Biomarkers of farm animal microbiota status and uses thereof

Abstract

The present invention relates to the use of one or more ratios of the amount of bacteria in the microbiota of a farm animal as a marker to determine the status of the intestinal microbiota of a farm animal and whether this status is satisfactory, or to the use of an extract for selecting a satisfactory intestinal microbiota of a farm animal, intended to preserve or improve the gut microbiota of different farm animals in need thereof, said ratio being selected from the group consisting of Phylum Thallungiae / Phylum Proteinae; clostridium IV and XlVa cluster / Enterobacteriaceae; a ruminaceae family and a hispiraceae / enterobacteriaceae family; butyrate-producing bacteria / phylum of Thalictrum; aerobic bacteria / anaerobic bacteria.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of livestock animals, and more particularly, to an alternative method to improve the health of livestock animals. Reducing health problems in farm animals is a core concern for any livestock farmer, as it is clearly linked to high performance in terms of production and meat quality, and from an ethical point of view, to better animal welfare. BACKGROUND

[0002] Growth performance of animals is usually characterized by measuring zootechnical parameters such as body weight (BW), body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR). Regarding the assessment of animal health, parameters are measured on the animals or their environment. Commonly used parameters are based on the detection of plasma proteins in lumen fluids, short chain fatty acid (SCFA) production in the gut microbiota, lipopolysaccharide (LPS) in serum, bacteria crossing the intestinal epithelium (bacterial translocation), etc. (Ducatelle, R.; Goossens, E.; De Meyer, F.; Eeckhaut, V.; Antonissen, G.; Haesebrouck, F.; van Immerseel, F. (2018) Biomarkers for monitoring intestinal health in poultry. Present status and future perspectives. In : Veterinary research, vol. 49, n° 1, p. 43). Regarding external parameters, litter and footpad dermatitis scores are usually measured (Eichner, G.; Vieira, S. L.; Torres, C. A.; Coneglian, J. L. B.; Freitas, D. M.; Oyarzabal, O. A. (2007) Litter Moisture and Footpad Dermatitis as Affected by Diets Formulated on an All-Vegetable Basis or Having the Inclusion of Poultry By-Product. In : The Journal of Applied Poultry Research, vol. 16, n° 3, p. 344-350).

[0003] Gastrointestinal (GIT) health, and more particularly intestinal health, is a key factor of the overall health and well-being of animals, and thus of production.

[0004] In the case of livestock, animals face significant challenges, in particular due to the increase in farm size, the increasing acceleration of growth, sensitive physiological stages, for which their performance can significantly decrease. Resilience is the ability of an animal to adapt to this challenging environment by greater resistance. More resilient animals will have more regular production and better welfare.

[0005] MC Costa et al. Different antibiotic growth promoters (AGPs) induce specific changes in the cecal microbiota membership of broiler chicken, PiosOne, vol. 12, no. 2 (2017) studied the impact of five antibiotic growth promoters (AGP) on broiler chickens. The evaluation was carried out in the cecal microbiota and the changes in specific bacterial members present in the chicken cecum were observed. To do this, the ratio of Firmicutes / Proteobacteria was calculated and the treated groups showed lower values compared to the control group. However, these results demonstrate that these AGPs have a strong impact on rare bacterial species present in the cecal microbiota, but not on commensal bacteria, and in particular on Firmicutes such as Clostridium, Lactobacillus and Ruminococcus. SUMMARY

[0006] According to the present application, it was found that the determined microbial ratios of the intestinal microbiota can be used on the one hand as biomarkers of the state of this microbiota and on the other hand for the selection of microbial inocula, such as for maintaining or improving the GIT health of farm animals or for breeding the resilience of young animals that will grow under agricultural conditions.

[0007] The present application thus consists in the use of one or more ratios of the amount of bacteria in the microbiota of farm animals, the ratios being chosen from the following: Firmicutes / Proteobacteria; Clostridium IV and XlVa clusters / Enterobacteriaceae; Ruminococcaceae and Lachnospiraceae / Enterobacteriaceae; butyrate-producing bacteria / Firmicutes; aerobes / anaerobes.

[0008] In one aspect, the present application relates to the use of said ratios as markers to determine the status of the gut microbiota of a farm animal and to determine whether said status is satisfactory. In one embodiment, the present application relates to the use of at least two, at least three, at least four or all of said ratios.

[0009] In another aspect, the present application relates to the use of said ratios for the selection of an extract of a satisfactory gut microbiota of a farm animal, said extract being intended to maintain or improve the gut microbiota of a farm animal in need thereof (different from the farm animal from which the extract comes).

[0010] In one embodiment, the object of the present application is a biomarker of the status of the microbiota of a farm animal, said biomarker being selected from the ratios of the amounts of the following bacteria,

[0011] Firmicutes / Bacteroidetes,

[0012] Clostridia IV and XlVa clusters / Enterobacteriaceae,

[0013] Ruminococcaceae and Lachnospiraceae / Enterobacteriaceae,

[0014] Butyrate-producing bacteria / Firmicutes and

[0015] Aerobes / anaerobes.

[0016] The calculation of the ratios is done by dividing the populations of the different microbiota, which are detected and analyzed by any appropriate method such as 16S sequencing. The numerator and the denominator of the formula have the same unit since they are both relative abundance data, so the result of the ratio has no unit.

[0017] This new biomarker directly reflects the intestinal health of a farm animal and can find many applications in the breeding industry in view of its impact on production.

[0018] Thus, according to the present application, there is provided a method for determining in vitro or ex vivo the status of the gut microbiota of a farm animal, said method comprising at least the following steps:

[0019] detecting and quantifying in a biological sample of said farm animal bacteria selected from the group consisting of Firmicutes, Bacteroidetes, Clostridia IV and XlVa clusters, Enterobacteria, Ruminococcaceae, Lachnospiraceae, butyrate-producing bacteria, aerobes and anaerobes; and

[0020] calculating at least one ratio selected from the group consisting of:

[0021] Firmicutes / Bacteroidetes,

[0022] Clostridium genus IV and XlVa clusters / Enterobacteriaceae

[0023] Rumenaceae and Trichophyceae / Enterobacteriaceae

[0024] Butyrate-producing bacteria / Firmwallis and

[0025] Aerobic / anaerobic bacteria.

[0026] In another embodiment of the invention, a method is provided for selecting a satisfactory extract of the gut microbiota of farm animals, the method comprising the following steps:

[0027] The amount of each type of bacteria selected from Firmicutes, butyrate-producing bacteria, aerobic bacteria and anaerobic bacteria was detected and quantified in biological samples of the farm animals, and at least one ratio selected from the following was calculated: butyrate-producing bacteria / Firmite (a1), aerobic bacteria / anaerobic bacteria (a1).

[0028] The amount of each type of bacteria selected from Firmicutes, butyrate-producing bacteria, aerobic bacteria and anaerobic bacteria was detected and quantified in biological samples of different farm animals, and the ratio of at least one of the following was calculated: butyrate-producing bacteria / Firmite (a2), aerobic bacteria / anaerobic bacteria (b2).

[0029] Compare the calculated ratios (a1) and (b1) and / or (a2) and (b2); and

[0030] Select the satisfactory extract from the comparison.

[0031] According to another embodiment of the present invention, a non-therapeutic method is provided for maintaining or improving the gut microbiota of farm animals in need, the method comprising the following steps:

[0032] Select a satisfactory microbiome extract according to the method described above, and

[0033] The animal is treated with the satisfactory microbial extract or its derivatives produced by fermentation, the animal being different from the animal from which the satisfactory extract or its derivatives are selected.

[0034] A healthier gut environment will ensure gut health, which in turn benefits an animal's resilience to challenges.

[0035] Prior to the disclosure of this invention, some terms used herein are defined.

[0036] A microbiome extract comprises a collection of microorganisms, components, or products derived from a microbiome. The extract or any sample of the extract is obtained by any means capable of recovering microorganisms, components, or products from the microbiome, suitable for a specific microbiome source, such as the gut. The sample collection methods employed may include those recognized and established in the art. These methods are based on the contents of different gastrointestinal (GIT) segments and feces, their fermentation, combinations, or mixtures of different animal contents. The mixtures need to originate from the same GIT segment.

[0037] Microbial transfer (MT) is the process of administering a microbial extract (also known as a microbial inoculum) from a selected animal to an animal in need. Intestinal microbial transfer (IMT) is equivalent to fecal microbial transfer (FMT), but extends to other types of inoculum besides fecal matter, such as cecal and ileal material and their fermentation products. According to the invention, this microbial transfer is carried out via animal feed or drinking water.

[0038] A microbial inoculum is a microbial community from a selected animal that is considered resistant to adverse conditions compared to the animal to which the inoculum was administered, either because the animal suffers from dysbiosis or because it is a young animal with a poor and / or immature microbiome. This microbial community can be isolated from and / or derived from samples such as feces, intestinal contents (cecum or ileum), for example, through their fermentation.

[0039] In this document, farm animals with a satisfactory gut microbiota are understood to be farm animals with a mature gut microbiota; for poultry, this typically begins to occur from 3 weeks of age. This characteristic is inherently age-dependent and is a concept well known to those skilled in the art; as explained in the background of this invention, farm animals with a satisfactory gut microbiota are farm animals for which a satisfactory gut microbiota extract is not required. The expressions “satisfactory gut microbiota extract” and “satisfactory extract of gut microbiota” have the same meaning.

[0040] Farm animals that need to maintain or improve their gut microbiota are those whose resilience and resistance are enhanced when challenged to improve their abilities and reduce performance loss; in the gut, there are interactions between the gut microbes and the host, which can be mutually beneficial or harmful, thereby regulating health or disease states; these interactions are stronger in young animals because the gut is not yet mature and defense mechanisms are still forming; therefore, such animals are likely to be young animals with immature microbiota; in poultry, they are chickens 10 days old or younger.

[0041] By means of controls or control values, it includes values ​​that can be measured on unchallenged animals, and in this case, can be specific values ​​or can belong to a range of values; it also includes values ​​measured on challenged animals, but whose ratios are more satisfactory than those from other challenged animals (also known as resilient animals).

[0042] The invention will be disclosed and detailed below with reference to the gut health and microbiome of poultry, but from the perspective of production issues, the invention can be extended to any other animal, especially any farm animal; such farm animals are primarily monogastric animals, such as pigs.

[0043] According to one embodiment of the present invention, a method for determining the gut microbiota status of farm animals in vitro or in vitro further includes the following steps:

[0044] The ratio or each of the biological samples from the animal is compared with a control value determined for the ratio or each of the ratios.

[0045] The state of the microbial community is determined from the comparison.

[0046] Preferably, at least one ratio selected from Firmicutes / Proteobacteria, Clostridium IV and XlVa clusters / Enterobacteriaceae, Ruminaceae and Trichophyceae / Enterobacteriaceae, and Butyrate-producing bacteria / Firmicutes is calculated, and

[0047] If the ratio or each calculated ratio is greater than or equal to the control value determined for the corresponding ratio, the state of the microbiome is assessed as satisfactory; if the ratio or each of the ratios is lower than the control value, the state of the microbiome is assessed as unsatisfactory.

[0048] In addition, or in another embodiment, the redox ratios associated with aerobic and anaerobic bacteria are calculated together with the aforementioned ratios. If the redox ratio is less than or equal to a control value determined for the corresponding ratio, the state of the microbiome is assessed as satisfactory, while if the ratio or each of the ratios is greater than the control value, the state of the microbiome is assessed as unsatisfactory.

[0049] The above method may include calculating at least two, at least three, at least four, or all five of the ratios, and optionally comparing them with a control value.

[0050] Biological samples are advantageously selected from cecal contents, ileal contents, fecal contents, and any fermentation derivatives thereof.

[0051] Bacterial abundance is preferably measured by relative abundance obtained from sequencing analysis of 16S rRNA sequencing data, but in practice it can be measured by any other suitable analysis known to those skilled in the art, such as by any other metagenomic sequencing such as whole-genome shotgun (WGS ST Park and J. Kim. Trends in Next-Generation Sequencing and a New Era for Whole Genome Sequencing, Int. Neurourol J. 2016 Nov; 20(Suppl 2): ​​S76-83), or by quantitative PCR (qPCR).

[0052] In a specific implementation of this method, the state of the chicken's microbiota is determined and a control value of 60 is set for any of the following ratios: Firmicutes / Proteobacteria; Clostridium IV and XlVa clusters / Enterobacteriaceae; Rumenaceae and Trichophyceae / Enterobacteriaceae; Butyrate-producing bacteria / Firmicutes; Aerobic / Anaerobes; and / or the control value for the aerobic / anaerobic ratio is measured in chickens under farm conditions that are unchallenged or less challenged compared to the challenges faced by chickens whose gut microbiota state is determined.

[0053] As described above, in a further application of the biomarkers of the present invention, a method is provided for selecting extracts of a satisfactory gut microbiota from farm animals.

[0054] In a preferred embodiment, the method can be performed as follows:

[0055] Among all the measured values ​​of the test samples, the extract is considered satisfactory if the ratio of butyrate-producing bacteria to Firmicutes is the highest, and / or if the ratio of aerobic bacteria to anaerobic bacteria is the lowest, and therefore the extract is selected.

[0056] According to one embodiment of the invention, as described above, the non-therapeutic method for maintaining or improving the gut microbiota of farm animals in need is intended for young farm animals, such as chickens, whose gut microbiota is not yet mature.

[0057] For example, if the farm animal is a chick, then apply 1-9.10. 7 The extract contains an amount of CFU (colony forming units).

[0058] The extract can be used immediately or after being stored under stable conditions. In a preferred embodiment, the extract is fermented. Any fermentation can be performed on the extract, provided it helps to promote the desired ratio.

[0059] Another subject of the invention is the use of one or more ratios of bacterial quantities in the microbiota of farm animals as markers for determining the state of the gut microbiota of farm animals, said ratios being selected from: Firmicutes / Proteobacteria; Clostridium IV and XlVa clusters / Enterobacteriaceae; Rumenaceae and Trichophyceae / Enterobacteriaceae; Butyrate-producing bacteria / Firmicutes; Aerobic / Anaerobic bacteria.

[0060] Another subject of the invention is the use of extracts of satisfactory gut microbiota from farm animals as feed additives for maintaining or improving the gut microbiota of various farm animals in need of this. Advantageously, the satisfactory extract is selected using the method described above for selecting extracts of satisfactory gut microbiota from farm animals. In a further preferred embodiment, the selected extract is then fermented.

[0061] The invention is illustrated in the examples below, thereby revealing its objectives and benefits.

[0062] The solution described below is the final example of these examples.

[0063] Chickens were divided into several groups, one group fed a standard diet (STD), while the other groups were treated with the treatments defined below. Cecums of the chickens were extracted, and the microbiome was analyzed by 16S sequencing, with ratios calculated based on a taxonomic table generated from amplicon sequence variants (ASVs).

[0064] Pigs were divided into several groups, one group fed a standard diet (STD), while the other groups were treated with the treatments defined below. Colons were extracted from piglets, and the microbiome was analyzed by 16S sequencing. Ratios were calculated based on a taxonomic table generated from amplicon sequence variants (ASVs).

[0065] The following studies show a sample of the different challenges animals can face:

[0066] Nutritional issues, such as the presence of rye and fat (mild dysbiosis, MLD); or the addition of galactomannan (GAL);

[0067] Challenges can arise from pathogen (viral or bacterial) or parasitic infections, such as severe dysbiosis feeding regimens (the same diet as MLD but supplemented with bacteria, SV), necrotizing enteritis (NE), or Salmonella infection.

[0068] In all these studies, we can see how the expected trends occur when animals are challenged. Attached Figure Description

[0069] The following embodiments are described with reference to the following figures. Figures 1-9 Involves research on chickens, andFigures 10-1 4. Research involving piglets:

[0070] Figure 1 Box plots of the ratio 2.1 calculated in four studies (MLD, SV, GAL, and NE) and compared with STD.

[0071] Figure 2 Box plots of the ratio 2.3 calculated in four studies (MLD, SV, GAL, and NE) and compared with STD.

[0072] Figure 3 Box plots of the ratio 2.4 calculated in four studies (MLD, SV, GAL, and NE) and compared with STD.

[0073] Figure 4 Box plots of the ratio 2.15 calculated in four studies (MLD, SV, GAL, and NE) and compared with STD.

[0074] Figure 5 Box plot of the ratio 2.17 calculated in 4 studies (MLD, SV, GAL and NE) and compared with STD.

[0075] Figure 6 Box plots of redox ratios calculated in four studies (MLD, SV, GAL, and NE) and compared with STD.

[0076] Figure 7 Box plots of redox ratio 2 (Ln) calculated in four studies (MLD, SV, GAL, and NE) and compared with STD.

[0077] Figure 8 SCFA producer ratio and redox ratio of inoculum A and inoculum B.

[0078] Figure 9 Effect of inoculum on body weight of 7-day-old chickens (p: 0.007).

[0079] Figure 10 Box plot of the ratio 2.3 calculated in the Salmonella challenge and compared with STD.

[0080] Figure 11 Box plot of the ratio 2.4 calculated in the Salmonella challenge and compared with STD.

[0081] Figure 12 Box plot of the ratio 2.15 calculated in the Salmonella challenge and compared with STD.

[0082] Figure 13 Box plot of the ratio 2.17 calculated in the Salmonella challenge and compared with STD. Detailed Implementation

[0083] Example 1: Describing the selection ratio

[0084] 1.1) Ratio 2.1: Firmicutes / Proteobacteria

[0085] In four studies of chickens, the rates varied as expected, with challenges based on malnutrition (changes in dietary fat, fiber, viscosity, and generally nutritional status) and necrotizing enteritis. Figure 1 Because the confidence level is 60%, it is considered relevant to assessing gut health. Since it is difficult to establish a reference value for "health / resilience," the values ​​for this ratio vary across different studies.

[0086] 1.2) Ratio 2.3: Clostridium IV and XlVa clusters (CC) / Enterobacteriaceae

[0087] Clostridium IV and XlVa clusters (CC) are numerators because they can utilize large amounts of nutrients that are indigestible by the host and produce large quantities of short-chain fatty acids (SCFAs), which play a significant role in intestinal homeostasis. They can reduce inflammation because of their unique biological activities, such as the production of butyrate, secondary bile acids, and indolepropionic acid.

[0088] Under standard conditions, this ratio should be higher when no challenge is induced than when challenge is induced, reflecting a greater ratio of CC than Enterobacteriaceae, or a reduction in Enterobacteriaceae.

[0089] In chickens; the change in the ratio of 2.3 was in line with expectations in all studies, except for mild dysbiosis (MLD) and severe dysbiosis (SD) in the dysbiosis group on day 20. The corresponding confidence index was 80% ( Figure 2 ).

[0090] like Figure 10 As shown, the ratio of 2.3 in the Salmonella challenge in piglets is in line with the expected trend.

[0091] 1.3) Ratio 2.4: Rumenaceae and Trichophyceae / Enterobacteriaceae

[0092] The main difference between ratios 2.4 and 2.3 lies in the utilization of different taxa. While ratio 2.3 is used to assess the amount of CC in the microbiota, ratio 2.4 allows for the assessment of the proportions of two major SCFA-producing families: *Clostridium* and *Rumenococcus*, and specifically butyrate producers. *Clostridium* is a phylogenetic and morphologically heterogeneous taxa belonging to the genus *Clostridium* XlVa within the phylum Firmicutes; *Rumenococcus* belongs to cluster IV. Compared to the challenge, this ratio is expected to increase in the control group, reflecting a greater proportion of *Rumenococcus* and *Clostridium* than Enterobacteriaceae, or a decrease in Enterobacteriaceae (as seen in chicken studies). Figure 3 and piglet research Figure 11 The trend is similar to 2.3 because the Clostridium genus clusters from ratio 2.3 belong to the Ruminaceae and Trichophyceae families. The difference between these two ratios is that, within families, the ratio uses more genera compared to CC. Ratio 2.4 represents an 80% confidence level.

[0093] 1.4) Ratio 2.15: Butyrate producers / Firmwallis

[0094] Ratio 2.15 assessed the proportion of butyrate-producing genera within the Firmicutes phylum. Bacteroidetes (Gram-negative) and Firmicutes (Gram-positive) are the most abundant phyla in the gut; Bacteroidetes primarily produce acetate and propionate, while Firmicutes primarily produce butyrate in the human gut. Therefore, under good health conditions (standard diet), this ratio would be higher than the ratio in the challenge diet, manifested as an increase in butyrate-producing bacteria, which have beneficial effects on colonic cells, strengthening the epithelial barrier, reducing inflammation, and increasing the production of mucin and antimicrobial peptides. Compared to other ratios, ratio 2.15 varied between 0 and 1 because it evaluated the proportion of multiple genera capable of butyrate production across all phyla. Figure 4 The ratio of 2.15 is consistent with the expected trends observed in four studies conducted in chickens, including those involving dysbiosis (MLD) and necrotic enteritis challenge (NE) caused by dietary fat. This resulted in a 60% confidence index ( Figure 4 ).

[0095] like Figure 12 As shown, the ratio of 2.15 in the Salmonella challenge in piglets is in line with the expected trend.

[0096] 1.5) Ratio 2.17: Butyrate Producers (based on Vital, 2017 and those microorganisms from ratio 2.15) / Firmicutes

[0097] Ratio 2.17 completes the list of butyrate producers presented in ratio 2.15 (based on Vital, M.; Karch, A.; Pieper, DH (2017) Colonic Butyrate-Producing Communities in Humans: an Overview Using Omics Data. In: mSystems, vol. 2, n° 6), adding 6 new taxa to the previous ratio. This ratio is expected to be higher under standard conditions than under challenges, implying an increase in butyrate-producing genera under standard conditions. The arbitrary units of this ratio are defined as ranging from 0 to 1. Minor differences were noted compared to ratio 2.15. However, the ratio varied as expected across all challenges. The confidence index for this ratio is 60% ( Figure 5 (In chicken research).

[0098] like Figure 13 As shown, the ratio of 2.15 in the Salmonella challenge in piglets is in line with the expected trend.

[0099] 1.6) Reduction / oxidation (Red / Ox) ratio [Aerobic / Anaerobic bacteria]

[0100] Ratios and their Napier logs associated with redox activity were proposed. These ratios link aerobic and anaerobic bacteria. Based on Million, M.; Raoult, D. (2018) Linking gut redox to human microbiota. In: Human Microbiome Journal, vol. 10, p. 27-32. It divides the microbiome into aerobic and anaerobic groups. In humans, these ratios link the gut's reducing / oxidative potential to the colonic microbiota and inflammatory states. High concentrations of aerobic bacteria are associated with oxidative stress through increased reactive oxygen species (ROS), which preferentially proliferates in the gut microbiota and is detrimental to anaerobic bacteria. These ratios are based on a comprehensive and precise list of specific aerobic or anaerobic species. Due to a lack of species data and to account for homogeneity, the ratios were redesigned, and the relative abundance of species was replaced by the relative abundance of genera (see Million et Raoult 2018, Annexe 4, cited above). Figure 6This shows how the redox ratio 1 changed with the expected trend on days 13, 28, and 35 of Experiment 1, and in Experiments 3, 4, and 5. The confidence index for this ratio is 66%. The redox ratio 2 (Ln ratio) followed the expected trend in the same study as redox ratio 1, with the confidence index decreasing to 55%. Figure 7 ).

[0101] Example 2: Selection Ratio

[0102] The microbial inoculum should be selected based on the following microbial ratios:

[0103] 2.1) Ratio of short-chain fatty acid (SCFA) producers to Enterobacteriaceae

[0104]

[0105] SCFAs are molecules that improve gut health through various mechanisms, such as providing energy to intestinal cells, maintaining the integrity of the intestinal barrier, producing mucus, and preventing inflammation. This ratio contains rumen bacteria and spirochetes capable of producing SCFAs, as well as Enterobacteriaceae members that cause gastrointestinal infections, such as Escherichia coli or Salmonella.

[0106] 2.2) Redox ratio

[0107]

[0108] Inoculants containing a low redox ratio can lead to better resistance to challenges. This ratio is based on the optimal environment for cecal bacteria to properly perform their activities, which is typically anaerobic. Many pathogens are aerobic, and measuring the number of bacteria that are considered aerobic or anaerobic can reflect the health of the gut.

[0109] To calculate these ratios, 16S rRNA sequencing was performed on the samples, and the ratios were calculated using a relative abundance table obtained from the sequencing.

[0110] The following inoculum was selected:

[0111] Inoculum A: An inoculum with a high SCFA producer ratio and a low redox ratio;

[0112] Inoculum B: Inoculum with a low SCFA producer ratio and a high redox ratio; such as Figure 8 As shown.

[0113] Example 3: Determining the optimal concentration of microbial inoculum.

[0114] In vivo experiments were conducted to determine the inoculum concentration at which animals did not lose performance. Animals were fed a standard diet, and different groups received inoculum concentrations at 10...5 Up to 10 8 CFU / animal inoculation.

[0115] When birds are given a high dose (10) 8 When CFU / birds were vaccinated, compared to the unvaccinated control, body weight (BW) decreased by 10% on day 7 (D7), and livestock performance was significantly reduced. In contrast, when animals received 10... 6-7 At CFU / bird dosage, their body size (BW) was similar to that of the uninoculated group (see [reference needed]). Figure 1 ).

[0116] In summary, the optimal microbial dose for IMT and thus reducing animal stress and performance loss is 10. 6-7 CFU / Bird.

[0117] Example 4: Using inoculum A and inoculum B from Example 2

[0118] 4.1) Scenario 1: No challenge conditions

[0119] The animals were fed a standard diet. The results are shown in Table 1 below.

[0120] Table 1: Effect of inoculum on control group* (%)

[0121]

[0122] *Control group = Unvaccinated group fed with standard daily diet (STD)

[0123] Table 1 shows that, under no-challenge conditions, the chickens receiving inoculated agent A showed no difference in livestock parameters compared to the uninoculated group. In contrast, inoculated agent B had adverse effects on the animals receiving it, reducing body weight (BW) by 4% and increasing feed conversion ratio (FCR) by 2.23%, reflecting the higher feed intake (Fl) in this group.

[0124] 4.2) Scenario 2: Nutritional challenges of brooder and grower diets based on rye and pork fat

[0125] Chickens were fed brooder and grower diets based on wheat, rye, and pork fat. Rye contains a high amount of non-starch polysaccharides, which increase the viscosity of digestibles and reduce the motility of the small intestine, thus posing a challenging condition for the birds (Challenge 1).

[0126] The results are listed in Tables 2 and 3 below.

[0127] Table 2: Effect of inoculum from the challenge control group* on brooder diets (%)

[0128]

[0129] *Control group = Unvaccinated group receiving Challenge Diet 1

[0130] Following a rye-pork fat diet challenge, administration of inoculum A or B to birds increased their performance on day 14, with animals performing better when receiving inoculum A. This was manifested in a 4.69% increase in body weight (BW), a 5.13% increase in body weight gain (BWG), and a 1.54% decrease in free body rate (FCR) when inoculum A was administered. On the other hand, inoculum B resulted in a lower 3% increase in BW and a significant increase in free body rate (Fl) (6.22%), which reduced the efficiency of chicken production by 2.76% in the FCR (Table 2).

[0131] Table 3: Effect of inoculum from the challenge control group* on growing diets (%)

[0132]

[0133] *Control group = Unvaccinated group receiving Challenge Diet 1

[0134] On day 28, only animals receiving inoculated agent A showed a 3.34% improvement in body weight gain (BWG), while no improvement was recorded in animals receiving inoculated agent B (0.61%). Considering the overall parameters, the BWG of animals receiving inoculated agent A was 3.43, compared to 0.62 for animals receiving inoculated agent B. Animals receiving inoculated agent A had a similar free flow rate (FCR) to uninoculated animals (0.72%), compared to the 2.17% increase seen in animals receiving inoculated agent B (Table 3).

[0135] 4.3) Situation 3. Inflammatory and anti-nutritional growth diets (galactomannan).

[0136] To induce anti-nutritional and inflammatory challenges in animals (Challenge 2), 2% guar gum was added to the standard growing diet. Galactomannan induces localized inflammation in chickens, increases iNOS, K2O3, and IL-1, and has systemic effects on SAA, α1GP, IL-10, and IL-6 (unpublished data from Adisseo), and reduces glucose uptake, thus decreasing animal body weight (Rainbird et al., 1984). It also increases the viscosity of digestate and slows intestinal motility, resulting in a greater opportunity for the proliferation of unwanted microorganisms. These microorganisms utilize nutrients that could otherwise be used for animal growth.

[0137] The inoculum was administered on day 0 and day 14.

[0138] The results are shown in Table 4 below.

[0139] Table 4: The impact of inoculum on growth challenge diets (%)

[0140]

[0141] *Control group = Unvaccinated group receiving Challenge Diet 2

[0142] Animals that received inoculation A (BW and BWG +4.07% and +7.08%, respectively) performed better than animals that received inoculation B (+1.42% and +5.53%) (Table 4).

[0143] In summary, a method for inoculating chickens with IMT has been developed to enhance the animals' resistance to challenging conditions. The method comprises (1) administering a microbial inoculum at a concentration that ensures no loss of performance due to inoculation (10). 7 (CFU / bird), and (2) the microbial inoculum was characterized by the microbial ratio calculated from 16S sequencing data. In short, 10 6 Up to 10 7 Animals with CFU / birds inoculated with microbial inoculum that have a high SCFA producer ratio and a low redox ratio will have less performance loss when faced with challenges in juvenile or later life (in terms of BW, BWG, Fl, and FCR).

Claims

1. Use of one or more ratios of the amount of bacteria in the microbiota of a farm animal, said ratios selected from the group consisting of: Firmicutes / Bacteriotes; Clostridia IV and XlVa clusters / Bacteroidales; Ruminococcaceae and Lachnospiraceae / Bacteroidales; butyrate producing bacteria / Firmicutes; aerobes / anaerobes, for determining the status of the intestinal microbiota of a farm animal and whether said status is satisfactory.

2. Use of one or more ratios of the amount of bacteria in the microbiota of a farm animal, for selecting an extract of a satisfactory intestinal microbiota of a farm animal, aimed at maintaining or improving the intestinal microbiota of a different farm animal in need thereof.

3. The use according to claim 1 or 2, which is the use of at least two, at least three, at least four or all of said ratios.

4. A method for determining in vitro or ex vivo the status of the intestinal microbiota of a farm animal, comprising at least the steps of: detecting and quantifying bacteria selected from the group consisting of Firmicutes, Bacteriotes, Clostridia IV and XlVa clusters, Bacteroidales, Ruminococcaceae, Lachnospiraceae, butyrate producing bacteria, aerobes and anaerobes in a biological sample of said farm animal; and calculating at least one ratio selected from the group consisting of: Firmicutes / Bacteriotes, Clostridia IV and XlVa clusters / Bacteroidales, Ruminococcaceae and Lachnospiraceae / Bacteroidales, butyrate producing bacteria / Firmicutes and aerobes / anaerobes. further comprising the steps of: comparing said or each of said ratios of said biological sample of said animal to a control value determined for each of said ratios, and determining the status of said microbiota from said comparison. comprising calculating at least one ratio selected from the group consisting of Firmicutes / Bacteriotes, Clostridia IV and XlVa clusters / Bacteroidales, Ruminococcaceae and Lachnospiraceae / Bacteroidales, butyrate producing bacteria / Firmicutes, and if said or each calculated ratio is greater than or equal to the control value determined for the corresponding ratio, the status of said microbiota is rated as satisfactory, and if said or each of said ratios is lower than said control value, the status of said microbiota is rated as unsatisfactory. comprising calculating at least the ratio of aerobes / anaerobes, and if said ratio is less than or equal to said control value, the status of said microbiota is rated as satisfactory, and if said ratio is higher than said control value, the status of said microbiota is rated as unsatisfactory. comprising calculating at least two, at least three or at least four or five of said ratios, and optionally comparing to a control value. said biological sample is selected from the group consisting of caecal content, ileal content and faecal content. the amount of bacteria is measured by relative abundance obtained from data generated from sequencing analysis of 16s rRNA sequencing. said farm animal is a chicken, and the control value for any of the following ratios is determined for the same chicken that is not the same chicken: Firmicutes / Bacteriotes; Clostridia IV and XlVa clusters / Bacteroidales; Ruminococcaceae and Lachnospiraceae / Bacteroidales; butyrate producing bacteria / Firmicutes; aerobes / anaerobes.

5. The method of claim 4, wherein ​ ​ ​ 6. The method of claim 5, wherein ​ ​ ​ 7. The method of claim 5, wherein ​ ​ ​ 8. The method according to any one of claims 4 to 7, characterized in that ​ ​ 9. The method according to any one of claims 4 to 8, characterized in that ​ 10. The method according to any one of claims 4 to 9, characterized in that ​ 11. The method according to any one of claims 4 to 10, characterized in that ​ 12. The method according to any one of claims 4 to 11, characterized in that Said farm animal is a chicken and said control value of the ratio aerobes / anaerobes is measured in a chicken under farm conditions which are not challenged or less challenged than the chicken for which the state of the gut microbiota is determined.

13. A method for selecting an extract of a satisfactory gut microbiota of a farm animal comprising the steps of: detecting and quantifying in a biological sample of said farm animal the amount of each bacteria selected from the group consisting of Firmicutes, butyrate-producing bacteria, aerobes and anaerobes, and calculating at least one ratio selected from the group consisting of butyrate-producing bacteria / Firmicutes (al) and aerobes / anaerobes (a2); detecting and quantifying in a biological sample of different farm animals the amount of each bacteria selected from the group consisting of Firmicutes, butyrate-producing bacteria, aerobes and anaerobes, and calculating at least one ratio selected from the group consisting of butyrate-producing bacteria / Firmicutes (bl) and aerobes / anaerobes (b2); comparing the calculated ratios (al) and (bl) and / or ratios (a2) and (b2); and selecting from said comparison said satisfactory extract.

14. The method of claim 13, wherein, said extract is selected if the ratio of butyrate-producing bacteria / Firmicutes (al) is greater than or equal to the ratio (bl) and / or if the ratio of aerobes / anaerobes (a2) is less than or equal to the ratio (b2).

15. A non-therapeutic method of maintaining or improving the gut microbiota of a farm animal in need thereof, characterized in that comprising the steps of: selecting a satisfactory microbiota extract according to the method of claim 13 or 14, and administering said satisfactory microbiota extract or a derivative thereof produced from fermentation to said farm animal.

16. The method of claim 15, wherein said farm animal is a youngling.

17. The method of claim 16, wherein The farm animal is a broiler chicken, and the extract is administered in an amount of 1-9.10 7 CFU of the extract.

18. A biomarker of the state of the microbiota of a farm animal selected from the group consisting of the following ratios of amounts of bacteria: Firmicutes / Firmicutes; Clostridium IV and XlVa cluster / Enterobacteriaceae; Ruminococcaceae and Lachnospiraceae / Enterobacteriaceae; butyrate-producing bacteria / Firmicutes; aerobes / anaerobes.

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