Compositions and methods for modulating and / or stimulating immune responses in humans and / or animals

Abstract

The present invention provides a composition comprising a wall polysaccharide from at least one Candida species and a wall polysaccharide from at least one different yeast species. The composition can be used to modulate and / or stimulate an immune response in an animal or a human.

Description

[0001] This application is a divisional application of CN201680040529.3. Field of the Invention

[0002] The present application relates to compositions and methods for modulating and / or stimulating immune responses in humans and / or animals. Background of the Invention

[0004] Yeast and its derivatives are increasingly used as nutritional supplements to improve intestinal health in animals and humans by activating antifungal immune responses initiated at the intestinal mucosal surface, primarily through the Dectin 1 and β-glucan pathways, or by directly binding to unwanted bacteria in the digestive tract. Both effects rely on the specificity of the expression of immune antigens within the yeast cell wall and the expression of antigen receptors within the intestinal epithelial cells. However, it should be noted that in addition to the well-described β-glucan / Dectin 1 interaction, other "yeast ligand"-receptor interactions mediating the activation of immune responses (and more specifically antifungal immune responses) may have a significant role and are directly dependent on the composition and structure of the yeast cell wall. There are currently market options for products using nonspecific, inactive yeasts that claim to stimulate immune responses through these pathways; however, these products lack specificity and consistency in composition and results. It would be highly desirable to provide improved yeast-based products that are able to enhance the stimulation of immune responses in animals or humans. Overview of public content

[0006] The present disclosure provides a composition comprising wall polysaccharides from at least one Candida species and wall polysaccharides from at least one different yeast species. Surprisingly, when combined with wall polysaccharides from at least one different yeast species, wall polysaccharides from at least one Candida species can enhance stimulation of animal or human immune response even at low inclusion rates. In some embodiments, the amount of wall polysaccharides from at least one Candida species is 1 to 99 dry weight % based on the total weight of wall polysaccharides in the composition. In embodiments, the amount of wall polysaccharides from at least one Candida species is 10 to 50 dry weight % based on the total weight of wall polysaccharides in the composition. In alternative or supplementary embodiments, the amount of wall polysaccharides from at least one Candida species and wall polysaccharides from at least one different yeast species is 1 to 100 weight % based on the total weight of the composition. In embodiments, the amount of wall polysaccharides from at least one Candida species and wall polysaccharides from at least one different yeast species is 20 to 80 weight % based on the total weight of the composition. In another embodiment, the amount of wall polysaccharides from at least one Candida species and wall polysaccharides from at least one different yeast species is 30 to 60 weight percent based on the total weight of the composition. In an embodiment, the at least one Candida species is Candida utilis. In another embodiment, the wall polysaccharides from at least one different yeast species are from Saccharomyces sp, Hanseniaspora sp, Hansenula sp, Kluyveromyces sp, Metschnikowia sp, Pichia sp, Starmerella sp and Torulaspora sp or a mixture thereof. The at least one different yeast species also comprises interspecies hybrids derived from any of these yeast species.

[0007] The present disclosure also provides a use as defined in the preceding embodiments for modulating and / or stimulating an immune response in an animal and / or a human. In another embodiment, the animal is a domestic animal.

[0008] The present disclosure also provides methods for regulating and / or stimulating an immune response in an animal by administering to the animal a composition comprising a wall polysaccharide from at least one Candida species and a wall polysaccharide from at least one different yeast species. In an embodiment, the combination is in the form of a food additive, an animal feed, or a pharmaceutical product. In another embodiment, the pharmaceutical product can be administered orally or parenterally. In yet another embodiment, the food additive, animal feed can be added to a complete animal or human diet, or presented as a tablet, pellet, or bead added separately for direct consumption. In yet another embodiment, the animal is a domestic animal. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Having thus generally described the nature of the present invention, reference will now be made to the accompanying drawings showing, by way of example, preferred embodiments thereof, and in which:

[0011] Figure 1 showing the effect of inclusion of laminarin on the in vitro production of reactive oxygen species (ROS) by mononuclear cells incubated with wall polysaccharides from different strains of Saccharomyces cerevisiae according to Assay 1 of Example 1;

[0012] Figure 2 shows the in vitro production of ROS by monocytes incubated with a blend of wall polysaccharides from different strains of Saccharomyces cerevisiae at different concentrations according to Assay 2 of Example 1;

[0013] Figure 3 shows the comparative effect of laminarin on the in vitro production of ROS by monocytes incubated with a wall polysaccharide from a strain of Candida utilis or a wall polysaccharide from a strain of Saccharomyces cerevisiae according to Assay 3 of Example 1;

[0014] Figures 4a and 4b show the in vitro production of ROS by monocytes incubated with different concentrations of wall polysaccharides from a Saccharomyces cerevisiae strain alone or in combination with a wall polysaccharide from a Candida utilis strain (a) without laminarin and (b) with 100 μg of laminarin according to Assay 4 of Example 1;

[0015] Figure 5 shows the survival function of penaed shrimp infected with Vibrio parahaemolyticus and treated with different combinations of wall polysaccharides according to the Kaplan Meyer statistical procedure of Example 2;

[0016] Figures 6a and 6b show the microvillus density of the distal small intestine of sea bass by electron microscopy at weeks 5 and 10 after feeding the experimental diet according to the present disclosure according to Example 3; and

[0017] Figure 7 Shown is the expression of two immune genes (IL1β and IL10) in the distal small intestine after feeding the experimental diet according to the present disclosure at week 10 according to Example 3.

[0018] Figure 8 Shown are the survival rates of shrimp 14 days after challenge according to Example 6 (mean±SD); patterns indicated with the same capital letters were not different between treatments (Fisher's test, 5% probability).

[0019] Fig. 9 The directions of the spatulas for Atlantic salmon and rainbow trout according to Example 9 are shown.

[0020] Fig.10 It is shown that according to Example 9, when feeding supplemented with 60SC wall polysaccharide (PARIETAL POLYSACCHARIDES) and (50SC / CU wall polysaccharide TM (PARIETAL POLYSACCHARIDES TM Relative expression levels of genes related to mucus production in skin samples of rainbow trout (O. mykiss) fed the experimental diet of ) for 8 weeks. Data are presented as box plots (n=10 / treatment). *- indicates significant upregulation; P<0.0001

[0021] Fig.11 Shown is the growth performance of fish fed a control diet according to Example 9 and rainbow trout fed an experimental diet for 56 days.

[0022] Fig.12 Shown are examples of intestines of seabass fed control (A & B), 60 SC wall polysaccharide (C & D), 50 SC / CU wall polysaccharide (E & F) for 10 weeks according to Example 10. Scale bars are 10 μm (panels A, C and E) and 1 μm (panels B, D and F).

[0023] Fig.13 Illustrate the feeding control and experimental diets according to Example 10 ( Fig.13 A) 5 weeks and ( Fig.13 B) Microvilli density measurements in the hindgut of 10-week old sea bass. Asterisks indicate significant differences (P<0.001) between fish fed the control diet and fish fed the experimental diet.

[0024] Fig.14Illustrate gene expression data of sea bass hindgut samples after 10 weeks of feeding the experimental diet. Box plots show data for HSP70, PCNA, IL-1β and IL-10 according to Example 10. Asterisks indicate that gene expression is significantly up-regulated or down-regulated compared to control-fed fish (P<0.05).

[0025] Fig.15 Shown are the average intestinal lesion scores of broiler chickens challenged with Eimeria acervulina, E. maxima and E. tenella according to Example 11 on day 20 (day 6 post-challenge).

[0026] Fig.16 The cumulative mortality (in %) of broiler chickens at the end of the trial according to Example 11 is shown.

[0027] Fig.17 Shown are oocyst fecal excretion (occidia Oocysts per Gram Fecal Material (OPG)) of broiler chickens challenged with Eimeria acervulina, Eimeria maxima and Eimeria tenella according to Example 11 on day 20 (day 6 post-challenge). Details

[0029] The cell wall of fungi is mainly composed of wall polysaccharides including chitin, mannose-based structures and beta-glucans. Glucans make up up to 50% of the cell wall and are bioactive agents used therapeutically to modify the immune response. Experimentally, these wall polysaccharides can confer protection against various attacks including tumor development and infection. Many advances have been made recently in understanding the host's immune response to infection. New cell surface molecules that regulate the host's response to microorganisms have been discovered. These receptors are called pathogen-associated pattern recognition receptors (PRRs) and are mainly known as Toll-like receptors, C-type lectin receptors, scavenger receptors and complement receptors.

[0030] These immune receptors are activated by specific ligands encountered on the surface of bacteria or fungi. In the case of fungi, glucose units linked by β-1,3 and β-1,6 glycosidic bonds, known as β-glucans and mainly extracted from Saccharomyces cerevisiae, have been shown to induce antimicrobial, fungicidal and antitumor activities. The mechanism by which these molecules induce protection involves immune receptors; one of them is Dectin 1, which is the main β-glucan receptor on monocytes. It contains a single extracellular C-type lectin-like wall polysaccharide recognition domain that is able to recognize yeast cells or derivatives and help mobilize immune responses. Thus, the C-type lectin receptor Dectin 1 (Marakala et al., Mamm Genome (2011) 22: 55-65) is described to bind β-glucans from Saccharomyces cerevisiae and participate in the induction of various pathways, in particular leading to the phagocytosis and elimination of particles such as zymosan (cell wall extracts of Saccharomyces cerevisiae and other yeast derivatives obtained from strains of Saccharomyces cerevisiae (examples of other yeast derivatives are but not limited to yeast cell walls, inactive yeast or autolyzed yeast)). Threat detection mechanisms and direct responses to infection are highly conserved, and the immune response is triggered in part by the binding of these pathogen ligands (e.g., β-glucans) to PRRs on the surface of host cells. Within a few hours, the nonspecific innate immune system is activated, accompanied by inflammation. Local inflammation plays an important role in the immediate response to infection. Many participants in the innate immune system (including neutrophils, monocytes, macrophages, complement factors, cytokines, antimicrobial peptides, and acute phase proteins) are rapidly mobilized in a complex and highly regulated response to provide immediate defense against infection. There are currently market options for inactive yeast-based products that claim to stimulate immune responses through these pathways; however, these products lack specificity and consistency of results. It would be highly desirable to provide improved yeast-based products that can enhance the stimulation of animal or human immune responses.

[0031] The present disclosure provides a composition comprising a wall polysaccharide from at least one Candida species and a wall polysaccharide from at least one different yeast species. In the context of the present disclosure, "wall polysaccharides" are chitin, manno-oligosaccharides, β1,3 glucans and β1,6 glucans. Unexpectedly, when combined with a wall polysaccharide from at least one different yeast species, the wall polysaccharide from at least one Candida species can enhance the stimulation of an animal or human immune response even at low inclusion rates.

[0032] In some embodiments, the amount of wall polysaccharide from the at least one Candida species is 1 to 99 dry weight percent based on the total weight of wall polysaccharides in the composition. In embodiments, the amount of wall polysaccharide from at least one Candida species is 5 to 90 dry weight percent based on the total weight of wall polysaccharides in the composition. In another embodiment, the amount of wall polysaccharide from at least one Candida species is 5 to 80 dry weight percent based on the total weight of wall polysaccharides in the composition. In yet another embodiment, the amount of wall polysaccharide from at least one Candida species is 10 to 75 dry weight percent based on the total weight of wall polysaccharides in the composition. In yet another embodiment, the amount of wall polysaccharide from at least one Candida species is 10 to 60 dry weight percent based on the total weight of wall polysaccharides in the composition. In embodiments, the amount of wall polysaccharide from at least one Candida species is 10 to 50 dry weight percent based on the total weight of wall polysaccharides in the composition. In another embodiment, the amount of wall polysaccharide from at least one Candida species is 10 to 20 dry weight percent based on the total weight of wall polysaccharides in the composition. In yet another embodiment, the amount of wall polysaccharides from at least one Candida species is 10 to 15 dry weight % based on the total weight of wall polysaccharides in the composition. In yet another embodiment, the amount of wall polysaccharides from at least one Candida species is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 dry weight % based on the total weight of wall polysaccharides in the composition. In another embodiment, the amount of wall polysaccharides from at least one Candida species is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 dry weight % based on the total weight of wall polysaccharides in the composition. In yet another embodiment, the amount of wall polysaccharides from at least one Candida species is at least 10, 11, 12, 13, 14 or 15 dry weight % based on the total weight of wall polysaccharides in the composition.

[0033] In alternative or supplementary embodiments, the amount of wall polysaccharides from at least one different yeast species is 1 to 99 dry weight % based on the total weight of wall polysaccharides in the composition. In embodiments, the amount of wall polysaccharides from at least one different yeast species is 50 to 90 dry weight % based on the total weight of wall polysaccharides in the composition. In another embodiment, the amount of wall polysaccharides from at least one different yeast species is 70 to 90 dry weight % based on the total weight of wall polysaccharides in the composition. In yet another embodiment, the amount of wall polysaccharides from at least one different yeast species is 80 to 90 dry weight % based on the total weight of wall polysaccharides in the composition. In yet another embodiment, the amount of wall polysaccharides from at least one different yeast species is at least 50, 55, 60, 65, 70, 75, 80, 85 or 90 dry weight % based on the total weight of wall polysaccharides in the composition. In another embodiment, the amount of wall polysaccharides from at least one different yeast species is at least 70, 75, 80, 85 or 90 dry weight % based on the total weight of wall polysaccharides in the composition. In yet another embodiment, the amount of wall polysaccharide from at least one different yeast species is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 dry weight % based on the total weight of wall polysaccharides in the composition.

[0034] In alternative or supplementary embodiments, the amount of wall polysaccharides from at least one Candida species and from at least one different yeast species is 1 to 100 weight % based on the total weight of the composition. In embodiments, the amount of wall polysaccharides from at least one Candida species and from at least one different yeast species is 20 to 80 weight % based on the total weight of the composition. In another embodiment, the amount of wall polysaccharides from at least one Candida species and from at least one different yeast species is 30 to 60 weight % based on the total weight of the composition. In yet another embodiment, the amount of wall polysaccharides from at least one Candida species and from at least one different yeast species is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 weight % based on the total weight of the composition. 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,67,70,73,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121

[0035] In one embodiment, the amount of wall polysaccharides from at least one Candida species is 10 to 50 dry weight % based on the total weight of wall polysaccharides in the composition; and the amount of wall polysaccharides from at least one Candida species and wall polysaccharides from at least one different yeast species is 30 to 60 weight % based on the total weight of the composition.

[0036] In another embodiment, the amount of wall polysaccharides from at least one Candida species is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 dry weight % based on the total weight of wall polysaccharides in the composition; and the amount of wall polysaccharides from at least one Candida species and wall polysaccharides from at least one different yeast species is 30 to 60 weight % based on the total weight of the composition.

[0037] In yet another embodiment, the amount of wall polysaccharides from at least one Candida species is 10 to 50 dry weight % based on the total weight of wall polysaccharides in the composition; and the amount of wall polysaccharides from at least one Candida species and wall polysaccharides from at least one different yeast species is at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 weight % based on the total weight of the composition.

[0038] 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weight % based on the total weight of the wall polysaccharides in the composition.

[0039] In embodiments, the at least one Candida species is Candida utilis. In the present disclosure, it is understood that Candida utilis includes two forms of sex (Cyberlindnera jardinii) and asexual (Candida utilis). In another embodiment, the at least one different yeast species is from Saccharomyces, Hansenula, Hansenula, Kluyveromyces, Metschnikowia sp, Pichia, Starmerella sp and Torulaspora sp or a mixture thereof. The at least one different yeast species also includes interspecies hybrids derived from any one of these yeast species. In yet another embodiment, the at least one different yeast species is Saccharomyces. In yet another embodiment, the at least one different yeast species is Saccharomyces cerevisiae. Saccharomyces cerevisiae also includes subspecies, such as but not limited to Saccharomyces boulardii. In yet another embodiment, the at least one different yeast species is a blend of two or more Saccharomyces cerevisiae. In yet another embodiment, the at least one different yeast species is a blend of at least one Saccharomyces cerevisiae and at least one Saccharomyces boulardii.

[0040] In an embodiment, the composition as defined in the preceding embodiment can be used to regulate and / or stimulate an immune response of an animal or a human. When used herein, the term "regulate" will be understood to refer to any measurable increase or decrease in an immune response. In another embodiment, the composition as defined in the preceding embodiment can be used to improve animal or human health and resist infection. In yet another embodiment, the composition as defined in the preceding embodiment can be used to improve animal or human resistance to pathogenic microorganisms. In yet another embodiment, the composition as defined in the preceding embodiment can be used to promote animal breeding performance. In an embodiment, the animal is a domestic animal. Exemplary domestic animals include but are not limited to fish, shrimp, calves or piglets.

[0041] In an embodiment, the composition as defined in the preceding embodiment can be used to promote and / or improve the intestinal health, intestinal integrity and intestinal morphology of an animal. In another embodiment, the composition as defined in the preceding embodiment can be used to stimulate an anti-inflammatory response in an animal. In yet another embodiment, the composition as defined in the preceding embodiment can be used to promote and / or improve growth performance parameters and feed conversion in farmed animals. In yet another embodiment, the composition as defined in the preceding embodiment can be used to reduce morbidity, particularly during antibiotic treatment and mortality in farmed animals. In an embodiment, the composition as defined in the preceding embodiment can be used to reduce mortality in farmed animals. In another embodiment, the composition as defined in the preceding embodiment can be used to reduce diarrhea in animals. In yet another embodiment, the composition as defined in the preceding embodiment can be used to reduce sensitivity to intestinal pathogens. In yet another embodiment, the composition as defined in the preceding embodiment can be used to reduce the sensitivity of animals to parasitic infections and related conditions. In one embodiment, the composition as defined in the preceding embodiment can be used to promote and / or improve the production or quality of mucus in the skin of aquatic animals.

[0042] In yet another embodiment, the composition as defined in the preceding embodiments can be used as an ingredient in conventional animal feed.

[0043] In yet another embodiment, the composition as defined in the preceding embodiments is typically presented in a form for addition to a complete animal or human diet, or separately as tablets, pellets or beads for direct consumption.

[0044] The present disclosure provides a method for regulating and / or stimulating an immune response in an animal or a human by administering to the animal or human a composition comprising a wall polysaccharide from at least one Candida species and a wall polysaccharide from at least one different yeast species. Also provided is a method for improving the health and / or resistance to infection of an animal or a human by administering to the animal or a human a composition comprising a wall polysaccharide from at least one Candida species and a wall polysaccharide from at least one different yeast species. Also provided is a method for improving the resistance of an animal or a human to pathogenic microorganisms by administering to the animal or a human a composition comprising a wall polysaccharide from at least one Candida species and a wall polysaccharide from at least one different yeast species. Also provided is a method for promoting animal reproductive performance by administering to the animal or a human a composition comprising a wall polysaccharide from at least one Candida species and a wall polysaccharide from at least one different yeast species.

[0045] In embodiments, the combination is in the form of a food additive, a feed additive, a feed material / ingredient, an animal feed or a pharmaceutical product. In another embodiment, the pharmaceutical product can be administered orally or parenterally. In yet another embodiment, a food additive, an animal feed can be added to a complete animal or human diet, or presented as a tablet, a pellet or a pearl separately added in the form of direct consumption. In yet another embodiment, the animal is a domestic animal. Exemplary domestic animals include but are not limited to fish, shrimp, calves or piglets.

[0046] The following examples are used to further describe and define the present invention, but are not intended to limit the present invention in any way. Example

[0047] Embodiment 1:

[0048] Dectin 1 is not the only PRR that recognizes yeast wall polysaccharides. The structure and architecture of the cell wall may play an important role. We hypothesized here that different yeast species, even with similar wall polysaccharide composition, may exhibit different architectures of their wall polysaccharide network, leading to the activation of different sets of PRRs, not necessarily associated with the activation of Dectin 1. An in vitro competition assay using laminarin, a specific soluble ligand with high affinity for Dectin 1, as described in Example 1 was performed to evaluate this hypothesis.

[0049] In this example, the inventors have made the choice to evaluate the effects of different combinations of wall polysaccharides extracted from different yeast strains and species on the innate immune response. The cell model used in these experiments is a primary culture of human monocytes. The ability of monocytes to produce oxygen species was evaluated: thanks to chemiluminescence, the production of anionic superoxide was studied. This molecule is produced by NADPH oxidase, an enzyme complex encountered on the surface of monocytes. Oxygen species are involved in killing live bacteria or fungi engulfed by monocytes.

[0050] Materials and methods

[0051] Cell culture

[0052] Peripheral blood mononuclear cells (PBMC) are obtained from healthy blood donor buffy coats by standard Ficoll-Hypaque gradient method. Human monocytes are separated from monocytes by adhering to plastic for 2 hours in SFM medium optimized for macrophage culture at 37°C in a humid atmosphere containing 5% CO2. They are seeded into multi-well culture plates and non-adherent cells are removed by washing with HBSS that does not contain calcium or magnesium. The remaining adherent cells (monocytes>85%) are cultivated in SFM before stimulation.

[0053] Determination of oxygen species production

[0054] Monocytes were plated in 96-well microplates. Reactive oxygen species (ROS) production was measured by chemiluminescence using a thermostatically (37°C) controlled photometer (Wallac 1420 Victor 2, Finland) in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol, Sigma). Chemiluminescence production was continuously monitored for 60 minutes after incubation of cells with luminol (66 μM) in the presence of 100 μg / ml of different wall polysaccharides alone or in the presence of gradient levels of laminarin (0, 1, 10 and 100 μg / ml, depending on the assay). 100 μg / ml of zymosan was used as a positive control.

[0055] Yeast wall polysaccharides used in this study:

[0056] Wall polysaccharides extracted from different yeast strains:

[0057] Saccharomyces cerevisiae strains: L60, L62, L69, L72

[0058] Candida utilis strain: L75 (NRRL-900)

[0059] Levels of wall polysaccharides tested in different assays:

[0060] Candida utilis strains: Wall polysaccharide levels were standardized to 30 dry weight % based on the total weight of the composition.

[0061] Saccharomyces cerevisiae strains: Polysaccharide levels were standardized to 30, 35, 45 and 60 dry weight % based on the total weight of the composition.

[0062] Combinations of Candida utilis and Saccharomyces cerevisiae: Wall polysaccharide levels were standardized to 30, 35, 40, 45 and 50% dry weight based on the total weight of the composition. The content of wall polysaccharides from Candida utilis in these combinations was 15, 15, 10, 15 and 5% dry weight based on the total weight of the composition or 50, 43, 25, 33 and 10% dry weight based on the total weight of wall polysaccharides in the composition, respectively (Figures 4a, 4b)

[0063] result:

[0064] Assay 1: In vitro production of ROS by monocytes incubated with wall polysaccharides from different strains of Saccharomyces cerevisiae and different concentrations of laminarin. Each wall polysaccharide fraction from four different strains of Saccharomyces cerevisiae was normalized to 60% dry weight based on the total weight of the fraction and tested for their affinity to Dectin 1. Figure 1 As shown, wall polysaccharides from all strains were able to induce ROS production, whereas laminarin inhibited this induction in a linear dose-response manner.

[0065] Assay 2: Dose response of wall polysaccharides from Saccharomyces cerevisiae on ROS production in vitro by monocytes. Blends of wall polysaccharides from two different strains of Saccharomyces cerevisiae (L62 and L69) were prepared and standardized to 30, 35, 45 and 60 dry weight % based on the total weight of the blend and tested for their ability to induce ROS production. Figure 2 As shown, a linear dose response was obtained over the concentration range tested.

[0066] Assay 3: Comparative effect of laminarin on the in vitro production of ROS by monocytes incubated with wall polysaccharides from a strain of Candida utilis or wall polysaccharides from Saccharomyces cerevisiae. The wall polysaccharide fraction from a proprietary strain of Candida utilis was normalized to 30% dry weight based on the total weight of the fraction and tested for its affinity to Dectin 1. Similarly, the wall polysaccharide fractions from a strain of Saccharomyces cerevisiae (L62) were normalized to 30 and 60% dry weight based on the total weight of the fraction, respectively, and tested for their affinity to Dectin 1. Figure 3 As shown, in the absence of laminarin, the wall polysaccharide fractions of Candida utilis and Saccharomyces cerevisiae were able to induce the production of ROS. Figure 3 Increasing laminarin concentrations increased the inhibition of ROS production by the wall polysaccharide fraction of S. cerevisiae. However, increasing laminarin concentrations did not show any inhibition of ROS production by the wall polysaccharide fraction of Candida utilis, even at a concentration of 100 μg / ml.

[0067] Assay 4: In vitro production of ROS by monocytes incubated with different concentrations of wall polysaccharides from Saccharomyces cerevisiae strains alone or in combination with wall polysaccharides from Candida utilis strains (a) without laminarin and (b) with 100 μg of laminarin;

[0068] As shown in Figures 4a and 4b, the presence of wall polysaccharides from Candida utilis in different compositions resulted in enhanced monocyte ROS production even at low inclusion rates (i.e., 5% by dry weight based on the total weight of the composition). As shown in Figure 4b, compositions comprising wall polysaccharides from Candida utilis and wall polysaccharides from at least one Saccharomyces cerevisiae were not subject to the inhibitory effect of laminarin on the polysaccharide from Saccharomyces cerevisiae alone (e.g., laminarin). Figure 1 and 3 ), even at lower inclusion rates (i.e. 5 dry weight % based on the total weight of the composition or 10 dry weight % based on the total weight of the wall polysaccharides in the composition).

[0069] Embodiment 2:

[0070] A composition comprising wall polysaccharides from Candida utilis (CU) and wall polysaccharides from Saccharomyces cerevisiae (SC) was tested as a preventive feeding strategy to reduce the susceptibility of shrimp to Vibrio disease. The amount of wall polysaccharide was 50 wt% based on the total weight of the composition (50 SC / CU).

[0071] To evaluate the benefits of 50SC / CU wall polysaccharide over SC wall polysaccharide alone, we applied an in vivo disease challenge to juvenile shrimp. The infection method was immersion and we used a virulent strain of Vibrio parahaemolyticus capable of inducing acute hepatopancreatic necrosis syndrome.

[0072] The wall polysaccharides tested were:

[0073] - 50SC / CU wall polysaccharide containing 6 % dry weight (based on the total weight of the composition, or 12 % dry weight based on the total weight of wall polysaccharides in the composition) of wall polysaccharides from Candida utilis NRRL-900: 3 doses tested: 0.04%; 0.08% and 0.12% of the feed

[0074] - 60SC wall polysaccharide from a strain of Saccharomyces cerevisiae (CNCM I 1079): Dose tested: 0.12% of the feed

[0075] The experiment was conducted with juvenile Litopenaeus vannamei weighing 2.07 ± 0.05 g, fed a diet supplemented with or without the candidate to be tested for 21 days, and then treated with 100 ml of a 1.1 × 10 9CFU / ml culture immersion challenge of a virulent strain of V. parahaemolyticus that induces AHPNS (grown in TSB + 2% NaCl (TSB +) at 28°C for 18 hours). Mortality was monitored for 10 days and the cumulative survival of each diet was statistically compared to the control within each trial. Kaplan and Meier survival curves (Kaplan and Meier, 1958) were also constructed for each diet and the curves were compared using the log-rank test to determine differences between the curves and whether the survival trends between the curves were different. The significance level was set at P = 0.05.

[0076] The 50SC / CU treatment showed a linear dose response to reduce pathogen strain-induced mortality (LogRank (Mantel-Cox), p < 0.01). An inclusion rate of 0.04% still significantly increased survival compared to the positive control (infected). 60SC / CU showed only a marginal improvement of 0.12%.

[0077] Operation plan:

[0078] A small number of juvenile Litopenaeus vannamei were fed a commercially formulated shrimp diet mixed with the test product for 21 days and then bath exposed to Vibrio parahaemolyticus (the agent that causes acute hepatopancreatic necrosis disease - AHPND) to determine if the feed additive affected survival and infection rates.

[0079] Aquaria and experimental design:

[0080] The SPF (specific pathogen-free) animals used in this example have been examined for important infectious diseases using histopathology and PCR techniques, including WSSV (white spot syndrome virus), TSV (Taura syndrome virus), IMNV (infectious myonecrosis virus), AHPND (acute hepatopancreatic necrosis disease). Postlarvae at 12 days of age were transferred from the hatchery to a wet laboratory and cultured for another 45 days under strict biosafety to reach a size of 1-2 grams. One day before the start of the study, each 1-2 gram SPF Litopenaeus vannamei was transferred to 27 90L tanks (35 shrimps / tank). At the end of the trial, all shrimps in each tank were weighed in-group before stocking to calculate growth rate. All animals in these tanks were fed their respective test baits for 21 days (Table 1).

[0081] Four replicate tanks per treatment were fed with formulated feed containing the test product. Four tanks were designated as positive controls and the other four tanks were used as negative controls. The negative control tanks were also fed commercial granular shrimp feed, but were not challenged with Vibrio parahaemolyticus, which causes EMS / AHPND. All tanks received their respective feeds at approximately 5% of their body weight daily.

[0082] Sixteen treatment tanks were challenged with V. parahaemolyticus 21 days after the feed containing the feed additive was administered. At the same time, the positive control tank was fed the same commercial shrimp feed without any additions and challenged with V. parahaemolyticus on day 21 of the trial. The challenged shrimp were kept for another 10 days in order to record survival and monitor growth rate.

[0083] Attack Methods:

[0084] In this study, shrimp were subjected to immersion challenge. Tryptic soy broth + 2% (TSB+) sodium chloride inoculated with a virulent strain of Vibrio parahaemolyticus for 18 hours was added directly to the tank. The bacterial suspension was added to the tank to obtain bacterial density measured by optical density absorption (OD600nm). During the challenge period, the shrimp continued to maintain their respective feed treatment for another 10 days.

[0085] result

[0086] The final survival rates at the end of the experiment after statistical treatment are listed in Table 1. Figure 5 The distribution of survival rates of different groups of shrimp during the experiment according to the Kaplan Meyer procedure is reported.

[0087] Table 1: Final survival: summary of observed treatments

[0088]

[0089] Tables 2 and 3 show the statistical differences between the survival distributions of shrimps in different groups.

[0090] As shown in Table 2, all tests applied showed highly significant treatment effects between the survival rate distributions.

[0091] Table 2: Statistical comparison: testing the equality of survival distributions among treatment levels.

[0092]

[0093] As shown in Table 3, bacterial challenge significantly affected the survival of shrimp in all groups when the death kinetics of each infected group were compared to the negative control (non-infected group) using the Kaplan-Meier method and the log-rank (Mantel-Cox) test. In addition, these in vivo results confirm the beneficial effects of including wall polysaccharides from Saccharomyces cerevisiae and Candida utilis in modulating and / or stimulating natural defenses and improving the performance of animals under challenge conditions (Table 3).

[0094] Table 3: Statistical comparison of distributions using the log-rank (Mantel-Cox) test: Testing for equality of survival distributions between different treatments.

[0095]

[0096] Example 3

[0097] The experimental results of this example in sea bass showed that "50SC / CU wall polysaccharide" versus "60SC wall polysaccharide" had a positive effect on fish performance and response to stress.

[0098] Sea bass (Dicentrarchus labrax) with an average weight of 15.4 g were fed with 0.08% of "50SC / CU wall polysaccharide" versus 0.2% of "60SC wall polysaccharide" for 10 weeks.

[0099] As shown in Table 4, feeds containing high levels of soybean meal (40%) were formulated.

[0100] Table 4: Bait recipe

[0101]

[0102] 3 tanks with 25 fish per treatment. Salinity stress was applied at week 8. Parameters recorded: Growth performance / gut morphology / gut microbial flora.

[0103] Table 5 shows the final performance in week 10.

[0104] Table 5: Week 10 performance

[0105]

[0106] All data are expressed as mean ± standard deviation (SD). Data were converted when necessary and statistically analyzed using SPSS statistical software version 18 for Windows (SPSS Inc., Chicago, IL, USA) and P < 0.05 level was accepted as significant. All data were analyzed using one-way analysis of variance. Mann-Whitney's nonparametric test was used to determine significant differences between the control group and the experimental group.

[0107] Figures 6a and 6b show the scanning electron microscopy results of the microvillus density of the distal small intestine of seabass after feeding the experimental diets at the 5th and 10th weeks, respectively. Data are expressed as mean ± SD (n = 3). Significant differences from the corresponding controls are expressed as (*) P < 0.01, (**) P < 0.001. Data were converted where necessary and statistically analyzed using SPSS statistical software version 18 for Windows (SPSS Inc., Chicago, Il, USA) and the level of P < 0.05 was accepted. One-way analysis of variance was used to analyze the data, and Tukey's post hoc test was used to determine significant differences between the control group and the experimental group.

[0108] Table 6: Light microscopy examination at week 5 and week 10

[0109]

[0110] All data are presented in Table 6 as mean ± standard deviation (SD). Data were converted when necessary and statistically analyzed using SPSS statistical software version 18 for Windows (SPSS Inc., Chicago, IL, USA) and the level of P < 0.05 was accepted. One-way ANOVA was used to analyze all data. Post hoc Tukey HSD test was used to determine the significant differences between the control group and the experimental group.

[0111] Figure 7 Shown is the expression of two immune genes (IL1β and IL10) in the distal intestine of seabass after feeding the experimental diet according to the present disclosure at week 10.

[0112] Embodiment 4:

[0113] This example demonstrates the effect of supplementing weaned piglets with a combination of wall polysaccharides according to the present disclosure in the second phase feed.

[0114] Experimental setup

[0115] Duration of trial: 42 days

[0116] Animal materials and housing conditions:

[0117] Weaned pigs were housed in pens of 12 piglets. In one building, 6 pens were assigned to one of the two treatments (control and treatment). Two series of experiments were repeated over time, resulting in 12 replicate pens per treatment when the two series were analyzed together.

[0118] Test product: 50SC / CU wall polysaccharide with 6% CU

[0119] Treatments: The experiment consisted of 2 treatments:

[0120] T-1 - Control - Normal diet without yeast derivatives

[0121] T-2-control diet supplemented with 50SC / CU wall polysaccharide at 0.08%

[0122] Bait

[0123] The animals were fed a two-phase diet as shown in Table 7.

[0124] Table 7: Two-phase diet

[0125]

[0126] Zootechnical parameters measured:

[0127] Body weight / pen at the beginning and end of each feeding period,

[0128] Feed consumption in the second feeding phase / circle

[0129] Mortality and culling rates

[0130] Statistical analysis

[0131] The results were analyzed using the GLM (generalized linear model) univariate model using SPSS.

[0132] result :

[0133] A. Growth performance

[0134] Significant effects were detected on final body weight and average daily gain as shown in Table 8. This effect was mainly due to a significantly better performance in the second phase, with significantly higher daily growth rates and feed conversion rates in the treatment groups.

[0135] Table 8:

[0136]

[0137] Other Observations: Only one piglet died in one of the treatment pens, while three (3) died in the control pen, all due to meningitis. Fewer ear bites were observed in the treatment groups. Feces were more liquid in the control group.

[0138] Embodiment 5:

[0139] This example evaluates the effects of a 50SC / CU wall polysaccharide composition on the zootechnical and health performance of pre- and post-weaning calves when supplemented via solid feed.

[0140] Duration of the study : 6 weeks.

[0141] Materials and methods

[0142] Animals involved:

[0143] Total Animals : 129 at the beginning and 125 at the end.

[0144] gender :male

[0145] variety : Friesian (Source: from different farms in France)

[0146] Experimental treatment

[0147] Two dietary treatments were compared, using the same dietary meal supplemented with different products:

[0148] - As T0: control diet without any product.

[0149] -T1: same diet supplemented with 50 SC / CU wall polysaccharide

[0150] ○ First 2 weeks: 5 g / animal / day = 2.7 g / kg starter feed

[0151] ○ Then: 3 g / animal / day = 1.6 g / kg starter feed

[0152] Experimental design

[0153] After arrival at the farm, the animals were randomly assigned to pens. Six days later, the experiment was started and the animals were weighed to homogenize between pens with mean body weight and mean body weight standard deviation. All pens were run simultaneously. The average starting body weight was 53.9 ± 4.3 kg, and the average starting age was 27.3 ± 4.4 days.

[0154] Site description and husbandry

[0155] All animals were housed in the same building, 10-11 animals in pens with straw. Each treatment involved four pens: three pens with 11 animals each and one pen with 10 animals = 43 animals per treatment. Each pen had a feeder, and there was no contact between feeders in the pens. The straw was shared between the two pens, but there was no cross contamination between treatments. Milk was distributed twice a day, at 8:00 am and 6:00 pm. Calf concentrate feed was distributed manually once a day in the feeding trough, and the amount distributed was weighed daily before distribution. All animals had free access to the straw.

[0156] The animals were fed according to the following schedule:

[0157] ○ Ds arrival - 2 weeks: 2 x 1.5 L / day of colostrum milk dilution (25% CP and 19.5% fat), then milk replacer (22% CP and 18% fat) + solid feed (powdered diet) ad libitum + straw ad libitum + water ad libitum

[0158] ○2 weeks to weaning (34 days): 1×2L / day milk replacer (19% CP and 15% fat) + solid feed (powdered meal) ad libitum + straw ad libitum + water ad libitum

[0159] ○ Weaning-Leaving: Solid feed (powdered meal) ad libitum + straw ad libitum + water ad libitum

[0160] Calves are weaned when they show good body condition and start eating more than 1 kg / day / animal.

[0161] Recorded parameters and applied methods

[0162] Individual live weight: Calves were weighed individually every two weeks at 10 am from the start of the study until the end of the study (4 weighings in total). Feed consumption: Feed consumption (concentrate feed) was recorded weekly per pen = summed up as the weight of feed offered each day - the last day of the week to weigh the rejected feed.

[0163] result

[0164] Body weight (BW) and average daily gain (ADG)

[0165] As shown in Table 9, the mean body weight (BW) of animals fed T1 tended to be better than that of animals fed T0. No interaction was found between treatment and time period. The initial and final BW (BW0 and BW42, respectively) were numerically higher for T1.

[0166] Table 9: Body weight during the trial

[0167]

[0168] As shown in Table 10, although animals from T1 had numerically better weight gain than animals from T0, there was no difference in average daily gain between treatments.

[0169] Table 10: Average daily weight gain during the trial

[0170]

[0171] The standard deviation within the pen is a good reflection of the heterogeneity of weight and gain as farmers seek better uniformity after more uniform intake. The standard deviation at T1 was significantly lower than at T0, meaning that control animals had greater heterogeneity in weight than treated animals (Table 11). No statistical significance was observed with regard to heterogeneity in growth, but numerically, control animals had more heterogeneous growth than treated animals.

[0172] Table 11: Standard deviation of BW and ADG during the test (circled)

[0173]

[0174] Concentrate feed intake and feed efficiency (FE) and feed conversion ratio (FCR)

[0175] Statistical analysis Total concentrate feed intake (total intake / number of animals over total period of time in the pen) did not show any differences from week to week. Analysis of feed conversion ratio did not show any significant differences between treatments.

[0176] Diarrhea and morbidity

[0177] As shown in Table 12, animals from T1 showed fewer days of diarrhea and required a lower number of antibiotic treatments compared to the control T0.

[0178] Table 12: Diarrhea days and number of antibiotic treatments

[0179]

[0180] The Mann-Whitney test applied to the percentage of animals treated at least once for diarrhea showed a significant decrease at T1 compared to T0, and a trend towards a decrease in the percentage of animals treated with antibiotics.

[0181] Percentage of animals treated at least once with antibiotics and animals treated at least once for diarrhea.

[0182]

[0183] Embodiment 6:

[0184] Juvenile Litopenaeus vannamei were fed a commercially formulated shrimp feed (mixed with a feed additive product supplied by LALLEMAND) for 14 days and then challenged with the microsporidian parasite Enterocytozoon hepatopenaei for 14 days to determine if any dose of the feed additive had a positive effect on survival, reduced infection rates and parasite load.

[0185] Materials and methods

[0186] SPF (specific pathogen free) juvenile shrimp were used in this study. These shrimp were examined for WSSV (white spot syndrome virus), TSV (taura syndrome virus), IMNV (infectious myonecrosis virus), EMS / AHPND (early mortality syndrome / acute hepatopancreatic necrosis syndrome) and EHP (enterohepatocellular carcinoma). One day before the start of the study, each 1.50 ± 0.09 g of Litopenaeus vannamei was transferred to a 120 L tank (30 shrimp / tank) containing 90 L of water at a salinity of 20 ppt. Each tank was equipped with a submerged biofilter containing activated corals.

[0187] Experimental design

[0188] The experiment was set up as a completely randomized design, where each treatment was randomly designed with a different tank. The total length of the trial: 29 days, according to Table 13, including 1 day of acclimatization, 14 days of feed additive application, and then 14 days of EHP challenge under the same feeding treatment. During the trial, shrimp in all tanks received their respective feeds four times a day (Table 14) at full capacity. Five tanks were filled with formulated feed containing feed additives. Five tanks were designated as positive controls for cohabitation challenge and five tanks as negative controls.

[0189] Table 13: Experimental schedule

[0190]

[0191] Table 14 Definitions of all 120 L aquariums used in the EHP study

[0192]

[0193] Shrimp bait manufacturing

[0194] The feed additive was mixed with feed powder and thickener (CMC-carboxymethylcellulose) and water were added, and then extruded using a pressurized meat grinder. The feed was then dried at 50 degrees Celsius for 6 hours. The feed was crushed to the desired size (1.5-2 mm in length) for the study.

[0195] Attack Methods

[0196] Small juvenile shrimp were challenged with the enteric hepatocellular carcinoma causing EHP (a microsporidian parasite that mimics the natural route of infection) using a standard cohabitation challenge method.

[0197] SPF and infected shrimp were separated by a net divider that allowed free exchange of water. The cohabitation challenge method was designed as follows: a 50-cubic-liter rectangular net was installed in each 120-liter plastic tank. During the challenge period, 20 EHP-infected shrimp were stocked in the hanging net and 30 SPF shrimp were stocked outside the hanging net. Both EHP-infected shrimp and SPF shrimp were fed with the experimental feed of their respective treatments for another 14 days. The negative control (F) was also treated with the same challenge method. However, 20 SPF shrimp were stocked in the hanging net instead of 20 EHP-infected shrimp. During the challenge period, both the shrimp in the net and the shrimp outside the net of the negative control were fed commercial shrimp bait.

[0198] The parasite burden density of EHP-infected shrimp was 5.05E+06 CFU / g of Enterohepaticocystis at the start of challenge.

[0199] Sampling and observation

[0200] qPCR testing for EHP was done on 5 shrimp / tank / time point at 3 time points: on day 14 (just before challenge) to confirm the EHP-free status of SPF shrimp, on day 15 (24 hours after challenge) and on day 18 (96 hours after challenge) to quantify parasite load. At the end of the challenge study, all live animals were counted as survivors.

[0201] Statistical analysis

[0202] Mean values ​​from the seven treatments were compared with analysis of variance using Fisher's LSD test, and differences were considered significant when P < 0.05.

[0203] result

[0204] Shrimp Performance Parameters

[0205] Average production parameters during the 28-day experimental period are given in Table 15. Values ​​represent the mean of five replicates.

[0206] Table 15

[0207]

[0208] Survival rate of challenge test

[0209] Survival rate of treatments 14 days after challenge

[0210] The survival rate of treated shrimp 14 days after challenge was Figure 8 Mean ± SD are given in ; means indicated with the same capital letter were not different between treatments (Fisher's test, 5% probability).

[0211]

[0212] The results of qPCR testing for EHP parasite load are given in Table 16. At the beginning of the challenge experiment (just before the challenge), there was an EHP-free state in the treatments. 48 hours after the challenge, the experimental shrimp in treatment A and the positive control (G) were attacked by the disease, while in the negative control (F) there was an EHP-free state. Therefore, this shows that the experimental setup was acceptable and that no cross-contamination occurred in the negative control. The EHP parasite load in treatments A and G was similar after 48 hours. However, after 120 hours after the challenge, the EHP parasite load in 4 out of 5 tanks in treatment A was below the detection limit, while all tanks from the negative control were EHP positive (5 / 5).

[0213] Example 7

[0214] Evaluation of the effect of 50SC / CU wall polysaccharide on heat stress resistance in broiler chickens

[0215] Heat stress is one of the most important environmental stressors challenging poultry production worldwide. Heat stress negatively affects poultry performance, reducing growth and product safety. During heat stress, broiler chickens undergo extensive thermoregulatory adaptations to prevent death from heat exhaustion, which has a negative impact on performance.

[0216] Experimental design

[0217] Place :Private experimental farm (France)

[0218] Experimental design : 2 groups:

[0219] Control (C): no supplementation; 50SC / CU wall polysaccharide (Y): 800g / ton and 400g / ton of 50SC / CU wall polysaccharide in the initial and growth phases, respectively. No 50SC / CU wall polysaccharide was supplemented during the fattening period (800-400-0).

[0220] Diet:

[0221] 3 phases (starting, growing and finishing). The three feeding phases are: Days 0-10, 11-25, 26-35.

[0222] animal:

[0223] The trial was conducted on broiler chickens of the Ross PM3 breed, which were randomly allocated to 13 pens upon arrival, with a density of 20 per pen (C: 6 pens, Y: 7 pens).

[0224] Heat stress attack:

[0225] Heat stress challenge was performed on day 20: the room temperature was raised to 31 °C during the night between days 19 and 20.

[0226] Measurement: Number and date of deaths per lap.

[0227] Statistical analysis:

[0228] The differences in mortality between the two groups during the trial (and especially the resistance of the birds to heat stress) were analyzed by the Kaplan-Meier test. The differences between the groups were considered significant at p values ​​of p < 0.1. Otherwise the statistical analysis showed no significance (NS).

[0229] result

[0230] Mortality and resistance of birds to heat stress challenge: The results are given in Table 17

[0231] Table 17

[0232]

[0233] Forty birds died during the heat stress challenge: nine birds in the 50SC / CU wall polysaccharide group and 31 birds in the control group. The total number of deaths in the 50SC / CU wall polysaccharide group was 17 and in the control group 38. The Kaplan-Meier analysis (a statistical test comparing the mean survival time of two groups) was highly significant (p<0.001) and gave the following estimates: The mean survival time was 29.6 and 32.9 days for groups C and Y, respectively. Therefore, the 50SC / CU wall polysaccharide group was more resistant to the acute heat stress challenge than the control group.

[0234] In conclusion, feeding 50SC / CU wall polysaccharide resulted in greater resistance to heat stress.

[0235] Example 8

[0236] Introduction and Purpose

[0237] The aim of this study was to test the effects of 50 SC / CU PARTILE polysaccharide on performance, morbidity and incidence of diarrhea in weaned piglets after drug withdrawal.

[0238] Experimental design

[0239] Animals and housing

[0240] A total of 480 weaned piglets (dam line: Large White x Landrace, sire line: Danbred Duroc) from 4 consecutive batches were distributed in 8 nurseries with 4 pens per nursery and 20 piglets per pen. They were distributed so that the initial body weight was as uniform as possible within a pen and between treatments, and 10 gilts and 10 boars were placed in each pen.

[0241] Piglets were vaccinated against Mycoplasma at 7 and 21 days of age and against Circovirus at 21 days of age.

[0242] Time period

[0243] The trial started with weaning at an average age of 19 days and lasted for 55 days.

[0244] deal with

[0245] Piglets were fed a two-phase feeding program and had ad libitum access to feed and water throughout the experimental period. There were two treatments (Table 18): Control (C) and Trial 1 (T1).

[0246] The current antibiotic strategy used on the farm is: Prestarter: 120ppm colistin + 300ppm amoxicillin + 2400ppm ZnO; Starter: 1600ppm ZnO.

[0247] For the test, to challenge the piglets, the drugs became: Preparation period: 2400 ppm ZnO; Initial period: blank feed.

[0248] Table 18 Processing

[0249]

[0250] Experimental setup and observations

[0251] - Body weight: Individually. At the beginning and end of the experiment, and when changing the diet.

[0252] - Feed intake: per round. When changing the diet, this is the difference between the feed supply for the day and the remaining feed.

[0253] - Morbidity: Individual and group health care interventions, including regular herd prevention.

[0254] - Mortality: date, weight and apparent cause.

[0255] - Diarrhea: Every lap. Check the incidence of diarrhea every day.

[0256] statistics

[0257] The variance was analyzed using the general linear model in SPSS 22.0 (IBM) according to the following model:

[0258] Y jk =μ+ax IBW+batch j +Processing k +Batch j *deal with k+e jk

[0259] in:

[0260] Y = final body weight (FBW), average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (FCR), mortality; IBW = initial body weight used as covariate; Batch = fixed effect due to batch (j = 1, 2, 3, 4); Treatment = fixed effect due to treatment (k = C, T1); Batch*Treatment = interaction between experimental treatment and batch; e = error. Experimental unit is circle. Significance was declared from P < 0.05.

[0261] No interaction was found between treatment and batch. Therefore, it was removed from the model.

[0262] result

[0263] Performance

[0264] There were significant differences in FBW (P<0.01), starter and overall growth (P<0.05), and starter FCR (P<0.05), with piglets in T1 having higher BW, faster growth, and lower FCR. In addition, T1 piglets grew faster in the starter period (P<0.1) and ate more in the starter period (P<0.1). The performance results are shown in Table 19.

[0265] Table 19 Results of the period and overall performance of piglets in treatments C and T1

[0266] Comparison T1 SEM P-value IBW(kg) 6.40 6.40 0.089 - BW preparation period (kg) 11.33 11.63 0.123 0.101 FBW(kg) 30.43 31.58 0.276 0.008 ADG preparation period (g / d) 240 255 6 0.098 ADG start period (g / d) 581 607 7 0.015 Total ADG (g / d) 449 470 4 0.012 ADFI preparation period (g / d) 350 382 13 0.090 ADFI start period (g / d) 960 963 17 0.915 Overall ADFI (g / d) 724 737 14 0.484 FCR preparation period (g / g) 1.457 1.499 0.042 0.496 FCR start period (g / g) 1.657 1.585 0.023 0.041 Overall FCR (g / g) 1.614 1.566 0.023 0.160

[0267] The fact that the differences were larger during the start-up period than during the run-in period could be due to the fact that the health status of the farm was good and piglets fed the blank diet were more aggressive during the start-up period than during the run-in period.

[0268] Mortality was low during the experimental period and did not differ between treatments. It averaged 1.8% over the entire period. A total of 13 piglets died during the trial.

[0269] in conclusion:

[0270] The main conclusions of this trial are:

[0271] -Supplementation of the post-weaning diet with 50 SC / CU wall polysaccharide improved final body weight, overall average daily gain and overall feed conversion ratio in terms of quantity compared to the limited drug control diet.

[0272] - The impact seems to be greater during the start-up period than during the preparation period, which may depend on the health of the farm.

[0273] - 50SC / CU wall polysaccharide may be a good alternative to ZnO and / or antibiotics during the start-up period.

[0274] It is recommended to test the possibility of 50SC / CU wall polysaccharide replacing antibiotics, compare the 50SC / CU wall polysaccharide treatment with the positive control, and the possible complementary effect of adding 50SC / CU wall polysaccharide immediately after antibiotics.

[0275] Example 9

[0276] Facility, sample description, and experimental baits

[0277] Experiments were conducted with rainbow trout (Oncorhynchus mykiss) fry. After four weeks of acclimatization, 480 fish (23.07 ± 0.2 g) were randomly assigned to 16 × 150-l fiberglass tanks (30 fish per tank) containing aerated recirculating fresh water. Fish were fed a fixed diet at 3% body weight for 56 days during three feeding times (0900, 1300 and 1700 h). Fish were weighed in batches every two weeks after a 24-h starvation period and were housed at 14.5 ± 0.5 °C with a 12:12 h light:dark photoperiod. Water pH was maintained at 6.8-7.5, dissolved oxygen at 7.5-8 mg / l, ammonium at 0.04-0.08 mg / l, nitrite at 0.02-0.06 mg / l, and nitrate at 54-58 mg / l. For each treatment, rainbow trout were fed one of four diets in quadruplicate tanks: 1] control, 2] 60 SC wall polysaccharide, 3] 50 SC / CU wall polysaccharide - continuous feed and 4] 50 SC / CU wall polysaccharide - pulsed for 56 days (see Table 21 for diet composition).

[0278] Table 21. Formulations of experimental diets used in the Plymouth experiment. Each ingredient component is expressed in g / kg of each diet.

[0279]

[0280] method

[0281] Mucus collection

[0282] Fish were randomly selected and humanely killed according to station operating procedures. MS222 was the anesthetic of choice. Care must be taken when netting the fish to complete the process as quickly as possible to minimize damage to the mucosal surface of the fish. Immediately after death, mucus was collected by scraping one side of the fish with a small spatula from the edge of the gill cover to the anus ( Fig. 9 : Directions for Atlantic salmon and rainbow trout spatula). Transfer the accumulated mucus at the tail end of the fish into a 1 ml pre-weighed syringe. Store the mucus in 1 ml eppendorf tubes and immediately freeze at -80 °C until analysis.

[0283] Real-time qPCR sample collection

[0284] Skin samples (<100 mg) were collected from rainbow trout, transferred to 1 mL of RNAlater solution (Applied Biosystems, Warrington, UK), and stored at 4°C for 24 h and then at −80°C until RNA extraction. After RNA extraction, qPCR was applied to observe the expression of specific biomarkers of mucus production.

[0285] Growth performance calculation

[0286] Growth was assessed by weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and fatness (K). The following formulas were used for calculation: NWG (g) = FW-IW; SGR (% BW / day) = 100 ((lnFW-lnIW) / T); FCR (g / g) = FI / WG; PER = WG / PI; K = FW / (FL 3 ). Where FW = final body weight (g), IW = initial body weight (g), T = feeding duration (days), WG = wet weight gain (g), FI = feed intake (g), PI = ingested protein (g) and FL = final length (cm).

[0287] result

[0288] Mucus sample analysis

[0289] The data on mucus production are given in Table 22. Compared to the control, mucus production was increased in fish fed 60SC and 50SC / CU, with a numerical advantage in favor of 50SC / CU.

[0290] Table 22. Skin mucus production (mg / cm) in fish after 28 and 56 days of feeding on the experimental diets.

[0291] Data are expressed as mean ± SD.

[0292]

[0293] Real-time qPCR analysis

[0294] Gene expression analysis of rainbow trout skin samples showed that the expression of mucus production-specific biomarkers was significantly upregulated in fish fed 50 SC / CU wall polysaccharide continuously (2.31 ± 1.26 NEL; P = 0.0003) and pulsed (2.09 ± 0.58 NEL; P < 0.0001) compared with control-fed fish (1.16 ± 0.48 NEL). Fig.10 It shows that feeding supplemented with 60SC wall polysaccharide and (50SC / CU wall polysaccharide TMRelative expression of this gene in the skin samples of rainbow trout (O. mykiss) fed an experimental diet of ) for 8 weeks. Data are presented as box plots (n=10 / treatment). *- indicates significant upregulation; P<0.0001

[0295] Growth performance

[0296] No significant differences in growth performance were found in rainbow trout fed the experimental diet for 56 days compared to fish fed the control diet. Fig.11 In the figure, the growth performance of rainbow trout fed the experimental diet for 56 days is shown.

[0297] Embodiment 10:

[0298] Fish, experimental design, and bait formulation

[0299] After acclimation for 4 weeks, 225 seabass (15.45 ± 0.1 g) were randomly assigned to 9 × 110-l fiberglass tanks (25 fish per tank). Seabass were fed a fixed ratio of 2.0% body weight per day for 10 weeks during 3 feeding periods at 9:00, 13:00, and 16:30 h. After a 24-h starvation period, fish were weighed in batches weekly and housed at 24.14 ± 0.85 °C, salinity 26.58 ± 4.32, and a 12:12 h light:dark photoperiod. The water pH was maintained at 6.8-7.5, dissolved oxygen at 7.5-8 mg / l, ammonium at 0.04-0.08 mg / l, nitrite at 0.02-0.06 mg / l, and nitrate at 54-58 mg / l.

[0300] The bait was formulated to contain a high inclusion level of soybean meal (inclusion 40%) and the bait formulation is shown in Table 23.

[0301] Table 23. Formulation of experimental diets. Each ingredient component (% inclusion level) is indicated.

[0302]

[0303] method

[0304] Light microscopy analysis (LM)

[0305] The distal intestine of the fish was removed, fixed with 10% marine formalin and kept at 4°C for 48 hours, then transferred to 70% ethanol for storage. Prior to analysis, intestinal samples were dehydrated (Leica TP 1020) and embedded in paraffin according to Dimitriglou et al. (Journal of animal science, 2009, 87, 3226-3234).

[0306] The samples were sliced ​​at 5 μm thickness (Leica RM2235 microtome), dried in an oven overnight, and then automatically stained with hematoxylin and eosin (HE) (Leica Autostainer XL). The slides were mounted with coverslips using DPX and dried. Photos of the stained samples were then taken (Leica DMIRB microscope and Olympus E410 digital SLR camera). Image analysis was performed using Image J 1.47v (National Institutes of Health, Bethesda, Maryland, USA) software. In addition to goblet cells (GC), LM images were used to calculate the length of the folds (FL) and the width of the lamina propria (LP-W), measuring more than 200 μm. For the perimeter ratio (PR), after conversion to 8 bits, the image threshold was adjusted. The perimeter ratio was calculated by dividing the inner perimeter by the outer perimeter. Data are expressed as mean ± standard deviation (SD) and statistical analysis was performed using SPSS Statistics Version 15 for Windows (SPSS Inc., Il, USA) and accepted at P < 0.05. Histological data were analyzed using one-way ANOVA. Post hoc Tukey HSD tests were used to determine significant differences between the control and treatment groups.

[0307] Scanning electron microscopy (SEM)

[0308] The samples were fixed in 0.1 M sodium cacodylate buffer with 2.5% glutaraldehyde (1:1 volume ratio, pH 7.2). The samples were then dehydrated in a gradient ethanol series (30% alcohol, 50%, 70%, 90% and 100% x2) for 15 minutes at each step and critical point dried (K850 Emitech) with ethanol as the intermediate fluid and CO2 as the transition fluid. The samples were then sputter coated with gold (K550 Emitech) and observed with a JSM 6610LV scanning electron microscope. For each sample, multiple images were captured at a magnification of x 500-x 20,000 to assess the overall integrity of the intestine. Microvilli density measurements were performed using Image J (V1.45) using images at a magnification of x 20,000 ( Fig.13 ). One-way ANOVA was used to analyze SEM data. Post hoc Tukey HSD test was used to determine significant differences between the control and treatment groups.

[0309] Real-time qPCR sample collection

[0310] Hindgut samples (<100 mg) were collected from European seabass, transferred to 1 mL of RNAlater solution (Applied Biosystems, Warrington, UK), and stored at 4°C for 24 h and then at -80°C until RNA extraction.

[0311] RNA extraction and cDNA synthesis

[0312] Total RNA was extracted using TRI reagent (Ambion, Life technologies, UK) according to the manufacturer's instructions with some modifications. Briefly, 50-100 mg of intestinal sample was removed from RNAlater solution and excess solution was removed by pressing the sample between sterile paper towels. The sample (<100 mg) was transferred to a test tube containing 1 mL of TRI reagent and homogenized for 10 minutes. 200 μl of chloroform was then added and, after mixing, the sample was centrifuged at 12,000 × g for 15 minutes. The upper aqueous phase was transferred to a tube containing an equal volume of isopropanol. The mixture was vortexed and centrifuged at 14,000 × g for 15 minutes. The supernatant was discarded and the precipitated RNA precipitate was washed with 1 ml of 75% ethanol. Total RNA was dissolved in diethyl pyrocarbonate (DEPC) and purified using RNeasy Plus Mini Kit according to the manufacturer's instructions (Qiagen, UK) to remove any contaminating genomic DNA. The concentration and quality of RNA in each sample were determined by measuring the absorbance ratio at 260 / 280nm and 260 / 230nm (NanoDrop Technologies, Wilmigton, USA). The integrity of RNA was confirmed by running the samples on a Bio-analyser, with an RNA integrity number (RIN) range of 8.0-9.5 (Agilent technologies, UK), and the samples were stored at -80°C. A total amount of 1 μg of RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad, UK). The reactants were placed at 25°C for 5 minutes, then at 42°C for 30 minutes, and inactivated at 85°C for 5 minutes. The iScript cDNA synthesis kit contains a combination of oligo dT and random hexamers, which can be used for a variety of targets.

[0313] Real-time PCR assay

[0314] Using StepOne Plus TMPCR reactions were performed using the SYBR green method using a real-time PCR thermocycler (Applied Biosystems). Duplicate PCR reactions were performed for each sample analyzed. 2 μl of diluted (1 / 10) cDNA was mixed with 5.5 μl of 2× concentrated iQ TM SYBR Green Supermix (Bio-Rad), 0.3 μM forward primer and 0.3 μM reverse primer were mixed and each PCR reaction was placed on a 384-well plate. The primers used and their sequences are shown in Table 1. The thermal curve for all reactions was 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 59°C. Fluorescence was monitored at the end of each cycle. Additional dissociation curve analysis was performed and showed a single peak in all cases. β-Actin and GAPDH were used as reference genes in each sample to normalize the results by eliminating variations in the quantity and quality of mRNA and cDNA (Bustin et al., 2009). In fact, according to geNorm TM The algorithm used by the software (Vandesompele et al., Genome biology, 2002, 3(7), research 0034) confirmed the stability and suitability of GAPDH and β-actin as reference genes.

[0315] An expression stability value "M" was generated for each reference gene. No amplification product was observed in the negative control and no primer dimer formation was observed in the control template. Modifications in gene expression are expressed relative to controls sampled simultaneously with the treatments.

[0316] The cycle threshold (Ct) was defined as the point at which fluorescence rose significantly above background fluorescence and was determined manually for each run. The PCR efficiency of each primer set was determined using the equation E(PCR efficiency) = 10(-1 / slope) using serially diluted cDNA (n = 3) and the resulting Ct versus log cDNA input curve. The normalized expression level (NEL) of the target gene was calculated based on the Ct deviation (ΔCt) of the unknown sample and the control sample, and the geNorm TM Calculations outlined in the manual (http: / / medgen.ugent.be / ~jvdesomp / genorm / ) and Vandesompele et al. (Genome biology, 2002, 3(7), research 0034) are presented in comparison to the reference genes GAPDH and β-actin.

[0317] All statistics for RT-qPCR data were performed using permutation tests in R.

[0318] Growth performance calculation

[0319] Growth was assessed by weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and fatness (K). The following formulas were used for calculation: NWG (g) = FW-IW; SGR (% BW / day) = 100 ((lnFW-lnIW) / T); FCR (g / g) = FI / WG; PER = WG / PI; K = FW / (FL 3 ). Where FW = final body weight (g), IW = initial body weight (g), T = feeding duration (days), WG = wet weight gain (g), FI = feed intake (g), PI = ingested protein (g) and FL = final length (cm).

[0320] result

[0321] Optical Microscope

[0322] Light microscopy showed that sea bass fed the control and experimental diets displayed an intact epithelial barrier and a mucosal arrangement of organized villous mucosal folds. The intestinal mucosa is composed of a simple epithelial tissue and a lamina propria (LP) containing scattered IELs (see Fig.12 A). The circumference ratio (a higher circumference ratio indicates a higher absorptive intestinal surface caused by more and / or longer mucosal folds) was significantly increased in fish fed the 50 SC / CU wall polysaccharide diet at weeks 5 and 10 of the experimental period compared to fish fed the control diet (see Tables 24A and B). In contrast, fish fed the 60 SC wall polysaccharide diet regimen showed a significant increase in PR after only 10 weeks of feeding compared to fish fed the control (see Table 24B).

[0323] Table 24A. Light microscopy data after 5 weeks of feeding the experimental diets. Data are presented as mean ± standard deviation (n=6 / treatment). a-b Superscripts indicate significant differences using Tukey's HSD post hoc test

[0324]

[0325] Table 24B. Light microscopy data after 10 weeks of feeding the experimental diets. Data are presented as mean ± standard deviation (n=18 / treatment). a-b Superscripts indicate significant differences using Tukey's HSD post hoc test

[0326]

[0327] SEM analysis

[0328] The epithelial surface of all fish appeared healthy, with uniform enterocyte formation and dense microvilli, with no signs of cell or microvilli disruption or necrosis. Representative images of fish from each treatment are presented in Fig.12 In this study, microvilli density measurements were performed. Fig.13 A and 13B. After 5 weeks of feeding the experimental diet (see Fig.13 A), fish fed 60SC wall polysaccharide (2.90±1.0au) and 50SC / CU wall polysaccharide (4.81±1.0au) showed a significant increase in microvilli density (P<0.001) compared with fish fed the control diet (1.58±0.5au). Fig.13 B), Fish fed 60SC wall polysaccharide (3.51±0.4 au) and 50SC / CU wall polysaccharide (3.48±0.7 au) showed a significant increase in microvillus density (P<0.001) compared to fish fed the control diet (1.66±0.2).

[0329] Real-time qPCR

[0330] Gene expression data showed that fish fed the 60SC wall polysaccharide (0.17±0.07NEL) and 50SC / CU wall polysaccharide (0.11±0.08NEL) diets showed a significant down-regulation of heat shock protein 70 (HSP70) compared to fish fed the control diet (0.48±0.15NEL) (P=0.03). Similarly, fish fed the 60SC wall polysaccharide (0.14±0.1NEL) and 50SC / CU wall polysaccharide (0.12±0.04NEL) diets showed a significant down-regulation of proliferating cell nuclear antigen (PCNA) gene expression compared to fish fed the control diet (0.47±0.14NEL) (P=0.03). In contrast, gene expression data for the inflammatory effector cytokine interleukin-1β (IL-1β) showed significant upregulation in fish fed the 50SC / CU wall polysaccharide diet (1.74±0.4 NEL) compared to fish fed the control diet (0.51±0.4 NEL) (P=0.03). Similarly, gene expression data for the anti-inflammatory effector cytokine interleukin-10 (IL-10) showed significant upregulation in fish fed the 50SC / CU wall polysaccharide diet (2.79±2.0 NEL) compared to fish fed the fish feed control diet (0.36±0.2 NEL) (P=0.05). All data are as follows Fig.14 shown.

[0331] Example 11: Anticoccidial efficacy of 50SC / CU wall polysaccharide in commercial broiler chickens exposed to a mixed challenge of Eimeria acervulina, Eimeria maxima and Eimeria tenella

[0332] Purpose

[0333] The objective of this study was to measure the anticoccidial efficacy / susceptibility of 50SC / CU wall polysaccharide against a mixture of Eimeria acervulina, Eimeria maxima and Eimeria tenella.

[0334] Experimental design

[0335] The study started with 45 cages of 10 birds each. Eight treatments were replicated nine times.

[0336] deal with

[0337]

[0338] animal

[0339] Male broiler chickens (strain Ross 708) were used for this experiment on the day of hatching. At the hatchery, the birds were sexed and received routine vaccinations. Only healthy chickens were used in this study. At the beginning of this study, the birds were housed (day 0), at which time they were assigned to experimental cages. No birds were replaced during the study. The bird cages were weighed on days 0, 14, 20, and 28.

[0340] Poultry House

[0341] Upon arrival, the chickens were housed in battery cages. The floor area per animal was 0.51 square feet per bird. A thermostatically controlled gas furnace / air conditioner maintained uniform temperature. Even continuous lighting was provided by fluorescent lamps hung vertically along the walls. Feed and water were provided ad libitum.

[0342] Biological Agents

[0343] The coccidia inoculum consisted of a mixed culture of three species of Eimeria. The species were Eimeria acervulina, Eimeria maxima and Eimeria tenella. The coccidia inoculum was administered to the birds by oral gavage in the infection treatment.

[0344] On study day 14, each bird in the uninfected treatment received 1 ml of distilled water by mouth pipette (po). Birds in the infected treatments received a coccidian inoculum diluted to a titer of 1 ml volume (mean oocyst counts of 75,000, 25,000 and 50,000 for E. acervulina, E. maxima and E. tenella, respectively) (po).

[0345] Injury score

[0346] Five (5) birds per cage were humanely euthanized and group weighed on day 20. Birds were scored for coccidiosis lesions based on the area of ​​the intestinal tract infected.

[0347] Attack timeline

[0348] ■D14: Coccidiosis challenge: 75,000, 25,000 and 50,000 E. acervulina, E. maxima and E. tenella, respectively

[0349] ■D20: Number of oocysts in fecal material (mixed per cage)

[0350] Intestinal lesion score (5 birds / cage)

[0351] result

[0352] exist Figures 15 to 17 The results are given in

[0353] like Fig.15 As shown, animals fed 50 SC / CU wall polysaccharide showed lower specific lesions associated with infection with E. acervulina, E. maxima and E. tenella. This effect resulted in significantly lower mean lesion scores.

[0354] Interestingly, if Fig.16 As shown, the mortality of broiler chickens infected with Eimeria sp. was also numerically reduced in a dose-dependent manner during the experiment.

[0355] like Fig.17 As shown, pathogen oocyst excretion at day 20 (6 days post infection) was significantly reduced in birds fed 50 SC / CU wall polysaccharide in a dose-dependent manner.

[0356] These results support the fact that feeding broiler chickens with 50SC / CU wall polysaccharide can alleviate the negative effects associated with Eimeria infection.

[0357] Although the invention has been described in conjunction with specific embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is intended to cover any variations, uses or modifications of the invention in accordance with the principles of the invention and including those departing from the present disclosure as known or customary practice in the field to which the invention pertains and as applicable to the basic features set forth above and as in accordance with the scope of the appended claims.

Claims

1. A composition comprising a wall polysaccharide from Candida utilis and a wall polysaccharide from Saccharomyces cerevisiae, wherein the wall polysaccharides from Candida utilis and the wall polysaccharides from Saccharomyces cerevisiae are chitin, manno-oligosaccharides, β1,3 glucan and β1,6 glucan, and the amount of the wall polysaccharide from Candida utilis and the wall polysaccharide from Saccharomyces cerevisiae is 20 to 80% by weight based on the total weight of the composition.

2. The composition according to claim 1, wherein the amount of wall polysaccharide from Candida utilis is 10 to 50 dry weight % based on the total weight of wall polysaccharides in the composition.

3. A composition according to claim 1 or 2, wherein the amount of wall polysaccharide from Candida utilis is 10 to 15 dry weight % based on the total weight of wall polysaccharides in the composition.

4. The composition of claim 2, wherein the amount of wall polysaccharide from Candida utilis is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 dry weight % based on the total weight of wall polysaccharides in the composition.

5. The composition of claim 3, wherein the amount of wall polysaccharide from Candida utilis is at least 10, 11, 12, 13, 14 or 15 dry weight % based on the total weight of wall polysaccharides in the composition.

6. The composition according to any one of claims 1 to 5, wherein the amount of the wall polysaccharide from Candida utilis and the wall polysaccharide from Saccharomyces cerevisiae is 30 to 60% by weight based on the total weight of the composition.

7. A composition according to any one of claims 1 to 5, wherein the amount of wall polysaccharides from Candida utilis and from Saccharomyces cerevisiae is at least 20, 23, 25, 27, 30, 33, 35, 37, 40, 43, 45, 47, 50, 53, 55, 57, 60, 63, 65, 67, 70, 73, 75, 77 or 80% by weight based on the total weight of the composition.

8. The composition of any one of claims 1 to 5, wherein the amount of wall polysaccharides from Candida utilis and wall polysaccharides from Saccharomyces cerevisiae is at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 weight % based on the total weight of the composition.

9. The composition according to claim 1, wherein the amount of wall polysaccharide from Candida utilis is 10 to 50 dry weight % based on the total weight of wall polysaccharides in the composition; and the amount of wall polysaccharide from Candida utilis and wall polysaccharide from Saccharomyces cerevisiae is 30 to 60 weight % based on the total weight of the composition.

10. The composition of claim 1, wherein the amount of wall polysaccharides from Candida utilis is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 dry weight % based on the total weight of wall polysaccharides in the composition; and the amount of wall polysaccharides from Candida utilis and wall polysaccharides from Saccharomyces cerevisiae is 30 to 60 weight % based on the total weight of the composition.

11. The composition of claim 1, wherein the amount of wall polysaccharides from Candida utilis is 10 to 50 dry weight % based on the total weight of wall polysaccharides in the composition; and the amount of wall polysaccharides from Candida utilis and wall polysaccharides from Saccharomyces cerevisiae is at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 weight % based on the total weight of the composition.

12. The composition of claim 1, wherein the amount of wall polysaccharides from Candida utilis is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 dry weight % based on the total weight of wall polysaccharides in the composition; and the amount of wall polysaccharides from Candida utilis and wall polysaccharides from Saccharomyces cerevisiae is at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 weight % based on the total weight of the composition.

13. A composition according to any one of claims 1 to 12 for use in modulating and / or stimulating an immune response in an animal.

14. A composition according to any one of claims 1 to 12 for use in improving intestinal health, intestinal integrity and intestinal morphology in an animal.

15. A composition according to any one of claims 1 to 12 for use in stimulating an anti-inflammatory response in an animal.

16. A composition according to any one of claims 1 to 12 for use in improving growth performance parameters and feed conversion in farmed animals.

17. A composition according to any one of claims 1 to 12 for use in reducing morbidity and mortality in farmed animals, and in particular antibiotic treatment.

18. A composition according to any one of claims 1 to 12 for use in reducing mortality in farmed animals.

19. A composition according to any one of claims 1 to 12 for use in reducing diarrhoea in an animal.

20. A composition according to any one of claims 1 to 12 for use in reducing susceptibility to enteric pathogens.

21. A composition according to any one of claims 1 to 12 for use in reducing the susceptibility of an animal to parasitic infections and related conditions.

22. A composition according to any one of claims 1 to 12 for use in improving skin mucus production in aquatic animals.

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