DESCRIPTION

The present description refers to the use of one or more strain of the species Lactobacillus mucosae in the treatment and/or prevention of gastroenteritis from £ coli in animals including humans and pharmaceutical or veterinary compositions, probiotics food, additives and food supplements containing the same.

STATE OF ART

Escherichia coli is a commensal bacterium making up the microflora of the gastrointestinal track of many animals, including humans. However, there are many £ coli strains with high pathogenicity that are responsible of enteritis, toxemia and septicemia.

Several studies have shown that some strains of E.coli are able to adhere to the intestinal mucosa, by adhesins and to subsequently produce enterotoxin causing diarrhea in mammals including humans.

£. coli infection is, in fact, described as one of the major causes of enteritis. In particular the £ coli strain 0157:H7 has been for the first time identified as a pathogenic agent in 1982 following an enteric epidemic in the United States, associated with the consumption of contaminated hamburgers, having even fatal implications.

The infection by pathogenic strains of £ coli is seriously affecting the gastrointestinal tract mostly of young people, with very short incubation times, whereas in adults £ coli acts by infecting various organs and systems.

Amongst this strain there are recognised 171 serotypes characterized by different combinations of antigens O, H, K, F . It is one of the main species of bacteria living in the lower intestines of warm-blooded animals (including birds and mammals).

Although it represents a common intestine inhabitant and plays a key role in the digestive process, there are situations in which £ coli can cause diseases in humans and animals. Some £ coli strains are the causative agent of intestinal and extra intestinal diseases such as urinary tract infections, meningitis, peritonitis, septicaemia and pneumonia.

Some £ coli strains are toxigenic, i.e. producing toxins that can cause diarrhoea. Dysentery from £ coli is a common food-borne poisoning, because is mainly contracted by contaminated food. The contamination can take place from poorly cooked infected meat, from unpasteurized milk and cheese products, and from other food contaminated with faeces. £ coli produces four types of toxins that are differentiated by their different sensitivity to heat treatment, in heat-labile and thermostable, and for the toxigenic action (shiga toxin and haemolytic toxin, HlyA).

The heat-labile toxin, called LT, is very similar in its structure and function to cholera toxin. It contains one 'A' subunit and five 'B' subunits in an olotoxin. The B subunits contribute to the adhesion and to the entry of the toxin into the host intestinal cells, whereas the A subunit stimulates the cells to release water causing diarrhoea.

Many £ coli strains are known to have characteristics that can be harmful to a host animal. Although in most adults (animals or humans) the probability of a pathogenic strain causing a disease more severe that diarrhoea or being completely asymptomatic is relatively high, this is not true for puppies or young children or in people/animals that are ill or that are debilitated by a recent illness, or in people/animals under special treatments.

There are in fact known pathogenic strains of £. coli that can cause serious illness and even death. An example of a particularly virulent strain of £ coli is E.coli 0157:1-17. The enteropathogenic £ coli (EPEC) was the first pathogenic agent associated with diarrheal diseases and is still a major cause of serious diseases in paediatric patients and in young animals that have not yet reached the stage of adulthood. The disease is caused by the fact that these strains are able to adhere to the small intestine epithelium and to interfere with the absorption of the substances thus causing the formation of a hyperosmolar environment in the intestine lumen, and a subsequent capture of water and finally diarrhoea.

The framework of the adhesion to the epithelium and destruction of microvilli justifies the definition of A E histopathology (attachment/effacement). Another classification of pathogenic £ coli recognizes the enterotoxigenic £ coli (ETEC). These strains of £ coli can cause very serious gastroenteritis, in particular in travellers (such as humans or an imals when transferred from one farm to another) and in individuals who ingested foods or liquids contaminated by faeces. I n th e USA about 80,000 cases a year of i nfections caused by ETEC are registered. Furthermore, the strains of enterohaemorrhagic £ coli (EHEC) are the main responsible for diseases in industrialized countries. It is estimated that these bacteria cause infection in about 73,000 human beings and nearly 600 deaths each year in the USA. About 50 serotypes cause disease; however the primarily responsible is the serogroup 0157:1-17. The ingestion of less than 100 bacteria can cause the disease, that has associated with the consumption of poorly-cooked beef, unpasteurized milk, contaminated fruit juice (e.g. from bovine faeces) and raw vegetables. The disease discloses itself in the large intestine after an incubation period of 3-4 days during which non-bloody diarrhoea appears. After about three days, the disease has an acute phase with severe abdominal pain and bloody diarrhoea. The appearance of blood in the diarrhoea is caused by toxins (Stx-1 and Stx-2) secreted by EHEC bacteria that cause the destruction of enterocytes. The production of Stx-2 is often associated (in 10% of children with diarrhoea caused by EHEC) to the haemolytic uremic syndrome (HUS), characterized by: acute renal failure, thrombocytopenia, haemolytic anaemia, microangiopathy.

In fact, Stx-2 is able to bind with higher affinity to Gb3 expressed by renal cells, causing their destruction. Stx toxins are also capable of stimulating the TNF-a ad interleuchina-6 production that while supporting the inflammatory situation, promote the exposure of Gb3. £ coli 0157:H7, in contrast to other serotypes, does not ferment sorbitol, this allows its identification in Mac Conkey agar containing sorbitol (evaluating the colonies that lack the fermentation activity). However, the bacterial culture analysis must be accompanied by the identification of toxins by commercial immunoassay. The 0124, 0143 and 0164 strains (enteroinvasive £ coli, El EC) can cause bloody diarrhoea (are also white blood cells are present), abdominal cramps and fever. Enteroaggregant £. coli Pathogenic strains (EAEC) are involved in a persistent watery diarrhoea in travellers and infants in developing countries. These strains express both Bfp and AAF/1 , AAF/2 and AAF/3 (all encoded by a plasmid), that are adhesion factors promoting colonization of the small intestine, with stimulation of mucus production. This forms a bio film that is able to isolate and aggregate bacteria. The aggregation is followed by a reduction of the microvilli length, mononucleata infiltration and haemorrhage.

The £ coli pathogenic strains of are also defined as verocytotoxic (VTEC), this definition seems to overlap, in the literature data, to that of EHEC, since it is reported, about VTEC strains that the infections by £ coli 0157 and other VTEC pathogens are an emerging problem of public health for humans. In fact, they have been found responsible of epidemic in several countries, with many persons involved, and causing seriou s path ologies as hemorrhagic colitis and emoliticuremic sindrom (EUS) with fatal outcome in the children and in the elderly. Strains diffusely adherent of £ coli (DAEC) cause a benign watery diarrhoea in children aged 1 to 5 years. The clinical picture shows a lengthening of the microvilli of the enterocytes of the small intestine, with incorporation of bacteria in the enterocytes. These represent particularly serious clinical manifestations of infection by £ coli reported, in particular, on paediatric subjects (definition expanded to not- adult animals). Enteritis caused by £ coli, which as mentioned above are the most common manifestations of £ coli infection in young individuals, are characterized by inflammation of the intestinal mucosa. The higher susceptibility in young subjects appears to be due to the absence of antibodies at birth, whose acquisition occurs only through ingestion of colostrum. Therefore an £. coli infection in the first hours or days of life can become fatal, in particular in newborn humans or animals, due to the excessive dehydration caused by diarrhoea.

£ coli infections in humans are mainly caused by the consumption of contaminated food (ground beef, unpasteurized milk, cured sausages, and cheese from raw milk). The highest contamination risk is relative to undercooked meat and unpasteurized milk. In addition, a significant risk is due to the contamination of acid foods, as pathogenic £ coli strains are known that have a marked acid tolerance which allows them to survive for long periods in the foods.

£ coli contamination of foods is mainly due to the fact that livestock is the major reservoir of the £ coli bacterium. In particular, cattle such as calves are the most susceptible to infections caused by this bacterium. As already mentioned above, this infection in young individuals is characterized by high mortality due to enteritis during the first months of life, resulting in considerable economic losses for the farmer. The therapy against £ coli infections by consists in the administration of electrolyte solutions to restore the proper water and salt balance made preca riou s by dehyd ration with concom itant use of active anti biotics or, alternatively, in carrying out a prophylactic vaccination. I t must be stressed, however, that antibiotic therapy is expensive, always determines a worsening of the zootechnic performances for the animals (lower weight gain) and requires a withdrawal time that is not always respected with a subsequent persistence of the molecules in the meat and in the derived products (e.g. milk, cheeses, yoghurt and others).

To that, it should be added that the continuous use of antibiotics facilitates the onset of drug resistance phenomena and the need of new antibiotic molecules, more and more difficult to find.

Since the major source of human infection is caused, as mentioned previously, by contaminated food, one of the strategies currently used to limit and/or eliminate £ coli infections is the systematic vaccination of adult animals in the last third of pregnancy and the administration of the vaccine to all young animals around 5-6 month of age when maternal antibodies have fallen below the threshold of protection . However, this procedure is time consuming and in particular considerable economic investments for farmers. ln the recent years an increasing interest has been directed to the use of probiotics as agents capable to restore the balance of the gastrointestinal flora and beyond.

US 5,965,128 patent describes a method for the treatment and prevention of £ coli 0157:1-17 infection in ruminants by using probiotics. The inventors isolate a number of bacterial strains from the gastrointestinal tract and faeces of adult bovines and report as a probiotic against £. coli 0157:1-17 infections at least one strain of £ coli selected from: £ coli 271 ATCC 20220, £ coli 786 ATCC 202018 and £ coli 797 ATCC 202019. Such strains, belonging to the same pathogenic species are, according to the inventors among those isolated, the only ones that are capable to significantly inhibit the growth of £ coli 0157:H7.

The patent application US 2006/0134082 describes a specific probiotic for the treatment and prevention of £. coli infection in calves. It is recalled here that in the state of the are, the calves appear to be particularly susceptible to £. coli gastrointestinal infections. The inventors selected strains to be used, among those present in the faeces of adult cattle, and show the combination of strains of Streptococcus bovis and Lactobacillus gallinarum as the one effective for reducing the presence of £ coli bacteria in the gastrointestinal tract of calves.

The patent application WO2009/15571 1 claims a probiotic for the treatment and/or prevention of enteritis caused by £ coli in mammals, consisting of bacteria selected from Lactobacillus, Streptococcus, Lactococcus, Bifidobacterium In particular among the lactobacilli the £ coli species L. acidophilus, L. fermentum, L. rhamnosus are shown to be effective in infections.

There is therefore a need in the art, as previously mentioned, to identify new strains of probiotics that have beneficial effects and/or therapeutic effects in case of a gastrointestinal infection by £ coli.

SUMMARY OF THE INVENTION

The present invention relates to one or more strain belonging to the Lactobacillus mucosae species to be used in the treatment and/or prevention in animals including humans of pathogenic £. coli bacillosis , pharmaceutical and/or veterinary compositions comprising one or more strain of the Lactobacillus mucosae species for use in the treatment and/or prevention of gastroenteritis caused by pathogenic £ coli in animals including humans, methods of preparing such compositions, probiotics foods or food supplements comprising one or more strain of the Lactobacillus mucosae species and methods of preparation of such foods or food supplements.

In particular the invention relates to: - one or more strain of the Lactobacillus mucosae species for use in the treatment and/or prevention in animals including humans of pathogenic E. coli bacillosis ;

- a pharmaceutical and/or veterinary composition comprising at least one strain belonging to the Lactobacillus mucosae s peci es a n d o n e or m ore pharmaceutically acceptable excipient for use in the treatment and/or prevention in animals including humans of pathogenic £ coli bacillosis;

- a process for the preparation of the pharmaceutical or veteri nary composition as herein defined and as defined in the claims, comprising the step of mixing at least one bacteria strain belonging to the Lactobacillus mucosae species with one or more pharmaceutically acceptable excipient and/or other bacterial strains;

- a probiotic food or a probiotic food supplement, comprising at least one strain belonging to the L. mucosae species and suitable food ingredients and/or preservatives and/or acidifying and/or antioxidants and/or colouring agents and/or substance stimulating the immune response and/or further probiotic bacteria and/or dietary supplements and/or excipients;

- a process for the preparation of the probiotic food or probiotic food supplement according to any one of claims from 14 to 16 comprising the step of mixing at least one strain belonging to the Lactobacillus mucosae species with preservatives and/or acidifying agents and/or antioxidant agents and/or further probiotic bacterial strains and/or dietary supplements and/or excipients suitable and the storage thereof in frozen or freeze-dried form.

DETAILED DESCRIPTION OF THE TABLES

Table 1. Preventive/healing effect of L. mucosae in colibacillosis of calves.

Table 1 shows the efficacy results of L. mucosae CCCF 3706 in preventing infections from pathogenic £. coli in calves to which L. mucosae was administered within the first 30 min after birth, after mixing with colostrum that can be maternal or artificial colostrum, (e.g., whole colostrum by Sloten).

Table 2. L. mucosae effect in gastroenteritis caused by pathogenic £ coli in calves treated with probiotic for 20 days.

Table 2 shows the efficacy results of L. mucosae CCCF 3706 in gastroenteritis caused by pathogenic £ coli in calves to which freeze-dried L. mucosae was mixed with the artificial milk and administered for 20 days at the indicated dose.

Table 3. L. mucosae effect in gastroenteritis caused by pathogenic £ coli in calves treated with probiotic for 60 days. Table 3 shows the efficacy results of L. mucosae CCCF 3706 in gastroenteritis caused by pathogenic E. coli in calves to which freeze-dried L. mucosae was mixed with the artificial milk and administered for 60 days at the indicated dose.

GLOSSARY

By Lactobacillus mucosae spp. (L. mucosae spp.) or species of Lactobacillus mucosae (L. mucosae) are intended all the strains belonging to the L. mucosae specie.

By not adult animal it is meant animals that are in a developmental stage corresponding to the paediatric age for men, a cow, for example, is defined not adult if aged between 0 and about 8 months of age.

Probiotic bacteria: bacteria that positively influence the health of the host, improving his intestinal microbial balance.

By Bacillosis caused by pathogenic E. coli those pathological forms developed by £ coli which fall into three types of infections in humans are meant: u ri nary tract i nfections (UTI ), neonatal men ingitis , and intestinal d iseases (gastroenteritis).

Pathogenic E. coli: E. coli strains that are causative agents of intestinal and extra intestinal diseases such as urinary tract infections, meningitis, peritonitis, septicaemia and pneumonia.

Included in the definition of pathogenic £ coli are:

- Enteropathogenic £ coli (EPEC)

- Enterotoxigenic £. coli (ETEC).

- Enterohemorrhagic £ coli (EHEC)

- Enteroaggregative £ coli (EAEC)

- Diffusely adhering £ coli (DAEC)

- Enteroinvasive £ coli (EIEC)

- Avian pathogenic £ coli (APEC)

- Verocytotoxic £ coli (VTEC)

By probiotic food/feed or probiotic dietary supplement it is meant a food/feed product containing living micro-organisms capable of producing a beneficial effect to the person who ingests it.

By fragments or biologically active derivates of lactoferrin it is meant parts of the molecule or modified forms of the molecule (such as deletion, mutation, addition of amino acids) that maintain the biological activity of lactoferrin (e.g., immunomodulating, in particular immunostimulatory). DETAILED DESCRIPTION OF THE INVENTION

The present invention reports for the first time the effectiveness of strains of the species Lactobacillus mucosae as active principles in the treatment and/or prevention of gastroenteritis caused by pathogenic £ coli such as, for example, enteropathogenic £ coli (EPEC); enterotoxigenic £ coli (ETEC); enterohaemorrhagic £. coli (EHEC); enteroaggregative £ coli (EAEC); diffusely adherent £ coli (DAEC); enteroinvasive £ coli (EIEC); avian pathogenic £ coli (APEC); verocytotoxic £ coli (VTEC).

The executors of the present invention have in fact highlighted how strains of the bacterial species Lactobacillus mucosae when administrated even alones experimentally to individuals, show the ability to effectively fight and prevent infection by pathogenic £ coli.

The Lactobacillus mucosae species was firstly isolated in the gastrointestinal tract of pigs (Roos S et al. Lactobacillus mucosae sp. nov., "A new species with in vitro mucus-binding activity isolated from pig intestine." International Journal of Systematic and Evolutionary Microbiology 2000, 50: 251 -258) and only in recent years its presence has been described among the species constituting the micro flora of the gastrointestinal tract of calves (Busconi M, Reggi S, Fogher C. "Evaluation of biodiversity of lactic acid bacteria microbiota in the calf intestine tracts." Antonie Van Leeuwenhoek. 2008, 94:145-155).

According to the present invention the Lactobacillus mucosae species can be isolated from biological samples such as faeces, gastrointestinal tissue samples and any other suitable sample known by the skilled person, belonging to animal species for which the presence of said species in the gastrointestinal micro flora has been described, such as, e.g., calves or pigs. Alternatively, they may be isolated, where present, from samples obtained from other animals, including humans according to standard procedures as, for example, described in Busconi M, Reggi S, Fogher C. Antonie Van Leeuwenhoek. 2008. 94:145-155. By way of example if the starting sample is represented by gastrointestinal tissue samples obtained from calves, the averagely skilled person can isolate and identify the L. mucosae species following what is described in Material and Methods section of the scientific article of Busconi et al (Busconi M, Reggi S, Fogher C. Evaluation of biodiversity of lactic acid bacteria microbiota in the calf intestine tracts. Antonie Van Leeuwenhoek. 2008. 94: 145-155). In particular the biological identification of the isolated species in the L. mucosae species can be carried out by analysis of gene sequence homology using, for example, the 16S ribosomal gene.

According to the present description one or more strains of Lactobacillus mucosae spp. can be used in treatment and/or prevention of gastroenteritis caused by pathogenic £ coli in animals, including humans.

This means that bacteria from a single strain of Lactobacillus mucosae, or a combination of two or more different strains from the same species can be used.

The association of two strains according to the present invention may also result in a synergistic effect of the two, i.e., the sum of the antipathogenic £ coli activity of each individual strain, is inferior to the antipathogenic activity of the strains used together.

The strains can be selected from any L. mucosae strain known or isolated from samples and characterized using techniques known in the literature such as, for example, the one described in the materials and methods chapter (pp 147-148) in Busconi M, Reggi S, Fogher C. Antonie Van Leeuwenhoek. 2008. 94 herein incorporated by reference, or they can be selected among the ones deposited on March, 1 st 2010 at DSMZ, according to the Budapest Treaty with deposit receipt dated March 2 2010, and identified by deposit numbers DSM 23409 and DSM 23408, depositor Italstarter Sri. with deposit receipt of DSMZ dated March 2 2010, Such strains are identified in the present description, respectively, as L. mucosae CCCF 3706 and CCCF 3464.

According to an embodiment of the invention the above indicated strains can be in combination with human or animal, native or recombinant, lactoferrin or biologically active fragments thereof or biologically active derivatives thereof. Advantageously, an association with lactoferrin, fragments or derivatives thereof from the species in which they will perform prevention or the treatment for bacillosis from pathogenic £ coli will be made.

Hence, for the prevention or the treatment of adult or infant humans, the association with natural or recombinant lactoferrin or with fragments thereof or biologically active derivatives thereof of human origin will be preferred; or the treatment, e.g., of cattle or calves the association with natural or recombinant lactoferrin or fragments thereof or biologically active derivatives thereof cattle origins will be preferred etc.

Among the different classes of animals potentially susceptible to pathogenic £. coli gastrointestinal infections are included in general warm-blooded animals, in particular birds, breeding mammals and humans. Poultry, cattle, pigs, equines, sheep, primates and pets are particularly affected, and are described in the state of art as the species among which the highest mortality due to pathogenic £ coli gastrointestinal infections has been recorded. In one of the possible embodiments, the mammals are selected in the group of: cattle, pigs, equines, sheep, primates (humans), canines, felines, rodents and goats. In infants and in young animals, not adults (for humans in the paediatric age) infections from pathogenic £ coli can cause severe enteritis. In particular in breeding animals, as for example cattle, the mortality in young individuals, not adults (e.g.. calves) due to those gastroenteritis is really frequent.

Data obtained by the inventors demonstrated that one or more strain of the L. mucosae species are more effective in restoring the gastrointestinal balance in individuals affected by pathogenic £ coli enteritis when compared to commonly known probiotic bacterial strains for said use, such as, by way of example Streptococcus faecium.

As evident in the reported tables, the administration of L. mucosae to calves in the first minutes after birth (table 1 ), for twenty days (table 2) and for sixty days (table 3) determines both a significant decrease of the bacterial £ coli in the faeces and a decrease in the percentage of episodes of diarrhoea in the animals affected by enteritis, and in the death of animals. The correlation between total number of £ .coli and the percentage of diarrhoea episodes highlights how L. mucosae is effective against pathogenic £ coli gastroenteritis.

L. mucosae strains, can hence be used for the prevention and the therapy of the above reported diseases caused by £ coli in a particularly advantageous way in humans of paediatric age and in subjects having an analogous age among animals, in physically debilitated subjects, in patients undergoing special medical treatment rendering them more susceptible to pathogenic £. coli infections and in elderly subjects that are less resistant to the above mentioned bacterial infections.

In these more vulnerable subjects, the use of L. mucosae strains as herein described is of particular importance as they are those most exposed to damages, even lethal, by the pathogenic microorganism.

The use of Bacteria belonging to the L. mucosae species, as herein described, for said patients is one of the best embod iments of the present invention.

Any strain or combinations of strains belonging to the Lactobacillus mucosae species can be used, in particular, in the treatment or in the prevention of pathogenic £ coli gastroenteritis in paediatric age for humans and for not adult subjects for the remaining animals.

The preventive use of strains as explained above allows avoiding, for example in animal breeding, or in epidemics, the propagation of the infection from other affected subjects or from healthy carriers of the pathogenic £ coli strain.

In one embodiment the possible strains belonging to the L. mucosae species can be one or more among those deposited (CCCF 3706 and CCCF 3464) on March the 1 st, 2010 at DSMZ, according to the Budapest Treaty, and identified by deposit numbers DSM 23409 and DSM 23408, depositor Italstarter Sri. with deposit receipt of DSMZ dated March 2 2010, Such strains are identified in this description as L. mucosae CCCF 3706 and CCCF 3464.

In this case, one or more strain belonging to this group, optionally in combination with other L. mucosae strains can be used.

Th e strains according to the description are useful and effective in preventing and treating in general pathogenic E. coli infections , for example, they can be useful in the treatment of infections by pathogenic £ coli such as enteropathogenic £ coli (EPEC); enterotoxigenic £. coli (ETEC); enterohaemorrhagic £ coli (EHEC); enteroaggregative £ coli (EAEC); diffusely adherent E. coli (DAEC); verocytotoxic £ coli (VTEC); enteroinvasive £ coli (EIEC); avian pathogenic £ coli (APEC), strains belonging to these groups of £ coli are, for example, £ coli 0157: H7; 01 :K1 : H7; 045:K1 :H7; O78: K80: H9; 06:K2:H1 ; 04:K54:H5; 018:K1 :H7; 02:K1 :H5; 02:K1 :H7, etc.

According to the invention, one or more strain belonging to the L. mucosae species can also be in freeze-dried and/or frozen form. Freeze-drying and freezing are to be intended as means for the temporary inactivation of the bacteria, that do not prevent the restoration of cell division, and that can be carried out in any one of the ways known to the skilled person that are reported in detail in laboratory manuals.

Other forms of temporary inactivation of bacteria, other than freeze-drying and freezing, are included in the embodiments of the invention.

Object of the present invention are also pharmaceutical (including paediatric pharmaceutical compositions) or veterinary compositions comprising at least one strain belonging to the L. mucosae species for use in the treatment and/or prevention of gastroenteritis caused by pathogenic £ coli in animals including humans. Such compositions will comprise an amount of L. mucosae bacteria in the range from 100 to 100,000 x 10 ⁶ UFC per each single dose.

Such compositions include also paediatric pharmaceutical compositions and compositions suitable for administration to baby animals.

Together with the aforementioned bacteria, one or more pharmaceutically acceptable excipients may be present in the compositions. Suitable excipients can be: culture medium, calcium carbonate, silicic acid, talc, magnesium stearate, anhydrous lactose, gelatine, trehalose.

Together with the bacteria and the other mentioned excipients substances capable of stimulating the immune response or capable of chelating chelate iron may be present in the compositions. A suitable stimulating substance could be the protein lactoferrin or peptides derived thereof.

Th e compositions may comprise, as active ingredients, solely bacterial strains belonging to the L. mucosae species or can further comprise one or more bacterial strains belonging to probiotic bacterial species, that is to say, strains belonging to probiotic bacterial species other than L. mucosae.

By way of example these species may be Lactobacillus acidophilus, L. rhamnosus, L. delbrueckii, L. helveticus, L. plantarum, L. gassei, L. casei, L. reuteri, Enterococcus faecium, Bifidobascterium animalis, B. breve, B. infantis, B. longum, etc.

The bacterial strains belonging to the L. mucosae species useful for the preparation of the pharmaceutical or veterinary compositions can be selected from any L. mucosae strain known or isolated by samples and characterized using techniques known in the literature such as, for example, the one described in the chapter material and methods (pp 147-148) in Busconi M, Reggi S, Fogher C. Antonie Van Leeuwenhoek. 2008. 94 herein incorporated by reference or selected by: the ones deposited (DSM 23409 and DSM 23408, depositor Italstarter Sri, respectively CCCF 3706 and CCCF 3464) on 1 st of March 201 0 at DSMZ, with deposit receipt of DSMZ dated March 2 2010, according to the Budapest Treaty.

Th e bacteria according to the present description may be suspended, freeze-dried or frozen, provided they are not killed because their vitality is essential for the preventive or therapeutic effect here disclosed bound to the probiotic activity of such bacteria.

The preparation of the compositions of the invention can be carried out by freeze-drying of bacterial cultures, by a dmixing freeze-dried cultures or by suspension of bacterial pellets or colonies with water or with further suitable excipients, preservatives, flavouring, colouring agents.

The bacteria can also be supplied in frozen form, stored according to standard techniques.

The excipients can be commonly used excipient for bacterial therapeutic or preventive preparations , of veterinary or pharmaceutical grade.

The compositions according to the invention are for use in the treatment and/or prevention of gastroenteritis caused by E. coli in animals including humans. I n one of the possible embodiments these animals are mammals such as for example: cattle, pigs, equines, sheep, primates, canines, felines, goats, rodents and farm mammals, for breeding, competition or as pets. In particular, these pharmaceutical compositions comprising one or more Lactobacillus mucosae strains are used, in gastroenteritis caused by E. coli in humans of paediatric age, and their correspondent veterinary compositions can be particularly suitable for use with non adu lt animals in a development stage corresponding to the paediatric age of humans (puppies, for example calves).

In particular, the described compositions will be of considerable utility when administered to new born or in the first months of age.

The pharmaceutical veterinary compositions herein described can be provided in the form of powder, freeze-dried, in tablets, capsules, liquid, thick liquid, semisolid, gel, suspension, emulsion, slurry, syrup, elixir, lozenge, hard or soft gelatine capsule, granulate.

I n case of paediatric or veterinary compositions, these could include suitable flavouring agents that rendering their administration to kids or puppies easier.

These flavouring agents are obviously to be selected form the ones commonly used according to the desired patient. I n the case of children there flavouring agents having fruit flavour, honey flavour or other flavours commonly used by the skilled person can be used. For veterinary compositions, flavouring agents which make the composition more palatable for the desired animal can be used. Obviously these flavouring agents can be different if the patient is an herbivore or a carnivorous as known in the state of the art.

The compositions do be administered to newborns or before weaning, can conveniently be tasteless and odourless so to be, administrated together with maternal milk or alone, when necessary or desirable, without being rejected by the patient.

Object of the present description are also probiotic foods and supplements or probiotic food additives comprising at least one strain belonging to the L. mucosae species.

For food/supplement/probiotic additive it is meant a foodstuff containing microorganism capable of reaching the intestines, multiply and exert a beneficial effect on health of the organism.

That food or supplement, besides containing as probiotic microorganism at least one strain of L. mucosae, will contain suitable food ingredients and/or activators of the immune response and/or preservatives and/or acidifying and/or antioxidant and/or colouring agents and/or further probiotic bacteria strains and/or food supplements (mineral salts, vitamins or others) and/or excipients.

As for the compositions of the invention, the bacteria of the L. mucosae species present in the food or supplement can belong to one or more strain, selected from any L. mucosae strain known or isolated by samples and characterized following techniques known in literature, such as, for example the one described in the chapter materials and methods (pp 147-148) in Busconi M, eggi S, Fogher C. Antonie Van Leeuwenhoek. 2008. 94 herein incorporated by reference. Also the strains CCF3706 (deposit number DSM 23409) and CCF3464 (deposit number DSM 23408) sent on the 1 st march 2010 at DSMZ, with deposit receipt of DSMZ dated March 201 0, depositor Italstarter Sri according to the Budapest Treaty can be used. The food and supplements can contain one or more strain (for example two) belonging to the L. mucosae species as sole probiotic strains. Furthermore, in particular embodiments such foods or supplements can comprise also strains belonging to different probiotic species for example the species Lactobacillus acidophilus, L. rhamnosus, L. delbrueckii, L. helveticus, L. plantarum, L. gassei, L.casei, L. reuteri, Enterococcus faecium, Bifidobascterium animalis, B. breve, B. infantis, B. longum.

The foodstuff, the supplement or the additive can conveniently be, as of example and not as a limitation, in the form of liquid, thickened liquid, semisolid, solid, freeze-dried, powder, granules, paste, emu lsion , suspension, slurry, croquettes, chewable bars, yoghurt, cheese, juice or fruit compote, drink or dairy- food or non dairy food, gelatine, syrup, elixir, natural colostrum, artificial colostrum, condensed milk, powdered milk, flavoured powder, infant milk formula.

Probiotic foods of particular interest can be foods intended for paediatric patients such as, for example, natural colostrum, artificial colostrum, condensed milk, powdered milk, flavoured powder, infant milk formula and homogenized food. In a particular embodiment the foodstuff is probiotic foods for use with animals, for example, croquettes.

The foods of the invention can be prepared according to any of the methods known by the skilled person mixing at least one of the bacterial strains of L. mucosae in fresh, freeze-dried or frozen from, with suitable food ingredients and optionally preservatives and/or acidifying and/or antioxidant agents.

Furthermore, they can be combined also with further probiotic bacterial strains not belonging to the L. mucosae species and/or supplements such as for example, vitamins, food salts, amino acids, immunostimulants and/or suitable excipients.

In one embodiment, together with said bacteria and suitable pharmaceutical excipients one or more substance stimulating the immune response or capable of chelating iron may be present in the compositions or in the probiotic foods comprising as unique active ingredients or probiotic strains one or more L. mucosae strains as indicated above (optionally in combination with one or more further probiotic strain indicated above).

Various immu nostimulants that the skilled person can add to the compositions of the invention are known. I n one embodiment, the present description provides pharmaceutical compositions as described above comprising effective doses of lactoferrin (a known immunostimulant), known to the skilled person. This lactoferrin can be human lactoferrin, recombinant human lactoferrin (in the art many ways of producing recombinant human lactoferrin are known such as, for example, in plant seeds), fragments or derivatives thereof that retain the biologic immunostimulating activity of lactoferrin. The fragments can be part of the immunostimulating protein, and the derivatives can be forms of the protein, in which one or more amino acids can be deleted, added or mutated, that retain the immunostimulating characteristics of the lactoferrin . Derivatives in which the mutations are neutral, in which one or more amino acid is replaced by amino acids with the same characteristics (for example basic, acids etc.) are preferred.

The lactoferrin can be human or animal lactoferrin, even if not necessary, a species-specific use of the lactoferrin is preferred, hence human lactoferrin (or derivatives or fragments thereof as described above) for pharmaceutical compositions and an imal lactoferrin (or derivatives or fragments thereof as described above) for veterinary compositions. I n a preferred embodiment the veterinary compositions can comprise lactoferrin (or derivatives or fragments thereof as described above) of the species to which they are designed.

The amount of bacteria to be inserted in the foodstuffs can be any amount considered suitable by the skilled person. By way of example foods and probiotic supplements can comprise from 100 to 100,000 x 10 ⁶ CFU per gram. These can be grown directly in the foodstuff or added in any preparation stage of the foodstuff.

The present description reports below the effects induced by one ore more strains belonging to the L. mucosae species when administered on birth or to subjects with gastrointestinal infections or, as a prevention, before the infections occur. In particular, these strains have demonstrated, in experiments carried out by the inventors, the capability of restoring the physiological gastrointestinal balance i n pathological conditions essentially consisting of enteritis. I n particular i n conditions wherein the mostly common pathological agent of said enteritis was represented by pathogenic forms of the E. coli bacteria. Some of the experimental results obtained are reported in the following examples and are provided for a better understanding of the invention and are not to be intended as a limitation of the same.

Preliminary data have shown that the association of one, two or more L. mucosae strains, as for example one or both deposited strains DSM 23409 and DSM 23408, depositor Italstarter Sri, is more effective in the prevention and also in the treatment of the bacillosis of pathogenic E. coli in animals, including humans, than using only one L. mucosae strain.

EXAMPLES

1. Preventive effect of L. mucosae in gastroenteritis caused by pathogenic E. coli in calves.

Test carried out on calves i n cattle shed with constant presence of diarrhoea and animal death episodes due to pathogenic E. coli. The administration was carried out at birth without subsequent challenge with the pathogen, considering the environmental load sufficient for the validation of the product.

The marked effect of L. mucosae, at very high doses, on the intestine physiology worth consideration. The skilled person can easily distinguish a diarrhoea due to a pathogenic from an alimentary diarrhoea due to high doses of L. mucosae that resolves in few days, without any antibiotic therapy and without consequences for the animal. It is the first time that a phenomenon of this kind is observed after the administration of a probiotic and this testifies the physiological relevance of this bacterial species (L. mucosae) in the intestinal balance of the animal. The quantity to be administered can be calibrated, for each animal species, to avoid episodes of physiological imbalance that however have no consequences on the animal, as they resolve spontaneously in a few days and are not evidenced with adequate dosages (100 x 10 ⁶ ).

L mucosae strain used: CCCF 3706 (deposit number DSM 23409) deposited at DSMZ, depositor Italstarter Sri according to the Budapest Treaty on th e 1 st of March 2010 with deposit receipt of DSMZ dated March 2 2010 Formulated in a blended frozen product mixing the bacterial biomass harvested after centrifugation of the fermented in a suitable cryoprotectant. The preparation thus obtained in administrated within the first 30 minutes from birth to the calves. Different concentrations of L. mucosae where tested and the effectiveness of the treatment has been evaluated (i) in terms of reduced percent of diarrhoea episodes in early life period (1 st-10th day), (ii) as reduction of coliform bacterial load in the faeces of the treated animals, (iii) with assessment of the health status of the animal after 30 days from the treatment. As shown in table 1 it is possible to highlight that L. mucosae, when administered in a dose comprised between 100 and 100,000 X 10 ⁶ CFU) within 30 minutes from birth can (i) completely contrast the onset of diarrhoea phenomena due to colibacillosis, (ii) induce a significant decrease of the presence of coliform in faeces, (iii) determine a good state of health after 30 days from the treatment with higher weight gain than the one of non treated animals or of animals treated with antibiotics. For the control animals, for which diarrhoea and an high bacterial load were observed, it has been necessary take action with a antibiotic therapy (gentamicin) on the 10th day after birth to remove the infection. The evaluation of the effectiveness of L. mucosae in the treatment of pathogenic E. coli enteritis has been confirmed repeating the protocol described above on calves born in different months (August, September and October 2009) and in different farms.

TABLE 1

Test with a L. mucosae strain in a colostrum.

The composition has been formulated as frozen 8 ml single dose bacterial product of the strain of the L. mucosae stored at -20°C and administered, after defrost at 30°C, in the first minutes from birth mixing it with 50/100 ml of colostrum.

N° Dosage Diarrhoea episode (%) Bacterial Observations Calves ¹ x 10 ^¾ 1 °-10° gg; 1 1 °-20° gg; 21 °-30° count

flfl faeces ⁵ x 10 ⁶

10 100,000 0 100 0 45 Alimentary diarrhoea solved spontaneously with no death of the animals.

8 10,000 0 100 0 23 Alimentary diarrhoea solved spontaneously with no death of the animals.

15 1 ,000 0 100 0 34 Alimentary diarrhoea solved spontaneously with no death of the animals.

100 0 0 0 38 No diarrhoea episode and no death.

8 control 50 80 0 550 Antibiotic therapy started the 10 ^th day

(gentamicin). No death but intestinal problems.

10 ^J 100,000 0 100 0 65 Alimentary diarrhoea solved spontaneously with no death of the animals.

25 ^J 10,000 0 100 0 50 Alimentary diarrhoea solved spontaneously with no death of the animals.

20 ^J 1 ,000 0 80 0 28 Alimentary diarrhoea solved spontaneously with no death of the animals.

18 ^J 100 0 0 45 No diarrhoea episode and no death.

6 ^J control 100 50 0 820 Antibiotic therapy started the 10 ^th day

(gentamicin). One veal death and intestinal problems. N° Dosage Diarrhoea episode (%) Bacterial Observations Calves ¹ x 10 ^¾ 1 °-10° gg; 1 1 °-20° gg; 21 °-30° count

gg faeces ⁵ x 10 ⁶

22 ⁴ 100,000 0 100 0 78 Alimentary diarrhoea solved spontaneously with no death of the animals.

15 ⁴ 10,000 0 100 0 65 Alimentary diarrhoea solved spontaneously with no death of the animals.

14 ⁴ 1 ,000 0 100 0 55 Alimentary diarrhoea solved spontaneously with no death of the animals.

20 ⁴ 100 0 0 0 37 No diarrhoea episode and no death.

12 ⁴ control 100 30 0 650 Antibiotic therapy started the 10 ^th day

(gentamicin). No death but intestinal problems.

¹ Calves treated on birth day also in different farms

² Calves born on August 2009

³ Calves born on September 2009

⁴ Calves born on October 2009

⁵ CFU of coliformi (on Violet Red Bile Agar) of faeces samples from rectal ampoule on the 10th day of life referred as g of fresh weight.

2. Effect of L. mucosae on gastroenteritis from a pathogenic E. coli in calves treated with probiotic for 20 or 60 days.

Test made on calves in cattle shed with presence of diarrhoea and death of animals due to pathogenic E. coli. The administration has been carried out for 20 or 60 days without challenge with the pathogen, considering the environmental load sufficient for the verification of the product.

Cells of L mucosae strain CCCF 3706 (number of deposit DSM 23409) deposited on 1 st of March 2010 at DSMZ, with DSMZ deposit receipt on March 2 2010 depositor Italstarter Sri according to the Budapest Treaty has been lyophilized and diluted, using suitable inert substances, at a CFU concentration desired for the administration at the dose of 1 0 g for each animal per day, after mixing in milk. In particular the different freeze-dried preparations were suitable, at the dosage of 10 g day/animal, for the administration of several bacterial concentrations between 100 and 100,000 x 1 0 ⁶ CFU. The administration to the calves has been continued for 20 days or 60 days starting from the first day after birth without preliminary administration of the colostrum containing L. mucosae in the frozen form. As highlighted from the obtained results reported in table 2 and table 3 the administration of L. mucosae is effective at the different concentrations used against the pathogenic E. coli infections. Several concentrations of L. mucosae have been tested and the efficacy of the treatment has been evaluated (i) in terms of reduced percent of diarrhoea episodes in early life (1 st-10th day), (ii) as reduction of coliform bacteria in the faeces of the treated animals, (iii) with assessment of the health status of the animal after 30 days from the treatment. As shown in table 2 and 3 it is possible to highlight that L. mucosae, when administered in a dose between 100 and 100,000 X 10 ⁶ CFU) for 20 or 60 days after birth can (i) completely contrast the onset of diarrhoea phenomena due to colibacillosis, (ii) induce a significant decrease of the presence of coliform in faeces, (iii) determine a good state of health at 30 and 60 days from the treatment with higher weight gain than with respect to non treated animals or to animals treated with antibiotics. For the control animals, for which diarrhoea and high bacterial load were observed, it has been necessary take action with an antibiotic therapy (gentamicin) on the 10th day after birth to remove the infection. The evaluation of the effectiveness of L. mucosae in the treatment of pathogenic E. coli enteritis has been confirmed repeating the protocol described above on calves born in different months (August, September and October 2009) and different farms. The effective concentration of L. mucosae, without physiological lack of balance at intestinal level, results to be the dose of 100 x 10 ⁶ CFU for which an efficacy of 100% compared to the control was observed. For the, controls, from the 1 0 ^th day of life an antibiotic treatment (gentamicin) has been necessary because of sever intestinal problems. It is worth to note that in few cases in spite of the antibiotic therapy the pathogenic E. coli infection has led to the death of some calves. Normally if the antibiotic therapy is not started when colibacillosis diarrhoea phenomena are present, the mortality is between 10 to 30 %. In cattle sheds of intensive farm the morbidity of neonatal colibacillosis is around values of 80-100%. The evaluation of the effectiveness of L. mucosae in the treatment of pathogenic E. coli enteritis has been confirmed by repeating the protocol described above on calves born in different months (August, September and October 2009) and different farms.

TABLE 2

Test with a freeze-dried L. mucosae strain administered in milk for 20 days.

N° Dose ⁴ Diarrhoea episode (%) Bacterial Observations

Calves ¹ x 10 ⁶ 1 °-10° gg; 1 1 °-20° gg; 21 °-30° count feces ⁵

x 10 ⁶

23" 1 ,000 0 100 0 135 Alimentary diarrhoea solved spontaneously with no death of the animals.

100 0 0 0 80 No diarrhoea episode and no death.

12" Control 70 60 0 1200 Antibiotic therapy started the 10 ^th day

(gentamicin). One veal death and intestinal problems.

20 ^J 10,000 0 100 0 70 Alimentary diarrhoea solved spontaneously with no death of the animals.

25 ^J 1 ,000 0 80 0 85 Alimentary diarrhoea solved spontaneously with no death of the animals.

16 ^J 100 0 0 55 No diarrhoea episode and no death.

12 ^J Control 100 50 0 920 Antibiotic therapy started the 10 ^th day

(gentamicin). One veal death and intestinal problems.

¹ Calves treated from the first day after birth

² Calves born on August 2009

³ Calves born on September 2009

⁴ Freeze-dried product administrated at a dose of 10 g per day per animal starting with products of different cell number

⁵ CFU of coliformi (on Violet Red Bile Agar) of faeces samples from rectal ampoule on the 10th day of life referred as g of fresh weight.

TABELLA 3

Test with a freeze-dried L. mucosae strain administered in milk for 60 days.

N° Dosage ⁴ Diarrhoea episode (%) Bacterial Observations

Calves ¹ x 10 ⁶ 1 °-10° gg; 1 1 °-20° gg; 21 °-30° count feces ⁵

x 10 ⁶

25 ^J 1 ,000 0 80 0 68 Alimentary diarrhoea solved spontaneously with no death of the animals.

100 0 0 65 No diarrhoea episode and no death.

12 ^J control 100 50 0 620 Antibiotic therapy started the 10 ^th day

(gentamicin). One veal death and intestinal problems.

¹ Calves treated from the first day after birth

² Calves born on August 2009

³ Calves born on September 2009

⁴ Freeze-dried product administrated at a dose of 10 g per day per animal starting with products of different cell number

⁵ CFU of coliformi (on Violet Red Bile Agar) of faeces samples from rectal ampoule on the 10th day of life referred as g of fresh weight.

The tests reported above have been carried out (data not shown) also with other strains besides to one above indicated (example CCF3464, deposit number DSM 23408) deposited at DSMZ, depositor Italstarter Sri according to the Budapest Treaty on the 1 st of March 2010 with DSMZ deposit receipt of March 2 2010, and other L. mucosae not deposited strains (example. CCCF3720, 3517, 3713, 3755, 3742, 3140, 3273, 3309, 3467, 3435, 3264, 3705, 3260, 3274, 3419, 3266, 3066, 3430, 3191 , 3390, 3389, 3539, etc.), obtaining similar results with all the tested strains.

The L. mucosae strains used in the experiment reported above have been isolated from sample coming from different breeding farms.

The preliminary tests carried out on humans have been performed on a limited number of persons with gastrointestinal symptoms caused by E. coli. These persons have been treated with commercial probiotic preparation based on lactic acid bacteria containing also L. mucosae of unknown origin and preparations based on L. mucosae CCCF 3706 and CCCF 3464 in association or singularly, with the addition of human lactoferrin at a dose of 100 mg per administration. The administration of the preparations based on L. mucosae CCCF 3706 and CCCF 3464 singularly or in association with the addition of human lactoferrin has always determined a rapid restoration of the intestinal functionality. Extensive tests are in progress. BIBLIOGRAPHY

- US5965128

- US2006/0134082

- WO2009/15571 1

- Roos S et al. Lactobacillus mucosae sp. nov., a new species with in vitro mucus-bi nd ing activity isolated from pig i ntestine. I nternational Journal of Systematic and Evolutionary Microbiology 2000, 50: 251 -258

- Busconi M, Reggi S, Fogher C. Evaluation of biodiversity of lactic acid bacteria m icrobiota i n the calf intestine tracts. Antonie Van Leeuwenhoek. 2008;94:145-155