The present invention relates to compositions for use in the treatment and/or nutrition of poultry, such as broiler chickens {Gallus gallus domesticus).

Broiler chickens are the most widely farmed animals. Around 50 billion chickens are reared each year for global consumption. Chicken farming on an industrial scale presents significant challenges both of a practical and animal welfare nature. Birds which are densely stocked, even in a free-range environment, will be apt to transmit bacterial disease. Enteric bacterial infections such as Campylobacter jejuni are both prevalent and undesirable in broilers. One of the major indications for the use of antibiotics in broilers is enteric disease (Journal of Antimicrobial Chemotherapy, Vol 61, Issue 4, Pp947-952).

Studies have demonstrated that certain commensal bacteria present in the microbiota of poultry such as broilers can have a beneficial effect upon their rearing, by improving their gut health and thereby their performance in terms of feed conversion ratio (FCR) and rate of weight gain (see for example, "Microbiota of the chicken gastrointestinal tract: influence on health, productivity and disease", Stanley et al Appl. Microbiol. Biotechnol. (2014) 98:4301-4310; and Stanley D, Hughes RJ, Geier MS and Moore RJ(2016) Bacteria within the gastrointestinal tract microbiota correlated with improved growth and feed conversion: Challenges presented for the identification of performance enhancing probiotic bacteria. Front. Microbiol. 7: 187. doi: 10.3389/fmicb.2016.00187).

Reference herein to the bacteria as being commensal refers to their presence within the gastrointestinal tract of the majority of the broiler populations. However, it is the case that because of the environment, diet, broiler stock or other factors that either a particular broiler population or, for whatever reason, a proportion of broilers within a population, have an altered microbiota or lack one or more of those bacteria.

It would therefore be desirable to identify specific probiotics for poultry such as broilers comprising one or more such bacteria. The use of such a probiotic will, therefore, result in an improvement in the profile of commensal bacteria within a broiler chicken, since it will then include one or more of these bacteria shown to be beneficial to rearing, which have a beneficial effect upon broiler health and performance.

Therefore, according to the present invention, there is provided a composition comprising:

(i) a probiotic selected from one or more of the bacteria Bifidobacterium animalis, Collinsella tanakaei, Lactobacillus reuteri, Anaerostipes, Lactobacillus crispatus, Pediococcus acidilactici, Lactobacillus pontis, Faecalibacterium prausnitzii, Coprococcus catus, Roseburia intestinalis, Anaerostipes butyraticus, Butyricicoccus, Lactobacillus johnsonii, and Ruminococcus sp. ; and (ii) a prebiotic material.

The composition of the invention may comprise the specific probiotic bacterial strain Lactobacillus crispatus DC21.1 (NCIMB 42771), deposited on 23 June 2017 at NCIMB Limited, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA,. United Kingdom.

The composition of the invention may comprise the specific probiotic bacterial strain Lactobacillus johnsonii DC22.2 (NCIMB 42772), deposited on 23 June 2017 at NCIMB Limited, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA,. United Kingdom.

The composition of the invention may comprise the specific probiotic bacterial strain Lactobacillus reuteri DC1B4 (NCIMB 42773), deposited on 23 June 2017 at NCIMB Limited, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA,. United Kingdom.

The composition of the invention may comprise the specific probiotic bacterial strain Ruminococcus sp. DC3A4 (NCIMB 42774), deposited on 23 June 2017 at NCIMB Limited, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA,. United Kingdom.

The probiotic bacteria used in the invention are typically commensal bacteria.

An example of the performance objectives for broiler chickens can be found in Aviagen Ross 308 Broiler Performance Objectives 2014 documentation.

An example of the nutrition specifications for broiler chickens can be found in Aviagen Ross 308 Broiler Nutrition Specifications 2014 documentation.

Prebiotic materials are defined by the US Food and Drug Administration as being non- digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon.

The definition provided by the US Food and Drug Administration has been reviewed and modified based on three criteria:

(a) resistance to gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption;

(b) fermentation by intestinal microflora;

(c) selective stimulation of the growth and/or activity of intestinal bacteria associated with health and well-being; In view of this, prebiotic materials are defined by Gibson et al. (2004) (Gibson, G. R, Probert, H. M., Loo, J V., Rastall, R A., and Roberfroid, M. B. (2004) "Dietary modulation of the human colonic microbiota: updating the concept of prebiotics " Nutrition Research Reviews, 17(2) 259-275) as being a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health.

Examples of prebiotics are inulin, fructo-oligosaccharides (also known as oligofructose) which is a partial hydrolysate of inulin, galacto-oligosaccharides (GOS) (also known as transgalacto-oligosaccharides), lactulose, lactosucrose, isomalto-oligosaccharides, xylo- oligosaccharides, arabinoxylo-oligosaccharides, gluco-oligosaccharides, mannan oligosaccharides (MOS), soyabean oligosaccharides, and pectic oligosaccharides.

Prebiotic definitions

The following definitions provided by Gibson et al. (2004) are accepted as standard definitions of the above-mentioned examples of prebiotics:

Inulin and fructo-oligosaccharides

Inulin, or the hydrolysed fructo-oligosaccharides, are described as either an:

• a-D-glucopyranosyl-[P-D-fmctofuranosyl]n-i-P-D-fmctofuranosi de (GF _n ); or a

• P-D-fructopyranosyl-[P-D -fmctofuranosyl] _n -i-P-D-fructofuranoside.

The fructosyl-glucose linkage is always β(2<— 1) as in sucrose, but the fructosyl-fructose linkages are β(1<— 2).

There are a number of sources of inulin. A major source of inulin is chicory. Chicory inulin is composed of a mixture of oligomers and polymers in which the degree of polymerisation (DP) varies from 2 - 60 with an average DP ~ 12.

Fructo-oligosaccharides (oligofructose) are formed by the partial (enzyme catalysed or chemical) hydrolysis of inulin giving a mixture of both a-D-glucopyranosyl-[P-D- fmctofuranosyl]n-i-P-D-fructofuranoside (GF _n ) and P-D-fructopyranosyl-[P-D -fructofuranosyl] _n - ι-β-D-fructofuranoside molecules with a DP of 2 - 7.

Galacto-oligosaccharides (GOS)

GOS are a mixture of oligosaccharides formed by the enzyme (β-galactosidase) catalysed transglycosylation of lactose and subsequent galacto-oligosaccharides. The oligosaccharides are often considered to be of the form (gal)n-glc with DP = 2 - 8 and β(1→6), β(1→4) and β(1→4) mixed linkages; however, galactans with the same linkages can be present. The product mixtures depend upon the enzymes used and the reaction conditions. GOS (galacto-oligosaccharide) is sold by Dairy Crest Ltd under the trade name Nutrabiotic® GOS for animal feed applications.

Lactulose

Lactulose is manufactured by the isomerisation (often chemical isomerisation) of lactose to generate the disaccharide galactosyl-P(l→4)-fructose.

Lactosucrose

Lactosucrose is_produced from a mixture of lactose and sucrose in an enzyme (for example β-fructofuranosidase) catalysed transglycosylation reaction. The fructosyl residue is transferred from sucrose to the C 1 position of the glucose moiety in the lactose, producing a non-reducing oligosaccharide.

Isomalto-oligosaccharides

Isomalto-oligosaccharides are_manufactured from malto-oligosaccharides, or maltose (both of which are produced from starch by the combined reactions catalysed by a-amylase and pullulanase, or β-amylase and pullulanase). The malto-oligosaccharides and maltose are converted into a(l→6)-linked isomalto-oligosaccharides by enzyme (a-glucosidase or transglucosidase) catalysed transglycosylation reactions.

Xylo-oligosaccharides and arabinoxylo-oligosaccharides

Xylo-oligosaccharides and arabinoxylo-oligosaccharides are_made from wood or cereal non- starch materials (corn cobs, wheat bran etc.). Depending upon various xylan sources used, and the method of production, the structures vary in degree of polymerization, monomeric units, and types of linkages. Generally, xylo-oligosaccharides are mixtures of oligosaccharides formed from xylose residues, typically DP = 2 - 10, linked through P(l→4)-linkages. Xylan is usually found in combination with other side groups such as a-D-glucopyranosyl uronic acid or its 4-O-methyl derivative, acetyl groups or arabinofuranosyl (giving arabinoxylo-oligosaccharides) residues.

Xylo-oligosaccharides and arabinoxylo-oligosaccharides are produced by chemical methods, enzyme catalysed hydrolysis (e.g. the hydrolysis of arabinoxylans catalysed by combinations of endo-l,4"P-xylanases, β-xylosidases, arabinofuranosidases and feruloyl esterases) or a combination of chemical and enzyme catalysed treatments.

Gluco-oligosaccharides

Gluco-oligosaccharides are often referred to as a-GOS. These are mixed a-gluco- oligosaccharides produced in reactions catalysed by dextran sucrase in fermentation processes (fermentation of Leuconostoc mesenteroides) or in the enzyme catalysed transglycosylation reactions involving sucrose in the presence of maltose. This gives oligosaccharides with a range of a-linkages (e.g. glucosyl-a(l→2)-glucosyl-a(l→6)-glucosyl-a(l→4)-glucos e). Mannan oligosaccharides (MOS)

Mannan oligosaccharides are normally obtained from the cell walls of the yeast Saccharomyces cerevisiae. and presented as products of different levels of purity. In the yeast cell wall, mannan oligosaccharides are present as:

· complex molecules that are linked to the cell wall proteins as -O and -N glycosyl groups;

• a-D-mannans made up of an a-(l,6)-D-mannose backbone to which are linked a-(l,2)- and a-(l,3)- D-mannose branches (1 - 5 mannosyl groups long).

Soyabean oligosaccharides

Soyabean oligosaccharides are a-galactosyl sucrose derivatives (e.g. raffinose, stachyose, verbascose). They are isolated from soya beans and concentrated for the final product formulation.

Pectic oligosaccharides

Pectic oligosaccharides (POS) are obtained by pectin depolymerization by either enzyme (pectin hydrolases and lyases) catalysed reactions or acid (typically) hydrolysis. Given that pectins are complex ramified heteropolymers made up of:

• a smooth region of linear backbone of a(l→4)-linked D-galacturonic acid units which can be randomly acetylated and/or methylated);

• hairy regions of rhamnogalacturonan type I and rhamnogalacturonan type II;

the structural diversity of the pectic oligosaccharides from pectin hydrolysis is high. The prebiotic materials useful in the invention may be naturally or non-naturally occurring. The probiotics are responsive to prebiotics, with the populations of the probiotics increasing due to the presence of the prebiotic material, and the presence of the prebiotic material correlates with improved broiler performance, including weight gain during rearing.

The one or more bacteria are typically selected from the more specific bacterial strains, as identified as nearest cultural examples: Bifidobacterium animalis subsp. lactis str. V9, Collinsella tanakaei str. YIT 12064, Lactobacillus reuteri str. BCS136, Anaerostipes sp. str. 35-7, Lactobacillus crispatus str. STl, Lactobacillus crispatus str. DC21, Lactobacillus crispatus str. DC21.1 (NCIMB 42771), Lactobacillus johnsonii str. DC22.2 (NCIMB 42772), Lactobacillus reuteri str. DC1B4 (NCIMB 42773), and Ruminococcus sp. str. DC3A4 (NCIMB 42774).

The probiotic bacteria used in the invention were identified as being up-regulated in a broiler trial treatment that contained galacto-oligosaccharides (GOS) in the feed, compared to a control feed.

The one or more bacteria may be selected from their nearest (based on sequence) equivalents. Identification of the bacteria included in the composition of the invention is based on Operational Taxonomic Units (OTUs) identified from 16S rDNA sequences from the V4 region of the microbiome. Specifically, 16S rRNA gene sequences were aligned against a reference alignment based on the SILVA rRNA database and clustered into OTUs with an average neighbor clustering algorithm. The nearest 16S rRNA gene sequence identities to the OTUs are reported on the basis of BLASTn searches if data matches are from type cultures with a BLAST identity >99%. The laboratory and bioinformatic techniques used to identify the bacteria included in the composition of the invention is described as follows:

Histology

Samples of ileum for histological assessment were examined from birds from each relevant treatment. The fixed tissue samples were dehydrated through a series of alcohol solutions, cleared in xylene, and finally embedded in paraffin wax (Microtechnical Services Ltd, Exeter, UK). Sections (3 to 5 μπι thick) were prepared and stained with modified hematoxylin and eosin (H&E) using standard protocols. After staining, the slides were scanned by NanoZoomer Digital Pathology System (Hamamatsu, Welwyn Garden City, UK). Measurements of villus height and crypt depth were made using the NanoZoomer Digital Pathology Image Program (Hamamatsu) of 10 well-oriented villi scanned at 40 X magnification. Villus height was measured from the tip of the villus to the crypt opening and the associate crypt depth was measured from the base of the crypt to the level of the crypt opening. The ratio of villus height to relative crypt depth (V:C ratio) was calculated from these measurements.

RNA Isolation and RT-qPCR of the Cytokines and Chemokines

RNA was isolated from cecal and ileal tissue biopsies using NucleoSpin RNA isolation kit

(Macherey-Nagel, GmbH & co. KG, Diiran DE) according to the manufacturer's protocol with the following modifications. Tissue samples were homogenized in Lysis buffer with 2.8 mm ceramic beads (MO BIO Laboratories Inc., Carlsbad, USA) using TissueLyser II (Qiagen, Hilden, DE) prior to subsequent purification as described in the protocol. RNA was eluted in DEPC treated water (Ambion ThermoFisher Scientific, UK) and stored at -80°C. RNA quality and concentration were assessed using Nanodrop ND-1000 Spectrophotometer (Labtech International Ltd, Uckfield, UK). The ratio 260/280 nm was in the range of 1.79 to 2.17 with the mean of 2.12 ±0.01 for all RNA samples used. Reverse Transcription was performed with 1 μg of RNA using Superscript II (Invitrogen Life Technologies, Carlsbad, USA.) and random hexamers (Untergasser's Lab 2008 accessed online 16/12/2016; URL http://www.untergasser.de/lab/protocols/cdna_synthesis_super script_ii_vl_0.htm). Quantitative PCR reaction was performed with cDNA template derived from 4 ng of total RNA in triplicate using SYBR Green Master mix (Applied Biosystems, ThermoFisher Scientific). Cytokines and chemokines fold change were calculated using the "comparative Cycle threshold (Ct) method" established by the manufacturer as described by Livak, K.J., and Schmittgen, T.D. (2001). Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 ^~aac T Method. Methods 24, 402-408.. The average of the triplicate Ct values was used for analysis and the target genes Ct values were normalized to those of the housekeeping gene encoding Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The RNA level of expression was determined by qPCR using the Roche Diagnostics LightCycler 480 (Hoffmann La Roche AG, CH). The primers used for qPCR of GAPDH, IFN-γ, IL-Ιβ, IL-4, IL-6, IL-10, IL-17A, IL-17F, CXCLil and CXCLi2 are presented in Table 6.

DNA Extraction and PCR Amplification of 16S rRNA Gene Sequences and Microbiota Diversity Analysis

Bacterial DNA was isolated from 0.25 g cecal content using the PowerSoil DNA Isolation Kit (MO Bio Laboratories) according to the manufacturer's instructions. Using the isolated DNA as a template the V4 region of the bacterial 16S rRNA gene was PCR amplified using primers 515f (5' GTGCCAGCMGCCGCGGTAA 3 ) and 806r (5' GGACTACHVGGGTWTCTAAT 3 ) as described by Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A., Turnbaugh, P. J., et al. (2011). Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. U S A. 108 Suppl 1, 4516-4522.

doi: 10.1073/pnas.1000080107.

Amplicons were then sequenced on the Illumina MiSeq platform using 2 x 250 bp cycles. Prior to metagenomic analysis sequence reads with a quality score mean below 30 were removed using Prinseq (Schmieder, R., and Edwards, R. (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27, 863-864. doi: 10.1093/bioinformatics/btr026.). The 16S rRNA sequence analysis was performed using Mothur v. 1.37.4 (Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B., et al. (2009). Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537-7541. doi: 10.1128/AEM.01541-09.). Analysis was performed as according to the MiSeq SOP (accessed online 08/12/2016; Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S.K., and Schloss, P.D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl. Environ. Microbiol. 79, 5112-5120.) with the exception that the screen, seqs command used a maxlength option value similar to that of the 97.5 percentile length. The 16S rRNA gene sequences were aligned against a reference alignment based on the SILVA rRNA database (Pruesse, E., Quast, C, Knittel, K., Fuchs, B.M., Ludwig, W.G., Peplies, J., et al. (2007). SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucl. Acids Res. 35, 7188-7196 doi: 10.1093/nar/gkm864) for use in Mothur (available at:

https://www.mothur.org/wiki/Silva_reference_files), and clustered into operational taxonomic units (OTUs) with an average neighbor clustering algorithm. The nearest 16S rRNA gene sequence identities to the OTUs are reported on the basis of BLASTn searches if data matches are from type cultures with a BLAST identity >99%. If not, the consensus taxonomy of the OTUs is reported as generated using the classify. otu command in Mothur with reference data from the Ribosomal Database Project (version 14) (Cole, J. R., Wang, Q., Fish, J.A., Chai, B., McGarrell, D. M., Sun, Y., et al. (2014). Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucl. Acids Res. 42(Database issue), D633-D642.; Wang, Q., Garrity, G. M., Tiedje, J. M., and Cole, J.R. (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol . 73, 5261-5267. doi:

10.1128/AEM.00062-07) adapted for use in mothur (available at:

https://www.mothur.org/wiki/RDP_reference_files).

Data Analysis

ANOVA followed by Tukey's multiple comparisons test and Kruskal-Wallis test followed by Dunn's multiple comparisons test was performed using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, USA, www. graphpad. com) . Metastats were

implemented within Mothur (White, J.R., Nagarajan, N., and Pop, M. (2009). Statistical methods for detecting differentially abundant features in clinical metagenomic samples. PLoS Comput. Biol. 5:el000352. doi: 10.1371/journal.pcbi.1000352). Data processing and ordination were performed using R project (R Development Core Team, 2008. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3- 900051-07-0; URL http : //www. R-proj ect . org) . Heatmaps were plotted using the heatmap.2 function of R package gplots (Warnes, G.R., Bolker, B., Bonebakker, L., Gentleman, R., Huber, W., Liaw, A., et al. (2016). gplots: Various R Programming Tools for Plotting Data. R package version 3.0.1. https://CRAN.R-project.org/package=gplots).

Ethics Statement

Studies were carried out under license and in accordance with UK Animals (Scientific

Procedures) Act 1986. All procedures were approved by the Local Ethics Committee of the University of Nottingham.

The most preferred one or more bacteria are selected from the specific bacterial strains Lactobacillus crispatus str. DC21.1 (NCIMB 42771), Lactobacillus johnsonii str. DC22.2 (NCIMB 42772), Lactobacillus reuteri str. DC1B4 (NCIMB 42773), and Ruminococcus sp. str. DC3A4 (NCIMB 42774).

Preferable compositions of the present invention are Lactobacillus crispatus str. DC21.1 (NCIMB 42771) with a galacto-oligosaccharide, such as Nutrabiotic® GOS, Lactobacillus johnsonii str. DC22.2 (NCIMB 42772) with a galacto-oligosaccharide, such as Nutrabiotic® GOS, Lactobacillus reuteri str. DC1B4 (NCIMB 42773) with a galacto-oligosaccharide, such as Nutrabiotic® GOS, and Ruminococcus sp. str. DC3A4 (NCIMB 42774) with a galacto- oligosaccharide, such as Nutrabiotic® GOS.

The strains Lactobacillus crispatus str. DC21.1 (NCIMB 42771), Lactobacillus johnsonii str. DC22.2 (NCIMB 42772), Lactobacillus reuteri str. DC1B4 (NCIMB 42773), and Ruminococcus sp. str. DC3A4 (NCIMB 42774), are all commensal to Ross 308 broilers grown on a standard wheat-based feed that also contains Nutrabiotic® GOS (galacto-oligosaccharide) and produced in the poultry facility, University of Nottingham, Sutton Bonington campus, and were isolated from digesta taken from the caecum.

The specific bacterial strains, as well as the bacteria (not sequenced), have been identified from the microbiome of the same.

It is reported in Stanley, D., Hughes, R. J, and Moore, R J. (2014) "Microbiota of the chicken gastrointestinal tract: influence on health, productivity and disease " Applied Microbiology and Biotechnology, 98 4301-4310 and Stanley D, Hughes RJ, Geier MS and Moore RJ(2016) Bacteria within the gastrointestinal tract microbiota correlated with improved growth and feed conversion: Challenges presented for the identification of performance enhancing probiotic bacteria. Frontiers in Microbiology, 7:187. doi: 10.3389/fmicb.2016.00187 that the bacteria and specific bacterial strains are associated with good outcomes, and/or are associated with the microbiota of broilers that display high performance.

According to one embodiment of the invention, the composition may comprise two or more probiotics. For example, a first probiotic preparation may be taken from a group comprising specific facultative anaerobic commensal bacteria, for example Lactobacillus spp. and Bifidobacterium spp., which produce acetate and lactate when acting on a prebiotic, and a second probiotic preparation may be taken from a group comprising specific strictly anaerobic commensal bacteria which produce butyrate "feeding" on the acetate and lactate produced by the probiotic preparation of the first group. A 'probiotic preparation' is considered to comprise one or more probiotic bacteria taken from the respective facultative anaerobic or strictly anaerobic group.

According to another embodiment of the invention, the composition may comprise the two or more probiotics in combination with only one prebiotic material. An example of a potential combination of a composition according to this embodiment may be a first probiotic, for example Lactobacillus spp. or Bifidobacterium spp., taken from a group comprising specific facultative anaerobic commensal bacteria which produce acetate and lactate when acting on the prebiotic, and a second probiotic taken from a group comprising specific strictly anaerobic commensal bacteria which produce butyrate "feeding" on the acetate and lactate produced by the first probiotic, in combination with a prebiotic, for example, Nutrabiotic® GOS.

The bacteria may comprise facultative anaerobic bacteria or strictly anaerobic bacteria

According to another embodiment of the invention, the composition may comprise facultative anaerobic bacteria in combination with a prebiotic. The combination may create acetate and lactate.

According to another embodiment of the invention, the composition may comprise strictly anaerobic bacteria in combination with acetate and lactate. The combination may create organic acids. The organic acids may be, for example, butyrate.

The prebiotic material used in the composition of the invention is typically substantially indigestible in the gastrointestinal system of a chicken.

Another aspect of the present invention was to identify specific probiotics which respond favourably to the use of polymeric saccharide, such as an oligosaccharide sugar, as a prebiotic material; and whose populations with the broiler gastrointestinal tract can, therefore, be increased by the use of such prebiotics. Therefore, the prebiotic material is typically a polymeric saccharide, such as an oligosaccharide.

The oligosaccharide used in the composition of the invention may be selected from one or more of fructooligosaccharide (also known as oligofructose) which is a partial hydrolysate of inulin, mannanoligosaccharide (MOS), galactooligosaccharide (GOS), xylooligosaccharide, arabinoxylanoligosaccharide, soyoligosaccharide, lactulose, lactosucrose, isomalto- oligosaccharides, gluco-oligosaccharides, pectic oligosaccharides, and inulin. Typically, the oligosaccharide is a galactooligosaccharide.

Galactooligosaccharides (GOS) have the general form (galactosyl)n-lactose and typically range in size from trisaccharides to octasaccharides. Structural complexity is introduced by the different intermolecular bonds. Products said to comprise GOS therefore typically contain a mixture of galactooligosaccharides, lactose, glucose and galactose, and the term GOS is used herein in a manner intended to encompass such products.

GOS (galacto-oligosaccharide) is sold by Dairy Crest under the trade name Nutrabiotic® GOS for animal feed.

Typically, Nutrabiotic® GOS L is used as the prebiotic in the composition of the present invention. Nutrabiotic® GOS L complies with UK and EU Regulations and recommended purity specifications, including heavy metals, for feed and food ingredients. An analysis of

Nutrabiotic® GOS L is provided in Table 21.

The recommended inclusion, or dose, rate of Nutrabiotic® GOS in animal feed diets depends on a number of factors. For example:

• the animal (e.g. broiler (chicken for fattening) or piglet);

• life cycle and the feeding regime (e.g. the different feeds being used and the duration of their use; use, and commencement of use, of a creep (pre-starter) feed;

age of piglets at weaning etc.);

· formulation of the Nutrabiotic® GOS product and, to a lesser extent, the batch of the

Nutrabiotic® GOS product being used.

The data presented in Table 22 are recommendations based on typical feeding regimes and ones that have been used in both research and commercial trials. They can be modified as required.

The data presented in Table 24 provides an estimate of the metabolizable energy values of Nutrabiotic® GOS L in broilers and piglets.

Nutrabiotic® GOS contains no significant quantities of protein or fat, or vitamins, minerals etc. as shown in Table 21. Nutrabiotic® GOS contains a range of carbohydrates that are either digested as sugars, or fermented as soluble fibre. In the context of energy value for animal feed applications and specific animals, the definition of what is considered fibre is complicated as an appreciable number of disaccharides present in Nutrabiotic® GOS L are fermented. Moreover the proportion of disaccharides that are fermented will differ depending on the animal (e.g. piglets compared to poultry).

Starter, grower and finisher refer to the diets at the different stages of the broiler production cycle. The diets correspond to the following periods (day 0 is defined as the day the broiler chicks are "placed" in the poultry shed, although at day 0 the broiler chicks are usually 1 day old):

• 0 - 10 days Starter feed (sieved crumb, but can alternatively be in the form of a mash feed)

• 11-24 days Grower feed (pellets 3 mm diam.)

· 25-35 days Finisher feed (pellets 3 mm diam.)

The feeds, after the mixing of all the raw materials are pelleted (after steam injection and treatment) are extruded through a defined die to typically give a 3 mm pellet, that is the final broiler feed.

The pelleting process may follow typical methods known to a person skilled in the art.

Suitable feed and pellet size may be known to a person skilled in the art.

Crumb refers to a crumbed (broken into crumb) pelleted feed - typically to give smaller feed pieces that the broiler chicks can manage. A mash feed (a feed mixture that has not been pelleted) may be used instead of a crumb feed for the started feed.

The production cycle in this example is 35 days, which is reasonably common for experiments involving male (we only use the faster growing males to decrease the statistical variation in experimental systems) Ross 308 birds. Poultry cycles are more complex with birds being "harvested" at 35 - 42 days to get different weight ranges for commercial purposes.

Typically, the production cycle is 35 days, which is reasonably common for experiments involving male Aviagen Ross 308 birds, as typically used in the present invention.

The composition of the invention typically includes an amount of between about 10 ⁴ colony forming units (cfu) to 10 ¹² cfu, typically between about 10 ⁵ cfu to 10 ¹⁰ cfu, more typically between about 10 ⁶ cfu to 10 ⁸ cfu, and most typically 10 ⁷ cfu. CFU is essentially the number of live bacteria added at day 9 of a trial. Preferably, the addition of CFU should not preclude the probiotic being added at different times, or continuously, as part of the feed, in a commercial operation.

The composition of the invention includes a prebiotic, typically Nutrabiotic® GOS. Typically, a starter feed includes an amount of prebiotic, for example Nutrabiotic® GOS, between about 55% to 95% (w/w) solids concentration syrup, typically between about 65%> to 85%) (w/w) solids concentration syrup, more typically between about 70% to 80%> (w/w) solids concentration syrup, and most typically about 75% (w/w) solids concentration syrup.

Typically, in the starter feed the prebiotic, for example Nutrabiotic® GOS, is added at a dose rate between about 0.50%> to 5.00%> (w/w complete starter feed), typically between about 1.50%) to 3.50%) (w/w complete starter feed), more typically between about 2.00% to 3.00%, even more typically between about 2.20% to 2.60% (w/w complete starter feed), even more typically between about 2.40% to 2.50%, and most typically about 2.47% (w/w complete starter feed).

Typically, a grower feed includes an amount of prebiotic, for example Nutrabiotic® GOS, between about 55% to 95% (w/w) solids concentration syrup, typically between about 65% to 85%o (w/w) solids concentration syrup, more typically between about 70% to 80% (w/w) solids concentration syrup, and most typically about 75% (w/w) solids concentration syrup.

Typically, in the grower feed the prebiotic, for example Nutrabiotic® GOS, is added at a dose rate between about 0.20% to 5.00% (w/w complete grower feed), typically between about 0.60%) to 3.50%) (w/w complete grower feed), more typically between about 0.90% to 2.80%, even more typically between about 1.10% to 2.00% (w/w complete grower feed), even more typically between about 1.15% to 1.60%, even more typically between about 1.20% to 1.40%, and most typically about 1.24% (w/w complete grower feed).

Typically, the prebiotic, for example, Nutrabiotic® GOS, is not added to the finisher feed.

In a typical trial experiment, the addition of the bacteria is typically made in 0.10ml of MRD (Maximum Recovery Diluent), giving 10 ⁷ cfu (colony forming units) or viable cells, by cloacal gavage.

A further aspect of the present invention relates to a composition as defined hereinabove for the treatment and/or nutrition of poultry, such as broiler chickens, to which at least one of the probiotics responds to produce an increase in population.

The composition of the invention may also further comprise a nutrient food source. The nutrient food source may contain a source of protein, starch, amino acids, fat, or a combination of any two or more thereof. The nutrient food source may also contain one or more food additives which can be found in poultry feed, such as, but not limited to, vaccines, antibiotics, and coccidio stats, or a combination thereof. The antibiotics may be those used in treatment or as growth promoters.

The composition of the invention, containing the probiotic bacteria which are responsive to the prebiotics, and whose presence correlates with improved broiler performance, is able to impart benefits to the development of the poultry compared with poultry which is not exposed to the composition, such as an increased rate of growth, and/or a higher final weight, and/or a larger ratio of kilograms of feed required per kilogram of growth of the poultry.

The inventors have been able to show that gastrointestinal populations of the probiotic bacteria respond to the administration of prebiotics, such as oligosaccharides; and that increases in populations of one or more of the probiotic bacteria correlate to improved weight gain within broilers.

Also provided by the present invention is a composition for use in the treatment of enteric bacterial disease in poultry, the composition comprising:

(i) a probiotic selected from one or more of the bacteria Bifidobacterium animalis,

Columella tanakaei, Lactobacillus reuteri, Anaerostipes, Lactobacillus crispatus, Pediococcus acidilactici, Lactobacillus pontis, Faecalibacterium prausnitzii, Coprococcus catus, Roseburia intestinalis, Anaerostipes butyraticus, Butyricicoccus, Lactobacillus johnsonii, and Ruminococcus sp.; and

(ii) a prebiotic material.

The definitions and embodiments defined above for the composition of the invention also apply to the composition for use in the treatment of enteric bacterial disease in poultry.

The enteric bacterial disease is infection by one or more of the following: Clostridium perfringens, Salmonella spp, pathogenic and toxigenic Escherichia coli (EPEC and ETEC).

The composition of the invention may be administered in any suitable manner, including, but not limited to, orally (via feed, which may need to be encapsulated in order to protect the probiotic from the acidic environment in a chicken's stomach), via intracloacal delivery (Arsi, Donoghue, Woo-Ming, Blore and Donoghue: Intracloacal Inoculation, an Effective Screening Method for Determining the Efficacy of Probiotic Bacterial Isolates against Campylobacter Colonisation in Broiler Chickens, Journal of Food Protection, Vol 78, No. 1 2015, Pages 209- 213), or via a spray, such as onto chicks so they consume the composition by licking their feathers.

A further aspect of the present invention is a composition for the treatment and/or nutrition of poultry, such as a broiler chicken, comprising one or more specific probiotics and a prebiotic material which produce organic acids in the gastrointestinal tract, which impart benefits to the health of the broiler chickens. All of the probiotics listed hereinabove are able to act in an strictly anaerobic manner, while some are also able to act in a facultative anaerobic manner.

It is those (e.g. Lactobacillus spp. and Bifidobacterium spp.) which act in a facultative anaerobic manner which produce organic acids, such as acetic and lactic acids, in the gastrointestinal tract such as acetic and lactic acids, when fermenting the prebiotic. The probiotics which are strict anaerobes, produce butyrate and other organic acids when supplied with a prebiotic and the acetate and lactate. These probiotics include, for example, Coprococcus catus,

Roseburia intestinalis, and Anaerostipes butyraticus, Ruminococcus sp., Butyricicoccus, and

Faecalibacterium prausnitzii.

These bacteria are known to feed upon fibre in the gastrointestinal tract of a broiler chicken. That feeding process generates the organic acids which are beneficial in at least two ways. Firstly, they reduce the pH within the tract which, generally speaking, tends to assist the growth of beneficial gut flora whilst simultaneously inhibiting the growth of more harmful flora.

Secondly, the acids are directly beneficial per se as nutrients to the broiler and so the presence of one or more of these bacteria produces useable sources of energy.

The probiotics used in the invention serve the additional benefit of reducing populations of harmful gut flora. Examples of such harmful flora are Clostridium perfringens which is known to cause necrotic enteritis, and Salmonella whose presence is extremely harmful to humans and so desirably eliminated from broilers.

Although one or more of the compositions set out above can be used to treat, for example, the presence of undesirable gut flora in broiler chickens, they may advantageously also be used in feed compositions for prophylactic purposes.

The invention will now be described further by way of example with reference to the following examples, which are intended to be illustrative only and in no way limiting upon the scope of the invention.

Examples

An example of a trial experiment using the composition of the invention included the following:

· prebiotic (Nutrabiotic® GOS) at the following dose rates:

^■ starter feed: Nutrabiotic® GOS a 75 %(w/w) solids concentration syrup added at a dose rate of 2.47 %(w/w complete starter feed)

° grower feed: Nutrabiotic® GOS a 75 %(w/w) solids concentration syrup added at a dose rate of 1.235 %(w/w complete grower feed)

° finisher feed: Nutrabiotic® GOS is not added to the finisher feed;

• Single addition of a probiotic preparation of 10 ⁷ cfu, added at day 9 of the trial.

Addition was made in 0.10 ml of MRD (Maximum Recovery Diluent) by cloacal gavage. This method of addition was solely for the purpose to ensure proof of concept. It is not envisaged that this method of addition would be used in a production environment.

Table 1 provides a list of the ingredients in a commercially available poultry feed mixture, with which the composition of the invention may be combined for administration to the poultry.

Table 1

Ronozyme® WX (Xyl) 0.020 0.020 0.020

Key

RM - Raw material

Ext. Hipro Soya Meal - Extruded Hipro soya meal (and extruded high protein soybean meal)

Lysine HC1 - (lysine hydrochloride) and Methionine DL (a racemic mixture of the methionine D and L isomers) amino acids Threonine - an amino acid

Dicalcium phosphate, sodium bicarbonate, and salt (sodium chloride) are commonly used nutrients

TM - Blank Premix for Broiler Formulation is the premix of vitamins and trace elements listed in Table 2.

Ronozyme® P5000 (CT) and Ronozyme® WX (Xyl) are commercial names for enzymes that are commonly used in wheat-based feeds, specifically:

^! Ronozyme® P5000 (CT) is a coated phytase enzyme

* Ronozyme® WX is a xylanase

Reference is also made to Aviagen Ross 308 Broiler Nutrition Specifications 2014 documentation as examples of broiler diets

Table 2 provides the details of the TM Blank Premix for Broiler Formation listed in the ingredients in Table 1.

Table 2

PANTO 15.0000

FOLIC 1.5000

BIOTIN 251.0000

CHOLCHL 250.0000

FE 20.0000

MN 100.0000

CU 10.0000

ZN 80.0000

I 1.0000

SE 0.2500

MO 0.5000

*CA/USA 24.9103

*ASH/USA 74.3901

Tables 3 - 8 provide information regarding a trial experiment (Trial 1) carried out by the Applicant. Trial 1 concerned the performance and the up-regulation of certain commensal bacteria in GOS test treatments.

Trial 1

Trial design, measures and analysis

• Objective(s): Indicate optimum %(w/w) inclusion rate of galacto-oligosaccharides, reduce and vary the galacto-oligosaccharides %(w/w) inclusion rate in the different feed periods over the lifetime of the bird. The initial objective of the trial was to investigate the effect of Nutrabiotic™ GOS L on the broiler microbiota by NGS and metagenomic analysis (along with analyses of gut morphology and changes in immune function response).

° Samples for the analysis of gut morphology are stored in formaldehyde awaiting analysis.

° Results from NGS and metagenomic analysis of the caecal microbiota, along with changes in immune function response (as determined through the up/down regulation of cytokines and chemokines) will be available in the coming weeks.

• Product: Nutrabiotic® GOS L - a GOS 50% syrup containing approximately 72 %(w/w) dry solids

• Base diet: wheat-based (xylanase and phytase included), no coccidiostat • Type of bird: male Ross 308 (good chicks from strong 35 week old breeders)

• Number of treatments: 1 x control + 5 x GOS tests

Quality Control

• Feed screened for Salmonella prior to arrival of birds to ensure no contamination at feed mill.

• Birds screened on arrival for Salmonella and during the trial for both Campylobacter and Salmonella.

Relevant facts/observations

General

• Bird health was good with one bird suffering from hip dislocation and another suffering from sudden death. No comments were received concerning gut lesions.

Performance

· Nutrabiotic™ GOS L improved performance in terms of rate of weight gain with overall the best performance appearing to be for the higher GOS inclusion rate being fed throughout the growth period (P < 0.05). These improvements are maintained in the Test treatments.

° The confidence intervals of the weight data are quite wide, especially when sample numbers are decreased.

• It appears Nutrabiotic™ GOS L improves FCR for all treatments.

Standard microbiological analyses

• Standard microbiological methods were used to analyse on caecal samples, by standard microbiological methods:

° Campylobacter counts (CCDA plates, micro-aerobic incubation at 42 °C for 48 h, using the Miles and Misra method);

° lactic bacteria counts (MRS plates, anaerobic incubation at 30 °C for 48 h);

° coliform counts (MacConkey no.3 plates, incubation at 37 °C for 24 h). Microbiota analyses

• DNA was extracted from the caecal microbiota, targeted amplicon sequencing was employed using 16S RDNA (the gene for bacterial 16S rRNA) as a marker and molecular phylogenetic methods (amplification, sequencing, grouping sequences into OTUs, and the identification of OTUs) are used to infer the composition of the microbial community.

• Alpha-diversity (number or richness) of taxa were quantified by the Simpson Index for each treatment with good precision (as shown by asymptotic rarefaction curves) and showed no difference between each treatment, as was expected.

• Beta-diversity, which describes how many taxa are shared between treatments (a similarity score and represented by the Yue and Clayton theta similarity coefficient), gave different results:

° there was a significant difference (P < 0.0010) was found between the GOS[+] and

GOS[-] groups (taken as a whole); ;

° other measures, including AMOVA (analysis of molecular variance) confirmed these significant differences with the magnitude of the diversity being: GOS

3.37% > GOS 1.685% > control with corresponding significance: (GOS 3.37% - GOS 1.685%) > (GOS 1.685% - control);

° graphical representation of dissimilarities were shown as non-metric multidimensional scaling plots based on dissimilarity matrices built from the Yue and Clayton theta coefficients.

• Metastats (White et al. , 2009) was used to determine whether there are any OTUs that are differentially represented between the different treatments:

° between the GOS[+] and GOS[-] groups (taken as a whole) 42 OTUs were identified as significant.

Subsequent bioinformatics analyses

The major different OTUs in GOS[+] and GOS[-] groups have been identified, with the following candidate organisms identified as being GOS responsive. Identification was based on OTUs identified from 16S rDNA sequences from the V4 region of the microbiome. It is not possible to obtain more information of exact bacterial subspecies, and in some cases species, without a more complete analysis of the specific bacterial genome. The identification provided represents the nearest match from the SILVA rRNA database (16S rRNA gene sequences were aligned against a reference alignment based on the SILVA rRNA database and clustered into operational taxonomic units (OTUs) with an average neighbor clustering algorithm. The nearest 16S rRNA gene sequence identities to the OTUs are reported on the basis of BLASTn searches if data matches are from type cultures with a BLAST identity >99%.):

Bifidobacterium animalis subsp. lactis str. V9

Columella tanakaei str. YIT 12064

Ruminococcus torques str. ATCC 27756

Lactobacillus reuteri str. BCS136

Anaerostipes sp. str. 35-7

Lactobacillus crispatus str. ST1

Pediococcus acidilactici

Faecalibacterium prausnitzii

Conclusions

In conclusion it was shown that:

• there was an improvement in performance data, against the control, was seen in test treatments containing Nutrabiotic® GOS Syrup, particularly at the higher dose rate of 3.37 %(w/w);

• there was no significant difference between the "richness" of taxa (alpha-diversity) for each treatment, which is to be expected;

• there was a significant different between the number of taxa shared between between groups (beta-diversity) based on the inclusion of GOS in the diet, this allowed identification of bacteria that were "responsive to Nutrabiotic® GOS.

Table 3 provides a list of ingredients used in a poultry feed as part of Trial 1 Table 3

6.84 8.67 8.71 6.28 8.09 8.13

Oil 'Β' 5.707 7.526 7.566 4 2 3 0 9 9

Crude Protein (CP) 22.00 21 .00 19.01 21 .9 20.9 19.0 21 .9 20.9 19.0

(%) 2 4 9 91 90 08 95 97 12

2.63 2.60 2.61 2.70 2.67 2.68

Fibre (%) 2.775 2.745 2.749 7 8 1 6 7 0

5.81 5.23 4.85 5.80 5.23 4.85

Ash (%) 5.808 5.236 4.858 0 9 0 9 8 9

1 .42 1 .23 1 .09 1 .42 1 .23 1 .09

Lysine (%) 1 .430 1 .240 1 .091 9 9 1 9 9 1

0.69 0.58 0.52 0.69 0.58 0.52

Methionine (%) 0.691 0.582 0.521 5 6 4 3 4 2

Methionine + 1 .07 0.95 0.86 1 .07 0.95 0.86 Cystine (M+C) (%) 1 .070 0.950 0.861 0 0 0 0 0 0

0.27 0.26 0.23 0.27 0.26 0.23

Tryptophan (%) 0.270 0.261 0.235 1 2 6 0 1 5

0.94 0.82 0.74 0.93 0.83 0.74

Theonine (%) 0.940 0.830 0.741 0 9 0 9 0 0

1 .05 0.89 0.83 1 .05 0.88 0.83

Calcium (%) 1 .047 0.886 0.834 2 2 9 0 9 6

Total Phosphorus 0.67 0.60 0.56 0.67 0.61 0.56 (T:PHOS) (%) 0.677 0.613 0.565 2 7 0 5 0 2

Available

Phosphorus (A:PHOS) 0.50 0.45 0.42 0.50 0.45 0.42 (%) 0.500 0.450 0.420 0 0 0 0 0 0

0.32 0.32 0.32 0.32 0.32 0.32

Salt (%) 0.319 0.322 0.326 3 6 1 1 4 8

0.16 0.16 0.15 0.15 0.16 0.16

Sodium (%) 0.158 0.160 0.161 0 2 9 9 1 2

2.90 3.84 3.89 2.61 3.54 3.59

Linoleic acid (%) 2.318 3.251 3.302 3 0 1 3 5 7

0.96 0.92 0.82 0.95 0.92 0.82

Potassium (%) 0.955 0.920 0.822 2 7 9 8 4 5

0.20 0.20 0.19 0.19 0.20 0.20

Chloride (%) 0.198 0.200 0.201 1 2 8 9 1 3

Broiler ME inc.

enzyme contribution 12.65 13.20 13.40 12.6 13.2 13.4 12.6 13.2 13.4 (MJ) 2 4 3 49 03 04 52 04 03

Degussa poultry

digestible amino acid

values

1 .30 1 .12 0.98 1 .30 1 .12 0.98

Lysine (%) 1 .306 1 .122 0.984 5 1 4 6 2 4

0.63 0.53 0.47 0.63 0.52 0.47

Methionine (%) 0.635 0.528 0.473 8 1 5 6 9 4

Methionine + 0.94 0.83 0.75 0.94 0.83 0.75 Cystine (M+C) (%) 0.949 0.834 0.761 7 2 7 8 3 9

0.78 0.68 0.61 0.78 0.68 0.61

Theonine (%) 0.790 0.686 0.614 8 3 1 8 4 3

0.24 0.23 0.20 0.24 0.23 0.20

Tryptophan (%) 0.239 0.230 0.205 0 2 7 0 1 6

0.82 0.79 0.70 0.81 0.78 0.70

Isoleucine (%) 0.814 0.785 0.703 0 0 8 7 8 5

0.87 0.84 0.76 0.87 0.84 0.76

Valine (%) 0.874 0.843 0.759 7 7 2 5 5 0 0.49 0.48 0.43 0.49 0.48 0.43

Histidine (%) 0.496 0.479 0.429 9 1 2 7 0 1

1.29 1.24 1.09 1.28 1.23 1.08

Arginine (%) 1.275 1.225 1.077 3 3 5 4 4 6

Table 4 provides a comparison of the difference in speciation and Degussa poultry digestible amino acid values from Table 3 Table 4

0.00292 0.00291 0.00296 0.00145 0.00145 0.00145

Chloride (%) 3 6 3 9 7 9

Broiler ME inc. enzyme contribution 0.00298 0.00074 0.00066 0.00030 0.00043 0.00037

(MJ) 5 1 1 5 6 9

Degussa poultry digestible amino

acid values

0.00082 0.00085 0.00042 0.00042 0.00042

Lysine (%) 9 5 4.8E-05 6 9 3

0.00296 0.00198 0.00148 0.00148 0.00049

Methionine (%) 0.00298 4 8 4 1 1

0.00234 0.00333 0.001 18 0.001 19 0.00217

Methionine + Cystine (M+C) (%) 3 0.00238 3 8 2 8

0.00205 0.00304 0.00153 0.00153 0.00153

Theonine (%) 3 0.00307 3 4 6 2

0.00171 0.00170 0.00171 0.00085 0.00085 0.00085

Tryptophan (%) 7 7 8 4 3 5

0.00506 0.00510 0.00253 0.00253

Isoleucine (%) -0.0051 4 4 5 0.00253 8

0.00336 0.00332 0.00337 0.00166 0.00166

Valine (%) 8 8 2 6 2 0.00167

0.00251 0.00249 0.00251 0.00124 0.00124 0.00125

Histidine (%) 7 6 9 9 7 1

0.01809 0.01809 0.00902 0.00902 0.00903

Arginine (%) 3 0.01805 7 7 3 1

Table 5 provides a summary of the treatments used in Trial 1

Table 5

Group 3 3.37%(w/w)GOS starter 1 to 10 days

3.37%(w/w)GOS grower 11 to 24 days finishe

Control feed r 25 to 35 days

Group 4 3.37%(w/w)GOS starter 1 to 10 days

3.37%(w/w)GOS grower 11 to 24 days finishe

3.37%(w/w)GOS r 25 to 35 days

Group 5 Control feed starter 1 to 10 days

Control feed grower 11 to 24 days finishe

3.37%(w/w)GOS r 25 to 35 days

Group 6 1.685 %(w/w)GOS starter 1 to 10 days

1.685 %(w/w)GOS grower 11 to 24 days finishe

1.685 %(w/w)GOS r 25 to 35 days

Table 6 provides the weight (g) of the broilers used in Trial 1

Table 6

Table 7 provides the feed consumption of the broilers used in Trial 1

Table 7

Table 8 provides the cumulative feed consumption ratio of the broilers used in Trial 1

Table 8 Weight

Group (g)

0 8 15 22 28 35 Days

Gl 0.890 1.000 1.240 1.434 1.549 Total

G2 0.850 0.962 1.220 1.363 1.507 Total

G3 0.870 0.953 1.210 1.353 1.596 Total

G4 0.860 0.948 1.220 1.393 1.524 Total

G5 0.850 0.990 1.330 1.442 1.537 Total

G6 0.880 0.897 1.230 1.396 1.536 Total

Tables 9 - 20 provide information regarding a trial experiment (Trial 2) carried out by the Applicant. Trial 2 concerned the use of Lactobacillus crispatus DC21.1 (NCIMB 42771) as a probiotic

Trial 2

Objectives

To test the persistence and efficacy of Lactobacillus crispatus was provided as a probiotic to male Ross 308 broilers fed a standard wheat-based feed in the presence and absence of the galacto-oligosaccharide contain product - Nutrabiotic® GOS. Design

4 treatments each containing 20 -24 male Ross 308 broiler that were fed a standard wheat-based starter, grower and finisher feed. The feeds contained no antibiotic or coccidiostat products, but Nutrabiotic® GOS and Lactobacillus crispatus DC21.1 (NCIMB 42771). Details of the feed are given below and in the associated files. The trial was carried out for 35 days, and the Lactobacillus crispatus was added on day 9 by cloacal gavage with 10 ⁷ cfu (viable cells) being administered in 0.10 ml MRD (maximum recovery diluent) from a syringe that had been preloaded in an anerobic cabinet. • Group 1 : Pen 6 Nutrabiotic® GOS Lactobacillus crispatus

• Group 2: Pen 7 Nutrabiotic® GOS not added

• Group 3 : Pen 8 not added not added

• Group 4: Pen 9 not added Lactobacillus crispatus

Results

There was only a single addition of the Lactobacillus crispatus was added on day 9 after bird placement. Persistence of the Lactobacillus crispatus was determined as follows:

• DNA extractions were made from from caeca contents (MPBio kit) with concentration ranges of 80-250 ng/μΐ;

• DNA concentrations were normalised;

• qPCR was used, with absolute quantification using a standard curve based on extracted Lactobacillus crispatus DNA at different dilutions.

The concentration of the Lactobacillus crispatus, which is a commensal strain, when administered on day 9 after bird placement was present at the end of the trial at 1.9 - 2.9 x the concentration in treatments where it had not been added by oral gavage.

Whilst this was not a large trial, lacking statistical power, and the results were not significant in that P > 0.05, the increase in bird weight at 35 days was greatest for group 1 (Nutrabiotic® GOS + Lactobacillus crispatus) with, in some comparisons P < 0.10.

Conclusions

Lactobacillus crispatus DC21.1 (NCIMB 42771) persists in the broiler caecum at the end of the experiment period, at day 35, when administered at day 9. The probiotic was present a concentrations of 1.9 - 2.9 x the concentration in control treatments. Whilst the trial lacked statistical power, and the results were not significant in that P > 0.05, the increase in bird weight at 35 days was greatest the test group (Nutrabiotic® GOS + Lactobacillus crispatus) with, in some comparisons P < 0.10.

Table 9

Table 9 provides the performance data of Trial 2 - Group 1, Pen 6 Group Lactobacillu

1 Pen 6 GOS2 s crispatus

08/11/1 18/11/1 28/11/1 05/12/1 13/12/1

Date 6 15/11/16 6 6 6 6

Time (days) 0 7 10 20 27 35

Bird Weight Weight Weight Weight Weight Weight

(g) (g) (g) (g) (g) (g)

1 41 214 262 740

2 40 198 345 985 1321 1931

3 35 189 323 803

4 39 198 313 876 1159 1745

5 38 219 332 937 1432 2270

6 37 181 297 715

7 40 183 282 758 1298 1803

8 41 185 302 792 1379 2240

9 34 218 299 782

10 41 171 292 742 1240 1958

11 43 173 313 925 1202 1998

12 38 191 272 746

13 35 186 276 717

14 39 203 230 862

15 39 196 298 867

16 37 147 204 617

17 39 177 263 862 1372 2260

18 41 171 319 986 1361 2080

19 40 196 330 828 1294 2040

20 41 149 251 779

average weight 38.9 187.3 290.2 816.0 1305.8 2032.5 st. dev. 2.4 19.6 35.8 96.5 85.5 184.4

RSD (%) 6.1% 10.5% 12.3% 11.8% 6.5% 9.1% cum. feed per

bird (g) 163 339 1023 1896 3196

FCR 0.870 1.168 1.254 1.452 1.572

Table 10 provides the performance data of Trial 2 - Group 2, Pen 7 Table 10 _P 2

15/11/201 18/11/201 28/11/201 05/12/201 13/12/201

Date 08/11/16 6 6 6 6 6

Time (days) 0 7 10 20 27 35

Bird Weight Weight Weight Weight Weight Weight

(g) (g) (g) (g) (g) (g)

1 36 178 295 782

2 33 148 248 621

3 35 179 262 757 1042 1567

4 35 148 234 628

5 38 179 256 799 1317 2018

6 40 189 254 892

7 41 168 256 766 1112 1787

8 41 175 263 792 1282 1946

9 38 168 299 717

10 38 172 275 778

11 41 164 329 788

12 37 161 308 746 1082 1632

13 38 166 282 781

14 39 201 320 862 1192 1769

15 40 197 324 898 1186 1738

16 39 154 294 728 1132 1670

17 38 166 242 719

18 43 207 276 986 1372 2149

19 42 204 355 978 1524 2289

20 40 176 251 779

averag

e

weight 38.6 175.0 281.2 789.9 1224.1 1856.5 st. dev. 2.5 17.4 33.3 95.7 149.3 236.2 RSD

(%) 6.6% 9.9% 11.8% 12.1% 12.2% 12.7% cum.

feed per

bird (g) 158 336 939 1699 2945

FCR 0.900 1.193 1.189 1.388 1.586

Table 11 provides the performance data of Trial 2 - Group 3, Pen 8

Table 11

14 42 203 314 893

15 38 196 258 753 1161 1803

16 41 147 310 857

17 42 177 303 870 1294 1720

18 41 171 287 802

19 40 196 297 840 1424 2123

20 38 169 283 781

21 37 149 249 589

22 41 182 287 802

23 30 190 282 858 1482 2163

24 42 171 267 799

average

weight 39.5 185.7 287.5 790.8 1310.2 1933.2 st. dev. 3.0 18.5 25.8 107.3 141.0 177.6

RSD (%) 7.6% 10.0% 9.0% 13.6% 10.8% 9.2% cum. feed per

bird (g) 158 328 999 1760 2932

FCR 0.848 1.142 1.263 1.344 1.517

Table 12 provides the performance data of Trial 2 - Group 4, Pen 9 Table 12

40 198 230 715 1084 1718

35 189 244 739

39 198 315 703

38 219 261 658 1078 1700

37 181 266 679 1061 1676

40 183 237 691

41 185 274 668 1180 1890

34 218 306 714 1089 1525

41 171 315 802

43 173 287 791 1324 2015

38 191 343 920 1422 2080

35 186 350 952 1548 2300

39 203 330 872 1361 2052

39 196 290 819 1248 1825

37 147 272 719 1261 1932

39 177 263 720

41 171 297 752

40 196 280 742 1214 1840

41 149 281 779

38 185 309 799 1312 1970

36 152 244 791

40 188 276 801 1328 1890

41 207 311 895

average

weight 38.9 186.5 285.5 768.4 1250.7 1886.6 st. dev. 2.3 19.7 32.3 79.6 144.4 197.2

RSD (%) 5.9% 10.6% 11.3% 10.4% 11.5% 10.5% cum. feed per

bird (g) 158 323 938 1719 2872

FCR 0.847 1.131 1.221 1.375 1.522 Table 13 provides the t-Test data from Trial 2

Table 13

t-Test

Table 14 provides the feed consumption data from Trial 2 - Group 1, Pen 6

Table 14

18/11/2016 10 14600 920 3520 6780 20 176 339

28/11/2016 20 12000 3274 13680 20460 20 684 1023

05/12/2016 27 16000 3000 8726 29186 10 873 1896

13/12/2016 35 13000 42186 10 1300 3196

Table 15 provides the feed consumption data from Trial 2 - Group 2, Pen 7 Table 15

Group 2 Pen 7 GOS2

Table 16 provides the feed consumption data from Trial 2 - Group 3, Pen 8 Table 16

Group 3 Pen 8

Table 17 provides the feed consumption data from Trial 2 - Group 4, Pen 9 Table 17

Group 4 Pen 9 Lactobacillus crispatus 08/11/2016 0 4000 210

15/11/2016 7 5000 1040 3790 3790 24 158 158

18/11/2016 10 16200 1433 3960 7750 24 165 323

28/11/2016 20 12000 1063 14767 22517 24 615 938

05/12/2016 27 20000 3865 10937 33454 14 781 1719

13/12/2016 35 16135 49589 14 1153 2872

Table 18 is the feed formulation used in Trial 2, days 0 - 10 Table 18

Group 0 - 1 0 days

Star ter feed (sieved

cru I nb)

L. crispatus GOS No. Approx. intake Cumm. intake birds

(kg/te) ( ^' kg/bird) (kg/trt) (kg/bird) (kg/trt)

1 23.H O 2i > i > 2<>4 5 88 n.2 ^l M 5 88

2 23.860 20 0.294 5.88 0.294 5.88

3 - 24 0.294 7.06 0.294 7.06

4 + 24 0.294 7.06 0.294 7.06

Totals 88 25.9 25.9

Table 19 provides the feed formulation used in Trial 2, days 11 - 24 Table 19

L. No.

crispatus GOS birds Intake Cumm. intake

(kg/te) (kg/bird) (kg/trt) (kg/bird) (kg/trt)

1 + 11.93 20 1.312 26.24 1.606 32.12

2 - 11.93 20 1.312 26.24 1.606 32.12

3 - 24 1.312 31.488 1.606 38.544

4 + 24 1.312 31.488 1.606 38.544

Totals 88 115.456 141.328

Table 20 is the feed formulation used in Trial 2, days 25 - Table 20

Table 21 provides a description of Nutrabiotic® GOS L with which the composition of the invention may comprise as a prebiotic.

Table 21

Nutrabiotic® GOS L Description : galacto-oligosaccharide syrup.

Typical analysis : dry matter: 75 %(w/w) of which galacto-oligosaccharides: 59 %(w/w DM), lactose: 17%(w/w DM), glucose: 17%(w/w DM), galactose: 7%(w/w DM)

Sensorial : clear yellow to colourless liquid syrup, slightly sweet taste.

Viscosity

1000-5000 mPa.s HAAKE

pH

3.1 - 3.8 ISO 10523 (1994), potentiometric (10 % w/w)

Microbiological:

Total plate count < 1000 cfu/g IDF 100B (1991), PCMA

72h 30°C

Yeasts < 50 cfu/g IDF 94B (1990), OGYE 5 days 25°C

Moulds < 50 cfu/g IDF 94B (1990), OGYE 5 days 25°C

Enterobacteriaceae absent in 1 g BDI 23, VRBG 24h 30°C

Escherichia coli absent in 5 g IDF 170A-1 (1999), LSTB

48h 37°C, ECB 48h 44°C

Salmonelleae absent in 25 g IDF 93B (1995)

Packaging: 1200 kg IBC

Storage:

keep in clean, dry and dark

conditions, keep away from

strongly odorous materials.

Shelf life:

18 months after production

date. Table 22 provides recommendations based on typical feeding regimes and ones that have been used in both research and commercial trials. They can be modified as required. A

comparison between broilers and piglets is also provided. Table 22

Batch number - Nutrabiotic® GOS L batch no. AQ6215

Dry matter 74.2 %(w/w)

Water 25.8 %(w/w)

Broilers

Starter feed day 0 - 10 24.70 kg per metric tonne of complete feed

Grower feed day 11 - 24 12.35 kg per metric tonne of complete feed

Finisher feed day 25 - not generally required

Piglets

Creep (pre- starter) feed day 10 - weaning 15.1 kg per metric tonne of complete feed

Weaning day 28

Starter feed day 28 - 35 9.1 kg per metric tonne of complete feed

Link feed day 35 - 49 9.1 kg per metric tonne of complete feed

Grower feed day 49 - 63 as required

Notes

Dose rates for Nutrabiotic® GOS L are given for the syrup product as is.

Table 23 provides primers sequence 5 '-3' for the genes expression determined by qPCR.

Table 23

Target gene Primer sequence (5 '-3') Product size NCBI Accession Reference

(bp) number

343 NM_204305.1 Nang

GACGTGCAGCAGGAACACTA (2011) R: TCTCCATGGTGGTGA

AGACA IFN-γ F: 152 M 205149.1

Nang

TGAGCCAGATTGTTTCGATG

(2011)

R: CTTGGCCAGGTCCATGATA

IL-Ιβ F: 272 NM 204524.1

GGATTCTGAGCACACCACAGT

Nang

R:

(2011)

TCTGGTTGATGTCGAAGATGT C

IL-4 F: 186 NM 001007079.1

GGAGAGCATCCGGATAGTGA Nang et al. R: (2011)

TGACGCATGTTGAGGAAGAG IL-10 F: 203 NM 001004414.2

Nang et

GCTGCGCTTCTACACAGATG

(2011)

R: TCCCGTTCTCATCCATCTTC

IL-6 F: GCTCGCCGGCTTCGA NM_204628.1 Kaiser et

R: (2003)

GGTAGGTCTGAAAGGCGAAC AG

IL17-A F: 68 NM 204460.1 Reid

CATGGGATTACAGGATCGATG A

R: GCGGCACTGGGCATCA

IL17-F F: 78 XM 426223.5 Reid et al.

TGACCCTGCCTCTAGGATGAT (2016)

C

R:

GGGTCCTCATCGAGCCTGTA

ChCXCLil F: CCGATGCCAGTGCATAGAG 191 NM_205018.1 Rasoli et al.

R: (2015)

CCTTGTCCAGAATTGCCTTG ChCXCLi2 F: CCTGGTTTCAGCTGCTCTGT 128 M_205498.1 Rasoli

R: (2015) GCGTCAGCTTCACATCTTGA

Table 24 provides an estimate of the metabolizable energy values of Nutrabiotic® GOS L in broilers and piglets.

Table 24

Batch number

Nutrabiotic® GOS L batch no. AQ6215

Dry matter 74.2 %(w/w)

Water 25.8 %(w/w)

Net Metabolizable Energy (NME): broilers

6.06 kJ/g Nutrabiotic® GOS L syrup product 1.45 kcal/g Nutrabiotic® GOS L syrup product

Net Metabolizable Energy (NME): piglets

7.26 kJ/g Nutrabiotic® GOS L syrup product 1.74 kcal/g Nutrabiotic® GOS L syrup product

Notes

The NME values are expressed per weight of the Nutrabiotic® GOS L product as is, i.e. the syrup product that is added.

It is of course to be understood that the present invention is not intended to be restricted to the foregoing examples which are described by way of example only.

The present invention relates to compositions for use in the treatment and/or nutrition of poultry, such as broiler chickens {Gallus gallus domesticus). However it is not beyond the scope of the invention that the present invention may also relate to game birds such as grouse, pheasant or quail, for example.