The invention relates to a composition for use in the prevention and/or treatment of mycotoxic disease caused by sporidesmin. In particular, the invention relates to a composition comprising at least one phyllosilicate mineral for use in the prevention and or/treatment of pithomycotoxosis (facial eczema).

Background of the Invention

Pithomycotoxicosis - commonly known as facial eczema (and occasionally called facial dermatitis) - is a mycotoxic disease causing photosensitisation in ruminants that graze pasture contaminated with spores from the fungus Pithomyces chartarum.

Pithomyces chartarum grows on litter at the base of pasture. When sporulation occurs, usually in warm, moist conditions, it produces a hepatotoxin called sporidesmin. Animals grazing the pasture, (such as sheep, cattle, alpaca, goats or deer) ingest the harmful toxin, which is absorbed in the upper gut and passes to the liver, where it is predominantly excreted into the bile ducts.

Inflammation and blockage may occur in the bile ducts and eventually fibrosis may lead to cirrhosis of the liver. Sporidesmin is often excreted to a lesser extent into the urinary tract, where it may also cause cystitis.

The characteristic lesions of facial eczema are caused largely by the blockage of the bile ducts. Phylloerythrin, a breakdown product of chlorophyll in the rumen, is absorbed into the bloodstream but is not properly excreted in the bile. Failure to eliminate these photosensitising pigments from the circulating blood causes lesions in unpigmented and hairless areas of skin when exposed to sunlight.

An incubation or lag period of around 7 to 20 days from ingestion of sporidesmin to the appearance of symptoms.

Inflammation and oedema occur not only on the face, but also commonly the ears, lips, vulva, escutcheon and udders or teats. Thus, "facial eczema" is something of a misnomer, as it is suggestive of only part of the clinical picture but is nevertheless the term commonly used to describe the condition (which does not always have visible symptoms). In addition to the "eczema" type lesions, animals can also become very stressed and irritated in sunlight. Weakness and weight loss often occur. In affected animals having severe liver damage or those suffering additional stressors, facial eczema commonly ultimately leads to death several weeks later.

Management of facial eczema is further complicated from differing seasonal weather conditions (affecting spore number) and also by an incubation or lag period of around 7 to 20 days from ingestion of sporidesmin to the appearance of any symptoms. In dairy cows, an immediate reduction in milk yield and diarrhoea is sometimes observed before more visible symptoms appear.

Although sporidesmin is not generally considered to be a problem in milk, signs of toxicosis have been reported in calves that had no access to pasture but that were suckling cows that had clinical signs of sporidesmin toxicosis.

Facial eczema in ruminants is a long-standing problem, particularly in New Zealand, where Pithomyces chartarum grows in perennial ryegrass. However, there are increasing reports of the disease in other countries where ruminants are intensively grazed on pasture, such as Australia, France, South America and South Africa.

Various solutions have been proposed and trialled to address the problem of mycotoxic disease caused by Pithomyces chartarum.

Many proposed solutions to prevent and control facial eczema have focussed on avoiding or reducing fungus growth in pasture. The fungus is usually invisible to the human eye and early attempts to mitigate its detrimental effects on animals have included avoiding grazing toxic pasture by restricting seasonal grazing, or measurement and monitoring of spore count. Other measures include reduction of flock density and sowing of P. chartarum resistant pasture. Fungicides such as thiabendazole have also been used to spray the ryegrass before spore counts rise. Fungicide use is obviously undesirable for environmental reasons.

At the level of the animal, it has been found that activated carbon adsorbs sporidesmin. When dosed to sheep, the effect was dependent on the amount of carbon and a short time interval between carbon dosing and sporidesmin poisoning. Although activated carbon is highly effective at adsorbing a large number of mycotoxins, it is unsuitable as a dietary supplement because it tends to also adsorb other agents such as important vitamins and minerals in the digestive tract.

A number of other adsorbents are known to bind certain mycotoxins. However, the efficacy of mycotoxin binders differs significantly due to differing and complex chemical structures of both adsorbent and toxin, as well as pH. For example, whilst aluminosilicates such as bentonite (and many other clays) will bind aflatoxin B1 with high efficiency, problematic mycotoxins such as deoxynivalenol (DON) are not readily adsorbed by most clays.

Binding and removal of other mycotoxins in the digestive tract is therefore complex. Many mycotoxins will not bind efficiently at normal in vivo concentrations, or can remain toxic even after binding. Not all mycotoxins will be bound by every adsorbent and the degree of binding can also vary significantly. Where adsorbents are provided as animal feed additives, their concentrations in feed may also be restricted to inefficient dosage amounts due to undesirable binding of essential nutrients.

In view of the relative inefficacy of clay binders in respect of mycotoxins other than aflatoxins, other natural and synthetic binders have been proposed. For example, there has been some research into microorganisms that may be utilised to biotransform mycotoxins into less toxic metabolites.

Many species of bacteria and fungi have been shown to enzymatically degrade mycotoxins. Toxicity of products of enzymatic degradation and undesirable effects of fermentation remain concerns.

Yeasts and lactic acid bacteria have been shown to bind different mycotoxins strongly to cell wall components. Often, additional enzymatic components will be required with different adsorbents in mixtures. US9901108 (Mann) discloses an exemplary method of reducing toxicity of a mycotoxin in a foodstuff using a composition comprising a combination of enzymes, a trichothecene binding agent and a yeast.

US8153737 discloses a biosynthetic (molecularly imprinted) polymer for selectively sequester and/or adsorb specific target mycotoxins, describing sequestration capabilities in respect of sporidesmin. Such synthetic polymers are expensive and are not currently approved as feed additives. Utilising synthetic products may raise certain safety concerns for animal use and as such, there is a desire for natural solutions.

There are over 400 known mycotoxins and there is a significant body of research on those mycotoxins well known to have impacted food safety, economic trade and human health. These include aflatoxins (AF), zearalenone (ZEA), Fusarium toxins such as deoxynivalenol/vomitoxin (DON), ochratoxin (OTA), fumonisins (FUM), trichothecenes (TCT) such as T-2 toxin. US8951533 (Alltech Inc) describes a method for reducing bioavailability of mycotoxins using a clay- interlaced yeast cell wall extract incorporated into a feedstuff. The data shows various experimental testing of Fusarium mycotoxins including DON, FUM and OTA but does not disclose binding of sporidesmin.

Despite advances in efficient binding of the well known mycotoxins, Sporidesmin poisoning of ruminants grazing on pasture remains a significant problem. To date, facial eczema remains problematic on a large scale.

Some studies have indicated that zinc reacts with sporidesmin to form a mercaptid, which aids elimination of sporidesmin from an animal. Feeding low levels of zinc resulted in some improvement in animal recovery, but near toxic levels of zinc are surprisingly commonly administered to sheep and cattle orally in an attempt to prevent and treat facial eczema. Such acceptance of near toxic dosing of zinc in agricultural practice highlights the scale of the problem of facial eczema.

In summary, it is generally accepted that known treatments other than careful husbandry are ineffective in preventing and treating facial eczema. Selective breeding of more resistant animals by selection following sporidesmin challenge is considered the best long term method of control.

It would be desirable to provide an improved composition for use in treatment and/or prevention of pithomycotoxicosis (facial eczema).

Summary of the Invention

One aspect of the invention provides a composition for use in the prevention and/or treatment of pithomycotoxosis, said composition comprising at least one phyllosilicate mineral.

Optionally, the at least one phyllosilicate mineral comprises a magnesium phyllosilicate.

In certain embodiments, the magnesium phyllosilicate comprises sepiolite.

The composition may consist substantially a heterogeneous mixture of naturally occurring phyllosilicate minerals.

Advantageously, the composition may consist substantially of naturally occurring minerals (minerals of natural origin ie elements or chemical compounds that have been formed as a result of geological processes). In certain embodiments the composition comprises an aluminium phyllosilicate.

Optionally, the at least one phyllosilicate mineral may comprise a smectite,

The smectite may comprise bentonite.

The smectite may comprise a natural sodium dioctahedral smectite.

The aluminium phyllosilicate may comprise bentonite.

Optionally, the bentonite may comprise structurally or chemically modified bentonite.

Optionally, the composition comprises the at least one phyllosilicate mineral at between around 75% and 100% by weight of said composition.

Optionally, the composition comprises the at least one phyllosilicate mineral at around 90% or more by weight of said composition.

Optionally, the composition comprises the at least one phyllosilicate mineral at 94% or more by weight.

In certain embodiments, the composition comprises sepiolite at more than around 50% by weight.

In certain embodiments the composition comprises sepiolite at between around 50% to 95% by weight.

In certain embodiments the composition comprises sepiolite at around 90% or more by weight.

In certain embodiments, the composition comprises bentonite at between around 30% to 50% by weight.

Optionally, the composition comprises sepiolite and bentonite in a ratio of around 2:8.

Optionally, the composition comprises sepiolite and bentonite in a ratio of around 5:4.

Optionally, the composition comprises sepiolite and bentonite in a ratio of around 5:5.

Optionally, the composition comprises sepiolite and bentonite in a ratio of around 6:4.

In certain embodiments, the composition comprises bentonite at around 40% by weight.

In certain embodiments, the composition may comprise diatomaceous earth.

The composition may be for use in binding/adsorbing an epipolythiodioxopiperazine mycotoxin.

The pithomycotoxicosis may be mycotoxic disease caused by sporidesmins. Advantageously, the composition may adsorb more than 70% of the sporidesmins at a pH of approximately 2.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

The sporidesmins may comprise congeners sporidesmin A, sporidesmin D, sporidesmin E and sporidesmin B.

In certain embodiments the composition adsorbs more than 28% of the sporidesmin A at a pH of approximately 2.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 70% of the sporidesmin A at a pH of approximately 2.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 28% of the sporidesmin A, sporidemin D, sporidesmin E and sporidesmin B.at a pH of approximately 2.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 70% of the sporidesmin A, sporidemin D, sporidesmin E and sporidesmin B.at a pH of approximately 2.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 13% of the sporidesmin A at a pH of approximately 6.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 50% of the sporidesmin A at a pH of approximately 6.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 70% of the sporidesmin D at a pH of approximately 2.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 77% of the sporidesmin D at a pH of approximately 2.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 20% of the sporidesmin D at a pH of approximately 6.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

In certain embodiments the composition adsorbs more than 47% of the sporidesmin D at a pH of approximately 6.5 when at a toxin level of 0.5pg/ml and adsorbent dosage of 0.5% w/v.

The composition may be for use in reducing the symptoms of pithomycotoxosis.

The composition may be for preventing sporidesmin induced hepatopathy.

The composition may be for preventing sporidesmin induced photosensitisation. Advantageously, the treatment may comprise preventing or reducing risk of further symptoms, or symptoms of increased severity, following a sporidesmin challenge to the animal.

Optionally, the composition may be for use in animals.

The animals may comprise ruminant animals such as cattle, sheep, goats, buffalo, deer.

In certain embodiments, the animals comprise camelids, such as alpacas and llamas.

In certain embodiments, the composition is for use in binding sporidesmin mycotoxin in the gastrointestinal tract of an animal.

In certain embodiments, the composition is for use in a method of reducing bioavailability of sporidesmins in an animal.

Optionally the composition comprises less than about 1% by weight of activated carbon.

Optionally, the composition comprises about 0.75% by weight of activated carbon.

Optionally, the composition comprises zinc oxide or zinc sulphide.

Another aspect of the invention comprises a method of preventing and/or treating pithomycotoxosis, comprising administering the composition in an effective amount to an animal.

Optionally, the composition may be administered orally at a daily dosage of between about lOg and 500g daily.

Optionally, the composition may be administered orally at a daily dosage of between about 80g and 160g daily.

In certain embodiments, the composition may be administered orally at a daily dosage of between about 250g and 500g daily.

The composition may be administered orally at a daily dosage of between about 250g and 350g daily.

The composition may be administered orally at a daily dosage of about 320g daily.

In certain embodiments, the composition is administered as a feed additive.

Optionally, the composition is administered as a substantially dry feed.

Advantageously, the composition may be administered at a dosage of around 2% of the animal's daily dry matter intake. In certain embodiments, the method comprises administering the composition to an animal at a daily dosage of between about 250g and 350g; and administering elemental zinc to the animal at a daily dosage of 20mg/kg liveweight or less.

Optionally, the elemental zinc is administered by a controlled release zinc bolus.

In certain embodiments, the method comprises administering elemental zinc to an animal at a dosage of lOOmg/kg of dry feed or less.

In certain embodiments, the method comprises administering the composition to the animal at a first dosage amount for a first time period and administering a second dosage amount to the animal for a second time period, wherein the second dosage amount is higher dosage than the first dosage amount.

In certain embodiments, the method comprises administering the composition to the animal in a first formulation for a first time period and administering a second formulation to the animal for a second time period, wherein the first formulation comprises a higher sepiolote content and the second formulation comprises a lower sepiolite content and comprises bentonite.

Optionally, during the first time period, the animal's local pasture Pithomyces chartarum spore count is between around 0 and 20,000 spores/g.

Optionally, during the second time period the animal's local pasture Pithomyces chartarum spore count is around 20,000 spores/g or more.

Advantageously, the method may reduce, remove and/or eliminate sporidesmin from the gastrointestinal tract of the animal.

The method may reduce the bioavailability of the sporidesmin in the gastrointestinal tract.

Another aspect of the invention comprises a feed or feed additive comprising the composition of the invention.

Another aspect of the invention provides a composition for use in the prevention and/or treatment of pithomycotoxosis, said composition comprising sepiolite, bentonite and activated carbon.

In certain embodiments, the sepiolite may be present in the composition at around 19-75% and more preferably around 20% to 50% by weight.

In certain embodiments, the bentonite may be present in the composition at between around 25- 85% and more preferably around 5% to 80% by weight. Optionally, activated carbon may be present in the composition at less than 1% by weight of total mixture. The activated carbon may comprise activated vegetable charcoal.

Brief Description of the Figures

In the Figures, which illustrate embodiments of the invention by way of example only:

Figure 1A illustrates absorption spectra of sporidesmin congeners indicated as peaks 1-5.

Figure IB is a graphical representation of peak 1 sporidesmin adsorption data (sporidesmin D)

Figure 2 is a graphical representation of peak 2 sporidesmin adsorption data (sporidesmin A)

Figure 3 is a graphical representation of peak 3 sporidesmin adsorption data (unconfirmed sporidesmin congener).

Figure 4 is a graphical representation of peak 4 sporidesmin adsorption data (sporidesmin E)

Figure 5 is a graphical representation of peak 5 sporidesmin adsorption data (sporidesmin B)

Figure 6 is a graphical representation comparing adsorption data at differing pH and between compositions at adsorbent dosage of 0.05% w/v.

Figure 7 is a graphical representation comparing adsorption data at differing pH and between compositions at adsorbent dosage of 0.25% w/v.

Figure 8 is a graphical representation comparing adsorption data at differing pH and between compositions at adsorbent dosage of 0.5% w/v.

Figure 8B is a graphical representation comparing adsorption data in relation to peaks 1, 2 and 4 at differing pH and between compositions at adsorbent dosage of 0.5% w/v.

Figures 9A-9C show secondary electron images of sepiolite(9A), bentonite(9B) and diatomaceous earth (9C) utilised in certain embodiments of the invention.

Figures 10A and 10B show results of adsorption by Composition 2 of sporidesmin congeners.

Figures 11 to 15 show results of investigations into the dosage effects of compositions on sporidesmin adsorption.

Figures 16 to 20 show results of investigation into toxin concentration effect on adsorption of sporidesmins by the composition.

Figure 21 to 25 show results of an investigation int time contact effect on adsorption of sporidesmins. Description

As illustrated in the Figures and Tables 1-5 below, it has been found that Composition 1 and Composition 2 adsorb sporidesmins in liquid medium. Sporidesmins adsorption by Compositions 1 and 2 was significantly affected by adsorbent dosage, with increased % adsorption at 0.5% (w/v).

For both compositions, the adsorption of sporidesmins was significantly affected by medium pH. In all cases, the adsorption of sporidesmins was more efficient at pH 2.5. When tested at 0.5% (w/v), Composition 2 adsorbed more than 70% of each sporidesmin compound at pH 2.5, and between 27 and 61% at pH 6.5.

In the specific illustrative examples that follow, Composition 1 is a sepiolite composition (Anpro ^® Anpario pic, UK). Sepiolite is a magnesium rich phyllosilicate clay mineral that is often used as pellet binder. Diatomaceous earth (DE) is a sedimentary mineral formed from fossilized diatoms (a type of microscopic algae). It typically contains an assortment of trace minerals, with the main component being silica. Diatomaceous earth has a number of industrial applications, one of which is as an anti caking and pelleting agent in feed processing. It is also useful as a natural insecticide and as a filtration material.

Composition 2 is a sepiolite and bentonite composition (Anpro Advance ^® , Anpario pic, UK).

Bentonite clay is a clay mineral typically formed from volcanic ash. The two most common types are calcium bentonite and sodium bentonite. Sodium bentonite may consist largely of smectite and is commonly used as a binding agent in food pellets. Bentonite lm558 (an EU authorised feed additive) which may be in structurally modified form as shown in Figure 9B, may be utilised in the composition of the invention. Typical content analysis of the sepiolite (by x-ray fluorescence) +/- around 2% indicates SiC : 55%, AI2O3: 7.2%, MgO: 18.0%. For diatomaceous earth typical content is Si0 ₂ : 71%, AI2O3: 15.4%, MgO: 1.4%.

Content analysis of Composition 1 (by x-ray fluorescence) +/- around 2% indicates typical content is S1O2: 57%, AI2O3: 7.5%, MgO: 16.3%. Content analysis of Composition 2 (by x-ray fluorescence) +/- around 2% indicates Si0 ₂ : 57.6%, Al ₂ 0 ₃ : 11.6%, MgO: 11.4%.

The combination of bentonite and sepiolite together has an unexpected synergistic effect in relation to adsorption of sporidesmin. As illustrated in Figures 9A and 9B, sepiolite and bentonite have different structural properties, with the former having a rod and needle type structure and the latter having a folded layered structure. Having the surprising ability to effectively adsorb sporidesmin, the composition of the invention has utility in therapeutic and/or preventative treatments, including the treatments of side effects or reduction of symptoms associated with sporidesmin mycotoxicosis.

In this document, the terms pithomycotoxicosis and facial eczema are used interchangeably to describe a condition or disease having any of a range of downstream effects or symptoms caused by the mycotoxin sporidesmin, including but not limited to photosensitisation and hepatopathy.

Method

In vitro adsorption trials were performed by testing a fixed amount of each material towards the mycotoxin at known concentration (0.5pg/mL). The materials were tested at different dosages ie 0.05, 0.25 and 5.0 g/kg. Adsorption experiments were performed at constant temperature (37°C) and contact time (90 min). pH value of the medium was be set at 2.5 and 6.5.

Adsorption experiments were performed in triplicate and included mycotoxin controls and blank samples. Mycotoxin controls were buffered solutions of the toxin (without the adsorbent materials) processed as the test tubes and used to measure mycotoxin reduction and stability of the toxin during the incubation time or any unspecific binding of the toxin to the vessels. Blank samples were buffered suspensions of the materials (without the toxin) processed as the test tubes and used to assess any compound of the matrix that could interfere with the HPLC/UHPLC analysis of the mycotoxin.

The mycotoxin was analysed by High Performance Liquid Chromatography (HPLC) or Ultra High Performance Liquid Chromatography (UHPLC) with UV detection.

In vitro experiments were performed with two adsorbing compositions and activated carbon as a control. In vitro tests were performed with concentration of Sporidesmin equal to 0.5 pg/ml; buffer solutions at pH 2.5 and 6.5; adsorbent dosages equal to 0.5, 0.25 and 0.05% (w/v) (corresponding to 5, 2.5 and 0.5 Kg/Ton).

Sporidesmin (a mycotoxin produced by fungus Pithomyces Chartarum) was isolated and purified by AgResearch in New Zealand and 1 mg of total Sporidesmins dissolved in approximately 1 mL of methanol was received.

The standard solution of Sporidesmins was diluted with methanol to a final volume of 5 mL to obtain a stock solution at 200 pg/mL. Sporidesmin stock solution was stored in the dark at 4°C. The Sporidesmin stock solution was properly diluted with buffers at different pH values (2.5 and 6.5) to prepare the mycotoxin working solutions for adsorption experiments.

Results

As illustrated in Figure 1A, the analythical method of the present invention utilised optimized (UHPLC-PDA), which was able to separate five different sporidesmin congeners (indicated as peaks 1-5) all showing similar absorption spectra. Peak number 2 corresponds to sporidesmin A, which is the most abundant congener. Sporidesmins are composed of multiple congeners. These are sporidesmin A (often simply termed "sporidesmin") and sporidesmins B-J. The basic structure common to all sporidesmin congeners is a 2,5-dioxoiperazine skeleton formed from tryptophan and alanine. The dioxopiperazine ring is bridged by a disulfide chain in sporidesmin A and B, where sporidesmin E has a trisulfide chain and sporidesmin G a tetrasulfide chain. In C, D and F the sulfide bridges are modified or eliminated.

Sporidesmin A (which term is often used interchangeably with sporidesmin) is the chief toxic component that makes up over approximately 90% of the sporidesmin congeners produced in culture, the remainder being smaller quantities of the less toxic components B, C, D, E, F, G, FI and J.

With reference to Figure IB and Table 1 below, at a toxin level of 0.5 pg/ml 71.9% of Sporidesmin D was adsorbed by Composition 1 (the DE/Sepiolite composition) at pH 2.5, using an adsorbent dosage of 0.5% (w/v). 77.2% was adsorbed using Composition 2. The percentage adsorption in both cases was lower at pH 6.5.

Table 1 (Peak 1 Results - Sporidesmin D)

*Adsorbent dosage (w/v) Values are meanisd of three independent experiments

Toxin level = 0.5 pg/ml With reference to Figure 2 and Table 2 below, at a toxin level of 0.5 pg/ml 28.4 % of Sporidesmin A was adsorbed by Composition 1 (the DE/Sepiolite composition) at pH 2.5, using an adsorbent dosage of 0.5% (w/v). 70.6% was adsorbed using Composition 2 (ie with the addition of further components bentonite and activated carbon). The percentage adsorption in both cases was lower at pH 6.5 but Composition 2 still achieved binding of over 50% of sporidesmin at pH 6.5.

Table 2 (Peak 2 Results - Sporidesmin A)

*Adsorbent dosage (w/v) Values are meanisd of three independent experiments

Toxin level = 0.5 pg/ml

Table 3 (Peak 3 Results)

*Adsorbent dosage (w/v) Values are meanisd of three independent experiments

Toxin level = 0.5 pg/ml Table 4 (Peak 4 Results - Sporidesmin E)

*Adsorbent dosage (w/v) Values are meanisd of three independent experiments

Toxin level = 0.5 pg/ml

Table 5 (Peak 5 Results - Sporidesmin B)

*Adsorbent dosage (w/v) Values are meanisd of three independent experiments

Toxin level = 0.5 pg/ml

Figures 6 to 8 illustrate and summarize comparative adsorption results of Compositions 1 and 2 at adsorbant dosages of 0.05 w/v (Figure 6), 0.25 w/v (Figure 7) and 0.5 w/v (Figure 8). Referring to Figure 8, the adsorption of sporidesmins by Composition 2 was significantly higher than the adsorption recorded by Composition 1 at pH 2.5. The adsorption of sporidesmins by Composition 2 was also higher than the adsorption recorded by Composition 1 at pH 6.5 (although the difference was not so marked in respect of most congeners adsorbed).

Although some clays and other similar minerals have been utilised in mycotoxin binders for common mycotoxins such as aflatoxin, it has been surprisingly found that a composition comprising a mixture of naturally derived clay (and optionally clay-type) sedimentary minerals is able to effectively bind sporidesmin over a range of pH concentrations.

The difficulties in predicting binding capabilities of adsorbents in relation to sporidesmin is evidenced by the Results, which clearly show that particular dosage affected the outcome, different congeners were bound with significantly different levels of efficacy and that pH significantly affected binding of all sporidesmins.

As shown in Figure 6, at inclusion of 0.5% (w/v), Composition 2 effectively binds over 70% of Sporidesmin A and all other sporidesmin congeners tested at pH 2.5. Composition 2 also binds more than 50% of sporidesmin A and E at pH 6.5.

This is important in ruminants, where sporidesmin A is believed to be the most abundant and main causative agent of facial eczema (with other sporidesmin congeners having a lesser but nonetheless contributary role), and wherein it is desirable to bind sporidesmin effectively both in the rumen, (typically having a pH or around 6-6.5) and the intestine (having much lower pH).

Figures 10A and 10B show results of adsorption by Composition 2 of sporidesmins represented by peak 2, 1 and 4 (corresponding to sporidesmins A, D and E respectively) as ng of toxin per mg of product. Peak 2, corresponding to Sporidesmin A, was the most adsorbed compound, followed by Peak 4 (sporidesmin E) and Peak 1 (sporidesmin D).

Sporidesmin Adsorption Results - Isotherm Study

Adsorption isotherms were used to calculate the parameters (maximum adsorption capacity, adsorption affinity) related to the mycotoxins adsorption, calculate the C ₅ o and Cioo parameters and evaluate the effect of pH on adsorption process.

Two sets of Adsorption isotherms were obtained at constant temperature (37 °C) and constant pH (6.5 or 2.5) by testing a fixed amount of sporidesmin with increasing amounts of adsorbent (ISOTHERMS 1) or testing a fixed amount of adsorbent towards standard solutions containing increasing concentrations of sporidesmins (ISOTHERMS 2).

Adsorption isotherms 1 were obtained by plotting the amount of mycotoxin adsorbed in percentage as a function of the ratio of Adsorbent amount/Sporidesmin concentration

Adsorption isotherms 2 were obtained by plotting the amount of bound mycotoxin per unit mass of product as a function of sporidesmin concentration at equilibrium.

The curve fitting of experimental data was performed using non-linear regression method. Experimental data were analysed by mathematical models to calculate adsorption parameters

Kinetic study

A kinetic study was utilised to evaluate the kinetic model of mycotoxin adsorption (pseudo first order and pseudo second order model) and calculate the specific rate constants.

The experiments were performed using a constant adsorbent amount (0.2 or 2% for Composition 2 (Anpro Advance, Anpario pic) and Composition 1 (Anpro, Anpario pic), respectively), pH (6.5), temperature (37°C) and sporidesmin concentration (100 ng/mL), and by varying the contact time of the products with the toxins. The amount of sporidesmin adsorbed per mg of product was plotted as function of time. Experimental data were plotted using both the non-linear pseudo first order model (PFO) and the non-linear pseudo second order model (PSO).

Results - Dosage Effect

As shown in Tables 6A to 6C below and as illustrated in Figures 11 to 15, it was found that sporidesmin adsorption was affected by adsorbent dosage.

Figures 12A to 12C illustrate the dosage effect results corresponding to Composition 2. Figures 13A to 13C illustrate dosage effect results corresponding to Composition 1. Composition 2 confirmed its higher efficacy in adsorbing sporidesmins with respect to Composition 1.

Sporidemins adsorption by Composition 2 was slightly higher at pH 2.5 (lower values for C ₅ o and Cioo parameters). The efficacy of Composition 2 in adsorbing sporidesmins congeners decreased in the following order: Peak2 > Peak4 > Peakl. Sporidesmin A was the most adsorbed congener.

Taking into account the values of C ₅ o and Cioo parameters, 10 and 14 mg of Composition 2 adsorbed 50% of 1 pg of sporidesmin A (Peak2) at pH 2.5 and 6.5, respectively. A =5 fold higher dosage (50 mg of product) can adsorb all toxin (1 pg) as illustrated by comparison of the results shown in Figure 14. Figure 15 illustrates the overall results for C ₅ o values. At pH 2.5 the amount of Composition 2 product able to reduce 50% of Sporidesmins was significantly lower (up to 7 times) than Composition 1. At pH 6.5 the amount of Composition 2 product able to reduce 50% of Sporidesmins was significantly lower (10-15 times) than Composition 1 product.

Table 6A Peak 1 Results

Table 6B Peak 2 Results Table 6C Peak 4 Results

Results - Toxin Concentration Effect

Figure 16 illustrates the effect of Peak 2 (sporidesmin A) concentration on the amount of sporidesmin A adsorbed per mg of product under experimental conditions of : pH: 2.5 and 6.5, Composition 2 dosage: 2 mg/mL, Composition 1 dosage: 20 mg/mL, Toxins concentrations: 0.025 - 0.5 pg/mL, (7 experimental data points), Triplicate independent experiments. Where:

B _max : maximum adsorption capacity

Ki_: affinity (its reciprocal is the concentration to have the surface half-occupied from the toxin)

Figures 17A to 17B show results for Composition 2 adsorption of peaks 1, 2 and 4.

Figures 18A to 18B show results for Composition 2 adsorption of peaks 1, 2 and 4 Figures 19 and 20 illustrate a summary of results of the investigation into toxin concentration effect. Composition 2 confirmed its higher efficacy in adsorbing sporidesmins with respect to Composition 1 product (higher B _max values). Maximum adsorption of sporidesmins by Composition 2 was significantly higher at pH 2.5 (higher B _max values). Affinity for sporidesmins adsorption by Composition 2was significantly higher at pH 6.5 (higher KL values). Sporidesmin A was adsorbed with high capacity and affinity. The values of B _max calculated for the combination AA/sporidesmin A, i.e. 54 and 26 ng tox adsorbed/mg of product at pH 2.5 and 6.5, are quite close to those obtained by ISOTHERMS 1. Results - Time Contact Effect - Kinetic

Figure 21 illustrates time contact effect on adsorption of Sporidesmin A per mg of product under experimental conditions of : pH: 6.5, Composition 2 dosage: 2 mg/mL, Composition 1 dosage: 20 mg/mL, [Sporidesmin]: 100 ng/mL, Contact Time: 2 - 120 min, (10 experimental data points.

Triplicate independent experiments. Where: q : binding capacity at time t (mg toxin/g binder) q _e : maximum binding capacity (mg toxin/g binder) ki: pseudo first order specific rate constant (1/min) l< : pseudo second order specific rate constant (g binder/mg toxin*min)

AA: Composition 2

Figures 22 to 24 show results for adsorption of sporidesmins by Composition 2 over time.

Higher values of R ² and lower values of X ² indicate a better fitting of experimental data. Figure 25 shows results for adsorption by Composition 1 under experimental conditions: pH: 6.5, Composition 1 dosage: 20 mg/mL, [Sporidesmin]: 100 ng/mL, Contact Time: 2 - 120 min , (9 experimental data points). Triplicate independent experiments. Using a dosage of Composition 1 (2%, w/v), maximum adsorption was reached after few minutes of contact.

Sporidesmins maximum adsorption onto Composition 2 required about 60 min. Kinetics followed the pseudo second order model (PSO), which explains the adsorption kinetic when initial adsorbate concentration in solution is not too high and the adsorption onto the active sites is the main mechanism and rate-controlling step of the adsorption process. Maximum adsorption values (q _e ) are in accordance with previous trials. Sporidesmin A was the congener adsorbed with highest efficiency, being k the lowest. In accordance with previous trials (isotherms 1 and 2), sporidesmins adsorption efficiency decreased in the following order: peak 2 > peak 4 > peak 1 at pH 6.5 (highest q _e and lowest k ).

Results - Conclusions

Maximum Adsorption Values of Anpro Advance calculated for sporidesmin A at different pH values by different adsorption experiments and expressed as ng toxin adsorbed / mg of product Composition 2 confirms its higher efficacy in adsorbing sporidesmins with respect to Composition 1 product. Maximum adsorption of sporidesmins by Composition 2 was significantly higher at pH 2.5 (higher B _ma x values obtained by Isotherms 2); while affinity for sporidesmins adsorption was higher at pH 6.5 (higher K _L values). Sporidesmin A was adsorbed with high capacity / affinity and was the most adsorbed congener followed by peaks 4 and 1. Maximum adsorption occurred in almost 60 min. Taking into account the values of B _ma x calculated for the combination Composition 2 /sporidesmin A, i.e. 54 and 26 ng tox adsorbed/mg of product at pH 2.5 and 6.5, it can be calculated that about 20 - 40 mg of Anpro Advance can adsorb 1 pg of sporidesmin A. From a practical point of view, considering that ruminants (such as sheep) are susceptible to sporidesmins (especially sporidesmin A) on a pg/kg basis (ppb), the results suggest that 1 g of Composition 2 /kg feed (0.1%) should be effective in a feed containing ca. 20-50 pg sporidesmin A/kg feed, once consumed.

Based on the results, it is envisaged that higher dosage amounts would lead to higher levels of binding. The results suggest that both compositions 1 and 2 are effective for adsorbing toxins and/or reducing bioaccessibility of toxins in the liquid rumen, taking into account thermodynamic parameters related to the adsorption process in vivo.

It is further envisaged that the composition of the invention may be utilised in combination with conventional zinc dosing treatments for pithomycotoxicosis. Advantageously, when used in combination with the present invention, zinc dosing levels can be reduced, such that zinc toxicity may be avoided, even when attempting to treat established outbreaks.

At present, dietary intake of elemental zinc at 20mg/kg liveweight animal per day is recommended a few weeks before pastures are at risk of becoming toxic, in order to maintain a protective blood serum zinc level of between 20-30pmol/L. This is known as "prevention dosing".

Elemental zinc may be administered to animals as zinc oxide in feed or oral drench. Intra-ruminal boluses may provide sustained release of zinc oxide. Zinc sulphate can also be given in drinking water. "Crisis dosing" during danger periods suggests that dietary intake of elemental zinc at 25- 28mg/kg liveweight animal per day may be appropriate where the animals have not previously been treated. Such "danger periods" may be identified or predicted by monitoring spore count, either in the pasture, or as faecal spore counts. Also, certain weather conditions that may be favourable to germination or sporulation can be monitored. Germination can be year round and a low number of P. chartarum spores may persist even in winter. However, most development of P. chartarum is from mid-summer until the death of the ryegrass pasture and sporulation is mainly seasonal. Sporidesmin is concentrated in the spores during sporulation and persists for some time after. The "danger period" in some cases may be over 100 days.

It is recommended that when pasture spore counts are upwards of around 20,000 spores/g and weather conditions favour sporulation, farmers should implement prevention strategies.

Even with close monitoring of increasing spore counts, cumulative effect of ingesting low levels of sporidesmin over longer time periods can be equally dangerous for the animals. For example, grazing a pasture with a spore count of around 10,000 for ten days could have the same effective toxicity as an animal grazing a pasture with a spore count of 20,000 for just one day.

Advantageously, the present invention provides a wholly natural composition that can be administered long term to an animal, as part of a daily feed, without any adverse effects or cumulative adverse effects over time. The composition thus is able to provide a cost effective level of protection against sporidesmin all year round and the dosage and/or formulation may be modified in "danger periods" for increased adsorption. In one example, Composition 1 (which provides a particularly low cost composition having a moderate level of sporidesmin adsorption) is administered to animals daily when Pithomyces chartarum spore count is low (eg 0-20,000 spores/g of pasture) and Composition 2 is administered in an effective amount when pasture spore count rises to between 20,000-80,000 spores/g. Composition 2 may be administered in a high dosage (optionally in addition to some low level elemental zinc ie much lower than in current zinc dosing recommendations) during periods when spore count is very high (eg 80,000-100,000 spores/g). Optionally, zinc oxide could be included as part of the formulation.

The composition may be provided as a feedstuff or feed additive. Various types of feedstuffs are envisaged. These include but are not limited to a pellet, mash, forage, concentrate, soluble, grain, grass, hay, fibre, molasses or meal. The composition may be administered in troughs in the field/pasture, or collecting yards at milking time (for cattle). It may be incorporated into a compound feed (eg meal or pellet) for feeding at pasture or in the parlour at milking, and/or mixed with some other portion of the daily diet of the animal.

Vets currently recommend six zinc boluses are administered substantially simultaneously to a 500kg cow to give six weeks of protection against sporidesmin. Each bolus is 132g and generally contains around 116.2g elemental zinc. This equates to 697.2g of zinc per 42 days - or 16,600mg of zinc per cow per day. If a 500kg cow consumes 16kg of dry matter, this equates to around lOOOmg Zn/kg of dry feed. Certain legal limits in cattle feed require lOOmg/kg dry feed or less and an upper tolerable limit established at 500mg/kg of dry feed for cattle. This highlights the problems associated with zinc dosing as a preventative measure for sporidesmin mycotoxicosis. It is envisaged that the composition of the invention may be administered alone or in combination with a much lower level of zinc, such as less than lOOmg/kg of dry feed per day.

The invention provides a number of advantages, which constitute a real breakthrough in the prevention and/or treatment of pithomycotoxosis (facial eczema).

Utilising the composition of the invention, a safe and effective animal feed (or feed additive) consisting of minerals all of natural origin and proven to be safe and non toxic for consumption can easily administered orally. Harmful sporidesmin metabolites can be reduced, removed and excreted from the affected animals, thereby reducing or negating harmful effects of sporidesmin. The composition advantageously is able to bind sporidesmin(s) over a wide ranging pH, which provides binding capabilities in both the rumen and intestine.

Thus, prevention and treatment of the symptoms of pithomycotoxosis can be achieved at low cost, with minimal environmental impact and without the need for supplementation with additional enzymes.

Further, the dosage required to prevent and treat facial eczema provides efficacy at levels with low risk of toxicity or sequestration of essential vitamins and minerals from the animal's digestive tract.

In fact, natural clays have many additional benefits for animal welfare and productivity. Ingesting clays can reduce the speed of food passage through the digestive tract (allowing increased digestion) and can reduce gastrointestinal gases and pathogens.