Microbial compositions

Abstract

The disclosure provides a composition comprising two or more of (a) a bacterium of species Lactobacillus johnsonii (L. johnsonii); (b) a bacterium of species Limosilactobacillus reuterii (L. reuterii); and (c) a bacterium of species Megasphaera elsdenii (M. elsdenii). The disclosure further provides related pharmaceutical compositions, animal feeds and animal feed supplements and bacteria. The disclosure also concerns use of the composition, pharmaceutical composition, feed or feed supplement or bacterium to prevent or treat post-weaning diarrhoea in an animal.

Description

[0001] MICROBIAL COMPOSITIONS

[0002] Field of the disclosure

[0003] The disclosure provides a composition comprising two or more of (a) a bacterium of species Lactobacillus johnsonii (L. johnsonii (b) a bacterium of species Limosilactobacillus reuterii (L. reiilerii) and (c) a bacterium of species Megasphaera elsdenii (M elsdenii). The disclosure further provides related pharmaceutical compositions, animal feeds and animal feed supplements and bacteria. The disclosure also concerns use of the composition, pharmaceutical composition, feed or feed supplement or bacterium to prevent or treat post- weaning diarrhoea in an animal.

[0004] Background

[0005] Post-weaning diarrhoea (PWD) is a disease that affects piglets after weaning, for example during the first two weeks after weaning. PWD causes diarrhoea, dehydration and growth retardation, and even death in 20-30% of cases.

[0006] Farmed pigs are often weaned at 21 days of age. In intensive production systems, weaning may even occur as early as 1-2 weeks of age. In contrast, in non-farmed pig populations, weaning tends to occur around 12 to 17 weeks after birth. The early age of weaning in farmed pigs likely contributes to the development of PWD.

[0007] PWD is a multifactorial disease. Stress associated with removal from the sow, dietary changes and adaptation to a new environment may contribute to gut dysfunction in PWD. However, the main contributory factor is the presence of a particular pathogen, namely enterotoxigenic Escherichia coli (ETEC) reviewed by Kaper et al. 2004. A reduced feed intake initially after weaning contributes to intestinal inflammation, with changes in intestinal morphology such as villus height and crypt depth. This facilitates the colonisation of the intestines with ETEC from the environment (e.g. from mammary glands, from the farrowing room, or from the pen, originating from the gut of piglets with ETEC diarrhoea).

[0008] The pathogenicity of ETEC is due to the production of enterotoxins and adhesins. These virulence factors are often found on transmissible plasmids. Enterotoxins impair enterocyte function, increasing secretion and reducing absorption of fluids and electrolytes. ETEC produce two types of enterotoxins: large heat-labile (LT) and small heat-stabile (ST). Both are able to start a cascade of events leading secretion of CT and inhibition of Na+with a subsequent release of water into the intestinal lumen for osmosis and finally, diarrhoea. Some ETEC strains can also bear genes codifying for Shiga toxin 2e (Stx2e). Adhesins are cell-surface components which facilitates adhesion of the bacteria to other cells. In E. coli they are located at the tip of the fimbriae.

[0009] Different strains of ETEC exist, characterised by different combination of toxins and fimbria. The ETEC types most commonly causative of PWD in piglets produce fimbriae F4 and Fl 8. ETEC F4 seem to be involved in the aetiology of diarrhoea during the first 2-3 weeks post- weaning, while the Fl 8 strain predominates in later post- weaning periods. F4 and Fl 8 fimbriae are recognized by F4 and F18 specific glycoprotein receptors present on the villi of the small intestine of pigs. The presence of these receptors determines the susceptibility of pigs to ETEC infections and thus development of disease.

[0010] The pig gut microbiota is very complex because of its dynamic composition and diversity both over time and along the entire gastrointestinal tract. However, ETEC may be found in faeces from healthy, non-diarrhoeic piglets. In such piglets, there is often a high diversity of E. coli strains. In contrast piglets, with PWD tend to have only a small number of strains, which are pathogenic. It is well-known that the gut microbiota plays a role in many processes linked to host health including development of the immune system and defence against pathogens. It follows that dysbiosis (most typical features of dysbiosis are a decrease in the diversity of the microbiota, a loss of beneficial microbiota, or an overgrowth of harmful microbiota (Hrncir T 2022)) is an important factor in the development of PWD, and that a probiotic approach may assist in its prevention. However, there is a need for effective probiotics to implement this approach.

[0011] Summary of the disclosure

[0012] The present inventors have demonstrated that a certain bacterial composition may be used to prevent or treat post-weaning diarrhoea (PWD) in animals, such as pigs. By administering the bacterial composition prior to or around the time of weaning, the onset of PWD may be suppressed. Similarly, administering the bacterial composition following the onset of PWD may alleviate symptoms of the condition. In this way, animal welfare and productivity may be improved.

[0013] Accordingly, the disclosure provides: a composition comprising two or more of (a) a bacterium of species Lactobacillus johnsonii (L. johnsonii (b) a bacterium of species Limosilactobacillus reuterii (L. reuterii),' and (c) a bacterium of species Megasphaera elsdenii (M. elsdenii) a pharmaceutical composition comprising:

[0014] (i) the composition of the disclosure; or

[0015] (ii) (a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 2; (b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608 , optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 1; (c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of M. elsdenii DSM 20460, optionally wherein the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 3; (d) a bacterium of species V. magna, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857 , optionally wherein the bacterium of species V. magna comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 4; or (e) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T), optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 5; and a pharmaceutically acceptable carrier or excipient; an animal feed or feed supplement comprising:

[0016] (i) the composition of the disclosure; or

[0017] (ii) (a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 2; (b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 1; (c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of M. elsdenii DSM 20460, optionally wherein the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 3; (d) a bacterium of species V. magnet, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857, optionally wherein the bacterium of species V. magna comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 4; or (e) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T), optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 5; a bacterium, which is:

[0018] (a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2;

[0019] (b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1;

[0020] (c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome oiM. elsdenii DSM 20460, optionally wherein the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 3; or

[0021] (d) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R bovis JE7A12(T) , optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 5; a method of alleviating post-weaning diarrhoea in an animal, comprising administering to the animal the composition, pharmaceutical composition, feed or feed supplement or bacterium of the disclosure; and the composition, pharmaceutical composition, feed or feed supplement or bacterium of the disclosure for use in a method of alleviating post-weaning diarrhoea in an animal, wherein the method comprises administering the composition, pharmaceutical composition, feed or feed supplement or bacterium to the animal.

[0022] Brief description of the Figures

[0023] Figures 1 Sample plots from sPLS-DA (sparse Partial Least Square Discriminanat Analysis) performed on 16S data at ASV level, displaying piglet samples grouped by LT toxin levels. Samples included rectal microbiome across various time points: (A) 7d from birth, (B) 3d before weaning, (C) weaning and (D) 4d post-weaning. Samples are projected into the space spanned by the first two components of the sPLS-DA model with 95% confidence level ellipse plots.

[0024] Figure 2 Sample plots from sPLS-DA (sparse Partial Least Square Discriminanat Analysis) performed on 16S data at ASV level, displaying piglet samples grouped by LT toxin levels. Samples included intestinal content microbiome at the moment of euthanasia: (A) mid-jejunum, (B) ileum and (C) colon. Samples are projected into the space spanned by the first two components of the sPLS-DA model with 95% confidence level ellipse plots.

[0025] Figures 3 and 4 Loading values of the top 25 contributing ASVs to the first (Figure 3) and second (Figure 4) components of a sPLS-DA on 16S data from rectal and intestinal piglet samples. ASVs primarily contributing to group discrimination (high-LT vs. low-LT) at each time point and body site, are ranked in ascending order according to their contribution weight to components 1 and 2 of the sPLS-DA. ASVs are colored based on the group in which that ASV is more abundant

[0026] Figures 5 and 6 Loading values of the top 25 contributing genera to the first (Figure 5) and second (Figure 6) components of a sPLS-DA on 16S data from rectal and intestinal piglet samples. Genera primarily contributing to group discrimination (high-LT vs. low-LT) at each time point and body site, are ranked in ascending order according to their contribution weight to components 1 and 2 of the sPLS-DA. Genera are colored based on the group in which that genus is more abundant.

[0027] Figure 7 Inhibition effect of different bacterial combinations on the count of ETEC in CFU / ml (colony-forming units per milliliter) (log2 transformed data). Dashed lines, in black and dark gray, represent the mean values of the negative and positive control, respectively, p values: p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *.

[0028] Figure 8 Inhibition effect of supernatant of different bacterial combinations on the count of ETEC in CFU / ml (colony-forming units per milliliter) (log2 transformed data). Dashed lines, in black and dark gray, represent the mean values of the negative and positive control, respectively.

[0029] Figure 9 Inhibition effect of bacterial mixture cultured in two variants (MV1, MV2) on the count of ETEC in CFU / ml (colony -forming units per milliliter) (log2 transformed data). Dashed lines, in black and dark gray, represent the mean values of the negative and positive control, respectively, p values: p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *, p>0.05 = ns

[0030] Figure 10 Inhibition effect of the supernatant of bacterial mixture cultured in two variants (MV1, MV2) on the count of ETEC in CFU / ml (colony -forming units per milliliter) (log2 transformed data). Dashed lines, in black and dark gray, represent the mean values of the negative and positive control, respectively

[0031] Brief description of the sequence listing

[0032] SEQ ID NO: l is a genomic nucleic acid sequence of a bacterium of species L. r enter ii.

[0033] SEQ ID NO: 2 is a genomic nucleic acid sequence of a bacterium of species L. johnsonii.

[0034] SEQ ID NO: 3 is a genomic nucleic acid sequence of a bacterium of species AT. elsdenii.

[0035] SEQ ID NO: 4 is a genomic nucleic acid sequence of a bacterium of species V. magna.

[0036] SEQ ID NO: 5 is a genomic nucleic acid sequence of a bacterium of species R. bovis.

[0037] SEQ ID NOs 6-35 are nucleic acid sequences of primers. Detailed description

[0038] It is to be understood that different applications of the disclosed methods and products may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only, and is not intended to be limiting.

[0039] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0040] General definitions

[0041] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs.

[0042] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a bacterium” includes “bacteria”, reference to “an animal” includes two or more such animals, and the like.

[0043] In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “composition comprising a bacterium” should be interpreted to mean that the composition contains the bacterium but may also contain additional components.

[0044] In some aspects of the disclosure, the word “comprising” is replaced with the phrase “consisting of’. The term “consisting of’ is intended to be limiting. For example, the phrase “a composition consisting of a bacterium” should be understood to mean that the composition contains the bacterium and no additional components.

[0045] The terms “protein” and “polypeptide” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0046] For the purpose of this disclosure, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotide residues at nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide residue as the corresponding position in the second sequence, then the nucleotides are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0047] Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence has a certain percentage identity to SEQ ID NO: X, SEQ ID NO: X would be the reference sequence. For example, to assess whether a sequence is at least 80% identical to SEQ ID NO: X (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: X, and identify how many positions in the test sequence were identical to those of SEQ ID NO: X. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: X. If the sequence is shorter than SEQ ID NO: X, the gaps or missing positions should be considered to be nonidentical positions.

[0048] The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0049] Composition

[0050] The disclosure provides a composition comprising two or more of (a) a bacterium of species Lactobacillus johnsonii (L. johnsonii (b) a bacterium of species Limosilactobacillus reuterii (L. reiilerii) and (c) a bacterium of species Megasphaera elsdenii (M elsdenii). As demonstrated in the Examples, this core combination of bacteria is especially effective in inhibiting ETEC. The core combination may, for example, reduce the survival of ETEC and / or reduce the growth of ETEC. Reduction of ETEC survival may, for instance, be relative to ETEC survival in the absence of the core combination. Reduction of ETEC growth may, for instance be relative to ETEC survival in the absence of the core combination. In any case, inhibition of ETEC may, for example, be mediated directly by the presence of the bacteria and / or by factors secreted by or otherwise released from the bacteria. As ETEC is the main contributory factor to the development of PWD, inhibition of ETEC leads to prevention and / or treatment of PWD. The composition may comprise any two or more of (a) a bacterium of species L. johnsonii, (b) a bacterium of species L. reuterii and (c) a bacterium of species M. elsdenii in any combination. The composition may, for example, comprise (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c). Preferably, the composition comprises (a) a bacterium of species L. johnsonii (b) a bacterium of species L. reuterii,' and (c) a bacterium of species M. elsdenii.

[0051] In any case, (i) the bacterium of species L. johnsonii may comprise a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200; (ii) the bacterium of species L. reuterii may comprise a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608; and / or (iii) the bacterium of species M. elsdenii may comprise a nucleic acid sequence having at least 70% identity to the genome of AT. elsdenii DSM 20460. Any combination of (i), (ii) and (iii) may apply, such as: (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii) and (iii). For example, the composition may comprise: (i) a bacterium of species L. johnsonii that comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200; (ii) a bacterium of species L. reuteri that comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608; and (iii) a bacterium of species M. elsdenii that comprises a nucleic acid sequence having at least 70% identity to the genome of AT. elsdenii DSM 20460.

[0052] When the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, the bacterium of species L. johnsonii may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to the genome of L. johnsonii ATCC 33200. The bacterium of species L. johnsonii may, for example, comprise a nucleic acid sequence comprising or consisting of the genome sequence of L. johnsonii ATCC 33200. In any case, the nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species L. johnsonii . The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species L. johnsonii. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species L. johnsonii.

[0053] When the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, the bacterium of species L. reuterii may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to the genome of L. reuterii ATCC 53608. The bacterium of species L. reuterii may, for example, comprise a nucleic acid sequence comprising or consisting of the genome sequence of L. reuterii ATCC 53608. In any case, the nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species L. reuterii. The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species L. reuterii. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species L. reuterii.

[0054] When the bacterium of species AT. elsdenii comprises a nucleic acid sequence having at least 70% identity to the genome of AT. elsdenii DSM 20460, the bacterium of species AT; elsdenii may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to the genome c M. elsdenii DSM 20460. The bacterium of species AT. elsdenii may, for example, comprise a nucleic acid sequence comprising or consisting of the genome sequence of M. elsdenii DSM 20460. The nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species M. elsdenii. In any case, the nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species AT; elsdenii. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species AT. elsdenii.

[0055] Additional bacteria

[0056] The composition may comprise one or more additional bacteria. That is, the composition may comprise one or more bacteria in addition to the two or more of (a) a bacterium of species Lactobacillus johnsonii (L. johnsonii) (b) a bacterium of species Limosilactobacillus reuterii (L. reuterii),' and (c) a bacterium of species Megasphaera elsdenii (M elsdenii). For example, the composition may comprise two or more, three or more, four or more, or five or more additional bacteria. The composition may, for example, comprise two, three, four or five additional bacteria. The composition may, for example, comprise two to three, two to four, two to five, three to four, three to five, or four or five additional bacteria. In a preferred aspect of the disclosure, the composition comprises two or more additional bacteria, such as two additional bacteria. In any event, the one or more additional bacteria may comprise (d) a bacterium of species Veillonella magna (V. magnet) and / or (e) a bacterium of species Ruminococcus bovis (R. bovis). For example, the one or more additional bacteria may comprise (d) a bacterium of species V. magna. The one or more additional bacteria may comprise (e) a bacterium of species R. bovis. The one or more additional bacteria may comprise (d) a bacterium of species V. magna and (e) a bacterium of species R bovis.

[0057] In any case, (iv) the bacterium of species V. magna comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857; and / or (v) the bacterium of species R bovis comprises a nucleic acid sequence having at least 70% identity to the genome of R bovis JE7A12(T). Any combination of (iv) and (iv) may apply, such as: (iv); (v); or (iv) and (v).

[0058] When the bacterium of species V. magna comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857, the bacterium of species V. magna may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to the genome of V. magna DSM 19857. The bacterium of species V. magna may, for example, comprise a nucleic acid sequence comprising or consisting of the genome sequence of V. magna DSM 19857. In any case, the nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species V. magna. The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species V. magna. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species V. magna.

[0059] When the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T), the bacterium of species R. bovis may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to the genome of R bovis JE7A12(T). The bacterium of species R bovis may, for example, comprise a nucleic acid sequence comprising or consisting of the genome sequence of R bovis JE7A12(T). In any case, the nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species R. bovis. The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species R bovis. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species R. bovis.

[0060] The one or more additional bacteria may comprise, or further comprise, (1) one or more Prevotella strain, (2) one or more Blautia strain, and / or (3) one or more Subdoligranulum strain. Thus, the composition may comprise two or more of (a) a bacterium of species Lactobacillus johnsonii (L. johnsonii), (b) a bacterium of species Limosilactobacillus reuterii (L. reuterii), and (c) a bacterium of species Megasphaera elsdenii (M elsdenii) plus (1) one or more Prevotella strain, (2) one or more Blautia strain, and / or (3) one or more Subdoligranulum strain. The composition may, for example, comprise two or more of (a) a bacterium of species Lactobacillus johnsonii L. johnsonii), (b) a bacterium of species Limosilactobacillus reuterii (L. reuterii), and (c) a bacterium of species Megasphaera elsdenii (M. elsdenii),' plus (d) a bacterium of species Veillonella magna (V. magno) and / or (e) a bacterium of species Rumonicoccus bovis (R. bovis),' plus (1) one or more Prevotella strain, (2) one or more Blautia strain, and / or (3) one or more Subdoligranulum strain. In any case, the one or more additional bacteria may comprise: (1); (2); (3); (1) and (2); (1) and (3); (2) and (3); or (1), (2) and (3).

[0061] In a preferred aspect of the disclosure, the composition comprises: (a) a bacterium of species L. johnsonii,' (b) a bacterium of species L. reuterii,' (c) a bacterium of species M. elsdenii,' (d) a bacterium of species V. magna, and (e) a bacterium of species R. bovis. In a more preferred aspect of the disclosure, the composition comprises: (a) a bacterium of species L. johnsonii that comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200; (b) a bacterium of species L. reuterii that comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608; (c) a bacterium of species M. elsdenii that comprises a nucleic acid sequence having at least 70% identity to the genome of M. elsdenii DSM 20460; (d) a bacterium of species V. magna that comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857; and (e) a bacterium of species R. bovis that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T).

[0062] Exemplary strains Any strain of the bacteria described above may be comprised in the composition. That is, the composition may comprise any strain of L. johnsonii. The strain of L. johnsonii may, for example, comprise a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200. Numerous strains of L. johnsonii are known in the art. The composition may comprise any strain of L. reuterii. The strain of L. reuterii may, for example, comprise a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608. Numerous strains of L. reuterii are known in the art. The composition may comprise any strain of A7. elsdenii. The strain of A7. elsdeni may, for example, comprise a nucleic acid sequence having at least 70% identity to the genome of A7. elsdenii DSM 20460. Numerous strains of A7. elsdenii are known in the art. The composition may comprise any strain of V. magna. The strain of V. magna may, for example, comprise a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857. Numerous strains of V. magna are known in the art. The composition may comprise any strain of R. bovis. The strain of R. bovis may, for example, comprise a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T). Numerous strains of R bovis are known in the art.

[0063] The composition may comprise any Prevotella strain. The composition may comprise any Blautia strain. The composition may comprise any Subdoligranulum strain.

[0064] When the composition comprises a bacterium of species L. reuterii4the bacterium of species L. reuterii may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 1. For instance, the bacterium of species L. reuterii may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1. The nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species L. reuterii. The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species L. reuterii. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species L. reuterii. SEQ ID NO: 1 is the nucleic acid sequence of the genome of a L. reuterii strain that the inventors have found to be particularly effective at inhibiting ETEC.

[0065] When the composition comprises a bacterium of species L. johnsonii4the bacterium of species L. johnsonii may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 2. For instance, the bacterium of species L. johnsonii may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2. The nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species

[0066] L. johnsonii. The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species johnsonii. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species L. johnsonii. SEQ ID NO: 2 is the nucleic acid sequence of the genome of a L. johnsonii strain that the inventors have found to be particularly effective at inhibiting ETEC.

[0067] When the composition comprises a bacterium of species M. elsdenii , the bacterium of species M. elsdenii may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 3. For instance, the bacterium of species M. elsdenii may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 3. The nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species

[0068] M. elsdenii. The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species M. elsdenii. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species AT. elsdenii. SEQ ID NO: 3 is the nucleic acid sequence of the genome of a AT. elsdenii strain that the inventors have found to be particularly effective at inhibiting ETEC.

[0069] When the composition comprises a bacterium of species V. magnatthe bacterium of species V. magna may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 4. For instance, the bacterium of species V. magna may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 4. The nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species V. magna. The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species V. magna. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species V. magna. SEQ ID NO: 4 is the nucleic acid sequence of the genome of a V. magna strain that the inventors have found to be particularly effective at inhibiting ETEC.

[0070] When the composition comprises a bacterium of species R. hovis the bacterium of species R. bovis may comprise a nucleic acid sequence having at least 70% identity to SEQ ID NO: 4. For instance, the bacterium of species R. bovis may comprise a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 4. The nucleic acid sequence may, for example, be comprised in the genome of the bacterium of species R. bovis. The nucleic acid sequence may, for example, be comprised in the bacterial chromosome of the bacterium of species R. bovis. The nucleic acid sequence may, for example, be comprised in a plasmid comprised in the bacterium of species R. bovis. SEQ ID NO: 4 is the nucleic acid sequence of the genome of a R. bovis strain that the inventors have found to be particularly effective at inhibiting ETEC.

[0071] Function of the composition

[0072] As explained above, the composition inhibits ETEC and may therefore be used to prevent or treat PWD. Prevention or treatment of PWD encompasses any beneficial modulatory effect on ETEC in the context of the development or alleviation of PWD, as discussed further below. The composition may, for example, inhibit ETEC by inhibiting the growth of one or more ETEC. The composition may, for example, inhibit ETEC by inhibiting the survival of one or more ETEC. The composition may, for example inhibit ETEC by inhibiting the growth of one or more ETEC and inhibiting the survival of one or more ETEC. The composition may reduce the severity and / or spread of an ETEC infection. The composition may reduce faecal ETEC shedding. The ETEC whose growth is inhibited by the composition may be the same strain as the ETEC whose survival is inhibited by the composition. The ETEC whose growth is inhibited by the composition may be the same strain as the ETEC whose survival is inhibited by the composition.

[0073] Any or all of the bacteria comprised in the composition may be capable of inhibiting ETEC. For instance, the bacterium of species L.johnsonii may be capable of inhibiting ETEC. The bacterium of species L. reuterii may be capable of inhibiting ETEC. The bacterium of species AT. elsdenii may be capable of inhibiting ETEC. The bacterium of species V. magnet may be capable of inhibiting ETEC. The bacterium of species R. bovis may be capable of inhibiting ETEC. A Prevotella strain comprised in the compositions may be capable of inhibiting ETEC. A Blautia strain comprised in the compositions may be capable of inhibiting ETEC. A Subdoligranulum strain comprised in the compositions may be capable of inhibiting ETEC. The ability of the composition, or a given bacterium, to inhibit the growth and / or survival of ETEC may be assayed in vitro as set out in the Examples. In brief, the composition may be co-cultivated with ETEC in suitable conditions. Suitable conditions may, for example, comprise anaerobic co-cultivation for around 24 hrs (e.g. 12 to 36 hours) at 37°C. Following co-cultivation, serial dilutions of the co-culture may be plated (e.g. on MacConkey agar) and incubated at 37°C, for instance overnight. ETEC colonies can then be counted and numbers of pathogens (CFU per milliliter) determined for each.

[0074] The composition may, for example, promote clearance of one or more ETEC by the immune system. This may contribute to inhibiting survival of ETEC. For instance, the composition may promote clearance of one or more ETEC by the immune system in vivo. This may contribute to inhibiting survival of ETEC in vivo. Clearance of ETEC by the immune system may, for example, occur locally in the intestines. For example, ETEC may be phagocytosed by macrophages in the intestine, such as those underling intestinal epithelial cells. Components of the active immune system may promote phagocytosis. Such promotion may, for instance, be mediated by antibodies produced by ETEC-specific B cells. For instance, antibody opsonisation may mark the ETEC for phagocytosis. ETEC-specific T cells may lend T cell help to antibody production. Assays for ETEC phagocytosis, ETEC-specific antibodies, and / or ETEC-specific T-cells may be used to determine the effect of the composition on clearance of ETEC by the immune system. Such assays are well-known in the art.

[0075] Any or all of the bacteria comprised in the composition may be capable of promoting clearance of ETEC. Clearance of ETEC encompasses any extent of clearance of ETEC, including partial clearance of ETEC or substantial clearance of ETEC. For instance, the bacterium of species L. johnsonii may be capable of promoting clearance of ETEC. The bacterium of species L. reuterii may be capable of promoting clearance of ETEC. The bacterium of species M. elsdenii may be capable of promoting clearance of ETEC. The bacterium of species V. magnet may be capable of promoting clearance of ETEC. The bacterium of species R. bovis may be capable of promoting clearance of ETEC. RPrevotella strain comprised in the compositions may be capable of promoting clearance of ETEC. Blautia strain comprised in the compositions may be capable of promoting clearance of ETEC. A Subdoligranulum strain comprised in the compositions may be capable of promoting clearance of ETEC. The bacterium of species L. johnsonii, the bacterium of species L. reuterii, and / or the bacterium of species M. elsdenii may be particularly capable of promoting clearance of ETEC.

[0076] The composition may, for example, promote the function of the intestinal barrier. The composition may, for example, promote the integrity of the intestinal barrier. The composition may, for example, promote the function and integrity of the intestinal barrier. Promotion of the function and / or integrity of the intestinal barrier may counteract intestinal inflammation, such as intestinal inflammation commonly encountered at weaning. The skilled person is aware of how to evaluate promotion of the function and / or integrity of the intestinal barrier, and suitable assays are provided in the examples and literature discussed below. For example, changes in morphology commonly seen with intestinal inflammation, such as changes in villus height and / or crypt depth, may be reduced or eliminated in the presence of the composition. Longer villi and shallower crypt depth have been shown to improve the uptake of nutrients that pass through the gastrointestinal tract. Shorter villi and / or deeper crypt depth may be associated with intestinal inflammation. Villus height and / or crypt depth may be determined histologically, for instance on a biopsy. Promotion and / or integrity of the intestinal barrier may be evaluated using one or more assays described in Leblanc et al (2022); Qiao et al (2020); or Sudan et al (2022), incorporated by reference herein. By promoting of the function and / or integrity of the intestinal barrier, the composition may reduce or prevent colonisation of the intestine with ETEC (such as pathogenic ETEC) thereby treating or preventing PWD.

[0077] It is known that various bacteria, including bacteria of the Veillonella, Megasphaera, Lactobacillus (such as L. johnsonii) and Ruminococcus genera are able to produce SCFAs (Zhang & Huang (2023; He et al (2019); Flint & Bayer (2008). Any or all of the bacteria comprised in the composition may be capable of promoting the function and / or integrity of the intestinal barrier. For instance, the bacterium of species L. johnsonii may be capable of promoting the function and / or integrity of the intestinal barrier. The bacterium of species L. reuterii may be capable of promoting the function and / or integrity of the intestinal barrier. The bacterium of species AL elsdenii may be capable of promoting the function and / or integrity of the intestinal barrier. The bacterium of species V. magnet may be capable of promoting the function and / or integrity of the intestinal barrier. The bacterium of species R. bovis may be capable of promoting the function and / or integrity of the intestinal barrier. A Prevotella strain comprised in the compositions may be capable of promoting the function and / or integrity of the intestinal barrier. A Blautia strain comprised in the compositions may be capable of promoting the function and / or integrity of the intestinal barrier. A Subdoligranulum strain comprised in the compositions may be capable of promoting the function and / or integrity of the intestinal barrier. The bacterium of species L. johnsonii, the bacterium of species L. reuterii, and / or the bacterium of species M. elsdenii may be particularly capable of promoting the function and / or integrity of the intestinal barrier.

[0078] The composition may, for example, produce short chain fatty acids (SCFAs). For instance, the composition may produce acetate, propionate, and / or butyrate. SCFAs such as these are small organic monocarboxylic acids with a chain length of up to six carbons atoms, which are the main products of the anaerobic fermentation of indigestible polysaccharides in the large intestine. SCFAs are well-known to improve gut health through a number of local effects, such as maintenance of intestinal barrier integrity, mucus production, and protection against inflammation. SCFA production may, therefore, promote the function and / or integrity of the intestinal barrier, and / or reduce colonisation with ETEC. Production of SCFAs may be determined using methods known as the art, such as mass spectrometry or gas chromatography.

[0079] Any or all of the bacteria comprised in the composition may be capable of producing SCFAs. For instance, the bacterium of species L. johnsonii may be capable of producing SCFAs. The bacterium of species L. reuterii may be capable of producing SCFAs. The bacterium of species M. elsdenii may be capable of producing SCFAs. The bacterium of species V. magnet may be capable of producing SCFAs. The bacterium of species R. bovis may be capable of producing SCFAs. A Prevotella strain comprised in the compositions may be capable of producing SCFAs. A Blautia strain comprised in the compositions may be capable of producing SCFAs. A Subdoligranulum strain comprised in the compositions may be capable of producing SCFAs. The bacterium of species V. magna and / or the bacterium of species R. bovis may be particularly capable of producing SCFAs.

[0080] One or more of the bacteria comprised in the composition may, for example, consume lactate. Lactate is formed by many species of intestinal bacteria, and can accumulate to high levels in intestinal inflammation. Lactate may promote the fitness of E. coli species, such as ETEC. Therefore, consumption of lactate by bacteria in the composition may inihibit ETEC. Furthermore, lactate may be metabolised by lactate- consuming bacteria to produce SCFAs. As set out above, SCFAs improve gut health and may promote the function and / or integrity of the intestinal barrier, and / or reduce colonisation with ETEC. Lactate consumption may therefore provide a two-pronged approach to preventing or treating PWD.

[0081] Any or all of the bacteria comprised in the composition may be capable of consuming lactate. For instance, the bacterium of species L.johnsonii may be capable of consuming lactate. The bacterium of species L. reuterii may be capable of consuming lactate. The bacterium of species M. elsdenii may be capable of consuming lactate. The bacterium of species V. magnet may be capable of consuming lactate. The bacterium of species R. bovis may be capable of consuming lactate. Prevotella strain comprised in the compositions may be capable of consuming lactate. Blautia strain comprised in the compositions may be capable of consuming lactate. A Subdoligranulum strain comprised in the compositions may be capable of consuming lactate. The bacterium of species M. elsdenii, and / or the bacterium of species V. magna may be particularly capable of consuming lactate.

[0082] Manufacture of the composition

[0083] A composition described above may be provided by mixing or otherwise combining the different bacteria together. The different bacteria used to provide the composition may be cultured or incubated separately prior to being mixed or combined together. Alternatively, two or more of the different bacteria may be co-cultured or coincubated. All of the bacteria used to provide the composition may be co-cultured or coincubated. The disclosure also relates to a method of providing a composition of the disclosure, comprising co-culture or co-incubation of the relevant bacteria. The co-culture or co-incubation may be for at least one day, at least two days or greater. The disclosure further relates to a composition obtained or obtainable by the above described method.

[0084] Pharmaceutical composition

[0085] The disclosure provides a pharmaceutical composition comprising the composition of the disclosure, and a pharmaceutically acceptable carrier or excipient. Any of the aspects described above in connection with the composition of the disclosure may also apply to the pharmaceutical composition.

[0086] The disclosure further provides a pharmaceutical composition comprising (a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 2; (b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 1; (c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome oiM. elsdenii DSM 20460, optionally wherein the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 3; (d) a bacterium of species V. magnet, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857, optionally wherein the bacterium of species V. magna comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 4; or (e) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T), optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 5; and a pharmaceutically acceptable carrier or excipient. In other words, the disclosure provides a pharmaceutical composition comprising (i) any one of (a) to (e), and (ii) a pharmaceutically acceptable carrier or excipient. The bacterium of species L. johnsonii may be any L. johnsonii bacterium described above in connection with the composition of the disclosure. The bacterium of species L. reuterii may be any L. reuterii bacterium described above in connection with the composition of the disclosure. The bacterium of species M. elsdenii may be any M. elsdenii bacterium described above in connection with the composition of the disclosure. The bacterium of species V. magna may be any V. magna bacterium described above in connection with the composition of the disclosure. The bacterium of species R. bovis may be any R. bovis bacterium described above in connection with the composition of the disclosure. Pharmaceutically acceptable carrier or excipient

[0087] The pharmaceutical composition may be formulated using any suitable method. Formulation of bacteria with standard pharmaceutically acceptable carriers and / or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the cells to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19thEdition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0088] Pharmaceutically acceptable carriers and excipients are well-known in the art. Typically, the pharmaceutical composition of the disclosure is prepared as a liquid suspension of bacteria in the carrier or excipient. Suitable carriers or excipients include, for example, water, saline, dextrose, glycerol, oor the like and combinations thereof. In addition, if desired, the pharmaceutical compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and / or pH buffering agents.

[0089] Feed and feed supplement

[0090] The disclosure provides an animal feed or feed supplement comprising the composition of the disclosure, and a pharmaceutically acceptable carrier or excipient. Any of the aspects described above in connection with the composition of the disclosure may also apply to the pharmaceutical composition.

[0091] The disclosure further provides an animal feed or feed supplement comprising (a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 2; (b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 1; (c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome oiM. elsdenii DSM 20460, optionally wherein the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 3; (d) a bacterium of species V. magnet, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857, optionally wherein the bacterium of species V. magna comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 4; or (e) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T), optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 5; and a pharmaceutically acceptable carrier or excipient. In other words, the disclosure provides a pharmaceutical composition comprising (i) any one of (a) to (e), and (ii) a pharmaceutically acceptable carrier or excipient. The bacterium of species L.johnsonii may be any L. johnsonii bacterium described above in connection with the composition of the disclosure. The bacterium of species L. reuterii may be any L. reuterii bacterium described above in connection with the composition of the disclosure. The bacterium of species M. elsdenii may be any M. elsdenii bacterium described above in connection with the composition of the disclosure. The bacterium of species V. magna may be any V. magna bacterium described above in connection with the composition of the disclosure. The bacterium of species R. bovis may be any R. bovis bacterium described above in connection with the composition of the disclosure.

[0092] Animal feed

[0093] In the context of the present disclosure, the term “ animal feed" may refer to a food that is specifically designed, formulated or prepared as a diet for a non-human animal. The animal feed may, for example, be adapted to meet some or all of the nutritional needs of a non-human animal.

[0094] The non-human animal may, for example, be a mammal. The non-human animal may, for example, be a companion animal, such as a dog, cat, horse or rabbit. Preferably, though, the non-human animal is a farm animal, such as a pig, cow, or sheep. More preferably, the animal is a pig.

[0095] The non-human animal may, for example, be an adult. Preferably, though, the nonhuman animal is a juvenile. In the context of the present disclosure, the term “juvenile” may refer to an animal that is not yet mature, such as a piglet (an immature pig). The juvenile animal may not yet be weaned, or may be in the process of being weaned.

[0096] The animal feed may, for example, be a compound feed. A compound feed is a feed that is adapted to the needs of an animal based on its growth stage, physiological requirements and / or its production use. For example, a weaner pig and a grower pig are at different growth stages, and therefore have different nutritional needs. A non-pregnant sow and a pregnant sow have different physiological requirements, and therefore different nutritional needs. A pig reared for meat production and a pig used for breeding have different production uses, and therefore different nutritional needs. By way of another example, nutritional needs vary between dairy cattle and beef cattle due to their different production uses (milk vs meat). Types of compound feed include complete formula feed, concentrated feed, concentrate mixture, premixture, artificial milk and milk substitute feed, for example. A compound feed may be provided as a mash, pellet, crumb, cube or powder, for instance.

[0097] Animal feed supplement

[0098] An animal feed supplement may also be known as an animal feed additive. An animal feed supplement or additives may be defined as a substance other than a feed material which is intentionally added to feed or water. The substance may, for example, be added to feed or water to assist in meeting and animal’s nutritional requirements. The substance may, for example, be added to feed to improve the quality (such as appearance, shelf-life, or palatability) of the feed. The substance may, for example, be added to feed or water to improve the quality of obtained from the animal consuming the feed or water (for instance meat, milk or eggs). A key reason for adding the substance to feed or water is to improve the performance (e.g. growth or fertility) or health of the animal consuming the feed or water. This is an aim of the animal feed supplement of the present disclosure. The animal feed supplement improves health by preventing or treating PWD.

[0099] The feed supplement may, for example, be a solid. The solid may, for example, be a powder, pellet, block or lick. The feed supplement may, for example, be a liquid or suspension.

[0100] The bacteria provided in the composition, pharmaceutical composition, animal feed or feed supplement of the disclosure are typically live bacteria. The disclosure also relates to a supernatant obtained or obtainable from a culture or co-culture of bacteria of the disclosure, which has one or more functional properties of a composition of the invention as described above.

[0101] Bacterium

[0102] The disclosure further provides a bacterium, which is:

[0103] (a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2;

[0104] (b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1;

[0105] (c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome oiM. elsdenii DSM 20460, optionally wherein the bacterium of species AT. elsdenii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 3; or

[0106] (d) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T) , optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 5.

[0107] The bacterium may be provided in any form. The bacterium is typically an isolated bacterium. The bacterium is typically provided in live form. Further described herein is a culture of a bacterium of the disclosure and a co-culture of two or more bacteria of the disclosure, such as a co-culture of all bacteria used to provide a composition of the disclosure. A culture of a bacterium of the disclosure is typically a biologically pure culture. Also described herein is a supernatant obtained or obtainable from a culture or co- culture of bacteria of the disclosure, which has one or more functional properties of a composition of the disclosure as described above.

[0108] Prevention or treatment of PWD

[0109] The disclosure provides a method of preventing or treating post-weaning diarrhoea in an animal, comprising administering to the animal the composition, pharmaceutical composition, feed or feed supplement or bacterium of the disclosure. The disclosure also provides the composition, pharmaceutical composition, feed or feed supplement or bacterium of the disclosure, for use in a method of preventing or treating post-weaning diarrhoea in an animal, wherein the method comprises administering the composition, pharmaceutical composition, feed or feed supplement or bacterium to the animal. The disclosure further provides the composition, pharmaceutical composition, feed or feed supplement or bacterium of the disclosure, for use in the manufacture of a medicament for preventing or treating post- weaning diarrhoea in an animal.

[0110] Preventing PWD

[0111] To prevent PWD, the animal is typically administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium prior to and / or at weaning. For instance, the animal may be administered with the pharmaceutical composition, feed or feed supplement or bacterium prior to weaning. The animal may be administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium at weaning. The animal may be administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium prior to and at weaning.

[0112] “Prior to w eaning" may, for example, mean before removal of the animal from its mother. In the context of the composition or pharmaceutical composition or bacterium, “prior to w eaning" may, for example, mean before the introduction of the animal to solid food. “At w eaning" may, for example, mean at or around (e.g. within 2, 4, 6 or 12 hours of) removal of the animal from its mother. “At w eaning" may, for example, mean at or around (e.g. within 2, 4, 6 or 12 hours of) the animal’s first introduction to solid food.

[0113] Preventing PWD encompasses any beneficial modulatory effect on the development of PWD, including suppression or reduction of PWD. Preventing PWD may, for example, refer to reducing the clinical signs or symptoms associated with the onset of PWD. That is, preventing PWD may mean that clinical signs or symptoms of PWD that develop in an animal administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium are less severe, for example in comparison to an animal that is not administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium. Reducing the severity of the clinical signs or symptoms may minimise the impact of PWD on welfare and / or productivity (e.g. growth).

[0114] Preventing PWD may, for example, refer to delaying the onset of PWD. That is, preventing PWD may mean that clinical signs or symptoms of PWD occur later in an animal administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium, for instance compared to an animal that is not administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium. Delaying the onset of PWD may, for example, mean that the animal is older and / or has a more robust immune system at onset, such that the impact of PWD on welfare and / or productivity (e.g. growth) is minimised.

[0115] In some aspects, preventing PWD may refer to completely avoiding the onset of PWD. That is, preventing PWD may mean that clinical signs or symptoms of PWD never occur in an animal administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium.

[0116] Treating PWD

[0117] To treat PWD, the animal is typically administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium at and / or after weaning. For instance, the animal may be administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium at weaning. The animal may be administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium after weaning. The animal may be administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium at and after weaning.

[0118] “ After w eaning" may, for example, mean after (e.g. 12 or more, 24 or more, or 48 or more hours after) removal of the animal from its mother. “ After w eaning" may, for example, mean after (e.g. 12 or more, 24 or more, or 48 or more hours after) the animal’s first introduction to solid food.

[0119] Treating PWD encompasses any beneficial modulatory effect on PWD, including alleviation of PWD. That is, treating PWD may mean that clinical signs or symptoms of PWD are abrogated or eliminated in an animal administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium.

[0120] Treating PWD may, for example, refer to reducing the clinical signs or symptoms associated with PWD. That is, treating PWD may mean that clinical signs or symptoms of PWD that develop in an animal administered with the composition, pharmaceutical composition, feed or feed supplement or bacterium become less severe, for example compared to before treatment. Reducing the severity of the clinical signs or symptoms may minimise the impact of PWD on welfare and / or productivity (e.g. growth).

[0121] In one aspect, treating may refer to completely resolving the clinical signs or symptoms of PWD.

[0122] Administration and formulation

[0123] The composition or pharmaceutical composition or bacterium may be administered by any suitable route. Suitable routes may, for example, result in bacterial inoculation of the intestines. Suitable routes may include, for, example, the oral, buccal and rectal routes. Preferably, the composition or pharmaceutical composition or bacterium is administered orally. The usual administration route for the feed or feed supplement is oral. Oral administration to animals is well-described and may, for example, include direct administration (i.e. introducing the product to the mouth, or to the oesophagus or stomach by gavage) or administration in feed.

[0124] Formulation of bacteria for oral administration is well-known in the art. The composition, pharmaceutical composition, feed or feed supplement or bacterium may for example be formulated as a solid (e.g. a pill or pellet) or a liquid. The composition, pharmaceutical composition or feed supplement may, for example, comprise a delivery vehicle that optimises delivery of the bacterial to the intestines. The composition, pharmaceutical composition or feed supplement may, for example, be in a gastro-protected form. The composition, pharmaceutical composition, feed or feed supplement or bacterium are administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective. The quantity to be administered depends on the animal to be treated. Precise amounts of composition, pharmaceutical composition, feed or feed supplement or bacterium required to be administered may depend on the judgement of the practitioner and may be particular to each animal.

[0125] Animal

[0126] The animal may, for example, be a mammal. The mammal may, for example, be a human. Preferably, the mammal is a non-human mammal. The non-human animal may, for example, be a companion animal, such as a dog, cat, horse or rabbit. Preferably, though, the non-human animal is a farm animal, such as a pig, cow, or sheep. Most preferably, the animal is a pig.

[0127] The animal may, for example, be an adult. Preferably, though, the animal is a juvenile. In the context of the present disclosure, the term “juvenile” may refer to an animal that is not yet mature, such as a piglet (an immature pig). The juvenile animal may not yet be weaned, or may be in the process of being weaned. In this instance, the composition, pharmaceutical composition, feed or feed supplement or bacterium may be administered to prevent PWD. Alternatively, the juvenile animal may be weaned. In this case, the composition, pharmaceutical composition, feed or feed supplement or bacterium may be administered to treat PWD. Prevention and treatment are described in detail above.

[0128] The following Examples illustrate the invention.

[0129] Examples

[0130] Materials and Methods

[0131] Animals and Sample collection

[0132] Samples were collected from eight Belgian farms during one farrowing cycle each between December 2019 and February 2021. The farms for this study were selected based on specific criteria, including weaning piglets between 3 and 4 weeks of age, not using zinc oxide, and not vaccinating against post-weaning diarrhoea or oedema disease with limited or no use of antibiotics throughout the experiment. Preference was given to farms with prior incidents of post-weaning diarrhea; however, two farms with no such history (Farm3 and Farm5) were also included to establish a control group for our analysis. Finally, eight farms were chosen for field sampling. These selection criteria were chosen to ensure that the farms had similar management practices and to reduce potential confounding factors in the study. For each of the eight farms, 8 sows and their respective litters were randomly selected and included in the study, based on similarity in parturition days. Animal sampling comprised tail collection at 3 days post-birth, rectal swab collection at four time points, and collection of intestinal digesta at the end of the experiment. These samples encompassed all piglets from the participating litters, ranging between 99 to 112 individuals per farm, while from the sows only rectal swabs were collected. Intestinal content collection was performed on a selected number of animals (n=64).

[0133] Briefly, at 3 days after birth, tails were collected from all piglets of the litters involved in the study. Tail docking, which is a routine practice on the farm, had been performed prior to tissue collection and thus, the tails were truncated and stored at -20°C until further analysis. These analyses included DNA extraction and screening for genetic susceptibility to F4 and F18 ETEC strains.

[0134] Rectal swabs from piglets were collected at 7 days of birth, 3 days before weaning, day of weaning and 4 days-post-weaning. Rectal swabs from sows were also collected at 7 days after giving birth and 3 days before weaning of the piglets. Due to varying farm management practices, including different weaning times, the ages of piglets at the time of sampling varied according to their respective farms. However, for the purpose of this study, our primary focus lies on post-weaning diarrhea. Consequently, we prioritized the weaning day as a more crucial parameter compared to age. A total of 3444 piglet rectal swabs and 128 sow rectal swabs were collected across 4 timepoints pre and post weaning. Rectal swabs were collected using dry swabs and stored immediately at -20°C for microbiota composition analysis (16S rRNA gene sequencing).

[0135] Intestinal digesta collection occurred at varying intervals across farms, determined by the onset of post-weaning diarrhea, spanning from 4 to 7 days post-weaning. In cases where farms had no prior history of diarrhea (Farm3 and Farm5), collection was scheduled arbitrarily at 5 and 4-days post-weaning, respectively, following the timeline of euthanasia established in previously sampled farms. For Farm8, collection took place at 7 days postweaning, once it was evident that the piglets were not prone to developing diarrhea, despite the farm's history of infections. Intestinal sample collection included only eight piglets per farm that, at the end of the study, were selected based on a range of factors including genetics, litter, weaning pen, healthy status and treatment received during the entire study. The selected 64 piglets were euthanized by intravenous injection of an overdose of sodium pentobarbital (24 mg / kg, Nembutal, Sanofi Sante Animale, Brussels, Belgium). Mid- jejunal, ileal and colonic contents were collected in Eppendorf tubes and stored immediately at -20°C for for microbiota composition analysis. Cotton swabs from each segment of the piglets' intestines (i.e. duodenum, proximal, middle and distal jejunum, ileum, colon, caecum) were collected and subsequently plated on blood agar plates for bacterial isolation.

[0136] Additionally, an intestinal segment of 15 cm was excised from the mid jejunum and washed three times with Krebs-Henseleit buffer (0.12 M NaCl, 0.014 M KC1, 0.001 M KH2PO4, 0.025 M NaHCO3, pH 7.4) as previously reported (Nguyen U V. et al., 2017). Subsequently, the segment was opened and incubated with Krebs-Henseleit buffer containing 1% (v / v) formaldehyde (Krebs-formol) for 15 min at room temperature. Next, the villi were scraped off with glass slides and stored in Krebs-formol at 4 °C until further analysis. Jejunal villi were collected to perform a villus adhesion assay and to determine the phenotypical susceptibility to ETEC F4 and Fl 8.

[0137] From each farm, eight piglets were selected for intestinal sample collection at the end of the study, based on a range of factors including genetics, pen of weaning, healthy status and treatment received during the entire study. The time of euthanasia was dependent on the development of diarrhoea, as this is a key indicator of ETEC infection in piglets. Selected piglets (N=64 in total) were euthanized by sodium pentobarbital injection for sampling. Mid-jejunal, ileal and colonic contents were collected were stored at -20°C for microbiota composition analysis (16S rRNA gene sequencing). Jejunal villi of each piglet were also collected to perform a villus adhesion assay and to determine the phenotypical susceptibility to ETEC F4 and Fl 8.

[0138] DNA extraction from piglet tails and genotyping of MUC13 and FUT1 polymorphisms Piglet tails were subjected to DNA extraction by using the DNase blood and tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Piglets DNA was used to test their genetic susceptibility to F4 and Fl 8 ETEC strains to include in the study piglets with different genetic background. The genetic susceptibility to ETEC F4 strain was determined by genotyping the MUC13 gene in the piglets DNA as described previously (Ren J. et al., 2012). Briefly, primers F7 / R7 (F7: 5'-TTC TAC TCT GAT TCC AC A TCA CG-3'; R7: 5'-TGG TCA TGT CT A GGA CTC TTT GAG-3') were used to amplify the MUC13 gene. The MUC13A allele was indicated by amplicons of 151 bp and marked as R (resistant), and the MUC13B allele was represented by amplicons of 83 bp and marked as S (susceptible). The genetic susceptibility to ETEC F18 strain was determined by genotyping the FUT1 gene in the piglets DNA as previously described (Meijerink et al., 1997). Briefly, primers M307_fwd (5’-CTG-CCT-GAA-CGT-CTA- TCA-AGA-TC-3’) and M307_rev (5’-CTT-CAG-CCA-GGG-CTC-CTT-TAA-G-3’) were used to amplify the FUT1 gene. The resulting amplicons were digested with Cfol restriction enzyme resulting in fragments of 241, 93 and 87 bp for the susceptible genotype (SS); fragments of 328, 241, 93, 87 bp for the heterozygous susceptible (RS) and fragments of 328 and 93 bp for the resistant (RR).

[0139] Isolation and characterization of ETEC strains from farms

[0140] Cotton swabs from segments of the piglets' intestines were collected as described above and used to isolate different wild ETEC strains. Swabs were immediately plated on blood agar and incubated overnight at 37° C. Phenotypically different colonies were purified and subjected to DNA extraction. Genomic DNA was isolated from all strains using the alkaline lysis method by resuspending a single bacterial colony in 20 pl lysis buffer (0.25% SDS, 50 mM NaOH). After boiling at 95°C for 5 min, 180 pl of nuclease- free water was added and centrifuged for 5 min at maximum speed. The DNA was stored at -20°C until further use. The virotype of each colony was determined by multiplex PCR as previously described by Casey et al. (Casey & Bosworth, 2009) by using the indicated primers for the detection of the following virulence factors: STb, STa, LT, STx2e, F4, Fl 8. Each colony was also subjected to an E.coli specific PCR by using primers XanQ_2_fw (5’-GCAGATGGTCTGGTTTCTGT-3’) and XanQ_2_rev (5’- CGACGCCAGTCATCTGAATAA-3’) (Leurs et al., 2022) to confirm the isolates were E. coli. Bacterial strains were stored at -80°C in glycerol until further use.

[0141] In vitro villous adhesion assay

[0142] The in vitro villus adhesion assay was performed as previously described (Girardeau, 1980). Briefly, the jejunal villi were first washed with Krebs buffer and subsequently suspended in PBS with 1% D-mannose, which prevents adhesion of E. coli by type 1 pili. Then, 3.2 x 1OA7 F4+ and F18+ bacteria were added to 450 pl of PBS containing 1% D-mannose and the villi. These samples were then incubated for 45 min at room temperature while rotating and were subsequently analyzed by phase contrast at a magnification of 400x. The number of adherent bacteria was determined by counting the bacteria adhering to a 50-pm brush border length in 20 different areas. Adhesion of less than 5, between 5 and 30 and more than 30 bacteria per 250 pm villous brush border length was noted as negative, weak or strongly positive, respectively.

[0143] DNA extraction. Library preparation, 16s rDNA sequencing

[0144] A total of 416 rectal swabs and 192 intestinal contents belonging to the 64 euthanized piglets, and 124 rectal swabs from the sows were selected for DNA extraction and 16S analysis. Rectal swabs were cut in small pieces and collected in Eppendorf tubes while, lOOmg were used for each intestinal content. DNA was extracted using the hexadecyltrime-thylammonium bromide (CTAB) method described by Griffiths et al. (Griffiths et al., 2000) with modifications described by Aguirre et al. (Aguirre et al., 2019). The resulting DNA was resuspended in 50 pL of water and the quality and concentration of the DNA was examined spectrophotometrically (NanoDrop, Thermo Fisher Scientific, Merelbeke, Belgium).

[0145] Bacterial community composition was assessed by amplifying the V3-V4 hypervariable region of the 16S rRNA gene by using the gene-specific primers S-D-Bact- 0341-b-S-17 and S-D-Bact-0785-a-A-21 (Klindworth et al., 2013). The PCR amplifications were performed as described by Aguirre et al. (Aguirre et al., 2019). The final PCR products were purified using CleanNGS beads (CleanNA, Gouda, The Netherlands). The DNA concentration of the final barcoded libraries was measured with a Quantus fluorimeter and Quantifluor dsDNA system (Promega, Madison, WI, USA). The libraries were combined to an equimolar 5 nM pool and sequenced with 30% PhiX spike-in using the Illumina MiSeq v3 technology (2 * 300 bp, paired-end) at the Oklahoma Medical Research center (Oklahoma City, OK, USA).

[0146] Bioinformatics and statistical analysis of 16S rRNA gene amplicon data

[0147] Demultiplexing of the amplicon dataset and deletion of the barcodes was done by the sequencing provider. Quality of the raw sequence data was checked with the FastQC quality-control tool (Babraham Bio-informatics, Cambridge, United Kingdom). Subsequently, the sequences were trimmed, quality-filtered and dereplicated using the DADA2 algorithm (vl.22) within R (v4.1.2) (Callahan et al., 2016). An initial amplicon sequence variant (ASV) table was constructed before chimaeras were identified using the removeBimeraDenovo function. Finally, taxonomy was assigned using DADA2’s native naive Bayesian classifier against the Silva database (vl38) (Quast et al., 2013). To select the appropriate subsampling depth, alpha rarefaction curves were generated. Further analysis were performed using the phyloseq (vl.38) (McMurdie & Holmes, 2013) pipeline in R. qPCR ETEC and data analysis

[0148] DNA extracted from rectal swabs and intestinal content were subjected to quantitative PCR. Absolute quantification PCRs were developed to target ETEC virulence factor genes STb, STa, LT, F4, Fl 8 and the overall population of E. coli in each sample. To design the primers targeting ETEC virulence factor genes, the reference genomes of Escherichia coli UMNK88 for the F4 strain and UMNF18 for the F18 strain were used as template (Shepard et al., 2012). The primers were designed using the Benchling primer wizard (Benchling, San Francisco, CA) and were further assessed for specificity towards E. coli through Primer Blast analysis (Ye et al., 2012). Table 1 presents the list of the primers developed and utilized in this study for conducting qPCR against ETEC virulence factor genes, along with their corresponding annealing temperature.

[0149] Table 1. Primers developed in this study for quantitative PCR of ETEC virulence factors F18, F4, LT, STa, STb and of their respective standard fragment

[0150] To determine the efficiency of the developed primer pairs, a standard curve was generated for each gene using respectively the standard fragment primer pairs and DNA from isolated wild type ETEC with the de-sired corresponding virotype. The resulting PCR products were purified (MSB Spin PCRapace, Stratec Molecular, Berlin, Germany) and DNA concentration was determined spectrophotometrically (NanoDrop, Thermo Fisher Scientific, Merelbeke, Belgium). The concentration of the linear dsDNA standard fragments was adjusted to 10A9 copies / pl, with ten-fold dilution steps.

[0151] The specificity and efficiency of each of the primer pairs were assessed using a SYBRgreen qPCR assay. Each 12 pl qPCR reaction consisted of 2pl template DNA, 6 pl SensiMix™ SYBR® & Fluorescein Kit (Bioline), 0.5 pM forward primer and 0.5 pM reverse. Cycling was performed on a real-time PCR thermal cycler (Biorad, Hercules, CA, USA) and conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 45 s and 62°C for 1 min. The fluorescent products were detected at the last step of each cycle. To confirm the specificity of the reaction, PCR products were subjected to melt analysis using a dissociation protocol comprising 95°C for 15 s, followed by 0.5°C incremental temperature ramping from 65 °C to 90°C.

[0152] The overall population of E. coli was quantified by using the primer pair XanQ_2_fw (5’-GCAGATGGTCTGGTTTCTGT-3’) and XanQ_2_rev (5’- CGACGCCAGTCATCTGAATAA-3’) as previously described (Leurs et al., 2022).

[0153] The qPCR data were analyzed using CFX Maestro Software from Bio-Rad (BioRad Laboratories, Inc., Hercules, CA). Standard curves for each target bacteria were generated using 10-fold (i.e., 101- 108) serial dilutions of generated standard fragments as templates. Samples, standards and non-template controls in triplicate were included in each run. The qPCR efficiency ranged between 96 and 100% with a regression coefficient value (RA2) systematically above 0.98. Results were expressed in logio copies of 16S rRNA genes per gram of intestinal content or per rectal swab.

[0154] Categorization of the piglets based on LT severity

[0155] Based on the qPCR data, piglets were classified into two groups based on the levels of the LT toxin gene post-weaning, therefore piglets were divided depending on their LT severity. Piglets with no detectable levels of the LT toxin in any of their samples were assigned to the lowLT loads group, while piglets with detectable levels of the LT toxin in at least one of the analyzed samples belonged to the high LT group. The group was defined as TowLT’ instead of ‘noneLT’ to account for undetectable level of the genes by qPCR or bacterial isolation. The ETEC strains isolated from each intestinal segment of the piglets were also included in the classification into the two groups. If a strain exhibited a virotype that included the LT toxin, the piglets from which this strain was isolated, were assigned to the highLT group. sPLS-DA - Identification of bacterial taxa associated with lowLT count post- weaning

[0156] A sparse Partial Least Square Discriminant Analysis (sPLS-DA) was as used to estimate the contributive microbial taxa in lowLT vs. highLT classification. The analysis was done at 2 levels: ASV level and genus level for each timepoint including 7 days of birth, 3 days before weaning, weaning, 4 days post-weaning, and for different body sites such as rectal swabs, mid-jejunum, ileum, colon. This analysis was carried out in R (v4.1.2) using the mixOmics package (v6.19.1) as previously described (Le et al., 2016). Briefly, the raw ASV data were used as predictors and the LT severity classification as response vector. An initial tuning of sPLS-DA parameters was performed to determine the main variables (i.e. ASVs or genera) and number of components that enable discrimination of lowLT and highLT piglets with the lower possible error rate. To obtain a reliable estimation of the error rate a 10-fold, 100-repeat design was utilized for both selection of the number of components and the number of variables. Following this tuning, the final sPLS-DA model was constructed using these optimized values. When the optimal component number resulted in 1, it was changed to 2 components, needed for graphical purposes.

[0157] The optimal number of variables (genera or ASVs) that were selected at each time point and body site for the final sPLS-DA model are illustrated in Tables 2 and 3.

[0158] Table 2. Optimal number of features (genera) selected for the final sPLS-DA model at each time point and body site

[0159] Table 3. Optimal number of features (ASVs) selected for the final sPLS-DA model at each time point and body site plotlndiv(), cim() and plotLoadings(), functions within the mixOmics package were used to generate images from the sPLS-DA such as, sample plots, clustered image maps and loading plots (Le Cao & Welham, 2021).

[0160] Bacteria isolation, DNA extraction, 16S Sanger sequencing, selection of candidates Fresh fecal samples were collected from healthy farms from sows and piglets. Samples were collected in a plastic bag that was hermetically closed with a GasPak™ EZ anaerobe sachet inside to obtain an anaerobic environment and allow the survival of the targeted anaerobic bacteria. Fecal samples were serially diluted and each dilution was plated on different selective and general agars.

[0161] Agar used for the bacterial isolation are: 5% horse blood-enriched Columbia agar (Neogen), Rogosa agar (Oxoid), MRS agar (Oxoid), Wilkins-Chalgren Anaerobe agar (Oxoid), Fastidious anaerobe agar (Neogen), Schaedler anaerobe agar (Oxoid).

[0162] A total of 260 colonies were selected, purified and subjected to genus identification with MALDI-TOF MS. Bacterial DNA was extracted following the alkaline lysis method by resuspending a single bacterial colony in 20 pl lysis buffer (0.25% SDS, 50 mM NaOH). After boiling at 95°C for 5 min, 180 pl of nuclease-free water was added and centrifuged for 5 min at maximum speed. The DNA was stored at -20°C until further use.

[0163] Then, DNA was subjected to Sanger sequencing of the entire 16S rRNA gene by using the primers fDl (5’-AGAGTTTGATCCTGGCTCAG-3’) and rDl (5’- AAGGAGGTGATCCAGCC-3’), as previously described (Weisburg et al., 1991). PCR products were directly sequenced using the service from Eurofins (Germany) service. The chromatogram of each sequence was visually inspected by using Geneious to assess for sequence purity, and aligned against the ASVs obtained from the sPLS-DA analysis, as reference sequences. Based on their similarity with the reference sequences, bacteria were selected for further analysis.

[0164] Co-cultivation of probiotic candidates with ETEC in vitro

[0165] Bacterial strains were individually cultivated anaerobically overnight at 37°C in 5ml of BHI broth supplemented with 1% D-glucose, lactose, L-tryptophan, soluble starch. The pathogenic ETEC strain (virotype: F4:LT:STb) and Enterococcus faecium (Oralin®) were cultivated in the same way. After cultivation, each probiotic culture and E. faecium were spinned down and the pellet was resuspended in 5 ml of fresh medium. Supernatant was collected for a subsequent experiment. 50 pl of each candidate were transferred in a 24-well plate with fresh medium and 50 pl of ETEC (diluted 1 / 1000) were added to each well with a final volume of 600 ul. The probiotic strains individually and in different combinations were co-cultivated with ETEC for 24 h at 37°C in anaerobic conditions. As a negative control, 50 ql of the pathogen without any addition was cultivated. The cultures were serially diluted 10-fold, plated on MacConkey agar and incubated overnight at 37°C. The next day, ETEC colonies were counted and numbers of pathogens (CFU per milliliter) were determined for each bacterial set-up.

[0166] The cell-free supernatant obtained from overnight bacterial cultures was collected and transferred in a 24-well plate with 150 pl of fresh medium and 50 pl of ETEC (diluted 1 / 1000) per each well. The supernatant of each strain was tested individually and in different combinations with other strains. The total volume of supernatant in each well was adjusted to reach a consistent volume of 400 pl, ensuring uniformity in the experimental conditions. ETEC colony counts was performed as described above.

[0167] Experiments were performed in duplicates.

[0168] In order to assess potential statistically significant variations among the different combinations in their effectiveness in reducing ETEC levels, we employed the analysis of variance (ANOVA) in R (v4.1.2) within the Rstudio environment (vl.4.1103). Subsequently, a post-hoc Tukey test was conducted to delve into specific pairwise differences among the combinations. The normality of the data was visually assessed using graphical methods (histograms and quantile-quantile plots). Additionally, the Shapiro test was applied to formally test the normality assumption. In cases where the data did not meet the assumption of normality, a log2 transformation was performed to approximate normality.

[0169] Optimization of fermentation conditions for the selected bacterial combination

[0170] Combination 41 - comprising Ruminococcus bovis, Veillonella magna, Lactobacillus johnsonii, Megasphaera elsdenii and Limosilactobacillus reuterii strains - was selected for the optimization phase. Two variants of the mixture were tested: (MV1) where each probiotic strain was cultured individually for 24 hours, followed by combining the strains when inoculated with ETEC; (MV2) involving the cultivation of the five bacteria together, using a two-step process spanning 48 hours. The experiment included both live bacteria and supernatant-only conditions, as described above, and was performed in duplicates.

[0171] The mixture variant 1 (MV1), E.faecium and ETEC were cultivated as previously described. For the mixture variant 2 (MV2), defined volumes from pure cultures of each bacterial strain were collected (1ml for L. johnsonii and R. bovis, and 0,1 ml for elsdenii, V. magna and L. reuterii), transferred in 100 ml of BHI-supplemented media and grown for 24 hours. After this period, 2 ml of the mixture were transferred in 100 ml of fresh media and incubated for an additional 24 hours. Following cultivation, 5 ml of the MV2 were centrifuged, and the pellet was resuspended in 5 ml of fresh medium. Supernantant was collected for a subsequent experiment. Subsequently, 250 pl of both mixture variants were transferred to separate wells in a 24-well plate with 300 pl fresh medium and 50 pl of ETEC (diluted 1 / 1000) added to each well. The 24-well plate was incubated for 24 hours at 37°C under anaerobic conditions. Negative and positive control setup, serial dilution, plating on MacConkey agar and the enumeration of ETEC colonies were conducted following the procedures described in the previous section.

[0172] In the supernatant-based experimental setup, 400 pl of supernatant from MV1, MV2 and E.faecium, was added to separate wells, along with 150 pl of fresh medium and 50 pl of ETEC (diluted 1 / 1000) in each well. For the ETEC-only supernatant control, 400 pl of Hank's Balanced Salt Solution (HBSS) was combined with 150 pl of fresh medium and 50 pl of diluted ETEC. ETEC colony counts was performed as previously described.

[0173] Example 1 - Screening for microbes correlating with low LT count post-weaning

[0174] The levels of the LT toxin gene (qPCR data) at the final time point in all piglet samples were observed to be elevated in farms with higher diarrheic scores (Kruskal-Wallis test; p<0.05). Therefore, the presence of the LT toxin gene was chosen as a variable for categorizing animals, implying its presence might be linked to post-weaning diarrhea. Piglets were sorted based on the presence of the LT toxin gene in any of their post-weaning samples, resulting in two groups: low-LT and high-LT. The distribution of piglets within these groups is described in Table 5. In total, 33 piglets were assigned to the low-LT group and 31 piglets to the high-LT group.

[0175] Table 4 Distribution of piglets into low-LT and high-LT groups across eight farms

[0176] Distinct bacterial features are linked with varying LT toxin levels. To identify microbiota members contributing significantly to the discrimination between the low-LT and high-LT groups across various time points and body sites, a supervised classification technique, known as sPLS-DA (sparse Partial Least Square Discriminanat Analysis), was employed. Figures 1 and 2 represent the analysis covered rectal swabs from four time points - 7 days from birth (Figure 1A), 3 days before weaning (Figure IB), weaning day (Figure 1C), 4 days after weaning (Figure ID) - and samples from the mid jejunum (Figure 2A), ileum (Figure 2B), and colon (Figure 2C), collected at the time of euthanasia. The sPLS-DA identifies the most discriminative features that best characterize each group, providing insights into the microbial composition dynamics across different anatomical locations and temporal stages.

[0177] The analysis revealed a clear distinction in the clustering of the rectal microbiome between piglets with low LT toxin levels and those with high LT levels (Figure 1). This distinction was evident as early as 7 days post-birth and became more pronounced over time, culminating in two distinctly separated clusters by 4 days post-weaning. Similarly, the jejunal, ileal, and colonic microbiomes also exhibited distinct separations between the two groups (Figure 2), suggesting a correlation between LT toxin-carrying enterotoxigenic Escherichia coli levels and specific microbial features.

[0178] The ASVs primarily contributing to the discrimination between the low-LT and the high-LT group in each set of samples are visualized with loadings plots (Figures 3 and 4). These plots illustrate the coefficient weight of their contribution to component 1 (Figure 3) and component 2 (Figure 4) of the sPLS-DA, ranked from the bottom to the top based on their importance and colored according to their abundance in the low-LT or high-LT group.

[0179] At phylum level, the ASVs characterizing the two groups belonged primarily to the Firmicutes phylum in both, with a greater presence in the low-LT group. Conversely, ASVs from the Bacteroidota, Actinobacteriota and Campylobacterota phyla were mostly associated with high-LT group. At the family level, both groups exhibited discriminative ASVs belonging to families such as Lachnospiraceae and Prevotellaceae . However, families including Oscillospiraceae , Ruminococcaceae, Streptococcaceae and Veillonellaceae. despite their high frequency in both groups, displayed a higher prevalence within the low-LT group. On contrary, families such as I.aclobacillaceae. Bacteroidaceae, and Rikenellaceae. although among the most frequent in both groups, exhibited greater abundance in the high-LT group. The redundancy of the families within both groups highlights the potential inaccuracies associated with family-level analysis, due to the broad range of bacterial genera and species within each family, each possessing diverse characteristics and functions.

[0180] At genus level, considering all time points and body sites together, ASVs belonging to the HT002, Blautia, Megasphaera, Subdoligranulum genera were discriminative for the low-LT group, while ASVs belonging to genera like Bacteroides, Alloprevotella, UCG-005, Christensenellaceae R-7 group were discriminative for the high-LT group.

[0181] In our study, we have thus far delineated the taxonomy of discriminant ASVs, including details on their phylum, family, and genus. However, delving into ASV-level description offers a deeper understanding of species or strain-level diversity, affording a high-resolution taxonomic classification crucial for precise assignments, and aiding in the identification of microbial members pivotal for ecological or functional roles.

[0182] Figures 3 and 4 illustrate the key ASVs associated with either low-LT or high-LT values across each time point and body site, spanning the first 2 components of the sPLS- DA. In the process of selecting and narrowing the potential bacterial candidates from the listed ASVs, it was crucial to identify bacterial features not only correlated with low levels of the LT toxin but also demonstrating universality, meaning they were shared among the majority of farms, and consistency across various time points and body sites.

[0183] The ASVs meeting these criteria included lactic acid bacteria such as, species belonging to HT002 genus - a candidate genus of Lactobacillaceae family - (ASV5 and ASV212), mostly present in the small intestine, and to Lactobacillus genus like L. johnsonii prophage law anensis (ASV3) and L. delbrueckii (AS VI 66), which are more important in the early life stages of piglet life; and short chain fatty acid producers such as, uncultured UCG-003 (candidate genus of Oscillospiraceae family, ASV393); uncultured Veillonella (ASV171); Blautia coccoides / hansenii / marasmi / producta (ASV433); Megasphaera elsdenii (ASV123 and ASV51); uncultured Prevotella (ASV153); uncultured Prevotellaceae (ASV375); uncultured Muribaculaceae (ASV15, ASV98), uncultured Intestinimonas (ASV392); uncultured Ruminococcus R NI Ty, uncultured Subdoligranulum (AS VI 57) which are important in supporting the gut of the piglets during the delicate phase of the weaning transition.

[0184] Moreover, analysis at genus level was performed to identify beneficial genera associated with low LT values and to confirm the findings of the ASVs level analysis (Figures 5 and 6). Genus level analysis confirmed genera such as, UCG-003, Veillonella, Blautia, HT002, Lactobacillus, Megasphaera, Intestinimonas, Colidextribacter were mostly linked with low-LT values.

[0185] Example 2 - Isolation of bacterial candidates associated with low LT loads post weaning from fecal isolates

[0186] Following the identification of candidate bacteria associated with low LT loads postweaning, we aimed to isolate these candidates from fecal samples of healthy pigs. A total of 260 bacteria isolates were obtained and subjected to DNA extraction, followed by Sanger sequencing of the entire 16S rRNA gene. Subsequently, the obtained sequences were compared with the 16S amplicon data of this study. The sequence analysis revealed that out of the 260 isolates, 64 unique bacteria were identified (Table 5), among which 17 were associated with low LT levels. Based on their sequence identity with the candidate bacteria described in the previous paragraph, their potential function from literature research, and their contribution role in the low-LT group, eleven bacteria were selected for subsequent analysis (Table 5, text in bold and underlined). Specifically, as no bacteria belonging to the Veillonella genus were isolated, a type strain (DSM19857) was purchased. Moreover, the isolate corresponding to AS VI 53 exhibited two distinct phenotypes. Consequently, the two phenotypes were designated as two separate bacteria, labeled AS VI 53.1 and AS VI 53.2.

[0187] Table 5 Taxonomic identification of bacteria isolated from feces of healthy pigs following sequence analysis. A total of 260 bacterial isolates was obtained, representing 64 unique ASVs (Amplicon Sequence Variants) derived from the 16S amplicon data of this study. Each unique bacterium is presented with its isolate frequency, taxonomic classification, and association with low or high LT levels. In bold are indicated the bacteria selected for further analysis.

[0188] Example 3 - Co-cultivation of probiotic candidates with ETEC in vitro Eleven bacterial strains were co-cultivated with ETEC individually and in different combinations to evaluate their effect on ETEC growth. Table 6 summarizes taxonomic information of the bacterial strains, while Table 7 presents each tested combination. Table 6- Taxonomic information on candidate bacterial strains

[0189] Table 7 - List of the combinations tested

[0190] The bacterial cultures / combinations were incubated together with ETEC, allowing for an evaluation of their potential synergistic effects on inhibiting ETEC growth (Figure 7).

[0191] Further results are shown in Table 8.

[0192] Table 8. Bacterial combinations statistically reducing ETEC CFU / ml compared to the negative control. Mean ETEC CFU / ml values, expressed in log2 units, along with standard deviations and decrease in log2 units compared to the negative control. Combinations represent different experimental setups to assess their inhibitory effect on ETEC proliferation. Sorted in descending order based on the magnitude of the decrease. P -values indicate the statistical significance of differences observed

[0193] Among the 66 combinations evaluated, 24 exhibited a statistically significant reduction in the number of ETEC colonies compared to the negative control (Table 8), ranging between 3.95 and 5.44 log2 units. On average, the collective impact of all eleven bacteria (combination 66) resulted in a substantial reduction of 4.94 log2 units in ETEC levels. A correlation analysis revealed a weak positive correlation (r=0.166) between the number of bacteria within each combination and the reduction in ETEC levels, implying subtle tendency for an increased number of bacteria to mildly coincide with a higher decrease in ETEC. However, the correlation is relatively weak, suggesting that the number of bacteria alone has limited predictive power for ETEC reduction and, other factors, such as specific bacterial interactions and the production of inhibitory substances, are likely more influential in shaping the observed variations in ETEC decrease across combinations. For instance, the combination that reduced the levels of ETEC the most (combination 30, reduction of 5.44 log2 units) included members of the Subdoligranulum and Prevotella genera, suggesting potential synergistic effects within specific bacterial compositions can influence more ETEC levels.

[0194] Delving deeper, specific bacterial strains emerge as potential influencers across combinations. Specifically, Limosilactobacillus reuterii (strains 'G' and 'H'), Lactobacillus johnsonii ('E'), and Megasphaera elsdenii ('F') were the most frequently occurring strains, present in 19-21 combinations out of the 24 combinations that significantly reduced ETEC numbers. This suggests a potential synergistic effect when these strains are present together. On the contrary, specific strains were less commonly observed in combinations that significantly reduced the target. Members of the Prevotella 9, Blautia obeum / wexlerae and Prevotella genera (indicated as ‘A’, ‘B’, ‘K’, respectively) were among the least frequent contributors. To further unravel the mechanisms underlying the observed inhibitory effects, a parallel experiment was conducted utilizing the supernatant of bacterial cultures, in order to understand the potential direct action of metabolites and / or antimicrobial compounds secreted by the candidate bacterial strains. Results are shown in Table 9.

[0195] Table 9 Supernatant combinations showing a reduction in ETEC CFU / ml compared to the negative control. Mean ETEC CFU / ml values, expressed in log2 units, along with standard deviations and decrease in log2 units compared to the negative control. Combinations represent different experimental setups aimed at assessing their inhibitory effect on ETEC proliferation. Sorted in descending order based on the magnitude of the decrease.

[0196] In contrast to the co-cultivation experiments involving live bacterial strains, the utilization of bacterial supernatants alone did not yield statistically significant reductions in ETEC levels (Figure 8). Despite this, notable reductions reaching up to 4.87 log2 units were observed (Table 9). Lactobacillus johnsonii ('E') emerged as the most influential when applied individually, demonstrating the highest reduction in ETEC levels, followed by combination 41 (comprising Ruminococcus bovis, Veillonella magna, Lactobacillus johnsonii, Megasphaera elsdenii and Limosilactobacillus reuterii strains, “CDEFG” respectively) which exhibited a reduction in ETEC levels by 4.47 log2 units, emphasizing the collaborative effect of these specific strains when their supernatants were combined. While the co-cultivation experiments highlighted live bacterial strains as potent inhibitors, the findings suggest that the efficacy of supernatant-mediated inhibition is nuanced, with individual strains and specific combinations playing pivotal roles in shaping the observed reductions in ETEC levels.

[0197] Overall, the screening of candidate bacteria highlighted the inhibitory potential of the combination 41, comprising Ruminococcus bovis, Veillonella magnet. Lactobacillus johnsonii, Megasphaera elsdenii and Limosilactobacillus reuterii strains. This combination, when used with live bacteria, demonstrated a statistically significant reduction in ETEC levels by 4.15 log2 units (p.value = 0.023). While the supernatant-only condition did not reach statistical significance, it exhibited one of the highest reductions, reaching 4.47 log2 units. This dual performance in inhibiting ETEC growth, both in the presence of live bacteria and through the potential bioactive compounds present in the supernatant, drove the selection of combination 41 for further investigations. Therefore, we moved from the screening phase to the optimization of fermentation conditions.

[0198] Example 4 - Optimisation of cultivation conditions

[0199] Following the screening phase, the optimization phase explored further in refining the cultivation conditions for the selected bacteria combination. This phase aimed to maximize the inhibitory potential of the bacterial consortium against ETEC through adjustments in the cultivation protocol. Two distinct approaches were explored: (MV1) individual cultivation of each of the 5 bacterial strains followed by combination moments before ETEC exposure, and (MV2) simultaneous co-cultivation of all selected bacteria for 48 hours before ETEC inoculation. The experiment was performed using both live-bacteria and supernatant-only setups. Results are shown in Tables 10 and 11.

[0200] Table 10 Optimization phase results with live-bacteria setup. Mean ETEC CFU / ml values, expressed in log2 units, along with standard deviations and decrease in log2 units compared to the negative control. MV1, MV2, positive control, and negative control represent different experimental conditions. P-values indicate the statistical significance of differences observed.

[0201] Table 11 Optimization phase results with supernatant-only setup. Mean ETEC CFU / ml values, expressed in log2 units, along with standard deviations and decrease in log2 units compared to the negative control. MV1, MV2, positive control, and negative control represent different experimental conditions.

[0202] In the live-bacteria setup, the highest reduction in ETEC CFU / ml compared to the negative control was observed with MV2, which reduced ETEC levels of 2.86 log2 units (p = 0.002), followed by MV1 (1.69 log2, p=0.016) and the positive control E. faecium (1.57 log2, p=0.021) (Table 10; Figure 9). In contrast, in the supernatant-only setup, no significant reduction in ETEC level was observed across any condition. The highest reduction was observed with MV2 with a decrease of 1.95 log2 units, followed by MV1 and the positive control (Table 11; Figure 10).

[0203] These results suggest that a prolonged co-incubation of bacteria, as observed with MV2, may enhance the inhibitory potential of bacterial consortia against ETEC proliferation. Despite the lack of significant reductions in the supernatant-only setup, the highest reduction observed with MV2 suggests the importance of bacterial interaction for ETEC inhibition. Therefore, a prolonged bacteria co-cultivation may facilitate the establishment of crossinteraction mechanisms, favoring collaborative microbial dynamics among bacterial strains. This cooperative interaction may potentially promote a more efficient inhibition of ETEC proliferation compared to individual bacterial cultivation.

[0204] Example 5 - Tolerance to low pH and bile salts

[0205] Resistance to low pH of the five strains was determined according to the method by Tokath et al. 2015.

[0206] Each strain was incubated anaerobically at 37°C for 24-48 hrs in a broth (time point 0). After incubation, the cells of each strain were harvested by centrifugation at 4000 rpm for 20 min. The pH was adjusted to the wanted value (4.0) to the initial volume. The supernatant was removed, and the pellets were suspended in sterile PBS (phosphate-saline buffer; 9 g / L NaCl, 9 g / L Na2HPO4-2H2O, 1.5 g / L KH2PO4). The mixture was incubated anaerobically at 37°C for 4 hrs (time point 4). At both time points, 0 and 4, tenfold serial dilutions were prepared in sterile saline solution (0.9% NaCl). Aliquots of samples were taken and bacterial counts were determined by spread plate method. All plates were incubated anaerobically at 37°C for 4-5 days.

[0207] The following growth media (broth and agar) were used:

[0208] Lactobacillus johnsonii: MRS broth (Oxoid) and MRS agar (Oxoid) Limosilactobacilus reuteri'. MRS broth (Oxoid) and MRS agar (Oxoid) Ruminococcus bovis'. Pras Chopped meat carbohydrate broth (Anaerobe Systems) and Fastidous anaerobe agar with 5% horseblood (FB agar) (Tammer-Biolab Oy) Veillonella magna: Fastidous anaerobe broth (F.A.B. broth) (Neogen) and FB agar

[0209] Megasphaera elsdenii: MRS broth and Fastidous anaerobe agar (FAA agar) (Neogen)

[0210] The percentage survival of the bacteria was calculated as follows: cf u of viable cells survived

[0211] ^survival -;- -r_ log ciu oi initial viable ceils inoculated x 100,

[0212] Results are shown in Table 12. Table 12 Acid tolerance of bacterial strains (pH 4.0)

[0213] 1Type strain obtained from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

[0214] 2Whole genome sequencing performed.

[0215] Resistance to bile salt of the five strains was determined according to the method by Tokath et al. 2015.

[0216] Each strain was incubated anaerobically at 37°C for 24-48 hrs in 9ml of a broth. After the incubation time, 0,1 ml were transferred to 10 ml broth (inoculum size of 1% (v / v) containing 0.3% bile salt (Oxgall) (time point 0). The mixture was incubated anaerobically at 37°C for 4 hrs (time point 4). At both time points, 0 and 4, tenfold serial dilutions were prepared in sterile saline solution (0.9% NaCl). Aliquots of samples were taken and bacterial counts were determined by spread plate method. All plates were incubated anaerobically at 37°C for 5 days.

[0217] The following growth media (broth and agar) were used:

[0218] Lactobacillus johnsonii: MRS broth (Oxoid) and MRS agar (Oxoid) Limosilactobacilus reuteri'. MRS broth (Oxoid) and MRS agar (Oxoid) Ruminococcus bovis'. Pras Chopped meat carbohydrate broth (Anaerobe Systems) and Fastidous anaerobe agar with 5% horseblood (FB agar) (Tammer-Biolab Oy)

[0219] Veillonella magna'. Brain Heart Infusion broth (BHI broth) and Fastidous anaerobe agar with 5% horseblood (FB agar) (Tammer-Biolab Oy)

[0220] Megasphaera elsdenii: MRS broth and Fastidous anaerobe agar (FAA agar) (Neogen)

[0221] The percentage survival of the bacteria was calculated as follows: log cfti of viable cells survived d

[0222] Results are shown in Table 13.

[0223] Table 13 Bile salt tolerance of bacterial strains (0.3%)

[0224] 1Type strain obtained from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

[0225] 2Whole genome sequencing performed.

[0226] Several bacteria showed considerable pH resistance and very high tolerance to bile salt under the relevant conditions.

[0227] Conclusions

[0228] Post-weaning diarrhoea poses significant challenges to the piglet industry, resulting in growth retardation, diarrhea, and mortality, leading to significant economic losses. While enterotoxigenic Escherichia coli (ETEC) pathogen is primarily implicated, other factors as host genetics, immune response, environmental stressors, and gut microbiota contribute to disease onset. With concerns over antibiotic resistance and the zinc oxide ban, alternative measures as microbiome-based solutions are needed and promising (Rhouma et al., 2017, Gresse et al., 2017). Our study comprehensively analysed the piglet gut microbiome and its association with ETEC shedding patterns to elucidate piglet susceptibility mechanisms and identify potential preventive strategies. Specifically, we identified microbial members correlated with low LT-carrying ETEC levels and developed a bacterial mixture for further investigation.

[0229] We included eight farms in our study, with different backgrounds in terms of genetics, management practices, and history of infections. This allowed us to account for the variability introduced by different farm environments and practices, ensuring a broad and universal perspective on the piglet gut microbiome.

[0230] ETEC strains are characterized by fimbriae, and enterotoxins which vary in virulence (Nagy & Fekete, 2005; Luppi, 2017; H. Wang H et al., 2019). We focused on the LT toxin due to its strong association with severe diarrhoea and to its possible involvement in facilitating bacterial adhesion, colonization and growth (Berberov et al., 2004; Duan et al., 2022, 2023; Fekete et al., 2013; Johnson et al., 2009). Our empirical observations further supported this, as we consistently observed a correlation between farms where ETEC isolates harbouring the LT toxin were present and instances of severe diarrhoea. Therefore, we proceeded with a comparative analysis of gut microbiota between piglets displaying high levels of LT toxin and those with low or undetectable levels of the toxin post-weaning. Notably, we observed distinct microbiome compositions in these two groups as early as one week after birth. This observation suggested that piglets with low LT levels already harbour a distinct microbiome, potentially consisting of beneficial bacteria capable of colonizing and impeding the expansion of ETEC strains carrying the LT toxin later in life. These results are consistent with previous findings, supporting the importance of the early gut microbiome colonization in influencing the development of diseases later in life and underlining its potential application in clinical practices (Dou et al., 2017).

[0231] Microbial members associated with low levels of LT colonization included bacteria belonging to taxa widely known for their beneficial contribute to host health, among which 15 ASVs were selected as ideal candidate for the development of the bacterial mixture. These bacteria included members of the Lactobacillaceae family as Lactobacillus and HT002 genera, which are more important in the early life stages of piglet life and mostly present in the small intestine; SCFA producers as members of Oscillospiraceae. Muribaculaceae and Prevotellaceae families and Veillonella, Megasphaera, Intestinimonas, Roseburia, Subdoligraniihim. Prevotella and Ruminococcus genera. By delving into the literature, we were able to formulate hypotheses regarding the functions, metabolism, and co-occurrence mechanisms of these bacteria, which may contribute to creating an environment capable of preventing the expansion of LT-carrying ETEC.

[0232] For instance, during the suckling phase, piglets digest lactose from sow milk via endogenous lactase and P-galactosidase from lactic acid bacteria (LAB) such as members of Lactobacillaceae family, reducing stomach pH and inhibiting pathogens (Zhao et al., 2021). Lactase activity declines at weaning, leading to incomplete lactose digestion and potential post- weaning diarrhoea due to excess of lactate. Balancing microbial populations to regulate the presence of lactate producers by adding lactate-consuming bacteria like Megasphaera and Veillonella can prevent excessive lactate production in the gut upon weaning and consequently alleviate post-weaning diarrhoea symptoms. These bacteria metabolize lactate to SCFAs as acetate, propionate and butyrate across various species, including humans (Hashizume et al., 2003; Zhang & Huang, 2023). Specifically, SCFAs are the main products of the anaerobic fermentation of indigestible polysaccharides like dietary fiber and resistant starch, by bacteria such as Ruminococcus, Subdoligranulum, Blautia and Prevotella. SCFAs produced by gut microbiota positively influence various physiological processes in the host. Consequentially, a stable colonization of SCFA- producing bacteria might mitigate enteric diseases symptoms, by promoting integrity of the intestinal barrier, enhancing mucosal defence mechanisms, maintaining gut homeostasis, and supporting the immature piglet gastrointestinal tract in the digestion of resistant starch. Therefore, these bacteria were involved in our selection for their functional role in SCFA metabolism and gut health.

[0233] Additionally, the selected bacteria might have roles in the defence mechanisms against pathogens, besides their role in host metabolism and digestion by production of SCFAs. Veillonella is also included in the gamma-aminobutyric acid (GABA) catalysation pathway. GABA is a neurotransmitter involved in fluid transport through luminal secretion of C1-, found at high levels in the jejunum of diarrheic pigs (Wu H et al., 2018), and it promotes lysosomal maturation and antibacterial responses, suggesting a role in resist ETEC infection (Xia et al., 2019; Zhang & Huang, 2023). However, the clear role of GABA and its metabolism by the gut microbiota in mitigating ETEC infection has yet to be fully understood and require further studies.

[0234] Many members of the Lactobacillaceae family are well-known bacteria, mainly for their probiotic roles and a variety of beneficial functions. In fact, beyond being involved in the lactose metabolism, these species can present different characteristics depending on the strain. They can be involved in reducing ETEC colonization and prevent diarrhoea through the production of antimicrobial compounds, or the modulation of inflammatory response, mucosal immunity and gut microbiota (Dell’anno et al., 2021; Gao et al., 2022; He et al., 2019; Kanmani et al., 2018; Li Y et al., 2019; Peng et al., 2022; Suda et al., 2022; Wang W et al., 2020; Wu J et al., 2022; Xie et al., 2021; Xin et al., 2020; Yang GY et al., 2020; Yang Y et al., 2015; Zheng et al., 2021). Moreover, many commensal bacteria including Lactobacilli can promote host immunity via indole production. Indole is a metabolite derived from microbial degradation of tryptophan, involved in various host physiological processes such as, enhance intestinal barrier function by up-regulating the expression of tight-junction proteins, promote intestinal homeostasis and protect the mucosa (Bansal et al., 2010; Li J et al., 2021, Alexeev et al., 2018; Shimada et al., 2013; Zelante et al., 2013).

[0235] Having explored and hypothesized the metabolic and cross-feeding mechanisms and functions of diverse bacteria, we then focused on refining our selection of optimal candidate bacteria. This list of ideal candidate bacteria served as a starting point for the isolation of pure cultures from field samples. Among all isolated cultures, 11 isolates were selected based on their similarity with the previously selected 15 bacteria, their contribution in our study in the low-LT group and their known role as beneficial bacteria extensively reported in the literature.

[0236] This list was further narrowed by testing these strains for their role in inhibiting the growth of ETEC in a co-culture experiment where different bacterial combinations were tested, both live and supernatant-only. Among the 66 combinations evaluated, 24 exhibited statistically significant reductions in ETEC levels, underscoring the potential of certain bacterial compositions to inhibit ETEC growth. The utilization of bacterial supernatants alone did not yield statistically significant reductions in ETEC levels, highlighting the nuanced efficacy of supernatant-mediated inhibition compared to live bacterial strains. Interestingly, a weak positive correlation was observed between the number of bacteria within each combination and the reduction in ETEC levels, suggesting that a higher number of bacteria may contribute to a greater decrease in ETEC, albeit to a limited extent. The higher reduction of ETEC colonies observed in combinations with a co-culture of various bacteria could potentially result from increased nutrient competition due to the presence of multiple strains, rather than direct defence mechanisms against ETEC. However, if this was the case, we would expect to observe the highest ETEC inhibition in combinations with a higher number of bacteria. Interestingly, the highest reduction was actually observed in combination 30, which included only three bacteria, suggesting that factors beyond bacterial abundance may be at play. On the other hand, our findings highlight the limited efficacy of individual strains in inhibiting ETEC growth. For instance, only L. johnsonii demonstrated a notable reduction in ETEC colonies in the cell-free supernatant (CFS) setup, but this effect was not statistically significant. This underscores the importance of synergistic interactions among bacterial strains in exerting effective inhibition against pathogens and confirm previous research supporting the superiority of multi-strain probiotic over individual strains (Hansen et al., 2021; Pupa et al., 2022).

[0237] Further investigation into the specific mechanisms underlying these synergistic effects is warranted to optimize the formulation of bacterial consortia for enhanced efficacy against ETEC infection.

[0238] The selection of the combination for further steps was not solely based on the reduction of ETEC levels, instead, we took a comprehensive approach. We carefully examined all the significant combinations and observed how specific bacterial strains, such as Limosilactobacillus reuterii, Lactobacillus johnsonii and Megasphaera elsdenii. emerged as potential influencers across many combinations. This indicates their potential cooperative effects in inhibiting ETEC proliferation.

[0239] Moreover, our goal in developing a microbiome-based product was not primarily focused on directly targeting the pathogen itself. Post-weaning diarrhea is a complex disease with multiple contributing factors, where the presence of the pathogen is just one aspect among many others. We aimed to address the multifaceted nature of the condition by considering various mechanisms of action for our product. Probiotics can exert their effects through three distinct mechanisms: (i) directly inhibiting the growth and virulence of pathogens by secreting antimicrobial substances, suppressing pathogen virulence genes, or competing for adherence sites; (ii) influencing the composition and activity of resident microbiota, like by producing SCFA; and (iii) enhancing the host immune system and promoting anti-inflammatory response and intestinal barrier function (Gresse et al., 2017).

[0240] By targeting these different pathways, we aim to develop a product that comprehensively addresses the complex nature of post-weaning diarrhea and promotes gut health in a multifaceted manner. Our primary focus was on selecting a combination that could effectively modulate the resident gut microbiota to establish early colonization of beneficial bacteria and foster a symbiotic ecosystem where they collaborate to maintain gut balance and health. We prioritized bacteria capable of cooperative interactions, such as lactic acid bacteria (LAB) and lactate consumer bacteria like Megasphaera and Veillonella, which work synergistically in lactate metabolism. Additionally, we included SCF As- producing bacteria such as Ruminococcus, Blautia, Intestinimonas, Prevotella and Subdoligranulum to further enhance gut health and provide support to piglet digestion of solid feed. However, we extended our focus beyond these functions to include bacteria with defensive capabilities against pathogens, such as Lactobacilli, and bacteria that can stimulate host immunity, as many species do through the production of metabolites like indole and its derivatives. Notably, the combination 41, comprising Ruminococcus bovis, Veillonella magna, Lactobacillus johnsonii, Megasphaera elsdenii and Limosilactobacillus reuterii strains, showed promising inhibitory potential both with live bacteria and supernatant. This combination aligns perfectly with our product structure, as it encompasses various functionalities essential for promoting gut health, as supported by findings from literature studies.

[0241] Based on this, we hypothesize the mechanism of action of our product as follows: L. johnsonii and L. reuterii contribute to the defence mechanism against pathogens, either through the production of antimicrobial substances or by stimulating the immune system through metabolite production. M. elsdenii and V. magna aid in gut resident modulation, particularly in conjunction with Lactobacilli, by producing SCF As that assist the host through various mechanisms and help reduce the pH of the environment, creating less favourable conditions for pathogen growth. Additionally, Ruminococcus plays a crucial role in supporting the digestion of solid feed during the weaning process. By facilitating the breakdown of solid feed components, Ruminococcus helps piglets adapt to dietary changes more effectively. We speculate that early colonization of this bacterial combination in the piglet gut could foster a healthy microbiome capable of supporting the animal later in life, particularly during the challenging weaning transition.

[0242] Subsequent optimization of fermentation conditions aimed to maximize the inhibitory potential of the bacterial consortium against ETEC. In this phase, the results revealed that a prolonged co-incubation of bacteria, rather than individual cultivation, may enhance the inhibitory potential of bacterial consortia against ETEC proliferation. Although the supernatant-only setup did not show significant reductions, the highest reduction observed with simultaneous co-cultivation suggests the crucial role of bacterial interaction for ETEC inhibition. These findings lead us to hypothesize that a prolonged coincubation of the five bacterial strains may maximize the effectiveness of the bacterial consortium against ETEC.

[0243] Overall, these findings underscore the complex dynamics involved in microbial interactions and their impact on ETEC growth inhibition. Further studies are warranted to elucidate the specific mechanisms underlying the observed inhibitory effects and to optimize the cultivation conditions for enhanced efficacy against ETEC infection.

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[0291] Xie W et al. (2021) A bovine lactoferricin-lactoferrampin-encoding Lactobacillus reuteri CO21 regulates the intestinal mucosal immunity and enhances the protection of piglets against enterotoxigenic Escherichia coli K88 challenge. Gut Microbes 13. Xin J et al. (2020) Probiotic Lactobacillus johnsonii BS15 Promotes Growth Performance, Intestinal Immunity, and Gut Microbiota in Piglets. Probiotics Antimicrob Proteins 12:184-193.

[0292] Yang GY et al. (2020) Anti-inflammatory effects of Lactobacillus johnsonii L531 in a pig model of Salmonella Infantis infection involves modulation of CCR6+ T cell responses and ER stress. Vet Res 51.

[0293] Yang Y et al. (2015) Feed Fermentation with Reuteran- and Levan -Producing Lactobacillus reuteri Reduces Colonization of Weanling Pigs by Enterotoxigenic Escherichia coli. Appl Environ Microbiol 81:5743-5752.

[0294] Zelante T et al. (2013) Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity 39:372-385.

[0295] Zhang S-M et al. (2023) The Commensal Anaerobe Veillonella dispar Reprograms Its Lactate Metabolism and Short-Chain Fatty Acid Production during the Stationary Phase. Microbiol Spectr 11.

[0296] Zhao J et al. (2021) The role of lactose in weanling pig nutrition: a literature and metaanalysis review. J Anim Sci Biotechnol 12:1-17.

[0297] Zheng D et al. (2021) Lactobacillus johnsonii activates porcine monocyte derived dendritic cells maturation to modulate Th cellular immune response. Cytokine 144.

Claims

CLAIMS1. A composition comprising two or more of:(a) a bacterium of species Lactobacillus johnsonii (L. johnsonii(b) a bacterium of species Limosilactobacillus reuterii (L. reuterii) and(c) a bacterium of species Megasphaera elsdenii (M elsdenii).

2. The composition of claim 1, wherein the composition comprises:(a) a bacterium of species L. johnsonii,'(b) a bacterium of species L. reuterii,' and(c) a bacterium of species M. elsdenii.

3. The composition of claim 1 or 2, wherein:(a) the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200;(b) the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608; and / or(c) the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70% identity to the genome of AL elsdenii DSM 20460.

4. The composition of any one of the preceding claims, wherein the composition comprises:(a) a bacterium of species L. johnsonii that comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200;(b) a bacterium of species L. reuteri that comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608; and(c) a bacterium of species M. elsdenii that comprises a nucleic acid sequence having at least 70% identity to the genome of AL elsdenii DSM 20460.

5. The composition of any one of the preceding claims, wherein the composition comprises one or more additional bacteria.

6. The composition of claim 5, wherein the one or more additional bacteria comprise:(d) a bacterium of species Veillonella magna (V. magnet) and / or(e) a bacterium of species Rumonicoccus bovis (R. bovis).

7. The composition of claim 5 or 6, wherein the one or more additional bacteria comprise:(d) a bacterium of species V. magnet and(e) a bacterium of species R. bovis.

8. The composition of claim 6 or 7, wherein:(d) the bacterium of species V. magna comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857; and / or(e) the bacterium of species R bovis comprises a nucleic acid sequence having at least 70% identity to the genome of R bovis JE7A12(T).

9. The composition of any one of claims 5 to 8, wherein the one or more additional bacteria comprise (i) one or more Prevotella strain, (ii) one or more Blautia strain, and / or (iii) one or more Subdoligranulum strain.

10. The composition of any one of the preceding claims, wherein the composition comprises:(a) a bacterium of species L. johnsonii that comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200;(b) a bacterium of species L. reuterii that comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608;(c) a bacterium of species M. elseienii that comprises a nucleic acid sequence having at least 70% identity to the genome o M. elseienii DSM 20460;(d) a bacterium of species V. magna that comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857; and(e) a bacterium of species R. bovis that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T).

11. The composition of any one of the preceding claims, wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 1.

12. The composition of any one of the preceding claims, wherein the bacterium of speciesL.johnsonii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO:2.

13. The composition of any one of the preceding claims, wherein the bacterium of speciesM. elsdenii comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO:3.

14. The composition of any one of claims 6-13, wherein the composition comprises a bacterium of species V. magnet, and wherein the bacterium of species V. magna comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 4.

15. The composition of any one of claims 6-13, wherein the composition comprises a bacterium of species R. bovis, and wherein the bacterium of species R bovis comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 5.

16. The composition of any one of the preceding claims, wherein the composition inhibits the growth of one or more enterotoxigenic E. coli bacteria (ETEC).

17. The composition of any one of the preceding claims, wherein the composition promotes clearance of one or more ETEC by the immune system.

18. The composition of any one of the preceding claims, wherein the composition promotes the function and / or integrity of the intestinal barrier.

19. The composition of any one of the preceding claims, wherein the composition produces short chain fatty acids.

20. The composition of any one of the preceding claims, wherein one or more of the bacteria comprised in the composition consumes lactate.

21. A pharmaceutical composition comprising:(i) the composition of any one of claims 1 to 20; or(ii) (a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2;(b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1;(c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of AT. elsdenii DSM 20460, optionally wherein the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 3;(d) a bacterium of species V. magnet, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857, optionally wherein the bacterium of species V. magna comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 4; or(e) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T), optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 5; and a pharmaceutically acceptable carrier or excipient.

22. An animal feed or feed supplement comprising:(i) the composition of any one of claims 1 to 14; or(ii) (a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2;(b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1;(c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome oiM. elsdenii DSM 20460, optionally wherein the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 3;(d) a bacterium of species V. magnet, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of V. magna DSM 19857, optionally wherein the bacterium of species V. magna comprises a nucleicacid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 4; or(e) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R. bovis JE7A12(T) , optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 5.

23. A bacterium, which is:(a) a bacterium of species L. johnsonii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. johnsonii ATCC 33200, optionally wherein the bacterium of species L. johnsonii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2;(b) a bacterium of species L. reuterii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome of L. reuterii ATCC 53608, optionally wherein the bacterium of species L. reuterii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1;(c) a bacterium of species M. elsdenii, optionally wherein the bacterium comprises a nucleic acid sequence having at least 70% identity to the genome oiM. elsdenii DSM 20460, optionally wherein the bacterium of species M. elsdenii comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 3; or(d) a bacterium of species R. bovis, optionally wherein the bacterium that comprises a nucleic acid sequence having at least 70% identity to the genome of R bovis JE7A12(T) , optionally wherein the bacterium of species R. bovis comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 5.

24. A method of preventing or treating post-weaning diarrhoea in an animal, comprising administering to the animal the composition of any one of claims 1 to 20, the pharmaceutical composition of claim 21, the feed or feed supplement of claim 22, or the bacterium of claim 23.

25. The composition of any one of claims 1 to 20, the pharmaceutical composition of claim 21, the feed or feed supplement of claim 22, or the bacterium of claim 23 for use in a method of preventing or treating post-weaning diarrhoea in an animal, wherein the method comprises administering the composition, pharmaceutical composition, feed or feed supplement, or bacterium to the animal.

26. The method of claim 24, or the composition, pharmaceutical composition, feed or feed supplement or bacterium for use of claim 25, wherein the animal is a pig, optionally a piglet.

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