemistry and immunoblotting, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for a toll-like receptor protein under investigation are used to detect protein abundance in the subject. The antibodies can be detected by direct labelling of the antibodies themselves, for example with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabelled primary antibody may be used in conjunction with a labelled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available. Antibodies can be produced by methods well known in the art, for example, by immunizing animals with the protein under investigation. Further detailed description is provided below. [0082] Also contemplated are traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays. Nephelometry is an assay performed in liquid phase, in which antibodies are in solution. Binding of a toll-like receptor protein to the antibody results in changes in absorbance, which are measured. In the SELDI-based immunoassay, a biospecific capture reagent for a toll-like receptor protein is attached to the surface of an MS probe, such as a pre-activated ProteinChip array (see below). The protein is then specifically captured on the biochip through this reagent, and the captured protein is detected by mass spectrometry (see below). A further technique for assessing protein levels using an antibody- based platform involves the versatile fibre-based surface plasmon resonance (VeSPR) biosensor, as described in PCT International Publication Number WO 201 1/1 13085.

[0083] Proteomics can also be used to analyse the expression level of a toll-like receptor protein of interest present in a sample at a certain point of time. In particular, proteomic techniques can be used to assess the global changes of protein expression in a sample (also referred to as expression proteomics). Proteomic analysis typically includes: (i) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (ii) identification of the individual polypeptides recovered from the gel, for example by mass spectrometry or N- terminal sequencing; and (iii) analysis of the data using bioinformatics.

[0084] Protein microarrays (also termed biochips) may also be used to determine the level of expression of a toll-like receptor protein under investigation in a sample. Many protein biochips are described in the art, including for example protein biochips produced by Ciphergen Biosystems, Inc. (Fremont, CA), Zyomyx (Hayward, CA), Invitrogen (Carlsbad, CA), Biacore (Uppsala, Sweden) and Procognia (Berkshire, UK). Examples of such protein biochips are described in the following patents or published patent applications: US Patent Numbers 6,225,047, 6,537,749, 6,329,209, and 5,242,828, and PCT International Publication Numbers WO 00/56934 and WO 03/048768.

[0085] The expression level of a toll-like receptor protein under investigation can also be measured by mass spectrometry, a method that employs a mass spectrometer to detect gas phase ions. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. The mass spectrometer may be a laser desorption/ionization (LDI) mass spectrometer. In laser desorption/ionization mass spectrometry, the toll-like receptor protein or proteins to be detected are placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present the protein or proteins to ionizing energy for ionization and introduction into a mass spectrometer. A laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer.

[0086] The analysis of proteins by LDI can take the form of MALDI or of SELDI. The SELDI method is described, for example, in US Patents Numbers 5,719,060 and 6,225,047. SELDI also encompasses affinity capture mass spectrometry, surface-enhanced affinity capture (SEAC) and immuno-capture mass spectrometry (icMS) as described by Penno MA et al., 2012 (Res. Vet. Sci., 93: 61 1 -617). These platforms involve the use of probes that have a material on the probe surface that captures proteins through a non-covalent affinity interaction (adsorption) between the material and the protein. The material is variously called an "adsorbent," a "capture reagent," an "affinity reagent" or a "binding moiety." Such probes can be referred to as "affinity capture probes" and as having an "adsorbent surface." The capture reagent can be any material capable of binding a protein. The capture reagent is attached to the probe surface by physisorption or chemisorption. The probes, which may take the form of a functionalised biochip or magnetic bead, may have the capture reagent already attached to the surface, or the probes are pre-activated and include a reactive moiety that is capable of binding the capture reagent, e.g. through a reaction forming a covalent or coordinate covalent bond. Epoxide and acyl-imidizole are useful reactive moieties to covalently bind protein capture reagents such as antibodies or cellular receptors. Nitrilotriacetic acid and iminodiacetic acid are useful reactive moieties that function as chelating agents to bind metal ions that interact non-covalently with histidine containing proteins. Adsorbents are generally classified as chromatographic adsorbents and biospecific adsorbents.

[0087] Another method of laser desorption mass spectrometry is called surface-enhanced neat desorption (SEND). SEND involves the use of probes comprising energy absorbing molecules that are chemically bound to the probe surface ("SEND probe"). The phrase "energy absorbing molecules" (EAM) denotes molecules that are capable of absorbing energy from a laser desorption/ionization source and, thereafter, contribute to desorption and ionization of analyte molecules in contact therewith. The EAM category includes molecules used in MALDI, frequently referred to as "matrix," and is exemplified by cinnamic acid derivatives, sinapinic acid (SPA), cyano-hydroxy-cinnamic acid (CHCA) and dihydroxybenzoic acid, ferulic acid, and hydroxyaceto-phenone derivatives. The energy absorbing molecule may be incorporated into a linear or cross-linked polymer, e.g. a polymethacrylate. SEND is further described in US Patent Number 6,124,137 and PCT International Publication Number WO 03/64594.

[0088] SEAC/SEND is a version of laser desorption mass spectrometry in which both a capture reagent and an energy absorbing molecule are attached to the sample presenting surface. SEAC/SEND probes therefore allow the capture of proteins under investigation through affinity capture and ionization/desorption without the need to apply external matrix. The CI 8 SEND biochip is a version of SEAC/SEND, comprising a CI 8 moiety which functions as a capture reagent, and a CHCA moiety which functions as an energy absorbing moiety.

[0089] Another version of LDI is called surface-enhanced photolabile attachment and Release (SEPAR). SEPAR involves the use of probes having moieties attached to the surface that can covalently bind a protein, and then release the protein through breaking a photolabile bond in the moiety after exposure to light, e.g. to laser light. SEPAR and other forms of SELDI are readily adapted to detecting a protein or protein profile, as required by the methods of the present invention.

[0090] MALDI is a traditional method of laser desorption/ionization. In one MALDI method, the sample to be tested is mixed with matrix and deposited directly on a MALDI chip. Depending on the sample being tested, the protein being tested is preferably first captured with biospecific (e.g. an antibody) or chromatographic materials coupled to a solid support such as a resin (e.g. in a spin column). Specific affinity materials that may bind the protein being detected are described above. After purification on the affinity material, the protein under investigation is eluted and then detected by MALDI.

[0091] Analysis of toll-like receptor proteins by time-of-flight mass spectrometry generates a time-of-flight spectrum. The time-of-flight spectrum ultimately analyzed typically does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses. This reduces noise and increases dynamic range. This time-of-flight data is then subject to data processing using specialized software. Data processing typically includes TOF-to-M/Z transformation to generate a mass spectrum, baseline subtraction to eliminate instrument offsets and high frequency noise filtering to reduce high frequency noise.

[0092] Data generated by desorption and detection of proteins can be analyzed with the use of a programmable digital computer. The computer program analyzes the data to indicate the number of proteins detected, and optionally the strength of the signal and the determined molecular mass for each protein detected. Data analysis can include steps of determining signal strength of a protein and removing data deviating from a predetermined statistical distribution. For example, the observed peaks can be normalized, by calculating the height of each peak relative to some reference. The computer can transform the resulting data into various formats for display. The standard spectrum can be displayed, but in one useful format only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling proteins with nearly identical molecular weights to be more easily seen. In another useful format, two or more spectra are compared, conveniently highlighting proteins that have varying expression levels between samples. Using any of these formats, one can readily determine whether a particular protein is present in a sample and to what level.

[0093] Analysis generally involves the identification of peaks in the spectrum that represent signal from a protein. Peak selection can be done visually, but commercial software can be used to automate the detection of peaks. In general, this software functions by identifying signals having a signal-to-noise ratio above a selected threshold and labelling the mass of the peak at the centroid of the peak signal. In one useful application, many spectra are compared to identify identical peaks present in some selected percentage of the mass spectra. One version of this software clusters all peaks appearing in the various spectra within a defined mass range, and assigns a mass (M/Z) to all the peaks that are near the mid-point of the mass (M/Z) cluster.

[0094] Software used to analyze the data can include code that applies an algorithm to the analysis of the signal to determine whether the signal represents a peak in a signal that corresponds to a protein under investigation. The software also can subject the data regarding observed protein peaks to a classification tree or ANN analysis, to determine whether a protein peak or combination of protein peaks is present that indicates the status of the particular clinical parameter under examination. Analysis of the data may be "keyed" to a variety of parameters that are obtained, either directly or indirectly, from the mass spectrometric analysis of the sample. These parameters include, but are not limited to, the presence or absence of one or more peaks, the shape of a peak or group of peaks, the height of one or more peaks, the log of the height of one or more peaks, and other arithmetic manipulations of peak height data.

[0095] With respect to measuring the level of activity of a toll-like receptor, the assay used will depend on the toll-like receptor under investigation. Generally, assays which measure for indicators of toll-like receptor signalling can be used. For example, assays which measure for NF-KB activation and/or nuclear translocation by an agonist of a toll-like receptor could be utilised. Alternatively, assays which identify a marker of NF-κΒ activation, for example the level of IKK degradation, could be used. Other assays may include measuring proinflammatory cytokine or chemokine production induced by an agonist of a toll-like receptor, or measuring the extent of MAPK activation/phosphorylation induced by an agonist of a toll-like receptor. However, as would be understood by a person skilled in the art, any assay which is based on any one or more down-stream mediators of toll-like receptor signalling could be used to measure the level of activity of the receptor.

[0096] The term "treating" as used herein, refers to partially or completely alleviating, inhibiting, delaying onset of, reducing the incidence of, ameliorating and/or relieving mastitis, or one or more symptoms of mastitis, suppressing the severity of mastitis, and/or reducing or eliminating the duration of mastitis. The term "preventing" as used herein, refers to reducing or eliminating the occurrence of mastitis.

[0097] As used throughout the specification the term "subject" refers to any mammal capable of, or presently, lactating. In most forms the subject will be a postpartum female mammal. This includes, but is not limited to, primates, including humans, Bovidae species, including cattle, sheep, goats, buffalo and camels. Species of particular relevance are humans and cattle (bovine). Whilst details regarding the genes and protein sequences for toll-like receptors in both humans and cattle have been referred to previously, it will be appreciated that the aspects of the present invention are not limited to these species and homologous genes for each of the toll-like receptors exists in most mammals.

[0098] In some embodiments, the method according to the first aspect of the invention involves administering to the subject an agent which inhibits the activity of at least one-tolllike receptor. As used throughout the specification the term "agent" refers to any substance or molecule that is capable of inhibiting the activity of at least one toll-like receptor. For example, the agent may be selected from one or more of the group consisting of a neutralizing antibody (or an antigen binding part thereof), an antisense nucleic acid that binds to toll-like receptor mRNA and which interferes with translation, a molecule that can specifically repress transcription of endogenous toll-like receptor mRNA such as a specific DNA or RNA binding protein, a nucleic acid capable of forming a triple helix structure, a small interfering RNA, a microRNA, a short hairpin RNA, a ribozyme that can cleave toll-like receptor mRNA, an aptamer, and an agent that interacts with or binds to the toll-like receptor protein (or a regulator of the toll-like receptor) and inhibits its activity, such as a drug, small molecule, protein, polypeptide or oligopeptide.

[0099] In some embodiments, the agent may be an antibody which specifically binds to a toll-like receptor. The term "antibody" as used herein is used in the broadest sense and encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as linear antibodies, single-chain antibody molecules, Fc or Fc' peptides, Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be one of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGI, lgG2, lgG3, lgG4, IgAI and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0100] Antibodies can be produced according to well-established techniques in the art, for example by immunizing animals with a toll-like receptor protein. Alternatively, given that the amino acid sequence for many toll-like receptors is available, the relevant receptor protein can be synthesized and used to generate antibodies by methods well-known in the art. For example, monoclonal antibodies to a toll-like receptor protein may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (for example, see Kohler et al., 1975, Nature 256: 495-497; Kozbor et al., 1985, J. Immunol. Methods 81 :31 -42; Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030; and Cole et al., 1984, Mol. Cell Biochem. 62: 109-120).

[0101] Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (for example, see Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA 86: 3833-3837; and Winter and Milstein, 1991 , Nature 349: 293-299). Antibodies may also be generated using phage display. For example, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g. human or murine). Phage expressing an antigen binding domain that binds to a toll-like receptor protein can be selected or identified using the relevant receptor protein, e.g. using labelled receptor protein or a portion thereof. Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilised Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies may include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182: 41 -50; Ames et al., 1995, J. Immunol. Methods 184: 177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24: 952-958; Persic et al., 1997, Gene 187: 9-18; Burton et al., 1994, Advances in Immunology 57: 191 - 280; PCT application number PCT/GB91/01 134; PCT publications numbers WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO 95/15982; WO 95/20401 ; and US Patent Numbers 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821 ,047; 5,571 ,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0102] Techniques which can be used to produce single-chain Fvs and antibodies include those described in US Patent Numbers 4,946,778 and 5,258,498; Huston ef al., 1991 , Methods in Enzymology 203: 46-88; Shu ei al., 1993, Proc. Natl. Acad. Sci. USA 90: 7995- 7999; and Skerra et al., 1988, Science 240: 1038-1040.

[0103] Antibody fragments which contain specific binding sites for a toll-like receptor protein may be generated using standard techniques known in the art. For example, F(ab')2 fragments may be produced by pepsin digestion of a toll-like receptor antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (for example, see Huse et al., 1989, Science 246: 1275-1281 ).

[0104] In some embodiments, inhibiting the activity of a toll-like receptor may be achieved by antisense or gene-targeted silencing strategies. Accordingly, such strategies utilise agents including antisense oligonucleotides, antisense RNA, antisense RNA expression vectors, small interfering RNAs (siRNA), microRNAs (miRNAs) and short hairpin RNAs (shRNAs) as referred to above. Still further, catalytic nucleic acid molecules such as aptamers, DNAzymes and ribozymes may be used for gene silencing. These molecules function by cleaving their target mRNA molecule rather than merely binding to it as in traditional antisense approaches. [0105] An "antisense oligonucleotide" corresponds to an RNA sequence as well as a DNA sequence coding therefor, which is sufficiently complementary to a toll-like receptor mRNA molecule, for which the antisense RNA is specific, to cause molecular hybridisation between the antisense RNA and the toll-like receptor mRNA such that translation of the mRNA is inhibited. Such hybridisation can occur under in vitro and in vivo conditions. The antisense molecule must have sufficient complementarity to the toll-like receptor gene so that the antisense RNA can hybridize to the toll-like receptor gene (or mRNA) and inhibit its expression regardless of whether the action is at the level of splicing, transcription, or translation. In some embodiments, the complementary antisense sequence is about 15 to 30 nucleotides in length, for example, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or longer or shorter, as desired. Antisense oligonucleotides can include sequences hybridisable to any of several portions of the toll-like receptor gene under investigation, including the coding sequence, 3 ' or 5' untranslated regions, or intronic sequences.

[0106] The terms "small interfering RNA" and "siRNA" interchangeably refer to short double- stranded RNA oligonucleotides that mediate RNA interference (also referred to as "RNA- mediated interference," or RNAi). RNAi is a highly conserved gene silencing event functioning through targeted destruction of individual mRNA by a homologous double- stranded small interfering RNA (siRNA) (Fire, A et al., 1998, Nature 391 : 806-81 1 ). Mechanisms for RNAi are reviewed, for example, in Bayne and Allshire, 2005, Trends in Genetics, 21 : 370-73; Morris, 2005, Cell Mol. Life Sci., 62: 3057-3066; and Filipowicz, et al., 2005, Current Opinion in Structural Biology, 15: 331 -3341.

[0107] For the purposes of the present invention, RNAi can be effected by introduction or expression in the subject of siRNAs specific for a toll-like receptor. The double stranded oligonucleotides used to effect inhibition of expression, at either the transcriptional or translational level, can be of any convenient length. siRNA molecules are typically from about 15 to about 30 nucleic acids in length, for example, about 19-25 nucleic acids in length, for example, about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleic acids in length. Optionally the dsRNA oligonucleotides can include 3' overhang ends. Exemplary 2- nucleotide 3' overhangs can be composed of ribonucleotide residues of any type and can be composed of 2'-deoxythymidine resides, which lowers the cost of RNA synthesis and can enhance nuclease resistance of siRNAs in the cell culture medium and within transfected cells (see Elbashir et al., 2001 , Nature 41 1 : 494-498). [0108] Longer dsRNAs of 50, 75, 100 or even 500 base pairs or more can also be utilised. Exemplary concentrations of dsRNAs for effecting toll-like receptor inhibition are about 0.05 nM, 0.1 nM, 0.5 nM, 1 .0 nM, 1 .5 nM, 25 nM or 100 nM, although other concentrations can be utilised depending upon the nature of the cells treated and other factors readily discernable to the person skilled in the art.

[0109] Exemplary dsRNAs can be synthesized chemically or produced in vitro or in vivo using appropriate expression vectors. Exemplary synthetic RNAs include 21 nucleotide RNAs chemically synthesised using methods known in the art. Synthetic oligonucleotides are preferably deprotected and gel-purified using methods known in the art (see for example Elbashir et al., 2001 , Genes Dev. 15: 188-200). Alternatively the dsRNAs can be transcribed from a mammalian expression vector. A single RNA target, placed in both possible orientations downstream of an appropriate promoter for use in mammalian cells, will transcribe both strands of the target to create a dsRNA oligonucleotide of the desired target sequence. Any of the above RNA species should be designed to include a portion of nucleic acid sequence represented in a target nucleic acid.

[0110] The specific sequence utilised in design of the siRNA oligonucleotides can be any contiguous sequence of nucleotides contained within the expressed gene message of a tolllike receptor target. Programs and algorithms, known in the art, may be used to select appropriate target sequences within a toll-like receptor gene (for example see the Ambion website at ambion.com). In addition, optimal sequences can be selected utilising programs designed to predict the secondary structure of a specified single stranded nucleic acid sequence and allow selection of those sequences likely to occur in exposed single stranded regions of a folded mRNA. Methods and compositions for designing appropriate siRNA oligonucleotides may be found, for example, in US patent number 6,251 ,588, the contents of which are incorporated herein by reference.

[0111] As would be understood by a person skilled in the art, ribozymes are enzymatic RNA molecules capable of catalyzing specific cleavage of RNA. The composition of a ribozyme molecule of the present invention should include one or more sequences complementary to a toll-like receptor mRNA, and the well-known catalytic sequence responsible for mRNA cleavage or a functionally equivalent sequence (see for example US patent number 5,093,246, which is incorporated herein by reference in its entirety). Ribozyme molecules designed to catalytically cleave a toll-like receptor mRNA transcript can also be used to prevent translation of toll-like receptor mRNA. While ribozymes that cleave mRNA at site- specific recognition sequences can be used to destroy target mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. Preferably, the target mRNA has the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art.

[0112] Toll-like receptor targeting ribozymes necessarily contain a hybridising region complementary to two regions, each of at least 5 and preferably each 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides in length, of the target toll-like receptor mRNA. In addition, the ribozymes should possess highly specific endoribonuclease activity, which autocatalytically cleaves the toll-like receptor sense mRNA.

[0113] With regard to antisense, siRNA or ribozyme oligonucleotides, phosphorothioate oligonucleotides can be used. Modifications of the phosphodiester linkage as well as of the heterocycle or the sugar may provide an increase in efficiency. Phophorothioate is used to modify the phosphodiester linkage. An N3'-P5' phosphoramidate linkage has been described as stabilising oligonucleotides to nucleases and increasing the binding to RNA. Peptide nucleic acid (PNA) linkage is a complete replacement of the ribose and phosphodiester backbone and is stable to nucleases, increases the binding affinity to RNA, and does not allow cleavage by RNAse H. Its basic structure is also amenable to modifications that may allow its optimisation as an antisense component. With respect to modifications of the heterocycle, certain heterocycle modifications have proven to augment antisense effects without interfering with RNAse H activity. An example of such modification is C-5 thiazole modification. Finally, modification of the sugar may also be considered. 2'-0-propyl and T- methoxyethoxy ribose modifications stabilize oligonucleotides to nucleases in cell culture and in vivo.

[0114] One of the initial steps in the development of therapeutic agents is the identification lead compounds that bind to the toll-like receptor. Many analogs of these lead compounds are synthesised to define key recognition elements for maximal activity. In this regard, large combinatorial libraries of peptides and oligonucleotides have been generated, and can be screened against a receptor to identify high-affinity ligands and inhibitors of the receptor. Inhibitory peptides and oligonucleotides can be delivered to a subject or the cell of a subject by direct transfection or transfection and expression via an expression vector. Appropriate expression vectors include mammalian expression vectors and viral vectors, into which has been cloned a nucleotide sequence encoding the peptide or an inhibitory oligonucleotide together with the appropriate regulatory sequences including a promoter. Suitable promoters can be constitutive or development-specific promoters. Transfection delivery can be achieved by liposomal transfection reagents, known in the art (e.g. Xtreme transfection reagent, Roche, Alameda, CA; Lipofectamine formulations, Invitrogen, Carlsbad, CA). Delivery mediated by cationic liposomes, by retroviral vectors and direct delivery are efficient.

[0115] In some embodiments, the agent may be a small (organic) molecule given that such molecules often have favourable pharmacokinetic properties. A small molecule is generally regarded as having a molecular weight of less than 900 Daltons (to allow diffusion across cell membranes and to enable oral bioavailability) and greater than 500 Daltons (given that clinical attrition rates are significantly reduced if the molecular weight is below this size). Small molecules may be natural in origin, such as being derived from a plant or may be synthetically produced. Examples of small molecule agents that are currently known to inhibit the activity of toll-like receptors include, but are not limited to, eritoran, (-)-naloxone, (+)- naloxone, (-)-naltrexone, (+)-naltrexone, LPS-RS, ibudilast, propentofylline, pentoxifylline, amitriptyline, ketotifen, cyclobenzaprine, desipramine, mesoridazine, rimcazole, harmine, tacrine, orphenadrine, diphenhydramine, venlafaxine, mianserin, imipramine, quercetin dehydrate, lidocaine, valproic acid, scopolamine, gabapentin, atropine, clonidine hydrochloride, aminophylline, capsaicin, mexletine hydrochloride, theophylline, minocycline, acetaminophen, tacrine, diphenhydramine, vitamin D2, mevinolin and hamine hydrochloride.

[0116] As used throughout the specification the term "administering" to a subject refers to the application to the subject, or to the cells of the subject, an external agent as referred to above. The agent may be administered any number of ways as would be understood in the art, and as described in further detail below. For example, methods of administering the agent includes, but is not limited to, providing an agent orally, providing an agent topically, providing an agent subcutaneously by way of injection, including an intravenous, intramuscular or intraperitoneal injection, providing an agent mucosally, providing an agent locally by way of intra-mammary administration (for example via the teat canal) or providing a prodrug of an agent by way of any one or more of the aforementioned routes. The agent may be formulated into a composition for the administration as described in further detail below.

[0117] Toll-like receptors are activated by the binding of an agonist/ligand to the receptor. Following activation, the toll-like receptor undergoes conformational changes and forms either a homo- or hetero-dimer and may further associate with other extracellular co-factors, or co-receptors, to fully activate toll-like receptor signalling. Accordingly, in some embodiments of the first aspect of the invention, inhibiting the activity of at least one toll-like receptor includes inhibiting the signalling of at least one toll-like receptor. [0118] "Signalling" of a toll-like receptor refers to any extracellular or intracellular events that are a requisite for the transfer of an extracellular signal (such as the binding of a ligand/agonist to a toll-like receptor) into an effect on the cell. In this regard signalling includes, but is not limited to engagement of the toll-like receptor by a ligand or agonist, association of the toll-like receptor with co-receptors/co-factors, association of the toll-like receptor with intracellular molecules, including adapter molecules, and downstream signalling of the toll-like receptor. For example, once activated toll-like receptors initiate downstream signalling to induce their effect on the cell. Accordingly, in some embodiments of the invention, inhibiting the signalling of at least one toll-like receptor may include inhibiting the downstream signalling of the at least one toll-like receptor.

[0119] As used throughout the specification, the term "downstream signalling" (also known as signal transduction) refers to any intracellular molecular change involved in the transfer of an extracellular signal into an effect on the cell. These include, but are not limited to, conformational changes in the intracellular domain of the receptor, association of the receptor with a downstream signalling molecule, and activation or inhibition of a downstream signalling molecule, which includes phosphorylation of a downstream signalling molecule. Downstream signalling molecules/mediators for toll-like receptors are known in the art and include, but are not limited to, molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R-associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor- associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a N F-KB transcription factor, TIR-domain-containing adapter-inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factors-activated kinase (TAK1 ), TAK-binding protein 1 (TAB1 ), and TAK-binding protein 1 TAB2, as well as other downstream signalling factors/mediators which are known in the art.

[0120] Of particular importance to the downstream signalling of toll-like receptors is the molecule MyD88. MyD88 acts as an adapter between the toll-like receptor intracellular domains and downstream signalling domains and as such provides a common convergent pathway for induction of signalling by the members of the toll-like receptor family. Further detail is provided below.

[0121] The activation and downstream signalling of toll-like receptor 4 (TLR 4) will be used as an example of the activation of a toll-like receptor. TLR 4 can be activated by a range of ligands which include, but are not limited to; lipopolysaccharide (LPS); taxol; fusion protein; envelope protein; heat-shock protein 60; heat-shock protein 70; Type III repeat extra domain A of fibronectin; oligosaccharides of hyaluronic acid; polysaccharide fragments of heparin sulphate; and fibrinogen. Following binding of a ligand (e.g. LPS), TLR 4 associates with the co-factors CD-14, MD-2 (LY96) and LPS-Binding Protein (LBP), which leads to intra-cellular conformational changes that allows the recruitment of downstream signalling molecules (adapter proteins). Subsequently, TLR 4 can activate either a MyD88-dependent or MyD88- independent pathway. The MyD88-dependent pathway involves the recruitment of MyD88 to the intracellular portion of TLR-4 and recruits IRAK. IRAK is then activated by phosphorylation and interacts with TRAF6 which ultimately leads to the activation of c-Jun NH2-terminal (JNK) kinases and NF-κΒ which in turn alters gene expression of the cell. The alternative pathway, the MyD88-independent pathway, relies on recruitment of TRIF to the adaptor protein TRIF-regulated adaptor molecule (TRAM) bound to the intracellular domain of toll-like receptor 4. Following localisation of TRIF with TRAM, inhibitor of nuclear factor kappa-B kinase subunit epsilon (ΙΚΚε) is activated and phosphorylates TANK-binding kinase 1 (TBK1 ) which in turn activates NF-κΒ ultimately altering gene expression of the cell.

[0122] The effects of toll-like receptor activation on cells are known in the art and include, production of cytokines and/or chemokines by the cell, phagocytosis by the cell, antigen presentation by the cell, reduction or arresting of protein synthesis, or apoptosis.

[0123] Ultimately the functionality of the toll-like receptors is dependent on many factors including the access of an activator (ligand/agonist) to the toll-like receptor, the ability of the necessary conformational changes of the toll-like receptor and co-receptors or co-factors to take place in order to activate the receptor and initiate downstream signalling, and the ability of the down-stream pathway to induce molecular changes with the cell.

[0124] In some embodiments, the method according to the first aspect of the invention further includes the step of administering to the subject at least one antimicrobial agent. Antimicrobial agents are known in the art, however for the sake of clarity the term "antimicrobial agent" as used throughout the specification refers to any agent that inhibits the growth of, and/or induces the death of, a microbe including bacteria, viruses and fungi. As such the term antimicrobial agent includes antibacterial agents, antiviral agents and antifungal agents.

[0125] In some embodiments, the method according to the first aspect of the invention further includes the step of administering to the subject at least one anti-inflammatory agent. Examples of suitable anti-inflammatory agents would be known in the art. For example, the anti-inflammatory agent may be selected from the group consisting of a non-steroidal antiinflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

[0126] In a second aspect the present invention provides a method of improving lactation in a mastitis affected subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject.

[0127] The term "improving lactation in a mastitis affected subject" as used herein means to improving the quantity, quality and/or composition of milk produced by the mammary glands of a mastitis affected subject when compared to the quantity, quality and/or composition of milk produced by the subject prior to performing the method. An improvement means a change toward a desired milk quantity, quality or composition. The desired milk quantity, quality or composition being that of a typical healthy lactating subject, or that of the mastitis affected subject prior to the advent of mastitis. Measures of milk quality and composition are known in the art, and in general represent any desirable trait in milk. This may include, but is not limited to, somatic cell counts, standard plate counts, concentration of nutrients including the concentration of fat, proteins, amino acids, and carbohydrate such as lactose, the concentration of minerals and ions such a potassium, calcium, zinc, magnesium, chloride, citrate and phosphate, the concentration of vitamins such as vitamins D, E, K, as well as the concentration of immune mediators such as tissue growth factors, interferons and immunoglobulins.

[0128] In one embodiment, milk production in the subject is increased. For example, and as described above, an increase in milk production may include an increase in the quantity and/or quality of milk when compared to the quantity and/or quality of milk produced by the subject prior to performing the method.

[0129] In a third aspect the present invention provides a method of treating and/or preventing mammary tissue damage in a mastitis affected subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject.

[0130] The term "mammary tissue damage" as used herein refers to any detrimental abnormal physical alteration, either permanent or temporary, of any tissue that constitutes the mammary gland. This includes, but is not limited to, alteration of the secretory structures such as alveoli, or the cellular structures that constitute the alveoli, alteration of the lobules, alteration of the ducts (lactiferous ducts), or alteration of any of the extracellular matrix, connective or adipose tissue that constitute the mammary gland.

[0131] With reference to the second and third aspects of the invention, methods for inhibiting the activity of at least one toll-like receptor have been described above. So too have the type of agents which may be used to inhibit this activity, and the methods that may be used to assay for the level of inhibition of the activity of a toll-like receptor. In some embodiments of the second and third aspect of the invention, the toll-like receptor may be selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9. Detail regarding these tolllike receptors is provided above. In some embodiments of the second and third aspects of the invention, the method further includes the step of administering to the subject at least one antimicrobial agent and/or at least one anti-inflammatory agent. Detail regarding these agents is provided above with respect to the first aspect of the invention.

[0132] In a fourth aspect, the present invention provides use of an agent that inhibits the activity of at least one toll-like receptor for the treatment and/or prevention of mastitis in a subject, for improving lactation in a mastitis affected subject, and/or for treating and/or preventing mammary tissue damage in a mastitis affected subject.

[0133] In a fifth aspect, the present invention provides use of an agent that inhibits the activity of at least one toll-like receptor in the manufacture of a medicament for the treatment and/or prevention of mastitis in a subject, for improving lactation in a mastitis affected subject, and/or for treating and/or preventing mammary tissue damage in a mastitis affected subject.

[0134] In a sixth aspect, the present invention provides a pharmaceutical composition when used for treating and/or preventing mastitis in a subject, improving lactation in a mastitis affected subject, and/or treating and/or preventing mammary tissue damage in a mastitis affected subject, the composition including an effective amount of an agent that inhibits the activity of at least one toll-like receptor in the subject.

[0135] An effective amount of the agent to be administered to the subject is not particularly limited, so long as it is within such an amount and in such a form that generally exhibits a useful or therapeutic effect. The term "effective amount" therefore means the quantity which, when administered to a subject in need of treatment, improves the prognosis and/or state of the subject. The amount to be administered to a subject will depend on the severity of mastitis, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors.

[0136] Examples of suitable agents have been described in detail above. The meaning of the terms "treating and/or preventing mastitis in a subject", "improving lactation in a mastitis affected subject", and/or "treating and/or preventing mammary tissue damage in a mastitis affected subject" have also been described in detail above with respect to the first aspect of the invention. Furthermore, the meaning of "inhibits the activity of at least one toll-like receptor in the subject" has also been described in detail above.

[0137] The delivery or administration of the agent in the various embodiments of the present invention may be delivery or administration of the agent alone, or delivery or administration of the agent formulated into a suitable pharmaceutical composition, as referred to above.

[0138] In this regard, the pharmaceutical composition may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the agent to be administered.

[0139] The preparation of such pharmaceutical compositions is known in the art, for example as described in Remington's Pharmaceutical Sciences, 18th ed., 1990, Mack Publishing Co., Easton, Pa. and U.S. Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton, Pa.

[0140] For example, the agent can be prepared into a variety of pharmaceutical compositions in the form of, for example, an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a gel, etc., and these preparations can be administered as intramuscular or subcutaneous injection or as injection to an organ, or as an embedded preparation or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc. The composition may be administered in the form of oral preparations (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions). Compositions containing the agent may also contain a preservative, stabiliser, dispersing agent, pH controller or isotonic agent. Examples of suitable preservatives are glycerin, propylene glycol, phenol or benzyl alcohol. Examples of suitable stabilisers are dextran, gelatin, a-tocopherol acetate or alpha-thioglycerin. Examples of suitable dispersing agents include polyoxyethylene (20), sorbitan mono-oleate (Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylene hydrogenated castor oil 60. Examples of suitable pH controllers include hydrochloric acid, sodium hydroxide and the like. Examples of suitable isotonic agents are glucose, D-sorbitol or D-mannitol.

[0141] The administration of the agent in the various embodiments of the present invention may also be in the form of a composition containing a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant or sweetener, taking into account the physical and chemical properties of the agent being administered. For these purposes, the composition may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, mucosally for example rectally, nasally, bucally, and vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, via intra-mammary administration (for example via the teat canal) or by any other convenient dosage form.

[0142] The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques. When administered parenterally, the composition will normally be in a unit dosage, sterile injectable form (solution, suspension or emulsion) which is preferably isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents, for example, as solutions in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or di- glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

[0143] The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti-oxidants, buffers and preservatives.

[0144] When administered orally, the agent will usually be formulated into unit dosage forms such as tablets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art. Such formulations typically include a solid, semisolid, or liquid carrier. Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.

[0145] A tablet may be made by compressing or moulding the agent optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.

[0146] The administration of the agent in the various embodiments of the present invention may also utilise controlled release technology. The agent may also be administered as a sustained-release pharmaceutical. To further increase the sustained release effect, the agent may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof (weight average molecular weight: ca. 80,000 to 2,000,000), carboxymethylcellulose sodium (weight average molecular weight: ca. 20,000 to 400,000), hydroxypropylcellulose (viscosity in 2% aqueous solution: 3 to 4,000 cps), atherocollagen (weight average molecular weight: ca. 300,000), polyethylene glycol (weight average molecular weight: ca. 400 to 20,000), polyethylene oxide (weight average molecular weight: ca. 100,000 to 9,000,000), hydroxypropylmethylcellulose (viscosity in 1 % aqueous solution: 4 to 100,000 cSt), methylcellulose (viscosity in 2% aqueous solution: 15 to 8,000 cSt), polyvinyl alcohol (viscosity: 2 to 100 cSt), polyvinylpyrrolidone (weight average molecular weight: 25,000 to 1 ,200,000).

[0147] Alternatively, the agent may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days. The agent may then be moulded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the agent over a prolonged period of time without the need for frequent re-dosing. Such controlled release films are well known to the art. Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene-vinyl acetate copolymer a degradable lactic acid-glycolic acid copolymers which may be used externally or internally. Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.

[0148] The carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time release characteristics and release kinetics. The agent may then be moulded into a solid implant suitable for providing efficacious concentrations of the agent over a prolonged period of time without the need for frequent re-dosing. The agent can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be moulded into a solid implant.

[0149] For topical and/or mucosal administration, the composition of the present invention may be in the form of a solution, spray, lotion, cream (for example a non-ionic cream), gel, paste or ointment. Alternatively, the composition may be delivered via a liposome, nanosome, or nutri-diffuser vehicle.

[0150] A cream is a formulation that contains water and oil and is stabilized with an emulsifier. Lipophilic creams are called water-in-oil emulsions, and hydrophilic creams oil-in- water emulsions. The cream base for water-in-oil emulsions are normally absorption bases such as vaseline, ceresin or lanolin. The bases for oil-in-water emulsions are mono-, di- and tri-glycerides of fatty acids or fatty alcohols with soaps, alkyl sulphates or alkyl polyglycol ethers as emulsifiers. [0151] A lotion is an opaque, thin, non-greasy emulsion liquid dosage form for external application to the skin, which generally contains a water-based vehicle with greater than 50% of volatiles and sufficiently low viscosity that it may be delivered by pouring. Lotions are usually hydrophilic, and contain greater than 50% of volatiles as measured by LOD (loss on drying). A lotion tends to evaporate rapidly with a cooling sensation when rubbed onto the skin.

[0152] A paste is an opaque or translucent, viscous, greasy emulsion or suspension semisolid dosage form for external application to the skin, which generally contains greater than 50% of hydrocarbon-based or a polyethylene glycol-based vehicle and less than 20% of volatiles. A paste contains a large proportion (20-50%) of dispersed solids in a fatty or aqueous vehicle. An ointment tends not to evaporate or be absorbed when rubbed onto the skin.

[0153] An ointment is an opaque or translucent, viscous, greasy emulsion or suspension semisolid dosage form for external application to the skin, which generally contains greater than 50% of hydrocarbon-based or a polyethylene glycol-based vehicle and less than 20% of volatiles. An ointment is usually lipophilic, and contains >50% of hydrocarbons or polyethylene glycols as the vehicle and <20% of volatiles as measured by LOD. An ointment tends not to evaporate or be absorbed when rubbed onto the skin.

[0154] A gel is usually a translucent, non-greasy emulsion or suspension semisolid dosage form for external application to the skin, which contains a gelling agent in quantities sufficient to impart a three-dimensional, cross-linked matrix. A gel is usually hydrophilic, and contains sufficient quantities of a gelling agent such as starch, cellulose derivatives, carbomers, magnesium-aluminum silicates, xanthan gum, colloidal silica, aluminium or zinc soaps.

[0155] The composition for topical and/or mucosal administration may further include drying agents, anti-foaming agents; buffers, neutralizing agents, agents to adjust pH; colouring agents and decolouring agents; emollients; emulsifying agents, emulsion stabilizers and viscosity builders; humectants; odorants; preservatives, antioxidants, and chemical stabilizers; solvents; and thickening, stiffening, and suspending agents, and a balance of water or solvent.

[0156] It should also be appreciated that other methods of delivery of an agent to inhibit the activity of a toll-like receptor are contemplated. For example, the agent may be delivered by way of a nucleic acid or vector that allows for expression of the agent in the appropriate target cells. For example, the agent may be delivered by way of a viral vector that causes expression of the agent in target cells.

[0157] In a seventh aspect, the present invention provides a combination product including an agent that inhibits the activity of at least one toll-like receptor and at least one antimicrobial agent, wherein the agent and at least one antimicrobial agent are provided in a form for co-administration or in a form for separate administration to a subject.

[0158] The components of the combination product may be packaged separately or together in suitably sterilized containers such as ampoules, bottles, or vials, either in multi-dose or in unit dosage forms. The containers are typically hermetically sealed. Methods are known in the art for the packaging of the components.

[0159] Co-administration of the agents in the combination product can be sequential or simultaneous and generally means that the agents are present in the subject during a specified time interval. Typically, if a second agent is administered within the half-life of the first agent, the two agents are considered co-administered.

[0160] In some embodiments of the seventh aspect of the invention, the agent inhibits the signalling of the at least one toll-like receptor as described in detail above. In some embodiments of the seventh aspect of the invention, the toll-like receptor may be selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9. Detail regarding these toll-like receptors is provided above. In some embodiments of the seventh aspect of the invention, the antimicrobial agent is an antibacterial agent as described above. The combination product may further include at least one anti-inflammatory agent as described above.

[0161] In a further aspect, the present invention provides a method of screening a candidate therapeutic agent useful for treating and/or preventing mastitis in a subject, the method including the step of assaying the candidate therapeutic agent for activity in inhibiting the activity of at least one toll-like receptor in the subject. Generally, an agent that decreases the expression of, the level of, or the activity of, one or more of the toll-like receptors when compared to an untreated, or non-inhibited, toll-like receptor is a candidate therapeutic agent useful for treating and/or preventing mastitis in the subject. Examples of suitable agents to screen are as described above. [0162] Screening assays may be performed in vitro and/or in vivo. For example, prospective agents may be screened to identify candidate therapeutic agents for the treatment of mastitis in a cell-based assay. In this regard, each prospective agent is incubated with cultured cells (for example cells obtained from a breast tissue sample of a subject suffering from mastitis, from cells obtained from a normal non-affected subject, from normal breast tissue of a subject suffering from mastitis, or from cell lines derived from a normal or affected subject), and the activity of a toll-like receptor under investigation is measured. In another example, candidate therapeutic agents may be screened in organ culture-based assays. In this regard, each prospective agent is incubated with either a whole organ or a portion of an organ derived from a non-human animal and the activity of a toll-like receptor under investigation is measured.

[0163] Screening methods may also employ administering prospective therapeutic agents to a subject suffering from mastitis. Accordingly, in one embodiment, the method includes measuring the activity of a toll-like receptor under investigation in the subject, wherein the activity is measured after administration of the candidate therapeutic agent to the subject. The activity of the toll-like receptor in the subject is then compared to a reference level of activity for the receptor. The reference level is generally the level of activity of the receptor in an untreated subject having mastitis, or the level of activity of a non-inhibited toll-like receptor. If the activity of the toll-like receptor under investigation in the subject is less than the reference level of activity, the candidate therapeutic agent can be said to be useful for the treatment of mastitis. The activity of a toll-like receptor may be measured by the methods described in detail above. A reference level of activity for a toll-like receptor under investigation may be derived from at least one untreated subject and is preferably derived from an average of untreated subjects (e.g. n=2 to 100 or more). A reference level of activity for a toll-like receptor under investigation can also be obtained from one or more samples taken from a subject having mastitis. For example, a reference level of activity for a toll-like receptor under investigation may be obtained from at least one sample from a subject and is preferably obtained from an average of such samples (e.g. n=2 to 100 or more), wherein the subject has mastitis.

[0164] The term "about" as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of +/- 10% or less, +/- 5% or less, +/- 1 % or less, or +/- 0.1 % or less of and from the numerical value or range recited or claimed. [0165] Reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention. See, for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory Press, 2001.

[0166] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0167] The invention is further illustrated in the following examples. The examples are for the purpose of describing particular embodiments only and are not intended to be limiting with respect to the above description.

EXAMPLE 1

Removal of TLR Signalling Resolves Mastitis

[0168] Utilising a toll-like receptor 4 (TLR 4) deficient mouse model, the inventors have established that mastitis progression is decreased and resolution of the disease is accelerated in the absence of TLR 4 signal induction. By introducing a focal blockage that contains an inflammatory signal the inventors have been able to create a unique model of mastitis induction that more accurately reflects natural mastitis development. Furthermore, it allows the inventors to probe and evaluate the immunological and functional changes in the mammary tissue at the point of mastitis induction, as well as systemic changes in a mastitis affected animal.

MATERIALS AND METHODS

Mice

[0169] All animal experiments were approved by the University of Adelaide Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th edition, 2004). Mice were maintained in specific pathogen-free conditions with controlled light (12 hr light, 12 hr dark cycle) and temperature at the University of Adelaide, Medical School Animal House. Female 8 week old Tlr4 null mutant mice (Tlr4-/-) on an inbred Balb/c background (provided by Prof Paul Foster, University of Newcastle) (ref) and wild type (Tlr4+/+) Balb/c mice (Laboratory Animal Services, University of Adelaide) were impregnated by wildtype stud males and pregnant female mice were housed individually. Induction of Mastitis

[0170] The day of parturition was designated Lactation Day 1 (LD1 ), at which time litter numbers were normalised to 6-8 pups/litter to ensure sufficient and similar lactation between dams. On LD8-LD10, dams were anaesthetised with 1 % Isoflurane. The 3rd right mammary teat was teat sealed as an involution control, the 3rd left mammary teat was left intact as a lactation control. 10 uL Matrigel containing 10 ug LPS (Sigma-Aldrich, St Louis, MO) was administered to the 4th right mammary gland via a 34G needle attached to a 100 uL glass syringe, inserted into the mammary gland teat. PBS control in matrigel was administered to the 4th left mammary gland. Dams recovered from anaesthetic for 30 minutes then were returned to their litter.

Blood Collection

[0171] Blood was collected for assessment of serum cytokine levels by retro orbital bleeding during the experiment or by cardiac puncture at termination. Each mouse was bled immediately prior to mastitis induction, at termination and at an intermediate time-point (TABLE 3). Blood was clotted at room temperature for 30 minutes then centrifuged at 2000 rpm for 10 minutes. Serum was carefully removed and stored at -80°C until analysis.

TABLE 3

Time points of blood collection post mastitis induction. The mice were bled prior to induction (0 h), at termination, and at an intermediate time point.

Cytokine Assays

[0172] Serum concentration of CXCL1 was measured using the ELISA Duo-Set kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Serum IL10 concentration was measured in the ELISA Ready-SET-Go kit according to the manufacturer's instructions (eBioscience, San Diego, CA). Serum concentrations of interleukins (IL) 1 B, 2, 4, and 12, tumor necrosis factor alpha (TNFa), interferon gamma (I FNY), CCL2, and IP10 (CXCL10) were measured by Luminex assay following manufacturer's instructions (MILIPLEX MAP kit; Millipore).

Litter Weights

[0173] Litters were weighed immediately post mastitis induction and at the same time daily until termination.

Tissue Collection and Processing

[0174] Ten to 1 1 mice per genotype were euthanized and tissue collected at either 8 hours (8 h), 1 day (D1 ), 3 days (D3) or 7 days (D7) post mastitis induction. At euthanasia the third and fourth pair mammary glands were dissected and fixed in 4% paraformaldehyde overnight then processed and embedded in paraffin. Five micron sections were mounted on glass slides (Colourfrost, HD Scientific Supplies) for H&E staining to examine histological changes and for immunohistochemical analysis.

Assessment of Affected Tissue

[0175] Sections of mammary gland were stained with H&E and images captured by the NanoZoomer (Hamamatsu, Japan). The affected area within the gland was defined as an area where there was apparent alveolar collapse or where the influx of leukocytes was such that it was unlikely that the area was functional. The affected area was determined (mm2) using NanoZoomer Digital Pathology Virtual Slide Viewer software (Hamamatsu, Japan) and percentage of total mammary gland area was calculated.

Immunohistochemistry

[0176] Mammary gland sections were dewaxed in Safsolvent rehydrated in decreasing concentrations of ethanol and rinsed in MQ water. Endogenous peroxidise activity was blocked with 1 .5% hydrogen peroxide, and then blocked with 15% normal rabbit serum (Sigma-Aldrich, St Louis, MO) to prevent non-specific binding. Tissue sections were immunostained for macrophages (F4/80 antibody at 1/100: rat monoclonal clone BM8, eBioscience, San Diego, CA) and neutrophils (RB6-Ly-6G antibody at 1/500: rat hybridoma supernatant, in house) at 4oC overnight. Sections were washed and incubated with a biotinylated rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) for 40 minutes at room temperature followed by washing and incubation with Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Positive staining was developed using DAB (DAKO Liquid DAB+Substrate Chromogen System, DAKO, Carpinteria, CA) and counterstained with hematoxylin. Tissue sections were then dehydrated in ethanol, cleared in Safsolvent and mounted in Entellan mounting media (Merck, Germany). Each stained section had a matched negative control, having the primary antibody incubation step with diluent alone. Antibody specificity was also confirmed using an isotype-matched negative control antibody.

Assessment of Immunostained Tissues

[0177] Immunostained sections were scanned by the NanoZoomer (Hamamatsu, Japan) and positive stained cells counted in 4 (RB6) and 6 (F4/80) randomly selected 40x magnification images using NanoZoomer Digital Pathology Virtual Slide Viewer software (Hamamatsu, Japan). Cells were counted manually by a blinded assessor and expressed as cells per mm².

Statistical Analysis

[0178] Data were analysed by ANOVA in IBM SPSS Statistics (IBM Corp, NY, USA). Data are presented as the mean ± SEM. P-values < 0.05 were considered statistically significant.

RESULTS

Model Development

[0179] Previous mouse models of mastitis have employed techniques involving excision of the nipple preventing feeding from the affected gland or infusion of the whole gland with stimulus and have failed to investigate the full course of the disease through to resolution and the consequences of inflammation on the functionality of the mammary gland. In contrast, our intention was to induce a focus of inflammation in an otherwise functional mammary gland reminiscent of the clinical case and to quantitatively characterise progression and resolution of the disease. Furthermore, it was essential to discern between the mere presence of inflammation in the mammary gland and the consequential physiological outcomes of mastitis such as glandular involution and alveoli cell function, both indicative of decreased mammary gland function and lactation insufficiency.

[0180] Lactating Balb/c mice were injected via the teat canal with LPS (10 pg/10 μΙ_) diluted in matrigel resulting in a small ductal blockage containing an inflammatory stimulus. There were no overt signs of poor health. Pup weight was measured daily as a proxy for maternal wellness, which demonstrated continuous weight gain by pups over the period of the study (Figure 1A).

[0181] Histological analysis of the LPS-treated glands showed a marked influx of inflammatory cells into the alveoli and milk ducts with the affected area increasing in size at 3 days post mastitis induction (Figures 1 B-D, 1 F). By 7 days post-induction the inflammation had either resolved, or the gland had become non-functional (Figure 1 E left and right respectively). This characteristic localised inflammation was not observed in untreated, teat- sealed or matrigel only vehicle mammary glands (Figure 1 G-I, respectively).

[0182] Specifically, Figure 1 displays representative photomicrographs of H&E stained mammary glands collected 8 h (B), 1 day (C), 3 days (D), 7 days (E) post mastitis induction. Normal uninterrupted lactation (G), involution induction via teat sealing (H) and matrigel-only vehicle (I) are shown in glands from the same cohort of mice 3 days post mastitis induction. A-C and I are 2.5x magnification (scale bars - 1 mm), the inset areas and E, G and H are 20x magnification (scale bars 100 pm). Quantification of the affected area as a percent of the total mammary gland is shown in Figure 1 E. Litters were weighed daily as a proxy indicator of maternal wellness and interference with lactation (Figure 1 F). Data are mean ± SEM of n = 10 - 1 1 mice per time-point. Asterisk denotes significant difference between LPS-matrigel and matrigel vehicle only (*p<0.05).

[0183] As shown in Figure 2, immunohistochemistry was utilised to examine the influx of neutrophils and macrophages into the gland following mastitis induction. RB6 positive neutrophils are rapidly recruited into the LPS-treated gland forming a dense region of staining (Figures 2A-D, representative of 8 hours, 1 day, 3 days and 7 days, respectively) compared to control (Figure 2E). Neutrophil staining was predominantly observed in the alveoli and in the milk ducts. This acute response reaches a peak 1 -day post mastitis induction (mean ± SEM: vehicle control 108 ± 36 neutrophils/mm², LPS 1875 ± 206 neutrophils/mm²; p<0.05) and has subsided by 7 days post induction (Figure 2F). There are very few neutrophils identified in untreated, teat-sealed or matrigel only vehicle control mammary glands (Figures 2G-I, respectively).

[0184] As shown in Figure 3, the spatial and temporal pattern of macrophage abundance differs to that of neutrophils (Figures 3A-D, representative of 8 hours, 1 day, 3 days and 7 days, respectively) compared to control (Figure 3E), with macrophage numbers only starting to increase 1 day post mastitis induction and peaking at 3 days (mean ± SEM: vehicle control 321 ± 57 macrophages/mm2, LPS 845 ± 73 macrophages/mm2; p<0.05) (Figure 3F). A smaller yet significant increase was observed in vehicle control glands at 3D and 7D post induction compared to 8 h (8h 105 ± 26 macrophages/mm2, 3D 321 ± 57 macrophages/mm2, 7D 549 ± 162 macrophages/mm2; p<0.05) This significant influx was not observed in the other control glands (Figure 3G - uninterrupted lactation; Figure 3H - teat sealed control; Figure 3I - matrigel vehicle only). Altered mastitis disease progression in Tlr4 null mutant mice

[0185] To investigate the contribution of TLR4 to mastitis disease progression and resolution, we utilised our mastitis model in Tlr4 null mutant (Tlr4-/-) mice, compared to wild type TLR4 replete (Tlr4+/+) mice. Haematoxylin and eosin staining of mammary glands permitted evaluation of the relative area in the gland that had undergone alveolar breakdown or damage. There were obvious areas of destruction in the glands of Tlr4-/- mice throughout the collection period.

[0186] As shown in Figure 4, a marked influx of RB6-positive neutrophils into the LPS- administered gland was observed in both genotypes of mice by 8 h post mastitis induction (Figures 4A-C), and was sustained at D1 (Figures 4D-F). Tlr4-/- mice exhibited a significant 2-fold higher abundance of neutrophils compared Tlr4+/+ at 8 h (mean ± SEM: Tlr4+/+ 1293 ± 313 neutrophils/mm², Tlr4-/- 2592 ± 384 neutrophils/mm²; p<0.05) (Figure 4M). The abundance of neutrophils remained high at D1 in mastitis-induced glands from both genotypes (Tlr4+/+ 1857 ± 206 neutrophils/mm², Tlr4-/- 2434 ± 422 neutrophils/mm²) (Figure 4M). Neutrophillic inflammation had substantially subsided by 3 days post-induction (Figures G-l) and was down to control levels by 7 days (Figures J-L). Low abundance of neutrophils was observed in untreated and teat sealed mammary glands from this same cohort of Tlr4-/- mice on D1 post mastitis induction (Figures 4N and 40, respectively).

[0187] The abundance of F4/80 positive macrophages in mastitic mammary glands from both Tlr4+/+ and Tlr4-/- mice increased at D1 and D3 post induction. At 8 h post-induction (Figures 5A-C), 1 day post-induction (Figures 5D-F), 3 days post-induction (Figures 5G-I) and 7 days post-induction (Figures 5 J-L), the macrophages were primarily located within the interstitial spaces between alveoli, and in close association with the basal laminar of the alveoli. In Tlr4+/+ mice, an increase in macrophage abundance was observed from 8 h, D1 and D3 post induction, plateauing thereafter (Figure 5M). In contrast, there was an 80% higher abundance of macrophages into Tlr4-/- glands at D1 (mean ± SEM: Tlr4+/+ 280 ± 39 macrophages/mm², Tlr4-/- 512 ± 41 macrophages/mm²; p<0.05), peaking at 3 days, and significantly reducing again by 7 days post mastitis induction (mean ± SEM:Tlr4+/+ 776 ± 136 macrophages/mm², Tlr4-/- 350 ± 53 macrophages/mm²; p<0.05). Low abundance of macrophages was observed in untreated and teat sealed mammary glands from this same cohort of Tlr4-/- mice on D1 post mastitis induction (Figures 4N and 40, respectively).

Attenuated serum cytokine concentrations in Tlr4 null mutant mice

[0188] To examine the systemic effects of the local mastitis induction, blood was collected and systemic changes in cytokine and chemokine concentrations were analysed. Specifically, the abundance of cytokines, including the anti-inflammatory cytokines interleukin 10 (IL10) and IL4, the pro-inflammatory cytokines IL1 B, IL2, IL6, TNFA and IFNG and the chemokine CXCL1 (KC), CCL2 and IP10 (CXCL10) were analysed. Serum CXCL1 concentrations in Tlr4+/+ mice following mastitis induction increased 240-fold from pre- treatment concentration 4 h post induction (Figure 6A). This declined rapidly but remained above basal levels until day 7. The acute increase in CXCL1 serum levels in Tlr4-/- mice exhibited a similar pattern however the magnitude of the response was attenuated by 14-fold compared to Tlr4+/+ mice at 4 h (mean ± SEM: Tlr4+/+ 22687 ± 920 pg/mL, Tlr4-/- 1424 ± 80 pg/mL; p<0.05) by 9-fold at 8 h (mean ± SEM: Tlr4+/+ 3937 ± 148 pg/mL, Tlr4-/- 432 ± 1 1 pg/mL; p<0.05), and 4-fold at D1 (mean ± SEM: Tlr4+/+ 769 ± 26 pg/mL, Tlr4-/- 169 ± 7 pg/mL; p<0.05).

[0189] Similarly, acute serum IL10 concentrations in Tlr4-/- mice were significantly attenuated compared to Tlr4+/+ mice following mastitis induction (Figure 6B), reduced by 7- fold at 4 h (mean ± SEM: Tlr4+/+ 422 ± 36 pg/mL, Tlr4-/- 62 ± 0 pg/mL; p<0.05), 7-fold at 8 h (mean ± SEM: Tlr4+/+ 450 ± 44 pg/mL, Tlr4-/- 62 ± 0 pg/mL; p<0.05) and 2.7-fold at D1 (mean ± SEM: Tlr4+/+ 168 ± 20 pg/mL, Tlr4-/- 62 ± 0 pg/mL; p<0.05).

[0190] Serum concentrations of a multitude of other cytokines and chemokines including IL1 B, IL6, TNFA, CCL2 and IP10 were also significantly decrease in Tlr4-/- mice compared to TLR4+/+ mice 4 h after induction of mastitis, whilst the abundance of IL2, IL4, IL12 and IFNG were consistent irrespective of the genotype of the mice (Figure 6C).

The Effect of Tlr4 signalling deficiency on Lactation Following Mastitis

[0191] Milk production by the mammary glands was assessed at various stages post mastitis induction in both Tlr4+/+ and Tlr4-/- mice. Anti-Whey Acidic Protein (WAP) antibody was utilised to assess the number of alveoli producing milk, whilst anti-phosphorylated-STAT5 (p- STAT5) antibody was used to identify functional alveoli epithelial cells. Furthermore, mammary gland tissue sections were assessed for signs of mammary gland involution.

[0192] One week following local induction of mastitis, areas of the gland populated by adipose tissue were observed in wild-type mice, suggesting that some areas of the gland had undergone involution; these were not observed in Tlr4-/- mice (Figure 7A and 7B, respectively). The percent area of the gland populated by glandular epithelium was quantified to assess the capacity for milk protein synthesis; this analysis was conducted in an area of the gland distal to the localized area where inflammation had been induced. On Days 1 and 3 (D1 and D3) following mastitis induction, the LPS-treated mammary gland from both genotypes was densely populated with glandular epithelium consistent with a high capacity for milk production. However, on Day 7 (D7), the glandular area was reduced in Tlr4+/+ mice; however, it remained high in Tlr4-/- mice (Figure 7C), indicating and increased capacity of Tlr4-/- mice to lactate relative to Tlr4+/+ mice.

[0193] In the alveolar epithelial structures observed on Day 7, the milk protein WAP was detected in both genotypes (Figure 7, D and E) and was not observed in negative controls (Figure 7F). Phosphorylated-STAT5 (p-STAT5) antibody was used to investigate activation of the alveolar epithelial cells to produce milk proteins. Mammary alveolar epithelium from both genotypes of mice stained positive for p-STAT5 at 7 days after mastitis induction (Figure 7G- 7I) and was not observed in negative controls (Figure 7J). The positive staining in mammary glands from Tlr4+/+ and Tlr4-/- mice was clearly observed in distinct locations within the epithelial cells: within the cytoplasm of the cell (Figure 7G), within the nucleus (Figure 7I), or both (Figure 7H). The distribution of p-STAT5 staining within epithelial cells was altered by the genotype of the mice. More Tlr4-/- mice exhibited p-STAT5 staining, predominantly in the nucleus (Figure 7K; P < 0.05), compared to Tlr4+/+ mice. In the latter genotype, less staining was observed in the nucleus and the predominance of staining was observed in the cytoplasm. When the percent of nuclear staining was quantified for each mouse, more nuclear staining was observed in Tlr4-/- mice compared to Tlr4+/+ (Figure 7K and 7L)

[0194] One week following induction of mastitis, Tlr4+/+ mice had reduced capacity for lactation compared to Tlr4-/- . Milk-secreting glandular area was reduced, and much of the gland was repopulated with adipose tissue, suggesting that partial mammary gland involution had occurred. Although the epithelial ducts that remained exhibited evidence of continued milk protein production, p-STAT5 was predominantly cytoplasmic in location. Phosphorylation of STAT5 and subsequent translocation to the nucleus is necessary for activation of milk protein synthesis, and the predominantly cytoplasmic location of p-STAT5 in Tlr4+/+ mice suggests a compromised capacity to produce milk in the remaining healthy glandular epithelium compared to Tlr4-/- mice.

[0195] As shown in Figure 8, static milk induces the production of CXCL1 from macrophages in a manner dependent on TLR 4 signalling. Macrophages isolated from the spleen of wild- type and Tlr4-/- mice, were incubated in triplicate with either fresh or static (4 hour incubation at 37°C) milk overnight and assessed for CXCL1 by ELISA. The results were normalised for the endogenous CXCL1 concentration of the milk samples. Prior to incubation, any contamination bacteria were removed by way of filtration and centrifugation of the samples. [0196] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS

1 . A method of treating and/or preventing mastitis in a subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject.

2. The method according to claim 1 , wherein the method includes administering to the subject an agent which inhibits the activity of the at least one toll-like receptor.

3. The method according to claim 1 or claim 2, wherein inhibiting the activity of at least one toll-like receptor includes inhibiting the signalling of the at least one toll-like receptor.

4. The method according to claim 3, wherein inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R-associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-κΒ transcription factor, TIR-domain- containing adapter-inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factors-activated kinase (TAK1 ), TAK-binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

5. The method according to any one of claims 1 to 4, wherein the toll-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9.

6. The method according to any one of claims 1 to 5, wherein the toll-like receptor is tolllike receptor 4.

7. The method according to any one of claims 1 to 6, further including the step of administering to the subject at least one antimicrobial agent.

8. The method according to claim 7, wherein the at least one antimicrobial agent is an antibacterial agent.

9. The method according to any one of claims 1 to 8, further including the step of administering to the subject at least one anti-inflammatory agent.

10. The method according to claim 9, wherein the at least one anti-inflammatory agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

1 1. The method according to any one of claims 1 to 10, wherein the subject is non- human.

12. The method according to any one of claims 1 to 1 1 , wherein the subject is bovine.

13. The method according to any one of claims 1 to 10, wherein the subject is human.

14. A method of improving lactation in a mastitis affected subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject.

15. The method according to claim 14, wherein the method includes administering to the subject an agent which inhibits the activity of the at least one toll-like receptor.

16. The method according to claim 14 or claim 15, wherein inhibiting the activity of at least one toll-like receptor includes inhibiting the signalling of the at least one toll-like receptor.

17. The method according to claim 16, wherein inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R-associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-κΒ transcription factor, TIR-domain- containing adapter-inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factors-activated kinase (TAK1 ), TAK-binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

18. The method according to any one of claims 14 to 17, wherein milk production in the subject is increased.

19. The method according to any one of claims 14 to 18, wherein the toll-like receptor is selected from the group consisting of toll-iike receptor 2, tolt-like receptor 3, toli-like receptor 4, toil-like receptor 5, toil-like receptor 7, tol!-iike receptor 8 and toli-like receptor 9.

20. The method according to any one of claims 14 to 19, wherein the toil-like receptor is toil-like receptor 4.

21. The method according to any one of claims 14 to 20, further including the step of administering to the subject at least one antimicrobial agent.

22. The method according to claim 21 , wherein the at least one antimicrobial agent is an antibacterial agent.

23. The method according to any one of claims 14 to 22, further including the step of administering to the subject at least one anti-inflammatory agent.

24. The method according to claim 23, wherein the at ieast one anti-inflammatory agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicy!ic acid), difiunisal, saisalate, tbuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, tndomethacin, tolmettn, suiindac, etodofac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxtcam, droxicam, iornoxicam, isoxicam, mefenamic acid, meclofenamic acid, fiufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, ftrocoxib, paracetamol, nimesulide, iicofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

25. The method according to any one of claims 14 to 24, wherein the subject is non- human.

26. The method according to any one of claims 14 to 25, wherein the subject is bovine.

27. The method according to any one of claims 14 to 24, wherein the subject is human.

28. A method of treating and/or preventing mammary tissue damage in a mastitis affected subject, the method including the step of inhibiting the activity of at least one toll-like receptor in the subject.

29. The method according to claim 28, wherein the method includes administering to the subject an agent which inhibits the activity of the at least one toll-like receptor.

30. The method according to claim 28 or claim 29, wherein inhibiting the activity of at least one toll-like receptor includes inhibiting the signalling of the at least one toll-like receptor.

31. The method according to claim 30, wherein inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R-associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-κΒ transcription factor, TIR-domain- containing adapter-inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factors-activated kinase (TAK1 ), TAK-binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

32. The method according to any one of claims 28 to 31 , wherein the toll-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9.

33. The method according to any one of claims 28 to 32, wherein the toll-like receptor is toll-like receptor 4.

34. The method according to any one of claims 28 to 33, further including the step of administering to the subject at least one antimicrobial agent.

35. The method according to claim 34, wherein the at least one antimicrobial agent is an antibacterial agent.

36. The method according to any one of claims 28 to 35, further including the step of administering to the subject at least one anti-inflammatory agent.

37. The method according to claim 36, wherein the at least one anti-inflammatory agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

38. The method according to any one of claims 28 to 37, wherein the subject is non- human.

39. The method according to any one of claims 28 to 38, wherein the subject is bovine.

40. The method according to any one of claims 28 to 37, wherein the subject is human.

41. Use of an agent that inhibits the activity of at least one toll-like receptor for the treatment and/or prevention of mastitis in a subject, for improving lactation in a mastitis affected subject, and/or for treating and/or preventing mammary tissue damage in a mastitis affected subject.

42. Use of an agent that inhibits the activity of at least one toll-like receptor in the manufacture of a medicament for the treatment and/or prevention of mastitis in a subject, for improving lactation in a mastitis affected subject, and/or for treating and/or preventing mammary tissue damage in a mastitis affected subject.

43. A pharmaceutical composition when used for treating and/or preventing mastitis in a subject, improving lactation in a mastitis affected subject, and/or treating and/or preventing mammary tissue damage in a mastitis affected subject, the composition including an effective amount of an agent that inhibits the activity of at least one toll-like receptor in the subject.

44. The composition according to claim 43, wherein the agent inhibits the signalling of the at least one toll-like receptor in the subject.

45. The composition according to claim 44, wherein inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R-associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen- activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-κΒ transcription factor, TIR- domain-containing adapter-inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factors-activated kinase (TAK1 ), TAK-binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

46. The composition according to any one of claims 43 to 45, wherein milk production in the subject is increased.

47. The composition according to any one of claims 43 to 46, wherein the toll-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, tolllike receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9.

48. The composition according to any one of claims 43 to 47, wherein the toll-like receptor is toll-like receptor 4.

49. The composition according to any one of claims 43 to 48, further including at least one antimicrobial agent.

50. The composition according to claim 49, wherein the at least one antimicrobial agent is an antibacterial agent.

51. The composition according to any one of claims 43 to 50, further including at least one anti-inflammatory agent.

52. The composition according to claim 51 , wherein the at least one anti-inflammatory agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

53. The composition according to any one of claims 43 to 52, wherein the composition is adapted to be administered orally.

54. The composition according to any one of claims 43 to 52, wherein the composition is adapted to be administered topically.

55. The composition according to any one of claims 43 to 52, wherein the composition is adapted to be administered as an injection.

56. The composition according to any one of claims 43 to 52, wherein the composition is adapted for mucosal administration.

57. The composition according to any one of claims 43 to 56, wherein the subject is non- human.

58. The composition according to any one of claims 43 to 57, wherein the subject is bovine.

59. The composition according to any one of claims 43 to 56, wherein the subject is human.

60. A combination product including:

(i) an agent that inhibits the activity of at least one toll-like receptor; and

(ii) at least one antimicrobial agent

the agent and the at least one antimicrobial agent provided in a form for co-administration to a subject or in a form for separate administration to a subject.

61. The combination product according to claim 60, wherein the agent inhibits the signalling of the at least one toll-like receptor in the subject.

62. The combination product according to claim 61 , wherein inhibiting the signalling of the at least one toll-like receptor includes inhibiting signalling mediated by one or more of molecular myeloid differentiation primary-response protein 88 (MyD88), IL-1 R-associated kinases (IRAKs), tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a mitogen-activated protein kinase (MAPK), an ΙκΒ kinase (IKK), a NF-κΒ transcription factor, TIR-domain-containing adapter-inducing interferon-β (TRIF), interferon regulatory factor (IRF) 3, IRF7, transforming growth factors-activated kinase (TAK1 ), TAK-binding protein 1 (TAB1 ), and TAK-binding protein 2 (TAB2).

63. The combination product according to any one of claims 60 to 62, wherein the toll-like receptor is selected from the group consisting of toll-like receptor 2, toll-like receptor 3, tolllike receptor 4, toll-like receptor 5, toll-like receptor 7, toll-like receptor 8 and toll-like receptor 9.

64. The combination product according to any one of claims 60 to 63, wherein the toll-like receptor is toll-like receptor 4.

65. The combination product according to any one of claims 60 to 64, wherein the at least one antimicrobial agent is an antibacterial agent.

66. The combination product according to any one of claims 60 to 65, further including at least one anti-inflammatory agent.

67. The combination product according to claim 66, wherein the at least one antiinflammatory agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), including aspirin (acetylsalicylic acid), diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketoprofen, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, tiaprofenic acid, celecoxib, parecoxib, etoricoxib, firocoxib, paracetamol, nimesulide, licofelone, lysine clonixinate, hyperforin, figwort, and calcitriol (vitamin D).

68. The combination product according to any one of claims 60 to 67, wherein the combination product is used for the treatment and/or prevention of mastitis in a subject, for improving lactation in a mastitis affected subject, and/or for treating and/or preventing mammary tissue damage in a mastitis affected subject.

69. The combination product according to claim 68, wherein milk production in the subject is increased.

70. The combination product according to any one of claims 60 to 69, wherein the subject is non-human.

71. The combination product according to any one of claims 60 to 70, wherein the subject is bovine.

72. The combination product according to any one of claims 60 to 69, wherein the subject is human.