LACTOBACILLI STRAIN COMBINATION AND ITS USE IN ANIMAL HEALTH

Abstract

The present invention relates to a mixture of microorganisms comprising a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius, its use in preparing an additive or food product for animal use and in preventing or treating necrotic enteritis.

Description

Title of the invention: COMBINATION OF LACTOBACILLI STRAINS AND ITS USE IN ANIMAL HEALTH FIELD OF INVENTION

[0001] The present invention describes a combination of lactobacilli (or lactic acid bacteria), namely a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius, and its use for the prevention and / or treatment of necrotic enteritis in animals, in particular chickens. PRIOR STATE OF TECHNOLOGY

[0002] In 2017, global meat production increased by 1.25% to 323 million tonnes (Mt) and is projected to grow by nearly 15% by 2027 (Organisation for Economic Co-operation and Development (OECD) / Food and Agriculture Organization (FAO), 2018). The growth in demand for meat products is primarily due to population growth, urbanization, and rising incomes in developing countries.

[0003] Poultry meat production represents the main sector in the meat products industry.

[0004] The term "poultry" refers to domestic birds belonging to the gallinaceous or web-footed families that are raised for their meat, eggs, and feathers. The term "poultry" covers a wide range of birds, from native and commercial breeds of chickens to Muscovy ducks, mallards, turkeys, guinea fowl, geese, quail, pigeons, ostriches, and pheasants. The most commonly consumed poultry meat is chicken.

[0005] Poultry meat is mainly produced in large-scale intensive farming systems. The global poultry industry has become a highly profitable sector whose success is closely linked to animal health, and more specifically to a healthy gastrointestinal tract, which ensures the efficient absorption of feed given to the poultry.

[0006] Indeed, avian diseases, that is, diseases affecting birds, can generate significant financial losses due to high mortality rates and low feed efficiency. These infections can be bacterial, fungal, viral, or parasitic in origin. Thus, the most common bacterial infections in birds are those caused by Escherichia coli, Salmonella spp., Clostridium perfringens, Pasteurella multocida, Staphylococcus aureus, Mycobacterium avium, Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis. Mycoplasma iowae, Clostridium sordellii, and Clostridium septicum are the cause of various diseases such as chronic respiratory disease of poultry, avian cholera, gangrenous dermatitis, necrotic enteritis, and avian tuberculosis.

[0007] Among the most important agents of enteric diseases in poultry are Escherichia coli and bacteria of the genus Clostridium.

[0008] Bacteria of the genus Clostridium (or clostridia) are Gram-positive, anaerobic bacilli that produce endospores. They are ubiquitous, meaning they are found in the environment and in the gastrointestinal tract of animals. It should be noted that most of these bacteria are non-pathogenic. Many of these bacteria are widely used in industrial fermentations, for example, in the synthesis of chemical compounds such as acetate, butyrate, lactate, ethanol, carbon dioxide, or solvents.

[0009] Nevertheless, certain bacteria of the genus Clostridium (C.) are known for their pathogenicity. These include C. botulinum, C. tetani, C. difficile, C. perfringens, C. novyi, and C. septicum. Clostridia cause several infections, including ulcerative enteritis caused by C. colinum and necrotic enteritis caused by C. perfringens.

[0010] Necrotic enteritis is a disease affecting poultry farms in all poultry-producing regions of the world. This disease is more common in broiler chickens, but laying hens and turkeys can also be affected.

[0011] The ability of C. perfringens to cause this pathology depends on the production of certain extracellular toxins and enzymes that cause degradation of intestinal cells, such as lecithinases or necrotizing toxins.

[0012] As an example, C. perfringens produces and secretes alpha-toxin (a-toxin), a lecithinase that causes hemolysis and tissue necrosis, or [3-toxin (or Necrotic enteritis toxin B-like or NetB) that causes the formation of endospores in the membrane of intestinal cells.

[0013] The bacterium C. perfringens is naturally present in the intestine. As an opportunistic bacterium, it requires a compromised intestinal balance to colonize, proliferate, and express its toxins, thereby causing necrotic enteritis. Studies have shown that simple C. perfringens infection alone is not sufficient to induce the pathology. The onset of necrotic enteritis requires the presence of conditions known as predisposing factors, such as coccidiosis, a disease caused by the parasite Eimeria spp., diet, or even immunosuppression or stress in animals.

[0014] Necrotic enteritis negatively affects the feed conversion ratio in livestock, meaning that the animals must consume more feed to achieve the same weight gain. This type of necrotic enteritis is problematic because it negatively impacts productivity and profitability, resulting in animals of reduced size and weight.

[0015] The most frequent signs of necrotic enteritis are drowsiness, lethargy, loss of appetite, diarrhea, dehydration, and loss of appetite. Chickens generally die within 1 to 2 hours of the onset of symptoms. Mortality rates associated with necrotic enteritis are generally between 2 and 10% but can sometimes reach 50%. The disease is characterized by a sudden increase in flock mortality, with birds usually dying without warning signs.

[0016] Necrotic enteritis is therefore responsible each year for colossal economic losses worldwide, estimated at over $6 billion in 2015. These losses are due to the costs of disease control measures and the decrease in production in terms of weight and mortality of animals on farms.

[0017] This is why strategies for the prevention and / or treatment of necrotic enteritis in farm animals are necessary.

[0018] One way to combat necrotic enteritis is prevention because, as mentioned previously, after infection, the development of the disease is very rapid and mortality is high.

[0019] The administration of low-concentration antibiotics, i.e., the administration of growth promoters (or AGPs for Antibiotic Growth Promoters) such as avoparcin, bacitracin, and virginiamycin, is very effective in the prevention and control of necrotic enteritis. These AGPs promote animal growth and improve feed conversion efficiency. It should be noted that these compounds are also used in human medicine, at higher concentrations.

[0020] However, the misuse of these antibiotics, particularly in human and veterinary medicine, has caused selective pressure that has accelerated the evolution and spread of resistant bacteria. Consequently, their use has been prohibited as antibiotics and retained solely for human medicine, particularly in Europe.

[0021] Following restrictions on the use of PGAs, alternatives have been developed to prevent and / or treat necrotic enteritis such as the use of NetB toxoids, or vaccination with Eimeria, a unicellular parasite, in order to reduce the prevalence of coccidiosis known to be an important predisposing factor for necrotic enteritis.

[0022] However, there remains a clear need to develop alternatives to overcome the increased mortality and morbidity rates associated with the ban antibiotics in animal feed, exhibiting similar performance to that obtained with antibiotics in the prevention and / or treatment of necrotic enteritis.

[0023] Due to their properties, lactic acid bacteria are widely used in the food industry as biological food preservatives or in industrial livestock farming for the prevention of infectious and zoonotic diseases. These bacteria are also used to improve livestock performance through the production of digestive enzymes, volatile fatty acids, and / or vitamins, which contribute to increased nutrient digestibility and improved feed conversion ratios. These lactic acid bacteria are commonly referred to as probiotics.

[0024] Thus, an alternative to the use of antibiotics or PGAs is to give animals additives or food products, in particular probiotics.

[0025] Generally speaking, probiotics are defined as live microorganisms that confer a health benefit to the host when consumed in sufficient concentrations. They can interact with the host to improve immunity, intestinal homeostasis, stimulate metabolism, or reduce the risk of infection by opportunistic pathogens.

[0026] Certain probiotic bacteria interfere with, or even eliminate, the pathogenicity of disease-causing microbial agents, for example, by eliminating or inhibiting the growth of pathogenic bacteria in the intestinal lumen. Some produce antibacterial substances capable of competing with pathogens for nutrients, growth factors, and attachment sites on the intestinal epithelium. They can also exert immunological functions by modulating the host's immune response, thus enabling it to better fight infections.

[0027] Most bacterial probiotics belong to the lactic acid bacteria. One of the main characteristics of this group of bacteria is their ability to produce lactic acid, in a strain-dependent manner, by homo- or hetero-fermentative fermentation of glucose.

[0028] Lactic acid bacteria belong to the phylum Firmicutes, the class Bacilli, and the order Lactobacillales. The taxon called lactic acid bacteria includes Gram-positive, non-spore-forming, anaerobic or facultative aerobic cocci, bacilli, or coccobacilli with a G+C percentage of less than 50%. These bacteria are acidophilic with an optimal growth pH between 3.5 and 6.5. Most strains have nutritional requirements and need rich media to grow. This group includes 10 genera, the best known of which are Lactobacillus, Pediococcus, Lactococcus, Enterococcus, Streptococcus, Leuconostoc and Camobacterium.

[0029] Among lactic acid bacteria, the most represented genus is Lactobacillus (L / x) with more than 253 species described to date (http: / / www.bacterio.net / lactobacillus.html).

[0030] Lactobacilli are widely used in the preparation of probiotics, in particular because of their ability to survive the extreme conditions found in the gastrointestinal tract, their good ability to adhere to intestinal cells allowing them to increase the retention of probiotics in the intestine, and their properties of eliminating or inhibiting the growth of pathogenic bacteria in the intestinal lumen.

[0031] Document EP 2 287 286 describes in particular that isolates of Lactobacillus (Lb. sakei or Lb. reuteri) possess anti-inflammatory and probiotic properties.

[0032] Document CN 105861399 also describes the use of a specific strain of Lb. plantarum to prevent necrotic enteritis in farmed chickens by inhibiting the growth of C. perfringens.

[0033] However, there is an obvious need to develop new effective solutions to combat C. perfringens responsible for necrotic enteritis of broiler chickens, for health and economic reasons. Description of the invention Definitions

[0034] The definitions below correspond to the meaning generally used in the context of the invention and are to be taken into account unless another definition is explicitly indicated.

[0035] The terms "about" or "approximately", used in reference to a measurable value such as a quantity, a duration, and other analogous values, should be understood as encompassing measurement uncertainties of ± 20% or ± 10%, preferably ± 5%, even more preferably ± 1%, and particularly preferably ± 0.1% of the specified value.

[0036] Intervals: Throughout this description, the various features of the invention may be presented as ranges of values. It should be understood that describing values ​​as ranges is solely for the purpose of simplifying reading and should not be interpreted as a rigid limitation of the scope of the invention. Accordingly, the description of a range of values ​​should be considered as specifically disclosing all possible intermediate intervals as well as each of the values ​​within that range. For example, the description of an interval from 1 to 6 should be considered as specifically describing each of the intervals it comprises, such as the intervals from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as each of the values ​​in that interval, for example, 1; 2; 2.7; 3; 4; 5; 5, 3 and 6. This definition is valid regardless of the range of the interval.

[0037] The term “isolated” should be understood in the context of the invention as synonymous with removed or extracted from its natural environment or state. For example, an isolated bacterial strain or peptide is a bacterial strain or peptide extracted from the natural environment in which it is usually found, such as a living plant or animal. Thus, a bacterial strain or peptide naturally present in a living animal is not an isolated bacterial strain or peptide within the meaning of the invention, whereas the same bacterial strain or peptide, partially or completely separated from the other elements present in its natural environment, is “isolated” within the meaning of the invention. An isolated bacterial strain or peptide may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

[0038] Surprisingly, the Applicant has identified a new specific combination of lactic acid bacteria, useful for combating necrotic enteritis, particularly in farm animals such as chickens.

[0039] Thus and according to a first aspect, the present invention relates to a mixture of microorganisms comprising a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius.

[0040] For the purposes of the invention, "mixture" means the association of at least 2 different species of microorganisms, advantageously of at least 2 distinct strains of bacteria, even more advantageously of at least 2 distinct strains of lactic acid bacteria (lactobacilli or Lactobacillus).

[0041] Thus, a mixture of microorganisms according to the invention may include one or more strains of Lb. reuteri and one or more strains of Lb. salivarius, and possibly other microorganisms, in particular other bacteria.

[0042] According to another aspect, the present invention relates to a mixture of lactic acid bacteria consisting of at least one strain of Lb. reuteri and at least one strain of Lb. salivarius. In one particular embodiment, the mixture of lactic acid bacteria consists of one strain of Lb. reuteri and at least one strain of Lb. salivarius, possibly two strains of Lb. salivarius. In another embodiment, the mixture of lactic acid bacteria consists of one strain of Lb. reuteri and one strain of Lb. salivarius.

[0043] As demonstrated in the present application, the specific mixture according to the invention is capable of reducing or inhibiting the growth and / or activity of the bacterium C. perfringens.

[0044] For the purposes of this invention, "growth of the bacterium C. perfringens" refers to a set of mechanisms leading to an increase in dry bacterial biomass. This includes the growth of the bacterial cell in size, mass and / or volume, as well as the growth of a population through cell division.

[0045] For the purposes of this invention, "activity of the bacterium C. perfringens" refers to the pathogenic or toxin-producing activity of this bacterium. This includes, in particular, the production or secretion of toxins, such as NetB and α-toxin, or enzymes. It may also refer to the ability of C. perfringens to adhere to or even colonize the gastrointestinal tract.

[0046] Advantageously, the mixture according to the invention allows a reduction or even an inhibition of the production and / or secretion of NetB and a-toxin.

[0047] According to a particular embodiment, the mixture according to the invention comprises the strain of Lb. reuteri deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 4, 2020.

[0048] According to another particular embodiment, the mixture according to the invention comprises the Lb. salivarius strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 4, 2020 and / or the Lb. salivarius strain deposited at the CNCM under number 1-5502 dated March 4, 2020.

[0049] Thus, and according to different embodiments, the mixture according to the invention comprises or is made up of: - the Lb. reuteri strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 on March 4, 2020, and the Lb. salivarius strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 on March 4, 2020; or - the Lb. reuteri strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 on March 4, 2020, and the Lb. salivarius strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 on March 4, 2020; or - the Lb. reuteri strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 4, 2020, the strain of Lb. salivarius deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 4, 2020 and the strain of Lb. salivarius deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 dated March 4, 2020.

[0050] According to a particular embodiment, the mixture contains other microorganisms, advantageously other probiotics. Preferably, the mixture contains other microorganisms selected from: bacteria of the genus Lactobacillus, bacteria of the genus Bifidobacterium, bacteria of the genus Streptococcus, bacteria of the genus Enterococcus, bacteria of the genus Pediococcus, bacteria of the genus Bacillus, yeasts, and combinations thereof.

[0051] Preferably, the Lactobacillus bacterium is chosen from the group consisting of: Lb acidophilus, Lb. lactis, Lb. helveticus, Lb. brevis, Lb. casei, Lb. plantarum, Lb. salivarius, advantageously another strain of Lb salivarius, Lb. reuteri, advantageously another strain of Lb reuteri, Lb. bifidus, Lb. bulgaricus, Lb. caucasicus, Lb. rhamnosus, Lb. gasseri, Lb. sakei, Lb. fermentum, and their combinations.

[0052] Preferably, the bacterium Bifidobacterium is chosen from the group consisting of: B. bifidum, B. longum, B. infantis, B. breve, B. adolescentis, B. animalis, B. lactis, and their combinations.

[0053] Preferably, the Streptococcus bacterium is chosen from the group consisting of: S. thermophilus, S. lactis, S. cremoris, S. diacetylcatis, and their combinations.

[0054] Preferably, the Enterococcus bacterium is chosen from the group consisting of: E. faecium, E. faecalis, and their combinations

[0055] Preferably, the Pediococcus bacterium is P. acidilactici.

[0056] Preferably, the bacterium Bacillus is chosen from the group consisting of: B. subtilis, B. velezensis, B. licheniformis, B. coagulans, B. pumilus, and their combinations.

[0057] Preferably, the yeast is chosen from the group consisting of: Candida Kefyr, Saccharomyces florentinus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var. boulardii, and their combinations.

[0058] According to another aspect, the present invention relates to a composition comprising a mixture of microorganisms as defined above.

[0059] Furthermore, the composition according to the invention may include the usual adjuvants or excipients used in the field concerned, such as hydrophilic or lipophilic thickeners or gelling agents, palatabilities, hydrophilic or lipophilic additives, preservatives, antioxidants, diluents, vitamins, minerals, suspending agents, cellulosic derivatives, absorbents, cryoprotectants or dyes.

[0060] Of course, a person skilled in the art will take care to choose this or these possible adjuvants or excipients, and to adjust their quantity, in such a way that the advantageous properties of the composition according to the invention are not, or substantially not, altered by the envisaged addition.

[0061] According to one embodiment, the composition according to the invention comprises nutrients usable as a carrier and / or prebiotic substances advantageously chosen from fructo-oligosaccharides, inulins, isomalto-oligosaccharides, lactitol, lactosucrose, lactulose, pyrodextrins, soy oligosaccharides, transgalacto-oligosaccharides, xylo-oligosaccharides, vitamins, in particular vitamin E.

[0062] According to another embodiment, the composition according to the invention comprises at least one compound selected from the following group: zeolites, calcium carbonate, calcium sulfate, magnesium carbonate, talc, trehalose, chitosan, shellac, albumin, starch, skimmed milk powder, whey, buttermilk powder, maltodextrins, lactose, inulin, dextroses, celluloses, clays including sepiolite, yeast and cereal derivatives, vegetable oils or a solvent selected from water or physiological saline.

[0063] According to a particular embodiment, the composition according to the invention comprises a coating material advantageously selected from maltodextrins, guar seed flour, gum arabic, alginates, modified starch and starch derivatives, dextrins, cellulose derivatives such as cellulose ester and cellulose ether, proteins such as gelatin, albumin, casein, gluten, gum arabic, tragacanth gum, lipids such as waxes, paraffin, stearic acid, mono- and diglycerides.

[0064] The mixture or composition according to the invention may be in the form of a powder, a capsule, a spray, a solution, an emulsion, a suspension or a dispersion.

[0065] Advantageously, the mixture or composition according to the invention is in dry or liquid form, in particular in freeze-dried, dried, pressed, liquid or frozen form, advantageously in freeze-dried form.

[0066] According to a preferred embodiment, the mixture or composition according to the invention is intended for oral administration. For this purpose, the mixture or composition may be in various suitable dosage forms, for example, as a lyophilized powder to be poured and dissolved in drinking water, as a liquid to be poured over food or into drinking water, as tablets, or as a powder packaged in capsules or any other suitable form. Advantageously, it is a powder or lyophilized powder to be poured and dissolved in the animals' drinking water.

[0067] According to a particular embodiment, the mixture or composition according to the invention contains the lactobacilli according to the invention at a final concentration of between 105 and 109 CFU (Colony Forming Unit) / mL of the mixture or composition according to the invention, advantageously between 106 and 108 CFU / mL, for example at a final concentration of 107 CFU / mL.

[0068] This concentration can be understood as the concentration of each microorganism, advantageously bacteria, more advantageously lactobacilli, present in the mixture or composition, advantageously in the animals' drinking water. Preferably, it refers to the concentration of all the microorganisms, advantageously bacteria, more advantageously lactobacilli, present in the mixture or composition. Thus, by way of example, for a final lactobacillus concentration of 10⁷ CFU / mL, a mixture according to the invention may comprise or be composed of a strain of Lb. reuteri and a strain of Lb. salivarius at a concentration of 0.5 x 10⁷ CFU / mL each.

[0069] According to another aspect, the invention relates to the use of a mixture or composition according to the invention as an additive or food product for animal use.

[0070] For the purposes of this invention, "food additive or product" refers to a composition intended to supplement the traditional diet and comprising nutrients or other substances having a nutritional or physiological effect. As already stated, this may be an additive to drinking water or to foods such as, for example, cereals and / or legumes like soybeans. By convention, the term "beverage" is used for ingested products in liquid form and the term "food" for those in solid form.

[0071] The mixture or composition according to the invention can be added to the beverage or food extemporaneously, or can be introduced in particular into the food at the time of its manufacture, for example by mixing or coating.

[0072] According to a particular embodiment, the mixture or composition according to the invention is added to the beverage or food so that the lactobacilli represent from 108 to 1014 CFU / kg of the beverage or food, advantageously from 1010 to 1012 CFU / kg.

[0073] According to another aspect, the invention relates to the use of a mixture or composition as described above, as well as drinks and food containing them, to combat necrotic enteritis in animals, namely to prevent and / or treat this pathology.

[0074] In a known manner and as described in the examples, the efficacy on necrotic enteritis can be evaluated by determining a lesion score in the intestines of the animals: - a score of 0 corresponds to a healthy gut; - a score of 1 corresponds to a thin and friable intestine; - A score of 2 corresponds to a thin and friable intestine with the appearance of small necrotic spots; and - A score of 3 corresponds to an intestine showing large lesions visible through the outer wall of the intestinal tract.

[0075] The prevention and / or treatment of necrotic enteritis can also be assessed by monitoring the animals' weight. As mentioned previously, intestinal lesions decrease feed conversion, which leads to weight loss in the animals.

[0076] According to a preferred embodiment, the animal targeted by the present invention is a fowl, advantageously a chicken, preferably a broiler chicken (or farm chicken).

[0077] As already stated and preferably, the mixture or composition according to the invention, possibly integrated into the beverage or food, is administered or ingested orally.

[0078] Treatment can be administered systematically to all animals from birth or can be initiated upon the appearance of symptoms or even deaths in the flock. Advantageously, treatment is carried out preventively, that is, before the appearance of any symptoms, either immediately after the animals are born or a few days later. Even more advantageously, treatment is continued until the animals die, which generally occurs at 40 days for poultry.

[0079] Furthermore, the dose can be taken once a day, or even with every drink or meal, or perhaps spaced several days apart. Advantageously, the dose is taken daily.

[0080] A preferred dosage corresponds to daily administration, in the form of a food supplement at a concentration of 107 CFU / mL (equivalent to 107 CFU / g), throughout the animal's life. EXAMPLES OF THE INVENTION'S IMPLEMENTATION

[0081] The manner in which the invention can be implemented and the resulting advantages will be more apparent from the following embodiment examples, given by way of illustration and not limitation, in support of the attached figure.

[0082] [Fig. 1] Fig. 1 represents the effectiveness of the bacterial mixture according to the invention in terms of chicken weight. The letters (a, b, c) indicate statistically significant differences.

[0083] [Fig. 2] Figure 2 represents the effectiveness of the bacterial mixture according to the invention in terms of chicken lesion score. The letters (a, b, c) indicate statistically significant differences.

[0084] Demonstration of the effect of the bacterial mixture according to the invention on necrotic enteritis

[0085] The experiments were carried out using the Lb. reuteri strain deposited with the CNCM (National Collection of Microorganism Cultures, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated March 4, 2020 and the Lb. salivarius strain deposited with the CNCM (National Collection of Microorganism Cultures, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 dated March 4, 2020.

[0086] The C. perfringens strain implemented in the example is a C. perfringens strain isolated from chicken with necrotic enteritis. 1 / Materials and methods:

[0087] The ability of the combination of Lb. reuteri and Lb. salivarius according to the invention to prevent necrotic enteritis was evaluated in vivo on broiler chickens from a cross of the Cobb 500 (female) and Hubbard M99 (male) lines.

[0088] The in vivo experiments were conducted over 17 days on 150 chickens (30 per experimental condition). The trial began on the day of hatching. During the trials, lactobacilli were administered alone (Lb. reuteri, Lb. salivarius) or in combination (Lb. reuteri + Lb. salivarius), by gavage once a day, on days 1 and 2, then from day 10 to day 13.

[0089] 1-1 / Chicken environment and diet

[0090] The chickens were raised in cages providing an average surface area of ​​432 cm² per chicken. The cages were placed on several levels in a climate-controlled room maintained at ambient temperature throughout the study, and approved for testing with a biohazard level 2. Lighting was provided 24 hours a day for the entire duration of the study. The birds received water and food ad libitum, i.e., they were fed until satiety.

[0091] The diet for the first 9 days is based on corn and soy. After day 10, the growth diet includes wheat (Tables 1 and 2). Both diets are administered in the form of a puree.

[0092] Table 1 shows the composition of the diets by ingredients (g / 100g)

[0093] [Table 1] Starter Diet (0-9 days) Growth Diet (10-17 days) Corn 60.83 34.87 Soybean Meal (48%) 33.91 28.14 Wheat - 20.00 Distillation Dried Grains - 10.00 Vegetable Fat Blend 1.17 3.06 Limestone 1.47 1.45 Monocalcium Phosphate 1.54 1.44 NaCl 0.44 0.28 L-Lysine HCl 0.15 0.26 DL-Metionine 0.21 0.21 L-Threonine 0.06 0.08 Vitamin Premix 0.18 0.18 Mineral Premix 0.05 0.05

[0094] Table 2 shows the approximate composition of the diets (g / 100g)

[0095] [Tables2] Starter diet (0-9 days) Growth diet (10-17 days) Dry matter 87.98 88.93 Protein 22.00 22.00 Fat 3.63 5.87 Fiber 2.17 2.89 Ash 5.45 5.54 Lysine 1.3 1.3 Calcium 0.88 0.88 Phosphorus 0.70 0.71 Apparent metabolizable energy value (kcal / kg) 3,000.00 3,000.00 1-2 / Vaccination

[0096] With the exception of the unchallenged group (which did not receive C. perfringens), the chickens were vaccinated on day 1 with "Advent@9X". This vaccine contains live oocysts (Veimeria acervulina, E. maxima, and E. tenell), gentamicin, and amphotericin B as preservatives. The objective is to help prevent avian coccidiosis caused by these pathogens. On day 9, the chickens were vaccinated intraocularly against infectious bursitis with Intervet, Bursal Vac-G603, which is a vaccine containing live attenuated viruses. 1-3 / Treatment of the different groups

[0097] Five different treatments were tested. Each treatment was repeated six times and each replica contained 5 chickens.

[0098] These treatments are summarized below:

[0099] - T- : Control group not supplemented with lactobacilli and not challenged with C. perfringens;

[0100] - T+ : Positive control group, not supplemented with lactobacilli and challenged with C. perfringens',

[0101] - Lb. reuteri: supplemented with Lb. reuteri and challenged with C. perfringens',

[0102] - Lb. salivarius: supplemented with Lb. salivarius and challenged with C. perfringens',

[0103] - Lb. reuteri + Lb. salivarius: supplemented by a mixture of Lb. reuteri and Lb. salivarius and challenged with C. perfringens. 1-4 / Administration of C. perfringens

[0104] The wild-type C. perfringens strain was cultured overnight in thioglycollate broth at 37°C. The perfringens strain was then administered at a concentration of 10⁷ CFU / mL as a 3 mL oral gavage dose in sterile thioglycollate broth, using a 20 mL syringe and a 20-gauge dosing needle, from 14 to 16 days of age. After administration, the birds were hand-restrained for 5 to 10 seconds to confirm delivery of the correct dose and the absence of stress. 1-5 / Administration of lactic acid bacteria

[0105] Lactic acid bacteria strains were cultured in MRS (Man-Rogosa-Sharpe) broth at 37°C overnight. On days 1 and 2 of age, 250 to 500 µL of Lactobacillus suspension at 10⁷ CFU / mL (either alone or the final concentration of the mixture of the two strains Lb reuteri and Lb salivarius, i.e., 0.5 x 10⁷ CFU / mL each for the mixture) were administered orally to the birds. On days 10 to 13 of age, the dosage was increased to 1 mL, while maintaining the concentration at 10⁷ CFU / mL. The control groups (T- and T+) received the same volume of sterile PBS solution. The negative control birds were treated first to reduce the risk of cross-contamination. 1-6 / Measurements taken on birds

[0106] The study ended on day 17. The birds' performance was measured on days 0, 10, 14, and 17 of the experimental period by recording the birds' weight (in g) ([Fig. 1]) and feed consumption for each cage. At the end of the trial, all birds were euthanized by CO2 asphyxiation. Necrotic enteritis lesions were sought in the intestines and analyzed as described by Prescott et al. (1978, Can. Vet. J. 19, 181-183) ([Fig. 2]).

[0107] The experimental protocol is summarized in Table 3 below. X corresponds to the day of administration of a product.

[0108] [Tables3] Trial Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Lactobacillus XXXXXX C. perfringe ns XXX Measurements XXX 1-7 / Statistical Analysis

[0109] Statistical comparisons between the different results obtained were performed by analysis of variance (ANOVA) using Statgraphics® Centurion XVI software. 2 / Results:

[0110] The results are presented in Table 4 below. [YES] [Tables4] Lesion score at 17 days Weight at 17 days Negative control (T-) 0.88 e 765.00“ Positive control (T+) 1.67 ab 699.58 e Lb. reuteri 1.29bc 709.79be Lb. salivarius 1.50ab 707.50be Lb. reuteri + Lb. salivarius 1.00 e 753.13ab

[0112] The index letters (a, b, c) indicate statistically significant differences. Groups with the same letter do not differ significantly.

[0113] The results of [Fig.1] show that the chickens in the group treated with Lb. Lb. reuteri or Lb. salivarius showed a non-significant increase in their weight (709.79g and 707.50g, respectively) compared to the positive control group (T+). This the increase does not allow the chickens to reach a weight similar to that of the negative control group (T-).

[0114] On the contrary, the chickens in the group treated with the mixture of Lb. reuteri and Lb. salivarius have similar weights (753.13g) to those in the group of unchallenged chickens (765g), which are significantly different from the infected group (699.58g).

[0115] Regarding intestinal lesions caused by the administration of C. perfringens ([Fig.2]), the data show that the administration of Lb. reuteri or Lb. salivarius alone to the challenged chicken groups does not significantly decrease the lesion score compared to the T+ group.

[0116] A significant reduction in intestinal lesions is obtained in the chickens in the group treated with the mixture of Lb. reuteri and Lb. salivarius which have a lesion score (1.00) similar to that of the unchallenged T- chicken group (0.88) and significantly different from that of the infected T+ group (1.67).

[0117] In conclusion, these in vivo data show that supplementation with combined Lb. reuteri and Lb. salivarius produces a protective effect against necrotic enteritis in chickens.

Claims

Demands

1. Mixture of microorganisms comprising: - a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius, said strain of Lb. reuteri being the strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 dated 04 March 2020; or - a strain of Lactobacillus reuteri and a strain of Lactobacillus salivarius, said strain of Lb. salivarius being the strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 on 04 March 2020 and / or the strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 on 04 March 2020; or - the Lb strain.reuteri deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5500 on March 4, 2020 and the Lb. salivarius strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5501 on March 4, 2020 and / or the Lb. salivarius strain deposited at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-5502 on March 4, 2020.

2. Mixture according to claim 1, characterized in that it consists of a strain of Lb. reuteri and a strain of Lb. salivarius.

3.

4. Composition comprising a mixture according to claim 1 or 2. Mixture according to claim 1 or 2 or composition according to claim 3 being in lyophilized form.

5. Food product comprising the mixture according to any one of claims 1, 2 or 4, or the composition according to claim 3 or 4.

6. Mixture according to any one of claims 1, 2 or 4, or composition according to claim 3 or 4, for use in the prevention and / or treatment of necrotic enteritis in animals.

7. 18 Mixture or composition for its use according to claim 6, characterized in that the mixture or composition is in a form suitable for oral administration.

8. Mixture or composition for its use according to claim 6 or 7, characterized in that the animal is poultry, advantageously a chicken, even more advantageously a broiler chicken.

Images