Means and methods for the management of digital dermatitis in hoofed animals

Abstract

The invention relates to the field of veterinary care of ruminants, in particular to means and methods for the management of Digital Dermatitis (DD). Provided is a method for the management of DD in a cloven-hoofed animal, such as a dairy cow, comprising contacting hooves of said cloven-hoofed animal with an aqueous solution comprising MgCl2.

Description

[0001] P137757PC00 Title: Means and methods for the management of Digital Dermatitis in hoofed animals. The invention relates to the field of veterinary care of ruminants. In particular, it relates to means and methods for the management of Digital Dermatitis. Digital dermatitis (DD), also known in the art as Mortellaro disease, is a significant infectious disease affecting the hooves of even-cloved animals, including dairy cows. DD is present in 70 to 95% of dairy herds, with herd prevalence rates averaging between 20 to 30% in Europe and the United States. The disease is characterized by painful lesions, lameness, and reduced milk production. Digital dermatitis is a circumscribed superficial ulceration of the skin along the coronary band, often on the plantar inter-digital ridge of the rear foot. It is a poly-microbial hoof infection, caused by multiple species of anaerobic spirochete bacteria, with the Treponemes as an important contributor (Treponema pedis). Effective prevention and treatment of DD are critical to maintaining herd health and productivity. To promote improved hoof health, milk production, and animal welfare, dairy producers use a variety of preventative and treatment measures. Preventive measures include footbaths with disinfectant solutions, hygiene management and nutritional supplements. For example, supplementing dairy cow diets with biotin and zinc has been shown to improve hoof health and potentially reduce the incidence of DD. Other preventive measures include not allowing infected animals to enter the herd, and ensuring that replacement animals are managed to prevent new infections. FR3022734A1 discloses an exemplary footbath for animals, consisting of at least one tank adapted to receive an antibacterial solution, characterized in that each of said tanks is oriented, and comprises an inlet zone, having a first end wall, called the inlet wall, and an outlet zone, having a second end wall, called the outlet wall, and in that said outlet wall has a height greater than said inlet wall. Dairy farmers use many different types of solutions in footbaths. Traditionally, the use of copper sulfate or formalin solutions in footbaths has been widely practiced. Whereas these substances are effective in controlling bacterial populations on hooves, they come with significant environmental and safety concerns. A major concern regarding the use of Copper sulfate footbath solutions is the accumulation of Copper on the soil. Footbath water is typically discarded into the manure lagoon, increasing the copper levels of the manure. Although the copper solution diluted and mixed with manure, could have implications for harm to wildlife when the manure is spread on the fields. High levels of copper in the soil may have toxic and negative effects on plant growth. Copper discharged into surface waters is of particular concern due to its high toxicity to aquatic species. Furthermore, copper sulfate is classified by the US Environmental Protection Agency as toxicity class 1, “highly toxic”. When handling copper sulfate, workers must take great care, because it is extremely corrosive to the skin and eyes and is quickly absorbed through the skin on contact. Formalin is a 37%-39% solution of formaldehyde. Formalin breaks downrapidly when exposed to air and organic matter such as manure. Tomaintain efficacy, it is necessary to renew the solution at regular intervals. After it has been used in the footbaths, is disposed with all the manure into manure lagoon, subsequently it is spread in the croplands. Whereas formaldehyde is extremely soluble in water and does not build up contaminant residues in plants and animals, it is a toxic substance and can affect human health causing irritation of eyes, nose, and throat, and burning of the skin. A carcinogenic effect of formalin in animals and humans has been reported (IARC, 2006). Continuous research and development in this field were aimed to improve the efficacy of existing methods and discover new solutions to ensure the health and productivity of dairy herds. Alternative disinfectant solutions that have been used include organic acids (e.g., acetic acid, lactic acid), disinfectants like quaternary ammonium compounds, sodium hypochlorite , zinc sulphate, and antibiotics such as lincospectin, tylosin , erythrocin or oxytetracycline. However, reports on the efficacy of these alternative footbath solutions is scarce and controversial. Furthermore, antibiotics are expensive and not accepted for use as a dairy cow footbath treatment in the European Union. Also, legislative restrictions on the sales of milk after antibiotic treatment exist. Therefore, the present inventors aimed at providing a novel approach for the management of the disease. More in particular, they sought to develop an alternative footbath solution which rely on ingredient(s) that are not only safe to the environment, handler and animal and not subject to legislative restrictions but also show an efficacy that is comparable to those containing copper sulfate or formalin. The novel approach must be non-carcinogenic and provide a safe profile for both animals and farm workers. Ideally, the active agent(s) is environmentally friendly, cost-effective and compliant with regulatory standards while maintaining its effectiveness in a wide range of environmental conditions. It was surprisingly found that at least some of these goals can be met by the use of an aqueous solution of magnesium chloride. As is shown herein below, comparing a footbath solution of magnesium chloride with a conventional 4% formalin footbath, it was found that the odds of curing Digital Dermatitis and the incidence of new lesions were statistically equal. More specifically, during a 12 week test period wherein dairy cows were weekly treated (twice within 24 h) with test solutions using a split footbath containing magnesium chloride in the left bath and formalin in the right bath. It was found that the presence of pre-existent lesions at the end of treatment period was higher for cows treated with formalin, and the incidence of new lesions was the same when comparing formalin versusMgCl2. Herewith, the invention provides a safe, effective and commerciallyattractive alternative to current footbath solutions. The invention therefore relates to a method for the management of interdigital dermatitis in a cloven-hoofed animal, comprising contacting hooves of said cloven-hoofed animal with an environmentally friendly aqueous solution comprising MgCl2. Also provided is an animal hoof cleaning system, comprising a footbath structure having a front wall, a rear wall, two side walls, and a floor, the footbath structure comprising a liquid containing region arranged to permit an animal to walk over the floor and through the foot bath in the liquid containing region, and wherein the liquid containing region comprises an aqueous solution of MgCl2. Still further, the invention provides a method for disinfecting animal hooves, comprising bathing the hooves of a cloven-hoofed animal in a bathing solution comprising MgCl2. In particular, the aqueous solution of MgCl2for use in the present invention preferably has not been subjected to electrochemical oxidation, thereby avoiding the formation of harmful components such as hypochlorites. The use of a magnesium chloride footbath solution which does not contain any toxic or harmful components for reducing the incidence of DD has not been taught or suggested in the art. DE102009057267 A1 relates to an ointment for treating Mortellaro's disease in cloven-hoofed animals and to a process for producing this ointment. The ointment is based on a mixture of cereal hydrolysate, acid whey and water, with the following substances being added to the mixture: a) 900 to 3,000 mg calcium chloride, b) 500 to 800 mg magnesium chloride, c) 85 to 190 ml iron saccharate, each based on 100 g of the base. It is suggested that the animals are treated with ointment in the hoof care station, where the ointment may be applied to the affected areas of the hoof with a brush to a thickness of 2 to 3 mm. DE102009057267 is silent about footbath treatments, let alone that it suggests the regular foot bathing of animals in a MgCl2 solution as a preventive measure to combat DD. RU2701857C1 and RU2187312C1 relate to the treatment of necrobacteriosis in sheep with an aqueous solution of natural bischofite, characterized in that an electrochemically structured aqueous solution of bischofite is used as the active substance using a copper anode. During electrolytic oxidation of a natural bischofite solution, chlorites, hypochlorites, magnesium and copper hypobromites are formed: Mg(ClO)2, Cu(ClO)2, Mg(BrO)2, Cu(BrO)2. The drawback of this approach is that hypochlorites, magnesium hypobromites, and copper hypobromites, while all effective oxidizing disinfectants, have notable drawbacks for use in hoof treatments. Hypochlorites can corrode metal surfaces in hoof baths and associated equipment, leading to increased maintenance costs, and their reaction with organic matter reduces effectiveness while creating toxic chlorinated byproducts that may harm beneficial microorganisms, persist in the environment, and threaten aquatic life. Magnesium and copper hypobromites share similar oxidative risks but can also produce brominated organic byproducts, such as bromoform, which are generally more persistent and often more toxic than chlorinated equivalents. Copper hypobromite introduces further environmental hazards, as copper ions are highly toxic to aquatic organisms even at very low concentrations and can accumulate in soils and sediments, while magnesium in large amounts can alter soil and water chemistry. All three compounds present safety concerns for handlers and animals, as they can irritate or damage skin, eyes, and mucous membranes, and hypobromites in particular can release bromine vapors that are especially irritating to the respiratory tract. In view of the known use of salt (NaCl) as a common household antiseptic that kills bacteria by dehydration, Speijers et al. (2010) investigated the efficacy of salt water as a footbath solution to help prevent or cure DD. To that end, either a 10% sodium chloride solution or tap water was used as part of a CuSO4 footbath regimen. However, no significant advantage was found when using salt on alternating weeks with 5% copper sulfate twice daily, for two days in a row, every two weeks. In contrast, using tap water in alternating weeks led to an absence of cows with active (M2) lesions at the end compared with the control (CuSO4only) and salt water treatments. These findings provide a teaching away from using magnesium chloride footbaths according to the present invention. Digital dermatitis (DD) is also known by several other terms, including Hairy Heel Warts, Strawberry Foot Rot, Mortellaro Disease, Heel Warts, and Papillomatous Digital Dermatitis (PDD). These synonyms are used interchangeably in different regions and contexts to refer to the same condition affecting cattle hooves. As used herein, the management of DD comprises controlling, preventing and / or reducing the incidence of DD. In one aspect, the invention provides a method for reducing the incidence of DD in a cloven-hoofed animal to a level equal to or better when compared to using a formalin footbath solution. For example, it comprises reducing the incidence of M2 lesions, preferably M2 lesions to the hind legs, of a dairy cow. As will be appreciated by a person skilled in the art, the invention is applicable to any type of cloven-hoofed animal. In one aspect, the animal is a cow, goat, sheep or pig. In a preferred embodiment, the animal is a cow, more preferably a dairy cow. Magnesium chloride is an inorganic compound with the formula MgCl2. It forms hydrates MgCl2. nH2O, where n can range from 1 to 12. These salts are colorless or white solids that are highly soluble in water. These compounds and their solutions, both of which occur in nature, have a variety of practical uses. Hydrated magnesium chloride is the form most readily available from various suppliers for different applications, including industrial use, agricultural purposes, and food processing. The aqueous footbath solution comprise MgCl2 in an amount effective to at least reduce the incidence and / or severity of digital dermatitis. This may depend on specific circumstances such as the type of animal, frequency of footbath treatment, specific needs and conditions of the herd. In a preferred embodiment, a footbath solution comprises or consists of Magnesium chloride brine, which is a highly concentrated solution of magnesium chloride (MgCl2) in water. It is typically derived from natural sources such as seawater, salt lakes, or underground deposits. Natural brine deposits found underground can be a source of magnesium chloride. Magnesium chloride can be extracted from brine or sea water. MgCl2brine may also be prepared by dissolving magnesium chloride flakes or pellets in water to achieve the desired concentration. Preferably, the magnesium chloride brine is used as such, and has not been subjected to any (electro)chemical treatment including electrochemical oxidation and / orelectroactivation. Electrochemical oxidation refers specifically to the anodicprocess in which chloride ions are oxidized to chlorine gas. That chlorine can then hydrolyze in water to form hypochlorous acid or hypochlorite depending on pH. Electroactivation is a broader concept. It describes the overall change in the solution caused by electrolysis, including not only the oxidation of chloride but also shifts in pH near the electrodes, the enrichment of the liquid with reactive chlorine and oxygen species, and the generation of metastable chemical states. In a specific embodiment, a footbath solution comprises MgCl2.6H2O (magnesium chloride hexahydrate) which is a salt hydrate that is also known as Bischofite. Bischofite is a crystalline salt hydrate left after evaporation of an ancient sea. Bischofite is extremely hygroscopic; it melts in the open air. As a mineral, Bischofite is encountered as a glomeroblastic salt rock. Pure Bischofite crystals are aquatic-transparent, but may also be of white, rose and fallow colour depending on impurities. Bischofite is encountered on many continents, in formations differing in age including modern ones. There were discoveries of small deposits in Western Ukraine, Byelorussia and Kazakhstan. All the discoveries were represented by thin (3 to 7 m) non-extended layers. Most notable potassium and magnesium salt deposits are found in Carlsbad, New Mexico; the Paradox Basin in Colorado and Utah; deposits in Strassfurt, Germany; the Perm Basin, Russia and the Williston Basin in Saskatchewan, Canada. Another natural source of Bischofite is the region around Veendam, The Netherlands. Nedmag B.V. mines the Bischofite salt from the Zechstein salts near Veendam with a unique solution mining method to produce a magnesium brine comprising at least 28 % MgCl2 by solution mining of magnesium at a depth of 1300 to 1800 m in the Zechstein III formation. In one aspect, the footbath solution comprises MgCl2in an amount of at least 15% w / w MgCl2. Good results, e.g. with dairy cows, may be obtained with at least 20%, at least 22%, at least 24%, at least 25%, at least 27%, at least 30%, at least 31%, or at least 32 % w / w MgCl2. Exemplary footbath solutions may comprise 10-35%, 15-35%, 20-35%, 25-35%, 10-32%, 15-32%, 20-32%, 25-32%, 28-32% or 30-32% MgCl2. In some embodiments, the solution comprises 10-30%, 15-30%, 17-30%, 20-30%, 25-30%, 10-25%, 15-25%, 17-25%, 20-25%, or 18-22% MgCl2. In some embodiments, the solutioncomprises 22-30%, 25-30%, 27-35%, 22-35%, 25-30%, 25-35%, 25-32%, 27- 34%, 30-35%, or 28-32% MgCl2. In one aspect, the solution is a brine comprising 30-35% MgCl2. In one aspect, the footbath solution contains minimal amounts or is essentially free of any carcinogenic agents and / or free of substances that are environmentally harmful. These include one or more of formalin, copper sulfate, hypochlorites and hypobromites. In one embodiment, the solution comprises less than 2 wt%, less than 1 wt% or less than 0.5 wt% copper sulfate, preferably less than 0.4wt%, less than 0.3 wt%, less than 0.2wt%, less than 0.1 wt% copper sulfate. More preferably, the aqueous footbath solution comprising MgCl2 comprises less than 0.05 wt%, less than 0.02 wt%, less than 0.01 wt% copper sulfate. In one aspect, the footbath solution is free of any carcinogenic agents, in particular free of formalin, and free of substances that are environmentally harmful, in particular copper sulfate. In a preferred embodiment, the footbath solution does not contain one or more of hypochlorites, magnesium hypobromites, and copper hypobromites. Additional active or beneficial agents may be added. These include one or more salts other than MgCl2, e.g. CaCl2, disinfectants, antiseptics, skin conditioning agents and organic acids. As is shown herein below, the effectiveness of MgCl2in killing T. pedis is not negatively impacted by the presence of typical manure ingredients. However, in some embodiments, the footbath solution comprises a germicidal additive to prevent that footbaths can become a breeding ground for bacteria, and can accelerate the spread of infectious hoof diseases, rather than prevent them. For example, the solution may comprise an effective amount of an N,N-bis(3-aminopropyl) C6-C18 alkyl amine, such as N,N- bis(3-aminopropyl)dodecylamine, bronopol, chlorhexidine salts, C6-C12 fatty acids, triclosan, glycolic acid, lactic acid, polyhexamethyl biguanide, polyhexamethylene guanidine hydrochloride, polyhexamethylene guanidine hydrophosphate, poly[2-(2-ethoxy)-ethoxyethyl]-guanidinium chloride, benzyl alcohol, benzoic acid and mixtures thereof. Skin conditioning agents may also be optionally used in the disclosed footbath solutions. Skin conditioning agents may provide extra protection for human or animal skin prior to or subsequent to being exposed to adverse conditions. For example, skin conditioning agents may include moisturizers, such as glycerin, sorbitol, propylene glycol, Laneth-5 to 100, lanolin alcohol, shea butter and coco butter; sunscreen agents, such as titanium dioxide, zinc oxide, octyl methoxycinnamate (OMC), 4-methylbenzylidene camphor (4-MBC), avobenzone, oxybenzone and homosalate; and itch-relief or numbing agents, such as aloe vera, calamine, mint, menthol, camphor, antihistamines, corticosteroids, benzocaine and paroxamine HCl. The contacting of hooves with a MgCl2 solution may be performed at regular intervals. For example, hooves are exposed to aqueous solution of MgCl2 at least once daily, weekly or biweekly. In some embodiments, the treatment is repeated within 24 hours. For example, the method may comprise contacting hooves with a MgCl2solution two times within 24 hours every 3-4 days, each week or every 2 weeks. It is also envisaged within the invention that a footbath solution of MgCl2 alternates with a footbath solution comprising other active ingredients. Contacting animal hooves with a MgCl2 solution is readily achieved using a walk-trough footbath. The footbath should be long enough for each animal to take several steps (usually around 10-12 feet) to ensure thorough contact with the solution. Suitably, the footbath structure has a length of at least 180 cm, preferably at least 200 cm, such as about 210, about 220 or about 230 cm. The width and depth must accommodate the animals comfortably. For example, the foot bath comprises an aqueous solution of MgCl2 to a height of at least 10 cm, preferably at least 12 cm, more preferably at least 15 cm. Thus, the invention also provides an animal hoof cleaning system, comprising a footbath structure having a front wall, a rear wall, two side walls, and a floor, the footbath structure comprising a liquid containing region arranged to permit an animal to walk over the floor and through the foot bath in the liquid containing region, and wherein the liquid containing region comprises an aqueous solution of MgCl2. In one embodiment, the cleaning system is integrated into an automatic milking station. The milking station is may be configured to automatically milk an animal housed therein. Typically, the automatic milking station comprises a set of teat cups that are configured to be attached to the teats of said animal for milking thereof. The footbath may comprise or consist of two or more separate sections or compartments. Each section can contain a different solution or clean water, providing a multi-step hoof care process for the cows. This design aims to improve the effectiveness of hoof treatments and enhance overall hoof health. In one aspect, the first compartment contains water or a cleaning solution to remove dirt and manure from the hooves, and the second compartment contains a MgCl2treatment solution to address and prevent hoof diseases. Hence, in some embodiments, a method for the management of DD comprises a footbath regimen wherein hooves are contacted with a prerinse footbath filled with water or other type of cleaning solution prior to contacting hooves with solution of MgCl2. In some embodiments, the footbath solution is comprised in a split footbath comprising a first and a second footbath structure each comprising a liquid containing region separated by a high ringed divider or narrow grill. By using a footbath with narrow grill, the footbath becomes dirty less quickly, this is because the manure and other substances fall through the grill and therefore do not enter the bath. The result is less soiling. In one aspect, a method or footbath system of the invention comprises a split footbath having a first liquid containing region arranged to receive the left hoofs of the animal and a second liquid containing region is arranged to receive the right hoofs of the animal. For example, each side can measure about 30-35 cm wide by 200-250 cm long, allowing for 70-100 L of MgCl2 solution per side. The magnesium chloride solution in the footbath is typically replaced regularly to maintain its effectiveness, and the footbath itself should be kept clean to prevent buildup of manure and other contaminants. The frequency with which animals are put through the footbath can vary, but it is often done once or twice a week. More frequent use might be necessary in herds with a high incidence of hoof problems. The footbath or animal hoof cleaning system is suitably placed in areas where animals frequently walk, such as in case of dairy cows in the exit lane from the milking parlor, to ensure regular use without requiring additional handling. LEGEND TO THE FIGURES Figure 1. Kaplan-Meier curve of the time to a first M2 lesions of digital dermatitis on the hind leg during a field trial on the effect of MgCl2 on the left in a split foot bath (LA) and 4% formalin on the right (RA) as a positive control. Figure 2. Cox regression analysis of the time to a first M2 lesion on a hind leg during a field trial into the effect of MgCl2on the left in a split foot bath (LA) and 4% formalin on the right as a positive control (RA) in dairy cows. EXPERIMENTAL SECTION Example 1: In vitro tests Digital Dermatitis is a polymicrobial hoof infection, caused by a mixture ofbacteria, with the spirochaete Treponema pedis as an important contributor.(Arrazuria et al. Microorganisms 2020, 8(10), 1507)). In this example, aqueous magnesium chloride solutions were evaluated for their in vitro efficacy to prevent the growth of Treponema pedis. Growing Treponema is very sensitive for oxygen, and has to be performed in an anaerobic environment. The strain that was used was GDB-2567, T. pedis. The culture medium was Anaerobic Systems OTEB (Oral Treponema Enrichment Broth): batch no: 283126, Expiry Date: 2023-04-10. Peracetic acid was 38-40%, 1L, Merck. Freshly collected cow’s manure was diluted 1:5 in peptone physiological saline, distributed into Greiner tubes, and centrifuged for 30 minutes at 4500 G. The supernatant was pipetted off and autoclaved. Afterwards, the supernatant was checked for sterility by placing the material on culture. The material was found to be sterile. T. pedis bacteria were thawed and placed in OTEB medium to grow. Culturing was performed in anaerobic tubes that are not opened, so the environment remains strictly anaerobic. Magnesium chloride solutions comprising 15, 10, 5, 2 or 1% MgCl2 were used for testing. Effects were determined after 2 days of incubation. All experiments were carried out in threefold. Phase contrast microscopy was used (x400 amplification) to count dead or alive Treponema. Sediment was determined macroscopically before vortex treatment. The amount of sediment is a measure for degraded T. pedis material. The results are shown in Tables 1 and 2.

[0002] Table 1 Exp % (w / w) T-pedis T-pedis Sediment MgCl2 Alive Dead cm A 5 0 12 1.0 B 2.5 0 17 0.5 C 1 0 7 0.3 D 0 400-500 0 0 Table 1 shows that all levels of Magnesium chloride work very well in killing T-pedis. The different sediment levels suggest that higher levels of Magnesium ions could precipitate with fragment molecules of the bacteria. In a second series of experiments (Table 2) a representative amount of sterile cow’s manure was added to the MgCl2solution (equivalent to 20 grams manure to one liter of footbath liquid), to study the efficiency of magnesium chloride in the presence of typical manure ingredients (i.e., coordination and hence potentially capturing of Mg2+ions in the presence of coordinating (sequestering) carboxylate units in manure). Table 2 Exp % MgCl2 manure % T-pedis T-pedis Sediment cm Alive Dead E 5 2 0 0-5 1.0 F 2.5 2 0 0-5 0.5 G 1 2 0 5-10 0.3 H 0 2 300-500 0 0 I 0 0 300-500 0 0 Table 2 suggests that with very low (1%) concentrations of magnesium chloride the presence of manure can diminish the activity of the Mg ions, possibly by coordinating cations. However, even in this situations there are no alive T-pedis anymore. The experiment H also shows that manure is not reducing the T-pedis activity. Example 2: Field study on dairy cows A field study was conducted to examine the effectiveness of a MgCl2footbath solution in the management of DD in lactating dairy cattle. The herd consisted of > 90% Holstein-Friesian dairy cows. The animals were housed in freestalls and had no meadow access. The study was designed as a non-inferiority study with a split foot bath comprising either a MgCl2test solution or a formalin as a positive control. The sample size was calculated prior to conducting the field study. Therein, a starting situation was chosen wherein 2% of the hind (rear) hooves has an M2 injury and wherein 4% of the 'at risk' rear legs monthly develop a new M2 injury with regular use of a 4% formalin foot bath (Holzhauer et al., 2012). With a one-sided confidence interval of 97.5% and a power of 80%, this resulted in a minimum of 160 hind limbs per treatment group to demonstrate a difference of 4 percentage points. With a one-sided confidence interval of 95% and a power of 70%, this resulted in a minimum of 150 rear limbs per treatment group to demonstrate a difference of 3 percentage points. One dairy farm was selected based on the following selection criteria: • willingness of the livestock farmer to participate in the study • milking parlor suitable for safely M-scoring the hind legs during milking • stable equipment suitable for installing a foot bath with a length of at least 233 cm • at least 150 lactating cows • cows that are used to regular use of foot baths The M- (Mortellaro) stages scoring system classifies cows showing signs of digital dermatitis (DD) into one of five disease stages: M1, M2, M3, M4 and M4.1. In addition, healthy digital skin is classified as M0. The M-stages scoring system is a highly effective way to diagnose and monitor DD. On this scale, M0 signifies a claw free of signs of digital dermatitis; M1 is a lesion <2 cm and is not painful; and M2 is a lesion in the ulcerative stage, with a diameter of >2 cm and painful to the touch. Although pain was not recorded in this study, when determining a lesion that was between the M1 and M2 stage, the lesion was gently touched to see if pain was evident. Stage M3 is the healing stage and is covered by a scab and M4 is the chronic stage and is characterized by dyskeratosis or proliferation of the surface that is generally not painful. An M4.1 was assigned to a new small lesion, as an M1, in the M4 lesion At the start of the trial, the hind legs of all lactating cows in the milking parlor were screened after rinsing the legs with water for the presence of active lesions of digital dermatitis (M1, M2 or M4.1 stage). Hind hooves with an active lesion of DD were treated the same or the next day as follows (without prior cleaning with water): M1 and M4.1 lesions with copper sulphate powder and copper and zinc chelate spray (Topro MicroDerm spray, REG NL 126591, Bio Enterprise B.V.) without bandage and M2 lesions with salicylic acid (Novaderma®, WDT) under a self- adhesive elastic bandage (Intracare B.V.). Treated legs were checked approximately 1 week later in the hoof trimming box after removal of any bandages. Lesions of DD that were still active at follow-up were treated with copper sulphate powder and copper and zinc chelates spray, without dressing and without prior cleaning with water. After this, the cows started using the foot bath weekly, twice within 24 hours, for a period of 12 weeks. Footbath solutions were replaced with fresh solutions every week. Two split foot baths were placed in series in the return aisle of the milking parlor. Both split footbaths were filled on the left with 32% magnesium chloride and on the right with 4% formalin, all to a depth of about 15 cm. On the fourth day after foot bathing, the hind legs, after rinsing with water, were scored for the presence of lesions of digital dermatitis using a telescopic mirror with integrated LED light source (HBM machines B.V.) in the milking parlour. Active lesions of DD were treated weekly on Tuesdays in the hoof trimming box by a hoof care professional. The actions during the trial are summarized in Table 3. Data was collected on paper and then digitized in an Excel file. The static unit was a hindlimb and statistical analyzes were performed in STATA (version 17.0). The prevalence of hind limbs with a digital dermatitis lesion was calculated per observation moment by dividing the number of legs with a digital dermatitis lesion by the total number of limbs with an M-score at that observation moment The incidence of hind limbs with a new M2 injury was calculated per week by dividing the number of paws with a new M2 injury by the number of limbs 'at risk' for developing a new M2 injury. For this purpose, only limbs were included per week with an M-score at the beginning and at end of that week, without an M2 injury at the beginning of that week. Significance in differences in prevalence and incidence between the treatment and control groups were tested one-sided with a proportion test in which the significance limit of 0.05 was adjusted to 0.001 according to Bonferroni correction. Table 3: of actions with dairy cows. Day Action Comment M-score in milkpit Zero scoring; 176 dairy cows scored 2 treatment active lesions according to protocol digital dermatitis 3 split footbath start study; footbath with magnesium chloride in left side and formalin in right side at afternoon- and evening milking 7 M-score in milkpit 164 dairy cows scored 8 treatment active lesions according to protocol digital dermatitis 10 split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking 11 split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking 14 M-score in milkpit 176 dairy cows scored 15 treatment active lesions according to protocol digital dermatitis 17 split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking 18 split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking 21 M-score in milkpit 187 dairy cows scored treatment active lesions according to protocol digital dermatitis footbath with magnesium chloride in left side and formalin in split footbath the right side at afternoon milking 23 split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking 24 split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking

[0003] split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking M-score in milkpit 190 dairy cows scored treatment active lesions according to protocol digital dermatitis split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking M-score in milkpit 196 dairy cows scored split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking treatment active lesions according to protocol digital dermatitis footbath with magnesium chloride in left side and formalin in split footbath the right side at afternoon milking split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking M-score in milkpit 198 dairy cows scored treatment active lesions according to protocol digital dermatitis split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking M-score in milkpit 199 dairy cows scored treatment active lesions according to protocol digital dermatitis footbath with chloride in left side and formalin in split footbath the right side at

[0004] split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking M-score in milkpit 203 dairy cows scored treatment active lesions according to protocol digital dermatitis split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking M-score in milkpit 197 dairy cows scored treatment active lesions according to protocol digital dermatitis split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking M-score in milkpit 199 dairy cows scored Treatment active lesions according to protocol digital dermatitis split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking M-score in milkpit 198 dairy cows scored Treatment active lesions according to protocol digital dermatitis split footbath footbath with magnesium chloride in left side and formalin in the right side at afternoon milking

[0005] 80 split footbath footbath with magnesium chloride in left side and formalin in the right side at morning milking

[0006] For the cumulative incidence of hindlimbs with a new M2 lesion during the entire study period, only hindlimbs with an M-score on 2 consecutive observation moments with no more than 16 days between the two observation moments were included. By means of logistic regression analysis, differences in the cumulative incidence of new M2 lesions in all 'at risk' measurement moments between the treatment and control groups were examined, correcting for repeated observations within the limb by including limb as a random effect. We indicated the time to a first M2 lesion of DD in this study period using a Kaplan-Meier curve. Differences in the time to a first M2 lesion of digital dermatitis between the treatment and control groups were examined using a Cox regression analysis provided that the proportional hazard assumption was met. An M-score could not be assigned to both hind legs for each cow at all observation moments because the typical anatomical location of DD dermatitis lesions could not be properly inspected due to, for example, too little heel height in the claws or the presence of a bandage. To correct for this, sensitivity analyses were performed for both the logistic regression analysis of the cumulative incidence of new M2 lesions of DD and for the Cox regression analysis for the time to a first M2 lesion of DD. The sensitivity analyses only included observation moments where an M-score was known for both hind legs per cow. RESULTS Prevalence of hindlimbs with lesions of digital dermatitis The prevalence of hindlimbs with digital dermatitis lesions averaged 27.6% during the study period and the prevalence of hindlimbs with M2 digital dermatitis lesions averaged 2.1% during the study period. At the start of the study, the prevalence of hindlimbs with a lesion of digital dermatitis, regardless of M stage, was 30.3% (100 / 330 hindlimbs), the prevalence in left hindlimbs was 29.7% (49 / 165 left hindlimbs; treatment group) and the prevalence in right hindlimbs was 30.9% (51 / 165 right hindlimbs; control group). The prevalence of hindlimbs with an M2 lesion of digital dermatitis was 2.1% (7 / 330 hindlimbs), the prevalence in left hindlimbs was 1.8% (3 / 165 left hindlimbs; treatment group) and the prevalence in right hindlimbs was 2.4% (4 / 165 right hindlimbs; control group). At the end of the study (04 / 29 / 2024), the prevalence of hindlimbs with a digital dermatitis lesion, regardless of M stage, was 21.2% (79 / 372 hindlimbs), the prevalence in left hindlimbs was 22.2% (41 / 185 left hindlimbs; treatment group) and the prevalence in right hindlimbs was 20.3% (38 / 187 right hindlimbs; control group). The prevalence of hindlimbs with an M2 lesion of digital dermatitis was 1.1% (4 / 372 hindlimbs), the prevalence in left hindlimbs was 1.6% (3 / 185 left hindlimbs; treatment group) and the prevalence in right hindlimbs was 0.5% (1 / 187 right hindlimbs; control group). Table 4 summarizes the prevalence of hindlimbs with digital dermatitis lesions during the study period. Notably, throughout the study period, there was no significant difference between treatment and control hindlimbs in the prevalence of digital dermatitis lesions regardless of M stage or M2 digital dermatitis lesions. Incidence of hindlimbs with new M2 lesions of digital dermatitis The cumulative incidence of hindlimbs with new M2 lesions of digital dermatitis with the total number of 'at risk' measurement moments as denominator was 1.6% (31 / 1930) in the treatment group and 1.0% (19 / 1952) in the control group ( P=0.08). The odds ratio of a new M2 lesion of digital dermatitis during all 'at risk' measurement moments in the study period for a hind paw in the treatment group is 2.76 (95% confidence interval 0.80 - 9.51) compared to a hindlimb in the control group (Table 5). When only cows with an M-score for both hindlimbs are included per observation moment, the odds ratio of a new M2 lesion of digital dermatitis during all 'at risk' measurement moments in the study period for the treatment group is 2.81 (95% confidence interval 0.73 – 10.81) compared to the hindlimbs in the control group (Table 5). Table 6 summarizes the weekly incidence of hindlimbs with a new M2 lesion of digital dermatitis since the previous M-score during the study period. The Kaplan-Meier curve shows how long it takes for a hindlimb in the treatment or control group to develop a first M2 injury (Figure 1). In the Cox regression analysis, hindlimbs in the treatment group had a hazard ratio for developing a first M2 injury of 1.74 (95% confidence interval 1.06 – 2.85) compared to hindlimbs in the control group (Figure 2). When only cows with an M-score for both hindlimbs per observation moment are included, the hazard ratio for developing a first M2 injury is 1.67 (95% confidence interval 1.00 - 2.76) for hindlimbs in the treatment group compared to with hind legs in the control group. In view of the above, it can be concluded that the use of a foot bath with an aqueous magnesium chloride solution (e.g. weekly, twice within 24 hours), is as effective as the same footbath protocol using 4% formalin in the prevention of new M2 lesions in 'at risk' hindlimbs of dairy cows. This makes MgCl2 an attractive alternative to formalin for use in foot baths in the management of digital dermatitis in cloven-hoofed animals.

[0007] Table 4. Overview of the prevalences of hind legs with lesions of digital dermatitis scored in the milking parlor during a field trial into the effect of MgCl2 on the left in a split foot bath and 4% formalin on the right as a positive control on the control of digital dermatitis in dairy cows. Prevalence hindlimb lesions % (N lesions / N scored) Day Digital dermatitis M2 lesions MgCl2 Formalin P-value1MgCl2 Formalin P-value11 29,7 (49 / 165) 30,9 (51 / 165) 0,595 1,8 (3 / 165) 2,4 (4 / 165) 0,649 7 21,1 (32 / 152) 21,7 (33 / 152) 0,556 3,9 (6 / 152) 0,7 (1 / 152) 0,028 14 20,0 (33 / 165) 26,2 (43 / 164) 0,910 0,6 (1 / 165) 1,2 (2 / 164) 0,721 21 24,6 (42 / 171) 28,1 (48 / 171) 0,769 2,9 (5 / 171) 2,9 (5 / 171) 0,500 29 25,0 (44 / 176) 30,1 (52 / 173) 0,855 2,8 (5 / 176) 3,5 (6 / 173) 0,631 35 22,8 (42 / 184) 27,1 (48 / 177) 0,827 1,6 (3 / 184) 1,1 (2 / 177) 0,342 42 33,3 (61 / 183) 36,0 (64 / 178) 0,700 1,6 (3 / 183) 1,1 (2 / 178) 0,338 49 40,2 (72 / 179) 36,9 (65 / 176) 0,262 2,2 (4 / 179) 0,6 (1 / 176) 0,091 55 28,2 (50 / 177) 26,9 (47 / 175) 0,385 2,8 (5 / 177) 1,7 (3 / 175) 0,242 62 22,7 (40 / 176) 24,6 (43 / 175) 0,658 3,4 (6 / 176) 1,1 (2 / 175) 0,077 69 27,5 (49 / 178) 23,0 (41 / 178) 0,165 0,6 (1 / 178) 0,6 (1 / 178) 0,500 76 19,5 (36 / 185) 20,8 (38 / 183) 0,623 2,2 (4 / 185) 0,0 (0 / 183) 0,023 83 22,2 (41 / 185) 20,3 (38 / 187) 0,332 1,6 (3 / 185) 0,5 (1 / 187) 0,155 1 Differences in the prevalence between the treatment and control groups were tested one-sided with a proportion test in which the significance limit of 0.05 was adjusted to 0.001 according to Bonferroni correction.

[0008] Table 5. Results of the logistic regression analysis for the association between MgCl2 or 4% formalin in a foot bath and the cumulative incidence of hindlimbs with new M2 lesions of digital dermatitis as outcome with leg as random effect Variable Odds ratio 95% Confidence Interval All ‘at risk’ hindlimbs during all ‘at risk’ scoring moments Treatment group 4% formalin 1 reference 32% MgCl2 2,76 0,80 – 9,51 Only ‘at risk’ hindlimbs of cows with an M-score for both hindlimbs per scoring moment Treatment group 4% formalin 1 reference 32% MgCl22,81 0,73 – 10,81

[0009] Table 6. Overview of the incidence of hind legs with a new M2 lesion of digital dermatitis since the previous M-score in the milking parlor during a field trial into the effect of magnesium chloride on the left in a split foot bath and on the right 4% formalin as a positive control on the control of digital dermatitis in dairy cows. Incidence of new M2 lesions Day % (N new M2 lesions / N hindlimbs ‘at risk’) MgCl2 Formalin P-value11n / a n / a7 3,5 (5 / 143) 0,7 (1 / 142) 0,050 14 0,7 (1 / 139) 0,7 (1 / 145) 0,48821 1,3 (2 / 157) 2,6 (4 / 154) 0,80228 1,9 (3 / 162) 1,9 (3 / 158) 0,512 35 1,2 (2 / 165) 0,6 (1 / 160) 0,29042 0,6 (1 / 171) 1,2 (2 / 167) 0,72649 1,2 (2 / 167) 0,0 (0 / 164) 0,08056 1,9 (3 / 159) 2,5 (4 / 159) 0,64963 2,5 (4 / 161) 1,2 (2 / 161) 0,205 70 0,0 (0 / 163) 0,6 (1 / 166) 0,84077 1,8 (3 / 170) 0,0 (0 / 172) 0,04084 1,7 (3 / 177) 0,5 (1 / 182) 0,1511 Differences in the incidence between the treatment and control groups were tested one-sided with a proportion test in which the significance limit of 0.05 was adjusted to 0.001 according to Bonferroni correction.

Claims

Claims 1. A method for the management of interdigital dermatitis (DD) in a cloven-hoofed animal, comprising contacting hooves of said cloven- hoofed animal with an aqueous solution comprising MgCl2, which solution has not been subjected to electrochemical oxidation.

2. A method for disinfecting animal hooves, comprising bathing the hooves of a cloven-hoofed animal in an aqueous bathing solution comprising MgCl2 which bathing solution has not been subjected to electrochemical oxidation.

3. The method according to claim 1 or 2, wherein said cloven-hoofed animal is a cow, goat, sheep or pig, preferably a cow, more preferably a dairy cow.

4. The method according to any one of claims 1 to 3, wherein the aqueous solution comprises at least 20w% MgCl2, preferably at least 25%, more preferably at least 30w% MgCl2.

5. The method according to any one of the preceding claims, wherein the aqueous solution comprising MgCl2 does not contain copper sulphate.

6. The method according to any one of the preceding claims, wherein the aqueous solution comprising MgCl2is a MgCl2brine, preferably a brine comprising bischofite (MgCl2.6H2O).

7. The method according to any one of the preceding claims, wherein the aqueous solution comprising MgCl2 does not contain formalin and / or hypochlorite.

8. The method according to any one of the preceding claims, wherein hooves are exposed to the aqueous solution of MgCl2 at least once weekly or biweekly.

9. The method according to any one of the preceding claims, comprising the use of a walk-through footbath.

10. An animal hoof cleaning system, comprising: a footbath structure having a front wall, a rear wall, two side walls, and a floor, the footbath structure comprising a liquid containing region arranged to permit an animal to walk over the floor and through the foot bath in the liquid containing region, and wherein the liquid containing region comprises an aqueous solution of MgCl2 which solution has not been subjected to electrochemical oxidation / activation.

11. The cleaning system according to claim 10, comprising a split footbath comprising a first and a second footbath structure each comprising a liquid containing region separated by a high ringed divider.

12. The cleaning system according to claim 10 or 11, wherein the cleaning system is integrated into an automatic milking station.

13. The use of a MgCl2 brine which has not been subjected to electrochemical oxidation as a footbath solution for a cloven- hoofed animal.

14. The use according to claim 13, wherein the MgCl2brine is a Bischofite (MgCl2.6H2O) brine.

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