Antimicrobial formulations for the improved treatment of veterinary hoof infections

Abstract

There is provided a composition comprising an organic material and at least a first surfactant which following addition of an aqueous solution to the composition can form a biphasic system, wherein the biphasic system is useful for the treatment of or prevention of hoof infections. Also provided is a biphasic system useful for the treatment of or prevention of hoof infections, wherein the biphasic system comprises the composition and an aqueous phase wherein the composition is dispersed as droplets within the aqueous phase.

Claims

1. A biphasic system comprising: a non-aqueous phase comprising: an organic material in the form of benzyl benzoate; and a surfactant in the form of polyethylene glycol dodecyl ether; and an aqueous phase comprising: a mixture comprising at least 60% formic acid, 10% to 30% acetic acid, and 3% to 7% ethanol; wherein the non-aqueous phase is dispersed as droplets within the aqueous phase and the biphasic system comprises an antimicrobial agent.

2. A composition capable of forming the biphasic system of claim 1 when mixed with an aqueous solution, the composition comprising: a. an organic material in the form of benzyl benzoate; b. a surfactant in the form of polyethylene glycol dodecyl ether; and c. a mixture comprising at least 60% formic acid, 10% to 30% acetic acid, and 3% to 7% ethanol; wherein the non-aqueous phase is dispersed as droplets within the aqueous phase and the composition comprises an antimicrobial agent; for treatment/prevention of hoof infections.

3. The composition as claimed in claim 2, wherein the composition comprises an aqueous solution at less than 90%.

4. The biphasic system as claimed in claim 1 wherein the antimicrobial agent is provided in the aqueous phase.

5. The biphasic system as claimed in claim 1 wherein the antimicrobial agent is provided in the non-aqueous phase.

6. The biphasic system as claimed in claim 1 for use in the treatment of hoof infections.

7. The biphasic system as claimed in claim 1 wherein the non-aqueous phase droplets are of a size 0.05 μm to 100 μm.

8. A kit of parts comprising: a. the composition of claim 1; and b. an aqueous solution; and wherein the composition and aqueous solution are capable of being mixed to provide the biphasic system of claim 1.

9. The kit of parts as claimed in claim 8 wherein the composition and aqueous solution are provided in amounts such that when mixed the aqueous solution is provided at 90% or more.

10. The kit of parts as claimed in claim 8, for use in the treatment of hoof infections.

11. A method of treating a hoof infection comprising the step of providing the biphasic system of claim 1 to an area to be treated on an animal in need thereof.

12. The method of claim 11, wherein the area to be treated is the rugae of a hoof.

13. The composition of claim 1 wherein, a hoof infection is selected from at least one of papillomatous digital dermatitis (PDD), interdigital dermatitis (stable hoof rot), interdigital phlegmon (hoof rot), laminitis, white line disease, and heel erosion.

14. The biphasic system of claim 1 wherein, a hoof infection is selected from at least one of papillomatous digital dermatitis (PDD), interdigital dermatitis (stable hoof rot), interdigital phlegmon (hoof rot), laminitis, white line disease, and heel erosion.

15. The method of claim 11 wherein, a hoof infection is selected from at least one of papillomatous digital dermatitis (PDD), interdigital dermatitis (stable hoof rot), interdigital phlegmon (hoof rot), laminitis, white line disease, and heel erosion.

Description

EXAMPLES OF FORMULATIONS

Example One

(1) A solution was provided that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™ [Formic Acid 60% to 100%, Acetic Acid 10% to 30% and Ethanol 3% to 7%]) and subsequent dilution with water.

(2) The biphasic system showed enhanced antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997.

(3) In particular a composition or predominately organic phase of the present invention comprising

(4) TABLE-US-00005 TABLE 1 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 38.8 Procetyl ® (alkoxylated cetyl alcohol) 10.4 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Isopropyl myristate 8.8 Blue liquid colour 0.22 Water 10.18
was mixed with the commercial product Hoofsure Endurance™ at a volume ratio of 1:9 and diluted with water to provide a final concentration of such embodiment of 0.375% v/v.

(5) In the final dilution, the provided components of a composition can be as listed below:

(6) Polyoxyethylene (4) lauryl ether, Propylene glycol-5-cethyl alcohol-20, polysorbate 80, Denatured Alcohol, Formic acid, Acetic acid, Isopropyl myristate, Water, Blue liquid colour, Benzoic acid, and oil of tea tree.

(7) The antimicrobial activity of this mixture, under both clean and dirty conditions (The clean condition doesn't have any bovine albumin in the tested sample, but the dirty condition contains 0.3% bovine albumin) is shown in table 2 below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(8) TABLE-US-00006 TABLE 2 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 1 Clean.sup.a 1 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 2.8 × 10.sup.6 1.5 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30 4.5 × 10.sup.4 3.7 × 10.sup.4 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 0 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

Example Two

(9) A further solution was provided that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™) and subsequent dilution with water and which shows enhanced antimicrobial activity both antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997.

(10) In the solution used in Example 2 Procetyl is replaced with the non-ionic surfactant Brij® 58 (Polyethylene glycol hexadecyl ether).

(11) TABLE-US-00007 TABLE 3 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 38.8 Brij ® 58 10.4 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Isopropyl myristate 8.8 Blue liquid colour 0.22 Water 10.18

(12) The solution (example 2) is mixed with the commercial product Hoofsure Endurance™ (1:9) and diluted with water to provide final concentration of 0.375% v/v.

(13) The antimicrobial activity of this mixture, under both clean and dirty conditions is shown in table 4 below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(14) TABLE-US-00008 TABLE 4 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 2 Clean.sup.a 2 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 6.8 × 10.sup.6 6.5 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30 4.2 × 10.sup.4 1.5 × 10.sup.4 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 0 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

Example Three

(15) A solution was provided that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™) and subsequent dilution with water and which shows enhanced antimicrobial activity both antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997. Example three illustrates the replacement of isopropyl myristate as the organic phase with ethyl ethanoate.

(16) TABLE-US-00009 TABLE 5 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 38.8 Procetyl ® 10.4 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Ethyl ethanoate 8.8 Blue liquid colour 0.22 Water 10.18

(17) The solution (example 3) is mixed with the commercial product Hoofsure Endurance™ (1:9) and diluted with water to provide final concentration of 0.375% v/v.

(18) The antimicrobial activity of this mixture, under both clean and dirty conditions is shown in the table below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(19) TABLE-US-00010 TABLE 6 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 3 Clean.sup.a 3 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 2.8 × 10.sup.6 1.8 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30 3.7 × 10.sup.4 2.2 × 10.sup.5 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 3.7 × 10.sup.3 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

Example Four

(20) A solution was provided that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™) and subsequent dilution with water and which shows enhanced antimicrobial activity both antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997. Example four illustrates the replacement of isopropyl myristate as the organic phase with sunflower oil.

(21) TABLE-US-00011 TABLE 7 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 38.8 Procetyl ® 10.4 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Sunflower oil 0.5 Blue liquid colour 0.22 Water 18.48

(22) The solution (example 4) is mixed with the commercial product Hoofsure Endurance™ (1:9) and diluted with water to provide final concentration of 0.375% v/v.

(23) The antimicrobial activity of this mixture, under both clean and dirty conditions is shown in the table below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(24) TABLE-US-00012 TABLE 8 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 4 Clean.sup.a 4 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 2.8 × 10.sup.6   3 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30 8.3 × 10.sup.3 1.7 × 10.sup.2 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 0 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

Example Five

(25) A solution was provided that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™) and subsequent dilution with water and which shows enhanced antimicrobial activity both antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997. Example five illustrates the replacement of isopropyl myristate as the organic phase with mineral oil.

(26) TABLE-US-00013 TABLE 9 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 38.8 Procetyl ® 10.4 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Mineral oil 0.5 Blue liquid colour 0.22 Water 18.48

(27) The solution (example 5) is mixed with the commercial product Hoofsure Endurance (1:9) and diluted with water to provide final concentration of 0.375% v/v.

(28) The antimicrobial activity of this mixture, under both clean and dirty conditions is shown in the table below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(29) TABLE-US-00014 TABLE 10 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 5 Clean.sup.a 5 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 2.8 × 10.sup.6 3 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30 1.7 × 10.sup.4 3 × 10.sup.3 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 0 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

Example Six

(30) A solution was provided that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™) and subsequent dilution with water and which shows enhanced antimicrobial activity both antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997. Example six illustrates the replacement of isopropyl myristate as the organic phase with tea tree oil.

(31) TABLE-US-00015 TABLE 11 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 38.8 Procetyl ® 10.4 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Tea Tree oil 0.5 Blue liquid colour 0.22 Water 18.48

(32) The solution (example 6) is mixed with the commercial product Hoofsure Endurance™ (1:9) and diluted with water to provide final concentration of 0.375% v/v.

(33) The antimicrobial activity of this mixture, under both clean and dirty conditions is shown in the table below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(34) TABLE-US-00016 TABLE 12 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 6 Clean.sup.a 6 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 2.8 × 10.sup.6 6.8 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30 7.0 × 10.sup.4 5.3 × 10.sup.5 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 0 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

Example Seven

(35) A solution was provided that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™) and subsequent dilution with water and which shows enhanced antimicrobial activity both antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997. Example seven illustrates the replacement of isopropyl myristate as the organic phase with thyme oil.

(36) TABLE-US-00017 TABLE 13 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 38.8 Procetyl ® 10.4 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Thyme oil 0.5 Blue liquid colour 0.22 Water 18.48

(37) The solution (example 7) is mixed with the commercial product Hoofsure Endurance™ (1:9) and diluted with water to provide final concentration of 0.375% v/v.

(38) The antimicrobial activity of this mixture, under both clean and dirty conditions is shown in the table below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(39) TABLE-US-00018 TABLE 14 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 7 Clean.sup.a 7 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 2.8 × 10.sup.6 6.5 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30 3.8 × 10.sup.4 1.8 × 10.sup.4 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 0 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

Example Eight

(40) A solution that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™) and subsequent dilution with water and which shows enhanced antimicrobial activity both antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997. Example eight illustrates the replacement of incorporation of the anionic surfactant, sodium dodecyl sulphate, into example one.

(41) TABLE-US-00019 TABLE 15 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 34.92 Procetyl ® 9.36 Sodium Dodecyl Sulphate 4.92 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Isopropyl myristate 8.8 Blue liquid colour 0.22 Water 10.18

(42) The solution (example 8) is mixed with the commercial product Hoofsure Endurance™ (1:9) and diluted with water to provide final concentration of 0.375% v/v.

(43) The antimicrobial activity of this mixture, under both clean and dirty conditions is shown in the table below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(44) TABLE-US-00020 TABLE 16 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 8 Clean.sup.a 8 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 2.8 × 10.sup.6   1 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30   6 × 10.sup.4 2.5 × 10.sup.6 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 6.2 × 10.sup.5 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

Example Nine

(45) A solution was provided that forms a biphasic system upon addition to a proprietary product (Hoofsure Endurance™) and subsequent dilution with water and which shows enhanced antimicrobial activity both antimicrobial activity both under clean and dirty (with the addition of 0.3% Bovine Serum Albumin) against Escherichia coli (NCTC12241). The rate of kill assay is based on the British and European standard test conditions BS EN 1276:1997. Example nine illustrates the replacement of incorporation of the cationic surfactant, benzalkonium chloride, into example one.

(46) TABLE-US-00021 TABLE 17 Component Concentration (% w/w) Brij ® L4 (also named Brij 30) 34.92 Procetyl ® 9.36 Benzalkonium chloride 4.92 Denatured ethanol 8.3 Formic acid 6.4 Acetic acid 16.9 Isopropyl myristate 8.8 Blue liquid colour 0.22 Water 10.18

(47) The solution (example 9) is mixed with the commercial product Hoofsure Endurance™ (1:9) and diluted with water to provide final concentration of 0.375% v/v.

(48) The antimicrobial activity of this mixture, under both clean and dirty conditions is shown in the table below. The initial inoculum of microorganism ranged between 10.sup.6-10.sup.7 colony forming units per mL. In addition, the comparative antimicrobial activities of Hoofsure Endurance™ (HSE) solution and copper sulphate (CS) solution, both at concentrations which are three-times their respective minimum inhibitory concentrations are shown.

(49) TABLE-US-00022 TABLE 18 Surviving Bacterial Count (cfu/mL) under clean and dirty conditions Contact Example Example HSE HSE CS Time (s) 9 Clean.sup.a 9 Dirty.sup.a Clean.sup.b Dirty.sup.b Clean.sup.c CS Dirty.sup.c 0 2.8 × 10.sup.6 1.0 × 10.sup.6 2.5 × 10.sup.6 6.5 × 10.sup.6 3.2 × 10.sup.6 1.4 × 10.sup.7 30 2.0 × 10.sup.4 4.0 × 10.sup.6 1.2 × 10.sup.6 6.5 × 10.sup.6 1.7 × 10.sup.6 1.1 × 10.sup.7 60 0 6.0 × 10.sup.5 2.3 × 10.sup.5 3.8 × 10.sup.6 6.2 × 10.sup.5 9.7 × 10.sup.6 120 0 0 5.0 × 10.sup.3 3.2 × 10.sup.5 3.3 × 10.sup.5 5.2 × 10.sup.6 300 0 0 0 0 5.0 × 10.sup.3 3.2 × 10.sup.4 .sup.aThe concentration was 0.375% v/v (3x MIC) .sup.bThe concentration was 0.375% v/v (3x MIC) .sup.cThe concentration of CS was 0.15% w/v (3x MIC)

(50) Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.