EP3250217A1

EP3250217A1 - Composition for use in preventing and/or treating diarrhea in animals

Info

Publication number: EP3250217A1
Application number: EP16702517.0A
Authority: EP
Inventors: Johann Bauer; Karin Schwaiger
Original assignee: Technische Universitaet Muenchen
Current assignee: Technische Universitaet Muenchen
Priority date: 2015-01-30
Filing date: 2016-01-28
Publication date: 2017-12-06
Also published as: WO2016120405A1

Abstract

The present invention refers to a composition for feeding animals comprising at least one type of microorganism of the species Lactobacillus reuteri, wherein the microorganism is species-specific for the animal fed with the composition. The composition is used in a method of preventing and/or treating diarrhea by compensating lack of these organisms in animals. In addition, microorganisms of the species Lactobacillus reuteri produce an antibacterial agent at a pH in the range of 3.5 to 5.5.

Description

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. Technical background

Diarrhea is one of the major problems in animals, particularly young animals, in particular farm animals such as calves, piglets, lambs, goats etc. Despite

improvements in management practices and prevention and treatment strategies, diarrhea is still the most common and costly disease affecting neonatal animals. A study at the U.S. Sheep Experiment Station (Dubois, ID) showed that diarrhea accounted for example for 46 % of lamb mortality. Diarrhea in animals is a complex, multi-factorial disease involving the animal, the environment, nutrition, and infectious agents.

In Germany, about 5 million calves are born per year, whereof still more than 10% of all newborn calves perish within the first half year despite intensive therapeutic measures in individual therapy and flock-prophylactic measures, respectively. About 75% of losses in calves are traced back to infectious diseases (DLZ, 2006; Bothmer and Budde, 1992). Therein, diarrhea plays an important role and is on top, with about 80% of infection- determined losses.

Most notably, 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. Providing that about 20% of all living-born calves come down with diarrhea (Metz und Metz, 1984), further damages of€ 58.2 million occur in Germany based on medical costs of€ 60 per diseased calf. Further subsequent damages which are difficult to be calculated, accrue via reduced breeding progress, diminished breeding selection options and reduced performance in adolescent or even adult cattle, as frequency and severity of calf disease strongly correlate to the later performance as dairy cow: adolescent animals which did not or only one time require veterinarian treatment during growing show a significant lower first birth age and a higher lactation performance than calves which required treatment. The importance of calfs health becomes in particular evident in view of the useful life: Of those calves being healthy during growing, only about 20% go off during first lactation, while the respective percentage of frequently treated calves accounts for 60% (Trilk und Miinch, 2004). Further, the necessary additional animal purchase bears the risk of germ introduction which is not to be underestimated (Brandle, 2006).

Animals suffering from strong diarrhea may lose up to 20% of their body mass in liquid. Along with said liquid electrolytes such as sodium, potassium, chloride and hydrogen carbonate are lost. Finally, following dehydration, acidosis and hypoglycemia the general condition is rapidly worsened extending to so-called "downer calves". Thus, early liquid and electrolyte substitution as well as coverage of the nutritional requirement are considered the most important measures. This is achieved by intravenous infusion in severe cases such as in insufficiently drinking calves or "downer calves". 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.

Enerlyte/Virbac; Glutellac/Bayer; Diatmix/Bewital; Medolyt/VetroStar MS; Diakur plus/Boehringer Ingelheim, Effydral/Essex; Floracid Novo/Albrecht). 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

consideration, which apply for animals in food production, various allaying agents are available such as adsorbents (aluminum silicate, carbon medicinalis), astringents (e.g. tannin or tannic acid), or motility inhibitors, whose application is, however, prohibited in food producing animals. Additionally, non-steroidal anti-inflammatory drugs (NSAID) can be employed for an anti-inflammatory effect (e.g. acetylic salicylic acid, ketoprofen), which, however, just serve as symptomatic therapy like all

aforementioned anti-diarrhea agents (Rosa Liste, "Rose list", 2009). Timely and sufficient first milking care is considered the most important factor in protection from intestinal infections. Vaccination of the dam prior to birth can further improve the colostrum's protective effect as the produced antibodies cross over to the milk and protect the calf from infections, which in turn prevents and reduces pathogen reproduction and excretion, respectively. Thereby, in combination with optimized keeping and hygiene conditions, infection stress on newborn calves should be reduced (Mansfeld et al., 2005). Probiotics are increasingly administered orally to support as well as to normalize the intestinal flora. These 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). In addition, prebiotics such as non-digestible oligosaccharides and fmctooligosaccharides are frequently used which serve as sole nutrition for probiotic microorganisms to selectively support the microorganisms. To date, only 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

"Cernelle 68" at a concentration of almost 10¹⁰ CFU/kg. Although diverse studies showed a certain positive effect of individual strains in calves for example (Beeman, 1985; Bonaldi et al. 1986; Umburger et al., 1989), losses caused by calves' diarrhea still remain a serious problem. Herein it is to be considered that the probiotic strains were originally not intended for specific employment in calves. For example, "Cernelle 68" was isolated from the human intestine. As the intestinal flora exhibits significant differences between various animal species and humans, related employment of arbitrarily chosen microorganisms not originating from the homologous animal species (i.e., non-species-specific) has to be evaluated critically. US 2011/0189132 Al refers to a composition comprising at least one probiotic microorganism inhibiting or reducing a population of pathogenic bacteria in or on an animal.

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.

Summary of the invention 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.

The 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.

The 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. Figures

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, and (^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. coli and Salmonella), Campylobacter, enterococci and lactobacilli were determined. The germ content was determined according to the surface- spatula- method on the basis of Gedek (1974). Thus, samples were streaked onto the following culture media after preparation of a dilution row and are counted out after incubation for 48 h at 37°C: Blood agar (aerobic mesophilic total germ count), Schaedler agar (anaerobic mesophilic total germ count), Gassner agar (Enterobacteriaceae), CATC agar (Enterococci) and LAMVAB agar (Lactobacilli). Additionally, Salmonella and Campylobacter, respectively, 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. Total germ count of aerobic microorganisms in healthy calves (-♦-) and calves which came down with diarrhea within 14 days post partum (--0--), the total germ count of anaerobic microorganisms in healthy calves (-■-) and in calves which came down with diarrhea (--□—), the count of enterococci in healthy calves (- ^A-) and in calves which came down with diarrhea within 14 days post partum (--Δ-), the count of Enterobacteriaceae in healthy claves (- ·-) and in calves which came down with diarrhea within 14 days post partum (--o--), as well as the count of lactobacilli in healthy calves (— x— ) and in calves which came down with diarrhea within 14 days post partum (--x--) were determined.

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 10⁹ Lactobacillus reuteri /dosage in a method of preventing diarrhea in calves. The suspension was administered to the claves (n=83) 24 h, 48 h, 72 h, and 96 h p.p., a control group of calves (n=83) received a placebo. 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, and 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, and 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. In the RAPD band profiles the species are identified, which are mainly expressed in healthy calves. 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.

Detailed description

In the present invention it was surprisingly identified that animals, particularly young animals right after birth, running the risk of suffering from severe diarrhea show a significant decrease of the content of specific types of microorganisms in the gastrointestinal tract even before the animal suffers from diarrhea. Even more surprising was that the most important microorganism, whose content is decreased in these animals, is a microorganism of the species Lactobacillus reuteri (see Fig. 1).

The great advantage of the present invention is that the composition comprises a microorganism of the species Lactobacillus reuteri, e.g., L. reuteri TH1 and/or L.

reuteri TH2, and is administered to the animal, e.g., early after birth (e.g., 12, 24, 36, 48, 60, 72, 84 or 96 h p.p.), even if the animal is not yet suffering from a diarrhea, i.e., a prophylactic administration. Further, 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.

Moreover, for example 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. Hence, 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 dense colonization of the intestine's inner wall by microorganisms, in particular bacteria which are at first missing in newborn mammals, is designated an intestine flora. 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. In ruminant animals, 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. In the colon, a total amount of germs up to 10¹¹ 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.

According to doctrine, 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 10⁸ CFU per g meconium. About 48 h after birth, animals reach a maximum germ load of 10⁸ CFU per g feces (Ducluzeau, 1983; Jimenez et al., 2008). However, the newborn's intestinal flora differs significantly from the one of grown-up animals. Primary settlers are mostly aerobic microorganisms which serve as "environment preparers" for subsequent colonization of anaerobic germs. Anaerobic microorganisms finally dominate in grown-up animals, and aerobic microorganisms account for just 10% of the intestinal flora (Isik, 2004). 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. In the beginning, the thin feces are of yellow color, later it turns aqueous, inter stratified with unmetabolized clotted milk compounds (Bothmer and Budde, 1992). The cause, in general, is considered to be strong colonization of the intestinal mucosa by E. coli with late or insufficient administration of colostrum. After oral take-up, Salmonella spp. migrates into the small intestine. Due to massive reproduction, they lead to an inflammation in the intestine associated with bad smelling, at first yellowish, later grey-greenish and finally dark brown to black diarrhea and temperature. Most animals die within a few days due to fluid and electrolytes loss or due to bacteria migrating via the intestinal mucosa into the blood, leading to central nervous disorders in connection with palsy. 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).

Animals' intestine flora exhibits differences in a qualitative and quantitative aspect already before the occurrence of the diarrhea, i.e. before the occurrence of clinical symptoms. An examination of calves' intestine flora for example showed this effect as demonstrated in Fig. 1 and 2. Based on these results it is presented that these differences are particularly significant within the first 24 to 48 h after a calf s birth. Some microorganisms, in particular bacteria, are missing or are reduced in number in the intestinal flora of diarrhea-affected animals, e.g., calves or of those that will suffer from diarrhea. These are in particular enterococci and/or lactobacilli (for example Fig. 2). It was surprisingly found out that 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..

Thus, the use of 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. In some embodiments, the 16S rRNA of the Lactobacillus reuteri strain comprises or consists of SEQ ID No. 1 (e.g., Lactobacillus reuteri THl) or of SEQ ID No. 2 (e.g., Lactobacillus reuteri TH2), which is amplified from 16 rRNA of the Lactobacillus reuteri strain using a primer pair Coml/Com2 (e.g., Fig. 4A and 5A). Moreover, in some embodiments 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).

The 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

Lactobacillus reuteri strains. MIC is 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 1

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 is a rapid, reliable, and high- throughput diagnostic tool for the identification of microorganisms. The technology is unique, e.g., in clinical microbiology, allowing laboratories to definitively identify bacterial and fungal isolates within minutes. In some embodiments the ratios of different Lactobacillus reuteri species such as L. reuteri THl and L. reuteri TH2, and/or 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. In some embodiments, 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). At a higher, 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.

In some embodiments, 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. Optionally, 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. In some embodiments, 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.). The 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.. In other embodiments, 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.

In one embodiment, 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.

In some embodiments, the composition of the present invention is added as dietary supplement to standard animal feed, or is administered to an animal as sole nutrition. Particularly advantageous, 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.

The 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,

Propionibacterium, Bifidobacterium, Eubacterium, Pediococcus, Clostridium, Pseudomonas, Proteus, Veillonella, Bacteroides and/or Escherichi. 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 casei, Lactobacillus coryniformis, Lactobacillus oris, Lactobacillus kefiri, Lactobacillus diolivorans, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus nantensis, Lactobacillus parakefiri, Lactobacillus fructivorans,

Lactobacillus kimchii, Lactobacillus manihotivorans, Lactobacillus perolens,

Lactobacillus ruminis, Leuconostoc lactis, Leuconostoc pseudomesenterioddes,

Enterococcus faecium, Enterococcous faecalis, Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus gallinarum, Enterococcus hirae, Enterococcus lactis, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus raffinosus, Enterococcus pseudoavium, Enterococcus cecorum, Enterococcus columbae, Enterococcus saccharolyticus, Enterococcus dispar, Enterococcus sulfureus,

Enterococcus asini, Enterococcus villorum, Enterococcus haemoperoxidus, Enterococcus moraviensis, Enterococcus ratti, Enterococcus pollens, Enterococcus gilvus,

Enterococcus seriolocida, Enterococcus solitarius, Enterococcus flavescens, Escherischa coli, Bifidobacterium adolescentis, Bifidobacterium animals subsp. animalis,

Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium ruminantium, Pediococcus acidilactici, Pediococcus cellicola,

Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans,

Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus.

In some embodiments the total number of microorganisms is 1 x 10⁵ to 5 x 10¹²/dosage, 3 x 10⁵ to 3 x 10¹²/dosage, 5 x 10⁵ to 10¹⁰/dosage, or 6 x 10⁹/dosage. In some embodiments, 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. 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. In some embodiments, the composition

additionally comprises for example binders, disintegrates, surfactants, adsorbing promoters, wetting agents, adsorbing agents, lubricants, foaming agents, fillers, extenders, humectants and similar diluents or excipients, aseptic agents, coloring agents, antioxidant agents or preservers. In a further embodiment, the rectal or intravaginal dosage form is supplied with an antibiotic. In most of the embodiments, the composition is liquid or solid. In case of prenatal prophylaxis of the animal, 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.

Administration of a 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

- production of substances such as exopolysaccharides which block the binding sides for microorganisms on the cell surface which is for example essential for the

reproduction of a virus or sporidium, and/or - modulation of the metabolism of the microorganism regarding the expression of virulence factors. 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).

Examples

The following examples represent specific embodiments of the present invention for a better and more detailed understanding of the invention. However, the present invention is not limited to these embodiments. 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 10⁹ / dosage. In this example 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.

The results of these experiments are shown in 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

Different 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. In one group 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. In the MRS-Boullion having pH 4.4 Lactobacillus reuteri THl and TH2 produced an antibacterial agent, which inhibited the growth of Escherichia coli, Salmonella Typhimurium as well as of Clostridium perfringens. In the MRS-Boullion having pH 6.5 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):

Table 3

^* Zone of inhibition in mm

Table 4

^* Zone of inhibition in mm

Example 4: MALDI-TOF spectra oi Lactobacillus reuteri THl and Lactobacillus reuteri TH2

A colony oi Lactobacillus reuteri THl and Lactobacillus reuteri TH2, respectively, after treatment with formic acid, is smeared directly on the sample target and overlaid with matrix to perform the MALDI-TOF spectra. 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. In one group (n = 83) a composition 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 10⁹ / dosage. To the animals of the other group (control group; n = 83) the composition of the present invention was not administered. 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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Claims

Composition for feeding animals comprising at least one type of

microorganism of the species Lactobacillus reuteri, wherein the microorganism is species-specific for the animal fed with the composition.

Composition of claim 1, wherein the Lactobacillus reuteri species is

Lactobacillus reuteri TH1 (6-15-5 Lac 2) and/or Lactobacillus reuteri TH2 (11- 8-5 Lac 1).

Composition of any of claims 1 or 2, wherein the gene of the 16S rRNA of the Lactobacillus reuteri strain comprises SEQ ID No. 1 or SEQ ID No. 2.

Composition of any one of claims 1 to 3 further comprising a microorganism of the genera Lactococcus, Lactobacillus, Leuconostoc Enterococcus,

Streptococcus, Propionibacterium, Bifidobacterium, Eubacterium, Pediococcus, Clostridium, Pseudomonas, Proteus, Veillonella, Bacteroides and/or

Escherichia.

Composition of claim 4, wherein one or more further microorganism(s) is/are 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 casei, Lactobacillus coryniformis, Lactobacillus oris,

Lactobacillus kefiri, Lactobacillus diolivorans, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus nantensis, Lactobacillus parakefiri, Lactobacillus fructivorans, Lactobacillus kimchii, Lactobacillus manihotivorans, Lactobacillus perolens, Lactobacillus ruminis, Leuconostoc lactis, Leuconostoc pseudomesenterioddes, Enterococcus faecium, Enterococcous faecalis, Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus gallinarum, Enterococcus hirae,

Enterococcus lactis, Enterococcus malodoratus, Enterococcus mundtii,

Enterococcus raffinosus, Enterococcus pseudoavium, Enterococcus cecorum, Enterococcus columbae, Enterococcus saccharolyticus , Enterococcus dispar, Enterococcus sulfureus, Enterococcus asini, Enterococcus villorum,

Enterococcus haemoperoxidus, Enterococcus moraviensis, Enterococcus ratti, Enterococcus pollens, Enterococcus gilvus, Enterococcus seriolocida,

Enterococcus solitarius, Enterococcus flavescens, Escherischa coli,

Bifidobacterium adolescentis, Bifidobacterium animals subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum,

Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium ruminantium, Pediococcus acidilactici, Pediococcus cellicola, Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus.

Composition according to any one of claims 1 to 5 further comprising an agent selected from the group consisting of an astringent, an adsorbent, inulin, a motility inhibitor, a lipid, a protein, a vitamin and a mineral.

Composition of any one of claims 1 to 6, wherein the composition is liquid or solid.

Composition of any one of claims 1 to 7, wherein the composition is a suspension, gel, powder, tablet, or ointment.

Composition of any one of claims 1 to 8, wherein the animal fed with the composition is a calf, a piglet, a foal, a goat, a lamb, a puppy, a cat, a chicken, a goose, or a duck.

10. Composition of any one of claims 1 to 9 for use in a method of preventing and/or treating diarrhea, wherein the composition is administered within 12, 24, 36, 48, 72, 84, or 96 h p.p..

11. Composition for use according to claim 10, wherein the pH is in the range of

3.5 to 5.5, where the microorganism of the strain Lactobacillus reuteri produces an antibacterial agent.

Composition for use according to claim 10 or 11, wherein the composition is administered orally, nasally, cutaneously, vaginally or rectally.

Lactobacillus reuteri THl (6-15-5 Lac 2; deposit no. DSM 29944) comprising SEQ ID No. 1 and/or 3.

14. Lactobacillus reuteri TH2 (11-8-5 Lac 1; deposit no. DSM 29945) comprising

SEQ ID No. 2 and/or 4.

15. Lactobacillus reuteri THl or Lactobacillus reuteri TH2 according to claim 13 or 14 further characterized by the MALDI-TOF spectrum of Fig. 7A.