The present invention falls within the veterinary field. In particular, the present invention relates to a composition which is effective in the therapeutic treatment of dysbiosis diseases in pet and farm animals belonging to the omnivorous mammal class through modulation of the intestinal microbiota.

To date, intestinal dysbiosis in the veterinary field is typically treated with antibiotics, in combination with diet, prebiotics and probiotics. However, the antibiotic acts locally, but is known to have transient efficacy and frequently induces long-term antibiotic resistance, resulting in a high risk of recurrence of the disease and of the dysbiosis associated therewith.

Therefore, there is a need for a new therapeutic approach for the treatment of dysbiosis diseases in the veterinary field, which does not exhibit the above-mentioned disadvantages.

In order to meet this need, the present inventors have developed a radically innovative approach, based on trans-species oral transplantation of intestinal microbiota from a healthy, i.e., not suffering from dysbiosis, monogastric herbivorous donor to an omnivorous recipient.

Therefore, the present invention relates to an intestinal microbiota preparation from a monogastric herbivorous mammal not affected by dysbiosis, for use in a method of therapeutically treating a dysbiosis disease in an omnivorous mammal in need thereof, wherein the therapeutic treatment method comprises orally administering to said omnivorous mammal said intestinal microbiota preparation from the monogastric herbivorous mammal.

In the context of the present invention, the expression "intestinal microbiota from a monogastric herbivorous mammal" refers to all the symbiotic microorganisms residing in the intestine of the monogastric herbivorous mammal, which play an essential role in maintaining the state of health of the host organism.

The novelty of the approach, which is the object of the present invention, is that, at present, as far as the inventors are aware, intestinal microbiota transplantation for the treatment of intestinal disorders has been limited to homologous transplants, i.e., from a donor to a recipient belonging to the same species. Moreover, to date, microbiota transplantation has been mainly carried out by direct intestinal instillation performed endoscopically, rather than orally as in the present invention.

The main advantages of the innovative approach, which forms the object of the present invention, are that it is a therapy inspired by a totally natural behaviour in omnivorous mammals - that is, the propensity to coprophagy - but which has gradually been lost with breeding and domestication in general. According to the present invention, this natural behaviour is advantageously translated into a therapeutic approach carried out in a totally controlled and safe way, thus eliminating the risks of transmission of pathogens, such as for example intestinal parasites.

The therapeutic approach developed by the present inventors allows the entire intestinal microbiota of the dysbiotic animal to be advantageously modulated, thus allowing the recovery of a health-promoting ecosystem configuration, as well as the concurrent resolution of the disease. In particular, it is an approach which, since it does not exhibit the risk of longterm resistance as in the case of antibiotics, allows satisfactory results to be obtained in the case of acute dysbiosis, as well as in the case of chronic and refractory dysbiosis, such as, by way of example, adverse food reactions, hemorrhagic gastroenteritis, bacterial enteritis, viral enteritis, inflammatory bowel disease (IBD), exocrine pancreatic insufficiency, primary or secondary motility disorders, race-related enteropathies.

A further advantage of the therapeutic approach, which forms the object of the invention, is that it allows the use of antibiotics to be reduced, both in the clinical practice for pet animals and in intensive breeding.

In a preferred embodiment of the invention, the monogastric herbivorous mammal used as a donor is a horse or a donkey.

In another preferred embodiment, the recipient omnivorous mammal is a pet or farm animal, more preferably a dog, a swine, or a non-human primate.

The beneficial action of the intestinal microbiota from the monogastric herbivorous donor is due to the conferment of health-promoting microorganisms it is enriched with, which include, in a preferred embodiment, bacteria capable of degrading fibres and producing short-chain fatty acids, such as butyrate, which have a powerful anti-inflammatory and immunomodulatory effect, and probiotic bacteria capable of exerting the so-called barrier effect, that is to counteract the growth of pathogenic microorganisms and opportunistic pathogens, thanks to the biosynthesis of natural antimicrobial compounds such as bacteriocins and acetate.

Therefore, in a preferred embodiment of the invention, the intestinal microbiota from the monogastric herbivorous donor comprises short-chain fatty acid-producing fibrolytic bacteria, more preferably of the genera Bacteroides, Parabacteroides, Prevotella, Fibrobacter, Lachnoclostridium, Ruminococcus, Ruminiclostridium, Subdoligranulum, Eubacterium, Butyricicoccus, Anaerotruncus, Blautia, Coprococcus, Lachnospira, Roseburia, Hungatella, Phascolarctobacterium, Saccharofermentans, Faecalibacterium, Oscillibacter, and/or Christensenella, and/or probiotic bacteria, more preferably of the genus Lactobacillus and/or Streptococcus.

In a further preferred embodiment, the intestinal microbiota preparation from the monogastric herbivorous donor consists of a stool sample from the monogastric herbivorous mammal not affected by dysbiosis dispersed in a pharmaceutically acceptable carrier, preferably sterile physiological solution. According to the invention, this preparation is administered orally, alone or, if necessary, in admixture with a feed suitable for the recipient omnivorous mammal. The person skilled in the art, in particular the veterinarian or breeder, shall be able to identify the feed suitable for this purpose, according to the species to which the recipient animal belongs and also according to other criteria, such as for example the age and state of health of the animal.

The following experimental examples illustrate, by way of example, a protocol for obtaining and using the intestinal microbiota preparation according to the invention, aimed at treating diseases of the gastrointestinal tract in an omnivorous recipient, which consists of a series of sequential steps, including the selection of the microbiota donor, the collection, storage and preparation of the sample for faecal transplantation, the administration to the recipient, and finally the monitoring of the engraftment and the effect on the recipient’s microbiota and state of health. The results obtained in an experimental trial are also described, the latter relating to the treatment of 5 dogs belonging to different breeds suffering from chronic dysbiosis administered orally with an intestinal microbiota preparation obtained from a horse as the donor.

The examples are provided purely by way of example, therefore are not intended to limit the scope of the invention as defined in the appended claims.

EXAMPLES

Example 1

Selection of the monogastric herbivorous donor

The microbiota donor is selected according to a protocol including the following criteria and steps:

(i) selecting a donor between 1 and 10 years old and in general good nutritional status;

(ii) clinical examination and blood-biochemical, urine and parasitological tests of the faeces must exclude organic and/or behavioural disease, certifying good health;

(iii) checking that vaccinations are in accordance with guidelines;

(iv) monitoring the donor's drug consumption and, in particular, the donor must not have taken antibiotics in the last 6 months; (v) checking the composition of the donor's intestinal microbiota, with particular reference to the presence of a state of eubiosis; and

(vi) checking the diet and lifestyle, which must include the consumption of hay and concentrate and breeding under general semi-wild conditions for the welfare of the animal.

Once the above requirements are met, the donor is certified as a universal donor for a period of 60 days, after which, to maintain this certification, testing and monitoring are repeated to re-attest compliance with these requirements.

Collection and storage of the donor ’s intestinal microbiota sample

For the collection of the intestinal microbiota sample, 300 g of fresh stool are collected using gloves and sterile containers, and a special collection bag, thus avoiding contact with the soil and/or litter. Immediately after collection, the stool is stored in the dark at a temperature of 4-8°C until preparation for microbiota transplantation, which will take place within 6 hours of fresh sample collection.

Sample preparation for microbiota transplantation

200 g of the stool sample are homogenized with sterile physiological solution - operating under sterile conditions - and brought to a temperature of 20°C. The preparation thus obtained, ready for oral administration, is administered within 1 hour of preparation.

Microbiota transplantation via oral administration

10 to 100 g of the preparation obtained, also depending on the recipient’s body weight, are offered to the recipient. If the intake is not spontaneous, the preparation is mixed with feed to facilitate its spontaneous intake.

Monitoring of the recipient's intestinal microbiota before and after transplantation

Monitoring of the recipient’s intestinal microbiota comprises molecular characterization of the recipient’s microbiota on the day before transplantation (Tl), the day after transplantation (T2) and 3 and 7 days after transplantation (T3, T4). Microbiota characterization is performed by taking a stool sample, extracting total microbial DNA and sequencing it using Next Generation Sequencing (NGS) techniques, also including targeted and untargeted metabolomics techniques.

Monitoring of the effects on the recipient ’s health Monitoring of the recipient’s state of health after treatment combines clinical data and stool consistency data according to the Bristol scale (1-7) collected through the completion of a questionnaire by the breeder or owner.

Example 2

The protocol described in Example 1 has been tested in small-scale experimental trials. In particular, 5 dogs belonging to different breeds and suffering from chronic dysbiosis were treated. The intestinal microbiota of the horse used as the donor was characterized by ultra- massive sequencing techniques (NGS) to verify the absence of pathogens and the general state of eubiosis. The donor was in good health, the diet was predominantly hay and extruded products, and the animal was living under semi-wild conditions. The 5 patients were treated as described in Example 1, and the intestinal microbiota was characterized by NGS prior to treatment (Tl), and 3 and 7 days after treatment (T2, T3). The state of health of the dogs, before and after treatment, was assessed by clinical examination, blood tests and stool examination. The treatment was shown to be immediately effective in terms of increasing stool consistency as early as 24 hours after administration, with a clear alleviation of symptoms from the day after treatment, which remained stable until the end of the observation period (Day 7). With regard to the intestinal microbiota data, the treatment was shown to be effective in facilitating the recovery to a eubiotic (health-promoting) condition, with particular reference to a general increase in diversity values and a concomitant reduction of pathogenic or opportunistic pathogenic components.

For 3 of the 5 treated dogs, abundance data on the main taxa of the intestinal microbiota at Tl (pre-treatment), T2 (3 days post-treatment) and T3 (7 days post-treatment) are given in Figures 1, 2 and 3. In particular, in the diagrams in Figures 1, 2 and 3, for each taxon of the intestinal microbiota, the white area corresponds to the range of abundance observed in a dataset of 30 healthy dogs and therefore represents the typical reference range for a eubiotic intestinal microbiota.

As for dog I, the data show that at T1 the subject was in a state of dysbiosis due to the increase in pathogenic and opportunistic pathogenic taxa of the intestinal microbiota, such as in particular Enterococcus and Clostridium sensu stricto 1. After treatment, already at time T2, the recovery of dog I to a eubiotic condition is evident from the reduction in the relative abundance of both taxa. At the same time, as evidence of the eubiotic transition after treatment, there is an increase in the relative abundance of certain short-chain fatty acidproducing taxa, such as Bacteroides, as well as an increase in probiotic microorganisms, such as Bifidobacterium, Lactobacillus, and Streptococcus.

As for dog II, the data show that at T1 the subject was in a state of dysbiosis due to the high ratio of opportunistic pathogenic taxa of the intestinal microbiota, such as Escherichia- Shigella. After treatment, already at time T2, the recovery of dog II to a eubiotic condition is evident from the strong reduction in the relative abundance of these taxa.

As for dog III, the data show that at T1 the subject was in a state of dysbiosis due to the increase in opportunistic pathogenic taxa of the intestinal microbiota, such as Escherichia- Shigella and Clostridium sensu stricto 1. After treatment, already at time T2, the recovery of dog III to a eubiotic condition is evident from the reduction in the relative abundance of these taxa.

The analysis of the diversity of the intestinal microbiota of dogs I-III shown in Figure 4 demonstrates that the treatment is effective in promoting convergence toward ecosystem diversity values typical of eubiotic microbiota, which are expressed as the number of species and their relative abundance distribution (Shannon Index) and phylogenetic diversity (Faith Diversity Index).

The data obtained herein show that the treatment is effective in promoting recovery to a healthy (eubiotic) intestinal microbiota in dogs with chronic dysbiosis. Treatment is effective in this context as early as 3 days after treatment and the eubiosis conditions remain stable until the end of the observation period. In particular, the treatment is effective in inducing a significant reduction in certain taxa referred to as pathobionts, which increase under dysbiotic conditions, thus consolidating chronic inflammatory states. In addition, the treatment is effective in promoting recovery to eubiotic intestinal microbiota diversity values.