NL2034971B1 - Composition for use in treatment and/or prevention of necrotic enteritis - Google Patents

Abstract

The current invention relates to a composition for use in prevention and/or treatment of necrotic enteritis in an animal, wherein the composition comprises a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier. The invention further relates to a feedstuff admixture comprising a feedstuff and a composition having a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier, wherein said composition is added to said feedstuff, such that the concentration in said feedstuff admixture of said saponin comprising extract is at least 10 ppm.

Description

COMPOSITION FOR USE IN TREATMENT AND/OR PREVENTION OF

NECROTIC ENTERITIS

FIELD OF THE INVENTION

The present invention pertains to the technical field of compositions for use in the treatment and/or prevention of necrotic enteritis.

BACKGROUND

Necrotic enteritis (NE) is a result of overproliferation of Clostridium perfringens, a sporulated Gram-positive bacteria that is considered a member of the normal intestinal microbiota. Spores are a dormant form of life extremely resistant to desiccation, heat, disinfectants, and UV radiation. Due to the resistant nature of spores, Clostridia are for instance virtually ubiquitous in poultry houses around the world.

Antibacterial drugs are commonly used to prevent or control the disease. In recent years, the European Union has banned the use of in-feed antimicrobials or growth promoters, leading to an increase in disease outbreaks in broiler flocks in European countries.

This has led to an explosion of interest in understanding the pathogenesis of NE and investigating how it can be prevented by approaches other than the use of antibiotics.

There remains a need in the art for an improved composition for use in the treatment and prevention of necrotic enteritis, thereby reducing the need for in-feed antimicrobials or growth promoters.

SUMMARY OF THE INVENTION

The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a composition for use according to claim 1. More particular, the invention relates to a composition for use in prevention and/or treatment of necrotic enteritis in an animal, wherein the composition comprises a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier. The present invention provides compositions for use in the treatment and prevention of necrotic enteritis,

thereby reducing the need for in-feed antimicrobials, coccidiostats or growth promoters in the production of animals (for instance those raised for human consumption).

Preferred embodiments of the composition for use are shown in any of the claims 2 to 10.

Claim 2 describes the above-mentioned composition for use, comprising at least 0.225% saponins (w/w).

Claim 3 describes the above-mentioned composition for use, comprising at least 50% of an aluminosilicate carrier (w/w).

Claim 4 describes the above-mentioned composition for use, wherein said aluminosilicate carrier is an aluminium phyllosilicate.

Claim 5 describes the above-mentioned composition for use, wherein said aluminium phyllosilicate is selected from the palygorskite-sepiolite group.

Claim 6 describes the above-mentioned composition for use, wherein said necrotic enteritis results from a prior or concomitant infection in said animal, wherein said infection is preferably an Eimeria infection, said infection enabling a proliferation of growth of Clostridium perfringens.

Claim 7 describes the above-mentioned composition for use, wherein said infection is an Eimeria infection, said infection damaging the intestinal epithelium of the animal, thereby allowing the leakage of plasma proteins into the intestinal lumen.

Claim 8 describes the above-mentioned composition for use, wherein the saponin comprising extract is a Quillaja saponaria plant extract.

Claim 9 describes the above-mentioned composition for use, wherein said composition is added to a feedstuff in order to obtain a feedstuff admixture, such that the concentration in said feedstuff admixture of said saponin comprising extract is at least 10 ppm.

Claim 10 describes the above-mentioned composition for use, wherein said animal is a non-human mammal, a fish, a bird or an amphibian.

In a second aspect, the present invention relates to a feedstuff admixture according to claim 11. More particular, the invention relates to a feedstuff admixture comprising a feedstuff and a composition having a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier, wherein said composition is added to said feedstuff, such that the concentration in said feedstuff admixture of said saponin comprising extract is at least 10 ppm. Preferred embodiments of the feedstuff admixture are shown in claims 12-13.

Claim 12 describes the above-mentioned feedstuff admixture, wherein the concentration in said feedstuff admixture of said aluminosilicate carrier is at least 125 ppm.

Claim 13 describes the above-mentioned feedstuff admixture, wherein the feedstuff admixture is provided on an ad libitum basis.

DESCRIPTION OF FIGURES

The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.

Figure 1 illustrates that birds receiving the composition for use according to an embodiment of the invention (T3) show a decrease in feed conversion ratio (FCR).

Figure 2 shows the scoring system for determining the extent of footpad lesions in the experimental groups in examples 1 and 2.

Figure 3 shows the scoring system for determining the litter quality of the experimental groups in examples 1 and 2.

Figure 4 shows the ingredient composition and calculated nutrient content of the diet to which the feed additives (including the composition for use according to an embodiment of the current invention) are added in Example 3.

Figure 5 shows the body weight gain (BWG) of the experimental groups (including the group receiving the composition for use according to an embodiment of the current invention) in example 3.

Figure 6 shows the mortality-corrected feed conversion ratio (FCRm) of the experimental groups (including the group receiving the composition for use according to an embodiment of the current invention) in example 3.

Figure 7 shows the amount of oocysts/ gram excreta (across all periods) of the experimental groups (including the group receiving the composition for use according to an embodiment of the current invention) in example 3.

Figure 8 shows the percentage of binding activity of the different materials (including the aluminosilicate of the composition for use according to an embodiment of the invention) to the alpha toxin in comparison to the ‘no material’ control group, where no material was added, and no binding is seen (0%) (Example 4).

Figure 9 shows the percentage of hemolytic activity of rNetB toxin pre-incubated with specific materials (including the aluminosilicate of the composition for use according to an embodiment of the invention) on chicken red blood cells (Example 5).

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns compositions for use in the treatment and prevention of necrotic enteritis, thereby reducing the need for in-feed antimicrobials, coccidiostats or growth promoters in the production of animals raised for human consumption.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

As used herein, the following terms have the following meanings:

TA”, “an”, and “the” as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a compartment” refers to one or more than one compartment.

“Comprise”, “comprising”, and comprises” and “comprised of” as used herein are synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

The expression “% by weight”, “weight percent”, “%wt", “w/w” or “wt%”, here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation. “Feed efficiency” as used herein refers to a measure of an animal's efficiency in converting feed mass into the desired output, e.g., weight gain, milk production. “Feed efficiency” also may be referred to as “feed conversion ratio (FCR)”, “feed conversion rate”, or “feed conversion efficiency”. Mortality-corrected feed conversion ratio (FCRm) as used will be calculated from body weight gain, mortality weight gain and feed intake. Feed intake is determined on a per pen basis. Body weight gain is equally expressed as a summation of body weight gain in the pen. Mortality weight gain will be determined by the difference of the mortality body weight minus the initial body weight and totaled for the pen. FCRm will be calculated as the ratio of total pen feed intake to the summation of body weight gain and morality weight gain on a pen basis.

As used herein, the term "feedstuff” refers to anything that may be consumed by an animal. The term "feedstuff” encompasses solid and liquid animal feeds (e.g. a feed ration), supplements (e.g. a mineral supplement, a protein supplement), a premix, water, feed additive carriers (e.g. molasses), and combinations thereof.

According to the AIPEA (Association ternational pour I'Etude des Argiles,

International Association for the Study of Clays) and CMS (Clay Minerals Study) nomenclature committees, the term "mineral clay” refers to a mineral that imparts plasticity to a clay and hardens upon drying or firing. Mineral clays include aluminosilicates, such as aluminium phyllosilicates. Mineral clays usually include minor amounts of impurities, such as potassium, sodium, calcium, magnesium, and/or iron. “Saponin” as used herein refers to a class of chemical compounds, one of many secondary metabolites found in natural sources, with saponins found in particular abundance in various plant species. More specifically, they are amphipathic glycosides grouped, in terms of structure, by their composition. In certain embodiments, saponin comprises one or more hydrophilic glycoside moieties combined with a lipophilic triterpene and/or steroidal derivative. “Therapeutically effective amount” as used herein refers to a quantity or concentration of a specified compound, composition or combination sufficient to achieve an effect in a subject. More specifically, it refers to the minimum amount or concentration of a compound or composition that is effective to prevent or reduce the symptoms or to ameliorate the condition of a disease. “Therapeutically effective amount” as used herein can thus be used in the context of an amount sufficient for prevention and/or for treatment of necrotic enteritis.

Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any 23, =4, 25, >6 or >7 etc. of said members, and up to all said members.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

Reference throughout this specification to "one embodiment” or "an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.

Thus, appearances of the phrases "in one embodiment” or "in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments.

Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

Description

Necrotic enteritis (NE) is a result of overproliferation of Clostridium perfringens (C. perfringens, CP), a sporulated Gram-positive bacteria that is considered a member of the normal intestinal microbiota. Spores are a dormant form of life extremely resistant to desiccation, heat, disinfectants, and UV radiation.

Antibacterial drugs are commonly used to prevent or control the disease. In recent years, the European Union has banned the use of in-feed antimicrobials or growth promoters, leading to an increase in disease outbreaks in broiler flocks in European countries. Globally, the economic impact of the disease is estimated at US$ 2 billion year through mortalities and poor performance and the cost of prevention and treatment. In EU countries, the profit of severely NE affected broiler flocks has been 33% less than flocks with low level of the disease.

In recent years there has been an explosion of interest in understanding the pathogenesis of NE and investigating how it can be prevented by approaches other than the use of antibiotics.

Most of the time Clostridium perfringens is present in animals’ intestines without causing disease. Predisposing factors are needed to exacerbate this bacterium, stimulate toxin production and cause the clinical disease. One such predisposing factor for development of necrotic enteritis is coccidial infection, resulting either from natural disease outbreak or from introduction at low levels through live coccidiosis vaccination. Coccidial infection can damage the intestinal epithelium, allowing the leakage of plasma proteins into the intestinal lumen —a rich nutrient substrate that

C. perfringens can exploit for oroliferation and toxin production. This can reduce performance and predispose animals to necrotic enteritis.

Probiotics and phytogenic feed additives, such as saponins, can help alleviate the negative effects of coccidial infection. For instance, they have been shown to reduce oocyst shedding, severity of intestinal lesions, and adverse effects on performance.

The inventors have unexpectedly observed that by providing a source of saponin (a phytogenic anticoccidial feed additive) on an aluminosilicate carrier effective prevention and treatment of necrotic enteritis can be obtained.

As such, in a first aspect, the invention relates to a composition for use in prevention and/or treatment of necrotic enteritis in an animal, wherein the composition comprises a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier.

The animal may be a land animal, an aquatic animal, an avian, or an amphibian. The animal may be a mammal, or a non-mammal. The non-human animal can be an animal raised for human consumption or a domesticated animal. Examples of animals that can be fed and/or administered the disclosed composition for use include, but are not limited to, ruminant species, such as a sheep, goat, cow, deer, bison, buffalo, elk, alpaca, camel or llama; ungulates, such as a horse, donkey, or pig; avians, such as chickens, including laying hens and broilers, turkey, goose, duck, Cornish game hen, quail, partridge, pheasant, guinea-fowl, ostrich, emu, swan, or pigeon; aquatic animals, such as an aquaculture species, such as fish (e.g., salmon, trout, tilapia, sea bream, carp, cod, halibut, snapper, herring, catfish, flounder, hake, smelt, anchovy, lingcod, moi, perch, orange roughy, bass, tuna, mahi mahi, mackerel, eel, barracuda, marlin, Atlantic ocean perch, Nile perch, Arctic char, haddock, hoki,

Alaskan Pollock, turbot, freshwater drum, walleye, skate, sturgeon, Dover sole, common sole, wolfish, sablefish, American shad, John Dory, grouper, monkfish, pompano, lake whitefish, tilefish, wahoo, cusk, bowfin, kingklip, opah, mako shark, swordfish, cobia, croaker, or hybrids thereof, and the like), crustaceans (e.g., lobster, shrimp, prawns, crab, krill, crayfish, barnacles, copepods, and the like), or molluscs (e.g., squid, octopus, abalone, conchs, rock snails, whelk, clams, oysters, mussels, cockles, and the like). Additionally, or alternatively, the animal may be a companion animal, such as canines; felines; rabbits; rodents, such as a rat, mouse, hamster, gerbil, guinea pig or chinchilla; birds, such as parrots, canaries, parakeets, finches,

cockatoos, macaws, parakeets or cockatiel reptiles, such as snakes, lizards, tortoises or turtles; fish; crustaceans; and amphibians, such as frogs, toads and newts.

In an embodiment, said animal is a non-human mammal, a fish, a bird or an amphibian. In a preferred embodiment, the animal is poultry, such as chickens, turkeys, ducks, and geese. Necrotic enteritis (NE) in either clinical or subclinical form is a major enteric poultry disease that has a detrimental effect on profitability in the poultry industry. Antibiotic growth promoters (AGPs) have been widely used in food animal production for decades. However, after observing the effects of AGPs on the development and expansion of drug-resistant bacteria, people started to understand its potential threat, and there was increased pressure to decrease antimicrobial use in poultry production. NE occurs in broilers aged between two and six weeks and mortality can reach 1% per day with a total mortality of 10-40%. Clinical signs include depression, dehydration, diarrhea, ruffled feathers and lower feed intake. The gross lesions of the small intestine range from thin and friable walls to frank and extensive necrotic lesions. Two forms of the disease are described, clinical and subclinical. The clinical form appears with the clinical signs and mortality noted above. The subclinical form presents as poor performance (reduced growth, reduced feed efficiency) without mortality. This form of the disease can be diagnosed by reduced feed conversion, by gross lesions in the small intestine and by bacteriology.

Most of the economic losses due to NE are related to the subclinical form and the high cost of preventing the disease with antibiotics and/or coccidiostats.

Toxins released by C. perfringens play a vital function in the incidence of NE. To date, more than 20 different types of C. perfringens toxins have been studied. For instance,

Poultry C. perfringens is categorized into 7 types (A-G). C. perfringens type A produces the a-toxin; type B produces the a-, B-, and e-toxins; type C produces the a- type, type E produce the a- and 1-toxins; and all types can produce the enterotoxin and B2-toxin. Each toxin type is associated with a particular human or animal disease, which suggests that C. perfringens virulence is linked to toxin and enterotoxin development. C. perfringens types A, C, and G are of particular interest to the poultry industry because they have been associated with diseases in avian species.

In a preferred embodiment, the invention relates to a composition for use in prevention and/or treatment of C. perfringens induced necrotic enteritis. In an embodiment, the invention relates to a composition for use in prevention and/or treatment of C. perfringens type A induced necrotic enteritis. In an embodiment, the invention relates to a composition for use in prevention and/or treatment of C.

perfringens type B induced necrotic enteritis In an embodiment, the invention relates to a composition for use in prevention and/or treatment of C. perfringens type C induced necrotic enteritis. In an embodiment, the invention relates to a composition for use in prevention and/or treatment of C. perfringens type D induced necrotic enteritis. In an embodiment, the invention relates to a composition for use in prevention and/or treatment of C. perfringens type E induced necrotic enteritis. In an embodiment, the invention relates to a composition for use in prevention and/or treatment of C. perfringens type F induced necrotic enteritis. In an embodiment, the invention relates to a composition for use in prevention and/or treatment of C. perfringens type G induced necrotic enteritis. In an embodiment, the invention relates to a composition for use in prevention and/or treatment of necrotic enteritis induced by a combination of any of the aforementioned types of Clostridium perfringens.

In an embodiment, said necrotic enteritis results from a prior or concomitant infection in said animal, wherein said infection is preferably an Eimeria infection, enabling a proliferation of growth of Clostridium perfringens.

It is estimated that C. perfringens colonizes over three-quarters of birds in any flock at any given time, but only small percentages develop NE. The intestinal number of

C. perfringens in healthy and in NE-affected birds are different. The C. perfringens population is found to be normally less than 10? to 10% colony-forming units (CFU) per g of the intestinal contents in the small intestine of healthy chickens compared to 107 - 10° CFU/g in diseased birds. An intact intestinal epithelium is the best defense against potential pathogens such as C. perfringens. The overgrowth of C. perfringens in the intestines has been suggested to occur because of a combination of events, including co-infection with coccidia. Not only Eimeria Spp., also other pathogens (e.g.

Salmonella Spp., Ascarid larvae, viruses) and agents, such as mycotoxins damaging the intestinal mucosa can pave the way for a C. perfringens infection.

In an embodiment, said necrotic enteritis results from a prior or concomitant Eimeria infection in said animal.

Coccidiosis is caused by protozoa of the phylum Apicomplexa, family Eimeriidae. Most species affecting poultry belong to the genus Eimeria and infect various intestinal sites. So far, seven species of Eimeria (E. acervulina, E. maxima, E. mitis, E. praecox,

E. necatrix, E. tenella, and E. brunetti) have been known to infect birds.

Eimeria species, such as E. acervuline, £ Tenia and E. maxima, are the most well- known predisposing factors for necrotic enteritis (NE) in chickens. Even though coccidiosis and NE are associated and have similar symptoms, coccidiosis usually develops before or during the NE phase. There is no clear mechanism showing how coccidia induces NE, and possible causes are proposed. Damage caused to the intestinal lumen, usually during coccidia propagation, leads to bleeding; thus, the leaked plasma proteins become a source of growth substrate for C. perfringens.

Furthermore, coccidiosis induces mucus production and provides a suitable environment for C. perfringens growth. By damaging the gut epithelium, Eimeria species give C. perfringens access to the intestinal basal domains of the mucosal epithelium. Then, the first phase of the pathological process takes place and from there, C. perfringens invades the lamina propria. Damage to the epithelium follows.

The plasma proteins leaking to the gut and the mucus produced are rich nutrient sources. This increase in available nutrients creates an environment favorable for the proliferation of C. perfringens. A further impact of Coccidiosis is shifting the microbial balance in the gut by decreasing the number of other bacterial species e.g.,

Candidatus savagella which activates the innate immune defense.

Saponins are a broad group of mostly plant-derived compounds that have been found to exert numerous biological effects in animal applications. They are typically composed of a hydrophobic aglycone, which is extensively decorated with functional groups prior to the addition of hydrophilic sugar moieties, to result in surface-active amphipathic compounds. Saponins are broadly classified as triterpenoids, steroids or steroidal glycoalkaloids, based on the aglycone structure from which they are derived. For more than 50 years saponins have been investigated for their potential application in animal production due to their abilities to reduce pathogen loads, influence nutrient uptake, regulate ammonia production, and affect the growth process. Saponins can be found in the botanical families of Quillajaceae,

Caryophyllaceae, Sapindaceae, Aceraceae, Hippocastanceae, Cucurbitancae,

Araliaceae or Asparagaceae. For instance, it is suggested that saponins derived from

Quillaja saponaria, the Chilean soap bark tree, and Yucca schidigera, a dessert plant of the American southwest, have great potential in poultry production due to these effects. Saponins have been shown to reduce oocyst shedding by Eimeria, reduce the severity of intestinal lesions and adverse effects on performance and are thus able to reduce coccidial infection and hence the predisposition towards necrotic enteritis.

The composition for use of the current invention comprises a therapeutically effective amount of such a saponin comprising extract on an aluminosilicate carrier.

In an embodiment, the composition for use comprises at least 1.5%, preferably at least 3%, more preferably at least 5%, more preferably at least 8%, more preferably at least 9%, more preferably at least 10%, more preferably at least 11%, more preferably at least 12%, more preferably at least 13%, more preferably at least 14%, such as 15% saponin comprising extract. In an embodiment, the composition for use comprises 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% saponin comprising extract.

In a preferred embodiment, the composition for use comprises at least 0.225%, preferably at least 0.50%, more preferably at least 0.75%, more preferably at least 1.00%, more preferably at least 1.25%, more preferably at least 1.50%, more preferably at least 1.75%, more preferably at least 2.00%, such as 2.25% saponins (w/w). In an embodiment, the composition for use comprises 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 2.00%, 2.25%, 2.50%, 2.75%, 3.00%, 3.25%, 3.50%, 3.75%, 4.00%, 4.25%, 4.50%, 4.75% or 5.00% saponins (w/w). In a preferred embodiment, the composition for use comprises between 2% and 2.5% (w/w), such as 2.25% (w/w) saponins.

In an embodiment, the saponin comprising extract is a Yucca schidigera plant extract.

In a preferred embodiment, the saponin comprising extract is a Quillaja Saponaria plant extract.

Preferably, the saponin is derived from the family of Quillajaceae. In particular, the saponin is derived from the genus Quillaja. Examples of quillaja include, but are not limited to, Quillaja brasiliensis, Quillaja lanceolata, Quillaja lancifolia, Quillaja molinae, Quillaja petiolaris, Quillaja poeppigii, Quillaja saponaria, Quillaja sellowiana,

Quillaja smegmadermos, or a combination thereof. Most preferably, the saponin is derived from the species Quillaja Saponaria.

Saponins can be found in various parts of the plant such as: leaves, stems, roots, bulbs, blossom and fruit. Methods of extracting the source of saponin from plants are known. A person of ordinary skill in the art will appreciate that, as used herein, a plant name may refer to the plant as a whole, or to any part of the plant, such as the roots, stem or trunk, bark, leaves, flower, flower stems, or seeds or a combination thereof. These plant parts may be used fresh, or dried, and may be whole, pulverized,

or comminuted. The name may also refer to extracts from any part or parts of the plant, such as chemical extracts, or extracts, including the residues, obtained by pressing, or any other methods of concentrating or extracting oils or other extracts known to those in the art or that are hereafter discovered.

In the composition for use according to the currentinvention, said saponin comprising extract is provided on an aluminosilicate carrier.

In a preferred embodiment, the aluminosilicate is processed/activated. “Activation” as used herein means that the aluminosilicate undergoes various processes such as extrusion, heat treatment (to fix the pore sizes), and particle size reduction to obtain specific desirable chemical and physical properties in accordance with its use.

In an embodiment, the aluminosilicate is extruded. In an embodiment, the aluminosilicate is thermally processed. In an embodiment, said thermal processing occurs at a temperature between 200°C and 400°C, preferably between 300°C and 400°C, such as 350°C. In an embodiment, the aluminosilicate is ultra finely milled.

Clays are found mixed or associated with other minerals and amorphous materials.

The identification of clays in a raw clay or soil always requires a purification step. This is because the presence of carbonates, iron oxides, organic materials, and the like interferes with the identification procedure. Purification is very important for the application of clays as a carrier material for other compounds. A general method for obtaining purified clays is fractionation by sedimentation after the removal of carbonates, hydroxides, and organic materials. In an embodiment, said aluminosilicate is fractionated, for instance through air classification. However, 100% pure clay may be achieved only at the laboratory scale rather than at an industrial scale. Even then, no more than 90% enrichment is usually achievable. In an embodiment, the aluminosilicate carrier has a purity of at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, such as 80%.

In an embodiment, the composition for use comprises at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, such as 85% of an aluminosilicate carrier (w/w). In an embodiment, the composition for use comprises 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,

75%, 76%, 77%, 78%, 79%, 80%, 31% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% of an aluminosilicate carrier (w/w).

Among all minerals, silicates constitute the largest and most interesting and complex class of minerals available on earth. One SiO4 tetrahedron can link to another and another and so forth. These silicate tetrahedrons can form complex structures. They can form single units (known as nesosilicates), double units (known as sorosilicates), chains (known as inosilicates), sheets (known as phyllosilicates), rings (known as cyclosilicates), and framework structures (known as tectosilicates).

In a preferred embodiment, the used clay belongs to the family of phyllosilicates, that is, layered or sheetlike structures more commonly referred to as layered silicates (LSs). In this subclass, rings of tetrahedrons are linked by shared oxygen atoms to other rings on a two-dimensional plane that produces a sheetlike structure. The silicon-to-oxygen ratio is generally 1:2.5 (or 2:5) because only one oxygen atom is exclusively bonded to the silicon and the other three are half-shared to other silicon atoms. The symmetry of the members of this group is controlled chiefly by the symmetry of the rings but is usually reduced to a lower symmetry by other ions and other layers. The typical crystal habit of this subclass is, therefore, flat, platy, and book like and always displays good basal cleavage.

In an embodiment, said aluminosilicate carrier is an aluminium phyllosilicate. The main structural components of phyllosilicates consist of silica dominating tetrahedral sheets and aluminium or magnesium dominating octahedral sheets bound together by shared oxygen atoms. Phyllosilicates can be divided into several categories based on the number of tetrahedral and octahedral sheets and the arrangement of these sheets. The sheet structure and arrangement will determine the physical and chemical properties of phyllosilicates.

In a preferred embodiment, said aluminium phyllosilicate is chosen from the palygorskite-sepiolite group. The palygorskite-sepiolite group consists of palygorskite, sepiolite, falcondoite, kalifersite, loughlinite, raite, tuperssuatsiaite, and yofortierite.

By providing such a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier, the amount of damaging C. perfringens toxins in the intestines is reduced, thereby providing a more effective prevention and treatment of necrotic enteritis. The current invention allows to simultaneously reduce one of the exacerbating factors (reduction of coccidin infection by means of the provision of a source of saponin) and reduce the amount of C. perfringens toxins itself (by means of the provision of an aluminosilicate carrier), thereby reducing the incidence of necrotic enteritis. A reduced number of Clostridium perfringens and/or Clostridium perfringens toxins within the intestinal tract may lead to the prevention or alleviation of necrotic enteritis.

The composition for use according to the current invention is able to reduce subclinical NE, as for instance evidenced by an improved Feed Conversion Ratio (FCR) (see for instance the experimental data provided in examples 1 and 2) and/or body weight gain in broilers. Furthermore, in broilers receiving a coccidiosis challenge (used/dirt litter containing oocysts shed by seeder birds), animals receiving the composition for use according to the current invention have a similar body weight gain and a similar FCR as animals not receiving the coccidiosis challenge and infection is kept under control with a similar amount of oocyst (see experimental data provided in example 3).

Subclinical NE can also be associated with deterioration in the litter material, increasing the risk of foot pad dermatitis, hock burn and breast blisters, three conditions that are large welfare problems for the industry. The composition for use according to the current invention is able to reduce subclinical NE, as evidenced by a reduced deterioration of the litter material and, related thereto, a reduced footpad score (see experimental data provided in examples 1 and 2).

As described above, C. perfringens often colonizes over three-quarters of birds in any flock at any given time, but only small percentages develop NE. The intestinal number of C. perfringens in healthy and in NE-affected birds are different. The C. perfringens population is found to be normally less than 10? to 10% colony-forming units (CFU) per g of the intestinal contents in the small intestine of healthy chickens compared to 107 - 10° CFU/g in diseased birds. Evidence shows that necrotic enteritis is produced when Clostridia reach high numbers within the intestine and that C. perfringens virulence is linked to toxin and enterotoxin development.

For instance, recent data has proposed a novel NetB toxin as the causative agent for necrotic enteritis in poultry. This finding was supported by a knockout mutant of the bacteria that was only able to reproduce the disease when complemented with the wild type NetB gene. NetB toxin is a pore forming protein able to bore 1.6 — 1.8 nm diameter hydrophilic pores into cell membranes. Another important toxin is the chromosomal-encoded alpha-toxin, a or ospholipase C sphingomyelinase that hydrolyzes phospholipids and promotes membrane disorganization.

In an embodiment, the composition for use is able to reduce or mitigate the action of one or more types of Clostridium perfringens toxins (eg alpha toxin or NetB toxin}, thereby preventing or treating necrotic enteritis in an animal at risk. In an embodiment, the composition for use is able to reduce or mitigate the action of one or more types of Clostridium perfringens toxins, by binding to the Clostridium perfringens toxins. In an embodiment, the composition for use is able to bind alpha

Clostridium perfringens toxin. In an embodiment, the composition for use is able to bind NetB Clostridium perfringens toxin. In an embodiment, the composition for use is able to reduce or mitigate the action of one or more types of Clostridium perfringens toxins, by inhibiting the hemolytic activity of the Clostridium perfringens toxins. In an embodiment, the composition for use is able to inhibit the hemolytic activity of NetB Clostridium perfringens toxins. The aluminosilicate carrier of the composition for use of the current invention is able to reduce or mitigate the action of one or more types of Clostridium perfringens toxins (eg alpha toxin or NetB toxin, see Examples 4 and 5).

The composition may be used to replace or supplement animal feedstuffs, or it may be administered separately from a feedstuff. In some embodiments, the feedstuff is a commercial feedstuff. The composition may be formulated in any form suitable for mixing with a feedstuff and/or replacing a feedstuff, including a powder, a granule, a pellet, a solution, or a suspension. Certain disclosed embodiments are formulated as a dry, free-flowing powder. This powder is suitable for direct inclusion into a commercially available feed, food product or as a supplement to a total mixed ration or diet. The powder may be mixed with either solid or liquid feed and/or with water.

In other embodiments, the composition is formed into pellets, and in further embodiments, the composition is formulated into granules, such as floating or sinking granules, suitable for feeding to aquatic animals. The composition for use according to the invention may be used in combination with a complete and balanced feed which provides all the recommended vitamins and minerals for the animal in question.

In some embodiments, the composition is administered daily to the animal at time intervals believed or determined to be effective for achieving a beneficial result. The composition may be administered in a single dose daily or in divided doses throughout the day. In an embodiment, the composition for use is administered on an ad libitum basis. Typically, a time period over which the combination is administered is sufficient such that the animal received a benefit from the composition.

In a preferred embodiment, said composition is added to a feedstuff in order to obtain a feedstuff admixture, said feedstuff admixture comprising an effective amount of the composition for use according to the current invention. In an embodiment, said composition for use may range from 0.001 to 100 kg per ton of feedstuff. In some embodiments, said composition for use is added in a sufficient amount such that the composition for use is added in amounts of from 0.001 to 10 kg per ton, such as 0.2 kg or 2 kg per ton of feedstuff. In a preferred embodiment, 200 gram of the composition is added to 1 ton of feedstuff. When expressed as a percentage of dry matter of feed, the composition for use is added in sufficient amount such that the composition may be added to animal feedstuffs in amounts ranging from 0.0001 to 2.5% by weight, such as from 0.0125% to 2% by weight, from 0.01 to 0.05 % by weight, such as 0.02% by weight.

In a preferred embodiment, said composition is added to a feedstuff in order to obtain a feedstuff admixture, such that the concentration in said feedstuff admixture of said saponin comprising extract is at least 10 ppm. In an embodiment, the concentration in said feedstuff admixture of said saponin comprising extract is 10 ppm, 11 ppm, 12 ppm, 13 ppm, 14 ppm, 15 ppm, 16 ppm, 17 ppm, 18 ppm, 19 ppm, 20 ppm, 21 ppm, 22 ppm, 23 ppm, 24 ppm, 25 ppm, 26 ppm, 27 ppm, 28 ppm, 29 ppm, 30 ppm, 31 ppm, 32 ppm, 33 ppm, 34 ppm, 35 ppm, 36 ppm, 37 ppm, 38 ppm, 39 ppm, 40 ppm, 41 ppm, 42 ppm, 43 ppm, 44 ppm, 45 ppm, 46 ppm, 47 ppm, 48 ppm, 49 ppm, 50 ppm, 75 ppm, 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 400 ppm, 450 ppm, 500 ppm or 1000 ppm.

In a second aspect, the invention relates to a feedstuff admixture comprising a feedstuff and a composition having a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier, wherein said composition is added to said feedstuff, such that the concentration in said feedstuff admixture of said saponin comprising extract is at least 10 ppm. In an embodiment, the concentration in said feedstuff admixture of said saponin comprising extract is 10 ppm, 11 ppm, 12 ppm, 13 ppm, 14 ppm, 15 ppm, 16 ppm, 17 ppm, 18 ppm, 19 ppm, 20 ppm, 21 ppm, 22 ppm, 23 ppm, 24 ppm, 25 ppm, 26 ppm, 27 ppm, 28 ppm, 29 ppm, 30 ppm, 31 ppm, 32 ppm, 33 ppm, 34 ppm, 35 ppm, 36 ppm, 37 ppm, 38 ppm, 39 ppm, 40 ppm, 41 ppm, 42 ppm, 43 ppm, 44 ppm, 45 ppm, 46 ppm, 47 ppm, 48 ppm, 49 ppm, 50 ppm, 75 ppm, 100 oom 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 400 ppm, 450 ppm, 500 ppm or 1000 ppm.

In an embodiment, the concentration in said feedstuff admixture of said aluminosilicate carrier is at least 125 ppm. In an embodiment, the concentration in said feedstuff admixture of said aluminosilicate carrier is 125 ppm, 130 ppm, 140 ppm, 150 ppm, 160 ppm, 170 ppm, 180 ppm, 190 ppm, 200 ppm, 210 ppm, 220 ppm, 230 ppm, 240 ppm, 250 ppm, 260 ppm, 270 ppm, 280 ppm, 290 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm or 1000 ppm.

In an embodiment, the invention relates to a feedstuff admixture comprising a feedstuff and a composition having a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier, wherein said composition is added to said feedstuff, such that the concentration in said feedstuff admixture of said saponin comprising extract is at least 10 ppm and the concentration in said feedstuff admixture of said aluminosilicate carrier is at least 125 ppm.

The feedstuff admixture of the invention is any composition which an animal may consume as (part of) its diet. It can be any feedstuff, such as dry product, semi moist product, wet food product or a liquid and includes a food supplement, a snack or a treat. Thus, the invention covers standard feed products including liquids, as well as pet food snacks (for example, snack bars, pet chew, crunchy treat, cereal bars, snacks, biscuits and sweet products) and supplements.

The composition having a therapeutically effective amount of a saponin comprising extract on an aluminosilicate carrier may be incorporated in a gelatinized starch matrix, or in any dry or wet feedstuffs or supplements. Methods of incorporation are known in the art.

The feedstuff admixture of the invention may be a dry product (with approximately 5 to approximately 15% moisture), a semi-moist product (with approximately 15 to approximately 70% moisture) or a wet product (with approximately 70 to approximately 90% moisture). The feedstuff comprised in the admixture is not essential to the invention and typical standard products can be included. The combined ingredients of the feedstuff can provide all of the recommended vitamins and minerals for the particular animal in question (a complete and balanced food).

The feedstuff admixture can be provided gl feed supplement. The feed supplement can be a granule, a powder, sauce, topping, biscuit, kibble, pocket or tablet that can be administered with or without an additional feedstuff. Where the feed supplement is administered with an additional feedstuff, the feed supplement can be administered sequentially simultaneously or separately. The feed supplement may be mixed with the feedstuff, sprinkled over the feedstuff or served separately. Alternatively, the feed supplement can be added to a liquid provided for drinking such as water or milk.

Additional components in the feedstuff admixture may be used for any desired purpose, such as a substantially biologically inert material added, for example, as a filler, or to provide a desired beneficial effect. For example, the combination may include a carbonate (including a metal carbonate such as calcium carbonate); a trace mineral, such as, but not limited to, chloride, fluoride, iodide, chromium, copper, zinc, iron, magnesium, manganese, molybdenum, phosphorus, potassium, sodium, sulfur, selenium, or a combination thereof; a bulking agent; a carrier; a colorant; a taste enhancer; a preservative; or a combination thereof. The preservative may be benzoic acid or a salt thereof, e.g. sodium benzoate; lactic acid or a salt thereof, e.g. sodium lactate, potassium lactate or calcium lactate; propionic acid or a salt thereof, e.g. sodium propionate; ascorbic acid or a salt thereof, e.g. sodium ascorbate; gallic acid or a salt thereof e.g. sodium gallate; sulfur dioxide and/or sulfites; nitrites; nitrates; choline, or a salt thereof, such as an anion salt of choline, e.g. choline halide, such as chloride, bromide, iodide, fluoride, or choline hydroxide; or any combination thereof.

Additionally, or alternatively, the combination may further comprise corn, soybean meal, soybean oil, wheat, barley, rye, rice hulls, canola, corn oil, limestone, salt, distillers dried grains with solubles (DDGS), dicalcium phosphate, sodium sesquicarbonate, methionine source, lysine source, L-threonine, mineral oil, biotin, folic acid, kelp, menadione dimethylpyrimidinol bisulfite, calcium aluminosilicate, or any combination thereof.

In a preferred embodiment, the feedstuff admixture is provided on an ad libitum basis.

The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

EXAMPLES AND/OR DESCRIPTION OF FIGURES

The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures.

Example 1. Evaluation of the effect of a composition for use according to an embodiment of the invention on broiler performance.

Introduction

The objective of the trial was to evaluate the effect of a composition for use according to an embodiment of the invention on broiler performance under normal conditions with a dietary challenge. The composition for use according to an embodiment of the invention included saponins (origine Quiflaja) with an aluminium phyllosilicate selected from the palygorskite-sepiolite group as carrier. The composition for use according to an embodiment of the invention was added to a feedstuff in a concentration of 2 kg/ton. The challenge consisted of a diet formulated by the addition of rye and removal of Non-Starch-Polysaccharides (NSP) enzymes.

Rapeseed meal was also added, and an increased crude protein level was implemented.

Materials and methods

Animals and housing

One day old, male broiler chicks were used in this trial. The broilers were housed in floor pens with each a total surface of 2.5 m2. Broilers were housed with 30 per pen at a density of 13.0/m2 and 9 pens per treatment. The floor was covered with fresh wood shavings (8 kg/pen). The experiment started the 16th of August 2021 and lasted 35 days (till the 20th of September 2021). Central heating and infrared bulbs {one per pen) provided optimal house temperature. The lighting program was 18L:6D from the age of 7 days till the end of the trial period (first days: 23L:1D). There was dynamic ventilation with air entrances at both sides and air extractions centrally at the top of the building. The ventilation rate depended on the measured temperature and age of the broilers thereby (1) keeping the temperature as close as possible to the optimal temperature schedule and (2) minimising the moisture, NH; and CO:- content of the inside air.

Diets

The experiment consisted of 3 treatments, each with 9 replicates of 30 broilers. The treatments were: e T1: Control = animals receiving the challenge e T2: Control + a composition comprising an aluminium phyllosilicate selected from the palygorskite-sepiolite group, wherein the composition is added to a feedstuff in a concentration of 2 kg/ton e T3: Control + a composition for use according to an embodiment of the invention (saponins (origine Quillaja) with an aluminium phyllosilicate selected from the palygorskite-sepiolite group as carrier, wherein the composition is added to a feedstuff in a concentration of (2 kg/ton)

Diets used were nutritional challenge diets: higher NSP level, no NSP enzyme added, higher crude protein level and addition of rapeseed meal. Diet compositions included a starter, grower and finisher diet. An in-feed coccidiostat was present, as well as a phytase. The diets were prepared at the Institute's feed milling facilities. A global batch of the basic diet was made so that the quantitative composition of all the experimental diets was exactly the same. Diets were mixed and pelleted with appropriate cleaning and flushing of the system. The diets were stored in barrels, and kept under dry conditions and at ambient temperature.

A three-phase feeding scheme was applied with a starter period of 9 days followed by a grower diet between 9 and 23 days of age and a finisher between 23 and 35 days of age. Feed was provided ad libitum as pellets by feed mangers, with sizes appropriate to the size of the animals. Drinking water was provided ad /ibitum by bell drinkers.

Additional treatments

The broilers were vaccinated the first day against Newcastle Disease. At 16 days of age the vaccination against Newcastle disease was repeated. Twice daily, the broilers and the housing facilities were inspected for the general health status, constant feed and water supply as well as temperature and ventilation, dead broilers, and unexpected events.

Average pen weight was recorded at the start and after 9, 23 and 35 days of age.

Feed intake was recorded in the respective periods: 0-9, 9-23, 23-35 days. Feed conversion ratio, daily growth rate, bird days and daily feed intake per bird were calculated for 0-9, 9-23 and 23-35 days and also for the cumulative periods: 0-23 and 0-35 days.

Daily mortality and culling were recorded per pen. Culled birds were not included in the mortality calculations, except when they were removed because of illness.

Corrections for mortality calculating zootechnical performances were done using the number of ‘broiler days’ (number of broilers x days alive).

Also the following parameters were determined at the end of the trial (day 35): e Scoring of footpad lesions and hock burn lesions. Eight broilers per pen were scored; for the footpad and hock lesions, the worst footpad and the worst hock of each broiler is scored. (see Figure 2 for the scoring system) e Scoring of the litter (see Figure 3 for the scoring system)

Data treatment

All zootechnical parameters were analysed by a General Linear Model (GLM) with treatment as a fixed factor and block as a random factor. A Tukey HSD test was used to compare the treatments if the GLM determined a significant (P<0.05) treatment effect or a trend (0.05<P<0.1).

Mortality was analyzed with logistic regression with treatment as a fixed factor and block as a random factor. Mortality stated in the table are the least squares means and their corresponding 95% confidence intervals, back-transformed to percentages with the inverse of the logit. The scoring data was analyzed using an ordered logistic regression model with treatment as a fixed factor. All analyses were performed using

R 3.5.1 for windows.

Results and discussion

Production results

At arrival, one-day old birds had an average body weight of 45.9 g. Broilers from the different transport boxes were randomly distributed over the different treatments.

Broilers that were removed out of the trial because of leg problems, were not taken into account for mortality, unless they were also weak.

Birds receiving the composition for use according to an embodiment of the invention (T3) show a numerical decrease in FCR compared to birds receiving the control treatment and compared to birds receiving a composition comprising only the carrier (see Figure 1). Feed intake was not significantly different between treatments (P =

0.860) with a mean of 94 g/bird/d for the Zontrol and 93 g/bird/d for the treatment supplemented with the composition for use according to an embodiment of the invention (T3).

Table 1: Litter scoring ree

Score0 [0.0 [00 [00

Score2 [444 [00 [00

Score3 [0.0 ~~ [00 [00

Table 2: Footpad scoring [mmm

Score3 [0.0 [0.0 [00

Score4 [0.0 [00 [00

Conclusions 1) Birds receiving the composition for use according to an embodiment of the invention (T3) show a numerical decrease in FCR compared to birds receiving the control treatment (T1) or compared to birds receiving a composition comprising only the carrier (T2) (p-value = 0.001) (see Figure 1). 2) Litter quality: birds receiving the composition for use according to an embodiment of the invention (T3) had a significantly better litter quality than birds receiving the control treatment (T1) (p-value = 0.004)(Table 1). 3) Footpad lesions: Birds receiving the composition for use according to an embodiment of the invention had a significantly lower occurrence of footpad lesions compared to birds receiving the control treatment (T1) (p-value = 0.001 ) (Table 2).

Example 2. Evaluation of the effect of a composition for use according to an embodiment of the invention on broiler performance.

A similar experiment as example 1 was performed, this time comparing: 25 . T1: Control = animals receiving the challenge . T2: Control + a composition comprising saponins (origine Quillaja), wherein the composition is added to a feedstuff in a concentration of 2 kg/ton ° T3: Control + a composition for use according to an embodiment of the invention (saponins (origine Quillaja) with an aluminium phyllosilicate selected from the palygorskite-sepiolite group as carrier wherein the composition is added to a feedstuff in a concentration of (2 kg/ton).

Results were similar as those of example 1, with birds receiving the composition for use according to an embodiment of the invention (T3) showing a significant decrease in FCR, a significant better litter quality and a lower occurrence of footpad lesions compared to birds receiving the control treatment (T1) or compared to birds receiving a composition comprising only saponins (T2).

Example 3: effect of a composition for use according to an embodiment of the invention on broiler performance under disease conditions with a coccidiosis challenge

Aim

The aim of this trial was to establish the effect of a composition for use according to an embodiment of the invention ("Composition A”) on broiler performance under disease conditions with a coccidiosis challenge. The composition for use according to an embodiment of the invention included saponins (origine Quillaja) with an aluminium phyllosilicate selected from the palygorskite-sepiolite group as carrier.

The composition for use according to an embodiment of the invention was added to a feedstuff in a concentration of 2 kg/ton. The challenge consisted of used/dirt litter containing oocysts shed by seeder birds (natural infection model).

Experimental set-up

In total, 1152 one-day-old male broilers were allocated to 12 replicate pens of 24 broilers. The animals had ad libitum access to experimental feed and water. The trial consisted of four treatments (see table 3 below): A positive control reared on clean litter (PC), with no additive or coccidiostats added to the feed; a negative control reared on used litter, with no additive or coccidiostats added to the feed (NC); a negative control reared on used litter, with a coccidiostat (Salinomycin) added to the feed (NC + Sal); a negative control reared on used litter, with the addition of a composition according to an embodiment of the invention on-top of the diet (NC +composition A). The feed and nutritional composition is displayed in Figure 4.

Table 3: treatment groups according to an embodiment of the invention

Results

The addition of composition A to the broiler diets during the whole rearing period improved final body weight (at 42 days of age) (P < 0.05) (Figure 5) and the feed conversion ratio (FCR, Figure 6) between CO and 42 days of age (FCR = 0.053) of coccidiosis infected broilers (used litter) to a similar level as broilers reared on clean litter. Furthermore, the number of oocysts shed by coccidiosis challenged broilers were similar to a treatment where broilers were reared on used litter and received

Salinomycin, an anticoccidial (Figure 7). Coccidiosis in poultry often pre-exists or occurs concurrently with field outbreaks of necrotic enteritis caused by Clostridium perfringens. Recent in vitro results show that the aluminosilicate carrier is able to bind the alpha- and NetB toxins produced by Clostridium perfringens (see examples 4 and 5, respectively). These results hint to a protective effect of the aluminosilicate in the development of Clostridium perfringens-associated necrotic enteritis. The supplementation of Quillaja saponins has the ability to improve broiler performance during a coccidiosis challenge and aid in the prevention and/or treatment of necrotic enteritis.

Conclusion

The composition for use according to an embodiment of the invention improved the performance of coccidiosis infected birds similar to the positive control (with clean litter). The composition for use according to an embodiment of the invention reduced the amount of oocysts shed comparable to the treatment with salinomycin. The distinctive combination of a standardized, highly concentrated Quillaja saponaria extract (comprising saponins) combined with a aluminosilicate carrier complements present necrotic enteritis control strategies and further promotes resilience to infection.

Example 4: Evaluation of the binding activity of the aluminosilicate in the composition for use according to an embodiment of the invention towards the C. perfringens alpha-toxin production.

Aim

The aim of this study was to test the binding activity of the aluminosilicate in the composition for use according to an embodiment of the invention towards the alpha- toxin. The alpha toxin is produced by Clostridium perfringens (CP) when culturing the strain.

Experimental set-up

In total 3 products/materials are included in the test, each of them are powder forms of clay materials: san aluminosilicate as used in the composition for use according to an embodiment of the invention (0.5%), “material A” + Yeast (0.5%), “material B” e Organic Zink preparation (appr. 9% Zn) (0.1%), “material C”

For all the experiments an in-house Clostridium perfringens strain is used. This strain was proven to produce alpha toxin. Starting from a C. Perfringens culture, with an approximately concentration of 10° CFU/ml, a dilution series was prepared. Then, 150 pl of each C. perfringens dilution was transferred in a 96 well plate. The aluminosilicate solution (dissolved in PBS) and other test products were added to each well in a volume of 150 pl to reach a 0.5% final solution. The plates were incubated for 90 min at 37°C, with continuous shaking. After incubation, the plate was centrifuged and supernatant is collected from each well, in order to determine the alpha-toxin concentration using the ELISA {via optical density measurements).

The controls included in the test were BHI (= culture medium of C. perfringens), PBS (= solvent of the aluminosilicate solution) and ELISA kit controls.

Materials and methods

Testing solubility of the materials

The solubility of the materials was tested by solving a percentage of material in the different solutions. The aim was to select the appropriate solvent to dissolve the materials. Following solvent were tested: bidi water, NaOH 0.5M, PBS and Clostridium perfringens culture medium.

Alpha toxin production and precipitation solved clay materials

In order to ensure an optimal contact between the materials and the CP culture, precipitation of the materials should be avoided by continuous shaking during incubation of CP culture and the materials. Additionally and after the prescribed incubation period, the solution (material + CP culture) was spinned, and the supernatant was collected for determination of alpha toxin presence using the ELISA kit.

Binding activity materials on CP alpha toxin production

The binding activity of the materials on he alpha toxin production by CP was tested by adding the different solutions of materials to serial dilutions of CP.

Controls were included, in order to test the effect on the background in the ELISA kit: e PBS = solvent of the clay material + BHI = broth for culturing CP + materials

Serial tenfold dilutions of CP culture were included as positive control for alpha toxin production. The serial dilutions were made from a starting culture of CP, with a concentration (CFU/ml) of 10°. Concentrations were tested up to CFU = 10°. Ten ml of the different materials was prepared, each were dissolved in PBS as determined in preliminary experiment. Starting from a CP culture, with appr. concentration of 10°

CFU/ml, a dilution series was prepared, and this up to a concentration of 1 CFU/ml

CP. Then, 150 pl of each CP dilution was transferred in a 96 well plate. The materials were added to each well in a volume of 150 pl. The plates were incubated for 90 min at 37°C, with continuous shaking. After incubation, the plate was centrifuged and supernatant was collected from each well, in order to determine the alpha toxin concentration using the ELISA. The controls included in the test were BHI (= culture medium of CP), PBS (= solvens of the materials) and ELISA kit controls. The ELISA test was performed in accordance with the manual. The supernatants were tested undiluted, in 100pL aliquots. The optical densities were read using a microplate spectrophotometer with a 450 nm filter.

Interpreting the results

The net optical density of each sample was calculated by subtracting from each sample well the optical density of the negative control.

Value= net OD sample=100 net OD positive control

Included in the ELISA kit the quality control defines the OD for validation and the percentage to conclude the status of each sample as positive or negative. For batch

ACP 18H20, the status is positive if the value > 7.00%, indicating Alpha toxin production.

Results

Solubility of the materials

Different solvents were tested for the different materials:

- Bidi water - NaOH 0.5M - PBS - CP medium (broth)

The materials were all soluble in PBS for the concentration needed in the further test i.e. 0.5%.

The percentage of binding activity was calculated in comparison to the ‘no material’ control group, where no material was added, and no binding is seen (0%). The results can be found below in table 5 and Figure 8. At the highest CP concentration (1,00E+10 CFU) the binding activity was the highest for the aluminosilicate as used in the composition for use according to an embodiment of the invention. When the

CP concentration decreased the binding activity of the materials increased. The highest binding activity was seen with the aluminosilicate as used in the composition for use according to an embodiment of the invention.

Table 5. Percentage binding alpha toxin production material material material material

A B C

Conclusions

In vitro results show that the aluminosilicate as used in the composition for use according to an embodiment of the invention is able to bind/adsorb alpha-toxin produced by Clostridium perfringens and this to a high extend in mild and severe

Clostridium perfringens concentrations. These results demonstrate a protective effect of the aluminosilicate in the development of Clostridium perfringens-associated necrotic enteritis.

Example 5: In vitro assessment of the binding capacity of materials on C. perfringens

NetB toxin resulting in the inhibition of the hemolytic activity assay.

Aim:

In chickens the netB gene, encoding necrotic enteritis B like (NetB) toxin has been recently shown to play a leading role in the virulence of necrotic enteritis and several experimental studies demonstrated that a reliable necrotic enteritis induction model mainly relied on the strain of C. perfingens that is netB positive.

The aim of this study was to test the binding activity of the aluminosilicate in the composition for use according to an embodiment of the invention towards the NetB toxin. The hemolytic activity of the NetB toxin results in lysis of red blood cells (RBCs). By binding to the NetB toxin the aluminosilicate inhibits the hemolytic activity of the toxin resulting in intact RBCs. For this, an in vitro assessment of the binding capacity of 0.5% or 0.1% materials, i.e., clay materials, yeast and organic material, on Clostridium perfringens NetB toxin was conducted. The binding capacity of the following materials was investigated: an aluminosilicate as used in the composition for use according to an embodiment of the invention (0.5%, “material A”), yeast (0.5%, “material B”), organic zinc preparation (0.1%, “material C”). The toxic effect of the NetB toxin of C. perfringens bound to the specific materials was assessed with a hemolytic activity assay. Materials that bind to the NetB toxin can inhibit the hemolytic activity of the toxin, resulting in intact RBCs.

Materials and methods

Overview procedure

The study can be divided in three experimental parts: 1) The assessment of the solubility of the different materials by solving a percentage of material in four different solutions (i.e., bidi water, 0.5 M NaOH (Sodium hydroxide), PBS (Phosphate buffered saline), and medium C. perfringens); 2) The assessment of the hemolytic activity of the recombinant NetB toxin by adding NetB toxin to chicken RBCs; and 3)

The in vitro assessment of the binding capacity of the specific materials on C. perfringens NetB toxin resulting in the inhibition of the hemolytic activity assay.

Solubility of the specific materials

The solubility of each specific material was tested in four different solutions (i.e., autoclaved bidi water, autoclaved 0.5 M NaOH, autoclaved PBS, and medium

Clostridium perfringens (MCP)).

A total volume of 60.0 mL was prepared of each solution, of which 10.0 mL was used per material. To prepare a 1.0% or 0.2% final concentration of the materials, 0.10 g or 0.02 g of the materials were weighed with a precision weighing balance (224i-1S

Sartorius balance) and added to each of the 10.0 mL solutions. The dissolved material solutions were incubated at 37.0 °C and shaked with 200 rotations per minute (rpm) in a shake incubator. The solubility was checked after 10 min, 20 min, 30 min and 1 hour of incubation.

Due to a possible hemolytic effect of 0.5 M NaOH on RBCs, a small test was performed to assess the hemolytic effect of 0.5 M NaOH. For this, 1% intact chicken RBCs (RBCs in PBS) were incubated for 30 min at 37.0 °C and shaked with 200 rpm in a shake incubator with 0.5 M NaOH or with PBS (negative control), and lysed chicken RBCs (RBCs in water) incubated with 0.5 M NaOH or PBS (positive control). Thereafter, 180.0 pL of the supernatant was transferred to a fresh flat-bottomed 96 well plate (without disturbing the pellet RBCs). The optical density (OD) was measured twice at 492 nm and 560 nm to determine the hemolytic activity of 0.5 M NaOH on RBCs.

The NetB toxin activity will be measured by the increase in absorbance due to the release of hemoglobulin from the RBCs.

Hemolytic capacity of Clostridium perfringens NetB toxin on red blood cells

The hemolytic activity of the recombinant NetB (rNetB) toxin is tested by adding the rNetB toxin to chicken RBCs. First, rNetB toxin (start concentration of 1800 pg/mL) was diluted by adding 0.053 mL rNetB toxin to 1.147 mL sterile PBS (stock solution for the whole study) to obtain a diluted concentration of 80 pg/mL, which was further incubated for 30 min at 37.0 °C and shaked with 200 rpm in a shake incubator. To check the hemolytic activity of the rNetB toxin, 100 uL diluted rNetB toxin, 100 pL 1% chicken RBCs, 100 pL 1% lysed chicken RBCs or 100 pL PBS was added to a flat- bottomed 96 well plate. After an incubation for 60 min at 37.0 °C and shaked with 200 rpm in a shake incubator, the plate was centrifuged at 1000g for 1 min at room temperature. Thereafter, 180.0 pL of the supernatant was transferred to a fresh flat- bottomed 96 well plate (without disturbing the pellet RBCs) to measure the optical density (OD) twice at 492 nm and 560 nm. The rNetB toxin activity will be measured by the increase in absorbance due to the release of hemoglobulin from the RBCs.

In vitro binding capacity of specific materials on Clostridium perfringens NetB toxin

The different materials are dissolved in PBS, of which 60 ul are added to a V-shaped bottom 96 well plate. In addition, 60 uL rNetB toxin with a concentration of 80 pg/mL or 60 pL sterile PBS are added to the same V-shaped bottom 96 well plate.

Thereafter, the V-shaped bottom 96 well plate is incubated for 30 min at 37.0 °C and shaked with 200 rpm in a shake incubator, and centrifuged at 1000g for 1 min at room temperature. To a flat-bottomed 96 well plate, 100 yL of 1% chicken RBCs, 100 pL of Lysed chicken RBC, 100 uL supernatant of the pre-incubated materials, and/or 100 pL PBS are added to the wells. The well plate is incubated for 60 min at 37.0 °C and shaked with 200 rpm in a shake incubator. Thereafter the plate is centrifuged at 1000g for 1 min at room temperature, and 180 pL supernatant was transferred to a fresh flat-bottomed 96 well plate to measure the OD twice at 492 nm and 560 nm. The rNetB toxin activity will be measured by the increase in absorbance due to the release of hemoglobulin from the RBCs.

Results

Solubility of the specific materials

To assess the solubility of the specific materials in the different solutions, the materials were first dissolved in the different solvent solutions. The weights of the different materials that were dissolved in the four different solutions are summarized in Table 6.

Table 6. Overview of the different materials, percentages, different solvents and weights.

Autoclaved bid water | 0.1005

OL | Autoclaved 0.5 M NaOH | 0.1008

Autoclaved PBS 0.1009 perfringens

Material B Autoclaved bidi water 0.1008

Lo | Autoclaved 0.5 M NaOH | 0.1007

OL | Autoclaved PBS 0.1005 perfringens

Material C Autoclaved bidi water 0.0208

OL [Autoclaved 0.5 M NaOH | 0.0209

OL | Autoclaved PBS 0.0203 perfringens

Based on the visual solubility of the different solvents, NaOH and PBS showed the most solubility of the test materials after 10 min, 20 min, 30 min and 1 hour. Even after exceeding the incubation time till 24 hours, similar results were observed. Due to the hemolytic activity of NaOH on RBCs, PBS was used further in this study as solvent solution.

Hemolytic capacity of Clostridium perfringens NetB toxin on red blood cells

The hemolytic capacity of the rNetB toxin on 1% chicken RBCs are summarized in

Table 8. No differences were observed between rNetB + Lysed RBC and PBS + Lysed

RBC (positive control) indicating that all lysed chicken RBCs that were used in this study were indeed lysed RBCs. Moreover, PBS + RBC had a low absorbance indicating no lysis of chicken RBCs (negative control). In addition, rNetB + RBC showed a high absorbance, even higher than the positive controls, indicating that rNetB toxin has a hemolytic capacity on the chicken RBCs.

Table 8 Mean OD values from rNetB toxin or PBS incubated with red blood cells or lysed red blood cells

Wavelength rNetB + RBC | rNetB + Lysed PBS + RBC PBS + Lysed

RBC (negative RBC (positive control control 0.212 £0.014 | 0.118 £0.006 0.080 £0.003 | 0.119 +0.001 [560 nm [0.274 +0.014 | 0.139 +0.007 0.079 £0.003 [0.142 £0.001

Data presented as mean tSD; RBC: 1% Chicken red blood cells dissolved in PBS; Lysed RBC: 1% Chicken red blood cells dissolved in water.

In vitro binding capacity of specific materials on Clostridium perfringens NetB toxin

Table 9 summarizes the mean OD values of the positive and negative controls used in the third part of the experiment, whereas the mean OD values of the specific materials incubated with rNetB toxin and RBCs or PBS are summarized in Table 10.

In this in vitro experiment, the material A (0.5%) (as used in the composition for use according to an embodiment of the invention) completely (100%) inhibits the hemolytic activity of rNetB toxin, which is shown in Figure 9. Organic zinc preparation (0.1%) does not inhibit the hemolytic capacity of the rNetB toxin (Figure 9). Another material that does not inhibit rNetB toxin activity is yeast (Table 10).

Table 9. Mean OD values of the positive and negative controls

Wavelength rNetB + PBS + PBS + Cells

Cells

RBC [492nm | 0.860.014 | 0.081 £0.003 560nm [0.397 £0.014 0.081 £0.003

Lysed RBC |492nm [0.162 +0.001 0.159 +0.001 560nm | 0.209 £0.000 0.205 £0.001

Data presented as mean £SD; RBC: 1% Chicken red blood cells dissolved in PBS; Lysed RBC: 1% Chicken red blood cells dissolved in water.

Table 10. Mean OD values of the specific materials incubated with rNetB toxin and RBCs or

PBS material material

Material A (0.5%) [492nm [0.086 0.006 0.092 +0.008 560nm | 0.086 x0.008 0.096 £0.010

Material B (0.5%) [492nm [0.316 +0.010 0.118 +0.001 560nm [0.415 + 0.010 0.106 +0.000

Material C (0.1%) 0.295 +0.000 0.086 £0.010 560mm | 0,408 0.001 0.087 £0.011

Data presented as mean SD; RBC: 1% Chicken red blood cells dissolved in PBS; Lysed RBC: 1% Chicken red blood cells dissolved in water.

Conclusion

The binding capacity of 3 materials was investigated. The toxic effect of the NetB toxin of C. perfringens bound to the specific materials was assessed with an hemolytic activity assay. Prior to the hemolytic activity assay two preliminary experiments were performed. First, the solubility of the different materials was assessed in four different solvent solutions (i.e., bidi water, 0.5 M NaOH, PBS, and medium C. perfringens), of which PBS was found to be the most suitable solvent solution. Second, the hemolytic activity of the recombinant NetB toxin was investigated by adding the toxin to 1% chicken RBCs, which indicated that the rNetB toxin was functional.

The in vitro hemolytic assay in which the binding capacity of the specific materials were tested on rNetB toxin showed that material A (0.5%) completely inhibited the rNetB toxin hemolytic activity, whereas organic zinc preparation (0.1%) itself does not inhibit the hemolytic capacity of the rNetB toxin. Another material that does not inhibit rNetB toxin activity is yeast. In conclusion, material A (0.5%) inhibits completely the hemolytic capacity of recombinant NetB toxin. The materials organic zinc preparation (0.1%) and yeast (0.5%) does not inhibit the rNetB toxin hemolytic activity. As such, the aluminosilicate of the composition for use according to an embodiment of the invention was able to completely inhibit the hemolytic capacity of recombinant NetB toxin.

The present invention is in no way limited to the embodiments described in the examples and/or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims (13)

CLAIMS 1. A composition for use in the prevention and/or treatment of necrotic enteritis in an animal, wherein the composition comprises a therapeutically effective amount of a saponin-containing extract on an aluminosilicate carrier. 2. The composition for use according to claim 1, wherein the composition comprises at least 0.225% saponins by total weight. 3. A composition for use according to any preceding claim, wherein the composition comprises at least 50% of an aluminosilicate carrier by total weight. 4. A composition for use according to any preceding claim, wherein the aluminosilicate carrier is an aluminium phyllosilicate. 5. A composition for use according to any preceding claim, wherein the aluminium phyllosilicate is selected from the palygorskite-sepiolite group. 6. A composition for use according to any preceding claim, wherein said necrotic enteritis is the result of a preceding or concurrent infection in said animal, wherein said infection is preferably an Eimeria infection allowing growth of Clostridium perfringens. 7. The composition for use according to claim 6, wherein said infection is an Eimeria infection, wherein said infection damages the intestinal epithelium of the animal, allowing plasma proteins to leak into the intestinal lumen. 8. A composition for use according to any preceding claim, wherein the saponin-containing extract is a Quillaja saponaria plant extract. 9. A composition for use according to any preceding claim, wherein the composition is added to an animal feed to obtain an animal feed mixture, such that the concentration of the saponin-containing extract in the animal feed mixture is at least 10 ppm (parts per million). 10. A composition for use according to any preceding claim, wherein the animal is a non-human mammal, a fish, a bird or an amphibian. 11. Animal feed mixture consisting of an animal feed and a composition comprising a therapeutically effective amount of a saponin-containing extract on an aluminosilicate carrier, wherein the composition is added to the animal feed such that the concentration of the saponin-containing extract in the animal feed mixture is at least 10 ppm. 12. Animal feed mixture according to claim 11, wherein the concentration of the aluminosilicate carrier in the mixture is at least 125 ppm. 13. Animal feed mixture according to any one of the preceding claims 11-12, wherein the animal feed mixture is provided ad libitum.