Teat sealant composition and method of preventing mastitis

Abstract

A teat sealant composition that does not contain a bismuth salt, a method of preparing same, and a method of preventing a new intramammary infection or mastitis.

Description

[0001] Teat Sealant Composition and Method of Preventing Mastitis

[0001] Related Applications

[0002] This application claims priority of Australian Patent Application No. 2023900582, filed 6 March 2023, the entire contents of which are incorporated herein by way of reference.

[0003] Technical Field

[0004] This invention relates to a teat sealant composition that does not contain a bismuth salt, a method of preparing same, and to a method of preventing a new intramammary infection or mastitis.

[0005] Background

[0006] In dairy cows, the incidence of mastitis caused by environmental pathogens is highest at the beginning of the 7-10 week dry-off period and again at calving at the end of the dry-off period. At dry-off cows develop a natural plug, a keratin plug, which seals the teat canal; however, this takes some time (several weeks) during which time entry of microorganisms may occur, hence causing mastitis.

[0007] Internal teat sealants (ITS) containing heavy metal bismuth salts are used at dry-off of the milking cow and in heifers to ‘seal’ the teat canal and thereby prevent, or at least reduce, the entry of environmental pathogenic microorganisms into the udder. Teat sealants may be used alone or in combination with an intra-mammary antibiotic (dry-cow therapy). Dry cow therapy containing antibiotics has been used for many years to reduce the incidence of mastitis during lactation in dairy cows.

[0008] Many studies have been published regarding the effectiveness of ITS to prevent new intramammary infections during the dry period and early lactation mastitis. They show an additional effectiveness if combined with antibiotic dry cow therapy. A targeted approached is being adopted across most countries where cows with infection at dry-off (end of lactation) receive antibiotic therapy to cure existing infections and then also receive a non-antibiotic teat sealant to prevent new infections from occurring during the dry-period; whereas cows with no infection at dry-off simply receive the non-antibiotic teat sealant.

[0009] Increasing concerns about the widespread use of antimicrobials in food-producing animals have changed attitudes to dry-cow therapy. Consequently, the use of non-antibiotic teat sealants is rising. Most ITS use bismuth subnitrate as their main component, namely up to 65% w / w. Bismuth subnitrate can be perceived as an undesirable heavy metal in milk by consumers. Milk processors in New Zealand were recently so concerned about the high levels of residues of bismuth that they looked into reducing the dose of the marketed products.

[0010] Increased uptake of bismuth-based products has led to an increase in bismuth residues in milk / milk products which is now receiving scrutiny from regulators and milk processors. However, with limited alternatives, and the continued pressure to reduce antibiotic use, there are no alternatives to bismuth-based sealants.

[0011] Detailed Description of the Invention

[0012] It is an object of one or more embodiments of the present invention to provide a teat sealant composition that does not contain a bismuth salt (ie., it is bismuth-salt-free), for preventing a new intramammary infection or mastitis, or, to provide the public with a useful commercial choice.

[0013] Composition

[0014] According to a first aspect of the present invention, there is provided a bismuth-salt-free teat sealant composition, formulated to occlude a teat canal and / or teat cistern.

[0015] According to a second aspect of the present invention, there is provided a teat sealant composition comprising a salt other than a bismuth salt, formulated to occlude a teat canal and / or teat cistern.

[0016] According to a third aspect of the present invention, there is provided a teat sealant composition comprising solid particles dispersed within a water-insoluble shear-thinning viscous fluid vehicle, wherein the solid particles are not a bismuth salt.

[0017] According to a fourth aspect of the present invention, there is provided a teat sealant composition comprising solid particles in a gel or in the form of a paste or in the form of a coarse suspension, formulated to occlude a teat canal and / or teat cistern.

[0018] Preferably, the composition is free of bismuth salts and bismuth compounds.

[0019] Preferably, the composition is formulated for administration to, and retention within, a teat canal and / or teat cistern of a cow.

[0020] Preferably, the composition is in the form of a gel, paste or coarse suspension.

[0021] Preferably, the composition has an adequately low viscosity so as to allow it to be administered via the teat canal and / or teat cistern by way of injection, preferably using a syringe. Preferably, the composition is administrable to a teat using a syringe (syringe utilizing a plunger), preferably an intramammary syringe.

[0022] Any suitable quantity can be administered. Typically, between approximately 2-5 g of composition is administered / injected.

[0023] Preferably, the syringeability force for injecting the composition into the teat does not exceed 4500 g at 25°C. Preferably, the syringeability force for injecting the composition into the teat is between about 1500 g and 3500 g at 25°C, including all numerical values between the upper and lower limits, as well as any subranges falling there within (inclusive of approximately 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3200, 3200, 3300, 3400, 3500). Note this force depends not only on the formulation of the composition but also on the design of the syringe. Preferably, the syringe is a standard intramammary syringe having a cannular / nozzle inner diameter of 1.5 mm, an outer diameter of 2.5 mm, an inner barrel diameter of 13.5 mm, a cannular / nozzle length of 12 mm, and a barrel length of 49 mm. Such syringes are sold by Hubert De Backer nv (HDB veterinary intramammary syringe) at https: / / www.hdb.be / _library / _files / Tabellen / 0.5.0 / Intramammary_0_5_0.pdf. Unless otherwise specified, syringeability studies described herein utilise this HDB veterinary intramammary syringe. Preferably, the composition thickens rapidly in the teat so as to be retained and occlude the teat canal and / or teat cistern.

[0024] Typically, the mass of the composition in the teat canal will be very small compared with that in the teat cistern.

[0025] Preferably, once located within the teat canal and / or teat cistern (or at least partially within the teat cistern), the composition has a viscosity high enough so as to be retained within the teat canal or teat cistern and occlude the teat canal or teat cistern.

[0026] Preferably, the composition has a bounce-back viscosity enabling it to be retained within the teat canal and / or teat cistern as a substantially coherent mass, wherein ‘bounce-back’ means that the viscosity increases fairly rapidly once the sealant is delivered into the teat.

[0027] Preferably, the composition has a rheology enabling the composition to be administered to a teat canal and / or teat cistern by way of injection, and to be retained within the teat canal and / or teat cistern and occlude the teat canal and / or teat cistern, or to prevent an intramammary infection.

[0028] Preferably, the composition has a density greater than about 1 g / cm3, and preferably between about 1.3 g / cm3and 1.6 g / cm3(inclusive of approximately 1.3, 1.4, 1.5 and 1.6).

[0029] The phrase ‘formulated to occlude’ means that the composition is capable of forming a physical barrier in the teat canal and / or teat cistern, thereby preventing the entry, or substantially preventing the entry, of pathogenic microbes such as bacteria into the udder.

[0030] Preferably, the composition shear-thins at the shearing forces applied during injection and stripping from the teat.

[0031] In some embodiments, the composition has antimicrobial activity, particularly antibacterial activity, particularly against bacteria that cause mastitis. The antibacterial activity can be antibacterial or bacteriocidal.

[0032] In some embodiments, the composition occludes a teat canal and / or teat cistern for a period of at least about 14 days, typically about 42 to about 90 days (including 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90 days and all sub-ranges between 42 and 90), or typically up to about 4 months (including about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, and 16 weeks).

[0033] In some embodiments, occlusion is for the full length of the dry period.

[0034] In some embodiments, occlusion is for approximately the first 14 days of the dry period until a natural keratin plug develops.

[0035] The composition is preferably used in a milking cow or heifer.

[0036] In some embodiments, the composition occludes the teat canal and / or teat cistern or substantially occludes the teat canal and / or teat cistern until a natural keratin plug has formed in the teat canal.

[0037] In some embodiments, the composition occludes the teat canal and / or teat cistern or substantially occludes the teat canal and / or teat cistern for about 4-6 weeks, while allowing the natural keratin plug to develop.

[0038] In some embodiments, the composition is used at dry-off of the cow.

[0039] In some embodiments, the composition is used when environmental pathogens are highest, at the beginning of the dry-off period.

[0040] In some embodiments, the composition is formulated such that no stripping from the teat canal and / or teat cistern is required at the end of the dry-period.

[0041] All ingredients of the composition can be veterinary-acceptable.

[0042] Preferably, some, most or all ingredients of the composition are biodegradable or environmentally safe.

[0043] In some embodiments, the composition is antimicrobial or antibacterial, including inhibiting growth of the microorganism or lethal to the microorganism (eg. bactericidal). In some embodiments, one or more ingredients of the composition may provide antimicrobial activity. In some embodiments, the solid particles may modify the rheology of the composition as well as provide antimicrobial activity.

[0044] The composition can have antimicrobial activity against mastitis causing bacteria. The composition can have antimicrobial activity against any one or more of the following bacteria: Streptococcus uberis, Staphylococcus aureus, and Escherichia coli (E. coli).

[0045] The solid particles, with respect to water, can be very soluble, freely soluble, soluble, sparingly soluble, slightly soluble, very slightly soluble, practically insoluble, or insoluble. Guidelines for levels of water-solubility are defined as follows (and are not to be taken as fixed exact figures):

[0046] Descriptive Level Parts of water (solvent) per 1 part of solute

[0047] Very Soluble Less than 1

[0048] Freely Soluble From 1 to 10

[0049] Soluble From 10 to 30

[0050] Sparingly Soluble From 30 to 100

[0051] Slightly Soluble From 100 to 1000

[0052] Very Slightly Soluble From 1000 to 10,000

[0053] Practically Insoluble, or Insoluble More than 10,000

[0054] Advantages of using water-soluble solid particles include: they can reduce the quantity of residues that are left in the teat canal (and udder) that will need to be stripped or filtered out during milking; and, some water-soluble solid materials can provide an antimicrobial effect around the teat sealant composition due to creating a hypertonic solution.

[0055] Any suitable type or types of solid particles (but excluding heavy-metal bismuth salts) can be used, in any suitable quantity or quantities.

[0056] In some embodiments, the solid particles have a solubility in water that exceeds about twice the iso-osmotic concentration.

[0057] In some embodiments, the solid particles comprise at least one type of salt that is not a bismuth salt, such as an organic salt or inorganic salt. These include, but are not limited to, halide salts, phosphates and carbonates, such as sodium chloride (which is water-soluble), potassium chloride (which is water-soluble), calcium phosphate (which is very slightly soluble, practically insoluble, or insoluble in water, depending on its form), and magnesium carbonate (which is sparingly soluble in water). The solid particles can comprise at least one type of heavy metal salt provided that it is not a bismuth salt and does not result in toxic or undesirable metal residues in milk or meat of the animal. Acceptable heavy metals may include calcium, magnesium and aluminium, but exclude toxic heavy metals.

[0058] The term ‘calcium phosphate’, as herein defined, context permitting, includes within its scope any compound comprising Ca2+combined with PO3−4, HPO2−4, or H2PO−4, including both anhydrous and hydrate forms (salt hydrates), including: monocalcium phosphate; dicalcium phosphate; tricalcium phosphate (which is practically insoluble or insoluble in water); calcium phosphate dibasic anhydrous (which is very slightly soluble) / dicalcium phosphate anhydrous; calcium phosphate dibasic dihydrate (which is very slightly soluble) / dicalcium phosphate dihydrate / dibasic calcium phosphate dihydrate; calcium phosphate monobasic anhydrous / calcium bis(dihydrogenphosphate) anhydrous / calcium dihydrogenphosphate anhydrous; calcium phosphate monobasic monohydrate / calcium bis(dihydrogenphosphate) monohydrate / calcium dihydrogenphosphate monohydrate; and calcium monohydrogen phosphate dihydrate (which is practically insoluble in water or very slightly soluble) / dicalcium phosphate dihydrate / calcium hydrogen phosphate dihydrate.

[0059] In some embodiments, the solid particles comprise at least one type of carbohydrate, such as a simple or complex sugar. These include, but are not limited to, sucrose (which is very soluble in water) and mannitol (which is freely soluble in water).

[0060] In some embodiments, the solid particles comprise at least one type of mineral, such as a clay, such as kaolin (which is generally insoluble in water), such as kaolinite, halloysite, dickite or nacrite.

[0061] In some embodiments, the solid particles comprise sodium chloride. In some embodiments, the solid particles comprise potassium chloride. In some embodiments, the solid particles comprise calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate). In some embodiments, the solid particles comprise calcium carbonate. In some embodiments, the solid particles comprise magnesium carbonate.

[0062] In some embodiments, the solid particles comprise sodium chloride or potassium chloride, and calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate). In some embodiments, the solid particles comprise sodium chloride or potassium chloride, and calcium carbonate. In some embodiments, the solid particles comprise sodium chloride or potassium chloride, and magnesium carbonate.

[0063] In some embodiments, the composition comprises solid particles in the following approximate amount ranges: 10% to 75%, 35% to 70%, 40% to 65%, 50% to 60%, w / w, including all numerical values between 10 and 75, including approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 and 75.

[0064] In some embodiments, the solid particles comprise approximately 25-60% w / w sodium chloride or potassium chloride (including approximately 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60).

[0065] In some embodiments, the solid particles comprise approximately 10-30% w / w magnesium carbonate (including approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30).

[0066] In some embodiments, the solid particles comprise approximately 10% w / w magnesium carbonate and approximately 25% w / w sodium chloride or potassium chloride.

[0067] In some embodiments, the solid particles comprise approximately 55% w / w sodium chloride or potassium chloride.

[0068] In some embodiments, the solid particles comprise approximately 35-60% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate) (including approximately 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60).

[0069] In some embodiments, the solid particles comprise approximately 60% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate).

[0070] In some embodiments, the solid particles comprise approximately 10% w / w sodium chloride or potassium chloride and approximately 50% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate).

[0071] In some embodiments, the solid particles comprise approximately 25% w / w sodium chloride or potassium chloride and approximately 35% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate).

[0072] In some embodiments, the solid particles comprise approximately 10% w / w sodium chloride or potassium chloride and approximately 50% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate).

[0073] In some embodiments, the solid particles can be milled and / or sieved. In some embodiments, the solid particles can be within a particle size range, or of a particular average particle size. In some embodiments, the average particle size can be less than approximately 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 µm in size. In some embodiments, the average particle size can be less than approximately 106 µm in size. In some embodiments, the average particle size can be less than approximately 80 µm in size. In some embodiments, the average particle size can be less than approximately 50 µm in size. In some embodiments, the average particle size can be less than approximately 30 µm in size. In some embodiments, the average particle size can be less than approximately 20 µm in size. In some embodiments, the average particle size can be less than approximately 10, 15, 20, 25, 30, 35 or 40 µm in size. In some embodiments, the average particle size can be approximately 10-40 µm in size (including approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40). In some embodiments, the average particle size can be approximately 10-50 µm in size (including approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50). In some embodiments, the average particle size can be approximately 20-30 µm in size (including approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30). In some embodiments, the average particle size can be approximately 1-10 µm in size (including approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10). In some embodiments, the average particle size can be greater than approximately 3 µm in size.

[0074] In some embodiments: the D90 (particle size distribution parameter of 90%) can be approximately 30-60 µm (including approximately 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60); the D50 can be approximately 6-12 µm in size (including approximately 6, 7, 8, 9, 10, 11 and 12); and / or the D10 can be 1-2 µm. In some embodiments, these D90, D50 and D10 particle size distribution parameters are suitable for sodium chloride. In some embodiments, these D90, D50 and D10 particle size distribution parameters are suitable for calcium phosphate as defined herein, preferably dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate. In some embodiments, the D90 of calcium phosphate as herein defined can be about 50 µm. In some embodiments, sodium chloride can be milled to an average particle size of approximately <10 µm. In some embodiments, sodium chloride can have an average particle size of approximately 20-30 µm.

[0075] Any suitable type of vehicle can be used. In some embodiments, the vehicle can comprise a water-insoluble carrier and a thickener. Typically, the thickener includes the solid particles. That is, the solid particles typically affect the rheology of the composition.

[0076] Any suitable type of water-insoluble carrier can be used. The water-insoluble carrier can comprise at least one type of carrier agent. In some embodiments, the carrier or carrier agent comprises at least one type of oil or oily liquid. Suitable examples include a vegetable oil, mineral oil, synthetic oil, medium chain triglyceride, and triglyceride. Preferred examples include paraffin oil (light or heavy), liquid paraffin, petrolatum (white or yellow), sesame oil, and miglyol (which is a medium chain triglyceride).

[0077] Any suitable quantity of carrier or carrier agent can be used. In some embodiments, the composition comprises carrier or carrier agents in the following approximate ranges: 20% to 80% w / w (including approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and 80), including all subranges and numerical values between 20 and 80%, including approximately 30-65% w / w (including approximately 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65).

[0078] Any suitable type of thickener can be used. The thickener can comprise at least one type of thickening agent. The at least one type of thickening agent can be water-soluble and / or water- insoluble. In some embodiments, at least one thickening agent can function both as a thickener and as an antimicrobial. An example of such an agent is lauric acid.

[0079] In some embodiments, at least one thickening agent can comprise a medium-chain saturated fatty acid, such as lauric acid. In some embodiments lauric acid is used

[0080] In some embodiments, the composition comprises approximately 1% to 10% w / w medium- chain saturated fatty acid, such as lauric acid, including all numerical values between 1 and 10 (including approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10), including approximately 1, 2.5 and 5%.

[0081] In some embodiments, at least one thickening agent can comprise a metal salt of a long chain fatty acid, such as a stearate salt. Suitable salts can include aluminium distearate, aluminium stearate, aluminium tristearate, ammonium stearate, barium stearate, butyl stearate, cadmium stearate, calcium stearate, cobalt stearate, copper stearate, glycol stearate, lithium stearate, magnesium stearate, manganese stearate, methyl stearate, potassium stearate, sodium stearate, strontium stearate, and zinc stearate. Preferably, the at least one thickening agent comprises aluminium stearate.

[0082] In some embodiments, the composition comprises approximately 1% to 10% w / w metal salt of a long chain fatty acid (including approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10), such as stearate salt, such as aluminium stearate, including all numerical values between 1 and 10 (including approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10), including approximately 4.3 and 5.3%.

[0083] In some embodiments, at least one thickening agent can comprise silicon dioxide, such as hydrophilic or hydrophobic silica. Suitable silicon dioxide agents include fumed silica, such as hydrophilic fumed silica or hydrophobic fumed silica. Hydrophilic fumed silica is sold under the trade mark Aerosil-200. Hydrophobic fumed silica is sold under the trade mark Aerosil R972.

[0084] In some embodiments, the composition comprises approximately 0.1% to 1% w / w silicon dioxide, such as hydrophilic fumed silica, including all numerical values between 0.1 and 1 (including approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1), including approximately 0.7%.

[0085] In some embodiments, at least one thickening agent can comprise a water-soluble fumed silica, such as the one sold under the trade mark Aerosil-200 by Evonik.

[0086] Any suitable quantity of thickener or thickening agent can be used. In some embodiments, the composition comprises thickener or thickening agents in the following approximate ranges: 0.1% to 10%, w / w, including all numerical values between 0.1 and 10 (including approximately 0.1, 0.2…1, 1.1, 1.2…2, 2.1, 2.2…3, 3.1, 3.2…4, 4.1, 4.2…5, 5.1, 5.2…6, 6.1, 6.2…7, 7.1, 7.2…8, 8.1, 8.2…9, 9.1, 9.2…10), including approximately less than 1%.

[0087] The composition can have antimicrobial activity against mastitis-causing bacteria. The composition can, for example, have antimicrobial activity against any one or more of the following bacteria: Streptococcus uberis, Staphylococcus aureus, and / or Escherichia coli (E. coli).

[0088] In some embodiments, the composition or component, agent or ingredient thereof can provide contact killing. In some embodiments, the composition or component, agent or ingredient thereof can provide solution killing. In some embodiments, the composition or component, agent or ingredient thereof can provide both contact and solution killing.

[0089] ‘Contact killing’ means that the composition’s surface has antimicrobial activity, when surface contact is made by a microbe such as a bacterium. ‘Solution killing’ means a chemical, ingredient or agent has leached from the composition and has antimicrobial activity when contact is made with a microbe such as a bacterium.

[0090] In some embodiments, the composition comprises at least one type of antimicrobial agent, particularly an antibacterial agent. Suitable agents include chlorhexidine, PVP-iodine, lacticin and lauric acid. Preferably, the composition does not contain an antibiotic. ‘Antibiotic’ in this context means a type of antibiotic typically / conventionally used in dry cow therapy. Any suitable quantity of antimicrobial agent can be used. In some embodiments, the composition comprises an antimicrobial agent in the following approximate ranges: 0.1% to 10%, w / w, including all numerical values between 0.1 and 10 (including approximately 0.1, 0.2…1, 1.1, 1.2…2, 2.1, 2.2…3, 3.1, 3.2…4, 4.1, 4.2…5, 5.1, 5.2…6, 6.1, 6.2…7, 7.1, 7.2…8, 8.1, 8.2…9, 9.1, 9.2…10), including approximately 1-5% for antimicrobial agents such as lauric acid (including approximately 1, 2, 3, 4 and 5), and approximately 0.2-2% for antimicrobial agents such as chlorohexidine (including approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2).

[0091] In some embodiments, antimicrobial activity is provided by the vehicle. In some embodiments, antimicrobial activity is provided by the solid particles. In some embodiments, antimicrobial activity is provided by both the vehicle and the solid particles. In some embodiments, antimicrobial activity is provided by an ingredient of the vehicle, such as the carrier or carrier agent thereof and / or the thickener or thickening agent thereof. For example, lauric acid can be both a thickening agent and can provide antibacterial activity. For example, a salt such as sodium chloride can be both a thickening agent and can provide antibacterial activity by way of creating a hypertonic solution around the composition within the teat canal and / or teat cistern.

[0092] The composition can comprise one or more other ingredients such as at least one antioxidant or preservative. Any suitable quantity or quantities can be added, such as approximately 0.01% w / w to 5% w / w (including approximately 0.01…0.05…0.1…0.5…2…2.5…3…3.5…4…4.5…5). Examples of antioxidant or preservative include alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, BHA (butylated hydroxy anisole), BHT (butylated hydroxy toluene), bronopol, butylparaben, cetrimide, chlorhexidine, chlorobutanol, chlorocresol, cresol, ethylparaben, fumaric acid, imidurea, malic acid, monothioglycerol, n-propyl gallate, parabens (methylparaben and / or propylparaben), phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, potassium sorbate, sodium ascorbate, sodium benzoate, sodium metabisulfite, sodium propionate, sorbic acid, and thimerosal.

[0093] The composition can comprise at least one colourant. Any suitable quantity or quantities can be added, such as approximately 0.01% w / w to 10% w / w (including approximately 0.01…0.1…1…10). Examples of colourants include dyes and pigments, including aluminium lake, caramel, methylene blue and titanium dioxide.

[0094] According to a fifth aspect of the present invention, there is provided a teat sealant composition comprising:

[0095] an oily carrier; and

[0096] a thickener that comprises solid particles and lauric acid,

[0097] wherein the solid particles are not a bismuth metal salt.

[0098] According to a sixth aspect of the present invention, there is provided a teat sealant composition comprising coarse filler material, in the form of solid particles, in a gelling agent, wherein the solid particles are not a bismuth metal salt.

[0099] According to a seventh aspect of the present invention, there is provided a teat sealant composition comprising:

[0100] liquid paraffin;

[0101] aluminium stearate;

[0102] fumed silica; and

[0103] water-soluble solid particles,

[0104] wherein said composition is in the form of a paste or suspension.

[0105] According to an eighth aspect of the present invention, there is provided a teat sealant composition comprising:

[0106] liquid paraffin;

[0107] aluminium stearate;

[0108] fumed silica; and

[0109] water-insoluble solid particles,

[0110] wherein said composition is in the form of a paste or suspension.

[0111] In some embodiments, the teat sealant composition comprises one of the following formulations shown in the Formulations Table (all ingredients shown are actual or approximate, and all are %w / w).

[0112] Formulations Table. Sodium Magnesium Calcium Aluminium Fumed Lauric Acid Paraffin Chloride Carbonate Phosphate, Stearate Silica Oil or (such as (Aerosil- Potassium Dicalcium 200) Chloride Phosphate Dihydrate) 55 4.3 0.7 5 35 60 4.3 0.7 5 30 55 4.3 0.7 2.5 37.5 10 50 4.3 0.7 5 30 10 50 4.3 0.7 5 (Added last 30 when preparing composition) 25 35 4.3 0.7 5 30 60 4.3 0.7 5 30 25 4.3 0.7 5 65 50 5.3 0.7 5 39 30 0.7 5 64.3 50 5.3 0.7 44 25 10 4.3 0.7 60

[0113] Preparation Method

[0114] According to a ninth aspect of the present invention, there is provided a method of preparing a teat sealant composition comprising solid particles dispersed within a water-insoluble shear-thinning viscous fluid vehicle, said method comprising the steps of:

[0115] (1) forming a water-insoluble gel by mixing at least one type of water-insoluble carrier agent with at least one type of thickening agent;

[0116] (2) adding solid particles to the gel to form a paste; and, optionally,

[0117] (3) adding at least a further type of thickening agent to the gel or paste, wherein:

[0118] a teat sealant composition comprising solid particles dispersed within a water-insoluble shear- thinning viscous fluid vehicle is prepared;

[0119] the solid particles are not a bismuth metal salt;

[0120] step (3) is optional; and

[0121] steps (2) and (3) need not be carried out in the stated order.

[0122] According to a tenth aspect of the present invention, there is provided a method of preparing a teat sealant composition, said method comprising the steps of:

[0123] (1) forming a water-insoluble gel by mixing at least one type of oily carrier with at least one type of thickening agent;

[0124] (2) adding solid particles to the gel to form a paste; and,

[0125] (3) adding lauric acid as a thickening agent to the gel or paste, wherein:

[0126] the solid particles are not a bismuth metal salt.

[0127] According to an eleventh aspect of the present invention, there is provided teat sealant composition when prepared by the method according to the ninth or tenth aspect of the present invention.

[0128] Unless stated to the contrary, ‘cow’ as used in this specification encompasses a ‘heifer’.

[0129] Method of Use

[0130] According to a twelfth aspect of the present invention, there is provided a teat sealant composition for preventing a new intramammary infection or mastitis in a cow, said composition being as described in respect of the first to eighth and eleventh aspects of the present invention.

[0131] According to a thirteenth aspect of the present invention, there is provided a teat sealant composition for use or when used for preventing a new intramammary infection or mastitis in a cow, said composition being as described in respect of the first to eighth and eleventh aspects of the present invention.

[0132] According to a fourteenth aspect of the present invention, there is provided use of a teat sealant composition in the manufacture of a medicament for preventing a new intramammary infection or mastitis in a cow, said composition being as described in respect of the first to eighth and eleventh aspects of the present invention.

[0133] According to a fifteenth aspect of the present invention, there is provided a method of preventing a new intramammary infection or mastitis in a cow, said method comprising the step of administering to at least one teat canal and / or teat cistern of the cow the composition as described in respect of the first to eighth and eleventh aspects of the present invention.

[0134] According to a sixteenth aspect of the present invention, there is provided use of the composition as described in respect of the first to eighth and eleventh aspects of the present invention for preventing a new intramammary infection or mastitis in a cow.

[0135] According to a seventeenth aspect of the present invention, there is provided a syringe containing the composition as described in respect of the first to eighth and eleventh aspects of the present invention.

[0136] According to an eighteenth aspect of the present invention, there is provided a kitset for use or when used in a method of preventing a new intramammary infection or mastitis in a cow, wherein the kitset comprises: a syringe capable of administering to at least one teat canal and / or teat cistern of a cow the composition as described in respect of the first to eighth and eleventh aspects of the present invention.

[0137] The composition is preferably used in a milking cow or heifer.

[0138] Preferably, the composition prevents a new intramammary infection or mastitis for a period of at least about 14 days, typically about 42 to about 90 days (including 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90 days and all sub-ranges between 42 and 90), or typically up to about 4 months (including about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, and 16 weeks).

[0139] In some embodiments, occlusion by the composition is for approximately the first 14 days of the dry period until a natural keratin plug develops.

[0140] In some embodiments, the composition occludes the teat canal and / or teat cistern or substantially occludes the teat canal and / or teat cistern until a natural keratin plug has formed in the teat canal.

[0141] Preferably, the composition protects the teat or teats of the cow from bacterial infection during an entire dry period.

[0142] Preferably, the composition is administered per quarter at the end of lactation.

[0143] Preferably, the composition is administered per quarter in maiden heifers approximately 4 weeks prior to calving.

[0144] Preferred embodiments of the invention are defined in the following numbered paragraphs.

[0145] 1. A bismuth-salt-free teat sealant composition, formulated to occlude a teat canal and / or teat cistern.

[0146] 2. A teat sealant composition comprising a salt other than a bismuth salt, formulated to occlude a teat canal and / or teat cistern.

[0147] 3. A teat sealant composition formulated for administration to, and retention within, a teat canal and / or teat cistern of a cow.

[0148] 4. The teat sealant composition of any one of the preceding paragraphs, wherein the teat sealant composition is in the form of a gel, paste or coarse suspension.

[0149] 5. The teat sealant composition of any one of the preceding paragraphs, wherein:

[0150] - the teat sealant composition has an adequately low viscosity so as to allow it to be administered via a teat canal and / or teat cistern by way of injection;

[0151] - approximately 2-5 g of the teat sealant composition is administered / injected into a teat canal and / or teat cistern;

[0152] - a syringeability force for injecting the teat sealant composition into a teat does not exceed 4500 g at 25°C, and preferably the syringeability force for injecting the teat sealant composition into a teat is between about 1500 g and 3500 g at 25°C;

[0153] - the teat sealant composition thickens rapidly in a teat so as to be retained and occlude a teat canal and / or teat cistern;

[0154] - once located within a teat canal and / or teat cistern, the teat sealant composition has a viscosity high enough so as to be retained within the teat canal or teat cistern and occlude the teat canal or teat cistern;

[0155] - the teat sealant composition has a bounce-back viscosity enabling it to be retained within a teat canal and / or teat cistern as a substantially coherent mass;

[0156] - the teat sealant composition has a rheology enabling the composition to be administered to a teat canal and / or teat cistern by way of injection, and to be retained within the teat canal and / or teat cistern and occlude the teat canal and / or teat cistern, or to prevent an intramammary infection;

[0157] - teat sealant composition has a density greater than about 1 g / cm3, preferably between about 1.3 g / cm3and 1.6 g / cm3;

[0158] - the teat sealant composition shear-thins at the shearing forces applied during injection and stripping from a teat;

[0159] - the teat sealant composition has antimicrobial activity, preferably antibacterial activity, preferably against bacteria that cause mastitis;

[0160] - the teat sealant composition is formulated to occlude a teat canal and / or teat cistern for a period of at least about 14 days, preferably about 42 to about 90 days;

[0161] - the teat sealant composition is formulated to occlude a teat canal and / or teat cistern for a full length of a dry period;

[0162] - the teat sealant composition is formulated to occlude a teat canal and / or teat cistern for at least approximately the first 14 days of a dry period until a natural keratin plug develops;

[0163] - the teat sealant composition occludes a teat canal and / or teat cistern or substantially occludes - the teat canal and / or teat cistern for about 4-6 weeks, while allowing a natural keratin plug to develop; or

[0164] - the teat sealant composition is formulated such that no stripping from a teat canal and / or teat cistern is required at the end of a dry-period.

[0165] 6. A teat sealant composition comprising solid particles dispersed within a water-insoluble shear-thinning viscous fluid vehicle, wherein the solid particles are not a bismuth salt.

[0166] 7. The teat sealant composition of paragraph 6, wherein: antimicrobial activity is provided by the vehicle; antimicrobial activity is provided by the solid particles; antimicrobial activity is provided by both the vehicle and the solid particles; antimicrobial activity is provided by an ingredient of the vehicle; or, the composition further comprises at least one type of antimicrobial agent, preferably an antibacterial agent.

[0167] 8. The teat sealant composition of paragraph 6 or paragraph 7, wherein the vehicle comprises a water-insoluble carrier and a thickener, and preferably the thickener includes the solid particles.

[0168] 9. The teat sealant composition of paragraph 8, wherein the water-insoluble carrier comprises at least one type of carrier agent, and the carrier agent comprises at least one type of oil or oily liquid.

[0169] 10. The teat sealant composition of paragraph 9, wherein the at least one type of oil or oily liquid comprises a vegetable oil, mineral oil, synthetic oil, medium chain triglyceride, or triglyceride, preferably paraffin oil, liquid paraffin, petrolatum, sesame oil, or a medium chain triglyceride.

[0170] 11. The teat sealant composition of paragraph 9 or 10, wherein the composition comprises approximately 20% to 80% w / w or 30-65% w / w of the at least one type of carrier agent.

[0171] 12. The teat sealant composition of any one of paragraphs 8 to 11, wherein the thickener comprises at least one type of thickening agent which is water-soluble and / or water-insoluble, preferably in an amount of approximately 0.1% to 10%, w / w.

[0172] 13. The teat sealant composition of paragraph 12, wherein the at least one thickening agent functions both as a thickener and as an antimicrobial.

[0173] 14. The teat sealant composition of paragraph 12 or paragraph 13, wherein the at least one thickening agent comprises a medium-chain saturated fatty acid, preferably lauric acid.

[0174] 15. The teat sealant composition of paragraph 14, wherein the composition comprises approximately 1% to 10% w / w of the medium-chain saturated fatty acid, preferably lauric acid.

[0175] 16. The teat sealant composition of paragraph 12 or 13, wherein the at least one thickening agent comprises a metal salt of a long chain fatty acid, preferably a stearate salt.

[0176] 17. The teat sealant composition of paragraph 16, wherein the metal salt of the long chain fatty acid comprises aluminium distearate, aluminium stearate, aluminium tristearate, ammonium stearate, barium stearate, butyl stearate, cadmium stearate, calcium stearate, cobalt stearate, copper stearate, glycol stearate, lithium stearate, magnesium stearate, manganese stearate, methyl stearate, potassium stearate, sodium stearate, strontium stearate, or zinc stearate, preferably aluminium stearate.

[0177] 18. The teat sealant composition of paragraph 17, wherein the composition comprises approximately 1% to 10% w / w of the metal salt of the long chain fatty acid.

[0178] 19. The teat sealant composition of paragraph 12 or paragraph 13, wherein the at least one thickening agent comprises silicon dioxide, preferably hydrophilic or hydrophobic silica.

[0179] 20. The teat sealant composition of paragraph 19, wherein the composition comprises approximately 0.1% to 1% w / w silicon dioxide, preferably hydrophilic fumed silica.

[0180] 21. The teat sealant composition of any one of paragraphs 6 to 20, wherein:

[0181] - the solid particles, with respect to water, are very soluble, freely soluble, soluble, sparingly soluble, slightly soluble, very slightly soluble, practically insoluble, or insoluble;

[0182] - the solid particles have a solubility in water that exceeds about twice the iso-osmotic concentration;

[0183] - the solid particles modify the rheology of the composition as well as provide antimicrobial activity preferably against mastitis causing bacteria;

[0184] - the solid particles comprise at least one type of salt that is not a bismuth salt, such as an organic salt or inorganic salt;

[0185] - the solid particles comprise a halide salt, phosphate or carbonate, preferably sodium chloride, potassium chloride, calcium phosphate as herein defined (such as dicalcium phosphate dihydrate or calcium phosphate dibasic dihydrate), or magnesium carbonate;

[0186] - the solid particles comprise at least one type of heavy metal salt provided that it is not a bismuth salt, preferably with the metal of the heavy metal salt comprising calcium, magnesium or aluminium, but excluding toxic heavy metals;

[0187] - the solid particles comprise at least one type of carbohydrate, such as a simple or complex sugar, preferably comprising sucrose or mannitol;

[0188] - the solid particles comprise at least one type of mineral, such as a clay, such as kaolin, such as kaolinite, halloysite, dickite or nacrite;

[0189] - the solid particles comprise sodium chloride;

[0190] - the solid particles comprise potassium chloride;

[0191] - the solid particles comprise calcium phosphate as herein defined;

[0192] - the solid particles comprise dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate;

[0193] - the solid particles comprise calcium carbonate;

[0194] - the solid particles comprise magnesium carbonate;

[0195] - the solid particles comprise sodium chloride or potassium chloride, and calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate);

[0196] - the solid particles comprise sodium chloride or potassium chloride, and calcium carbonate;

[0197] - the solid particles comprise sodium chloride or potassium chloride, and magnesium carbonate;

[0198] - the composition comprises solid particles in the following approximate amount ranges: 10% to 75%, 35% to 70%, 40% to 65%, or 50% to 60% w / w;

[0199] - the composition comprises 25-60% w / w sodium chloride or potassium chloride;

[0200] - the composition comprises 10-30% w / w magnesium carbonate;

[0201] - the composition comprises 10% w / w magnesium carbonate and approximately 25% w / w sodium chloride or potassium chloride;

[0202] - the composition comprises 55% w / w sodium chloride or potassium chloride;

[0203] - the composition comprises 35-60% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate);

[0204] - the composition comprises 60% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate);

[0205] - the composition comprises 10% w / w sodium chloride or potassium chloride and 50% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate);

[0206] - the composition comprises 25% w / w sodium chloride or potassium chloride and 35% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate); or

[0207] - the composition comprises 10% w / w sodium chloride or potassium chloride and 50% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate).

[0208] 22. The teat sealant composition of any one of paragraphs 6 to 21, further comprising one or more other ingredients such as at least one antioxidant or preservative.

[0209] 23. A teat sealant composition comprising solid particles in a gel or in the form of a paste or in the form of a course suspension, formulated to occlude a teat canal and / or teat cistern.

[0210] 24. A teat sealant composition comprising:

[0211] an oily carrier; and

[0212] a thickener that comprises solid particles and lauric acid,

[0213] wherein the solid particles are not a bismuth salt.

[0214] 25. A teat sealant composition comprising coarse filler material, in the form of solid particles, in a gelling agent, wherein the solid particles are not a bismuth salt.

[0215] 26. A teat sealant composition comprising:

[0216] liquid paraffin;

[0217] aluminium stearate;

[0218] fumed silica; and

[0219] water-soluble solid particles,

[0220] wherein said composition is in the form of a paste or suspension.

[0221] 27. A teat sealant composition comprising:

[0222] liquid paraffin;

[0223] aluminium stearate;

[0224] fumed silica; and

[0225] water-insoluble solid particles,

[0226] wherein said composition is in the form of a paste or suspension.

[0227] 28. The teat sealant composition of any one of paragraphs 23 to 27, wherein the average solid particle size is less than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 µm in size, or the average solid particle size is approximately 10-50 µm in size, or the average solid particle size is approximately 3-10µm in size.

[0228] 29. The teat sealant composition of any one of paragraphs 23 to 28, wherein:

[0229] - the solid particles, with respect to water, are very soluble, freely soluble, soluble, sparingly soluble, slightly soluble, very slightly soluble, practically insoluble, or insoluble;

[0230] - the solid particles have a solubility in water that exceeds about twice the iso-osmotic concentration;

[0231] - the solid particles modify the rheology of the composition as well as provide antimicrobial activity preferably against mastitis causing bacteria;

[0232] - the solid particles comprise at least one type of salt that is not a bismuth salt, such as an organic salt or inorganic salt;

[0233] - the solid particles comprise a halide salt, phosphate or carbonate, preferably sodium chloride, potassium chloride, calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate), or magnesium carbonate;

[0234] - the solid particles comprise at least one type of heavy metal salt provided that it is not a bismuth salt, preferably with the metal of the heavy metal salt comprising calcium, magnesium or aluminium, but excluding toxic heavy metals;

[0235] - the solid particles comprise at least one type of carbohydrate, such as a simple or complex sugar, preferably comprising sucrose or mannitol;

[0236] - the solid particles comprise at least one type of mineral, such as a clay, such as kaolin, such as kaolinite, halloysite, dickite or nacrite;

[0237] - the solid particles comprise sodium chloride;

[0238] - the solid particles comprise potassium chloride;

[0239] - the solid particles comprise calcium phosphate as herein defined;

[0240] - the solid particles comprise dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate;

[0241] - the solid particles comprise calcium carbonate;

[0242] - the solid particles comprise magnesium carbonate;

[0243] - the solid particles comprise sodium chloride or potassium chloride, and calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate);

[0244] - the solid particles comprise sodium chloride or potassium chloride, and calcium carbonate;

[0245] - the solid particles comprise sodium chloride or potassium chloride, and magnesium carbonate;

[0246] - the composition comprises solid particles in the following approximate amount ranges: 10% to 75%, 35% to 70%, 40% to 65%, or 50% to 60% w / w;

[0247] - the composition comprises 25-60% w / w sodium chloride or potassium chloride;

[0248] - the composition comprises 10-30% w / w magnesium carbonate;

[0249] - the composition comprises 10% w / w magnesium carbonate and approximately 25% w / w sodium chloride or potassium chloride;

[0250] - the composition comprises 55% w / w sodium chloride or potassium chloride;

[0251] - the composition comprises 35-60% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate);

[0252] - the composition comprises 60% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate);

[0253] - the composition comprises 10% w / w sodium chloride or potassium chloride and 50% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate);

[0254] - the composition comprises 25% w / w sodium chloride or potassium chloride and 35% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate); or

[0255] - the composition comprises 10% w / w sodium chloride or potassium chloride and 50% w / w calcium phosphate as herein defined (such as dicalcium phosphate dihydrate / calcium phosphate dibasic dihydrate).

[0256] 30. A teat sealant composition comprising one of the following formulations, wherein all ingredients shown are actual or approximate:

[0257] NaCl or KCl 55% w / w;

[0258] Aluminium stearate 4.3% w / w;

[0259] Fumed silica 0.7% w / w;

[0260] Lauric acid 5% w / w; and

[0261] Paraffin oil 35% w / w;

[0262] or

[0263] NaCl or KCl 60% w / w;

[0264] Aluminium stearate 4.3% w / w;

[0265] Fumed silica 0.7% w / w;

[0266] Lauric acid 5% w / w; and

[0267] Paraffin oil 30% w / w;

[0268] or

[0269] NaCl or KCl 55% w / w;

[0270] Aluminium stearate 4.3% w / w;

[0271] Fumed silica 0.7% w / w;

[0272] Lauric acid 2.5% w / w; and

[0273] Paraffin oil 37.5% w / w;

[0274] or

[0275] NaCl or KCl 10% w / w;

[0276] Calcium phosphate as herein defined 50% w / w;

[0277] Aluminium stearate 4.3% w / w;

[0278] Fumed silica 0.7% w / w;

[0279] Lauric acid 5% w / w; and

[0280] Paraffin oil 30% w / w;

[0281] or

[0282] NaCl or KCl 25% w / w;

[0283] Calcium phosphate as herein defined 35% w / w;

[0284] Aluminium stearate 4.3% w / w;

[0285] Fumed silica 0.7% w / w;

[0286] Lauric acid 5% w / w; and

[0287] Paraffin oil 30% w / w;

[0288] or

[0289] Calcium phosphate as herein defined 60% w / w;

[0290] Aluminium stearate 4.3% w / w;

[0291] Fumed silica 0.7% w / w;

[0292] Lauric acid 5% w / w; and

[0293] Paraffin oil 30% w / w;

[0294] or

[0295] Magnesium carbonate 25% w / w;

[0296] Aluminium stearate 4.3% w / w;

[0297] Fumed silica 0.7% w / w;

[0298] Lauric acid 5% w / w; and

[0299] Paraffin oil 65% w / w;

[0300] or

[0301] NaCl or KCl 50% w / w;

[0302] Aluminium stearate 5.3% w / w;

[0303] Fumed silica 0.7% w / w;

[0304] Lauric acid 5% w / w; and

[0305] Paraffin oil 39% w / w;

[0306] or

[0307] Magnesium carbonate 30% w / w;

[0308] Fumed silica 0.7% w / w;

[0309] Lauric acid 5% w / w; and

[0310] Paraffin oil 64.3% w / w;

[0311] or

[0312] NaCl or KCl 50% w / w;

[0313] Aluminium stearate 5.3% w / w;

[0314] Fumed silica 0.7% w / w; and

[0315] Paraffin oil 44% w / w;

[0316] or

[0317] NaCl or KCl 25% w / w;

[0318] Magnesium carbonate 10% w / w;

[0319] Aluminium stearate 4.3% w / w;

[0320] Fumed silica 0.7% w / w; and

[0321] Paraffin oil 60% w / w.

[0322] 31. A method of preparing a teat sealant composition comprising solid particles dispersed within a water-insoluble shear-thinning viscous fluid vehicle, said method comprising the steps of:

[0323] (1) forming a water-insoluble gel by mixing at least one type of water-insoluble carrier agent with at least one type of thickening agent;

[0324] (2) adding solid particles to the gel to form a paste; and, optionally,

[0325] (3) adding at least a further type of thickening agent to the gel or paste, wherein:

[0326] a teat sealant composition comprising solid particles dispersed within a water-insoluble shear- thinning viscous fluid vehicle is prepared;

[0327] the solid particles are not a bismuth metal salt;

[0328] step (3) is optional; and

[0329] steps (2) and (3) need not be carried out in the stated order.

[0330] 32. A method of preparing a teat sealant composition, said method comprising the steps of:

[0331] (1) forming a water-insoluble gel by mixing at least one type of oily carrier with at least one type of thickening agent;

[0332] (2) adding solid particles to the gel to form a paste; and,

[0333] (3) adding lauric acid as a thickening agent to the gel or paste, wherein:

[0334] the solid particles are not a bismuth metal salt.

[0335] 33. A teat sealant composition when prepared by the method according to paragraph 31 or paragraph 32.

[0336] 34. A teat sealant composition for preventing a new intramammary infection or mastitis in a cow, wherein the composition is as described in any one of paragraphs 1 to 30 and 33.

[0337] 35. A teat sealant composition for use or when used for preventing a new intramammary infection or mastitis in a cow, wherein the composition is as described in any one of paragraphs 1 to 30 and 33.

[0338] 36. Use of a teat sealant composition in the manufacture of a medicament for preventing a new intramammary infection or mastitis in a cow, wherein the composition is as described in any one of paragraphs 1 to 30 and 33.

[0339] 37. A method of preventing a new intramammary infection or mastitis in a cow, said method comprising the step of administering to at least one teat of the cow the composition as described in any one of paragraphs 1 to 30 and 33.

[0340] 38. Use of the composition as described in any one of paragraphs 1 to 30 and 33 for preventing a new intramammary infection or mastitis in a cow.

[0341] 39. A syringe containing the composition as described in any one of paragraphs 1 to 30 and 33.

[0342] 40. A kitset for use or when used in a method of preventing a new intramammary infection or mastitis in a cow, wherein the kitset comprises: a syringe capable of administering to at least one teat of a cow the composition as described in any one of paragraphs 1 to 30 and 33.

[0343] 41. The teat sealant composition of paragraph 34 or 35, the use of paragraph 36 or 38, the method of paragraph 37, or the kitset of paragraph 40, wherein the composition is used in a milking cow or heifer.

[0344] 42. The teat sealant composition of paragraph 34, 35 or 41, the use of paragraph 36, 38 or 41, the method of paragraph 37 or 41, or the kitset of paragraph 40 or 41, wherein:

[0345] - the composition prevents a new intramammary infection or mastitis for a period of at least about 14 days, about 42 to about 90 days, or up to about 4 months;

[0346] - occlusion by the composition is for approximately the first 14 days of a dry period until a natural keratin plug develops;

[0347] - the composition occludes the teat canal and / or teat cistern or substantially occludes a teat canal and / or teat cistern until a natural keratin plug has formed in the teat canal;

[0348] - the composition protects a teat or teats of the cow from bacterial infection during an entire dry period;

[0349] - the composition is administered per quarter at the end of lactation;

[0350] - the composition is administered per quarter in maiden heifers approximately 4 weeks prior to calving;

[0351] - the teat sealant composition has an adequately low viscosity so as to allow it to be administered via a teat canal and / or teat cistern by way of injection;

[0352] - approximately 2-5 g of the teat sealant composition is administered / injected into a teat canal and / or teat cistern;

[0353] - a syringeability force for injecting the teat sealant composition into a teat does not exceed 4500 g at 25°C, and preferably the syringeability force for injecting the teat sealant composition into a teat is between about 1500 g and 3500 g at 25°C;

[0354] - the teat sealant composition thickens rapidly in a teat so as to be retained and occlude a teat canal and / or teat cistern;

[0355] - once located within a teat canal and / or teat cistern, the teat sealant composition has a viscosity high enough so as to be retained within the teat canal or teat cistern and occlude the teat canal or teat cistern;

[0356] - the teat sealant composition has a bounce-back viscosity enabling it to be retained within a teat canal and / or teat cistern as a substantially coherent mass;

[0357] - the teat sealant composition has a rheology enabling the composition to be administered to a teat canal and / or teat cistern by way of injection, and to be retained within the teat canal and / or teat cistern and occlude the teat canal and / or teat cistern, or to prevent an intramammary infection;

[0358] - the teat sealant composition shear-thins at the shearing forces applied during injection and stripping from a teat; or

[0359] - the teat sealant composition occludes a teat canal and / or teat cistern or substantially occludes the teat canal and / or teat cistern for about 4-6 weeks, while allowing a natural keratin plug to develop.

[0360] Features described in respect of compositions / formulations / medicaments may also relate to methods / uses and vice-versa, context permitting. It is to be understood that the terms ‘formulation’, ‘medicament’ and ‘composition’ may be used interchangeably, context permitting.

[0361] Having broadly described the invention in its various embodiments, non-limiting examples of preferred embodiments will now be described.

[0362] Brief Description of the Figures

[0363] Figures of Example 1

[0364] Figure 1. Syringeability of F1 at various times of storage at 4, 25 and 40°C.

[0365] Figure 2. Syringeability of F2 at various times of storage at 25 and 40 °C.

[0366] Figure 3. Flow behaviour of Teatseal™ and calcium carbonate and calcium phosphate formulations.

[0367] Figure 4. Injection simulation of Teatseal™ and calcium carbonate and calcium phosphate formulations.

[0368] Figure 5. Flow behaviour of Teatseal™ and calcium phosphate formulations with higher aluminium stearate and 5% lauric acid.

[0369] Figure 6. Injection simulation of Teatseal™ and calcium phosphate formulations with higher aluminium stearate and 5% lauric acid.

[0370] Figure 7. Flow behaviour of Teatseal™ and prototype formulations.

[0371] Figure 8. Injection simulation of Teatseal™ and prototype formulations.

[0372] Figure 9. Recovery (%) of formulations tested in suspended udder study of Example 7.

[0373] Figure 10. Effect of storage at 25 °C on syringeability (injection force).

[0374] Figure 11. Effect of storage at 25 °C on syringeability (injection force).

[0375] Figure 12. Effect of temperature cycling (4 and 25 °C) on the syringeability of calcium phosphate and sodium chloride formulations.

[0376] Figure 13. Storage stability of calcium phosphate and sodium chloride formulations at 25 °C.

[0377] Figure 14. Flow behaviour of NaCl and KCl formulations.

[0378] Figure 15. Injection simulation behaviour of NaCl and KCl formulations.

[0379] Figures of Example 2

[0380] Figure 16. Flow behavior of commercial Teatseal™.

[0381] Figure 17. Injection simulation behavior of Teatseal™ batches.

[0382] Figure 18. Syringeability of Teatseal™ batches.

[0383] Figures of Example 3

[0384] Figure 19. Effect of method of addition of lauric acid on the rheology (viscosity versus shear rate) of the formulation: light green – bottom line in graph (Method 1), dark green -top line in graph (Method 2).

[0385] Figure 20. Effect of method of addition of lauric acid on the rheology during an injection simulation test.

[0386] Figure 21. Effect of lauric acid addition into room temperature formulation vs warm formulation on the rheology (viscosity versus shear rate) of the formulation: Blue (Room temp. formulation), Orange (Warm formulation).

[0387] Figure 22. Effect of lauric acid addition into room temperature formulation vs warm formulation on the rheology during an injection simulation test.

[0388] Figures of Example 4

[0389] Figure 23. Flow behaviour of various test teat sealant formulations versus Teastseal™ and Biobloc™.

[0390] Figure 24. Injection simulation for various test teat sealant formulations versus Teastseal™ and Biobloc™.

[0391] Figure 25. Flow behaviour of various test teat sealant formulations versus Teastseal™ and Biobloc™.

[0392] Figure 26. Injection simulation for various test teat sealant formulations versus Teastseal™ and Biobloc™.

[0393] Figure 27. Syringeability simulation for a test teat sealant formulation versus Biobloc™.

[0394] Figure 28. Flow behaviour of various test teat sealant formulations containing calcium carbonate versus Teastseal™ and Biobloc™.

[0395] Figure 29. Injection simulation for various test teat sealant formulations containing calcium carbonate versus Teastseal™ and Biobloc™.

[0396] Figure 30. Syringeability for various test teat sealant formulations containing calcium carbonate versus Biobloc™.

[0397] Figure 31. Flow behaviour of various test teat sealant formulations containing dibasic calcium carbonate versus Teastseal™ and Biobloc™.

[0398] Figure 32. Injection simulation for various test teat sealant formulations containing dibasic calcium carbonate versus Teastseal™ and Biobloc™.

[0399] Figure 33. Syringeability for a test teat sealant formulation versus Biobloc™ delivered from a BioBloc syringe or white HDB syringe.

[0400] Figure 34. Syringeability of Biobloc™ using its own syringe.

[0401] Figure 35. Syringeability of Biobloc™ using a white HDB syringe.

[0402] Figure 36. Syringeability of a test teat sealant formulation delivered using a white HDB syringe.

[0403] Figure 37. Syringeability of a test teat sealant formulation delivered using a Biobloc syringe.

[0404] Figure 38. Photograph of a test teat sealant formulation’s behaviour in a water-filled syringe, which is simulating a teat.

[0405] Figure 39. Photograph of a test teat sealant formulation’s behaviour in a water, which is simulating milk.

[0406] Figures of Example 5

[0407] Figure 40. Solution killing of E. coli and S. uberis was assessed for various paste formulations and is presented as bacteria cell survival plots. A. Solution killing of E. coli using various pastes over 24 h. B. Solution killing of E. coli using various pastes over 48 h. C. Solution killing of S. uberis using various pastes over 48 h.

[0408] Figure 41. Streptococcus uberis cell counts over time exposed to teat sealant formulations. A. S. uberis treated with control formulations (prototypes) over 5 days. B. S. uberis treated with NaCl 20% formulations (prototypes) over 5 days. C. S. uberis treated with NaCl 30% formulations (prototypes) over 5 days. D. S. uberis treated with NaCl 40% formulations (prototypes) over 5 days. E. S. uberis treated with NaCl 55% formulations (prototypes) over 5 days. F. S. uberis treated with various formulations (prototypes) over 5 days. G. S. uberis treated with KCl formulations (prototypes) over 5 days. H. S. uberis treated with kaolin formulations (prototypes) over 5 days.

[0409] Figure 42. Escherichia coli cell counts over time, exposed to different teat sealant formulations. A. Mean survival cure of E. coli over 20 days with different teat sealant formulations. B. E. coli treated with different teat sealant formulations, such as KCl, over 5 days.

[0410] Figure 43. Staphylococcus aureus cell counts over time, exposed to teat sealant paste formulations. A. Survival cure of S. aureus over 30 days with different teat sealant prototype formulations. B. Mean survival cure of S. aureus over 20 days with different teat sealant prototype formulations.

[0411] Figures of Example 6

[0412] Figure 44. Ultrasound image of Cow 302 front left teat, 1 hr (F2).

[0413] Figure 45. Ultrasound images of Cow 302 front right teat, 1 hr (F3).

[0414] Figure 46. Ultrasound images of Cow 302 rear right teat, 1 hr (Teatseal™).

[0415] Figure 47. Ultrasound image of Cow 302 rear left teat, 1 hr (F1).

[0416] Figure 48. Cow mammary glands at 28 days post-treatment (dry-off) showing udders with some milk present and have not completed involution (top row) and some udders that have undergo complete involution (bottom row).

[0417] Figure 49. Diagrammatic representation of teat sealant scoring. Teat sealant is depicted by the red shaded areas.

[0418] Figure 50.1hr-Samples; % recovery with respect to formulation dose injected.

[0419] Figure 51.48hr-Samples; % recovery with respect to formulation dose injected.

[0420] Figure 52.672hr-Samples; % recovery with respect to formulation dose injected.

[0421] Figure 53.672hr-Samples; % recovery with respect to formulation dose injected.

[0422] Figure 54.1hr-Samples; % recovery with respect to TS.

[0423] Figure 55.48hr-Samples; % recovery with respect to TS.

[0424] Figure 56.48hr-Samples; % recovery with respect to TS.

[0425] Figure 57.672hr-Samples; % recovery with respect to TS.

[0426] Figure 58.672hr-Samples; % recovery with respect to TS.

[0427] Figures of Example 7

[0428] Figure 59. Teat length grouped by treatment assigned to each teat. The bars represent median values, which are also depicted at the bottom within each bar. The error bars represent the range and the dots represent individual points (n = 12 / treatment).

[0429] Figure 60. Percentage of solids in milk samples grouped by treatment assigned to each teat. The bars represent median values, which are also depicted at the bottom within each bar. The error bars represent the range and the dots represent individual points (n = 12 / treatment).

[0430] Figure 61. Mass of formulation infused into the teats grouped by treatment. The bars represent median values, which are also depicted at the bottom within each bar. The error bars represent the range and the dots represent individual points (n = 12 / treatment).

[0431] Figure 62. Estimated recovery by dripping (A), stripping (B), scraping (C) and flushing (D), and total recovery (E) for the infused formulations. The bars represent median values, which are also depicted at the bottom within each bar. The error bars represent the range and the dots represent individual points (n = 12 / treatment).

[0432] Figure 63. Estimated recovery from the teat. The bars represent median values, which are also depicted at the bottom within each bar. The error bars represent the range and the dots represent individual points (n = 12 / treatment).

[0433] Figure 64. Scatterplot of teat length and percentage recovery from the teat with linear regression line (solid line) and 95% confidence intervals (broken lines).

[0434] Figure 65. Proportion of front (A) and rear (B) teats with recoveries from the teat greater than or equal to Teatseal™ (≥ 95.81% in front teats and ≥ 84.64% in rear teats).

[0435] Figure 66. Shows the cut open teats with the remaining formulation after stripping and before scraping.

[0436] Figures of Example 8

[0437] Figure 67. Locations of udder samples for histopathology.

[0438] Description of preferred embodiments

[0439] Example 1 - Development of teat sealant formulations and uses

[0440] Background

[0441] In dairy cows, the incidence of mastitis caused by environmental pathogens is highest at the beginning of the 7-10 week dry-off period and again at calving at the end of the dry-off period. At dry-off cows develop a natural plug, a keratin plug, which seals the teat canal; however, this takes some time (several weeks) during which entry of microorganisms may occur, hence the use of internal teat sealants.

[0442] Teat sealants are used at dry-off of the milking cow, and in heifers, to ‘seal’ the teat canal and thereby prevent, or at least reduce, the entry of environmental pathogenic microorganisms from entering the udder.

[0443] Formulation of a teat sealant presents the following challenges:

[0444] - It must be sufficiently liquid such that 2-5g can be infused from a syringe (no needle) though the single teat canal.

[0445] - It should flow into the epithelial folds of the mucosal lining of the teat to seal most effectively.

[0446] - It then must thicken so that it is retained in the teat cistern and perhaps the teat (streak) canal for the dry-period (up to 10 weeks).

[0447] - At the end of the dry-period, it must be capable of being stripped out.

[0448] - It should preferably remain a coherent mass in the teat; that is, bits of formulation should not break off and enter the udder and thereby contaminate first milkings after dry-off.

[0449] The ideal rheological properties of a teat sealant formulation are not known. Also, it is unclear if the sealant should be retained in the teat for the entire dry-period or whether it would be sufficient for the sealant to be present for, say, the first 4-6 weeks while allowing the natural keratin plug to develop. This would have the following advantages: no stripping at the end of the dry-period; and, less chance of first milkings beings contaminated with teat-sealant.

[0450] The majority of teat sealant formulations use high concentrations of bismuth subnitrate suspended in liquid paraffin. These have the following disadvantages:

[0451] - Contamination of first milkings after the dry period.

[0452] - Blockage of filters in milking lines.

[0453] - Problems with downstream processing, e.g. block-spot in cheese.

[0454] The aim of this research was to replace heavy metal bismuth subnitrate-containing teat sealants with water soluble or insoluble, environmentally acceptable materials. All formulations were highly concentrated suspensions of such materials in acceptable vehicles. The theory of such highly concentrated suspensions is of interest in some non-pharmaceutical areas (e.g. concrete manufacturer, oil drilling, etc) but predictive theory is poor so empirical research is essential.

[0455] Not wishing to be bound by theory, it seems that rheological properties of concentrated suspensions are influenced by:

[0456] - Concentration of solid.

[0457] - Particle size, size distribution, particle shape, particle surface properties, adhesion properties.

[0458] - Rheological properties (Newtonian, non-Newtonian) of the suspending liquid.

[0459] - History: method of preparation, storage conditions and time.

[0460] Teatseal™ is a commercial product known to have high efficacy. The present inventors’ aim was to understand the rheological properties of Teatseal™ (see Example 2), and then to use this information to guide the development of formulations using soluble and insoluble solid particles / fillers, instead of bismuth subnitrate.

[0461] Initial research attempted to make suitable viscoelastic systems using ethylcellulose, castor wax, chitosan, and alginate zein, to match the properties of Teatseal™ but this was unsuccessful. It was then decided to use highly concentrated oily suspensions rather than attempting to use gelling polymer systems. This was a staged iterative process in which materials were progressively screened as replacements for bismuth subnitrate, either eliminated based on their performance in the screening tests, or selected for further testing. Based on the assessments of Teatseal™ (see Example 2), aluminium stearate was retained as the gelling agent for the oil.

[0462] Initially Eudragit RLPO microparticles were trialled as the solid particles / filler but this was abandoned since it is a non-biodegradable polymer making it environmentally unacceptable. The inventors then moved to water-insoluble environmentally acceptable excipients such as magnesium carbonate, kaolin, microcrystalline cellulose (Avicel™), and keratin.

[0463] It was thought that the volume fraction of suspended solid would be important in trying to understand the rheological properties of concentrated oily suspensions. In order to determine the volume fraction of solid, the inventors needed to use a solid which did not absorb the oil as this would reduce the ‘free’ oil and thereby change the volume fraction of solid calculations. Therefore, the inventors decided to use sodium chloride as one of the solids. This led to the serendipitous discovery of the possibility of making a teat sealant using a water-soluble filler / solid particles.

[0464] The idea of using soluble fillers / particles is apparently novel and opens the possibility of designing slowly eroding teat sealants overcoming the need for stripping at the end of the dry-cow period.

[0465] The reasons for the efficacy of Teatseal™ is not clearly understood. It is assumed, in part at least, to act as a physical barrier to entry of bacterial into the teat. Additionally, it could be due to it having antibacterial properties. Consequently, another aspect of the development of the formulation was to test the antibacterial properties of formulations developed.

[0466] Materials / excipients

[0467] Paraffin oil-BP grade, lauric acid, magnesium carbonate, bismuth subnitrate (Sigma-Aldrich), aluminium stearate (Sterm Chemicals, USA), Aerosil (Aerosil-200 and Aerosil R972), Teatseal™ (Zoetis), NaCl (Dominion Salt Limited), Avicel PH 112 (FMC corporation), and kaolin (heavy) (Kempthorne Prosser & Co Limited) were obtained from commercial sources. NaCl was milled in a planetary ball mill (Retsch PM-100, balls size 9 mm) for 1 h (Speed-600 rpm) and sieved for 30 mins (90 µm).

[0468] Preparation method (beaker method) for lab tests

[0469] Weighed amounts of paraffin oil and aluminium stearate were added into a beaker and heated at 120°C for 30 mins. After melting / heating the mixture, the beaker was removed from the heat source and mixed with spatula until a gel like mixture was formed. The gel was cooled at room temperature followed by addition of solid particles / filler and mixed well with spatula. After mixing filler, Aerosil- 200 was added and mixed well with a spatula. Lauric acid was melted at 45°C and added to above mixture and mixed well with a spatula. Formulations were rested for 24 h in a beaker at room temperature before testing.

[0470] Preparation method for retention studies

[0471] Syringes, aluminium stearate, Aerosil-200, Aerosil-R972, lauric acid, kaolin and paraffin oil were sterilised by gamma irradiation. Heat sterilisation (160°C, 3 h) was used to sterilise magnesium carbonate, sodium chloride and glassware. 100 g of each formulation batch was prepared using the beaker method.

[0472] Syringeability

[0473] Unless otherwise specified, a standard 4.5 ml (or larger) intramammary syringe as sold by Hubert De Backer nv (HDB veterinary intramammary syringe) having a cannular / nozzle inner diameter of 1.5 mm, and an inner barrel diameter of 13.5 mm was used in each of the Examples described herein (https: / / www.hdb.be / _library / _files / Tabellen / 0.5.0 / Intramammary_0_5_0.pdf).

[0474] A texture analyser was used to measure the force and work required to expel a formulation from the syringe. The plunger of the syringe was advanced at a constant speed of 2 mm / s over a distance of 25 mm to expel the formulation in 12.5 s. The analyser continuously measured the force, providing a graph of force versus time.

[0475] Rheometry

[0476] A Discovery Hybrid Rheometer HR-3 (TA instruments) was used to test the rheology of the formulations. Sample (4-5 g) was placed on the plate of the rheometer. Cone / plate geometry was: 2°, 60 mm and a gap of 500 µm. Temperature was fixed at 32°C.

[0477] Shear thinning was tested by applying shear from 0 to 100 (1 / s). Soak time and duration was 300 s. To test the bounce back property, that is the rate of recovery of the viscosity after shear thinning, 550 (1 / s) initial shear rate was applied and this was followed by rates of 10 and 1 (1 / s).

[0478] Centrifugation / phase separation

[0479] 10 g of formulation was added into a 15 mL Falcon tube and centrifuged at 1800 G for 30 mins. After centrifugation, phase separation (oil layer) was visually assessed.

[0480] Dispersion / swelling

[0481] To test dispersion, each formulation (3-4 g accurately weighed) was added into a Falcon tube (50 mL) followed by addition of 40 mL distilled water. In another experiment, formulations were added into scintillation vials and 20 mL distilled water was added. Vials and tubes were shaken in a rotary shaker (150 rpm) at 37°C. In another experiment, 20 mL of milk was added instead of water.

[0482] The weight of remaining formulation was determined at various times by decanting the water, drying the vials with remaining formulation at 60°C for 24 h and weighing. In this case vials with milk, after decanting the milk the remaining formulation was gently rinsed three times with 10 mL of water to remove milk solids before drying as above.

[0483] In another screening test for swelling and dispersion, a formulation (3-4g) was added to a Falcon tube followed by the addition of water. Swelling of the formulation was assessed after 12-24 h and this was followed by gentle shaking. Some non-dispersing formulations were stored in Falcon tubes with excess water for a longer period to determine long-term coherence of the formulation in an aqueous environment.

[0484] To increase understanding of the dispersion of the formulation in aqueous media, formulation (0.5 g) was added into a 15 mL Falcon tube and 10 m milli Q water added. The tube was shaken at 150 rpm in an incubator at 37°C for 5 days after which the tube was centrifuged at 3000 rpm for 30 mins and supernatant removed. Supernatant was passed through a 0.22 um filter and analysed by inductive coupled plasma mass spectrometry (ICPMS). Milli Q water was used as a control and was analysed for sodium, bismuth and magnesium ions.

[0485] Retention studies

[0486] Formulations were administered to groups of cows in a balanced design as described herein in latter Examples. At 1 h, 2 day, and 2 or 4 weeks after administration, teats were stripped to recover the remaining teat sealant formulation. Recovered materials were quantified as follows.

[0487] Frozen sample tubes (Falcon) were received and stored at -18°C. For the analysis, Falcon tubes were removed from the freezer and equilibrated at room temperature overnight. The weight of tubes (wet weight) with sample was recorded. The weight of the empty petri dish was recorded, and sample was poured into the petri dish. The sample in tube and petri dish were dried at 50°C for 24 h. After drying, the weights of tube and petri dish were recorded. Total dry weight of sample was calculated after deducting the empty weights of tube and petri dish. The following equations were used to deduct milk solids in the dry sample and to calculate the weight of formulation recovered.

[0488] Weight of milk solids in the dry sample = (total weight of wet sample-total weight

[0489] of dry sample) / 0.9 x 0.1

[0490] Weight of formulation: Total weight of dry sample minus calculated weight of milk solids.

[0491] Note: The loss in mass (g) on drying at 50°C over the period of 24 h is attributed to water in the milk in the sample and this milk is assumed to be 10% solids and 90% water. It is also assumed there are no volatile components in the formulation which would be lost on heating to 50°C for 24 h.

[0492] Iteration 1:Lab tests to retention study

[0493] Screening and selection of material / filler

[0494] In these initial screening studies, formulations were prepared using the beaker method.

[0495] Table 1A. Initial screening of fillers / solid particles for the teat sealant formulations. Sr. No. Filler Aerosil-200 Aluminium stearate (%) (%) (%) 1 Keratin (50%) 1 2.3 2 MgCO3(35%) 1 2.3 3 NaCl (60%) 1 2.3 4 Kaolin (45%) 0.5 2.3 5 Avicel (50%) 1 2.3

[0496] All the formulations contain paraffin oil (to balance). Laboratory grade MgCO3was used.

[0497] The particle size of commercial sodium chloride was too large to make smooth formulations. It was milled using a planetary ball mill (Retsch PM-100) for 1 h followed by sieving to break the agglomerates. Milled and sieved NaCl was stored in an airtight container and was used in NaCl formulations.

[0498] Dispersion / swelling behaviour of formulations in water

[0499] The keratin formulation swelled and dispersed in water. The Avicel formulation swelled in water after one week. Keratin and Avicel were eventually deemed unsuitable.

[0500] Magnesium carbonate (35%), kaolin (45%) and NaCl (50%) formulations did not swell in water. NaCl formulations were found to be intact and surprisingly the sodium chloride did not leach out under the experimental conditions.

[0501] Parallel microbiological testing (see Example 5) indicated an antimicrobial agent would need to be added to some formulations to match the antimicrobial effect of Teatseal™. Hence lauric acid (5%, 10%) was included.

[0502] ICPMS analysis

[0503] ICPMS testing was done to check the dissolution of fillers / solid particles in water. The levels of bismuth ion from Teatseal™ and magnesium was low compared with Na+ions indicating some dissolution of the sodium chloride formulation. The dissolution of NaCl depended on the aluminium stearate percentage indicating that the dispersion of the sodium chloride formulation might be tuned by varying the percentage of aluminium stearate. Water (Milli Q) was used as control in this study and showed presence of magnesium and sodium ions in it which is why the Teatseal™ sample also showed these ions at low concentrations.

[0504] Table 1B. ICPMS analysis of the water from the dispersion tests. Formulation Bi Mg Na (ng / mL) (ng / mL) (ng / mL) Teatseal™ 6.9 27 1400 25% MgCO3+4.3% Aluminum stearate 0.013 25000 1800 50% NaCl+5.3%Aluminum stearate <0.1 170 2100000 50% NaCl+6.3%Aluminum stearate <0.1 240 1300000 Detection limit 0.005 5 5

[0505] All the formulations contained 5% LA and 0.7% Aerosil.

[0506] Formulations for retention study in cows (see Example 6)

[0507] After this screening work, magnesium carbonate, sodium chloride and Kaolin were selected as fillers / solid particles in formulations for assessment in cows. Six formulations were selected and screened on their dispersion, rheology and syringeability. Preliminary storage stability was assessed while the retention study was in progress.

[0508] Table 1C. Concentration (%w / w) of ingredients in the formulations used for the retention study of Example 6. Ingredient F1 F2 F3 F4 F5 F6 MgCO325% 30% 10% NaCl 50% 50% 25% Kaolin 45% Aluminium 4.3% 5.3% 4.3% 5.3% 4.3% stearate Aerosil 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% Lauric acid 5% 5% 5% 5%

[0509] F4 contained Aerosil-R972 and other formulations contained Aerosil-200 (‘Aerosil’).

[0510] Gravimetric loss of formulations in milk or water (gravimetric analysis)

[0511] Milk has slightly acidic pH (pH 6.6) hence prototype formulations were tested to determine dispersion / dissolution in this medium. In particular, the inventors were interested in testing the MgCO3formulation with milk. The inventors tested MgCO3and NaCl formulations for four weeks. NaCl formulations showed weight loss only with milk whereas the MgCO3formulation did not show weight loss with milk or water.

[0512] Table 1D. Loss of NaCl from F2 formulation. Days Media Initial weight of Final weight of Loss (g) formulation (g) formulation (g) Day=0 7 Milk 3.12 2.47 0.65 14 Milk 3.0 2.48 0.52 21 Milk 3.27 3.0 0.27 28 Milk 3.12 2.91 0.21 7 Water 3.26 3.18 0.08 14 Water 3.11 3.34 ---- 21 Water 3.0 3.0 ---- 28 Water 3.3 3.4 ----

[0513] Table 1E. Loss of MgCO3from F1 formulation. Days Media Initial weight of Final weight of Loss (g) formulation (g) formulation (g) Day=0 7 Milk 3.0 2.96 .04 14 Milk 3.1 3.1 ---- 21 Milk 3.0 3.1 ---- 28 Milk 3.12 2.9 0.21 7 Water 3.0 2.99 ---- 14 Water 3.0 3.0 ---- 21 Water 3.0 3.0 ---- 28 Water 3.0 3.0 ----

[0514] Rheology and Syringeability

[0515] The aim was to have a syringeability force below 4500 g. All formulations met this criterion. Two magnesium carbonate formulations F1 and F3 were chosen as F1 showed good rheology and syringeability, and F3 was selected as an example of poor formulation based on its viscosity and particularly its poor bounce back viscosity. Similarly, two water soluble salt NaCl containing formulations (F2 & F5) were selected. F2 included lauric acid whereas F5 was made without lauric acid. The inventors’ earlier work found that the kaolin formulation made with Aerosil R972 had better dispersion (no swelling) in water compared to formulation made with Aerosol-200. F6 formulation used a combination of NaCl and MgCO3.

[0516] Table 1F. Rheological properties and syringeability of retention study formulations. Filler Code Aluminium Aerosil LA Initial Bounce- Syringeability (%) stearate (%) (%) viscosity back (Force (g)) (%) (Pa.s) at viscosity shear (Pa.s) at rate 5 shear (1 / s) rate 1 (1 / s) MgCO3F1 4.3 0.7 5 286 279 2500 (25) NaCl (50) F2 5.3 0.7 5 254 212 3300 MgCO3F3 --- 0.7 5 166 89 3600 (30) Kaolin F4 4.3 0.7 5 258 379 2400 (45) NaCl (50) F5 5.3 0.7 --- 289 138 ND NaCl (25) F6 4.3 0.7 --- 176 350 ND + MgCO3 (10)

[0517] F4 contained Aerosil-R972 and other formulations contained Aerosil-200. ND = not determined.

[0518] Centrifugation (phase separation)

[0519] This test was done as a stress test since the inventors had noticed phase separation in Teatseal™ on storage. Teatseal™ and prototype formulations were centrifuged (18000 G force) for 30 mins and then visually observed. Teatseal™ showed separation of oil in this stress test whereas the prototype formulations did not.

[0520] Storage stability

[0521] Formulation F1 was stored at 4, 25 and 40°C for five weeks and syringeability was tested at various times during this period. F1 showed good stability (force below 4000 g) at 4°C, however hardening of the formulation was observed over time at 25 and 40°C. See Example 1 - Figure 1. It was therefore decided not to proceed with magnesium carbonate (but it continued to be tested in the current retention study).

[0522] Formulation F2 was stored for four weeks at 25 and 40°C. This sodium chloride formulation remained physically stable and showed hardening on storage. See Example 1 – Figure 2.

[0523] Retention study

[0524] Details of the methodology and analysis can be found in Example 6.

[0525] The retention study was conducted in dairy cattle. Six prototype formulations and Teatseal™ were tested in this trial. Formulations were tested in four treatment groups: Detailed discussion about the results is available in Example 6. In summary:

[0526] Table 1G. Summary treatment table. Treatment group Formulations Time (h) Group 1 F1-F3+ TS 1 Group 2 F1-F3 + TS 48 Group 3 F1-F3 + TS 672 Group 4 F4-F6 + TS 672

[0527] TS = Teatseal™

[0528] Table 1H. Recovery (%) of formulations at various times after administration. Formulation Recovery (%) with respect to dose injected 1 h 48 h 672 h Low High Low High Low High F1 10 64 3 136 3 30 F2 15 40 4 7 0 27 F3 9 110 2 39 9 50 F4 0 5.3 F5 0 50 F6 0 49

[0529] The data were highly variable. The recovery at 1 h was done as a test of the methodology. That is a low recovery at 1 h could indicate:

[0530] - Poor stripping of the formulation from the teat.

[0531] - Systematic error in the method of quantification (eg over-allowance for milk solids).

[0532] Low recovery at 1 h could also be due to:

[0533] - Dripping of formulation from the teat in the first 1 h.

[0534] - Some administration into the udder (although the teat was supposed to be pinched-off at the base during administration.

[0535] These recoveries at 1 h were lower than those found in several previous retention studies. The low recovery from some cows at 1 h meant caution should be exercised in the interpretation of data from this trial, but the following observations were made:

[0536] - F1 v F3: F3 was a supposedly a poor formulation (no aluminium stearate, poor bounce back) so perhaps the requirement for high bounce back could be relaxed.

[0537] - F1, F3 v F2, F5: formulations (F2, F5) with soluble sodium chloride were retained in some cows for 672 h as were those formulations with insoluble magnesium carbonate (F1, F3).

[0538] Iteration 2: Lab tests to suspended udder study

[0539] In this iteration additional solid particles / fillers were investigated, and the effect of method of manufacture was studied. A suspended udder test was conducted but the R&D was terminated before the planned retention study was conducted.

[0540] Method of manufacture

[0541] Beaker method

[0542] As above

[0543] Spatula method

[0544] Preliminary experiments demonstrated that the method of preparation affected the rheology of the formulation. Temperatures, times and order of addition of ingredients had to be standardised.

[0545] Aluminium stearate and paraffin oil were added into a beaker and heated in a preheated oven at 120°C for 40 mins to melt aluminium stearate. The beaker was removed from the oven and the mixture was mixed well with a spatula to form a gel. The gel was cooled to room temperature. The gel and Aerosil-200 were mixed with a spatula on a glass plate. Then filler (solid particles) was added and mixed well with spatula and spatulated until a smooth formulation was obtained. The formulation was placed into a beaker and melted lauric acid (45 °C) added. The mixture was returned to the slab and spatulated to break any lauric acid lumps.

[0546] Suspended udder formulations

[0547] Formulations were prepared by spatulation method. Suspended udder formulations were prepared with 0.3% methylene blue dye. The dye was added to aluminium stearate and oil gel and spatulated before adding Aerosil-200. The spatulation method was used to make these formulations.

[0548] Preparation of sterile formulations for antimicrobial testing

[0549] The laminar-flow hood was sterilised. All the cleaned glassware and spatulas were wrapped with aluminium foil and sterilised by heat sterilisation (160°C for 3 h). Sodium chloride was added into a glass beaker and was heat sterilised. Since calcium phosphate (dicalcium phosphate dihydrate which is calcium phosphate dibasic dihydrate) could not be heat-sterilised, a thin layer of calcium phosphate powder was sterilised by UV radiation for 30 mins in laminar-flow hood. All formulations were made by spatulation. After making the formulation, a thin layer of formulation was spread on the glass plate and sterilised by UV radiation for 30 min in the laminar-flow hood. After final sterilisation, formulations were packed in sterile Falcon tubes.

[0550] Analysis of samples from suspended udder study

[0551] Suspended udder sample were frozen when received and stored at –18°C prior to analysis. After overnight thawing, tubes were wiped off with tissue and wet weight of tubes with lid was recorded. Tubes were dried in a prewarmed oven at 50°C for 24 h. After drying, the weight of tubes with lids was recorded.

[0552] This study was terminated prematurely, and these udder samples were utilised in a subsequent study, as described in Example 7.

[0553] Density of fillers / solid particles

[0554] Since retention at the bottom of the teat could potentially be influenced by the density of the formulation, this was taken into consideration in choosing additional materials.

[0555] Table 1I: Reported densities of fillers. Filler Density (g / cm3) Bismuth Subnitrate 4.93 Magnesium Carbonate 2.16 Sodium Chloride 2.16 Calcium Carbonate 2.71 Calcium Phosphate (dicalcium 2.93 phosphate dihydrate)

[0556] Calculated density of formulations

[0557] Density of formulation was calculated by considering the mass of filler and oil. For example, Teatseal™ contains 65% w / w bismuth subnitrate and 35% w / w paraffin oil. The following method was used to calculate the density of prototype formulations:

[0558] Density of bismuth subnitrate= 4.93 g / cm3

[0559] Density of paraffin oil = 0.9 g / cm3

[0560] Density (ρ) = mass (m) / volume (V)

[0561] Bismuth subnitrate volume = 65 / 4.93 = 13.18

[0562] Paraffin Oil volume = 35 / 0.9= 38.8

[0563] Volume (total) = 51.98 - assuming volumes are additive

[0564] Density = 100 / 51.98 = 1.92 g / cm3

[0565] Table 1J. Calculated densities of formulations. Formulation Density (g / cm3) Teatseal™ 1.92 25%MgCO3+4.3% Aluminum stearate+0.7% Aerosil+5% LA (F1) 1.0 50% NaCl +5.3% Aluminium stearate+0.7% Aerosil-200+5% 1.2 Lauric acid (F2) 60% Calcium Phosphate+2.5% MgCO3+2% Aluminum 1.4 stearate+0.35% Aerosil+5% LA 55% Calcium Phosphate+5% MgCO3+2% Aluminum 1.35 stearate+0.35% Aerosil+5% LA 55% Calcium Phosphate+2.5% MgCO3+2% Aluminum 1.30 stearate+0.35% Aerosil+5% LA 55% Calcium Carbonate+3.0% Aluminum stearate+0.35% 1.34 Aerosil+5% LA 50% Calcium Phosphate+10% NaCl+3.0% Aluminum 1.37 stearate+0.35% Aerosil+5% LA 55% Calcium Phosphate+5% NaCl+3.0% Aluminum 1.41 stearate+0.35% Aerosil+5% LA

[0566] Note: microbiological testing indicated an antimicrobial agent would need to be added to some formulations to match the antimicrobial effect of Teatseal™. Hence lauric acid (5%, 10%) was included.

[0567] Based on these density calculations, calcium carbonate and calcium phosphate were included in the screening programme.

[0568] Effect of temperature on the syringeability

[0569] Syringes filled with formulation were stored at 4°C for 24 h. After 24 h, syringes were removed and syringeability was measured immediately. Other syringes were stored at 25°C prior to measurement of syringeability.

[0570] Dispersion

[0571] This test was a slight modification from that described above in Iteration 1. Scintillation vials (weighed) were each filled with 20 mL of distilled water. The syringe was used to add 3-4 g of the formulation into the vial. After adding formulations, vials were shaken in an orbital shaker at 37°C and 150 rpm. At predetermined times, vials were removed and the water layer was decanted. The vials were dried at 60°C for 24 h. After drying the weight of dry vial with remaining formulation was recorded.

[0572] Stability studies

[0573] Stability on storage

[0574] Formulations were prepared and rested at room temperature for 24 h before filling into HDB (white plunger and barrel) syringes. The syringes were stored tip side up in an incubator at 25°C for various time. To test the syringeability at different time points, syringes were removed from the incubator and mounted on a texture analyser and the force required to expel the formulations was measured.

[0575] Stability under temperature cycling

[0576] Formulations 3-4 g were filled into HDB syringes stored tip side up then exposed at 4 °C for 24 h and then at 25°C for 24 h. This temperature cycle was repeated for 10 days and then the syringeability of the formulations tested.

[0577] Results

[0578] The method of preparation had an influence on the physical properties, particularly syringeability, so methods were standardised as described above. Various preliminary studies on order of addition of lauric acid, temperature at addition of lauric acid underscored the need for standardisation. (See Example 3.)

[0579] Table 1K. Effect of method of preparation on physical properties. Filler Al. Aerosil- LA Method Initial Bounce- Syringeability stearate 200 viscosity back (Force (g)) (Pa.s) at shear viscosity rate 5 (1 / s) (Pa.s) at shear rate 1 (1 / s) 55% CaCO34.3% 0.7% 5% Beaker 110 67 5000 +2.5% MgCO355% CaCO34.3% 0.7% 5% Spatula 93 59 3700 +2.5% MgCO355% CaCO3---- 0.7% 5% Beaker 24 20 3200 +2.5% MgCO355% CaCO3---- 0.7% 5% Spatula 17 18 2700 +2.5% MgCO3

[0580] Rheology

[0581] Calcium carbonate and calcium phosphate were chosen as fillers / solid particles because of their higher densities (higher than sodium chloride and magnesium carbonate). However, formulations of these prepared by spatulation and with 2.3% aluminium stearate had low viscosity and / or poor bounce back behaviour compared to Teatseal™. Increasing the aluminium stearate concentrations and adding lauric acid improved the formulations. See Figure 3, Figure 4, Figure 5, and Figure 6.

[0582] Rheology of combination filler formulations

[0583] A combination of fillers was investigated in order to increase density while achieving rheological properties similar to Teatseal™. Calcium carbonate or calcium phosphate (higher densities) were used in combination with magnesium carbonate or sodium chloride. Calcium carbonate formulations with 5% magnesium carbonate and calcium phosphate formulations with 10% NaCl showed good rheology compared to commercial Teatseal™. Sodium chloride 55% and 60% formulations also showed good rheology and were selected as water soluble formulations whereas calcium carbonate formulations are examples of water insoluble formulations. Comparisons were made with two different batches of Teatseal™ and another bismuth subnitrate commercial product Biobloc™. See Figure 7 and Figure 8.

[0584] Suspended udder formulations and recovery

[0585] The following formulations were prepared by the spatulation method.

[0586] Table 1L. Formulations used in suspended udder study. Ingredients F6 F7 F8 F9 F10 MgCO35% 30% NaCl 10% 55% 60% Calcium Phosphate 50% Calcium Carbonate 50% Aerosil-200 0.7% 0.7% 0.7% 0.7% 0.7% Aluminium stearate 4.3% 4.3% 4.3% 4.3% Lauric acid 5% 5% 5% Methylene Blue 0.3% 0.3% 0.3% 0.3% 0.3%

[0587] Table 1M. Calculated density of the formulations. Formulation Calculated density (g / cm3) Teatseal™ 1.90 F6 1.41 F7 1.54 F8 1.32 F9 1.38 F10 1.09

[0588] Recovery of formulations from suspended udder study

[0589] The suspended udder study was conducted and the details of the study can be found in Example 7. In summary, nine udders were used, and four prototype formulations were tested. Teatseal™ and Biobloc™ were used as standard / control formulations. After infusing formulations, dripping from the teat was collected for gravimetric analysis. At one hour after infusion, teats were stripped then cut open and scrapped off to recover the residual formulations. Any formulations that went into the udder during infusion were also recovered when the teats were cut off. The formulations were flushed with the udder secretions or milk and labelled as flushed out.

[0590] Dripping

[0591] All the prototype formulations had good viscosity and only in one udder did dripping occur (F830% was dripped).

[0592] Stripping

[0593] Formulations were recovered by vigorous stripping. Most of the formulations were stripped out easily. The highest amount of prototype formulations recovered by stripping was almost 100% in udder-1 whereas the lowest was 10% recovered in udder-8. Formulations not recovered by stripping were either in the teat cistern or had gone up in the udder while infusing the formulations.

[0594] Scraping

[0595] Scraping was done to recover the remaining formulation which was not stripped from the teat. The highest amount of formulation by scraping was recovered in udder-2 whereas the lowest was recovered in udder-7. The inability to fully recover formulations from the teat by stripping may have contributed to the variability and the poor recovery in the retention study described above.

[0596] Flushed out

[0597] Variable amounts of the formulations, including Teatseal™, were flushed from the udder suggesting that it had been infused there, in spite of the teat being pinched off at the base during infusion. This also has implication for the retention study and for efficacy of teat sealant formulations. See Figure 9.

[0598] Storage stability studies

[0599] Storage stability of the formulations tested in the suspended udder study of Example 7 showed physical instability when stored at 25°C. The syringeability force increased to greater than 4500 g for the calcium carbonate and magnesium carbonate formulations at 25°C. Therefore, these two formulations were rejected for the future studies. Other formulations did not show hardening on storage. See Example 1 Figure 10 and Figure 11.

[0600] Effect of temperature cycle (4 and 25 °C) on storage stability

[0601] Temperature cycling did not affect the syringeability of calcium phosphate and sodium chloride (55%) formulations, but the 60% sodium chloride formulations showed slightly higher syringeability compared to day 0. See Figure 12.

[0602] Iteration 3 – Lab tests to suspended udder study

[0603] Based on results from Iteration 2, it was decided to progress the sodium chloride and calcium phosphate formulations. These formations were lab tested and also sent for antimicrobial testing. A description and results of this microbiological testing are shown in Example 5.

[0604] Sodium chloride and calcium phosphate formulations

[0605] Several sodium chloride and calcium phosphate formulations were tested for their rheological properties and storage stability.

[0606] Table 1N. Syringeability and rheological properties. Formulation Lauric Initial Bounce-back Syringeability acid (%) viscosity viscosity (g) (Pa.s) at (Pa.s) at shear shear rate 1 rate 5 (1 / s) (1 / s) 55% NaCl 1 376 183 ND 55% NaCl 2.5 302 196 3100 55% NaCl 5 52 95 ND 60% NaCl 1 594 240 ND 60% NaCl 2.5 576 257 4000 60% NaCl 5 211 145 ND 35% Calcium phosphate+25% 5 318 334 3000 NaCl

[0607] All the formulations contain 4.3% aluminium stearate and 0.7% Aerosil. ND = not determined.

[0608] Storage stability at 25 °C

[0609] Formulation filled syringes were stored tip side up assuming that if there is a phase separation it should be visible while measuring syringeability. The syringes were stored at 25°C and at predetermined time points formulations were observed for phase separation and tested for syringeability. There was no phase separation (formation of an oil layer). All the prototype formulations did not show any sign of hardening on storage up to six weeks. See Figure 13.

[0610] Effect of temperature on syringeability

[0611] Prototypes and commercial formulations were stored at 4°C for 24 h and syringeability was tested immediately after removing from the refrigerator. All the controlled formulation at room temperature showed good syringeability. However, prototype formulations had higher syringeability due to hardening of formulation at lower temperature. Teatseal™ showed unreliable results due to air pockets present in the syringe whereas Biobloc™ showed hardening of formulations on storage at 4°C. Teatseal™ from two different batches and Biobloc™ formulations were filled in HDB syringes and tested the syringeability after storage at 4°C for 24 h. Teatseal™ and Biobloc™ formulations also showed hardening of formulations at 4°C.

[0612] Table 1O. Syringeability of commercial and prototype formulations after 24 h storage at 4 °C. Formulation Temperature Syringe Syringeability (°C) Force (g) Teatseal RT Teatseal 650 Teatseal 4 Teatseal 1125 Teatseal RT HDB 3500 Teatseal 4 HDB 5500 Teatseal RT HDB 3700 Teatseal 4 HDB 6200 BioBloc RT BioBloc 2850 BioBloc 4 BioBloc 5500 BioBloc RT HDB 3900 BioBloc 4 HDB 5600 55%NaCl+2.5% LA- RT RT HDB 3400 55%NaCl+2.5% LA- 4C 4 HDB 8900 50% CP+5% LA- RT RT HDB 3300 50% CP+5% LA- RT 4 HDB 105000 35%CP+25%NaCl+5% LA- RT RT HDB 3100 35%CP+25%NaCl+5% LA- RT 4 HDB 7500

[0613] All prototype formulations contained 4.3% aluminium stearate and 0.7% Aerosil. RT= room temperature

[0614] Dispersion test

[0615] Calcium phosphate and sodium chloride formulations were tested for up to six weeks. After each time point, gravimetric analysis was done to calculate the loss of water-soluble salts. Calcium phosphate with 25% NaCl formulations lost around 100% after six weeks whereas 55% NaCl showed maximum loss around 30-40% and formulation with 60% NaCl showed 50% loss after six weeks. These results pointed to formulations for slowly eroding sealant formulations.

[0616] Table 1P. Loss (%) of water-soluble component (NaCl) of calcium phosphate formulations in dispersion test. Formulation Time Initial Initial mass Final Loss Loss Loss (%) (Days) weight of (g) of NaCl weight of (g) (%) of water- formulation in formulation soluble formulation after drying component (NaCl) 35% 14 4.05 1.0 3.85 0.2 5 20 CP+25% NaCl+5% LA (Vial-1) 35% 14 3.52 0.88 3.19 0.33 9.4 37.5 CP+25% NaCl+5% LA (Vial-2) 35% 28 4.20 1.0 3.24 0.96 23 96 CP+25% NaCl+5% LA (Vial-1) 35% 28 3.54 0.9 2.83 0.71 20 79 CP+25% NaCl+5% LA (Vial-2) 35% 42 4.10 1.0 3.30 0.8 19.5 80 CP+25% NaCl+5% LA (Vial-1) 35% 42 4.21 1.0 3.24 0.94 22 94 CP+25% NaCl+5% LA (Vial-2)

[0617] All the formulations contain 4.3% Aluminium stearate and 0.7% Aerosil.

[0618] Table 1Q. Loss (%) of NaCl formulations in dispersion test. Formulation Time Initial Initial Final Loss Loss Loss (Days) weight mass weight (g) of (%) of (%) of of (g) of of formul formul water- formul NaCl formul ation ation soluble ation in ation compo formul after nent ation drying (NaCl) 55% NaCl+2.5% LA 14 3.99 2.2 3.46 0.53 13 24 (Vial-1) 55% NaCl+2.5% LA 14 3.37 1.85 2.86 0.51 15 27 (Vial-2) 55% NaCl+2.5% LA 28 3.60 1.98 3.0 0.6 17 30 (Vial-1) 55% NaCl+2.5% LA 28 3.70 2.0 2.9 0.8 22 40 (Vial-2) 55% NaCl+2.5% LA 42 3.64 2.0 3.07 0.57 16 28 (Vial-1) 55% NaCl+2.5% LA 42 3.86 2.1 3.39 0.47 12 22 (Vial-2) 60% NaCl+2.5% LA 14 3.47 2.0 2.74 0.73 21 36 (Vial-1) 60% NaCl+2.5% LA 14 4.02 2.4 2.59 1.43 36 60 (Vial-2) 60% NaCl+2.5% LA 28 3.62 2.17 2.53 1.09 30 50 (Vial-1) 55% NaCl+2.5% LA 28 4.0 2.4 2.70 1.3 32 54 (Vial-2) 60% NaCl+2.5% LA 42 3.94 2.36 2.75 1.19 30 50 (Vial-1) 60% NaCl+2.5% LA 42 3.70 2.22 2.57 1.13 30 51 (Vial-2)

[0619] All the formulations contain 4.3% Aluminium stearate and 0.7% Aerosil.

[0620] Comparison of NaCl and KCl formulations (rheological properties)

[0621] Some initial tests were done with milled potassium chloride (KCl) formulations as an alternative soluble salt. Potassium chloride formulations showed similar rheological properties compared to NaCl (55%) formulation suggesting it to be a suitable alternative to sodium chloride. See Figure 14 and Figure 15.

[0622] Suspended udder study

[0623] Based on the lab-test and microbiological testing, the following formulations were selected for the suspended udder study as described in Example 7.

[0624] Table 1R. Formulations selected for suspended udder study. Ingredients F1 F2 F3 F4 F5 NaCl 55% 10% 25% 10% Calcium Phosphate 50% 35% 50% 60% Aerosil-200 0.7% 0.7% 0.7% 0.7% 0.7% Aluminium stearate 4.3% 4.3% 4.3% 4.3% Lauric acid 2.5% 5% 5% 5% 5%

[0625] Summary

[0626] Based on lab-based tests, suspended udder test and some preliminary data from a retention study, high concentrations of sodium chloride alone or in combination with calcium phosphate suspended in oil gelled with aluminium stearate and fumed silica (Aerosil-200), and containing lauric acid, show promise as teat sealant formulations overcoming the current reliance on bismuth subnitrate. Potassium chloride is likely a suitable alternative to sodium chloride. The idea of using soluble fillers / solid particles is truly novel and opens the possibility of designing slowly eroding teat sealants overcoming the need for stripping at the end of the dry-cow period.

[0627] The rheological properties (shear thinning, bounce back) of such formulations can be manipulated by varying the percentages of the sodium chloride and / or calcium phosphate and the concentration of aluminium stearate and Aerosil-200. The formulations can be adjusted to vary the dispersion rate in water indicating that it should be possible to design formulations to erode / disperse at tailored rates in the teat.

[0628] Short term stability studies suggest that sodium chloride and calcium phosphate formulation are physically stable (as assessed by syringeability and rheology), unlike formulations based on magnesium carbonate and calcium carbonate.

[0629] These bismuth subnitrate free teat sealant formulations have antimicrobial activity against mastitis causing bacteria in vitro (see Example 5).

[0630] Example 2 – Assessments of Teatseal™

[0631] Commercial Teatseal™ was assessed to:

[0632] - Determine inter-batch variability in rheological properties.

[0633] - Understand the influence of the excipients of the rheological behaviour.

[0634] - Provide some guidance on preferred rheological properties for an internal teat sealant.

[0635] Rheometry

[0636] In-house teat sealant formulation was prepared by the beaker method following the methods of PCT publication number WO 2010 / 065747 A2, the entire contents of which are incorporated herein by reference. Rheologies of commercial Teatseal™, in-house treat sealant formulations and different combinations (Table 2A) were tested is several ways:

[0637] - Shear thinning behaviour, ie. viscosity versus shear rate.

[0638] - Injection simulation, ie. a high initial shear rate simulating injection followed by sustained application of low shear rates to understand rate of recovery after injection.

[0639] A Discovery Hybrid Rheometer HR-3 (TA instruments) was used to test the rheology of the formulations. Sample (4-5 g) was placed on the plate of the rheometer. Cone / plate geometry was: 2°, 60 mm and a gap of 500 µm. Temperature was fixed at 32 °C.

[0640] Table 2A. Bismuth subnitrate containing formulations. Sr. No Filler (65%) Aerosil-200 (%) Aluminium stearate (%) 1 Bismuth subnitrate --- --- 2 Bismuth subnitrate 1 --- 3 Bismuth subnitrate --- 2.3 4 --- 1 2.3

[0641] All the formulations contain paraffin oil.

[0642] Syringeability

[0643] Batches and in-house formulations were also assessed using a texture analyser to measure the force and work required to expel a formulation from the syringe. The plunger of the syringe was advanced at a constant speed of 2 mm / s over a distance of 25 mm to expel the formulation in 12.5 s. The analyser continuously measured the force, providing a graph of force versus time.

[0644] Particle size and shape analysis

[0645] Since the rheology of concentrated suspensions is influenced by the size, shape and surface characteristics of the suspended particles, size and shape were assessed by scanning electron microscopy (SEM).

[0646] Teatseal™ (1 g) was added into a falcon tube (15 mL) followed by addition of hexanes (10 mL) to remove paraffin oil. Teatseal™ was mixed with hexanes using spatula and shaken (with hand) and centrifuged at 3000 rpm at 25°C for 30 mins. After centrifugation, supernatant was removed, and this step was repeated three times. Extracted bismuth subnitrate was dried at room temperature overnight. Scanning Electron Microscopy (SEM) was used to analyse the particle size and shape of bismuth subnitrate. Commercially available bismuth subnitrate (Sigma-Aldrich) was used as reference.

[0647] Results

[0648] Rheology of Teatseal™ batches

[0649] The six batches showed considerable inter-batch variability in shear thinning behaviour (Figure 16) Batches also varied in their behaviour in the injection simulation testing, particularly in the ‘bounce back’ that is the gelling behaviour when the shear rate was decreased (Figure 17).

[0650] Syringeability

[0651] The maximum force varied among batches from 1500 to 3300 g weight (Figure 18).

[0652] Particle size shape analysis

[0653] Commercial bismuth subnitrate and extracted bismuth subnitrate from Teatseal™ showed a range (10-40 µm) particle size and rod-shaped particles.

[0654] Effect of various excipients in Teatseal™

[0655] Investigation of Teatseal™ was conducted to understand the role of each ingredient on the rheological properties of Teatseal™. Different combinations were made, and rheology was tested (Table 2B).

[0656] Table 2B. Rheological properties of bismuth subnitrate containing formulations. Sr. No Filler (65%) Aerosil-200 Aluminium Initial viscosity Bounce-back (%) stearate (Pa.s) at shear viscosity (Pa.s) (%) rate 5 (1 / s) at shear rate 10 (1 / s) 1 Bismuth --- --- 11 10 subnitrate 2 Bismuth 1 --- 84 13 subnitrate 3 Bismuth --- 2.3 37 20 subnitrate 4 1 2.3 0.2 0.1 5 Bismuth 1 2.3 180 25 subnitrate 6 Commercial 143 33 Teatseal

[0657] 5 = in-house teat sealant formulation.

[0658] Flow behaviour – shear-thinning behaviour

[0659] Flow behaviour assesses the change in viscosity as the shear rate is increased. The formulation should be viscous when undisturbed but when a shearing force is applied the formulation should thin and flow like a liquid to make expulsion from the syringe easy. In another words when a shearing force is applied the sample shows shear-thinning. Aerosil-200 and aluminium stearate without bismuth subnitrate showed poor initial viscosity (Table A2). Bismuth subnitrate alone in paraffin oil showed higher but still poor initial viscosity. Addition of bismuth subnitrate in oil with aerosol showed improved viscosity (84 Pa.s). In-house teat sealant formulation showed good initial viscosity (Table A2) which was comparable with commercial Teatseal™: 180 Pa.s compared to commercial Teatseal™ (143 Pa.s). This difference is within the inter-batch variability in Teatseal™ and could be due to various factors: method of preparation, storage time, particle properties of bismuth subnitrate.

[0660] Injection simulation

[0661] This test is to mimic the injection of formulation into the teat. In this test, a high shear rate (550 (1 / s)) was applied initially for a few seconds followed by low shear rate (10 (1 / s)) after a few seconds to mimic the conditions in the teat in the period shortly after administration. At low shear rate the formulation should thicken quickly (so-called ‘bounce back’) in order to be retained in the teat. Preferably, the bounce-back should not be immediate but should allow time for the formulation to spread into crevices in the teat cistern. If the formulation does not bounce back it will drip from the teat. Samples without bismuth subnitrate and aluminium stearate showed poor bounce back (Table A2). This test indicated that the combination of aluminium stearate and bismuth subnitrate is important for the bounce back property.

[0662] Example 3 – Preparation of experimental teat sealant formulations containing lauric acid

[0663] In this Example, the inventors describe the general preparation of bismuth-salt-free teat sealant compositions / formulations, but otherwise with functional characteristics similar to Teatseal™.

[0664] The inventors discovered that bismuth metal-free teat sealant formulations could be formulated by way of the following steps:

[0665] - Forming a water-insoluble gel by mixing an oily carrier, such as paraffin oil, with a thickener (gelling agent), such as aluminium stearate.

[0666] - Adding a further thickener to the gel, such as fumed silica (Aerosil-200) to modify the flow behaviour of the gel.

[0667] - Adding solid particles (filler) to the gel, to obtain a paste containing coarse solid particles / filler. The particles can be water-soluble or water-insoluble. Various solid particle types can be used, including sodium chloride (which is water-soluble), potassium chloride (which is water-soluble), sucrose (which is very soluble in water), mannitol (which is freely soluble in water), dicalcium phosphate dihydrate (calcium phosphate dibasic dihydrate) (which is practically insoluble in water), magnesium carbonate (which is sparingly soluble in water), calcium carbonate (which is practically insoluble in water), and others. The solid particles may be milled to a particular average particle size. The particles modify the rheology of the formulation and may also provide antimicrobial properties.

[0668] - Adding a further thickener, such as lauric acid (which is practically insoluble in water), which provides antibacterial properties to the paste.

[0669] The rheological properties (shear thinning, bounce back) of such formulations can be manipulated by varying the percentages of the sodium chloride and / or calcium phosphate and the concentration of aluminium stearate and fumed silica (Aerosil-200). The formulations can be adjusted to vary the dispersion rate in water indicating that it is possible to design formulations to erode / disperse at tailored rates in the teat.

[0670] The teat sealant formulations were generally prepared as described below.

[0671] An oven was preheated to 120°C. Paraffin oil (Sigma-Aldrich) and aluminium stearate (Strem Chemicals, Inc.) were added to a beaker and mixed. The mixture was heated at 120°C for 40 mins to melt the aluminium stearate. The beaker was removed from the oven and mixed with a spatula to form a gel. The gel was cooled to room temperature over 30-40 mins. Aerosil-200 (Chemiplas (NZ) Ltd) was weighed and put onto a glass slab. The gel and Aerosil-200 were mixed with a spatula on the glass plate. Solid particles / fillers were added and mixed well with a spatula to obtain a smooth suspension / paste containing coarse filler / solid particles.

[0672] The suspension was added to a beaker and melted lauric acid (45 °C) was added and stirred in. The suspension was returned to the slab and spatulated for a few minutes to break any lauric acid lumps. The formulation was rested for 24h in a beaker at room temperature and then its rheological properties were tested.

[0673] Various coarse fillers / solid particles can be used: sodium chloride, potassium chloride, dicalcium phosphate dihydrate, and others. Calcium phosphate dibasic dihydrate was used (BP grade, Source-Sigma-Aldrich). NaCl (Pharmaceutical grade, Source-Dominion Salt Limited, NZ) was milled in a planetary ball mill (Retsch PM-100, balls size 9 mm) for 1 h (Speed-600 rpm) and sieved (90 µm). The size of milled NaCl particles was determined by electron microscope and was found to be <10µm (average size).

[0674] The method and timing of addition of lauric acid was found to affect the rheological properties of the formulation. For this reason, various methods for adding lauric acid were investigated, as described below.

[0675] Method 1-Lauric acid was added into the oil with aluminium stearate and heated at 120°C. After melting lauric acid and aluminium stearate, the mixture was cooled to room temperature. The mixture was mixed with the filler on a glass plate and spatulated until a smooth formulation was obtained.

[0676] Method 2-Lauric acid was melted at 45°C in the oven and added to the suspension (room temp.) followed by spatulation to break lauric acid lumps.

[0677] Formulations were rested for 24 h at room temperature before testing their rheological properties.

[0678] The flow behaviour of two ingredient-identical formulations is shown in Figure 19.

[0679] The effect of the method of addition of lauric acid on the rheology during an injection simulation test is shown in Figure 20.

[0680] Addition of lauric acid into room temperature formulation vs warm formulation

[0681] Method 3-Melted lauric acid was added to the room temp. formulation and the formulation was spatulated. (See method section.)

[0682] Method 4-To make this formulation the above-mentioned steps (method section) were followed. The formulation was placed in a beaker and lauric acid (solid) was added to the formulation and the mixture was heated in the oven at 45°C for 30 mins, followed by mixing in a beaker with spatula (warm formulation).

[0683] Formulations were rested for 24 h at room temperature before testing their rheological properties.

[0684] The effect of lauric acid addition into the room temperature formulation vs warm formulation on the rheology (viscosity versus shear rate) of the formulation - Blue (Room temp. formulation), Orange (Warm formulation) - is shown in Figure 21.

[0685] The effect of lauric acid addition into room temperature formulation vs warm formulation on the rheology during an injection simulation test is shown in Figure 22.

[0686] Example 4 – Rheological properties of teat sealant formulations

[0687] In this Example, the inventors show that teat sealant formulations described in the Examples above have similar rheological properties to the commercial bismuth-containing teat sealant product Teatseal™.

[0688] The flow behaviour of various test teat sealant formulations versus Teastseal™ and Biobloc™ is shown in Figure 23.

[0689] An injection simulation for various test teat sealant formulations versus Teastseal™ and Biobloc™ is shown in Figure 24.

[0690] The flow behaviour of various test teat sealant formulations versus Teastseal™ and Biobloc™ is shown in Figure 25.

[0691] An injection simulation for various test teat sealant formulations versus Teastseal™ and Biobloc™ is shown in Figure 26.

[0692] Syringeability for a test teat sealant formulation versus Biobloc™ is shown in Figure 27.

[0693] The flow behaviour of various test teat sealant formulations containing calcium carbonate versus Teastseal™ and Biobloc™ is shown in Figure 28.

[0694] BP Grade Calcium Carbonate Formulations

[0695] An injection simulation for various test teat sealant formulations containing calcium carbonate versus Teastseal™ and Biobloc™ is shown in Figure 29.

[0696] Syringeability for various test teat sealant formulations containing calcium carbonate versus Biobloc™ is shown in Figure 30.

[0697] The flow behaviour of various test teat sealant formulations containing dibasic calcium carbonate versus Teastseal™ and Biobloc™ is shown in Figure 31.

[0698] An injection simulation for various test teat sealant formulations containing dibasic calcium carbonate versus Teastseal™ and Biobloc™ is shown in Figure 32.

[0699] Syringeability for a test teat sealant formulation versus Biobloc™ delivered from a BioBloc™ syringe or white HDB syringe is shown in Figure 33.

[0700] The syringeability of Biobloc™ using its own syringe is shown in Figure 34.

[0701] The syringeability of Biobloc™ using a white HDB syringe is shown in Figure 35.

[0702] The syringeability of a test teat sealant formulation delivered using a white HDB syringe is shown in Figure 36.

[0703] The syringeability of a test teat sealant formulation delivered using a Biobloc™ syringe is shown in Figure 37.

[0704] Summary

[0705] Flow graphs, viscosity vs shear rate, are presented and simulation graphs, where the share rate starts high (simulating injection), lowers (simulating settling), to zero (simulating setting in the teat cistern).

[0706] All are compared with 2 marketed commercial teat sealants (TeatSeal™ and BioBloc™).

[0707] The present inventors’ lead formulation’s (50% CaHPO4+ 10% NaCl + 4.3% aluminium stearate + 0.7% fumed silica + LA added last) behavior in water (which is simulating milk) and syringe (which is simulating a teat) are shown in Figures 39 and 38, respectively.

[0708] Conclusions

[0709] The flow properties (rheology) of the teat sealant formulations are similar to commercial teat sealants.

[0710] Example 5 – Antimicrobial activity of teat sealant formulations

[0711] Summary

[0712] This Example shows that the commercially available bismuth-containing Teatseal™ formulation and new bismuth subnitrate-free teat sealant formulations / compositions developed by the present inventors have antimicrobial activity against mastitis causing bacteria in vitro. Teatseal™ was shown to be antimicrobial against Streptococcus uberis (S. uberis), however it had no effect on the growth of Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli). Sodium chloride (NaCl) 55% w / w with lauric acid (LA) 5% w / w was bactericidal against all three bacterial species. Potassium chloride (KCl) 50% w / w with LA 2.5% w / w was bactericidal against the two species tested:- S. uberis and E. coli. Calcium phosphate (dicalcium phosphate dihydrate which is calcium phosphate dibasic dihydrate) with several concentrations of NaCl and LA 5% w / w were bactericidal against S. uberis but not E. coli. The in vivo antimicrobial activity of these teat sealant formulations may be as good as or greater than that observed in vitro.

[0713] Background

[0714] A teat sealant is applied to the teat of a dairy cow at dry-off to prevent new mastitis infections. Internal teat sealants are typically composed of 65% w / w bismuth subnitrate, aluminium stearate (4.3% w / w), and Aerosil™ fumed silica (0.7% w / w) dispersed in a mineral oil vehicle, in the form of a paste. This antibiotic-free teat sealant paste is applied to the teat canal using a disposable syringe and functions by preventing bacteria from entering the udder. It is believed that the paste provides a physical barrier between the udder and the environment preventing bacteria entry.

[0715] The objective of this work was to evaluate in vitro the antimicrobial activity of teat sealant formulations against mastitis causing bacteria, S. aureu, S. uberis and E. coli. The antimicrobial activity of the paste was determined by counting live bacteria over time in media which had either been exposed to the paste prior (solution killing) or bacteria that has been exposed to the paste suspended in a medium (contact killing). Solution bacterial killing evaluates for any agents that have leached from the paste which impacts on bacteria survival, whereas contact killing evaluates whether the paste surface influences bacteria survival.

[0716] Materials and Methods

[0717] Materials

[0718] Bacteria strains; E. coli ATCC 10536, S. uberis ATCC 19436, S. aureus ATCC 6538 were kept in glycerol stocks (500µl culture mixed with 500µl of 87.5% glycerol) stored at -80˚C.

[0719] Methods

[0720] Bacteria culture

[0721] S. aureus cultures were grown in TSB (tryptic soy broth), S. uberis cultures were grown in Todd-Hewitt broth (THB) and E. coli cultures was grown in Lysogeny broth (LB).

[0722] Sterilisation of teat sealant formulations

[0723] Syringes, aluminium stearate, Aerosil™, lauric acid, kaolin and paraffin oil were sterilised by gamma irradiation. A heat sterilisation (160°C, 3 h) method was used to sterilise magnesium carbonate, sodium chloride and glassware.

[0724] Solution killing

[0725] Sterile bismuth subnitrate-free teat sealant compositions (1 g) were added directly into media only (THB, or LB) for a period of either 24, or 48 h at 32˚C with mixing at 100 rpm. After 24 h or 48 h a sample of the medium was removed for measurement of bacteria numbers. Antibacterial activity of the solution was measured either by spotting the solution onto bacterial cell lawns of either S. uberis, or E. coli, or transferred to a new universal medium and was inoculated with 1x106CFU / ml of either E. coli or S. uberis. Samples of the bacterial medium were taken at day 0, 1 and 3 after inoculation for cell survival calculations.

[0726] Contact killing

[0727] A sterile bismuth subnitrate-free teat sealant composition (1.0 g) was added aseptically into a 24-well plate, then 1 ml of medium inoculated with bacteria was added to each well and incubated at 32˚C, and mixed at 100 rpm. For each time point, a sample (50 µl) was removed from each well and serially diluted in PBS in a 96-well plate to 107cells / ml (time zero). Then 20 µl of dilutions 10 to 107cells / ml were plated onto TSA plates and grown overnight at 37˚C to calculate CFU / ml for each sample. Immediately after inoculation, a sample (10 µl) was removed from each universal medium and serially diluted in PBS in a 96-well plate to 107to determine the T = 0 h activity. Then 20 µl of dilutions 10-1to 10-7were plated onto TSA plates and grown overnight at 37˚C to calculate CFU / ml for each sample.

[0728] Results

[0729] Solution killing

[0730] Solution killing of E. coli and S. uberis was assessed for various paste formulations and is presented as bacteria cell survival plots (Figure 40A-C).

[0731] The antibiotic gentamycin (100 µg / ml) in the media was used as a positive control and was shown to be bactericidal for both E. coli and S. uberis. Media exposed to teat sealant formulations or the commercial TeatSeal™ for 24 h had no antibacterial effect on the growth of E. coli relative to the untreated control (Figure 40A). Of the media collected after 48 h exposure to the pastes, only 50% NaCl reduced the growth of E. coli relative to the untreated control as observed on day 1. However, the bacteria numbers recovered to the same cell density as the untreated control at day 3 (Figure 40B).

[0732] S. uberis was evaluated only in media that had been exposed to the pastes for 48 h. Media exposed to the formulation containing 50% NaCl and 5% LA for 48 h, killed all S. uberis at the 24 h time point and no regrowth of S. uberis was observed at 3 days (Figure 40C). This indicates for this formulation compound(s) have leached from this paste into solution which is bactericidal against S. uberis. In contrast for E. coli the 50% NaCl containing paste after 48 h exposure to media was bacteriostatic (Figure 40B). The formulation 25% MgCO3 + 5% LA was bacteriostatic for 24 h against S. uberis, but the cells recovered to the same level as the untreated and paraffin controls by the end of the experiment (Figure 40C).

[0733] Contact Killing

[0734] Streptococcus uberis

[0735] Media containing S. uberis was plated onto the pastes and at days 2 and 5 the bacteria numbers were evaluated. Figure 41 (A-H) shows bacterial survival over time for the teat sealant formulations. Paraffin oil, the vehicle used in making the paste, had no antimicrobial activity (Figure 41A). With the addition of lauric acid (LA) to paraffin oil, there was a strong antibacterial effect for LA loading of 2.5 and 5% w / w by day 2 whereas at a 1% w / w loading of LA it took 5 days to reach a zero bacterial count (Figure 41A). NaCl alone in paraffin oil at 20, 30, 40 and 55% (w / w) all showed some inhibition of bacteria cell counts compared to control at day 2, however at day 5 the number of bacteria was similar to control (Figure 41B-E). For all NaCl pastes that contained either 1, 2.5 or 5% LA there was strong reduction on bacteria cell count at day 2 and at day 5 there were still no bacteria (Figure 41B- F).

[0736] The antimicrobial activity of pastes made using dicalcium phosphate dihydrate (CaP) as the filling agent was also investigated (Figure 41F). These pastes all contained LA at 5% w / w with varying percentages of NaCl replacing the CaP; formulations mass % weight (CaP:NaCl:LA) 60:0:5, 50:10:5 and 35:25:5. All CaP based formulations showed no antimicrobial activity against S. uberis over the 5 day exposure with the paste. In the same experiment NaCl at 55% w / w with 2.5 % w / w LA demonstrated bactericidal activity against S. uberis at day 5 (Figure 41F).

[0737] KCl 55% w / w paste alone and with LA 1, 2.5 and 5% LA was effective at killing all the S. uberis at day 1 (Figure 41G).

[0738] Kaolin (heavy) is used in pharmaceuticals including topical and oral formulations. Kaolin is a layered silicate mineral, with an approximate chemical formula of H2Al2Si2O8(H2O) and is supplied as a fine powder. The antimicrobial activity of kaolin-based formulations against S. uberis was assessed without LA and with LA at either 1, 2.5 or 5% w / w. Kaolin based paste without LA showed a similar bacteria count to the control group, whereas all LA containing formulations showed antimicrobial activity against S. uberis (Figure 41H).

[0739] Escherichia coli

[0740] The antimicrobial activity of the different teat sealant formulations is presented as bacterial counts over time in Figure 42. Gentamycin, an antibiotic with known antibacterial activity against E. coli, was shown to kill all bacteria upon mixing on day 0 (Figure 42A). Teat sealant formulation pastes containing NaCl at 55 or 60% w / w were able to kill all E. coli at day 15 (Figure 42A) while NaCl paste at 55% w / w with LA at 5% w / w was able to kill all E.coli at the first time point at day 5 (Figure 42A). Teat seal paste containing CaP 50 % w / w and NaCl 10% w / w without and with LA at 5% w / w showed no inhibitory activity on E coli cell counts over the 20-day exposure (Figure 42A).

[0741] Paraffin oil (PO) containing no filler / solid particles but containing either 1, 2.5 or 5% w / w LA showed no antimicrobial activity against E.coli over the 20 day exposure period (Figure 42B). Teat sealant paste containing potassium chloride (KCl) filler at 55% w / w showed no antimicrobial activity against E. coli, however on the addition of LA at 2.5 % w / w there was a dramatic drop in bacteria cell counts at day 5 to zero counts (Figure 42B).

[0742] Staphylococcus aureus

[0743] S. aureus is a gram-positive bacterium which was completely inhibited by gentamycin on day 0, as seen in Figure 43. Paraffin oil had no impact on the growth of S. aureus (Figure 43A). Commercial teat sealant Teatseal™ and prototype teat sealant formulation pastes which contained NaCl 50, 55 or 60 % w / w only or MgCO325 % w / w with LA 5 % w / w showed no inhibitory activity against S. aureus (Figure 43A and B). Teat sealant prototype formulations NaCl 50 % w / w with LA 5 % w / w was shown to have strong antimicrobial activity with zero colony counts at day 5 (Figure 43A). Paste formulations which contained CaP 50 % w / w and NaCl 10 % w / w without LA and with LA at 5% w / w had no inhibitory activity towards S. aureus (Figure 43B).

[0744] Summary of results

[0745] Table 5A. Summary of solution killing after 48 h media exposure to different teat sealant prototype paste formulations. Teat sealant S. uberis E. coli Gentamycin + + Paraffin oil - - MgCO325% / LA 5% - - NaCl 50 % - - NaCl 50% / LA 5% + - TeatSeal™ - -

[0746] (-) < 3 log reduction (+) > 3 log reduction

[0747] Table 5B. Summary of contact killing of bacteria by different teat sealant prototype paste formulations. Teat sealant S. uberis S. aureus E. coli Paraffin oil - - - Paraffin oil / LA 1% + - Paraffin oil / LA 2.5 % + - Paraffin oil / LA 5 % + - TeatSeal + - - NaCl 20% - NaCl 20% / LA 1% + NaCl 20% / LA 2.5% + NaCl 20% / LA 5% + NaCl 30% - NaCl 30% / LA 1% + NaCl 30% / LA 2.5% + NaCl 30% / LA 5% + NaCl 40 % - NaCl 40 % / LA 1% + NaCl 40 % / LA 2.5% + NaCl 40 % / LA 5% + NaCl 55 % - NaCl 55 % / LA 1% + NaCl 55 % / LA 2.5% + + NaCl 55 % / LA 5% + + + NaCl 50% - NaCl 50% / LA 5% + + NaCl 55% - - + NaCl 60% + / + - + + KCl 55% / LA 0 % + - KCl 55% / LA 1 % + KCl 55% / LA 2.5 % + + KCl 55% / LA 5 % + MgCO340% - - MgCO325% / LA 5% - MgCO340% / LA 5% + - CaP 50% / NaCl 10% / LA - - - 0% CaP 60% / NaCl 0% / LA 5% + - CaP 55% / NaCl 5 % / LA 5% + CaP 50% / NaCl 10% / LA + - 5% CaP 45% / NaCl 15 % / LA + 5% CaP 35% / NaCl 25 % / LA + - 5% Kaolin 45% / LA 0 % - Kaolin 45% / LA 1 % + Kaolin 45% / LA 2.5 % + Kaolin 45% / LA 5 % +

[0748] (-) < 3 log reduction (+) > 3 log reduction

[0749] Conclusions

[0750] This Example shows that the commercial Teatseal™ and new bismuth subnitrate-free teat sealant prototypes developed by the present inventors have antimicrobial activity against mastitis causing bacteria in vitro.

[0751] The formulation containing sodium chloride (NaCl) 55% w / w with lauric acid (LA) 5% w / w was bactericidal against all three bacterial species.

[0752] The formulation containing calcium phosphate with several concentrations of NaCl and LA 5% w / w were bactericidal against S. uberis but not E. coli.

[0753] Based on the results from the laboratory work and based on the results from the clinical trials, two lead formulations were selected for a non-inferiority clinical field study (ie. whether a new experimental treatment is not unacceptably less efficacious than an active control treatment).

[0754] Properties that were considered:

[0755] - Rheology and syringability

[0756] - Antimicrobial activity

[0757] - Residues in milk (based on the selected compounds not actual residue data)

[0758] - Retention in suspended udder and live animals

[0759] - Tolerance in live animals

[0760] Following are the formulations that were trialled in a non-inferiority field efficacy study.

[0761] These were prepared as described in the earlier Examples.

[0762] Example 6 – Retention of novel teat sealant formulations administered at dry-off in dairy cattle

[0763] Summary

[0764] The objective of this retention study was to determine the mass and proportion of six teat sealant formulations F1-F6 recovered from the teats of four groups of cows scheduled for dry-off and compare recovery with the pioneer bismuth-containing product, TeatSeal™. This study was conducted in dairy cattle during autumn in Waikato, New Zealand.

[0765] A single application of six test formulations of teat sealant and one commercial product (Teatseal™; Zoetis (TS)) were administered via intra-mammary infusion at 1 tube per quarter. Animals were fed as per normal farm practice for dry-off (i.e. limited intake during the first week of dry-off to encourage milk production to cease). Sixteen cows were selected from a mob of approximately 20 cows presented for dry-off. Cows underwent udder health checks (rapid mastitis test to check for mastitis, udder palpation to check for inflammation and poor udder health and teat- end scoring to ensure healthy teat ends) and the first 16 to have negative RMT, and ≤ 1 with udder palpation and teat-end scores were enrolled. Cows were randomly allocated to Treatment Groups 1 (F1-F3 + TS; 1 hrs), 2 (F1-3 + TS; 48 hrs), 3 (F1-F3 + TS; 672 hrs) or 4 (F4-F6 + TS; 672 hrs).

[0766] On Day 0, cows were milked by the farmer and drafted out from the main mob for treatment. Cows were treated in the following order: Treatment Group 3, 4, 2, 1. Treatment Group 3 was treated and observed for approximately 1 hour prior to infusion of formulations in Treatment Group 4 cows. Approximately 1 hr after treatment, Group 1 was brought into the shed again, and teats were stripped individually after teats were examined by ultrasound. While the base of the teat was occluded, the infused formulations were carefully stripped out from the teats until no further excretions (milk and / or teat sealant) could be recovered. A second stripping was collected from Treatment Group 1 where the milk from the gland cistern was collected. This same process used for stripping one was used for the remaining treatment groups at 48 hrs (2 days) and 672 hrs (28 days). All samples were weighed and kept frozen until shipment for further examination. Ultrasound was also used to examine teats on Days 2 and 28.

[0767] Recovery of formulation was based on dry weight of recovered material after allowance for milk solids. The allowance for milk solids was done in two ways assuming 10% and 13% milk solids in the milk. There was a large amount of variation in amount of teat sealant recovered between formulations, animals and times. There were no test formulations that were consistently comparable to Teatseal™. There were also large variations in the amount (and proportion) of Teatseal™ recovered at the various times. There are several possible explanations as to the variations: cows being brought into the shed shortly after dry-off meant a let-down response was triggered. This would result in more milk in the teat canal and may have resulted in product ‘leaking’ with the pressure of the milk on top of the formulation. It was also relatively difficult to occlude the teat canal, hold the collection vessel and strip the teat. Physical measures (eg. a suitable clamp at the base of the teat) could be used to aid in the collection of formulation this way, and this would also aid in the stripping of the teat. Ultrasound examination revealed little about the amount of teat sealant within each treated cistern / canal.

[0768] In conclusion, recovery of all products (including Teatseal™) were variable with no clear performer in terms of teat sealant retention.

[0769] Study objective

[0770] The objective of the study was to determine the mass and proportion of six teat sealant formulations recovered from the teats of four groups of cows scheduled for dry-off and compare retention with the pioneer product, TeatSeal™, Zoetis).

[0771] Study design

[0772] This was a controlled, randomised study to determine the amounts of various novel teat sealant formulations retained in the teat in dairy cows at dry-off, and to compare them with those from a commercially available teat sealant (Teatseal™, Zoetis). Sixteen (16) healthy, pregnant lactating cows of variable ages schedules for dry-off on a commercial dairy farm were selected based on general examination, rapid mastitis tests and udder palpation scores.

[0773] Table 6A. Treatment details

[0774] Schedule of events

[0775] Table 6B. Proposed schedule of events.

[0776] Study animals

[0777] Species: Bovine

[0778] Breed: New Zealand Friesian

[0779] Number: Sixteen (16)

[0780] Sex: Pregnant females

[0781] Age: Mixed age

[0782] Treatment History: No mastitis, treated with dry cow therapy and teat sealant previous dry-off

[0783] Table 6CA. Investigational Veterinary Products (IVPs) / formulations. Name / Identity: 20TS R F1 (Formulation 1) Active ingredient & concentration: 250 mg / g Magnesium carbonate Dosage Form / Route: Intra-mammary Storage conditions: 4oC up to a minimum of 1 hr prior to treatment Packaging: Plastic intra-mammary syringe Name / Identity: 20TS R F2 (Formulation 2) Active ingredient & concentration: 500 mg / g Sodium chloride Dosage Form / Route: Intra-mammary Storage conditions: 4oC up to a minimum of 1 hr prior to treatment Packaging: Plastic intra-mammary syringe Name / Identity: 20TS R F3 (Formulation 3) Active ingredient & concentration: 300 mg / g Magnesium carbonate Dosage Form / Route: Intra-mammary Storage conditions: 4oC up to a minimum of 1 hr prior to treatment Packaging: Plastic intra-mammary syringe Name / Identity: 20TS R F4 (Formulation 4) Active ingredient & concentration: 450 mg / g Kaolin Dosage Form / Route: Intra-mammary Storage conditions: 4oC up to a minimum of 1 hr prior to treatment Packaging: Plastic intra-mammary syringe Name / Identity: 20TS R F5 (Formulation 5) Active ingredient & concentration: 500 mg / g Sodium chloride Dosage Form / Route: Intra-mammary Storage conditions: 4oC up to a minimum of 1 hr prior to treatment Packaging: Plastic intra-mammary syringe Name / Identity: 20TS R F6 (Formulation 6) Active ingredient & concentration: 250mg / g Sodium chloride & 100 mg / g magnesium carbonate Dosage Form / Route: Intra-mammary Storage conditions: 4oC up to a minimum of 1 hr prior to treatment Packaging: Plastic intra-mammary syringe Reference Product Name / Identity: Teatseal™ Active ingredient & concentration: Bismuth subnitrate 650 mg / mL 4 g per syringe Dosage Form / Route: Intra-mammary Storage conditions: Below 30oC (room temperature)

[0784] Table 6CB. Investigational Veterinary Products (IVPs) / actual formulations Ingredient F1 F2 F3 F4 F5 F6 MgCO325% 30% 10% NaCl 50% 50% 25% Kaolin 45% Aluminium 4.3% 5.3% 4.3% 5.3% 4.3% stearate Aerosil 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% Lauric acid 5% 5% 5% 5% Paraffin oil 65% 39% 64.3% 45% 44% 60%

[0785] F4 contained Aerosil-R972 and all other formulations contained Aerosil-200.

[0786] Treatment regime

[0787] Treatment Method and Timing

[0788] Cows were milked as per normal farm practice at the morning milking and were drafted from the milking herd upon release from the milking shed. They were drafted into a yard where they were held for treatment.

[0789] The IVP and control product was administered on a single occasion (Day 0). The IVPs were administered via the teats. The treatment plan for each cow is shown in Table 6D.

[0790] Cows in Tx Group 3 were treated first and monitored for approximately 1 hour for signs of acute adverse events. Following this, cows from Tx Group 4 were treated, then Tx Group 2. These cows were returned to the paddock where they were to remain for approximately two weeks (before joining the rest of the herd). Finally, cows in Tx Group 1 were treated and ending at approximately 6.5 hrs after milking finished, and released into a holding pen beside the shed. Approximately 40 minutes later, the cows in this group were brought back into the milking shed for stripping.

[0791] Table 6D. Treatment plan for each cow.

[0792] Udder health

[0793] Udder health was assessed prior to Day -1. Observations of turgidity and heat were made for each quarter when cows are in the milking shed for stripping. When conducting these observations, minimal pressure was applied to avoid shifting any IVP / CP around the teat / quarter.

[0794] Ultrasound procedure

[0795] A portable, small animal ultrasound (US) machine was used in an attempt to ascertain the amount (if any) of teat sealant was in the teat cistern. In order to have contact between the teat and the US probe, flexible plastic cups were filled with teat dip and the teat was immersed in the cup. Transverse and / or longitudinal images were captured on video clips and downloaded for viewing.

[0796] Specimen collection

[0797] Cows from Tx Group 1 were brought back into the milking shed for the 1 hr collection. The cow to be treated was sampled first (1:05-1:18 after administration of product). All cows were stripped between 1:06-1:14 hrs following administration of products.

[0798] Stripping of cows in Tx Group 2 was done 48:48-49:36 hrs following product administration.

[0799] While the base of the teat was occluded, the infused formulations were carefully stripped out from the teats until no further excretions (milk and / or teat sealant) could be recovered. The recovered milk, excretion and teat sealant from each teat was collected in individual plastic tubes. Cows from Group 1 had a second stripping to collect milk / product from the gland cistern.

[0800] Some extra “2nd stripping” samples were collected at Day 2 and Day 28.

[0801] Specimen analysis

[0802] The samples were dried and the dry masses determined. Since the formulations contain no volatile components, the difference between wet and dry masses will be due to aqueous secretions / milk in the wet samples. It was assumed that the aqueous secretions / milk contain 10% w / w solids. The recovered mass of formulations was calculated as:

[0803] Recovered mass = dry mass – milk solids

[0804] This was done for samples from both the first and second strippings. Percentage recoveries were calculated from recovered masses and mass administered, and compared with that for the Teatseal™ in each cow.

[0805] Statistical analysis

[0806] Means were calculated based on the raw data results. Raw data was presented in tables and graphs. Equations used were:

[0807] Recovery (%) relative to amount administered = (dry mass of formulation recovered / mass of formulation injected) x 100

[0808] Recovery (%) relative to Teatseal™ = (% recovery of formulation / % recovery for Teatseal™) x 100

[0809] Recovery after drying was measured by dry mass less estimated weight of milk solids

[0810] Conclusions

[0811] Recovery of all products (including TeatSeal™) were variable with no clear performer in terms of teat sealant retention.

[0812] Other finding

[0813] Because the variability was large and also retention of TeatSeal™ can be low, we interpreted these results as there is no clear underperformer among the experimental teat sealants.

[0814] Results

[0815] Teat strippings

[0816] Graphed teat stripping results are shown in Figures 50-58.

[0817] On Day 2 (48 / 49 hrs after treatment) four cows were observed to be dripping milk (leaking). These animals are listed in Table 6E. The ‘leaking’ was observed as the presence of drips of milk on the end of the teat.

[0818] Table 6E. Cows observed to be ‘leaking’ milk at Day 2 (48 / 49 hrs post-treatment). Cow ID Treatment group 337 2 375 2 639 3 396 4

[0819] The mean weight of each formulation at each Treatment Group (time) ranged from 2.6-2.9 g for test formulations and 4.0-4.1 g for Teatseal™ (Table 6F).

[0820] The mean weight of formulation recovered from the teats across the formulations and Treatment Group was 0.92 g, including Teatseal™ (Table 6G)^. The range of means for the test formulations with time was 0.12–2.93 g and 0.07-6.07 g for Teatseal™. The highest amount of product was recovered from the Teatseal™-treated quarters which is in line with a larger amount being administered. Of the formulations, F2 at 2 days and F3 at 1 hr had the highest recovery, although the variation for F2 (D2) was larger.

[0821] Table 6F. Mean weight (g ± standard deviation) of each formulation administered for each treatment group. TS F1 F2 F3 F4 F5 F6 1 hr 4.0 (0.1) 2.9 (0.14) 2.9 (0.08) 2.8 (0.14) - - - 2 days 4.0 (0.11) 2.9 (0.09) 2.8 (0.13) 2.7 (0.06) - - - 28 days 4.0 (0.08) 2.8 (0.06) 2.8 (0.05) 2.7 (0.07) 4.1 (0.09) 2.8 (0.13) 2.6 (0.18) 2.7 (0.24)

[0822] Table 6G. Mean weight (g) and range of formulation recovered at 1 hr (first stripping), 2 days and 28 days after administration. TS F1 F2 F3 F4 F5 F6 1 hr 2.94 1.03 0.75 1.13 - - - (0.32-6.07) (0.29-1.76) (0.46-1.10) (0.27-3.00) 2 days 1.00 1.13 0.12 0.36 - - - (0.07-3.57) (0.10-4.03) (0.09-0.19) (0.05-1.08) 28 1.75 0.45 0.39 0.69 days (0.43-4.42) (0.13-0.86) (-0.02-0.78) (0.24-1.39) 1.74 0.1 0.54 0.44 (0.43-4.41) (-0.01-0.16) (-0.02-1.40) (0.04-1.19)

[0823] Recovery relative to the amount of formulation administered was variable and ranged from 4.32-40.97% for the test formulations and 24.3-73.17% for Teatseal™ (Table 6G). Teatseal™ had the highest proportion of product recovered at 1 hr and 28 days (both Treatment Group 3 and 4). On Day 2, the highest proportion of product recovered was F1 with 37.82%, then Teatseal™ with 24.30%).

[0824] The mean mass of formulation recovered in an individual cow compared with Teatseal™ in that cow was variable with values from 15.57% (F4) to 844% (F1, Day 2). Generally, recovery was higher for F1-F3 compared with F4-F6.

[0825] For the second stripping, the variation was also large and in all cases more than product was ‘recovered’ (>114%) than was administered (>114% of data not shown). The large variation in this data made it very hard to interpret and therefore this data was not used and is not reported / discussed further.

[0826] Table 6H. Mean recovery (%) and range of formulation recovered at 1 hr (first stripping), 2 days and 28 days after administration relative to teat sealant percentage recovery. TS F1 F2 F3 F4 F5 F6 1 hr 100 128.33 119.2 71.01 - - - (27.91-389.45) (13.66-367.87) (22.18-109.19) 2 days 844.89 112.11 112.11 - - - 100 (6.75-2902.66) (3.88-193.95) (3.88-193.95) 28 100 113.27 100.30 155.42 days (3.47-254.97) (-0.57-179.69) (7.37-326.02) 15.57 55.9 40.37 100 (-0.70- (-1.39- (1.07- 40.81) 103.59) 68.45)

[0827] Table 6HA. Recovery (%) of formulations at various times after administration. Formulation Recovery (%) with respect to dose injected 1 h 48 h 672 h Low High Low High Low High F1 10 64 3 136 3 30 F2 15 40 4 7 0 27 F3 9 110 2 39 9 50 F4 0 5.3 F5 0 50 F6 0 49

[0828] Ultrasound

[0829] A portable, small animal ultrasound machine was used to determine the amount of teat sealant in the teats. A score 0 represents no teat sealant or only a small amount of teat sealant in the teat canal / teat cistern (Figure 49). A score of 1 represents a small amount of teat sealant sitting in the teat cistern but not sitting over the teat canal. A score of 2 represents a small amount of teat sealant sitting at the opening of the teat (i.e. over the inside of the teat canal, at the bottom of the teat cistern. A teat sealant score of 3 is ideal and provides >25% of the teat canal filled and sitting at the bottom of the teat cistern, covering the teat canal.

[0830] Cows underwent ultrasound scanning in the milking shed. Teat sealant scores were recorded for each teat of each animal that was to be stripped (e.g. Treatment Group 2 at 48 hrs, Treatment Group 3 and 4 at 28 days). Scores were averaged for each formulation (1- 6), and compared to each other and the control product. Data was presented as arithmetic and / or geometric means, and suitable statistical analysis was conducted.

[0831] Table 6I. Teat sealant scores and descriptions. (See Figure 49.)

[0832] Ultrasound (US) was captured on video clips and still images for cow 302 were obtained from those video clips (Figures 44-47). It was noted for teats containing Teatseal™ that sound waves did not penetrate the product. Unfortunately, it is not feasible to use US to measure the amount of teat sealant in each teat. It may, however, be possible, with more practice, to use US to ascertain the presence / absence of teat sealant.

[0833] Discussion

[0834] Recovery of product varied greatly between formulations, animals and time. There were no test formulations that were consistently comparable to the market leader, Teatseal™. In fact, there were also large variations in the amount (and proportion) of Teatseal™ recovered at the various times.

[0835] Cows were ‘dried-off’, i.e. treated, approximately 6.5 hrs after milking. Usually, cows are dried off in mobs to allow a shorter time between the end of milking and the drying-off treatment. Also, it is unusual for cows to be brought into the shed shortly (e.g. less than an hour or days) after drying off – normal practice is to put them in a paddock with little grass (and possibly hay) to reduce their energy consumption to aid in dry-off. The low recovery and lack of consistency within formulations could be attributed to milk drop when the cows’ let-down response was triggered when they were brought into the shed at 48 / 49 hrs. Cows have a psychological response to various noises, smells or events associated with milking. These vary amongst the cows and therefore vary with the time between let-down and milking. Some cows will see the shed and drop their milk, while others may be triggered by the sound of the milking machine. Without careful behavioural observations over a period of time, the triggers for individual cows cannot be ascertained with any great certainty. Stripping in yards could be considered, rather than bringing cows into the cow shed where their let- down trigger is activated.

[0836] Due to the cows coming into the shed several times, they may also have ‘leaked’ milk. When cows were brought in for 48 / 49 hr stripping, four cows were observed to be dripping milk (2, 1 & 1 from Tx Groups 2, 3 & 4, respectively). While the farmer presented the cows as ready for drying off, they still appeared to have a bit of milk (particularly at Day 2). Figure 48 shows some variation even at 28 days after dry-off. In future studies and although subjective, it would be useful to have a scoring system to indicate the degree of udder involution. It would also be worth recording which cows have milk dripping and which quarters were affected. The cows in the photos on the top row could have leaked milk as they had milk sitting in the teat canal and gland cistern.

[0837] Typical milk solid proportion is around 8-12% with larger differences between breeds (e.g. Jersey cows have higher milk fat and protein compared with Friesian cows (Holmes et. al 2003). Cows differ in their milk composition. Calculations were done assuming 10% milk solids and repeated assuming 13% milk solids. This made little difference to the results and this is because the milk volumes in the samples from the first stripping were not too large.

[0838] The variation may also be attributed to the difficulty involved in stripping the contents of the teat while occluding the base of the teat and holding the sampling pottle. If a device (e.g. band) can be applied to the base of the teat prior to the cow coming into (or near) the shed, this may help to prevent milk being dropped into the teat canal and will aid in the process of removing the content of the teat canal while avoiding the collection of milk from the gland cistern. This requires careful thought as the band / device cannot be too tight so-as to irritate the cow, nor can it be too loose to allow milk from the gland cistern to drop into the teat canal.

[0839] Ultrasound of teats proved relatively difficult in a moving animal. In order not to put pressure on the teats, a flexible cup filled with liquid was used. It may be possible to use the US probe without using the cup although consideration is needed with the amount of pressure: Too much may move the teat sealant around while too little pressure will irritate the cows as they do not react well to their teats being ‘tickled’.

[0840] Conclusions

[0841] Recovery of all products including Teatseal™ were variable with no clear performer in terms of retention. Some changes are recommended for any future work in this field in order help to potentially reduce some of the variation, such as:

[0842] • Milking cows immediately prior to administration of product

[0843] • Using a physical device to occlude the base of the teat and applying outside of the milking shed

[0844] • Stripping teats away from the milking shed

[0845] Ultrasound requires more practice and it may then be useful for assessing the presence / absence of teat sealant in the future.

[0846] References

[0847] Holmes, C. W., Brookes, I.M., Garrick, D.M., MacKenzie, D.D.S., Parkinson, T.J. and Wilson, G.F. Milk production from pasture (Principles and Practices). Massey University. Editor: D. Swain. ISBN 0473083086

[0848] Example 7 - Acute retention and recovery of teat sealant formulations in an ex vivo udder model (suspended udder study)

[0849] Objective

[0850] The objective of this study was to determine the retention and recovery of teat sealant formulations at approximately 60 minutes after intramammary infusion into excised cow udders.

[0851] Materials and methods

[0852] This study was conducted on two experimental days, separated by a week. On each day, udders from recently slaughtered dairy cows were retrieved from a nearby abattoir and transported to a necropsy room. This was a controlled temperature room set at 18 ± 1oC. Nine udders were selected each day (18 udders in total) on the basis of having four functional quarters with no gross evidence of mastitis. The udders were suspended from a frame in a close-to-natural position by penetrating them with a baling needle, threading synthetic string through the tissue and tying it to the frame. The length of each teat was then measured to the nearest millimetre from the base of the teat, at the insertion to the udder, to the teat meatus.

[0853] After discarding the first 3 streams of milk, approximately 10 mL of milk was collected from each teat into individual plastic containers, and stripping of the remaining milk out from each gland cistern was attempted. Then, treatments (21 OTTS S F1, 21 OTTS S F2, 21 OTTS S F3, 21 OTTS S F4, 21 OTTS S F5, and Teatseal™) were infused into the teats following a randomised design (Table 7A; https: / / www.random.org / lists / ). The infusion technique consisted of holding the base of the teat between the index finger and thumb of the non-dominant hand to close the teat cistern to prevent the treatment being infused into the gland cistern. Treatments were applied with the dominant hand after partial insertion (about 3 mm) of the syringe nozzle into the streak canal. Injection syringes were weighed before and after infusion into the quarters to determine the mass of product infused. The composition of the trialled treatments is depicted in Table 7B.

[0854] Individual plastic containers were positioned underneath each teat to collect any secretion / formulation that dripped spontaneously during approximately the first hour post-infusion. At approximately 60 minutes after being infused, treatments were stripped from the teats as follows. The base of the teat was held with the non-dominant hand between the thumb and the index finger and then stripped repeatedly with the dominant hand until no further product was recovered; the recovered product was collected in individual plastic containers.

[0855] After stripping each teat, the base of each teat was closed off with a Kelly forceps. Then, the teats were excised by cutting them transversally, dorsal to the Kelly forceps, and cut open longitudinally. Each teat cistern was visually inspected and photographed. A representative image of the longitudinal cross-section of each teat was taken and filed, but not analysed in this study. Immediately after obtaining photographs of the open teats, each teat was scraped and any recovered contents put into individual plastic containers.

[0856] The milk and any formulation flushed out by the rush of milk from the gland and gland cistern when cutting the teat was collected in two-litre plastic containers. Any visible formulation was placed into individual plastic containers and the milk discarded.

[0857] All the containers were properly identified, and weighed before and after collection of the samples. All samples were kept at -20oC until transported for analysis.

[0858] Table 7A. Treatment allocation in the 18 udders.

[0859] *FL = front left, FR = front right, RL = rear left, RR = rear right.

[0860] + F1 to F5 = Novel teat sealant formulations.

[0861] Table 7BA. Composition of the infused formulations.

[0862] Calcium phosphate here means calcium phosphate dibasic dihydrate.

[0863] Paraffin oil, to balance, was used in all formulations.

[0864] The samples were weighed (i.e., wet mass) and dried to constant weight, and the dry masses determined. Since the formulations contained no volatile components, the difference between wet and dry masses was due to the aqueous component in the wet samples (Equation 1).

[0865]

[0866] Samples collected before treatment infusion were used to determine the percentage of milk solids in secretions from individual glands (Equation 2). These percentage values were used to determine the mass of milk solids in samples from corresponding quarters (Equation 3). Then, the mass of formulation was calculated by subtracting the mass of milk solids to the dry mass of each sample (Equation 4).

[0867]

[0868] Results were expressed as percentage values in relationship to mass of infused treatment for samples collected by dripping, by stripping, by scraping, and by flushing, and for total treatment accounted for according to the following equations:

[0869]

[0870]

[0871] Statistical analysis

[0872] Data were analysed using the D’Agostino and Pearson normality test and, as appropriate, were presented as mean ± SD and / or median (range). Differences between treatments for teat length, milk solids in milk samples, mass of infused formulation, recovery by dripping, stripping, scraping, and flushing, and total treatment retrieved were analysed using a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. Differences between treatments for percentage recovery from the teat were analysed using a GLMM with udder as a random effect and formulation and front / rear teat as fixed effects. Differences between treatments in the proportion of teats for which percentage recovery from the teat was greater than or equal to Teatseal™ were compared using a chi-square test. The effect of teat length on the recovery of the different formulations was determined using linear regression analysis. Values of P < 0.05 were considered significant.

[0873] Results

[0874] The length of the teats from the 18 udders included in the study varied between 30 mm and 63 mm and median lengths were not significantly different when the teats were grouped by treatment (P = 0.3388; Figure 59). The estimated solids in milk samples obtained before infusing the formulations varied from 9.24% to 26.6% and median milk solids were not significantly different when the teats were grouped by treatment (P = 0.1342; Figures 60-65).

[0875] Conclusions

[0876] This study confirmed that teat sealant formulations can be recovered approximately 60 minutes after being infused into the teats of excised cow udders. The majority of the infused formulation was recovered by stripping followed by scraping and flushing, and the amount dripping was insignificant.

[0877] Further analysis of results from a previous suspended udder study showed that the recovery rate was lower in rear teats than in front teats, and that teat length had a moderate positive relationship with recovery rate from the teat (i.e., recovery by stripping + recovery by scraping; r = 0.4118). Similar to this previous study, the recovery rate from the teats was numerically lower in the rear teats than in the front teats, except for those teats treated with 21 OTTS S F2 (94.3% vs 88.22%). The recovery rate from the teats was significantly different in 21 OTTS S F1-treated teats only (P = 0.0078). Contrary to the previous suspended udder study, the current study found no association between teat length and recovery rate from the teat (r = 0.0011). Although it was the inventors’ impression that teats, probably from heifers, used in the previous suspended udder study were shorter than the teats used in the current study, probably from cows at the end of the lactation, the measured teat lengths were similar between studies (from 30 to 55 mm and from 30 to 63 mm, respectively). The different results may be due to methodological issues. In the previous suspended udder study, 27 teats were measured for length using the photographs of excised, open teats and the identifier label as reference. In the current study, 72 teats were measured using a calliper from the medial aspect of the base of the teat, at the insertion to the udder, to the teat meatus. Developing a methodology to accurately and consistently measure teat length is needed to determine the effect of this variable on the recovery of formulation from the teat.

[0878] Contrary to the previous suspended udder study, in which recovery rates from the teat varied from 1.44 to 126%, more consistent recovery rates were obtained in the current study with all the tested formulations (i.e., 30.96 to 124.57%). This may be attributed at determining milk solids in samples from each quarter instead of having an untreated quarter and assuming milk solids to be the same in the other three quarters for each udder. It may also be related to the apparently longer teats in udders from the current study (however, see preceding paragraph). The recovery rates from the teat for the tested formulations were also similar to those obtained with Teatseal™. This despite finding that a significant proportion of front teats had different recoveries from the teat for the infused formulation when considering a cut-off of at least equal to Teatseal™ (i.e., ≥ 95.81%). However, as an example, the three front teats infused with 21 OTTS S F1 that did not reach the cut-off mark had recovery rates between 94.2 and 94.75%. Similarly, all six 21 OTTS S F3-treated front teats had recovery rates between 90.38 and 95.46%. The cut-off benchmark may be considered as arbitrary and, thus, the results comparing the recovery rates from the teat to those for Teatseal™ should be interpreted in light of this limitation.

[0879] Some of the infused formulation entered the gland cistern. However, recovery by flushing was smaller in the current study (from 0.3% to 51.84%) than in the previous suspended udder study (from 23.4% to 87.87%). This may be due to partial tip insertion and apparently longer teats used in the current study as opposed to full tip insertion and apparently shorter teats in the previous study. Although it seems that syringe tip length insertion has an effect on the recovery rate of the infused formulation from the gland cistern, further studies are needed to test this hypothesis.

[0880] After strenuous stripping, some formulation was still present in the teat cistern as evidenced by the photographs in Figure 66. Formulation 21 OTTS S F5 and Teatseal™ had a more similar bioadhesion pattern with apparently more formulation remaining in the teat cistern than the other trialled formulations. This was confirmed by measuring recovery by scraping. Different rheological responses to shearing forces may contribute to the observed results. The distribution of the formulations in the teat cistern reported in the current study is not reflective of what may happen in undisturbed teats or those subjected to movement of udders in live cows.

[0881] In summary, the recovery rate of teat sealant formulations from the teat was comparable to that of Teatseal™ for both front and rear teats. Recovery by stripping, scraping, and flushing accounted for most of the recovery; recovery by dripping was insignificant.

[0882] Example 8 - Pilot local tolerance study with two novel teat sealant formulations in dairy cows at drying off

[0883] Summary

[0884] The scope of the study was to investigate the local and systemic tolerance of two teat sealant formulations (Teat Sealant 1 and Teat Sealant 2) at drying-off in dairy cows. In total 10 lactating cows (age in years: mean = 3.1; min = 2; max = 6) were included in the study in two study groups with each 5 animals on study day (SD) -1. On SD 0 two quarters of each cow of study group 1 were treated with Teat Sealant 1 (TS 1) and two quarters of each cow of study group 2 were treated with Teat Sealant 2 (TS 2) before drying-off. All remaining quarters were treated with OrbeSeal™ (composed of 65% bismuth subnitrate in a mineral oil vehicle) ad us. Vet. Local tolerance was assessed 1 h, 4 h, 12 h, once daily during the first week and once in the second week after application. Systemic tolerance was assessed during daily general observations, including a measurement of rectal temperature. On SD 14 after study closure examination cows were euthanized. Udder samples were taken and fixed in formalin 10% for histopathological evaluation.

[0885] Two adverse events occurred. Cow I 363 developed a severe clinical mastitis in the hind right udder quarter on SD 2, which required concomitant treatment. A relation to Staph. aureus, which was determined in the corresponding milk sample after arrival cannot be ruled out, even if the quarter was treated with Synulox LC Plus™ before inclusion. Cow I 366 developed a clinical mastitis in the front left quarter on SD 4. Both affected quarters were sealed with Orbeseal™ on SD 0. Besides milk dripping in three cows after drying-off no further findings were observed during local tolerance assessments.

[0886] Histopathologically the intramammary administration of TS 1 and 2 or Orbeseal™ did not reveal any Investigational Veterinary Product (IVP)-related local toxic effects. All cows were still markedly lactating after two weeks. Nevertheless, 2 cows (I 360 and I 366) showed chronic active inflammation in one or two udder quarters, where occurrence before treatment and / or exacerbation secondary to drying-off cannot be excluded. Reactive hypercellularity was seen in the associated lymph nodes. Cow I 363 revealed subacute abscesses in one quarter corresponding to clinical findings during the treatment period. Without major histopathological findings, many cows had increased granulocytes and / or mononuclear cells or debris in mammary alveoli, pointing out the mammary involution process.

[0887] In summary, TS 1 and TS 2 were well tolerated both systemically and locally.

[0888] Study scope

[0889] The scope of the study was to investigate the local and systemic tolerance of two teat sealant formulations (TS 1 and TS 2) at drying-off in dairy cows.

[0890] Animals and methods

[0891] In total 10 lactating cows (age in years: mean = 3.1; min = 2; max = 6) were included in the study in two study groups with each 5 animals on study day (SD) -1. During acclimatization and at inclusion approximately 1 month later, (SD -1) milk samples were taken for determination of somatic cell counts. A bacteriological examination was additionally performed.

[0892] Female Holstein cows (5 / test item) were treated intramammarily with TS 1 or TS 2 once at a dose volume of 5 g per quarter for an observation period of approximately 2 weeks. Randomly per cow, 2 quarters received the test item and the other 2 quarters were treated with a single dose of 4 g Orbeseal™ as reference substance.

[0893] On SD 0 two quarters of each cow of study group 1 were treated with TS 1 and two quarters of each cow of study group 2 were treated with TS 2 before drying-off. All remaining quarters were treated with OrbeSeal™ ad us. Vet. Local tolerance was assessed 1 h, 4 h, 12 h, once daily during the first week and once in the second week after application. Systemic tolerance was assessed during daily general observations, including a measurement of rectal temperature. Approximately 4 h after the treatment on SD 0 the cows were moved from the tie-stall into a nearby barn with loose-housing.

[0894] On SD 14 after study closure examination cows were euthanized. Udder samples were taken and fixed in formalin 10% for histopathological evaluation.

[0895] Results

[0896] At inclusion (on SD -1) the allocation of quarters to treatment with teat sealant formulation or reference item was determined by drawing lots according to study outline. The following treatment pattern, according to Table 8A, was determined.

[0897] Table 8A. Treatment pattern.

[0898] Table 8B. Determination of body weight [kg].

[0899] Milk sampling

[0900] Milk samples were taken according to protocol for determination of somatic cell counts (during acclimatization and on SD -1) and for bacteriological examination.

[0901] Table 8C. Bacteriological examination.

[0902] A major mastitis pathogen (Staph. aureus) was only determined in the front left quarter of cow I 366. Thereupon the quarter was treated with Synulox LC Plus™ as the Staph. aureus was susceptible for Amoxicillin. All other quarters were free of major pathogens.

[0903] Table 8D. Determination of somatic cell counts [x1000cells / mL].

[0904] Animals with quarters with somatic cell counts above 200,000 cells / mL were included in the study, even if defined otherwise in the inclusion criteria. All quarters and the milk from respective quarters were clinically unremarkable after arrival and at study inclusion. A major mastitis pathogen (Staph. aureus) was only determined in the front left quarter of cow I 366 and the quarter was treated with Synulox LC Plus™. All other quarters were free of major pathogens. Due to the clinical picture and the results from bacteriological examination after arrival and the subsequent treatment, it was decided to include all quarters into the study, despite of the increases in somatic cell counts.

[0905] Daily general health observation

[0906] During acclimatization and during the study until drying-off milk yields were determined twice daily. General health was determined daily during feeding and cleaning. During the study the severity score (SV) was assessed at least once daily. Cow I 363 was assigned to SV3 from SD 3 until SD 8 as she developed a severe acute mastitis, which required concomitant treatment. All other cows were assigned to SV2.

[0907] Physical examination

[0908] Physical examinations were conducted on SD -1 and before necropsy on SD 14. The following findings were observed, as per Table 8E.

[0909] Table 8E. Physical examination.

[0910] Investigational Veterinary Product (IVP) – TS 1 and TS 2

[0911] Table 8F. Teat Sealant 1 (TS 1) formulation. Ingredient TS1 MgCO325% Aluminium stearate 4.3% Aerosil 0.7% Lauric acid 5% Liquid paraffin 65%

[0912] Table 8G. Teat Sealant 2 (TS 2) formulation. Ingredient TS2 MgCO3NaCl 50% Kaolin Aluminium stearate 5.3% Aerosil 0.7% Lauric acid 5% Liquid paraffin 39%

[0913] Application

[0914] The intramammary application of the IVP and the reference item was conducted according to study outline. The complete dose was applied without any losses in every case. Teat Sealant 1 (TS 1) was difficult to apply and both hands were needed to push the complete content into the teat. Application of Teat Sealant 2 (TS 2) was comparable to the application of OrbeSeal™. Afterwards it seemed that the application (TS 2) provoked an unpleasant sensation in the area around the teat.

[0915] Local and systemic tolerance assessment

[0916] During local tolerance assessments all udder quarters were inspected for signs of inflammation (e.g. reddening, swelling, increased heat, pain). The following observations were made:

[0917] - I 359: milk dripped on SD 1

[0918] - I 360: milk dripped on SD 0 (12h) and SD 1

[0919] - I 362: milk dripped on SD 1

[0920] - I 363: mastitis of right hind quarter on SD 2, 3, 4 and 5. On SD 10 and 14 inflammation emanated from the right hind quarter towards right front quarter

[0921] - I 366: swelling of front left quarter on SD 4 and 5. On SD 14 swelling emanated from front left quarter towards left hind quarter.

[0922] Rectal temperature was measured once daily. Rectal temperature increased in some cows after drying-off without clinical relevance, except for cow I 363, who temporarily suffered from fever as she developed a severe clinical mastitis.

[0923] Adverse events

[0924] Two adverse events occurred during the study.

[0925] SD 2, I 363 (moderate adverse event): Right hind udder quarter severely swollen and painful.

[0926] SD 4, I 366 (mild adverse event): Swelling of front left udder quarter, no pain, general health undisturbed.

[0927] Clinical summary and concomitant treatment

[0928] Cow I 363 developed a severe mastitis and had to be treated.

[0929] SD 2: right hind quarter severely swollen and painful, rectal temperature: 39,7°C, treatment with Septomammin™ locally.

[0930] SD 2, evening: rectal temperature: 40.5°C, reduced general health, treatment with Septomammin™ locally and Metacam™ (12mL s.c.).

[0931] SD 3: rectal temperature: 41°C, milking out the respective quarter (yellowish fluid with remnants of teat sealant), treatment with Orbenin Extra™ (1 applicator intramammary), Septomammin™ locally, Vetalgin™ (40mL i.v.).

[0932] SD 4: in the morning and in the evening: milking out the respective quarter, treatment with Orbenin Extra™ (1 applicator intramammary), Septomammin™ locally.

[0933] SD 5: in the morning and in the evening: milking out the respective quarter, treatment with Orbenin Extra™ (1 applicator intramammary), Septomammin™ locally.

[0934] SD 6: general appearance reduced, ears dropped, animal lying a lot, rumen poorly filled, inappetence, udder quarter swollen, hard, no increased heat, painful during palpation, heart rate elevated, rectal examination: no feces, rumen moderately filled, treatment with infusion (1L of G40™, 60mL Catosal™, 13mL Metacam™ i.v.), drench (40L water + 2 bags of Rumentinol™ orally), milking out respective quarter (small amount of yellowish fluid and remnants of teat sealant), Orbenin Extra™ (1 applicator intramammary).

[0935] SD 7: rectal temperature: 37.8°C, drench (40L + 2 bags of Rumentinol™), swelling of quarter still severe, swelling moving towards front right quarter, feed intake after drenching.

[0936] SD 8: right hind quarter still hard and swollen, swelling moving towards front right quarter, animal fitter, rumen moderately filled, mucous nasal discharge, treatment with infusion (1.5L NaCl, 0.5L G40™, 30mL Catosal™, 250mL Calciumborogluconat™, 13mL Metacam™, 5mL Mederantil™ i.v.), after infusion shaking gait, but good feed intake.

[0937] SD 9: general appearance improved, good feed intake and rumination.

[0938] SD 14: general appearance good, right hind quarter still severely hard and swollen, right front quarter hard and swollen.

[0939] Necropsy of udder

[0940] All animals were euthanized via deep narcosis with Ketamin / Xylazin and subsequent application of T61™. Only the udder and udder lymph nodes were collected and examined. The body weights were recorded before study start.

[0941] After euthanasia, various samples were taken from each udder quarter from each cow, as shown in Figure 67.

[0942] Additionally, samples from areas with macroscopic findings and samples from both Lnn. mammarii were taken from each cow.

[0943] Necropsy revealed areas in the left udder in one cow of the TS 1 group. Change-in-contents and consistency change was observed in the right and left udder of single animals in the TS 2 group. Swelling of the mammary gland occurred in one cow of the TS 1 group. Udder lymph nodes were swollen in 2 cows of the TS 1 group and in one cow of the TS 2 group.

[0944] Histopathology

[0945] No treatment-related local toxic effect was found for TS 1, TS 2 or Orbeseal™. Three cows revealed chronic active suppurative or subacute abscessing mastitis. Many cows had increased granulocytic and / or monocytic cells or debris in one or more udder quarters.

[0946] The intramammary administration of TS 1 and 2 or Orbeseal™ once did not reveal any test item-related local toxic effects. All cows were still markedly lactating after two weeks. Nevertheless, 2 cows (I 360 and I 366) showed chronic active inflammation in one or two udder quarters, where occurrence before treatment and / or exacerbation secondary to drying cannot be excluded. Reactive hypercellularity was seen in the associated lymph nodes. Cow I 363 revealed subacute abscesses in one quarter corresponding to clinical findings during the treatment period. Without major histopathological findings, many cows had increased granulocytes and / or mononuclear cells or debris in mammary alveoli, pointing out the mammary involution process.

[0947] Mastitis was detected in 3 cows:

[0948] I 360 (group 01:TS 1 or Orbeseal™) had chronic active suppurative inflammation in the right front quarter and teat at moderate degree (treated with Orbeseal™), with slight focal fibroses in the glandular tissue. Correspondingly, the right udder lymph node grossly showed size increase and microscopically increased cellularity at slight degree. Slight focal glandular fibroses were also observed in the hind right and front left quarter (both treated with TS 1).

[0949] I 363 (group 01:TS 1 or Orbeseal™) revealed multiple subacute abscesses at marked degree with moderate bacteria in the right hind quarter (treated with Orbeseal™). The teat was affected by moderate inflammation. The right udder lymph node showed slightly increased cellularity.

[0950] I 366 (group 02:TS 2 or Orbeseal™) was affected by chronic active suppurative inflammation of gland and teat at slight to moderate degree in the left front (treated with Orbeseal™) and hind quarter (treated with TS 2) with focal glandular fibroses. The associated left lymph node showed slightly increased cellularity and lymphoid hyperplasia.

[0951] Noteworthy findings

[0952] Noteworthy findings that were considered to be test item related are summarized in the following Table 8H.

[0953] Table 8H. Summary of noteworthy findings given at lowest dose level of occurrence.

[0954] Summary

[0955] The intramammary administration of Teat Sealant 1 and 2 or Orbeseal™ once over 2 weeks did not reveal any test item-related local toxic effects. All cows were still markedly lactating. Nevertheless, 2 cows (I 360 and I 366) showed chronic active inflammation in one or two udder quarters where occurrence before treatment and / or exacerbation secondary to drying cannot be excluded. Reactive hypercellularity was seen in the associated lymph nodes. Cow I 363 revealed subacute abscesses in one quarter corresponding to clinical findings during the treatment period. Without major histopathological findings, many cows had increased granulocytes and / or mononuclear cells or debris in mammary alveoli which might be responsible for clinically increased cell counts in the milk.

[0956] Conclusion

[0957] The two occurred adverse events of clinical mastitis after drying-off were unlikely to be related to the IVP (ie. TS 1 and TS 2). Both affected quarters were sealed with Orbeseal™ on SD 0. Besides milk dripping in three cows after drying-off no further findings were observed during local tolerance assessments. Histopathological examination did not reveal any IVP-related local toxic effects. Both Teat Sealant 1 and Teat Sealant 2 were well tolerated systemically and locally.

[0958] Advantages of the present invention as exemplified

[0959] Some advantages of some embodiments of the present invention as exemplified include:

[0960] – the teat sealant formulation is bismuth-salt-free and has antimicrobial activity (but is ‘antibiotic-free’ in the sense that it does not include an antibiotic as used in dry-cow therapy);

[0961] - the teat sealant formulation is easy to administer;

[0962] - the teat sealant formulation can be infused from a syringe;

[0963] - the teat sealant formulation will remain with the teat throughout a large part of the dry period;

[0964] - the teat sealant formulation is easily removeable without concern for remaining heavy metal residues, like bismuth residues;

[0965] - at the end of the dry-period, the teat sealant formulation can be stripped out;

[0966] - the teat sealant formulation is effective and safe for the cow; and

[0967] - the teat sealant formulation is useful in the prevention of mastitis.

[0968] In the present specification, the word ‘comprising’ and its derivatives including ‘comprises’ and ‘comprise’ include each of the stated integers but does not exclude the inclusion of one or more further integers.

[0969] The terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10 %, preferably ±5 %, more preferably ±2 %, and even more preferably ±1 %.

[0970] Reference in this specification to any number range includes all possible numbers / numerical values falling within that range, and further includes all possible subranges falling within that range, context permitting.

[0971] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0972] Reference throughout this specification to ‘one embodiment’ or ‘an embodiment’ means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases ‘in one embodiment’ or ‘in an embodiment’ in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

[0973] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted by those skilled in the art.

Claims

Claims 1. A bismuth-salt-free teat sealant composition, formulated to occlude a teat canal and / or teat cistern.

2. A teat sealant composition comprising a salt other than a bismuth salt, formulated to occlude a teat canal and / or teat cistern.

3. A teat sealant composition formulated for administration to, and retention within, a teat canal and / or teat cistern of a cow.

4. The teat sealant composition of any one of the preceding claims, wherein the teat sealant composition is in the form of a gel, paste or coarse suspension.

5. The teat sealant composition of any one of the preceding claims, wherein: - the teat sealant composition has an adequately low viscosity so as to allow it to be administered via a teat canal and / or teat cistern by way of injection; - approximately 2-5 g of the teat sealant composition is administered / injected into a teat canal and / or teat cistern; - a syringeability force for injecting the teat sealant composition into a teat does not exceed 4500 g at 25°C, and preferably the syringeability force for injecting the teat sealant composition into a teat is between about 1500 g and 3500 g at 25°C; - the teat sealant composition thickens rapidly in a teat so as to be retained and occlude a teat canal and / or teat cistern; - once located within a teat canal and / or teat cistern, the teat sealant composition has a viscosity high enough so as to be retained within the teat canal or teat cistern and occlude the teat canal or teat cistern; - the teat sealant composition has a bounce-back viscosity enabling it to be retained within a teat canal and / or teat cistern as a substantially coherent mass; - the teat sealant composition has a rheology enabling the composition to be administered to a teat canal and / or teat cistern by way of injection, and to be retained within the teat canal and / or teat cistern and occlude the teat canal and / or teat cistern, or to prevent an intramammary infection; - teat sealant composition has a density greater than about 1 g / cm3, preferably between about 1.3 g / cm3and 1.6 g / cm3; - the teat sealant composition shear-thins at the shearing forces applied during injection and stripping from a teat; - the teat sealant composition has antimicrobial activity, preferably antibacterial activity, preferably against bacteria that cause mastitis;- the teat sealant composition is formulated to occlude a teat canal and / or teat cistern for a period of at least about 14 days, preferably about 42 to about 90 days; - the teat sealant composition is formulated to occlude a teat canal and / or teat cistern for a full length of a dry period; - the teat sealant composition is formulated to occlude a teat canal and / or teat cistern for at least approximately the first 14 days of a dry period until a natural keratin plug develops; - the teat sealant composition occludes a teat canal and / or teat cistern or substantially occludes the teat canal and / or teat cistern for about 4-6 weeks, while allowing a natural keratin plug to develop; or - the teat sealant composition is formulated such that no stripping from a teat canal and / or teat cistern is required at the end of a dry-period.

6. A teat sealant composition comprising solid particles dispersed within a water-insoluble shear- thinning viscous fluid vehicle, wherein the solid particles are not a bismuth salt.

7. The teat sealant composition of claim 6, wherein: antimicrobial activity is provided by the vehicle; antimicrobial activity is provided by the solid particles; antimicrobial activity is provided by both the vehicle and the solid particles; antimicrobial activity is provided by an ingredient of the vehicle; or, the composition further comprises at least one type of antimicrobial agent, preferably an antibacterial agent.

8. The teat sealant composition of claim 6 or claim 7, wherein the vehicle comprises a water-insoluble carrier and a thickener, and preferably the thickener includes the solid particles.

9. The teat sealant composition of claim 8, wherein the water-insoluble carrier comprises at least one type of carrier agent, and the carrier agent comprises at least one type of oil or oily liquid.

10. The teat sealant composition of claim 9, wherein the at least one type of oil or oily liquid comprises a vegetable oil, mineral oil, synthetic oil, medium chain triglyceride, or triglyceride, preferably paraffin oil, liquid paraffin, petrolatum, sesame oil, or a medium chain triglyceride.

11. The teat sealant composition of claim 9 or 10, wherein the composition comprises approximately 20% to 80% w / w or 30-65% w / w of the at least one type of carrier agent.

12. The teat sealant composition of any one of claims 8 to 11, wherein the thickener comprises at least one type of thickening agent which is water-soluble and / or water-insoluble, preferably in an amount of approximately 0.1% to 10%, w / w.

13. The teat sealant composition of claim 12, wherein the at least one thickening agent functions both as a thickener and as an antimicrobial.

14. The teat sealant composition of claim 12 or claim 13, wherein the at least one thickening agent comprises a medium-chain saturated fatty acid, preferably lauric acid.

15. The teat sealant composition of claim 14, wherein the composition comprises approximately 1% to 10% w / w of the medium-chain saturated fatty acid, preferably lauric acid.

16. The teat sealant composition of claim 12 or 13, wherein the at least one thickening agent comprises a metal salt of a long chain fatty acid, preferably a stearate salt.

17. The teat sealant composition of claim 16, wherein the metal salt of the long chain fatty acid comprises aluminium distearate, aluminium stearate, aluminium tristearate, ammonium stearate, barium stearate, butyl stearate, cadmium stearate, calcium stearate, cobalt stearate, copper stearate, glycol stearate, lithium stearate, magnesium stearate, manganese stearate, methyl stearate, potassium stearate, sodium stearate, strontium stearate, or zinc stearate, preferably aluminium stearate.

18. The teat sealant composition of claim 17, wherein the composition comprises approximately 1% to 10% w / w of the metal salt of the long chain fatty acid.

19. The teat sealant composition of claim 12 or claim 13, wherein the at least one thickening agent comprises silicon dioxide, preferably hydrophilic or hydrophobic silica.

20. The teat sealant composition of claim 19, wherein the composition comprises approximately 0.1% to 1% w / w silicon dioxide, preferably hydrophilic fumed silica.

21. The teat sealant composition of any one of claims 6 to 20, wherein: - the solid particles, with respect to water, are very soluble, freely soluble, soluble, sparingly soluble, slightly soluble, very slightly soluble, practically insoluble, or insoluble; - the solid particles have a solubility in water that exceeds about twice the iso-osmotic concentration; - the solid particles modify the rheology of the composition as well as provide antimicrobial activity preferably against mastitis causing bacteria; - the solid particles comprise at least one type of salt that is not a bismuth salt, such as an organic salt or inorganic salt; - the solid particles comprise a halide salt, phosphate or carbonate, preferably sodium chloride, potassium chloride, calcium phosphate as herein defined, preferably calcium phosphate dibasic dihydrate, or magnesium carbonate; - the solid particles comprise at least one type of heavy metal salt provided that it is not a bismuth salt, preferably with the metal of the heavy metal salt comprising calcium, magnesium or aluminium, but excluding toxic heavy metals;- the solid particles comprise at least one type of carbohydrate, such as a simple or complex sugar, preferably comprising sucrose or mannitol; - the solid particles comprise at least one type of mineral, such as a clay, such as kaolin, such as kaolinite, halloysite, dickite or nacrite; - the solid particles comprise sodium chloride; - the solid particles comprise potassium chloride; - the solid particles comprise calcium phosphate as herein defined; - the solid particles comprise calcium phosphate dibasic dihydrate; - the solid particles comprise calcium carbonate; - the solid particles comprise magnesium carbonate; - the solid particles comprise sodium chloride or potassium chloride, and calcium phosphate as herein defined, or calcium phosphate dibasic dihydrate; - the solid particles comprise sodium chloride or potassium chloride, and calcium carbonate; - the solid particles comprise sodium chloride or potassium chloride, and magnesium carbonate; - the composition comprises solid particles in the following approximate amount ranges: 10% to 75%, 35% to 70%, 40% to 65%, or 50% to 60% w / w; - the composition comprises 25-60% w / w sodium chloride or potassium chloride; - the composition comprises 10-30% w / w magnesium carbonate; - the composition comprises 10% w / w magnesium carbonate and approximately 25% w / w sodium chloride or potassium chloride; - the composition comprises 55% w / w sodium chloride or potassium chloride; - the composition comprises 35-60% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate; - the composition comprises 60% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate; - the composition comprises 10% w / w sodium chloride or potassium chloride and 50% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate; - the composition comprises 25% w / w sodium chloride or potassium chloride and 35% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate; or - the composition comprises 10% w / w sodium chloride or potassium chloride and 50% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate.

22. The teat sealant composition of any one of claims 6 to 21, further comprising one or more other ingredients such as at least one antioxidant or preservative.

23. A teat sealant composition comprising solid particles in a gel or in the form of a paste or in the form of a course suspension, formulated to occlude a teat canal and / or teat cistern.

24. A teat sealant composition comprising: an oily carrier; and a thickener that comprises solid particles and lauric acid, wherein the solid particles are not a bismuth salt.

25. A teat sealant composition comprising coarse filler material, in the form of solid particles, in a gelling agent, wherein the solid particles are not a bismuth salt.

26. A teat sealant composition comprising: liquid paraffin; aluminium stearate; fumed silica; and water-soluble solid particles, wherein said composition is in the form of a paste or suspension.

27. A teat sealant composition comprising: liquid paraffin; aluminium stearate; fumed silica; and water-insoluble solid particles, wherein said composition is in the form of a paste or suspension.

28. The teat sealant composition of any one of claims 23 to 27, wherein the average solid particle size is less than approximately 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 µm in size, or the average solid particle size is approximately 10-50 µm in size, or the average solid particle size is approximately 1-10 µm in size.

29. The teat sealant composition of any one of claims 23 to 28, wherein: - the solid particles, with respect to water, are very soluble, freely soluble, soluble, sparingly soluble, slightly soluble, very slightly soluble, practically insoluble, or insoluble; - the solid particles have a solubility in water that exceeds about twice the iso-osmotic concentration; - the solid particles modify the rheology of the composition as well as provide antimicrobial activity preferably against mastitis causing bacteria;- the solid particles comprise at least one type of salt that is not a bismuth salt, such as an organic salt or inorganic salt; - the solid particles comprise a halide salt, phosphate or carbonate, preferably sodium chloride, potassium chloride, calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate, or magnesium carbonate; - the solid particles comprise at least one type of heavy metal salt provided that it is not a bismuth salt, preferably with the metal of the heavy metal salt comprising calcium, magnesium or aluminium, but excluding toxic heavy metals; - the solid particles comprise at least one type of carbohydrate, such as a simple or complex sugar, preferably comprising sucrose or mannitol; - the solid particles comprise at least one type of mineral, such as a clay, such as kaolin, such as kaolinite, halloysite, dickite or nacrite; - the solid particles comprise sodium chloride; - the solid particles comprise potassium chloride; - the solid particles comprise calcium phosphate as herein defined; - the solid particles comprise calcium phosphate dibasic dihydrate; - the solid particles comprise calcium carbonate; - the solid particles comprise magnesium carbonate; - the solid particles comprise sodium chloride or potassium chloride, and calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate; - the solid particles comprise sodium chloride or potassium chloride, and calcium carbonate; - the solid particles comprise sodium chloride or potassium chloride, and magnesium carbonate; - the composition comprises solid particles in the following approximate amount ranges: 10% to 75%, 35% to 70%, 40% to 65%, or 50% to 60% w / w; - the composition comprises 25-60% w / w sodium chloride or potassium chloride; - the composition comprises 10-30% w / w magnesium carbonate; - the composition comprises 10% w / w magnesium carbonate and approximately 25% w / w sodium chloride or potassium chloride; - the composition comprises 55% w / w sodium chloride or potassium chloride; - the composition comprises 35-60% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate;- the composition comprises 60% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate; - the composition comprises 10% w / w sodium chloride or potassium chloride and 50% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate; - the composition comprises 25% w / w sodium chloride or potassium chloride and 35% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate; or - the composition comprises 10% w / w sodium chloride or potassium chloride and 50% w / w calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate.

30. A teat sealant composition comprising one of the following formulations, wherein all ingredients shown are actual or approximate: NaCl or KCl 55% w / w; Aluminium stearate 4.3% w / w; Fumed silica 0.7% w / w; Lauric acid 5% w / w; and Paraffin oil 35% w / w; or NaCl or KCl 60% w / w; Aluminium stearate 4.3% w / w; Fumed silica 0.7% w / w; Lauric acid 5% w / w; and Paraffin oil 30% w / w; or NaCl or KCl 55% w / w; Aluminium stearate 4.3% w / w; Fumed silica 0.7% w / w; Lauric acid 2.5% w / w; and Paraffin oil 37.5% w / w; or NaCl or KCl 10% w / w;Calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate 50% w / w; Aluminium stearate 4.3% w / w; Fumed silica 0.7% w / w; Lauric acid 5% w / w; and Paraffin oil 30% w / w; or NaCl or KCl 25% w / w; Calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate 35% w / w; Aluminium stearate 4.3% w / w; Fumed silica 0.7% w / w; Lauric acid 5% w / w; and Paraffin oil 30% w / w; or Calcium phosphate as herein defined, such as calcium phosphate dibasic dihydrate 60% w / w; Aluminium stearate 4.3% w / w; Fumed silica 0.7% w / w; Lauric acid 5% w / w; and Paraffin oil 30% w / w; or Magnesium carbonate 25% w / w; Aluminium stearate 4.3% w / w; Fumed silica 0.7% w / w; Lauric acid 5% w / w; and Paraffin oil 65% w / w; orNaCl or KCl 50% w / w; Aluminium stearate 5.3% w / w; Fumed silica 0.7% w / w; Lauric acid 5% w / w; and Paraffin oil 39% w / w; or Magnesium carbonate 30% w / w; Fumed silica 0.7% w / w; Lauric acid 5% w / w; and Paraffin oil 64.3% w / w; or NaCl or KCl 50% w / w; Aluminium stearate 5.3% w / w; Fumed silica 0.7% w / w; and Paraffin oil 44% w / w; or NaCl or KCl 25% w / w; Magnesium carbonate 10% w / w; Aluminium stearate 4.3% w / w; Fumed silica 0.7% w / w; and Paraffin oil 60% w / w.

31. A method of preparing a teat sealant composition comprising solid particles dispersed within a water-insoluble shear-thinning viscous fluid vehicle, said method comprising the steps of: (1) forming a water-insoluble gel by mixing at least one type of water-insoluble carrier agent with at least one type of thickening agent; (2) adding solid particles to the gel to form a paste; and, optionally, (3) adding at least a further type of thickening agent to the gel or paste, wherein: a teat sealant composition comprising solid particles dispersed within a water-insoluble shear- thinning viscous fluid vehicle is prepared;the solid particles are not a bismuth metal salt; step (3) is optional; and steps (2) and (3) need not be carried out in the stated order.

32. A method of preparing a teat sealant composition, said method comprising the steps of: (1) forming a water-insoluble gel by mixing at least one type of oily carrier with at least one type of thickening agent; (2) adding solid particles to the gel to form a paste; and, (3) adding lauric acid as a thickening agent to the gel or paste, wherein: the solid particles are not a bismuth metal salt.

33. A teat sealant composition when prepared by the method according to claim 31 or claim 32.

34. A teat sealant composition for preventing a new intramammary infection or mastitis in a cow, wherein the composition is as claimed in any one of claims 1 to 30 and 33.

35. A teat sealant composition for use or when used for preventing a new intramammary infection or mastitis in a cow, wherein the composition is as claimed in any one of claims 1 to 30 and 33.

36. Use of a teat sealant composition in the manufacture of a medicament for preventing a new intramammary infection or mastitis in a cow, wherein the composition is as claimed in any one of claims 1 to 30 and 33.

37. A method of preventing a new intramammary infection or mastitis in a cow, said method comprising the step of administering to at least one teat of the cow the composition as claimed in any one of claims 1 to 30 and 33.

38. Use of the composition as claimed in any one of claims 1 to 30 and 33 for preventing a new intramammary infection or mastitis in a cow.

39. A syringe containing the composition as claimed in any one of claims 1 to 30 and 33.

40. A kitset for use or when used in a method of preventing a new intramammary infection or mastitis in a cow, wherein the kitset comprises: a syringe capable of administering to at least one teat of a cow the composition as claimed in any one of claims 1 to 30 and 33.

41. The teat sealant composition of claim 34 or 35, the use of claim 36 or 38, the method of claim 37, or the kitset of claim 40, wherein the composition is used in a milking cow or heifer.

42. The teat sealant composition of claim 34, 35 or 41, the use of claim 36, 38 or 41, the method of claim 37 or 41, or the kitset of claim 40 or 41, wherein: - the composition prevents a new intramammary infection or mastitis for a period of at least about 14 days, about 42 to about 90 days, or up to about 4 months;- occlusion by the composition is for at least approximately the first 14 days of a dry period until a natural keratin plug develops; - the composition occludes the teat canal and / or teat cistern or substantially occludes a teat canal and / or teat cistern until a natural keratin plug has formed in the teat canal; - the composition protects a teat or teats of the cow from bacterial infection during an entire dry period; - the composition is administered per quarter at the end of lactation; - the composition is administered per quarter in maiden heifers approximately 4 weeks prior to calving; - the teat sealant composition has an adequately low viscosity so as to allow it to be administered via a teat canal and / or teat cistern by way of injection; - approximately 2-5 g of the teat sealant composition is administered / injected into a teat canal and / or teat cistern; - a syringeability force for injecting the teat sealant composition into a teat does not exceed 4500 g at 25°C, and preferably the syringeability force for injecting the teat sealant composition into a teat is between about 1500 g and 3500 g at 25°C; - the teat sealant composition thickens rapidly in a teat so as to be retained and occlude a teat canal and / or teat cistern; - once located within a teat canal and / or teat cistern, the teat sealant composition has a viscosity high enough so as to be retained within the teat canal or teat cistern and occlude the teat canal or teat cistern; - the teat sealant composition has a bounce-back viscosity enabling it to be retained within a teat canal and / or teat cistern as a substantially coherent mass; - the teat sealant composition has a rheology enabling the composition to be administered to a teat canal and / or teat cistern by way of injection, and to be retained within the teat canal and / or teat cistern and occlude the teat canal and / or teat cistern, or to prevent an intramammary infection; - the teat sealant composition shear-thins at the shearing forces applied during injection and stripping from a teat; or - the teat sealant composition occludes a teat canal and / or teat cistern or substantially occludes the teat canal and / or teat cistern for about 4-6 weeks, while allowing a natural keratin plug to develop.

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