WO2022090279A1

WO2022090279A1 - Saccharomyces cerevisiae yeast for the prevention and/or treatment of parasitic diseases

Info

Publication number: WO2022090279A1
Application number: PCT/EP2021/079759
Authority: WO
Inventors: Christine JULIEN; Karim ADJOU; Mohamed MAMMERI
Original assignee: Lesaffre Et Compagnie
Priority date: 2020-10-28
Filing date: 2021-10-27
Publication date: 2022-05-05
Also published as: FR3115455A1; WO2022090279A8; AR123934A1

Abstract

The present invention relates to the field of the prevention and/or treatment of parasitic diseases, and in particular the prevention and/or treatment of cryptosporidiosis in human beings and animals. The invention relates most particularly to the Saccharomyces cerevisiae strains deposited with the CNCM under number I-4407, I-5129 and/or I-5223, to a live yeast obtained by culturing said strains or else to a composition comprising same, for the use thereof in the prevention and/or treatment of parasitic diseases.

Description

Saccharomyces cerevisiae yeast for the prevention and/or treatment of parasitosis

Parent Patent Application

This patent application claims priority from French patent application number FR 20 11054 filed on October 28, 2020. The contents of the French patent application are incorporated by reference in their entirety.

Technical area

The present invention relates to the field of the prevention and/or treatment of parasitosis, and in particular to the prevention and/or treatment of cryptosporidiosis in humans and animals.

Technological Background

Cryptosporidiosis is a disease linked to a particular protozoan, Cryptosporidium. There are different species of Cryptosporidium, some of which only target certain animals. For example, Cryptosporidium baileyi or Cryptosporidium meleagridis are two species that affect poultry, while Cryptosporidium parvum does not affect poultry but rather mammals such as humans or ruminants.

Specifically, Cryptosporidium parvum is a parasitic protozoan that can infect a wide range of vertebrate hosts, including humans and ruminants. In newborn ruminants, cryptosporidiosis is a very widespread infection and the infection causes profuse diarrhoea, growth retardation and weight loss that can lead to the death of affected ruminants.

Indeed, the main clinical sign associated with cryptosporidiosis is acute diarrhea. Other associated clinical signs include dehydration, intense weakness, loss of appetite and colic. These different clinical signs can lead to death, especially for the most fragile animals, such as newborns.

In general, cryptosporidiosis therefore has very significant negative economic consequences in farms, due to the increase in mortality and growth delays, the cost of veterinary interventions and treatments it generates, but also the increase in working time for the management of sick animals.

Currently, few treatments are available in animals. For example, halofuginone is the only drug with marketing authorization in France for the prevention and/or treatment of cryptosporidiosis in calves.

However, not only is the efficacy of halofuginone partial, but it presents some toxicity and a risk of development of resistance of cryptosporidia to this compound has been reported (see Silverlas et al., Preventive Veterinary Medicine, 2009, 91: 73-84).

Furthermore, no drug is registered for the treatment of cryptosporidiosis in other animals, such as kids and lambs.

As mentioned previously, Cryptosporidium species may only be specific to certain animals, thus further complicating the emergence of new solutions for the treatment of cryptosporidiosis.

In ruminants, a multitude of compounds such as Azithromycin, Paromomycin, Nitazoxanide, Halofuginone Lactate, or Decoquinate have been tested against cryptosporidiosis but none of them has given satisfactory results for consistently control or eliminate cryptosporidiosis.

Lately, the use of alternative treatments has increased dramatically due to the worldwide increase in resistance to antimicrobial agents, including antiparasitic agents (Dinler, C et al., 2017. Cryptosporidiosis in Ruminants: Update and Current Therapeutic Approaches. Am. J. Anim. Vet. Sci. 12, 96-103).

Preliminary data obtained from testing cell cultures and newborn mice experimentally infected with Cryptosporidium parvum Iowa strain indicated prophylactic efficacy of some strains of the live yeast Saccharomyces cerevisiae. However, there is nothing to indicate that the results obtained in laboratories with young mice can be transposed to the scale of a farm in animals, such as goats, calves or lambs.

Document EP 13 818 279, of which the Applicant is the holder, describes chitin or chitin derivatives for their use in the prevention and/or treatment of parasitosis. In particular, compositions are described comprising, as active principle, a base agent chosen from chitin or a chitin derivative such as chitosan, N-acetyl-glucosamine or glucosamine. The compositions can also include an agent for boosting immunity.

This document also describes an example of treatment of cryptosporidiosis in kids by implementing a composition based on yeast cell walls, yeast extract, selenium-enriched yeast, live yeast and a blend of essential oils. The live yeast corresponds to the strain deposited by the Applicant at the CNCM under number 1-4407 and is used as an agent for stimulating immunity. In the compositions of this document, the active principle for use in the treatment of parasitosis is chitin or its derivatives. Nothing indicates or suggests that the live yeast deposited at the CNCM under the number I-4407 is effective as such without an active ingredient, and this, both for the prevention and for the treatment of parasitosis such as cryptosporidiosis.

Thus, although solutions are available, there is a real need for alternatives that are simpler to implement for the prevention and/or treatment of parasitosis, and in particular cryptosporidiosis, in particular in ruminant animals, and particularly in young animals. ruminant animals. Preferably, these alternative solutions have little or no toxic effects, are based on compounds of natural origin whose harmlessness in animals is known, and are simple to prepare and implement.

Summary of the invention

In general, the present invention is based on the demonstration that, quite surprisingly, the use of the live strain of Saccharomyces cerevisiae deposited on December 2, 2010 at the National Collection of Microorganism Cultures (CNCM) under number 1-4407 and/or the live strain of Saccharomyces cerevisiae deposited on August 31, 2016 at the National Collection of Microorganism Cultures (CNCM) under number 1-5129 and/or the live strain of Saccharomyces cerevisiae deposited on August 30, 2017 to the National Collection of Cultures of Microorganisms (CNCM) under number 1-5223, made it possible to propose an effective solution in the prevention and/or treatment of parasitosis thus responding to the growing need to limit infections by protozoa, particularly on farms.

A first object of the invention concerns the strain of Saccharomyces cerevisiae deposited on December 2, 2010 with the National Collection of Microorganism Cultures (CNCM) under number 1-4407 and/or the strain of Saccharomyces cerevisiae deposited on August 31, 2016 at the National Collection of Cultures of Microorganisms (CNCM) under number 1-5129 and/or the strain of Saccharomyces cerevisiae deposited on August 30, 2017 at the National Collection of Cultures of Microorganisms (CNCM) under number 1-5223, for its use in the prevention and/or treatment of parasitosis.

The invention also relates to compositions using, as sole active principle, specific strains of Saccharomyces cerevisiae for their use in the prevention and/or treatment of parasitosis. Thus, a second object of the invention relates to a composition comprising a strain of Saccharomyces cerevisiae deposited on December 2, 2010 with the National Collection of Microorganism Cultures (CNCM) under number 1-4407 and/or a strain of Saccharomyces cerevisiae deposited on August 31, 2016 at the National Collection of Cultures of Microorganisms (CNCM) under number 1-5129 and/or a strain of Saccharomyces cerevisiae deposited on August 30, 2017 at the National Collection of Cultures of Microorganisms (CNCM) under number 1- 5223, for use in the prevention and/or treatment of parasitosis.

These compositions according to the invention make it possible in particular to reduce mortality in young animals and/or diarrhea. In addition, these compositions only containing the strains according to the invention which are compounds of natural origin, the harmlessness in animals is known at the recommended doses of use. Therefore, these compositions have little or no toxic effects.

Finally, these compositions also have the advantage of being able to be incorporated into food compositions, in particular liquids, for use in the prevention and/or treatment of parasitosis, in particular in animals such as ruminants, and particularly in young animals such as goats for example. A third aspect of the invention relates to a method for treating parasitosis comprising the administration, to a patient, of an effective dose of the strain of Saccharomyces cerevisiae deposited by the Applicant Company on December 2, 2010 with the National Collection of Cultures of Microorganisms (CNCM) under number 1-4407 and/or the strain of Saccharomyces cerevisiae deposited on August 31, 2016 at the National Collection of Cultures of Microorganisms (CNCM) under number I-5129 and/or the live strain of Saccharomyces cerevisiae deposited on August 30, 2017 at the National Collection of Microorganism Cultures (CNCM) under number I-5223.

To the knowledge of the inventors, this is the first time that such strains have been used live and alone and that the effect on the prevention and/or treatment of parasitosis is obtained without the presence of one or more known active principles.

The Saccharomyces cerevisiae strain deposited under number 1-4407 has previously been described in application EP13818279, of which the Applicant is the holder. As developed previously, it was however neither known nor suggested that this strain could have an effect in the prevention and/or treatment of parasitosis even when it is used alone, without the presence of any other active ingredient.

Brief Description of Figures

Figure 1 shows the “anticryptosporidian” effect of Sc 1-4407 in vitro in an HCT-8 cell culture model (**: P < 0.01; ns: non-significant).

Figure 2 shows the "anticryptosporidian" effect of the yeast Sc 1-4407 in vivo by measuring the number of oocysts in the intestine (experiment representative of 4 independent experiments).

Figure 3 shows the "anticryptosporidian" effect of the yeast Sc 1-4407 in vivo by measuring the number of intracellular forms of the parasite on ileum sections (***; P < 0.001; ns: not significant) (a single isolated experience).

Figure 4 shows the daily mortality rate in groups 1, 2, 3 and 4 as a function of the number of days post-infection.

Figure 5 shows the hydration score in groups 1, 2, 3 and 4 according to the number of days post-infection. Detail of the hydration score: 1. Normal, 2. Moderately dehydrated, 3. Severely dehydrated. *: P < 0.05, **: P < 0.01, *** P < 0.001.

Figure 6 shows the evolution of the faecal score of the different groups according to the number of days post-infection. Diarrhea was scored on a score of 1 to 4. 1, normal; 2, pasty; 3, semi-liquid; 4, aqueous, ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 7 shows the duration of diarrhea in kids remaining alive throughout the experiment, ns: non-significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 8 shows the evolution of the health score of the different groups according to the number of days post-infection, ns: non-significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 9 shows the dynamics of C. parvum infection via the expression of the rate of unconcentrated oocysts released as a function of the number of days post-infection. P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 10 shows the prepatent period in the different infected groups. Aoocysts (method without concentration, detection by IF). The data is represented by a scatter plot (mean +/- SEM (standard-type mean)). One-way Anova and Tukey's post-test were used to compare the different groups. **: P<0.01, ***: P<0.001.

Figure 11 shows the duration of shedding of C. parvum oocysts in the different infected groups. Oocysts (method without concentration, detection by IF). The data is represented by a scatter plot (mean +/- SEM (standard-type mean)). One-way Anova and Tukey's post-test were used to compare the different groups. *: P<0.05, **: P<0.01.

Figure 12 shows the number of oocysts excreted (cumulated at 8 days postinfection). Oocysts (method without concentration, detection by IF). The data is represented by a boxplot (min-max). Mann-Whitney test was used to compare different groups. *: P<0.05, **: P<0.01.

Detailed description

A first object of the invention therefore relates to the Saccharomyces cerevisiae strain deposited by the Applicant Company on December 2, 2010 with the National Collection of Cultures of Microorganisms (CNCM) under number 1-4407 and/or the strain of Saccharomyces cerevisiae deposited on August 31, 2016 at the National Collection of Cultures of Microorganisms (CNCM) under number 1-5129 and/or the strain of Saccharomyces cerevisiae deposited on August 30, 2017 at the National Collection of Microorganism Cultures (CNCM) under number 1-5223, for their use in the prevention and/or treatment of parasitosis.

Quite surprisingly, and contrary to what was known hitherto, the strains according to the invention are used alone for the prevention and/or treatment of parasitosis. Therefore, it is therefore not necessary to add any active principle such as chitin or its derivatives such as chitosan, N-acetyl-glucosamine or glucosamine.

The Applicant has therefore in particular developed a means that is simpler to implement for the prevention and/or treatment of parasitosis.

The term "yeast strain" refers to a relatively homogeneous population of yeast cells. A yeast strain is obtained from the isolation of a clone, a clone being a population of cells obtained from a single yeast cell.

Culture of a yeast strain can be done by any suitable method. Yeast culture methods are known in the art, and those skilled in the art know how to optimize the culture conditions for each strain according to its nature. A yeast is obtained by cultivating a yeast strain in a culture medium, for example as described in the reference book "Yeast Technology", 2nd edition, 1991, G. Reed and T.W. Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8.

Thus, for example, on an industrial scale, yeast cells that can be used in the context of the present invention can be obtained by a process comprising the following steps: culturing the Saccharomyces cerevisiae 1-4407 strain and/or the strain of Saccharomyces cerevisiae 1-5129 and/or of the Saccharomyces cerevisiae 1-5223 strain, in a culture medium in several stages, first in semi-anaerobiosis, then in aerobiosis (medium/atmo sphere rich in oxygen) to obtain a multiplication of the starting yeast cells; and separation, by centrifugation, of the yeast cells thus produced to obtain a liquid yeast cream containing between 12% and 25% of yeast dry matter.

The yeast thus obtained is a living yeast.

According to one embodiment, the present invention relates to the Saccharomyces cerevisiae yeast obtained by culturing the Saccharomyces cerevisiae strain deposited with the CNCM under the number 1-4407 and/or the Saccharomyces cerevisiae yeast obtained by culturing the Saccharomyces cerevisiae strain deposited from the CNCM under number 1-5129 and/or the Saccharomyces cerevisiae yeast obtained by culturing the Saccharomyces cerevisiae strain deposited with the National Collection of Microorganism Cultures (CNCM) under number 1-5223, for their use in the prevention and/or treatment of parasitosis.

In the present invention, the expressions "yeast obtained by culture of the Saccharomyces cerevisiae strain deposited at the CNCM under the number 1-4407", "yeast obtained by culture of the strain 1-4407" and "yeast Sc 1- 4407", these three terms having the same meaning as given above.

Similarly, the expressions "yeast obtained by culturing the Saccharomyces cerevisiae strain deposited at the CNCM under number 1-5129", "yeast obtained by culturing the 1-5129 strain" and "yeast Sc 1-5129", these three terms having the same meaning as given above.

Similarly, the expressions "yeast obtained by culturing the Saccharomyces cerevisiae strain deposited at the CNCM under number 1-5223", "yeast obtained by culturing the 1-5223 strain" and "yeast Sc 1-5223", these three terms having the same meaning as given above.

According to the invention, the Sc 1-4407 yeast and/or the Sc 1-5129 yeast and/or the Sc 1-5223 yeast are used live in the prevention and/or treatment of parasitosis.

For the rest of the description, by "live yeast according to the invention", reference is made to the Sc 1-4407 yeast, the Sc 1-5129 yeast, the Sc 1-5223 yeast or mixtures thereof, unless the context does not make it possible to clearly identify whether it is one or the other of the said yeasts. By live yeast within the meaning of the present invention, is meant a yeast whose metabolism is active or reactivable or capable of multiplying, as opposed to a dead yeast for which the metabolism has been irremediably stopped.

According to one embodiment, the live yeast according to the invention may be in the form of a live dry yeast or a fresh live yeast, preferably in the form of a live dry yeast.

According to a preferred embodiment, the dry yeast according to the invention is chosen, for example, from active dry yeast, instant active dry yeast, and dry yeast with intermediate humidity, frozen or not, preferably the dry yeast is a dry yeast instant active.

By dry yeast is meant any yeast having a dry matter content greater than 90%, preferably ranging from about 92 to 96%. One advantage of dry yeast is its long shelf life.

An active dry yeast or an instant active dry yeast according to the invention comprises a yeast dry matter content greater than 90%, preferably a dry matter content of 92% to 96%.

Instant dry yeast has the particularity of not requiring rehydration before being incorporated into a product. It comes from dehydration by the action of a hot air gradient which makes it possible to transform a pasty product (pressed or liquid yeast) into fine vermicelli while remaining active.

Active dry yeast can be obtained by dehydrating pressed or liquid yeast by the joint action of heat (at low temperature) and mechanical activity, which make it possible to transform a pasty product (pressed or liquid yeast) into a dry product, in the form of spherules, while sparing its viability.

For example, active dry yeast can be obtained by extrusion and fluidized bed drying of pressed yeast or liquid yeast.

Active dry yeast is mainly in the form of spherules whose diameter can be generally ranged from 0.1 µm to 2.5 mm and H2O content from 4% to 8% by mass.

Instant active dry yeast can be obtained by dehydrating pressed or liquid yeast by the action of a hot air gradient which makes it possible to transform a pasty product (pressed or liquid yeast) into fine dry vermicelli. The instant dry yeast must then, in order to remain stable, be conditioned in the absence of oxygen.

The frozen or non-frozen intermediate moisture dry yeast comprises, for example, from 70% to 85% of yeast dry matter.

According to a particular embodiment, the live yeast according to the invention for their use in the prevention and/or treatment of parasitosis, are in the form of active dry yeast or in the form of instant dry yeast.

The live yeasts Sc 1-4407 and/or Sc 1-5129 and/or Sc 1-5223 for their use according to the invention are effective as the sole active ingredient in the prevention and/or treatment of parasitosis. Consequently, the live yeasts according to the invention are used alone without the presence of chitin or derivatives of chitin such as chitosan, N-acetyl-glucosamine or glucosamine.

A preferred embodiment, the live yeasts according to the invention are used in the prevention and/or treatment of parasitosis in animals. Preferably, the live yeasts are used in the prevention and/or treatment of parasitosis in ruminants, preferably in young ruminants, and more preferably, in the prevention and/or treatment of parasitosis in kids, calves and the lamb.

According to the invention, the live yeast Sc 1-4407 and / or Sc 1-5129 and / or Sc 1-5223 for its use in the prevention and / or treatment of parasitosis is implemented at the rate of a daily dose comprised of Ix10 ⁷ to Ix10 ¹¹ Colony Forming Unit (CFU)/animal. Preferably, the daily dose is from 1x10 ⁹ to 1x10 ¹¹ CFU/animal.

By "parasitosis" is meant within the meaning of the present invention a disease linked to a protozoan.

According to a preferred embodiment of the invention, the parasitosis is cryptosporidiosis. According to a variant of this embodiment, the cryptosporidiosis is caused by the protozoan Cryptosporidium, and preferably by the protozoan Cryptosporidium parvum. Thus, according to this embodiment, the invention relates to the strain of Saccharomyces cerevisiae deposited with the National Collection of Microorganism Cultures (CNCM) under the number 1-4407 and/or the strain Saccharomyces cerevisiae deposited with the National Collection of Cultures of Microorganisms (CNCM) under number 1-5129 and/or the Saccharomyces cerevisiae strain deposited with the National Collection of Cultures of Microorganisms (CNCM) under number 1-5223, for their use in the prevention and/or treatment cryptosporidiosis, preferably cryptosporidiosis caused by Cryptosporidium.

According to a first variant of this embodiment, the invention relates to the Saccharomyces cerevisiae strain deposited with the National Collection of Microorganism Cultures (CNCM) under number 1-4407, for its use in the prevention and/or treatment of cryptosporidiosis, preferably cryptosporidiosis caused by Cryptosporidium.

According to a second variant of this embodiment, the invention relates to the strain of Saccharomyces cerevisiae deposited with the National Collection of Microorganism Cultures (CNCM) under number 1-5129, for its use in the prevention and/or treatment of cryptosoridiosis, preferably cryptosporidiosis caused by the protozoan Cryptosporidium.

According to a third variant of this embodiment, the invention relates to the strain of Saccharomyces cerevisiae deposited with the National Collection of Microorganism Cultures (CNCM) under number 1-5223, for its use in the prevention and/or treatment of cryptosporidiosis, preferably cryptosporidiosis caused by the protozoan Cryptosporidium.

According to this particular embodiment, the animal is chosen from ruminants, preferably from young ruminants, and more preferably from calves, lambs and kids.

A second object of the invention relates to a composition comprising a live yeast Sc 1-4407 and/or a live yeast Sc 1-5129 and/or a live yeast Sc I-5223, for use in the prevention and/or treatment of parasitosis.

The composition according to the invention can be a pharmaceutical composition, a nutritional supplement with a health effect or optionally a food composition. According to a particular embodiment, the composition comprising the live yeast according to the invention, for its use in the prevention and/or treatment of parasitosis also comprises at least one pharmaceutically acceptable excipient.

As mentioned above, the effect on the prevention and/or treatment of parasitosis is obtained by the sole presence of live yeasts according to the invention. Consequently, the composition according to the invention is free of any other compound or active principle, with the exception of pharmaceutically acceptable excipients. Thus, the composition for its use according to the invention is in particular free of chitin or derivatives of chitin such as chitosan, N-acetyl-glucosamine or glucosamine.

According to a particular embodiment, the parasitosis is cryptosporidiosis. According to a variant of this embodiment, the cryptosporidiosis is caused by the protozoan Cryptosporidium, and preferably by the protozoan Cryptosporidium parvum.

These compositions used according to the invention make it possible in particular to reduce mortality in young animals and/or diarrhea. In addition, these compositions only containing the strains according to the invention which are compounds of natural origin, the harmlessness in animals is known at the recommended doses of use. Therefore, these compositions have little or no toxic effects.

Finally, these compositions also have the advantage of being able to be incorporated into food compositions, in particular liquids, for use in the prevention and/or treatment of parasitosis, in particular in animals such as ruminants, and particularly in young ruminants, such as for example kid, veal or lamb.

A third aspect of the invention relates to a method for treating parasitosis comprising the administration, to a patient, of an effective dose of the Saccharomyces cerevisiae strain deposited with the National Collection of Microorganism Cultures (CNCM) under number 1 -4407 and/or the Saccharomyces cerevisiae strain deposited with the National Collection of Microorganism Cultures (CNCM) under number 1-5129 and/or the Saccharomyces cerevisiae strain deposited with the National Collection of Microorganism Cultures (CNCM) under the number 1-5223. Live yeasts Sc 1-4407, Sc 1-5129 and Sc 1-5223 are as defined above.

Preferably, the patient is an animal. Preferably again, the animal is chosen from among a young ruminant, and most preferably, the young ruminant is a kid, a lamb or a calf.

According to a particular embodiment, the parasitosis is cryptosporidiosis. According to a variant of this embodiment, the cryptosporidiosis is caused by the protozoan Cryptosporidium, and preferably by Cryptosporidium parvum.

Examples

Example 1: In vitro inhibition of the parasite load induced by C. parvum.

The purpose of this example is to demonstrate via a cellular model the effectiveness of the strains according to the invention for the reduction of the parasite load induced by C. parvum.

Material and methods

Pests:

C. parvum oocysts (Iowa strain, Waterborne Inc., New Orleans, Louisiana, USA) were used in this study.

Products tested:

Live yeast obtained by culturing the Saccharomyces cerevisiae strain deposited with the CNCM under number 1-4407 (Sc 1-4407) was tested for this example, and is in the form of instant dry yeast.

Paromomycin sulphate (Olon S.p.A., Milano, Italy), considered a reference molecule for its “anti-cryptosporidian” effect (Schupfner et al., 2013), was used in this study as a “positive control”.

These two products were tested at a concentration of 500 pg/ml, equivalent to 10 ¹⁰ CFU/g of product.

Operating mode:

The cell cultures were carried out in 12-well culture plates (Nunc, France). Two types of experiments were carried out: A. Effect of live yeast on cells infected with Cryptosporidium: The cell cultures were all infected with 10 ⁴ oocysts of C. parvum when they were at 70% confluence.

Confluence should be understood as the percentage of the surface covered by the cells in culture/total surface of the culture well.

After 48 hours of incubation, with the exception of the negative control for the “anti-cryptosporidian” action, the live yeast Sc 1-4407 was tested at different concentrations: 250, 500 and 1000 pg/ml.

B. Direct effect of live yeast pre-incubated with C. parvum oocysts: 10 ⁴ C. parvum oocysts were incubated with 500 pg/ml of live Sc 1-4407 yeast.

For both experiments (A and B), and after 48 hours of incubation, the intracellular forms of Cryptosporidium were labeled with a specific polyclonal antibody conjugated to fluorescein (Sporo-Glo, Waterborne inc., New Orleans, LA, USA ).

The enumeration of cryptosporidia was made on 6 horizontal lines (each horizontal line corresponds to 50 random microscopic fields).

Negative controls were defined as 100% parasite growth. The average percentage of intracellular forms was used to show the intensity of infection.

Statistical analysis :

The statistical analysis was carried out with GraphPad Prism 5 (GraphPad Software, CA, USA) for the study of the “anti-cryptosporidian” effect of the live yeast Sc 1-4407 in vitro and in vivo. The Student's t test or the Mann-Whitney test was used to compare the intensity of infection between the control and the different products (paromomycin sulphate and live yeast Sc 1-4407). A difference was considered significant for P<0.05.

Results

As shown in Figure 1, the live yeast Sc 1-4407 significantly (P = 0.0016) reduced the percentage of intracellular cryptosporidia by 55% compared to the control, as did paromomycin sulphate. This example clearly demonstrates the anti-cryptosporidian effect of the Sc 1-4407 yeast according to the invention and consequently its effectiveness in the treatment of crypto sporidio se.

Example 2: In vivo inhibition in mice of the parasite load induced by C. parvum.

Material and methods

Pests:

Products tested:

Live yeast obtained by culturing the Saccharomyces cerevisiae strain deposited with the CNCM under number 1-4407 (Sc 1-4407) was tested for this example. Paromomycin sulphate (Olon S.p.A., Milano, Italy), considered a reference molecule for its “anti-cryptosporidian” effect (Schupfner et al., 2013), was used in this study as a “positive control”.

These two products were tested at a concentration of 1 mg (5.3×10 ⁴ CFU)/inoculation/animal.

Procedure:

CD-I mice (Charles River, France) used in this study were kept in isolators during all experiments. These experiments have been approved by the French Regional Ethics Committee number 16 Anses / ENVA / UPEC (Project number 14/04 / 15-4B) and by the Ministry of National Education, Higher Education and Research of France.

The CD-I mouse model is a model that can be extrapolated to the natural infection of newborn domestic ruminants (lambs, kids and calves) (Martin-Gômez et al., Parasitai Res (2006) 99: 1. https:// doi.org/10.1007/s00436-005-0055-12006).

The 5-day-old mice were experimentally infected by esophageal gavage with 10 ⁵ oocysts/animal of C. parvum, using plastic feeding tubes (Phymep 22-25, France). The mice were inoculated with 1 mg of live yeast Sc 1-4407 by gavage on D1, D+1 and D+4 post-infection (PI). Paromomycin sulfate was used as a positive control at the same dose (1 mg/inoculation).

The untreated control batch was inoculated with the diluent in which the products were dispersed (deionized water). The animals were euthanized by cervical dislocation on D+8 PI. The intestinal tract, from the duodenum to the rectum, of each animal was removed individually, completely opened by a longitudinal excision and its contents were suspended in a solution of PBS (Phosphate Buffered Saline®).

The intensity of infection was measured by two techniques:

(1). By counting the number of oocysts in the intestine using the Merifluor® Cryptosporidium / Giardia kit (Meridian Bioscience, Milan, Italy).

(2). By counting intracellular forms of Cryptosporidium from histological sections of ileum embedded in paraffin using the kit (Sporo-Glo, Waterborne inc., New Orleans, LA, USA).

Statistical analysis :

Results

As shown in Figure 2, the live yeast Sc 1-4407 significantly (P < 0.0001) reduced the percentage of C. parvum oocysts in the intestine compared to the control by 60%.

From FIG. 3, the yeast Sc 1-4407 significantly reduced (P<0.0001) by 57% the percentage of intracellular forms of cryptosporidia compared with the control.

This in vivo example on the CD-1 baby mouse model confirms the in vitro results of example 1. The implementation of the Sc 1-4407 yeast according to the invention allows thus reducing the parasite load of the treated mice and this model can be extrapolated to other animals such as lamb, kid or calf.

This example thus demonstrates the effect of live yeast Sc 1-4407 in the treatment of cryptosporidiosis in animals.

Example 3: in vivo test of the effect of Saccharomyces cerevisiae strains for the prevention of induced cryptosporidiosis in kids.

The objective of this example is to demonstrate the clinical and microbiological efficacy of live Saccharomyces cerevisiae yeast supplementation against experimental infection by C. parvum in goat kids.

The experimental model of kids is a model of choice for the study of cryptosporidiosis for several reasons: they are small compared to other models such as calves; they can all be grouped into a single group, thus expressing a large number of social behaviors.

Unlike the mouse model, which remains asymptomatic, this model reproduces exactly the clinical signs observed in humans and calves.

Neonates have the major advantage of being less expensive to use as a model to produce large numbers of oocysts and to illustrate the pathogenesis of cryptosporidian infections.

Thus, this model has been recommended and used to assess the therapeutic or prophylactic potential (Petermann et al., 2014. Efficacy of halofuginone lactate against experimental cryptosporidiosis in goat neonates. Vet. Parasitol. 202, 326-329).

Material and methods

Pests:

C. parvum oocysts (Iowa strain, Waterborne Inc., New Orleans, Louisiana, USA) were used in this example. They were purified from infected calves and used within 4 months of purification.

Strains used:

Two live strains of Saccharomyces cerevisiae were used:

The live strain Sc 1-4407 at a concentration of 1.2× ^{10 10} CFU/animal. The live strain Sc 1-5129 at a concentration of 1.94× ^{10 10} CFU/animal.

Experimental protocol :

All experiments were carried out in accordance with the European directive (2010/63/EU), and approved by the Loire Valley ethics committee (CEEA Val de Loire n° 19, dossier n° 2017120708306306306. V3 - 12556). Enrichment equipment was made available to the kids to ensure their well-being.

Forty one-day-old French male kids were randomly selected from farms known to have had no episodes of cryptosporidiosis in the years preceding the trial.

The faecal presence of Cryptosporidium oocysts was verified using the immunofluorescence and quantitative PCR (qPCR) techniques described below from the faeces of mother goats on the day of separation with their kid selected for l. 'trial.

The kids were separated from their mothers immediately after birth and fed three times with colostrum heated to 1 h at 56°C to prevent caprine arthritis-viral encephalitis (CAEV).

The one-day-old kids were transported to the experimental premises (Experimental infectiology platform of biosafety level 3, UE-1277-PFIE of INRA Nouzilly) where they were randomly divided into 4 groups (n =10 in each group) within the same room but in separate enclosures:

Group 1 (control): kids not infected with C. parvum,

Group 2: kids infected with C. parvum,

Group 3: kids infected with C. parvum and supplemented with live strain 1-5129,

Group 4: kids infected with C. parvum and supplemented with the live strain 1-4407.

The animals were fed ad libitum with a non-medicated commercial milk (Nectagneau®: 150 g for 850 ml of water) throughout the duration of the experiment.

During the first week, the kids were trained on artificial feeding. The kids in groups 3 and 4 received 1 g of live yeast diluted in 5 ml of non-medicated milk individually, orally using a syringe 4 hours before infection (OJ).

The kids in the groups not treated with the live yeast strains according to the invention (groups 1 and 2) received individually a placebo of 5 ml of non-medicated milk according to the same mode of administration.

For the infection, at D0, the kids of groups 2, 3 and 4 received orally individually 10 ⁶ oocysts of the C. parvum Iowa strain (diluted in 5 ml of sterile water).

As a control, the kids of group 1 received on D0, orally and individually, 5 ml of distilled water.

Supplementation with live yeast (groups 3 and 4) was repeated every day throughout the duration of the experiment (D25).

Clinical examination, scoring and sampling:

Continuous and individual recordings of the internal temperature of the kids were made by the Anipill® capsule throughout the duration of the experiment. This measurement method is non-invasive for kids. The kids received the capsule orally as soon as they arrived on the platform at JO.

Data on general health and hydration status were recorded throughout the study on a daily basis from the scoring grid described in the calf (Stebbins, E., et al 2018. Clinical and microbiology efficacy of the piperazine-based drug lead MMV665917 in the dairy calf cryptosporidiosis model. PLoS Negl. Trop. Dis. 12) and adapted as follows:

1.Normal,

2. moderately depressed/dehydrated,

3. severely depressed/dehydrated.

Weight and mortality rate were recorded during the daily clinical examination. Comatose kids or kids in very poor general health were euthanized.

Feces from each kid were collected daily using a panty system designed from a plastic bag that the kids wore at all times. The bags were removed and replaced when there was enough faeces. Each bag has been clearly identified (group / kid / day).

The samples of faeces collected were kept until their use either at 4°C to assess the parasite load, or at -80°C to study the faecal microbiota.

Fecal consistency and oocyst excretion

Fecal consistency was assessed on a scale of 0 to 4. 0: normal stool, without mucus; 1: pasty and thick, molded or not, glazed; 2: creamy; 3: semi-fluid; 4: liquid. A mean fecal consistency score was calculated daily for each group.

0.25 g of faeces from each kid was analyzed to determine the daily shedding of C. parvum oocysts. This amount was either suspended directly in 10 ml of PB S buffer or concentrated via diethyl ether - PBS concentration as previously described (Castro-Hermida, J. A et al., 2005. Prevalence of Giardia duodenalis and Cryptosporidium parvum in goat kids in western France. Small Rumin. Res. 56, 259-264) and whose pellet was resuspended in distilled water up to 400 pl.

Cryptosporidium oocysts were detected from 10 µl of unconcentrated oocyst solution by direct immunofluorescence assay (IFA) using the commercial Merifluor Cryptosporidium/Giardia immunofluorescence assay (Meridian Diagnostics, Inc., Milano, Italy) following the manufacturer's recommendations and with some modifications as described by Mohamed Mammeri et al., 2018 (Cryptosporidium immunolabeling in paraffin-embedded ileum of CD-I neonatal mice. Global science research journals 6, 197-202).

The stained slides were examined under a Leica fluorescent microscope using the Leica Application Suite software (version 4.5.0; Leica Microsystems; Inc., Switzerland).

The quantitative PCR technique (qPCR) was also used to quantify the parasite load of the kids at D0 post-infection (PI), from D+2 to D+13 PI, at D+15 PI and at D+20 PI.

Genomic DNA was extracted from 200 µL of the concentrated oocysts or unconcentrated oocysts using the QIAmp DNA Mini Kit (Qiagen, France) according to the manufacturer's instructions. Briefly, the samples were suspended in lysis buffer and the oocysts disrupted by subjecting them to an additional initial step of 10 freeze-thaw cycles (freezing in liquid nitrogen for 1 minute and heating in a water bath at 90°C for 1 minute) before proceeding with DNA extraction. The DNA was then stored at -20°C until used for molecular analysis. Then, the COWP gene was amplified by qPCR.

The parasite load was assessed by calculating the number of Cryptosporidium oocysts per gram of faeces (OPG) which was obtained by multiplying the total number of oocysts by the dilution factor.

Genetic analysis of a strain from Iowa:

Molecular characterization of the C. parvum Iowa strain was carried out before use and after each passage on at least three kids from each infected group (groups 2, 3 and 4).

This molecular characterization ensures that the animals have not been infected with another species of Cryptosporidium or another strain of C. parvum. Genomic DNA was also extracted from the C. parvum Iowa strain used to infect kids, as described below.

The genomic DNA extracted either from oocysts used to infect the kids, or from kids infected 5 days post-infection (3 animals for each of groups 2, 3 and 4) was analyzed by nested PCR targeting the subunit of fragment of the 18S rRNA gene as previously described (Xiao, L. et al., 1999, Genetic Diversity within Cryptosporidium parvum and Related Cryptosporidium Species. Appl. Environ. Microbiol. 65, 3386-3391). The amplification products were then visualized by electrophoresis in 2% agarose gels stained with ethidium bromide.

Cryptosporidium species were detected from positive 18S SSU-rRNA products by restriction fragment length polymorphism (RFLP) analysis using two endonucleases Sspl and MboII (New England BioLabs, France) as previously described (Feng, Y . et al., 2007. Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Vet. Parasitol. 144, 1-9.).

The amplification products were then visualized by electrophoresis on 3% agarose gels (MetaPhor - Ozyme, France) stained with ethidium bromide. C. parvum samples were subtyped by nested PCR sequence analysis of the 60 kDa glycoprotein locus (gp60) as previously described (Gatei, W et al., 2006. Development of a multilocus sequence typing tool for Cryptosporidium hominis. J Eukaryot. Microbiol. 53 Suppl 1, S43-48). The amplification products were then visualized by electrophoresis in 2% agarose gels stained with ethidium bromide.

In each PCR reaction, a positive control sample and a negative control sample were included.

The positive control was DNA extracted from 10 ⁶ oocysts of the C. parvum Iowa strain (Waterborne Inc., New Orleans, Louisiana, United States). The negative control was purified water.

DNA sequence analysis:

The PCR products of the two target genes (18S rRNA and gp60) were sequenced in both directions by Eurofins Genomics (France) and Genoscreen (France).

The consensus sequences were edited using the BioEdit Sequence Alignment Editor software and then compared with each other and with the sequences published in GenBank available in the Basic Local Alignment Search Tool (BLAST) via the National Center for Biotechnology Information (NCBI) .

C. parvum subtypes were recognized based on the number of trinucleotide repeats (TCA or TCG) or rare repeats (R) in the 60 kDa glycoprotein sequence as previously described (Sulaiman et al., 2005).

Histopathological examination:

At the end of the experiment, kids were killed by intravenous injection of a lethal dose of pentobarbital (10 ml per animal) after an intramuscular injection of zoletil®50 at a rate of 20 mg/kg. Ileum samples for histopathological examination were collected immediately after natural death or euthanasia of the kids.

Specimens were fixed in 10% buffered neutral formalin for one week, then stored in 70% ethanol before processing by routine histological methods. Briefly, the samples were embedded in paraffin and the blocks were cut into 5 µm sections. For each portion, two slides containing 3 sections spaced 1 cm apart were made: one slide was stained with conventional hematoxylin and eosin (HES) and then examined under an optical microscope according to standard procedures.

For specific verification of cryptosporidia, the second slide was stained by immunofluorescence (IF) using a commercial FITC-conjugated monoclonal antibody (Sporo-Glo™, Waterborne™ Inc, New Orleans, USA) as previously described (Mammeri, M., et al., 2018. Cryptosporidium immunolabeling in paraffin-embedded ileum of CD-I neonatal mice (Global Science Research Journals 6, 197-202).

IF sections were used to count endogenous parasites while HES sections were used primarily to observe pathological changes.

Data analysis and statistical methods:

Data were analyzed and graphs were edited for figure preparation using GraphPad Prism software version 5.00 (GraphPad Software, CA, USA). Data were expressed as mean ± standard deviation.

Analyzes of variance (ANOVA), or Bonferroni, Student and Kruskal-Wallis tests were used to analyze the data.

The temperature results were compared using a linear model in repeated data (mixed model) ANOVA, followed by a post-test in order to compare the groups with each other.

Comparison of survival curves was performed using the Mantel-Cox chi2 test. P values < 0.05 were considered statistically significant.

Results

Kid health and molecular characterization of Cryptospridium

Negative results were obtained from faeces of mother goats by IF and qPCR techniques. Additionally, no genetic changes from the Iowa strain were observed throughout the kid trial. This strain was characterized as a species of C. parvum by partial 18S rRNA and RFLP sequencing, and assigned to the IIaA17G2R1 subgenotype by sequencing of a locus encoding the glycoprotein gp60. No other species of Cryptosporidium or another strain of C. parvum was detected.

Mortality:

Deaths (euthanized due to severe illness or found dead) were observed in all groups. Mortality rates (Figure 4) were approximately 10% (1/10) lower for the uninfected control group (group 1) and group 4 (P = 0.9703) and approximately 80% (8/ 10) for groups 2 and 3 (P=0.6781) at the end of the experiment (D+26 PI).

Weight :

No statistical difference was observed between the various groups from the start of the experiment (D0) until D+6 PI (results not shown).

Then, the uninfected control group and group 4 showed continuous growth, while group 2 and group 3 showed a net weight loss until D+11 PI, then resumed their growth.

At the end of the experiment, the uninfected group and group 4 had the highest weight compared to the other groups.

Temperature :

The physiological temperature was recorded for all the groups from the start of the experiment until D+25 PI. The temperature remained normal throughout the experiment for groups 1 and 4, with no significant difference between these two groups.

On the other hand, hypothermia was recorded in group 2 with a significant difference with group 1 from D+3 PI to D+8 PI, then from D+14 PI to D+16 PI. The same observation was made with group 3 only on D+6 PI.

Hydration score:

For the uninfected control group and group 4, the hydration score was normal with no significant difference between these two groups throughout the experiment (Figure 5). For the infected group, this score increases from D+1 PI to D+1 1 PI, then to D+1 8 PI (significant difference with the uninfected control group). In group 3, this score increases significantly compared to that of the uninfected control group only on D+6 PI, D+8 PI and on D+9 PI before decreasing.

Fecal score:

Faeces quickly became watery in infected groups, with traces of mucus, and changed color from brown to yellow. The results of the faecal score (FIG. 6) show that supplementation with the live strains according to the invention reduced diarrhea in the two treated groups.

This reduction was significant with strain 1-4407 for almost the entire experiment.

Diarrhea lasted significantly longer in the infected groups (2, 3 and 4) than in the uninfected control group (Figure 7). In addition, the duration of the diarrhea is shorter (statistical difference) in the two treated groups 3 and 4 than in the infected control group (group 2).

This indicates that live yeast supplementation reduced the duration of diarrhea, with a greater effect of strain 1-4407.

Health Score:

Animals in the infected control group had less appetite and quickly became lethargic. Their health score (FIG. 8) increases from D+2 PI to D+11 PI, then on D+15 PI, D+16 PI and on D+20 PI.

The same observation was reported for group 3 where the health score increased significantly (compared to the uninfected group) from D+6 PI to D+1 l PL A normal score was reported for group 4 with no difference significant with the uninfected control group throughout the experiment.

Infection dynamics

The first results of the dynamics of the infection were obtained throughout the experiment using the immunofluorescence technique from unconcentrated oocysts (figure 9).

The excretion of oocysts began on D+4 PI in the infected control group with a peak on D+5 PI, before becoming undetectable from D+12 PI. The excretion of oocysts in the groups infected and supplemented with live yeast also started D+4 PI. However, the kids excreted significantly fewer oocysts (until D+9 PI) than in the infected control group.

Then, the excretion of oocysts decreases in all the infected groups until D+12 PI before becoming undetectable until the end of the experiment. Table 1 presents the percentage reduction in infection in these groups 3 and 4 compared to the control group using immunofluorescence.

Table 1

Unconcentrated oocysts

Days post infection (DPI) Group 4 (%) Group 3 (%)

3,100 (n= 8**) 100 (n= 9*)

4 99.78 (n= 8**) 90.62 (n= 10)

5 97.26 (n= 8**) 80.80 (n= 9)

6 97.59 (n=10) 73.99 (n=10)

7 89.25 (n=10) 66.27 (n=7)

8 83.18 (n=10) 33.87 (n=5*)

9 93.82 (n=10) 61.19 (n=5)

10 90 (n=10) 99.48 (n=4)

11 92.44 (n=10) 54 (n=3)

12 29.29 (n=9*) 100 (n= 2)

15 100 (n= 9) 100 (n= 2)

The asterisks indicate the impossibility of obtaining kid feces. *: one kid missing, **: two kids missing. The data are expressed as percentages of average oocysts compared to the untreated group (100%).

Although supplementation with strain 1-5129 was effective with respect to oocyst shedding, these results show that supplementation with strain 1-4407 was even more effective and reduced infection by more than 90% during the entire duration of the experience. With regard to the prepatent period (FIG. 10), the animals of the supplemented groups (groups 3 and 4) show a later excretion of oocysts compared to the infected control group using immunofluorescence from only unconcentrated oocysts.

In addition, for group 4, this supplementation significantly reduced the duration of oocyst excretion (Figure 11).

The cumulative number of oocysts excreted shows that the excretion is significantly reduced in groups 3 and 4 (FIG. 12) on D+8 PI (where there are at least 3 animals per group).

As presented in Table 2 below, supplementation with the two live yeasts according to the invention significantly reduces the rate of oocyst excretion over the first 8 days of supplementation (P<0.05).

Table 2 infection + infection + supplementation supplementation with 1-5129 with 1-4407

Detection of NC + IF 84% 96% oocysts _{C + IF 87% 97%}

Discussion

The objective of this example was the preventive effect of supplementation with live yeasts according to the invention in kids experimentally infected with 10 ⁶ oocysts of C. parvum (Iowa strain).

Only the presence of C. parvum species was detected, and of a single subtype also used for the experimental infection of animals. Moreover, this confirms that the kids were really free of cryptosporidiosis (mainly the wild strain of C. parvum) before being infected during the experimental test. The example demonstrates that daily supplementation with yeast I-4407 or I-5129 during the pre-weaning period of the newborn significantly modified the course of cryptosporidiosis.

The intensity of oocyst excretion was lower. There was less diarrhea in the groups supplemented with the two live strains according to the invention and a lower mortality rate in the groups supplemented with the live yeast strain 1-4407 compared to the control group infected and not supplemented with live strains .

In addition, in the groups supplemented with the live yeast strain 1-4407 according to the invention, the excretion of oocysts started later and the duration of excretion was shorter than in the control group.

Finally, the supplementation according to the invention seems to allow better control of the disease and prevent the resumption of parasite excretion (rebound phenomenon).

The live yeast strain 1-4407 also made it possible to obtain greater weight gain in kids compared to infected kids that had not received supplementation with said live strains.

Contrary to what was hitherto known for decoquinate (Mancassola, R., et al. 1997. Evaluation of decoquinate to treat experimental cryptosporidiosis in kids. Vet. Parasitol. 69, 31-37.) and halofuginone lactate (Petermann, J., et al. Efficacy of halofuginone lactate against experimental cryptosporidiosis in goat neonates. Vet. Parasitol. 202, 326-329), the administration of the live strains according to the invention is beneficial on the gain weight.

The products generally used in the prevention of cryptosporidiosis have several disadvantages.

For example, administration of nitazoxanide results in side effects due to acute toxicity resembling symptoms of cryptosporidiosis (Viel, H et al. 2007. Efficacy of nitazoxanide against experimental cryptosporidiosis in goat neonates. Parasitol. Res. 102, 163- 166.), a rapid rebound effect (re-excretion of oocysts) is observed after stopping treatment with a-cyclodextrin (Castro-Hermida, J. A et al., 2004a. Efficacy of a-cyclodextrin against experimental cryptosporidiosis in newborn goats. Vet. Parasitic. 120, 35-41). These disadvantages are not observed by the use of live strains according to the invention.

Unlike many ineffective products (Dinler, C., et al. 2017. Cryptosporidiosis in Ruminants: Update and Current Therapeutic Approaches. Am. J. Anim. Vet. Sci. 12, 96-103), the use of live strains according to the invention presents no risk of toxicity, said strains being able to be administered throughout the neonatal period of risk of diarrhea (4 weeks).

Thus, the administration of the live yeasts according to the invention in the feed of ruminants is found to be an alternative of choice for reducing the use of synthetic drugs in the control of cryptosporidiosis and for maintaining the growth performance of the animals.

In addition to being economically beneficial, controlling cryptosporidiosis also reduces the risk of zoonotic transmission from ruminants to humans.

In conclusion, the effectiveness of the supplementation with live strains according to the invention presented in the present example clearly demonstrates that the use of live strains 1-4407 and 1-5129 is an appropriate and attractive alternative for farmers for the prevention and /or the treatment of cryptosporidiosis in young animals.

Example 3 was reproduced under the same conditions but replacing the live strain 1-5129 with the live strain 1-5223. The results obtained (not presented here) with the live strain 1-5223 are strictly identical to those obtained with the strain 1-5129 and lead to the same conclusions.

(original in electronic form)

(This sheet does not form part of the international application or count as a sheet thereof)

(original in electronic form)

RESERVED FOR THE RECEIVING OFFICE

RESERVED FOR THE INTERNATIONAL OFFICE

Claims

1. Saccharomyces cerevisiae strain deposited with the CNCM under number 1-4407 and/or Saccharomyces cerevisiae strain deposited with the CNCM under number 1-5129 and/or Saccharomyces cerevisiae strain deposited with the CNCM under number 1-5223 , for its use in the prevention and/or treatment of parasitosis.

2. Live yeast obtained by culturing the Saccharomyces cerevisiae strain deposited with the CNCM under number 1-4407 and/or obtained by culturing the Saccharomyces cerevisiae strain deposited with the CNCM under number 1-5129 and/or obtained by culture of the Saccharomyces cerevisiae strain deposited with the CNCM under number I-5223, for its use in the prevention and/or treatment of parasitosis.

3. Strain or live yeast according to claim 1 or claim 2, for its use in the prevention and/or treatment of parasitosis in animals.

4. Strain or live yeast according to claim 3, for its use in the prevention and/or treatment of parasitosis in a young ruminant, and preferably in a kid, calf or lamb.

5. Strain or live yeast according to one of claims 1 to 4, for its use in the prevention and/or treatment of cryptosporidiosis.

6. Strain or live yeast according to claim 5, for its use in the prevention and/or treatment of cryptosporidiosis due to the protozoan Cryptosporidum, and preferably to the protozoan Cryptosporidum parvum.

7. Composition comprising a yeast obtained by culturing the Saccharomyces cerevisiae strain deposited with the CNCM under number I-4407 and/or obtained by culturing the Saccharomyces cerevisiae strain deposited with the CNCM under number 1-5129 and/or obtained by culture of the Saccharomyces cerevisiae strain deposited with the CNCM under number 1-5223, for use in the prevention and/or treatment of parasitosis. A composition according to claim 7, wherein said yeast is in the form of dry yeast, for daily use in an amount of 1x10 ⁷ to 1x10 ¹¹ CFU/animal.