WO2012024324A1

WO2012024324A1 - System and method for delivering germicidal agent to animal hoof

Info

Publication number: WO2012024324A1
Application number: PCT/US2011/047968
Authority: WO
Inventors: Howard Marginson; Maria Buchalova; Alex Skender; Thomas Hemling
Original assignee: DeLaval Holding AB
Current assignee: DeLaval Holding AB
Priority date: 2010-08-16
Filing date: 2011-08-16
Publication date: 2012-02-23
Anticipated expiration: 2013-02-16

Abstract

Methods and systems for treating or disinfecting animal hooves are provided. Generally, a germicidal composition comprising a water-immiscible liquid and a germicidal agent is applied to the animal hooves such as through spraying or using a footbath. In those embodiments employing a footbath, the germicidal composition may be used in conjunction with an aqueous liquid thereby forming a multi-phase system. In the context of a footbath, the germicidal composition forms a water-immiscible phase which floats on top of an aqueous phase comprising the aqueous liquid. The water-immiscible phase containing the germicidal agent preferentially adheres to the animal's hoof thereby forming a protective film thereon. The multi-phase system can also take the form of an emulsion that is applied to the animal's hooves via direct spraying or a footbath.

Description

SYSTEM AND METHOD FOR DELIVERING GERMICIDAL

AGENT TO ANIMAL HOOF

RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Patent Application No. 61/374, 170, filed August 16, 2010, which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is directed toward a germicidal composition comprising a germicidal agent dispersed within a water-immiscible liquid. The germicidal composition is particularly suited for use in treating animal hoof diseases. The germicidal composition may be directly applied to the animal's hooves such as through a spraying operation. However, the composition may also be applied by way of a footbath in which the composition is used alone or as a part of a multi-phase treatment system. In one embodiment of the multi-phase system, the germicidal composition comprises a first phase which floats on top of a second aqueous phase. As the animal passes through the footbath, the germicidal composition forms a protective film on the animal's hooves which permits continued delivery of the germicidal agent to the animal well after the animal has passed through the foot bath. In another embodiment, the water-immiscible phase and an aqueous phase can form an emulsion which is then applied to the animal's hoof.

Description of the Prior Art

Footbaths are commonly employed to cleanse animal hooves prior to entry of the animal into an enclosed area, such as a milking parlor. In addition, footbaths can also be used to deliver a medicament to the animal's hooves in the treatment of various bacterial diseases afflicting animal hooves, such as hairy-warts and hoof-rot. Heretofore, footbaths have used aqueous solutions comprising germicidal agents, such as copper sulfate, as the vehicle for delivery of the medicament. Aqueous solutions are relatively easy to use, but have demonstrated certain drawbacks. As can be appreciated, animal hooves are quite often soiled with manure that will readily contaminate the footbath. In traditional aqueous footbaths, the manure becomes dispersed in the aqueous footbath solution and reduces or eliminates the efficacy of the germicidal agent. Also, contact time between the animal and the germicide is quite limited with existing footbath solutions. Animals will step through the footbath in a few seconds. Additionally, aqueous germicidal solutions are readily washed off the animal's hooves after leaving the footbath thereby lessening the effectiveness thereof.

SUMMARY OF THE INVENTION

In one embodiment according to the present invention, there is provided a method of treating or disinfecting an animal hoof comprising the steps of providing a footbath containing a liquid antimicrobial composition and causing the animal to walk through the footbath. The composition comprises an aqueous phase and a water-immiscible phase. The water-immiscible phase is less dense than the aqueous phase and comprises a water- immiscible liquid and a germicidal agent dispersed therein. The animal walks through the footbath so that at least a portion of the water-immiscible phase is transferred onto the animal's hoof thereby forming a protective film.

In another embodiment according to the present invention, there is provided a system for use in treating or disinfecting an animal hoof. The system comprises an animal footbath that includes a multi-phase liquid composition. The liquid composition comprises an aqueous phase and a water-immiscible phase, with the water-immiscible phase having a germicidal agent dispersed therein.

In yet another embodiment of the present invention, there is provided a method of treating or disinfecting an animal hoof comprising the step of applying to the animal hoof a composition comprising a water-immiscible liquid and a germicidal agent dispersed therein. In certain embodiments, the composition comprises an emulsion, the emulsion comprising the water-immiscible liquid and an aqueous liquid. The emulsion may be applied to the animal's hooves in a number of ways, including by spraying or by way of a footbath. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an illustration of a footbath system according to the present invention; and Fig. 2 is an illustration of the footbath system depicting the water-immiscible phase floating on top of the aqueous phase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is directed toward methods and systems for treating animal hooves through application of a germicidal composition that comprises a germicidal agent dispersed or solubilized in a water-immiscible liquid. The germicidal composition may be applied to animal hooves in a number of ways, such as by spraying the composition directly onto the animal hoof, or using the composition in a footbath.

In one aspect, the present invention is generally directed towards methods and systems for treating animal hooves using the germicidal composition as a part of a multi-phase footbath system. As used herein, the term "multi-phase" includes those systems comprising two or more phases (i.e., three, four, etc. phases). Such multi-phase systems generally comprise an aqueous phase and one or more water-immiscible phases which include the germicidal composition.

Figs. 1 and 2 depict an exemplary footbath system employing the present germicidal composition. Generally, a footbath 10 configured for an animal, such as a cow, horse, goat, or sheep, to walk through is provided. Footbath 10 contains the germicidal composition 14 as a water-immiscible phase which floats upon an aqueous phase 16. As the animal walks through the footbath, the water-immiscible phase 14, which comprises the germicidal agent, forms a protective film over at least a portion of the animal hoof. In particular embodiments, the water-immiscible phase 14 has lower interfacial energy with the animal hoof than the aqueous phase thereby permitting the water-immiscible phase 14 to preferentially adhere to the hoof.

In certain embodiments of multi-phase systems according to the present invention, the use of surfactants or co-solvents that would destroy the separation between the various phases should be avoided, as in these embodiments the presence of distinct phases present a number of advantages. As noted above, animal hooves often contain a number of organic soils, especially manure. As the animal walks through the footbath, the manure and other soils may be washed off the hoof and into the footbath. In entirely aqueous footbath systems, the manure can reacts with the germicidal agent reducing its efficacy or rendering it completely ineffective. In multi-phase systems according to the present invention, at least a portion of the germicidal agent is contained in the water-immiscible phase. Therefore, when the manure from the animal's hooves is washed off into the footbath, the germicidal agent is effectively shielded from the manure, as the manure is dispersed primarily into the aqueous phase.

Another advantage is that because the germicidal agent is protected from deactivation by the manure, methods according to the present invention can be performed with or without a pre-wash step for the animal's hooves.

As noted above, the water-immiscible phase comprising the germicidal agent forms a protective film on the animal's hooves. This protective film is less susceptible from being washed off the animal and thereby remains in contact with the animal's hooves for much longer periods of time compared to purely aqueous solutions. Once applied, the germicidal agent, over time, migrates from the protective film thereby providing extended delivery of the germicidal agent to the animal. This extended delivery can result in increased efficacy or the need for fewer footbath treatments for the animal. In certain embodiments, the protective film remains on the animal's hooves for at least 15 minutes, or at least 1 hour, or at least 2 hours.

The aqueous layer may contribute to the germicidal effect on the animal's hoof. In certain embodiments according to the present invention, it is necessary that in order to be delivered to the animal's tissue the germicidal agent dispersed within the water-immiscible phase migrate to the interface between the water-immiscible phase and the aqueous phase. The germicidal agent may then be delivered from the water-immiscible phase to the animal's skin by passage through this interface. In such case, the water-immiscible phase acts as a reservoir for the germicidal agent. Thus, a high concentration of active germicide in the water-immiscible phase acts as a "reservoir" to "buffer" the concentration of the biocidal ingredient contained in the aqueous phase as the concentration of biocidal ingredient in the aqueous phase is depleted by the animal's passage or deactivated by manure contamination. The biocidal ingredient is replenished by the "buffer" contained in the water-immiscible phase. To provide the desired benefit a least a portion of the germicide should remain the water-immiscible phase.

In certain embodiments the water-immiscible phase has the ability to displace the water or moisture from the hoof when applied due to its lower interfacial energy with the tissue. The typical site of digital dermatitis infection is more prone to continually being moist, and wet environmental conditions are extremely important in the development of the disease.

In another aspect, the germicidal composition can be provided as an emulsion comprising a water-immiscible liquid phase having a germicidal agent dispersed therein and an aqueous liquid phase. In certain embodiments, the emulsion comprises discrete droplets of the water-immiscible phase dispersed throughout the aqueous phase. In alternate embodiments, the emulsion may comprise discrete droplets of the aqueous phase dispersed throughout the water-immiscible phase.

Formation and application of the emulsion to animal hooves may occur according to a number of mechanisms. In one embodiment, the emulsion may be created by simple mixing, such as through mechanical agitation, of the water-immiscible phase and aqueous phase followed by spraying of the emulsion onto the animal hoof. In other embodiments, the emulsion may be delivered by way of a footbath. In certain of these embodiments, the emulsion may be prepared within the footbath itself, or it can be separately prepared and then added to the footbath. If a germicidal agent is selected that has surfactant-like properties, such as quaternary ammonium compounds, tertiary alkyl amine and diamine compounds, an emulsion can be created between the water-immiscible phase and aqueous phase simply by adding the water-immiscible phase to the aqueous phase already present in the footbath. A minimal amount of mixing or agitation may be required to get the emulsion to form; however, once formed the emulsion may be quite stable, for at least one day, or in other embodiments for at least two days at 25°C. In some embodiments, the emulsion may float on top of a portion of the non-emulsified aqueous phase present in the footbath. If the emulsion is separately formed and then added to the footbath, it too may float on top of the aqueous phase present in the footbath, or the emulsion may be used as substantially the only liquid in the footbath thereby avoiding the use of a separate, non-emulsified aqueous phase. In still other embodiments, the footbath may comprise up to four distinct phases including any combination of the following: a water-immiscible phase, an emulsion phase in which the water-immiscible liquid is dispersed in an aqueous liquid, an emulsion phase in which the aqueous liquid is dispersed in a water-immiscible liquid, and an aqueous phase. Various emulsifying agents may be used to assist formation of the emulsion, such as, for example, non-ionic, anionic, cationic or amphoteric surfactants. In some embodiments, the emulsion formed is not highly stable, in that if it is permitted to rest, it will rapidly separate out into discrete phases. For example, in certain embodiments if an emulsion sample having a depth of between 3 to 12 inches and comprising a volume ratio of the water-immiscible phase to the aqueous phase of between about 1 : 100 to 1 :5 is permitted to rest (i.e., mechanical agitation is removed or animals are prevented from walking through a footbath containing the emulsion), at least 90% of the emulsion will separate into their respective aqueous and water-immiscible phases within 30 seconds at 2Cft.

In yet another aspect, the multi-phase system can be formed in situ by spraying the water- immiscible phase comprising the germicidal agent directly onto the animal's wet hoof. In these embodiments, a pre-wetting step may be employed to ensure that the animal's hooves are adequately moistened.

As noted above, the water-immiscible liquid comprises a germicidal agent. The germicidal agent may be present in the water-immiscible liquid at a level of between about 0.05% to about 100% (100% representing the use of a water-immiscible germicide, such as a water-immiscible fatty acid as the only component of the water-immiscible liquid), or between about 0.05 to about 49.9% by weight, or between about 0.05% to about 30% by weight, or between about 0.075% to about 20% by weight, or between about 0.1% to about 10% by weight. The germicidal composition, or water-immiscible phase as the case may be, presents a kinematic viscosity at 2dt of between about 0.5 to about 50 cSt, or between about 1 to about 30 cSt, or between about 1.1 to about 10 cSt. In particular embodiments, the germicidal composition, or water-immiscible phase, has a specific gravity of less than 1 , or between about 0.75 to about 0.95. In certain embodiments, the water-immiscible liquid has a flash point of at least 37.7t, or at least 93. t.

As discussed above, in certain embodiments the water-immiscible liquid is insoluble with water under normal usage conditions of the multi-phase system. Therefore, in order to assist in the clean up and disposal of spilled or unused water-immiscible germicidal composition, the water-immiscible liquid can, in certain embodiments, be rendered water soluble when desired. For example, the water-immiscible liquid can be rendered water soluble by addition of an acid, base, salt, or surfactant.

The water-immiscible liquid may comprise a polar or non-polar solvent. As used herein, "water-immiscible" means having a water solubility of less than 0.1% by weight at 25°C, or, in some embodiments less than 0.01% by weight at 25°C. In certain embodiments, the water-immiscible liquid is non-antimicrobial or substantially non-antimicrobial. By "non- antimicrobial or "substantially non-antimicrobial" it is meant that the liquid results in less than a 90% reduction (less than a 1-log order reduction) in the population of bacteria or spores of Bacillus species within 10 seconds at 60°C using the Germicidal and Detergent Sanitizing Action of Disinfectants, Official Methods of Analysis of the Association of Official Analytical Chemists, paragraph 960.09 and applicable sections, 15^th Edition, 1990 (EPA Guideline 91 -2). Thus, in these embodiments, the water-immiscible liquid is inert, and a separate germicidal component must be added in order to achieve the desired germicidal effects. Further, in certain applications it is desirable for the water-immiscible liquid to be less dense than the aqueous phase so that the water-immiscible liquid floats on top of the aqueous phase.

In particular embodiments, the water-immiscible liquid comprises a member selected from the group consisting of comprises a member selected from the group consisting of petroleum or mineral oils, vegetable oils (such as peanut and soya oil), polybutene, fatty alcohols (such as those having a carbon number of greater than 4), fatty acids (such as those having a carbon number greater than 4), methyl esters of fatty acids (such as methyl soyate, or CITRUSOY soy methyl ester available from Florida Chemical, Co.), methyl esters of triglycerides, silicon oil, d-limonene, substantially water-insoluble or lipophilic surfactants (such as defoaming, non-ionic surfactants, and low HLB surfactants in the range of 1-5), polypropylene glycols, water-insoluble glycol ethers, terpene oils (such as pine oil), biodiesel, water-insoluble anionic materials, olefins (such as hydrotreated isoparaffins and naphthenes including LPA 210 from Sasol and Exxsol D100 from Exxon), and combinations thereof. The water-immiscible liquid may also comprise certain materials that may be water miscible, but can be made water immiscible upon the addition of a salt, other electrolyte, acid or base. Examples of these types of materials include certain fatty acids, fatty amines, alkyl sulfates, alkylsulfonates polyacrylates, polycarboxylates, and polyamines. In certain embodiments, the water-immiscible liquid comprises a mixture of two or more water-immiscible fluids. In such embodiments, the primary or predominant water-immiscible fluid is present at a level of between about 25% to about 95% by weight, between about 40% to about 90% by weight, or between about 50% to about 80% by weight. The secondary, tertiary, etc. water immiscible liquids may be present at levels of between about 0.1% to about 45% by weight, between about 1% to about 30% by weight, or between about 5% to about 25% by weight. The secondary or tertiary water immiscible liquid may be incorporated for a variety of purposes including improving the solubility of the germicidal agent in the water immiscible phase, changing the partitioning of the germicidal agent between water the water immiscible phase, changing the viscosity or spreading properties of the water immiscible phase.

The germicidal agent used with the present invention can be any germicidal agent capable of being dispersed and remaining dispersed within the water-immiscible liquid for extended periods of time. In certain embodiments, the germicidal agent remains dispersed within the water-immiscible liquid for at least 3 months, or at least 6 months, or at least 1 year upon storage at 2$C. In still other embodiments, the germicidal agent is a solid at room temperature and is dissolved or dispersed in the water-immiscible liquid. In yet other embodiments, the germicidal agent can be any antimicrobial or sporicidal composition having the ability to achieve greater than a 99% reduction (2-log order reduction), greater than a 99.99% reduction (4-log order reduction), or greater than a 99.999%) reduction (5-log order reduction) of Bacillus species within 10 seconds at 60°C, using the above-mentioned Germicidal and Detergent Sanitizing Action of Disinfectants procedure. The germicidal agent may comprise a member selected from the group consisting of phenol derivatives, cationic germicidal agents, organic acids, oxidizing agents, halogen- containing compounds, essential oils, polychloro phenoxy phenols, imidazole compounds, polymeric reaction products of formaldehyde and acrolein, benzyl alcohols, iodine, iodine-containing compounds, pyrithione compounds, and combinations thereof. Exemplary phenol derivatives include chlorocresol and biphenyl-2-ol. Exemplary cationic germicidal agents include various quaternary ammonium compounds such as didecyl dimethyl ammonium chloride (DDAC), chlorhexidiene gluconate, and polyhexamethylene biguanide (PHMB). It is noted that certain cationic germicidal agents may normally be water soluble, however, alternative anions may be added to these compounds in order to render them soluble in the water- immiscible liquid. Exemplary organic acids include benzoic acid, salicylic acid, and various C7-C18 saturated or unsaturated fatty acids. Exemplary oxidizing agents include 6- (phthalimido)peroxyhexanoic acid (PAP), calcium hypochlorite, and oil-soluble peroxides such as 2-butanone peroxide. An exemplary polychloro phenoxy phenol is triclosan. An exemplary imidazole compound includes imazalil. Exemplary pyrithione compounds include omadine compounds. Exemplary essential oils include orange oil, terpene oil, and d- limonene. Chlorophene is an exemplary halogen-containing compound. In certain embodiments, the germicidal agents tend to migrate to the water-oil interface and distribute between the two phases. In particular embodiments, the germicide distributes between the two phases so there is a sufficient concentration in the aqueous phase to have a desired germicidal efficacy (bacteria log reduction of at least 5), but enough of the germicide remains in the water-immiscible phase to be deposited on the hoof. The germicidal agent may have a partition co-efficient, D (molar concentration in water-immiscible phase/molar concentration in aqueous phase), that is greater than 1, or at least 5, or between about 1 to about 300, between about 1.5 to about 250, between about 2 to about 200, between about 3 to about 150, between about 5 to about 100, or between about 10-50.

The water-immiscible germicidal composition may optionally include a number of additives such as adjuvants and coloring agents. Exemplary adjuvants which enhance skin penetration include menthol, limonene, linalool, cineole, nerolidol, farnesol, geraniol, carvone, terpiniolene, ascaridole, other terpenes, and essential oils. In certain embodiments, coloring agents can be added to the germicidal composition so as to provide a visual indicator of the presence of the germicidal composition in the footbath and, subsequently, on the animals hooves. In particular embodiments, the coloring agent can be any food-grade, or non-toxic coloring agent that is soluble in the water-immiscible liquid, but not soluble, or only sparingly soluble, in the aqueous phase. Other adjuvants can include viscosity modifying additives, viscosity index ingredients, tackifying agents, and antioxidants for water-immiscible phase stability, such as ascorbyl palmitate, tocopherols, and cathechin.

In certain embodiments of the multi-phase systems described above, the germicidal agent present in the water-immiscible liquid may migrate toward the interface between the water-immiscible phase and the aqueous phase so that it ultimately can be delivered to the animal's tissue. Thus, even prior to application to the animal, a portion of the germicidal agent may migrate from the water-immiscible phase into the aqueous phase thereby imparting some anti-microbial effect to the aqueous phase so as to counteract the deleterious effects of manure introduced into the system from the animal's hooves. Alternatively, or in addition, a further biocide may be added to the aqueous phase to enhance the germicidal efficacy of the system, or again, to counteract the effects of manure introduced into the system. These biocides include but are not limited to any water soluble biocides such as quaternary ammonium salts, formaldehyde, glutaraldehyde, peroxides, water soluble organic acids, metal salts such of copper or zinc and chlorhexidine. The aqueous phase may also comprise a water-soluble salt such as NaCl to regulate the distribution of ingredients within the multiple- phase system.

Additional materials may also be added to either or both of the water-immiscible or aqueous phases to improve the properties thereof. For example, phase transfer catalysts may be employed to transfer the germicidal agent from the aqueous phase to the water-immiscible phase. For example, quaternary ammonium compounds may be added to the system so as to effect transfer of peroxide anions from the aqueous phase to the water immiscible phase. Deflocculant compounds may be added to assist in manure separation. Exemplary deflocculants include polyvalent ions such as iron sulfate and aluminum compounds. Water displacing chemicals such as silicones may also be added. Cleaning agents such as sodium tripolyphosphate (STPP) may be added to assist with hoof cleaning. Chelating agents may be added to enhance germicidal efficacy for certain germicides and biocides present in the aqueous phase by binding iron.

Turning again to the figures, one aspect of the present invention, namely a footbath system employing the germicidal compositions described above, is shown and hereunder described in further detail and with reference to one or more specific embodiments. This description is by way of example and nothing contained herein should be viewed as limiting the scope of the present invention. As depicted in Figs. 1 and 2, footbath 10 capable of holding approximately 50-150 gallons of liquid is positioned in a location such that a cow, or any other animal that is desired to be treated, can be directed to walk therethrough. A two- phase liquid composition 12 is placed in footbath 10 comprising a water-immiscible phase 14 and an aqueous phase 16. The water-immiscible phase 14 is less dense than the aqueous phase 16 and as a result floats on top of the aqueous phase 16. In certain embodiments, the aqueous phase 16 is present in a greater amount than the water-immiscible phase 14. It is understood that as animals walk through the footbath 10, the liquid contained in the footbath may be sloshed and spilled outside of the footbath. However, in particular embodiments, the water-immiscible phase within the footbath during treatment of the animal is maintained within the range of between about 1 % to about 50%, or between about 1% to about 10%, or between about 1 % to about 5%, based on the weight of the entire footbath. Alternatively, the water-immiscible phase maintained within the footbath can be expressed as a ratio of the water-immiscible phase to the aqueous phase. In certain embodiments, the ratio of the water- immiscible phase to the aqueous phase is between about 1 : 100 to about 100: 1 , about 1 :50 to about 10: 1, about 1 :30 to about 2: 1, about 1 :20 to about 1 : 1 , about 1 : 10 to about 1 : 1.5, or about 1 :5 to about 1 :2. In even further embodiments, the water-immiscible phase 14 should be present within the footbath 10 in such quantity so that at least 75%, or at least 85%, or substantially the entire uppermost surface of the aqueous layer 16 (i.e., that portion of the aqueous layer forming the interface with the water-immiscible phase) is covered by the water- immiscible phase 14 when the liquid in the footbath is at rest.

The use of the germicidal compositions described herein in a footbath system provides an added benefit to the farmer in that no mixing of components is required. Simply, the germicidal composition may be poured or pumped into the footbath. In certain embodiments, the two-phase composition is intrinsically unstable and the aqueous phase and water- immiscible phase separate into discrete layers nearly instantaneously. Thus, in these embodiments it is desirable for the footbath liquid to be applied to the animal's hooves as separate phases, rather than being applied as a single phase or a pseudo-stable mixture. Thus, even upon agitation, such as caused by the passage of an animal through the footbath, the water-immiscible phase immediately substantially separates (i.e., in less than 10 seconds, or even less than 5 seconds) from the aqueous phase after the animal's passage.

In certain embodiments, an automatic dosing system can be used to replenish the water-immiscible phase as it is depleted. This dosing system can be activated following passage of one or more animals through the footbath. The dosing system may simply pump an operator- determined amount of the water-immiscible phase onto the surface of the bath. In particular embodiments, such automatic dosing is made at the entrance end of the bath. The use of an automatic dosing system can help ensure that a sufficient quantity of the water- immiscible phase is present in the footbath so as to provide adequate coverage of the water- immiscible phase on the animal's hooves.

In certain embodiments according to the present invention, it may be desirable to control the passage of animals through the footbath such that the liquid contained in the footbath may be in as less of an agitated state as possible before the next animal walks through. Allowing the waves of liquid within the footbath, due to the animal's movement, to settle permits the water-immiscible layer to spread out over the upper surface of the aqueous layer and avoid "pooling" in corners or isolated regions of the footbath.

Manure present on the animal's hoof is largely immiscible with the water-immiscible phase of the footbath, and if removed while walking through the footbath, is largely confined to the aqueous phase thereby preserving the activity of the germicidal agent contained within the water-immiscible phase.

Example 1

Table 1 illustrates exemplary germicidal composition which comprise a water- immiscible liquid in accordance with the present invention, and the germicidal efficacy thereof using a Modified EN 1040 and EN 1656 micro testing procedure in which the samples were mixed with bacteria media at the volume ratio 1 : 19, with the solutions continuously mixed during contact time. The tests in the presence of the manure were performed using a modified AOAC 960.09 sanitizer test. According to this test, manure was collected from a dairy farm, dispensed into Erlenmeyer flasks, homogenized and autoclaved at 121°C for 30 minutes. Manure samples were then stored in the freezer until needed. For testing, the manure samples were thawed to room temperature, and dispensed into testing tubes for desired challenge. The ratio of water immiscible phase and the combined bacteria challenge with manure and buffer was 1 : 19 by volume. The test was performed at room temperature.

Table 1

Ingredient % weight % weight % weight % weight % weight

Chlorocresol 3 5 6

Benzoic acid 1

Salicylic acid 0.36

Canola Oil 10 10

Peanut Oil 10

D Limonene 2

Soybean Oil 10 10

LPA 210 87 85 82 89 89.64

Total amount 100 100 100 100 100

Germicial efficacy test: EN 1040 and EN 1656, 3( ) min contact time at 10°C, and AOAC

960.09, 30 min contact time at room temperature;

results in Log reduction from bacteria count 10⁸

P. Aeruginosa 2.7 6.8 5.4* 4.5 5.4

E. Hirae 4.9 6.9 5.1 * 5.6 6.4

P. Hauseri 6.1 6.8 5.8* 4.8 5.4

S. Aureus 6.3 6.3 5.3* 6.7 6.7 E. Coli under 20% ** 6.6 6.6 3.2 3.1 manure challenge at

room temperature

S. Aureus under 20% ** 6.3 6.3 5.7 4.7 manure challenge at

room temperature

* EN 1656, high soil

** AOAC 960.09

Table 2 sets forth various germicidal, water-immiscible liquid compositions in accordance with the present invention. Particularly, the table reports various physical characteristics of water-immiscible compositions wherein the germicidal agent is benzoic acid or salicylic acid. The solubility of the germicidal agent in the water-immiscible liquid (oil) is given, along with partitioning data for the germicidal agent.

Table 2

* D values measured in acidified 300ppm hard water and specified oil system

Example 2

The germicidal agent present in water immiscible phase may be present at various concentrations. The maximum attainable concentration of germicidal agent in the water immiscible phase is largely dependent on the composition of the water immiscible phase and may be tailored to desired requirements by using different mixtures of water immiscible solvents. Table 3 reports the solubility characteristics of the germicidal agent chlorocresol in different water-immiscible phase formulations. The table also includes the data on the partition coefficients (D) of germicidal active between the aqueous hard water and the corresponding water immiscible phase.

Table 3: Solubility and Partition Coefficient Data for Chiorocresol in Various Solvents

In vitro efficacy of two-phase systems employing chlorocresol as the germicidal agent was tested using a modified EN1656 test method, the EN1656 test method being incorporated by reference herein. The modification was necessary due to the presence of oil phase (volume ratio at 1 part of oil : 19 parts of aqueous solution), and required continuous mixing of the solution during the contact time with bacteria. Additional modification in some instances included the use of MPN method for quantification of bacteria instead of plating on agar plate. The test was run under clean conditions as well as heavy soil conditions in the presence of albumin and yeast extract interfering substances as described in the EN 1656 test method. Contact time was 30 minutes, and contact temperature was 10°C. The additional in vitro efficacy under manure soil conditions was tested using the AOAC 960.09 method, incorporated by reference herein.

Table 4 presents the results of the modified EN 1656 test for chlorocresol showing the effect of the composition of the water immiscible solvent. A higher partition coefficient (D) indicates a system with a lower concentration of chlorocresol in the aqueous phase. Under identical conditions, systems with a D value of 7 and 14 are able to pass the requirements of EN1656 test, (log reduction>5) whereas systems with D of 28 and 58 do not pass the requirements for all organisms. It is suspected that the results of these in vitro tests may be enhanced by higher distribution of germicide into the aqueous phase. However, it is believed that a higher distribution of the germicide into the water-immiscible solvent (i.e., a higher D value) will provide greater germicidal performance in clinical applications.

Table 4: Effect of water immiscible media

Table 5 presents the results of modified EN 1656 test method showing the effect of the concentration of chlorocresol in water-immiscible phase. The concentration of chlorocresol was varied from 3 to 6 weight %. The oi aqueous volume ratio was 1 : 19, contact time was 30 minutes, and temperature was 10°C.

Table 6 presents the data of the AOAC 960.09 test in the presence of manure. The test was performed at ambient temperature (RT) or 10°C; and the manure challenge was modified from 10% up to 20% of the total testing volume. Log reduction >5 was achieved for all tested solutions, even in the presence of 20 % of manure.

Table 5: Results of Modified EN 1656 Method Showing Effect of Chlorocresol Concentration

Table 6: Results of AOAC 960.09 Method in Presence of Manure

Additional testing was performed under the modified EN 1656 test when the volume ratio of aqueous phase and water immiscible solvent was 2:8. The concentration of chlorocresol was held constant between the trials. The contact time for this testing was 30 minutes, and the temperature was 10°C. The results of this testing are shown in Table 7. Under these conditions all solutions show >5 log reduction for all tested organisms.

Table 8 presents the results of modified EN 1656 testing for varying amounts of chlorcresol at aqueous phase:oil volume ratio of 2:8. The contact time for this testing was 30 minutes, and the temperature was 10°C. The test was performed under clean and heavy soil conditions.

Table 7: Results of Modified EN 1656 Method for Aqueous:Oil Phase

Table 8: Results of Modified EN 1656 Method for Varying Chlorocresol Concentrations for Aqueous:Oil Phase

Example 3

In this example, salicylic acid was used as the germicidal agent and tested with different water-immiscible solvents. Table 9 below shows examples of Salicylic acid solubility in different composition solvents. The partition coefficient (D) was measured using acidified hard water (0.8% w/w HCl). Various two-phase compositions comprising salicylic acid were tested under the modified EN 1656 test as previously described. The tests were conducted at an oihaqueous phase volume ratio of 1 : 19, for a contact time of 30 minutes, at a temperature of 10°C. The results of these tests are provided in Table 10.

Table 9: Solubility and Partition Coefficient Data for Salicylic Acid in Various Solvents

Table 10: Results of Modified EN 1656 Method for Salicylic Acid

A salicylic acid-containing formulation was tested according to the modified AOAC 960.09 test in the presence of manure. The tests were conducted at room temperature and at an oikaqueous phase volume ratio of 1 : 19. Contact time was varied at either 5 or 30 minutes. The results of these tests are provided in Table 1 1.

Table 1 1

Example 4

The experimental formula of chlorocresol from Example 2, 6% chlorocresol, 25% soybean oil, 2% pine oil, LPA was tested on a commercial dairy farm. A commercially available product "4HOOVES," a quaternary ammonium-based composition from DeLaval, was used as the control for the study. 10 liters of above formula was poured in a hoofbath filled with 190 L of water. Cows walked through the hoofbath as they were leaving the milking parlor. A fresh hoofbath was prepared every day for 5 days a week for 2 months. At the start of the trial and every two weeks during the trial the hind hooves were scored and scores were recorded by veterinarian. The results of the trial are presented below. Table 12 provides the observed results of disease prevention, and Table 13 provides the observed results of disease cure. The experimental formula exhibited statistically similar preventive and curative properties indicating that good results can be obtained through a two-phase delivery system.

Table 12: Preventive Properties (P = 0.21)

Treatment Cows (n) Foot parts (n) Prevention

N %

4HOOVES 123 402 368 91.5

Two-phase 68 228 215 94.3

Total 265 630 583

Table 13 : Curative properties (P

The following table summarizes various germicidal compositions for use in multiphase systems according to certain embodiments of the present invention.

Table 14

Table 15 summarizes certain germicidal agents for use in particular embodiments of the present invention. The table sets forth ranges for the amount of each germicidal agent as a portion of the water-immiscible phase, and target D values for the combined multiphase system. It is contemplated that individual germicidal agents or mixtures of more than one germicidal agent may be used. Table 15

Germicidal agent Concentrations in D Values

water-immiscible

phase (as formulated)

Broad First Second Narrow

Intermediate Intermediate

Chlorocresol 0.5% - 45% >1 5 - 100 10-75 20-50

Chlorophene 0.01% -30% >1 5 - 100 10-75 20-50

Biphenyl-2-ol 0.05 %- 15% >1 5 - 100 10-75 20-50

DDAC 0.02% - 30 % >1 5 - 100 10-75 20-50

Chlorhexidine 0.02%- 10% >1 5 - 100 10-75 20-50

PHMB 0.2%- 15% >1 5 - 100 10-75 20-50

Benzoic acid 0.1%- 10% >1 5 - 100 10-75 20-50

Salicylic acid 0.1%- 10% >1 5 - 100 10-75 20-50

Triclosan 0.5% - 30% >1 5 - 100 10-75 20-50

Lonzabac 0.5% - 40% >1 5 - 100 10-75 20-50

C7- CI 8 fatty 5 - 100 10-75 20-50 acids 0.05% -49% >1

Iodine 0.1%- 15% >1 5 - 100 10-75 20-50

Phenoxyethanol 0.1% -20% >1 5 - 100 10-75 20-50

Essential oils 0.5% - 49% >1 5 - 100 10-75 20-50

Claims

We claim:

1. A method of treating an animal hoof comprising:

providing a footbath containing a liquid antimicrobial composition, said composition comprising an aqueous phase and a water-immiscible phase, said water- immiscible phase being less dense than said aqueous phase and comprising a water-immiscible liquid and a germicidal agent dispersed therein; causing said animal to walk through said footbath so that at least a portion of said water-immiscible phase is transferred onto said animal hoof thereby forming a protective film.

2. The method according to claim 1, wherein said animal hoof has not undergone a pre-washing step prior to said animal walking through said footbath.

3. The method according to claim 2, wherein said animal hoof, prior to passage through said footbath, comprises a manure soil.

4. The method according to any of claims 1 -3, wherein said aqueous phase comprises a biocide.

5. The method according to claim 4, wherein said biocide is different from said germicidal agent.

6. The method according to any of claims l -5,wherein said water- immiscible liquid comprises a member selected from the group consisting of petroleum or mineral oils, vegetable oils, polybutene, fatty alcohols, fatty acids, methyl esters of fatty acids, methyl esters of triglycerides, silicon oil, d-limonene, substantially water-insoluble or lipophilic surfactants, polypropylene glycols, water-insoluble glycol ethers, terpene oils, biodiesel, water-insoluble anionic materials, olefins, and combinations thereof.

7. The method according to any of claims 1 -5, wherein said water- immiscible liquid comprises one or more non-polar solvents.

8. The method according to any of claims 1 -7, wherein said germicidal agent comprises a member selected from the group consisting of phenol derivatives, cationic germicidal agents, organic acids, oxidizing agents, halogen-containing compounds, essential oils, polychloro phenoxy phenols, imidazole compounds, polymeric reaction products of formaldehyde and acrolein, benzyl alcohols, iodine, iodine-containing compounds, pyrithione compounds and combinations thereof.

9. The method according to any of claims 1-8, wherein said germicidal agent disperses between said water-immiscible phase and said aqueous phase in said footbath, said germicidal agent having a partition co-efficient, D (molar concentration in water- immiscible phase/molar concentration in aqueous phase), that is at least 5.

10. The method according to any of claims 1-9, wherein at least a portion of said water-immiscible phase and at least a portion of said aqueous phase form an emulsion within said footbath.

1 1. The method according to claim 10, wherein said emulsion floats on top of a non-emulsified portion of said aqueous phase.

12. The method according to claim 10, wherein said emulsion is formed by agitation of said water-immiscible phase and said aqueous phase within said footbath.

13. The method according to any of claims 1 -12, wherein said water- immiscible liquid is substantially non-germicidal.

14. A system for use in treating an animal hoof comprising: an animal footbath including a multi-phase liquid composition, said liquid composition comprising an aqueous phase and a water-immiscible phase, said water-immiscible phase having a germicidal agent dispersed therein.

15. The system according to claim 14, said water-immiscible phase being less dense than said aqueous phase.

16. The system according to claim 14, wherein said water-immiscible liquid comprises a member selected from the group consisting of petroleum or mineral oils, vegetable oils, polybutene, fatty alcohols, fatty acids, methyl esters of fatty acids, methyl esters of triglycerides, silicon oil, d-limonene, substantially water-insoluble or lipophilic surfactants, polypropylene glycols, water-insoluble glycol ethers, terpene oils, biodiesel, water-insoluble anionic materials, olefins, and combinations thereof.

17. The system according to claims 14 or 15, wherein said water- immiscible liquid comprises one or more non-polar solvents.

18. The system according to any of claims 14 to 17, wherein said germicidal agent comprises a member selected from the group consisting of phenol derivatives, cationic germicidal agents, organic acids, oxidizing agents, halogen containing compounds, essential oils, polychloro phenoxy phenols, imidazole compounds, polymeric reaction products of formaldehyde and acrolein, benzyl alcohols, iodine, pyridinethione compounds and combinations thereof.

19. The system according to any of claims 14-18, wherein said aqueous phase comprises a biocide.

20. The system according to claim 19, wherein said biocide is different from said germicidal agent.

21. The system according to any of claims 14-20, wherein said water- immiscible liquid is substantially non-germicidal.

22. A method of treating an animal hoof comprising:

applying to said animal hoof a composition comprising a water-immiscible liquid and a germicidal agent dispersed therein.

23. The method according to claim 22, wherein said application step comprises spraying said composition onto an animal hoof.

24. The method according to claim 23, wherein said method further includes the step of pre-wetting said animal hoof prior to application of said composition thereto.

25. The method according to claim 22, wherein said composition comprises an emulsion, said water-immiscible liquid comprising one phase of said emulsion.

26. The method according to claim 25, wherein said emulsion further comprises an aqueous phase.

27. The method according to claim 26, said method further comprising the step of forming said emulsion by adding said water-immiscible liquid and germicidal agent to a footbath containing said aqueous phase, and causing said animal to walk through said footbath so that at least a portion of said emulsion is transferred onto said animal hoof.

28. The method according to claim 26, said aqueous phase comprising an biocide dispersed therein.

29. The method according to claim 28, wherein said biocide is different from said germicidal agent.

30. The method according to claim 22-29, wherein said animal hoof comprises a manure soil prior to said application step.

31. The method according to any of claims 22-30, wherein said water- immiscible liquid comprises a member selected from the group consisting of petroleum or mineral oils, vegetable oils, polybutene, fatty alcohols, fatty acids, methyl esters of fatty acids, methyl esters of triglycerides, silicon oil, d-limonene, substantially water-insoluble or lipophilic surfactants, polypropylene glycols, water-insoluble glycol ethers, terpene oils, biodiesel, water-insoluble anionic materials, olefins, and combinations thereof.

32. The method according to claim 22, wherein said water-immiscible liquid comprises one or more non-polar solvents.

33. The method according to any of claims 22-32, wherein said germicidal agent comprises a member selected from the group consisting of phenol derivatives, cationic germicidal agents, organic acids, oxidizing agents, halogen containing compounds, essential oils, polychloro phenoxy phenols, imidazole compounds, polymeric reaction products of formaldehyde and acrolein, benzyl alcohols, iodine, pyridinethione compounds and combinations thereof.

34. The method according to any of claims 22-33, wherein said composition remains on said animal hoof for at least 15 minutes following said application step.

35. The method according to any of claims 22-35, wherein said composition forms a film on said animal hoof.

36. The method according to claim 35, wherein said germicidal agent migrates out of said film and into contact with said animal hoof.

37. The method according to claim 22, wherein said composition has a specific gravity of less than 1.

38. A liquid antimicrobial composition for use as a medicament in a footbath to treat an animal hoof, said composition comprising:

an aqueous phase and a water-immiscible phase, said water-immiscible phase being less dense than said aqueous phase and comprising a water-immiscible liquid and a germicidal agent dispersed therein.

39. The antimicrobial composition according to claim 38, wherein said water- immiscible liquid is substantially non-germicidal.

40. The antimicrobial composition according to claim 38, wherein said water- immiscible liquid comprises a member selected from the group consisting of petroleum or mineral oils, vegetable oils, polybutene, fatty alcohols, fatty acids, methyl esters of fatty acids, methyl esters of triglycerides, silicon oil, d-limonene, substantially water-insoluble or lipophilic surfactants, polypropylene glycols, water-insoluble glycol ethers, terpene oils, biodiesel, water-insoluble anionic materials, olefins, and combinations thereof.

41. The antimicrobial composition according to any of claims 38-40, wherein said germicidal agent comprises a member selected from the group consisting of phenol derivatives, cationic germicidal agents, organic acids, oxidizing agents, halogen-containing compounds, essential oils, polychloro phenoxy phenols, imidazole compounds, polymeric reaction products of formaldehyde and acrolein, benzyl alcohols, iodine, iodine-containing compounds, pyrithione compounds and combinations thereof.

42. The antimicrobial composition according to any of claims 38-41 , wherein said germicidal agent disperses between said water-immiscible phase and said aqueous phase in said footbath, said germicidal agent having a partition co-efficient, D (molar concentration in water-immiscible phase/molar concentration in aqueous phase), that is at least 5.