EP3250217A1 - Composition destinée à être utilisée dans la prévention et/ou le traitement de la diarrhée chez les animaux - Google Patents

Composition destinée à être utilisée dans la prévention et/ou le traitement de la diarrhée chez les animaux

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Publication number
EP3250217A1
EP3250217A1 EP16702517.0A EP16702517A EP3250217A1 EP 3250217 A1 EP3250217 A1 EP 3250217A1 EP 16702517 A EP16702517 A EP 16702517A EP 3250217 A1 EP3250217 A1 EP 3250217A1
Authority
EP
European Patent Office
Prior art keywords
lactobacillus
enterococcus
composition
reuteri
bifidobacterium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16702517.0A
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German (de)
English (en)
Inventor
Johann Bauer
Karin Schwaiger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technische Universitaet Muenchen
Original Assignee
Technische Universitaet Muenchen
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Filing date
Publication date
Application filed by Technische Universitaet Muenchen filed Critical Technische Universitaet Muenchen
Publication of EP3250217A1 publication Critical patent/EP3250217A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/20—Feeding-stuffs specially adapted for particular animals for horses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • composition for use in preventing and/or treating diarrhea in animals The present invention is directed to a composition comprising at least one type of microorganism of the species Lactobacillus reuteri, and its use in a method of preventing and/or treating diarrhea in an animal, particularly a young animal, suffering therefrom.
  • Diarrhea is one of the major problems in animals, particularly young animals, in particular farm animals such as calves, piglets, lambs, goats etc. Despite
  • Diarrhea in animals is a complex, multi-factorial disease involving the animal, the environment, nutrition, and infectious agents.
  • diarrheas within the first days of life constitute a particular severe problem worldwide: About 60% of losses occur in the first and an additional 30% within the second week of life (Pohlenz et al., 1978; Elze et al., 1994; Kohara et al., 1997). Calves having perished due to diarrhea cause an economic damage of about € 31.5 million per year in Germany. However, economic damages caused by the death of a calf are by far higher due to feed costs, wage costs and veterinarian costs. This calculation does not yet comprise costs caused by surviving diarrhea-affected calves which require treatment.
  • Non-invasive oral rehydration therapy is preferred as long as the calf can drink by itself (Rademacher et al., 2002), for what a multitude of electrolyte potions are available, whose composition is based on mineral materials and easy available carbohydrates, partly in combination with astringent or mucosa saving additives such as spruce needle extract, citrus marc, pectin or psyllium (e.g.
  • Administration of antibiotics to eliminate pathogens is recommended only if a complication or an additional disease (e.g. navel inflammation or pneumonia) is prevalent as the physiological intestinal flora is destroyed by this causal therapy, which may lead later on to a bacterial dysbiosis (Mansfeld et al., 2005). Taking restrictions into
  • adsorbents aluminum silicate, carbon medicinalis
  • astringents e.g. tannin or tannic acid
  • motility inhibitors whose application is, however, prohibited in food producing animals.
  • non-steroidal anti-inflammatory drugs e.g. acetylic salicylic acid, ketoprofen
  • acetylic salicylic acid, ketoprofen can be employed for an anti-inflammatory effect (e.g. acetylic salicylic acid, ketoprofen), which, however, just serve as symptomatic therapy like all
  • feed additives consist of living microorganisms which survive the craw's acidic environment in sufficient numbers to exercise a health promoting effect in the intestine.
  • Such microorganisms are mostly, lactic acid bacteria like Lactobacillus, Enterococcus and Bifidobacterium species.
  • These microorganisms which also belong to the physiologic intestinal flora, are considered to act preventively against diarrhea as pathogen growth-inhibiting competitive germs in addition to further positive characteristics (such as e.g. vitamin production, lactose disintegration or improvement of mineral material resorption) (Newbold, 1995).
  • prebiotics such as non-digestible oligosaccharides and fmctooligosaccharides are frequently used which serve as sole nutrition for probiotic microorganisms to selectively support the microorganisms.
  • prebiotics such as non-digestible oligosaccharides and fmctooligosaccharides are frequently used which serve as sole nutrition for probiotic microorganisms to selectively support the microorganisms.
  • bifidogenic oligosaccharides such as inulin and its hydrolysis product oligofructose and
  • galactooligosaccharides comply with these criteria.
  • Various commercially available products against claves' diarrhea are supplemented with probiotics. Floracid novo (Albrecht) or Mega Bac (Mega Sprint) contain e.g. Enterococcus faecium strain
  • the problem underlying the present invention was the development of a composition being highly efficient in the prevention and/or treatment of diarrhea, particularly in young animals such as calves, piglets, sheep, goats etc.
  • the present invention refers to a composition for feeding animals comprising at least one type of microorganism of the species Lactobacillus reuteri, wherein preferably the microorganism is species-specific for the animal fed with the composition.
  • the composition is for example administered to an animal within 12, 24, 36, 48, 72, 84 or 96 h post partum (p.p.). Examples of such Lactobacillus reuteri axe Lactobacillus reuteri TH1 or Lactobacillus reuteri TH2.
  • composition of the present invention further comprises or consists of one or more different types of microorganisms, which are for example of the genera Lactococcus, Lactobacillus, Leuconostoc Enterococcus, Streptococcus, Propionibacterium,
  • Bifidobacterium Eubacterium, Pediococcus, Clostridium, Pseudomonas, Proteus, Veillonella, Bacteroides and/or Escherichia.
  • composition of the present invention is used in a method of preventing and/or treating diarrhea in animals, wherein animals according to the present invention comprise any mammals or birds such as a calf or cow, a piglet or pig, a foal or horse, a goat, a lamb or sheep, a puppy or dog, a cat, a chicken, a goose, or a duck.
  • animals according to the present invention comprise any mammals or birds such as a calf or cow, a piglet or pig, a foal or horse, a goat, a lamb or sheep, a puppy or dog, a cat, a chicken, a goose, or a duck.
  • Fig. 1 shows that Lactobacillus reuteri is present in a high number in the feces of calves and that the number of Lactobacillus reuteri is significantly reduced in the feces of calves developing diarrhea in comparison to healthy calves, which do not develop diarrhea.
  • Fig. 1 further depicts that also the number of Lactobacillus mucosae significantly decreases in calves developing diarrhea.
  • Straight lines indicate the number of microorganisms in healthy animals and dotted lines indicate the number of microorganisms of animals developing or already suffering from diarrhea.
  • ( ⁇ ) shows the number of Lactobacillus reuteri
  • ( A ) shows the number of Lactobacillus mucosae (p ⁇ 0.05).
  • Fig. 2 refers to the intestinal flora of calves. Calf feces were taken rectally in aseptic conditions in definite, short intervals of a few hours in particular within the first three days of life, starting with the meconium sample. Optionally, a colostrum sample is obtained in addition. The feces samples are taken according to the following scheme: 0 h, 6 h, 12 h, 24 h, 48 h, and 3 d after birth. All samples were bacteriologically investigated, both qualitatively and quantitatively. Different selection enrichment methods guarantee the comprehensive overview on the prevalent germ diversity. The aerobic and the anaerobic mesophilic total germ count as well as Enterobacteriaceae (including E.
  • Salmonella and Campylobacter are selectively enriched according to DIN EN 12824 and on the basis of the method of the Bavarian state agency for health and food safety. A section of the investigation is depictured in Fig. 1. Evaluation of 24 calves shows a significantly reduced germ load of enterococci and lactobacilli within the first 48 h, in particular within the first 24 h after the calves' births.
  • Fig. 3 presents the results of the use of a composition, i.e., a suspension of the present invention comprising Lactobacillus reuteri THl and Lactobacillus reuteri TH2 in a ratio of 1:1 comprising 6 x 10 9 Lactobacillus reuteri /dosage in a method of preventing diarrhea in calves.
  • Fig. 3 shows a significantly decreased incidence for diarrhea in calves receiving the suspension in the first week p.p. (black column; p ⁇ 0.05), in comparison to calves receiving the placebo (white column). Also in the second week p.p., the incidence for diarrhea remains lower in calves receiving the suspension according to the present invention.
  • Fig. 4A depicts a partial sequence of the 16 rRNA gene oi Lactobacillus reuteri THl (SEQ ID No. 1) amplified using the primer pair Coml/Com2
  • Fig. 4B presents a species-specific partial sequence oi Lactobacillus reuteri THl amplified using the primer pair REUT1/LOWLAC (SEQ ID No. 3).
  • Fig. 5A depicts a partial sequence of the 16 rRNA gene oi Lactobacillus reuteri TH2 (SEQ ID No. 2) amplified using the primer pair Coml/Com2
  • Fig. 5B presents a species-specific partial sequence oi Lactobacillus reuteri TH2 amplified using the primer pair REUT1/LOWLAC (SEQ ID No. 4).
  • Fig. 6A presents the RAPD ("Random Amplified Polymorphic DNA") band profile of Lactobacillus reuteri THl and Fig. 6B shows the band profile for Lactobacillus reuteri TH2.
  • RAPD Random Amplified Polymorphic DNA
  • Fig. 7A shows the MALDI-TOF- spectra of Lactobacillus reuteri TH1 and TH2 after treatment with formic acid
  • Fig. 7B and 7C list the exact data forming the basis of the M ALD I-TOF-spectra of Fig. 7A.
  • Fig. 8 depicts the reduced detection frequency of rotaviruses and cryptosporidae, respectively, in calf faeces after administration of a composition of the present invention in comparison to untreated control calves.
  • composition comprises a microorganism of the species Lactobacillus reuteri, e.g., L. reuteri TH1 and/or L.
  • the composition is highly specific, in that the microorganisms of the composition are species-specific for the animal fed with the composition, at least the microorganism(s) of the species Lactobacillus reuteri.
  • microorganisms of the composition for feeding calves are calf- specific microorganism
  • microorganisms of the composition for feeding piglets are piglet- specific microorganisms
  • microorganisms of the composition for feeding goats are goat-specific microorganisms
  • microorganisms of the composition for feeding lambs are lamb-specific microorganisms
  • microorganisms of the composition for feeding puppies are puppy-specific microorganisms
  • microorganisms of the composition for feeding cats are cat-specific microorganisms
  • microorganisms of the composition for feeding birds are bird-specific microorganisms etc.
  • the composition of the present invention is specially balanced for the animal species to which it is administered and its specific intestinal flora, e.g., a calf-specific microorganism of the species Lactobacillus reuteri, optionally in combinations with different species of species-specific or non-species-specific, e.g., calf- specific, gram positive and/or negative microorganisms.
  • the intestinal flora of animals consists of and comprises, respectively, different species of gram positive and gram negative microorganisms whose appearance and composition is typical for the species of a specific animal's intestinal flora, as well as the amount of different
  • microorganisms related to the total amount per g of feces.
  • rumen and colon are the organs with the largest and most comprehensive population of microorganisms, wherein microorganisms comprise in general procaryotes and eukaryotes such as bacteria, archaea, amorphea in particular gram positive and negative bacteria, crenarchaeota, thaumarchaeota, euryarchaeota, fungi and algae.
  • a total amount of germs up to 10 11 CFU per g feces can for example be reached in ruminant animals (Dowd et al., 2008).
  • One important goal of the present invention is the substitution of missing and/or underrepresented microorganisms in the intestine of an animal, e.g., at very young age such as post partum for example as described above.
  • intestinal colonization of the animal such as a calf starts immediately after birth (Braegger, 2004; Nicolet, 1985). Colonization of the newborn first occurs via the oral cavity by germs of the birth channel, the immediate maternal environment, the mammary gland, the dam's feces and the germ species accidentally occurring in the immediate proximity of the newborn (Ducluzeau, 1983; Isik, 2004). This first colon colonization takes place very quickly, however, large differences are encountered: "sterile" samples of meconium, the newborn's first feces, exist as well as samples containing up to 10 8 CFU per g meconium.
  • the newborn's intestinal flora is very instable and numerous dominant colonies disappear after some days or are substituted by others (Favier et al., 2003, Lukas et al., 2007).
  • Administration of colostrum i.e. the administration of the dam's first milk, further nutrition with milk exchangers, stable hygiene, environmental germ flora and further management factors perform a strong influence on the development of the intestinal flora (Pfirrmann and Bohm, 2000).
  • Diarrhea pathogens in animals are, e.g., E. coli, Salmonella spp., Clostridium perfringens and Campylobacter spp., respectively.
  • E. coli belongs to the normal intestine inhabitants and only a part of its serotypes causes infectious diseases. These serotypes differ in so-called virulence factors from "harmless" E. coli. Virulence factors provide the ability to connect to intestinal cells and to produce certain toxins (Kaske und Kunz, 2003). E. coli often appears as secondary pathogen after Rota or Cornea virus infections and worsens the clinical picture (Bothmer and Budde, 1992). For example in newborn calves, diseases appear especially in the first two weeks of life.
  • Salmonella infections may occur in animals, e.g., in cattle of every age in principle, but calves are most sensitive due to their not yet fully developed immune system, wherein an epidemic-like course occurs frequently. The few surviving animals often permanently excrete (“permanent eliminators”) these microorganisms, and thus, constitute a threat to the whole pack or farm. In numbers, for example Salmonella by far play a less important role than E. coli in calves' diarrhea— but due to their zoonosis-like character as well as the gravity of their progression, Salmonella constitute a prominent diarrhea pathogen.
  • Clostridium perfringens can be isolated from the feces of animals, e.g., calves without clinically findings of the calf, and in addition, it is known as etiologic factor in enteric toxicemia (Rycke et al., 1986). Herein developing small intestine necroses are manifested in acute, severe, sometimes bloody diarrhea and heavy pain symptoms (Kaske and Kunz, 2003).
  • Campylobacter spp. are considered likewise as zoonosis pathogens and can be detected both in the feces of animals such as calves without clinical findings as well as in diarrhea-affected calves (Meylan, 2007). It is assumed that pathogens of older permanent eliminators without clinical findings are passed to newborn calves (Hofle, 2006). Upon section, an intense colonization of the colon is detectable; besides mucoid feces, ill calves do not exhibit clear symptoms of disease (Hofle, 2006).
  • the intestinal flora of animals e.g., calves
  • suffering from diarrhea primarily does not exhibit an excessive reproduction of pathogenic microorganisms such as bacteria, but rather that diarrhea is primarily caused by the absence or decline of protective microorganisms.
  • the absence of microorganisms strongly manifests in particular in the first 24 to 48 h after the animal's, e.g. calfs, birth so that this time frame is of high importance for conditioning the animal's such as the calfs intestinal flora and thus, for prevention and/or treatment of diarrhea.
  • the time frame for administering the composition to the animal in need thereof may expand up to 60, 72, 84 or 96 h p.p..
  • the composition of the invention counteracts and/or balances the imbalance of the animal's intestinal flora, which results in prevention and/or treatment of diarrhea.
  • the composition comprises or consists of at least one type of microorganism of the species Lactobacillus reuteri, for example Lactobacillus reuteri THl (6-15-5 Lac 2; deposit no. DSM 29944) or Lactobacillus reuteri TH2 (11-8-5 Lac 1; deposit no. DSM 29945), which have been isolated and characterized for the first time.
  • the 16S rRNA of the Lactobacillus reuteri strain comprises or consists of SEQ ID No.
  • SEQ ID No. 1 e.g., Lactobacillus reuteri THl
  • SEQ ID No. 2 e.g., Lactobacillus reuteri TH2
  • a primer pair Coml/Com2 e.g., Fig. 4A and 5A
  • species-specific partial sequences are identified and amplified using the primer pair REUTl/LOWLAC (e.g., Fig. 4B and 5B), which are for example comprised by Lactobacillus reuteri THl (SEQ ID No. 3) or Lactobacillus reuteri TH2 (SEQ ID No. 4).
  • strains Lactobacillus reuteri THl and Lactobacillus reuteri TH2 are further characterized via the MIC (Minimal Inhibitory Concentration) of antibiotics, wherein the different antibiotics have different inhibitory effect on the growth of the MIC (Minimal Inhibitory Concentration) of antibiotics, wherein the different antibiotics have different inhibitory effect on the growth of the MIC (Minimal Inhibitory Concentration) of antibiotics, wherein the different antibiotics have different inhibitory effect on the growth of the MIC (Minimal Inhibitory Concentration) of antibiotics, wherein the different antibiotics have different inhibitory effect on the growth of the MIC (Minimal Inhibitory Concentration) of antibiotics, wherein the different antibiotics have different inhibitory effect on the growth of the MIC (Minimal Inhibitory Concentration) of antibiotics, wherein the different antibiotics have different inhibitory effect on the growth of the MIC (Minimal Inhibitory Concentration) of antibiotics, wherein the different antibiotics have different inhibitory effect on the
  • Lactobacillus reuteri strains The lowest antibiotic concentration that prevents visible microorganism growth after overnight incubation. Table 1 shows characteristic MIC of the different antibiotics indicated in Table 1 for Lactobacillus reuteri THl:
  • Table 2 presents characteristic MIC of the different antibiotics indicated in Table 2 for Lactobacillus reuteri TH2: Table 2
  • Lactobacillus reuteri THl and Lactobacillus reuteri TH2 are further characterized by the MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) spectra depicted in Fig. 7A.
  • MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • Fig. 7A secretorescence mass spectrometry
  • the ratios of Lactobacillus reuteri species such as L. reuteri THl and/or L. reuteri TH2, and other types of microorganisms vary.
  • the ratios are for example 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the pH of the composition is in the range of 3.5 to 5.5, or 4.0 to 5.0, or at 4.4, where the microorganism of the strain Lactobacillus reuteri, e.g., L. reuteri THl and/or L. reuteri TH2, produces an antibacterial agent, which for example inhibits the growth of pathogens such as E. coli, Salmonella Typhimurium,
  • Clostridium perfringens etc. see for example Tables 3 and 4).
  • more neutral pH e.g., pH 6.1 to 7.4 or 6.5 which is for example the average pH in a calf intestine L. reuteri THl and/or L. reuteri TH2 for example does not show an antibiotic effect.
  • the composition comprises further one or more different types of microorganisms in addition to a microorganism of the species of Lactobacillus reuteri, which are particularly gram positive and/or negative bacteria.
  • the composition further or alternatively comprises an agent selected from the group consisting of an astringent, an adsorbent, inulin, a motility inhibitor, a protein, a lipid, a vitamin and a mineral material.
  • the composition is administered to the animal any time of its life for use in a method of preventing and/or treating diarrhea.
  • the composition is administered to the animal directly after birth, in particular in the first hours, the first days and/or first weeks after birth (p.p.).
  • composition of the present invention is administered for example within the first 1 to 48 h p.p., within the first 1 to 36 h p.p., within the first 1 to 24 h p.p., within the first 1 to 12 h p.p., within the first 1 to 6 h p.p., or within the first 1 to 2 h p.p..
  • the composition is administered within the first 1 to 6 months p.p., within the first 1 to 4 months p.p., within the first 1 to 2 months p.p., within the first 1 to 4 weeks p.p., within the first 1 to 2 weeks or within the first week p.p.
  • the composition is administered prenatally to the animal via the dam. In another embodiment the composition is administered to the animal prenatally and within the first 1 to 2 weeks p.p., within the first 24 to 48 h p.p., or within any time period indicated previously.
  • the composition of the present invention is added as dietary supplement to standard animal feed, or is administered to an animal as sole nutrition.
  • the composition results in the conditioning of the intestinal flora in still unborn or newborn animals, whereby a possible intestinal flora's imbalance of newborn calves is balanced by the microorganism of the composition, i.e. in view of the different species of microorganisms, and/or in view of the amount of individual microorganisms of different species in relation to the total amount of microorganisms.
  • a microorganism of the species Lactobacillus reuteri for example Lactobacillus reuteri TH1 or TH2 is essential in the composition.
  • composition further comprises or consists of one or more additional types of microorganisms, which are for example selected from the group consisting of genera Lactococcus, Lactobacillus, Leuconostoc, Enterococcus, Streptococcus,
  • Examples of these genera forming part of the composition according to the present invention are for example selected from the group consisting of Lactococcus chungangensis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. diacetilactis, Lactococcus lactis subsp. horidae, Lactococcus lactis subsp.
  • Lactis Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus garviae, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus parabuchneri, Lactobacillus ferintoshensis, Lactobacillus salivarius, Lactobacillus saerimneri, Lactobacillus buchneri, Lactobacillus curvatus, Lactobacillus agilis, Lactobacillus ingluviei, Pediococcus pentosaceus, Lactobacillus
  • Lactobacillus kimchii Lactobacillus manihotivorans, Lactobacillus perolens,
  • Lactobacillus ruminis Lactobacillus ruminis, Leuconostoc lactis, Leuconostoc pseudomesenterioddes,
  • Enterococcus asini Enterococcus villorum, Enterococcus haemoperoxidus, Enterococcus moraviensis, Enterococcus ratti, Enterococcus pollens, Enterococcus gilvus,
  • Pediococcus inopinatus Pediococcus parvulus and Pediococcus pentosaceus.
  • the total number of microorganisms is 1 x 10 5 to 5 x 10 12 /dosage, 3 x 10 5 to 3 x 10 12 /dosage, 5 x 10 5 to 10 10 /dosage, or 6 x 10 9 /dosage.
  • the composition of the present invention comprises or consists of a combination of characteristic microorganisms typical for the specific animal in amounts typical and characteristic for the animal.
  • composition is for example administered orally, nasally, cutaneously, rectally or intravaginally for example in form of a tablet, a suspension, a solution, a gel, a powder, an ointment or a suppository, wherein the components are for example blended in the ratio desired for the individual and particular needs of the animal.
  • the composition is for example administered orally, nasally, cutaneously, rectally or intravaginally for example in form of a tablet, a suspension, a solution, a gel, a powder, an ointment or a suppository, wherein the components are for example blended in the ratio desired for the individual and particular needs of the animal.
  • the composition is for example administered orally, nasally, cutaneously, rectally or intravaginally for example in form of a tablet, a suspension, a solution, a gel, a powder, an ointment or a suppository, wherein the components are for example blended in the ratio desired for the individual and particular needs
  • the rectal or intravaginal dosage form is supplied with an antibiotic.
  • the composition is liquid or solid.
  • the composition is administered to the dam for example intravaginally in some embodiments, e.g. 1 to 10 days prior to birth, preferably 1 to 5 days prior to birth, in particular preferably 1 to 2 days prior to birth.
  • composition of the present invention comprising at least one type of microorganism of the species Lactobacillus reuteri, e.g., L. reuteri THl and/or L. reuteri TH2, results for example in, but is not limited to, a specific significant reduction of the detection frequency of rotaviruses, a non-enveloped RNA virus belonging to the family Reoviridae, and/or a reduction of the detection frequency of Cryptosporidium, a genus of protozoans in calf faeces.
  • the reasons may be for example
  • Lactobacillus reuteri RC-14 for example is described to produce cyclic dipeptides suppressing the synthesis of toxic shock syndrome toxin- 1 in staphylococcus strains (e.g., Li et al., 2011: Lactobacillus rewterj-produced cyclic dipeptides quench agr-mediated expression of toxic shock syndrome toxin- 1 in staphylococci. PNAS, 108, 3360 - 3365).
  • Example 1 Use of a composition of the present invention for preventing diarrhea
  • a composition, in the present experiments a suspension, of the present invention comprising Lactobacillus reuteri TH1 and Lactobacillus reuteri TH2 in a ratio of 1:1 was administered to newborn calves within the first 24, 48, 72, and 96 h p.p.
  • the total number of Lactobacillus reuteri TH1 and TH2 was 6 x 10 9 / dosage.
  • the composition was administered to 83 calves and further 83 calves, forming the control group, received a placebo at the above mentioned time points.
  • the placebo did visually not differ from the suspension comprising Lactobacillus reuteri TH1 and TH2.
  • the status of the calves' health regarding the calves coming down with diarrhea was monitored for 14 days.
  • Fig. 3 Significantly less calves of the group receiving the composition comprising Lactobacillus reuteri TH1 and TH2 came down with diarrhea in comparison to the group of claves receiving placebo.
  • the incidence of diarrhea in the placebo group was 42.2 %, whereas the incidence of diarrhea in the group of calves treated with the composition according to the present invention was only 27.7 % in the first week p.p. This results in a significant reduction of 35 % of the risk for coming down with diarrhea.
  • Example 2 RAPD band profiles for Lactobacillus reuteri TH1 and TH2
  • RAPD band profiles were prepared for 57 isolates of Lactobacillus reuteri (according to standard methods), and only the profiles of healthy animals, not suffering from diarrhea, were used for the evaluation of the microorganisms mainly expressed in the feces of calves. These profiles show that Lactobacillus reuteri THl (6-15-5 Lac 2) and TH2 (11-8-5 Lac 1) are present in a significant number in healthy calves (see Fig. 6A and 6B).
  • Example 3 Production of antibacterial agents by Lactobacillus reuteri
  • Lactobacillus reuteri strains such as Lactobacillus reuteri THl and TH2 were isolated from feces of different calves, cultured in MRS-Boullion (Lactobacillus- Bouillon according to De Man, Rogosas und Sharpe) and the supernatants of these strains were tested in an agar diffusion test comprising Escherichia coli, Salmonella Typhimurium and Clostridium perfringens as test organisms according to standard proceedings.
  • the pH of the MRS-Boullion was kept stable with a buffer at pH 6.5, in the other group the MRS-Boullion reached a pH 4.4 due to the metabolism of the cultured Lactobacillus reuteri THl and TH2.
  • Lactobacillus reuteri THl and TH2 produced an antibacterial agent, which inhibited the growth of Escherichia coli, Salmonella Typhimurium as well as of Clostridium perfringens.
  • such antibacterial agent is not detectable.
  • Table 3 shows the antibacterial activity of the supernatant of a liquid- culture oi Lactobacillus reuteri THl against selected pathogens and Table 4 presents the activity of the supernatant a liquid- culture of Lactobacillus reuteri TH2 against selected pathogens (indicated in the tables):
  • Example 4 MALDI-TOF spectra oi Lactobacillus reuteri THl and Lactobacillus reuteri TH2
  • the mass spectra generated are analyzed by a Bruker Autoflex instrument and compared with stored profiles.
  • the MALDI-TOF spectra of Lactobacillus reuteri TH1 and Lactobacillus reuteri TH2 are shown in Fig. 7A, the raw data underlying the spectra are presented in Fig. 7B and 7C.
  • Example 5 Reduction of diarrhea causing microorganisms in calf faeces
  • New born calves were separated in two groups.
  • the faeces of the calves were investigated as well as the faeces of the untreated calves.
  • Microorganisms were detected in the faeces via ELISA and * shows significant differences (Chi-Quadrat-Test; p ⁇ 0.05).
  • the results are shown in Fig. 8: The number of the detection frequency of rotaviruses and the number of the detection frequency of crypto sporidae are reduced in the faeces of the calves after administration of a composition of the present invention in comparison to the number of the detection frequency of rotaviruses and crypto sporidae in faeces of the control animals.

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Abstract

La présente invention concerne une composition pour l'alimentation d'animaux comprenant au moins un type de micro-organisme de l'espèce Lactobacillus reuteri, ledit micro-organisme étant spécifique à l'espèce pour l'animal nourri avec la composition. La composition est utilisée dans une méthode de prévention et/ou de traitement de la diarrhée par la compensation de l'absence de ces organismes chez les animaux. De plus, les micro-organismes de l'espèce Lactobacillus reuteri produisent un agent antibactérien à un pH situé dans la plage allant de 3,5 à 5,5.
EP16702517.0A 2015-01-30 2016-01-28 Composition destinée à être utilisée dans la prévention et/ou le traitement de la diarrhée chez les animaux Withdrawn EP3250217A1 (fr)

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WO2022169933A2 (fr) * 2021-02-03 2022-08-11 Dupont Nutrition Biosciences Aps Compositions pour la santé intestinale
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CN114836358B (zh) * 2022-06-28 2023-02-24 中国农业科学院北京畜牧兽医研究所 罗伊氏乳杆菌sxdt-32及其应用
CN116286496B (zh) * 2023-02-19 2023-11-14 浙江大学 一种降低断奶仔猪腹泻的复合益生菌及其应用
CN115960775B (zh) * 2023-02-28 2024-03-12 吉林大学 一株增加雏鹅的生产性能并抗痛风的唾液乳杆菌

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