WO2014125154A1

WO2014125154A1 - Fungi as biological control agents

Info

Publication number: WO2014125154A1
Application number: PCT/ES2014/070109
Authority: WO
Inventors: Adolfo PAZ SILVA; Rita SÁNCHEZ-ANDRADE FERNÁNDEZ; María Sol ARIAS VÁZQUEZ
Original assignee: Universidade De Santiago De Compostela
Priority date: 2013-02-14
Filing date: 2014-02-13
Publication date: 2014-08-21
Also published as: ES2486166B2; ES2486166A1

Abstract

The invention relates to the use of fungi or reproductive forms thereof, preferably of the Mucor circinelloides and Duddingtonia flagrans species, as biological control agents against different viable forms of parasites capable of infecting pets and livestock. The biological control agent can be administered in the form of an additive in animal feed and/or as an anti-parasitic composition to the animal and/or applied to the animal's faeces and/or the animal's environment. The invention also relates to a method for the preventing the transmission of parasitic diseases in pets and livestock, involving treating the animals, the animals' faeces or the animals' environment with said fungi or reproductive forms thereof or with the biological control agent of the invention.

Description

FUNGES AS BIOLOGICAL CONTROL AGENTS

FIELD OF THE INVENTION The invention relates to the use of filamentous fungi, preferably of the genus Mucor and more preferably of the species M. circinelloides, alone or in combination with other species of fungi, preferably of the genus Duddingtonia, preferably D. flagrans, as parasiticides. against organisms such as cestodes, trematodes and nematodes, in pets and livestock.

STATE OF THE TECHNIQUE

Some parasites that affect both animals and people have stages of free life that develop in the soil. In humans, the infection is usually caused by the intake of contaminated food, or accidentally by the ingestion of soil, sand, etc. It contains parasite eggs, such as ascarids. On the contrary, animals are frequently infected during grazing, by ingesting eggs containing larvae inside them (ascarids, tricurids), metacercariae (trematodes), cysts (cestodes) or larvae (gastrointestinal and pulmonary nematodes). These infections can cause serious alterations, such as diarrhea, digestion disorders, infertility, weight loss, and death, to which significant economic losses should be added. The administration to said infected animals of treatments based on benzimidazoles, macrocyclic lactones or pyrantel salts, only reduces the burden of parasitic forms found in the definitive host, so that the possibilities of reinfection with infective phases found in the Ground are high. Another of the disadvantages that arise from the use of antiparasitic drugs is the risk of chemical residues in food of animal origin. Due to their metabolism and elimination through feces, they can also be harmful to some coprophagous insects, which affects the degradation of manure and thus the fertilization of the soil.

On the other hand, the storage of animal feces in a manure is not frequent in pig farms for fermentation, so the destruction of possible pathogens does not occur, especially the destruction of their eggs is not carried out. In these farms, fecal matter is temporarily stored in rafts, and its contents are emptied as they are filled. In this way, the spread of viable parasitic stages, which contaminate the soil, water, etc., is facilitated. A very common application of the fecal matter obtained in pig farms, is its use as fertilizer or fertilizer for pastures, fruit trees, orchards, etc., constituting an ideal vehicle for the dissemination of viable parasitic forms (eggs), assuming a risk to the safety of the crops themselves, of the cattle that can graze in the pastures paid with said fecal matter, as well as of the own human being, for the ease that exists to come into contact with the parasites present in these contaminated soils and that are responsible for zoonoses.

Among the most recent possibilities for deworming cattle, without using drugs, different biological control guidelines have been used. Biological control includes the use of natural enemies of parasites, mainly fungi or spores of fungi called saprophytes or predators, the best known are Arthrobotrys, Oligospora or Duddingtonia, also called trap-nematodes or nematophages (Larsen, M. Parasitology. 2000, 120: S121-S131; Gómez-Rincón, C, Uriarte, J., Valderrábano, J. Vet Parasitol. 2006, 141: 84-90). The concept of biological control is therefore aimed at the prophylaxis or prevention of parasitic infections, and its application is based on the use of microorganisms that can act in the free-life phases of these parasites. These fungi develop in the presence of parasites in the soil and are capable of destroying them to ensure the necessary supply of energy and nitrogen. These fungi are microscopic and do not develop fruiting bodies, but they have a form of growth that resembles a mold. Predatory fungi are special having developed organs that are capable of capturing and eliminating small nematodes, including the infective larvae of nematodes. Predatory fungi are originally telluric fungi, but it has been discovered that they can also grow in cow dung. It is precisely here that its beneficial effect should be used to eliminate a large number of infective larvae from parasitic intestinal worms. Although the development of larvicidal fungi has been associated with the presence of a set of substances (nemina) in the tegument of the nematode larvae, the mechanisms that determine their objective have not been clarified, so that tests are carried out in the that the fungal species and different parasitic stages (oocysts, eggs, larvae) are contacted. There is quite a suspicion that some fungi larvicides develop a series of structures such as traps (Duddingtonia), adhesive ends, constrictor rings (Arthrobotrys) ..., with which the larvae trap. In addition, these fungi are provided with hyphae called apresoria whose end or haustorium penetrates the larvae and feeds on them. It is unknown how some fungi (Pochonia) are capable of penetrating the parasite eggs cover, which in particular in ascarids is very strong due to its trilaminar composition based on chitin and mucopolysaccharides (Lysek H, Stérba J. Folia Parasitol (Praha ) 1991; 38 (3): 255-9). Some of the hypotheses point to the presence of haustorio, along with the release of some enzymes (Lysek H, Krajcí D. Folia Parasitol (Praha). 1987; (34): 57-60).

To use predatory fungi in practice, it is necessary to select fungal species that can pass through the intestinal tract of live cattle and thus also be useful in biological control. Of all the isolated fungi, the ideal candidate so far is the fungus of the species D. flagrans that, apart from being a highly efficient predator based on the formation of three-dimensional networks, produces abundant amount of resistance spores (clamidospores) that support the passage through the gastrointestinal tract of animals. This allows the fungus to be dosed easily and administered to animals, then appear in the feces and exert its predatory action on the larvae of parasites. In this regard, EP0603315 discloses the use of nematophage fungi, specifically fungi of the D. flagrans species, for the control of parasitic nematodes in cattle, sheep, pigs and horses. Said patent also discloses a method to reduce the population of parasitic nematodes in the feces of said animals by administering the D. flagrans fungus either as an additive in their diet or directly as an agent for the biological control of nematodes. parasites on the feces of these animals. Additionally, said patent discloses an additive for animal feed and a biological control agent, both comprising the fungus D. flagrans. Therefore, it is shown that D. flagrans is highly effective in reducing the number of larvae in fecal matter and, as a consequence, in pastures where animals feed. Also in patent application WO88 / 06407 a method for reducing the number of parasitic nematodes in domestic animals (pigs, dogs, cats, horses, sheep, goats, cows, etc.) and a method for reducing their transmission by administration is disclosed. to said animals of a composition comprising a hematophagous fungus belonging to the species Arthrobotrys spp. or Dactylaria spp. Said parasiticidal composition is capable of following exerting its nematophageal activity after passing through the gastrointestinal tract of infected animals, being therefore useful and, exercising its nematophage capacity, even in the feces of said animals. However, these fungal species with parasiticidal activity and capable of crossing the intestinal tract of animals possess only activity on the larvae of the nematodes, but not on other infective forms such as eggs of cestodes and trematodes.

Since these types of helminth parasites (cestodes, trematodes and nematodes), in all their infective forms, cysts, larvae and eggs, cause large losses to farmers, it is of great interest for them to control their growth and expansion. Therefore, it is necessary to find alternative methods to combat the different infective forms of the different parasites that infect domestic animals and livestock. These methods must have a wide spectrum of parasiticidal action that allows to adapt to the natural conditions in which mixed parasitic infections take place, having to have activity both on the different types of parasites that affect cattle and on the different infectious forms of them .

DESCRIPTION OF THE INVENTION Brief description of the invention

The present invention describes a biological control agent comprising at least one fungus or a reproductive form thereof, of the species M. circinelloides, the strain M. circinelloides CECT 20824 being preferred. Said biological control agent may further comprise other species of fungi or reproductive forms thereof and thus enhance their parasiticidal action, with fungal species belonging to Duddingtonia flagrans, also known as Arthrobotrys flagrans, preferably strain D. flagrans CECT 20823 being preferred. It should be noted that also, the biological control agent comprising the fungi or reproductive forms thereof, previously mentioned, alone or in combination, can be combined with other species of parasiticidal fungi or reproductive forms thereof, belonging to the genera selected from any of the following: Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or any of its combinations actions. It should be noted that the fungi or the reproductive forms thereof, which comprise the biological control agent described in the present invention are used in the form of live or viable spores. The term "Mucor", as used herein, refers to a genus of zygomycete fungus identified in the NCBI database by Taxonomy ID: 4830. The term "Mucor circinelloides", as used used in the present description, refers to a species of zygomycete fungus identified in the NCBI database by Taxonomy ID: 36080.

The term "Duddingtonia flagrans", as used herein, refers to a species of ascomycete fungus identified in the NCBI database by Taxonomy ID: 47257 or 97331.

The term "Trichoderma.", As used herein, refers to a genus of zygomycete fungus identified in the NCBI database by Taxonomy ID: 5543.

The term "Fusarium", as used herein, refers to a species of zygomycete fungus identified in the NCBI database by Taxonomy ID: 5506. The term "Verticillium", as used In the present description, it refers to a genus of ascomycete fungi identified in the NCBI database by Taxonomy ID: 5106, 264599 and 1036719.

The term "Arthrobotrys", as used herein, refers to a genus of ascomycete fungi identified in the NCBI database by Taxonomy ID: 13348.

The term "Pochonia", as used herein, refers to a genus of ascomycete fungus identified in the NCBI database by Taxonomy ID: 243023.

The term "biological control agent" refers to those compositions that comprise microorganisms, parts thereof, in particular, reproductive forms of said microorganisms, their metabolites or their by-products, which have natural or acquired activity in reducing or eliminating the harmful or adverse effects of parasites and / or pathogens, since they can act on the phases of free life thereof. For the purposes of the present invention the biological control agents described herein comprise fungi and / or reproductive forms thereof, such as, for example, live or viable spores, of fungi called saprophytes or predators. The fungi and / or preferred reproductive forms thereof described in the present invention belong to the species M. circinelloides and D. flagrans, preferably, the strains M. circinelloides CECT 20824 and D. flagrans CECT 20823, respectively.

The biological control agent described in the present invention is characterized in that it can take the form of an additive for animal feed, both for application together with the feed or even on water, or that of parasiticidal composition. Said biological control agent is capable of reducing the parasitic load present in animal feces and preventing the spread of larvae included in eggs that are excreted along with feces, thus preventing infection of animals. In a preferred embodiment, the biological control agent is an additive for animal feed. In a more preferred embodiment, the additive is added to a feed or food concentrate intended for animal feed, preferably during the industrial manufacturing process of the concentrate or feed, and more preferably during the mixing phase of the raw materials constituting the feed base or food concentrate. In another preferred embodiment, the biological control agent is a parasiticidal composition. The biological control agent, in the form of a parasiticidal composition, can be administered orally to the animals themselves, and / or on their feces and / or even on the grass, meadow or place where they live. The parasiticidal composition described in the invention may further comprise pharmaceutically acceptable carriers and / or excipients.

For the purposes of the present invention, the term "reproductive form" in relation to fungi refers to any element of sexual or asexual propagation of the fungus, and preferably spores. The spores are formed as a differentiation from the mycelium of fungi and are structures surrounded by a thick protective covering that are produced to ensure survival in their viable form (alive from fungi). In the present invention, the term spores includes chlamydiospores, sporangiospores, conidiospores, oospores, zigospores, ascospores, basidiospores, or any other type of spore known to those skilled in the art. The term "food additive", "food additive" or "additive for animal feed", for the purpose of the present invention, refers to a composition intended to be ingested together with the drink and / or with the normal diet of the animal, in particular mixed with the feed or food concentrate that is administered to the animal, and more preferably mixed during the manufacturing process thereof, and is intended, although not limited , a positive influence on the environmental repercussions of animal production, on the production, activity or welfare of animals, or on the characteristics of animal products, among others. More preferably, the additive described in the present invention has the purpose of reducing the parasitic load or amount of viable parasitic forms in animal feces and preventing the transmission of parasitic diseases in animals. In a preferred embodiment, the animal feed additive is added during the mixing phase of the raw materials that constitute the base of the feed or food concentrate intended for animal feed, during the industrial manufacturing process of the concentrate or feed. In the present invention, a way of adding the additive to the animal's feed or food concentrate is, for example, by producing spores of the fungus with parasiticidal activity in a culture medium, preferably liquid, and its subsequent incorporation into the feed of industrial processing in any of its possible presentations, including flour, granules and multiparticles, during the mixing phase of the raw materials that constitute the base of the feed or concentrate. For this, a culture medium, preferably liquid, is prepared comprising a carbon source, a nitrogen source and one or several mineral salts, among other possible components. After autoclave sterilization and subsequent cooling, an inoculum of the parasiticidal fungus is added which is desired to grow and is maintained in conditions of light and temperature suitable for the growth of the fungus and the production of spores, for example in dark conditions and room temperature (18-20 ° C) for preferably 20 days. After the incubation period, samples are collected and the spore concentration of each fungal preparation is determined, for example in a Neubauer chamber, to calculate the spore concentration / L of culture medium, and therefore to calculate the final concentration of spores / kg that will contain the feed. Next, the containers or drums containing the parasiticidal fungal spores are added to the food concentrate or feed during the mixing phase of the different raw materials. For this, food concentrates such as flour, granules or multiparticles can be used. Examples of such concentrates are DL Novillas 18® products (flour and granules), Pro-Horse Club® (granules) and Pro-Horse Eco Mix® (multiparticles). Therefore, in another aspect, the invention relates to a feed or feed concentrate for animal feed comprising the previously described additive.

The term "parasiticidal composition", for the purposes of the present invention refers to a composition capable of being administered orally to the animals themselves and / or on their feces and / or on the grass, meadow or place where they live or are. Said parasiticidal composition is intended, but not limited, to prevent the transmission of parasitic diseases and / or reduce the burden of parasites present in feces and / or pastures, thanks to the reduction of the parasitic load or number of viable parasitic forms in the animal feces and in the grass, meadow or place where these animals live.

It should be understood that the biological control agent described in the present invention, either in the form of an additive or in the form of a parasiticidal composition, is presented in a pharmaceutically acceptable form to be administered to an individual directly, preferably by oral administration, or by any other route, although the preferred route of administration to an individual is the oral route. The parasiticidal composition as previously mentioned, can also be administered on the feces of the subjects themselves, preferably mammalian animals, or on the soil in which the animals graze.

A "pharmaceutically acceptable carrier or excipient" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants. Such substances must be approved by a federal government regulatory agency or a state government or listed in the American Pharmacopoeia or the European Pharmacopoeia, or another pharmacopoeia generally recognized for use in animals, and more specifically in humans. In the same manner, suitable pharmaceutically acceptable excipients will vary depending on the particular dosage form selected. Suitable pharmaceutically acceptable excipients include the following types of excipients, not excluding others known in the state of the art: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, moisturizing agents, solvents, co-agents. solvents, suspending agents, emulsifiers, sweeteners, flavorings, taste masking agents, coloring agents, anti-cake agents, humectants, agents chelants, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants and buffering agents. The person skilled in the art will appreciate that certain pharmaceutically acceptable excipients can perform more than one function and can perform alternative functions depending on the amount of excipient present in the formulation and what other ingredients are present in the formulation. The specialists have the knowledge and skill in the art that allows them to select suitable pharmaceutically acceptable excipients in the appropriate amounts for use in the invention. In addition, there are several resources available to those skilled in the art that describe pharmaceutically acceptable excipients and may be useful in the selection of suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

In the context of the present invention, parasitic disease is preferably an infectious disease caused by viable forms, preferably eggs and larvae, of helminth parasites, preferably selected from any of the following: cestodes, trematodes, nematodes and / or any of your combinations The parasiticidal action exerted by the biological control agent described in the present invention, which comprises the fungus M. circinelloides CECT 20824, alone or in combination with other parasiticidal fungi, described above, exerts its parasiticidal action on subjects or individuals that are preferably animals mammals and more preferably, dogs, cats, bovids, ovids, suidae and equidae.

As used in the invention, the term "dog" refers to a domestic carnivorous mammal of the canid family, and in particular the subspecies Canis lupus familiaris. As used in the invention, the term "cat" refers to a small carnivorous mammal of the Felidae family, and in particular the domestic cat (Felis silvestris catus). As used in the invention, the term "bovid" refers to an animal of the Bovidae family, composed of ruminant mammals, with bone horns covered by corneal case, not caedizos, and that exist in both the male and the female . The bovid term includes bulls, cows, antelopes, sheep, rams, goats as well as other similar animals. The Bovidae family includes the subfamily Bovinae (also generically called "cattle"), which includes bovine animals with humps such as the zebu (Bos indicus) and no hump like the bull or cow (Bos taurus), as well as the yak (Poephagus grunniens), the mithan (Bibos frontalis), the banteng (Bibos banteng), the buffalo (Bos bubalus bubalis) and the bison, both the American (Bison bison) and the European (Bison bonasus). The Bovidae family includes the Caprinae subfamily (also generically referred to as "goat or sheep"), which includes sheep, rams and mouflon (genus Ovis, in particular, Ovis orientalis), goats (genera Capra and Oreamnos, among others), and chamois (genus Rupricapra), among others. In a preferred embodiment, the bovid is a cow or a bull. The term "ovid" refers to ruminant mammals of the Bovidae or Bovidae family, many of them covered with abundant wool, which have triangular section horns and spirally twisted or bent backwards, such as rams, goats and mouflon As used in the present invention, the term "suida" refers to an animal of the Suina suborder, which in turn comprises the Suidae family, composed of artiodactyla mammals with a snout ending in a disk-shaped nose, four fingers on each leg and strong and developed canines. The Suina suborder includes domestic pigs (Sus scrofa domestica), wild boars (Sus scrofa) and their closest relatives, such as American peccary (genus Tayassuidae) and hippos (genus Hippopotamidae). In a preferred embodiment, the suida is the domestic pig. As used in the present invention, the term "equid" refers to the Equidae family, composed of placental mammals of the order Perissodactyla, which comprises the genus Equus, and which includes horses (Equus ferus caballus), zebras (common zebra (Equus quagga), Grévy's zebra (Equus grevyi), mountain zebra (Equus zebra), etc.) and donkeys (Equus asinus, Equus africanus, Equus hemionus). In a preferred embodiment, the equine is a horse.

Another object described in the present invention relates to the use of at least one fungus or a reproductive form thereof, of the Mucor circinelloides species for the preparation of a biological control agent, with the strain M. circinelloides CECT 20824 being preferred. Alternatively, The present invention describes the fungus M. circinelloides, or a reproductive form thereof, preferably the strain M. circinelloides CECT 20824, for use as a biological control agent.

As previously described, said fungal strain or the reproductive form thereof, can be used alone or in combination with other parasiticidal fungal strains or their reproductive forms, fungal strains being preferred: D. flagrans, preferably strain CECT 20823, Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or any combination thereof. Such fungi are used in the form of reproductive forms, preferably live or viable spores. Another object described in the present invention relates to a cultivable fungal strain called CECT 20824 belonging to the species M. circinelloides.

Another object described in the present invention relates to a cultivable fungal strain called CECT 20823 belonging to the species D. flagrans.

Another object described in the present invention relates to a biologically pure culture of the fungi M. circinelloides CECT 20824 or D. flagrans CECT 20823. Another object described in the present invention relates to a method of reducing the load parasitic present in animal feces and in the places where they inhabit, such as meadows, pastures, etc., by administration, either to the feces themselves and / or to the place where they live, of an effective amount of at least one fungus or a reproductive form thereof, of the species M. circinelloides, preferably strain M. circinelloides CECT 20824, alone or in combination with any of the strains or reproductive forms of said strains, previously described throughout the invention, or of the biological control agent described previously, either as an additive for animal feed or parasiticidal composition, as defined throughout the present invention.

Another of the objects described in the present invention relates to a method of prevention of the transmission of parasitic diseases characterized by the administration to a subject susceptible to suffering from a parasitic disease and / or its feces and / or to the place in which the animals or subjects inhabit, of an effective amount of at least one fungus or a reproductive form thereof, of the species M. circinelloides, preferably the strain M. circinelloides CECT 20824 alone or in combination with any of the fungal strains or reproductive forms previously described throughout the invention, or of the biological control agent described previously, either in the form of an additive for animal feed or parasiticidal composition, as defined throughout the present invention. In a preferred embodiment, the method of preventing the transmission of parasitic diseases or the method of reducing the parasitic load in feces and / or pastures, is characterized in that parasitic diseases are caused by viable forms, preferably eggs and larvae , of parasites, preferably of helminth parasites, and more preferably of helminth parasites selected from any of the following: cestodes, trematodes, nematodes and / or any combination thereof and the subjects are animals, preferably mammals, which are selected from any from the list: dogs, cats, bovids, ovids, suidos and equidae.

The dosage of the fungus itself or of the biological control agent comprising it, described in the present invention, to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, age, weight, sex, tolerance, etc., of the mammal. Administration will also depend on the simultaneous treatment with other drugs and the tolerance of each subject to the drug administered. Persons skilled in the art may establish the appropriate dose using standard procedures. It is understood that the dose should be the effective amount of the fungus or fungal combinations capable of reducing the parasitic load in animal feces.

The term "subject" or "individual", as used in the description, refers to animals, preferably mammals, and more preferably, dogs, cats, bovids, ovids, suidae and equidae. The term "subject" or "individual" is not intended to be limiting in any way, and may be of any age, sex and physical condition.

Unless defined otherwise, all the technical and scientific terms used here have the same meaning as those usually understood by a person skilled in the field of the invention. Methods and materials similar or equivalent to those described herein may be used in the practice of the present invention. Throughout the description and the claims the word "comprises" and its variants, are not limiting and therefore are not intended to exclude other technical characteristics, additives, components or steps. On the contrary, the word "consists" and its variants, which do have a limiting character, referring exclusively to the technical characteristics, additives, components or steps that accompany it. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. Importantly, fungal species of the Fusarium and Trichoderma genera have already been suggested as potential biological control agents, especially plant nematodes (Radwan NA 2012. Applied Soil Ecology, 56: 58-62; Van Dessel, P. Nematropica 41 (2): 154-160) but also recently in the case of Trichoderma as a potential control agent for parasites that affect animals (Soto-Barrientos N., 201 1. Journal of Tropical Biology, 59 (1): 37- 52). However, in the examples of the present invention it is shown that these and other fungal species (such as species belonging to the genera Gliocladium, Penicillium, Sordaria and Verticillium), which a priori seemed good candidates as biological control agents for helminths, present an ovicidal activity much less than that demonstrated for the Mucor circinelloides species. In addition, although previous evidence indicated 30 ° C as the limit to observe the development of this fungus (de Hoog et al., 2000. Atlas of Clinical Fungí, 2nd ed, vol. 1. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands ), the present invention demonstrates for the first time the resistance of spores of this species to the high temperatures that occur during the manufacturing process of feed, as well as its ability to cross the digestive tract of animals while maintaining their viability and activity parasiticide. All this demonstrates for the first time the possibility of adding industrial spores of M. circinelloides to commercial feed and their suitability as an additive for animal feed, among other possible applications.

Additionally, it is known that some strains of nematophagous fungi, such as Trichoderma strains, inhibit the growth of other nematophage strains, such as Fusarium (Hernández Mansilla AA, Sierra Peña A, Carr Pérez A. Fitosanidad (Cuba) 10: 105- 108). In the present invention it is demonstrated for the first time that strains of M. circinelloides CECT 20824 and D. flagrans CECT 20823 can grow together, maintaining both their viability and their parasiticidal activity on different parasitic stages that can be found in the soil.

Description of the figures.

Figure 1. Photograph showing the parasiticidal activity of different nematophagous fungi (Mucor, Gliocladium, Trichoderma and Duddingtonia) on different viable parasitic forms (oocysts, eggs and larvae). Figure 2. Parasiticidal-ovicidal activity of different spore concentrations of the Mucor fungi (light gray bars) and Trichoderma (dark gray bars) on eggs of the parasitic nematode Parascaris equorum. Concentrations of fungi used are D1: 0.025x10 ^6, D2: 0.05x10 ^6, D3: 0.1x10 ⁶ D4: 0.2x10 ⁶ and D5: 0.4x10 ^6/5 g faeces. On the X-axis, the spore concentration of each fungus is shown and on the Y-axis, the percentage of the viability reduction of the parasitic nematode Parascaris equorum eggs. Figure 3. Parasiticidal-ovicidal activity of different spore concentrations of the Mucor fungi (light gray bars) and Trichoderma (dark gray bars) on eggs of the Trichuris spp. Concentrations of fungi used are D1: 0.025x10 ^6, D2: 0.05x10 ^6, D3: 0.1x10 ⁶ D4: 0.2x10 ⁶ and D5: 0.4x10 ^6/5 g faeces. On the X axis the spore concentration of each fungus is shown and on the Y axis the percentage of viability reduction of the eggs of the Trichuris spp parasite.

Figure 4. Parasiticidal-ovicidal activity after the addition of different concentrations of Mucor spores on eggs of the Calicophoron daubneyi parasite. The concentrations of fungi used are D1: 0.025x10 ⁶ (line with diamonds), D2: 0.05x10 ⁶ (line with squares), D3: 0.1x10 ⁶ (line with triangles), D4: 0.2x10 ⁶ (line with crosses) and D5: 0.4x10 ⁶ (line with asterisks) / 5 g. stool The number of days post-addition of the spores is shown on the X axis and on the Y axis the percentage of viability reduction of the Calicophoron daubneyi parasite eggs.

Figure 5. Parasiticidal activity on eggs of stray parasites / gram of feces, from spores of D. flagrans. The graph shows a group of animals treated with ivermecticin (dashed line), a group of animals treated with ivermectin and kept grazing in the area where the D. flagrans spores (continuous line with squares) have been poured and a third control group (Witness) that was not treated (continuous line). The number of weeks elapsed from the discharge of the spores to the grazing area is shown on the X axis and on the Y axis, the strungilated eggs / gram of feces.

Figure 6. Electron microscopy photograph showing the resistance of Duddingtonia spores to high heat treatment (72 ° C) for different periods of time.

Figure 7. Parasiticidal activity on eggs of spoon parasites of D. flagrans spores (2x10 ⁶ spores / animal) mixed with food concentrate (feed) against the prevention of reinfection of equine species: zebra (continuous line with triangles), domestic donkey (continuous line) and African donkey (dashed line with circles). The time expressed in months is shown on the X axis and on the Y axis the strungilated eggs / gram of feces. Figure 8. Photograph where it is observed that the species of fungi Mucor and Duddingtonia can grow together.

Figure 9. Parasiticidal activity of the combination of Mucor + Duddingtonia administered together with the feed in an equine hut. These animals were also given an anthelmintic (ivermectin). The solid line represents the group of animals treated with 2.5 kg of feed with spores / horse / day and the dashed line represents the group of control animals that was treated with 2.5 kg of feed without spores / horse / day (Witness ). On the X axis the time expressed in days is shown and on the Y axis the eggs of strungilates / gram of feces are shown.

Detailed description of the invention

The first object of the present invention relates to a biological control agent comprising at least one fungus or a reproductive form thereof, belonging to the species M. circinelloides, with the strain M. circinelloides CECT 20824 being preferred.

In a preferred embodiment, the biological control agent is characterized in that it also comprises at least one other fungus or a reproductive form thereof, belonging to the species D. flagrans, the strain D. flagrans CECET 20823 being preferred.

In another preferred embodiment, the biological control agent is characterized in that it also comprises at least one other fungus or a reproductive form thereof, belonging to a genus selected from any of the following: Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or any of its combinations.

In another preferred embodiment, the biological control agent is characterized in that it may comprise or be administered together with other components such as excipients and additives such as vitamins, amino acids, trace elements, binders, etc.

In another preferred embodiment, the biological control agent is characterized in that it can take the form of an additive for animal feed or parasiticidal composition. In a preferred embodiment, the biological control agent is an additive for animal feed. In a more preferred embodiment, the animal feed additive is added to a feed or food concentrate intended for animal feed, preferably during the industrial manufacturing process of the concentrate or feed, and more preferably during the mixing phase of the raw materials. which constitute the basis of feed or food concentrate. In another preferred embodiment, the biological control agent, either in the form of an additive or a parasiticidal composition, is characterized in that it is administered orally.

In another preferred embodiment, the biological control agent is a parasiticidal composition. In another preferred embodiment, the parasiticidal composition is administered, additionally or alternatively to oral administration, on the feces of the animals and / or on the soil where said animals live or are located.

In another preferred embodiment, the biological control agent is characterized in that the reproductive form used is spores, which are viable (live).

In another preferred embodiment, the biological control agent is characterized in that the fungus is able to pass through the digestive tract of the animal to which it is administered, survive in the feces of the animal, and exert its action therein reducing the amount of parasites in the stool

In another preferred embodiment, the biological control agent is characterized in that the reduction in the amount of parasites in the feces is at least 50%, preferably it is at least 60%, more preferably it is at least 66% and more preferably, it is at least 75%.

In another preferred embodiment, the biological control agent is characterized in that the biological control is exerted against viable forms of parasites, preferably of helminth parasites, and more preferably of helminth parasites selected from any of the following: nematodes, trematodes, cestodes and / or any of their combinations.

In another preferred embodiment, the biological control agent is characterized in that the biological control is exercised in animals selected from any of the list: dogs, cats, bovids, ovids, suidae and equidae. The second object described in the present invention relates to the use of at least one fungus or a reproductive form thereof belonging to the species Mucor circinelloides for the preparation of a biological control agent, the strain M. circinelloides CECT 20824 being preferred. Alternatively , the present invention also relates to a fungus or a reproductive form thereof belonging to the species Mucor circinelloides for use as a biological control agent.

In a preferred embodiment of the first use of the invention, it is characterized in that it also comprises the use of at least one other fungus or a reproductive form thereof belonging to the species Duddingtonia flagrans, the strain D. flagrans CECT 20823 being preferred.

In another preferred embodiment of the first use of the invention, it is characterized in that it also comprises at least one other fungus or a reproductive form thereof belonging to a genus selected from any of the following: Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or any of its combinations.

In another preferred embodiment of the first use of the invention, this is characterized in that the biological control agent may comprise or be administered together with other components such as excipients and additives such as vitamins, amino acids, trace elements, binders, etc.

In another preferred embodiment of the first use of the invention, this is characterized in that the biological control agent is an additive for animal feed. In another preferred embodiment of the first use of the invention, this is characterized in that the biological control agent is a parasiticidal composition.

In another preferred embodiment of the first use of the invention, this is characterized in that the biological control agent, either in the form of an additive for animal feed or in the form of a parasiticidal composition, is administered orally.

In another preferred embodiment of the first use of the invention, this is characterized in that the biological control agent in the form of a parasiticidal composition is administered additionally or alternatively to oral administration, on the feces of the animals and / or on the soil of the place where they live or where the animals are. In another preferred embodiment of the first use of the invention, this is characterized in that the reproductive form of the fungi used are spores, said spores being alive.

In another preferred embodiment of the first use of the invention, it is characterized in that the fungus, or a reproductive form thereof, is able to pass through the digestive tract of the animal to which it is administered, survive in the feces of the animal, and exert its action in them reducing the amount of parasites in the stool.

In another preferred embodiment of the first use of the invention, this is characterized in that the reduction in the amount of parasites in the feces is at least 50%, preferably it is at least 60%, more preferably it is at least one 66% and more preferably, it is at least 75%.

In another preferred embodiment of the first use of the invention, this is characterized in that biological control is exercised against viable forms of parasites, preferably helminth parasites, and more preferably helminth parasites selected from any of the following: nematodes, Trematodes, cestodes and / or any combination thereof.

In another preferred embodiment of the first use of the invention, it is characterized in that the biological control is exercised in animals selected from any of the list: dogs, cats, bovids, ovids, sweats and equidae. The third object described in the present invention relates to a cultivable CECT 20824 fungal strain characterized in that it belongs to the species M. circinelloides.

The fourth object described in the present invention relates to a biologically pure culture of the fungal strain M. circinelloides CECT 20824.

The fifth object described in the present invention relates to a cultivable CECT 20823 fungal strain characterized in that it belongs to the species D. flagrans.

The sixth object described in the present invention relates to a biologically pure culture of the fungal strain D. flagrans CECT 20823. The seventh object described in the present invention relates to a method of prevention of the transmission of parasitic diseases characterized by the administration to an animal susceptible to suffering from a parasitic disease and / or its feces and / or the environment where it is found, of a effective amount of at least one fungus or a reproductive form thereof belonging to the species M. circinelloides, preferably strain M. circinelloides CECT 20824, alone or in combination with at least one other fungus or a reproductive form thereof, belonging to one of strains or genera selected from any of the following: D. flagrans, preferably strain D. flagrans CECT 20823, Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or any combination thereof; or of a biological control agent as previously defined.

In a preferred embodiment, the prevention method described in the invention is characterized in that it is capable of reducing the parasitic load in the feces and / or in the environment in which the animals live or are.

In another preferred embodiment the prevention method described in the invention is characterized in that the reduction in the amount of parasites is at least 50%, preferably it is at least 60%, more preferably it is at least 66% and more preferably, it is at least 75%.

In another preferred embodiment, the prevention method described in the invention is characterized in that prevention is carried out on viable forms of parasites, more preferably of helminth parasites, and even more preferably of helminth parasites selected from any of the following : nematodes, trematodes, cestodes and / or any combination thereof.

In another preferred embodiment, the prevention method described in the invention is characterized in that the animals are selected from any of the list: dogs, cats, bovids, eggs, suidae and equidae.

Another object described in the present invention relates to a method of reducing the parasitic load present in animal feces and in the environment in which they are, by administration, either to the feces and / or the environment, of a effective amount of at least one fungus or a reproductive form thereof belonging to the species M. circinelloides, preferably strain M. circinelloides CECT 20824, only or in combination with at least one other fungus or a reproductive form thereof belonging to one of the strains or genera selected from any of the following: D. flagrans, preferably strain CECT 20823, Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or any of its combinations; or of a biological control agent as defined throughout the present invention.

In a preferred embodiment, the method of reducing the parasite load described in the invention is characterized in that the reduction in the amount of parasites is at least 50%, preferably it is at least 60%, more preferably it is at minus 66% and more preferably, it is at least 75%.

In another preferred embodiment, the method of reducing the parasitic load described in the invention is characterized in that the reduction of the parasitic load is carried out on the viable forms of parasites, preferably of helminth parasites, and more preferably of helminth parasites selected from among any of the following: nematodes, trematodes, cestodes and / or any combination thereof.

In another preferred embodiment, the method of reducing the parasitic load described in the invention is characterized in that the animals are selected from any of the list: dogs, cats, bovids, ovids, sweats and equidae.

Another aspect of the present invention relates to a feed or food concentrate for animal feed comprising the animal feed additive previously described. In a preferred embodiment, the feed or food concentrate is characterized in that it can further comprise other components such as excipients and additives such as vitamins, amino acids, trace elements, binders, etc.

In a preferred embodiment, the animal feed additive described in the present invention is additionally added to the feed or food concentrate preferably during the mixing phase of the raw materials that is produced during the manufacturing process thereof, and is intended, although without limited, a positive influence on the environmental impact of animal production, on the production, activity or welfare of animals, or on the characteristics of animal products, among others. More preferably, the feed or food concentrate described in the present invention, by understanding the additive for animal feed described in the invention, has the purpose of reducing the parasitic load or amount of viable parasitic forms in animal feces and prevention of the transmission of parasitic diseases in animals. In the present invention, a way of adding the additive to the animal's feed or food concentrate is, for example, by producing spores of the fungus with parasiticidal activity in a culture medium, preferably liquid, and its subsequent incorporation into the feed of industrial processing in any of its possible presentations, including flour, granules and multiparticles, during the mixing phase of the raw materials that constitute the base of the feed or concentrate. For this, a culture medium, preferably liquid, is prepared comprising a carbon source, a nitrogen source and one or several mineral salts, among other possible components. After autoclave sterilization and subsequent cooling, an inoculum of the parasiticidal fungus is added which is desired to grow and is maintained in conditions of light and temperature suitable for the growth of the fungus and the production of spores, for example in dark conditions and room temperature (18-20 ° C) for 20 days. After the incubation period, samples are collected and the spore concentration of each fungal preparation is determined, for example in a Neubauer chamber, to calculate the spore concentration / L of culture medium, and therefore to calculate the final concentration of spores / kg that will contain the feed. Next, the containers or drums containing the parasiticidal fungal spores are added to the food concentrate or feed during the mixing phase of the different raw materials. For this, food concentrates such as flour, granules or multiparticles can be used. Examples of such concentrates are DL Novillas 18® products (flour and granules), Pro-Horse Club® (granules) and Pro-Horse Eco Mix® (multiparticles).

Deposit of microorganisms according to the Budapest Treaty

The microorganisms used in the present invention were deposited in the Spanish Type Culture Collection (CECT) located in Building 3 CUE- Pare Científic Universitat de Valencia, Professor Agustín Escardino 9, 46980 Paterna (Valencia, Spain), with deposit number :

• CECT 20824: fungal strain of the species M. circinelloides deposited on December 18, 2012.

• CECT 20823: fungal strain of the D. flagrans species deposited on December 18, 2012. The examples described below are intended to illustrate the present invention, but without limiting the scope thereof.

Example 1. Isolation of parasiticidal fungi

The isolation of parasiticidal fungi used in the present invention was carried out from animal feces samples and soil samples from different livestock farms. Approximately 1 gram of each stool sample was seeded in Petri dishes containing culture medium consisting of wheat flour (25 g), agar (20 g), chloramphenicol (500 mg) and water (1 L). Once the fungus growth was observed, serial plate passes were made until a single species of fungus was achieved. In this way, 13 isolates were obtained, which were identified following different morphological keys as belonging to the genera: Gliocladium, Duddingtonia, Mucor, Fusarium, Trichoderma, Verticillium, Penicillium, Sordaria and Arthrobotrys. For this, it was taken into account if they were provided with columella, branched sporangiophore of hyaline nature, and spherical sporangium (Weitzman I, Whittier S, McKitrich JC, Della-Latta P. 1995. J. Clin. Microbiol. 33: 781-783 ).

As an example, the methodology followed for the identification of the isolates belonging to the genera Mucor and Duddingtonia is presented below. For the identification of said isolates, morphological and molecular techniques were used.

Morphological study:

The strains were inoculated into the culture media: PDA (Potato Dextroxe Agar; BD Difco ™ Potato Dextrose Agar, Madrid, Spain), MEA (Malt Extract Agar; BD Difco ™ Malta Extract Agar , Madrid, Spain), CMA (in its acronym Corn Meal Agar; BD BBL ™ Corn Meal Agar, Madrid, Spain) and two incubation temperatures were used: 26 ° C and 37 ° C.

The characteristics studied in the identification, after an incubation period of 7 days, were:

a) growth data: colony size, texture, color, exudate production, diffusible pigment production and colony reverse observation

b) detailed microscopic observation

c) formation of the teleomorphic state if any.

Molecular Study: Identification at the molecular level has been carried out by the following methods:

a) Amplification and subsequent sequencing (with two-way readings) of the ITS1 and ITS2 ribosomal DNA zone, which includes the 5.8S rDNA gene with itsl and its4 primers (White, TJ, et al. 1990. In MA Innis, et al. PCR protocols.

A guide to methods and applications. Academic Press, San Diego, Cali p. 315-322).

b) Amplification and subsequent partial sequencing of the β-Tubulin gene (with two-way readings), with primers Bt2a and Bt2b (Glass, NL, Donaldson, GC (1995). Appl Environ Microb 61: 1323-1330) .

c) Amplification and subsequent sequencing of the D1 / D2 domains of the 5 'end of the gene encoding the 28S rRNA with primers ni 1 and nl4 (Kurtzman, CP., & Robnett, CJ (1998). Antonie van Leeuwenhoek 73: 331 -371). The results of the morphological analysis, both microscopic and macroscopic, of the isolate identified as belonging to the species M. circinelloides showed rapid growth of the colonies in the different culture media used. These colonies showed a flocculent texture of a brown-brown color and the reverse of cream-colored. Positive growth is also observed at 37 ° C although with more restricted growth colonies. From the microscopic observation, the presence of a non-septate mycelium and asexual spores in large numbers, forming in sporangia, showing a morphology from spherical to sub-spherical and without apophysis. Sporangiophors appear branched sypodially and with rhizoids. Smooth sporangiospores, ellipsoidal ovoids, 4 x 5μηι, and clamidospores present in hyphae are also observed.

On the other hand, the results obtained from the molecular analysis, corroborated that the strain belonging to the genus Mucor, belonged specifically to the species M. circinelloides. To obtain these molecular results, 639 and 722 bp amplified were obtained for the ITS-5.8S rRNA and D1 / D2 ribosomal regions of the 28S rRNA gene, respectively, by the primers mentioned above. Next, a BLAST type analysis of the ribosomal sequences mentioned above was performed against the NCBI databases, obtaining the following results: • ITS1-5.8S-ITS2 region: M. circinelloides shows 99% identity (545/553) with strain CBS 195.68T belonging to the indicated species and 83% identity (440/533) with species M. hiemalis strain CBS 201.65NT.

• Region D1 / D2 of the 28S gene: M. circinelloides shows 99% identity (698/699) with strain CBS 195.68T belonging to the indicated species and a

90% (633/701) with the species M. hiemalis strain CBS 201.65NT.

Therefore, the results shown show that the isolate of the genus Mucor corresponds specifically to the species M. circinelloides.

On the other hand, the results of the morphological analysis, both microscopic and macroscopic, of the isolate identified as belonging to the species D. flagrans showed that these isolates did not grow at a temperature of 37 ° C in any of the culture media used, while if growth was observed at 26 ° C. The cultures obtained had the same characteristics at the macroscopic level in the PDA and MEA culture media: fast growing colonies, occupying the entire surface of the plate in 7 days, cottony texture, orange-brown in color. In the CMA culture medium, the colonies were smaller in diameter and showed a whitish cottony texture. The results obtained from the microscopic analysis showed the presence of hyaline hyphae, septate and branched, with an abundant production of clamidospores, intercalated in the hyphae or chained, presenting globular bumps on the surface. Conidia of different morphologies were also observed, ellipsoidal to cylindrical, with 0-1 septum, with a rounded end and the basal end with a slightly differentiated bottle shape, and a size of 20-39μηι x 1 1-15μιη. All these results asserted the belonging of the isolate of the genus Duddingtonia (Arthrobotrys) to the species D. flagrans.

For the molecular identification, amplifications of 644 and 577 bp were obtained for the ribosomal region ITS-5.8S rRNA and for the β-tubulin gene, respectively using the primers indicated previously. The BLAST analysis, performed as previously explained, showed that:

• Region ITS1-5.8S-ITS2: D. flagrans has 99.84% identity (639/640) with strain CBS 583.91 belonging to the species D. flagrans and 89% identity (442/499) with species D pseudoclavata strain AS 3.6756. • β-Tubulin gene: 100% identity has been obtained with both species D. flagrans and D. pseudoclavata, strain CBS 583.91 (442/442) and strain AS 3.6756 (532/532), respectively. Therefore, the results shown show that the isolate of the genus Duddingotine (Arthrobotrys) corresponds specifically to the species D. flagrans.

The preferred fungi used in the present invention belong to the genera Mucor and Duddingtonia, the species M. circinelloides and D. flagrans being preferred. The fungal strains belonging to the species M. circinelloides and D. flagrans were deposited on December 18, 2012 in the Spanish Type Culture Collection and were awarded the numbers CECT 20824 and CECT 20823, respectively.

Example 2. Analysis of parasiticidal activity of isolated fungal strains.

In order to analyze the parasiticidal activity, against the different forms of propagation of the parasites, of the different fungal strains isolated as described in Example 1, different methodologies were used. On the one hand, the percentage of reduction of the viable forms (cysts, eggs and larvae) of different species of parasites treated with the fungal strains isolated in Example 1 was analyzed. On the other hand, the capacity of said fungal strains for alter the different forms of spread of parasites.

To do this, plates were prepared with wheat-enriched culture medium, which were seeded with inoculums of the fungal isolates detailed in Example 1. Once growth was observed (10-12 days), various viable parasitic forms were added, as per example, Eimeria spp. oocysts, Parascaris equorum eggs or ciatostomino larvae. The possible parasiticidal effect of fungi on oocysts and eggs of parasites was analyzed according to the criteria established by Lysek (Lysek H, Faasatiová O, Cuero Pineda ON, Lorenzo Hernández N. 1982. Folia Parasitol. (Praha) 29: 265-270):

Activity Level alteration

Any

Breaking Deck II

Cover rupture and colonization of III

inside The larvicidal activity of fungi was determined by evaluating the ability to destroy the larvae through the formation of traps, constrictor rings, etc. The analysis of the activity of fungal plate isolates is summarized in Table 1.

Table 1. Parasiticidal activity of different fungal isolates against different viable parasitic forms.

-: absence of parasiticidal activity; +: presence of parasiticidal activity, the greater the number of + displayed, the greater parasiticidal activity.

None of the fungal isolates showed activity against oocysts. The fungi that presented the greatest ovicidal capacity were those belonging to the genera Mucor and Trichoderma, which showed the ability to break the cover of the eggs of ascarids, colonize their interior and destroy the embryo (activity III according to the criteria established by Lysek (1982 )). Duddingtonia presented the greatest larvicidal activity, thanks to the formation of trap rings. On the other hand, the isolates of the Gliocladium, Penicillium and Sordaria genera did not show any parasiticidal activity (Figure 1). Example 3. Parasiticidal-ovicidal activity of different species of nematophagous fungi on parasites grown in Petri dishes. 45 plates were prepared with wheat-rich agar culture medium, which were seeded with samples of each of the fungal isolates obtained in Example 1, at different concentrations. After 10-12 days (and observed growth), 200 eggs of different types of parasites / plaque were deposited, and the ovicidal effect of fungal isolates on the eggs of these parasites was evaluated at 30 days according to the criteria described in Lysek ( 1982; Lysek H, Faasatiová O, Cuero Pineda ON, Lorenzo Hernández N. 1982. Folia Parasitol. (Praha) 29: 265-270):

As control plates were used that did not contain fungi and to which the eggs of the parasites were added. The parasiticidal activity or efficacy of fungi was determined by applying the following formula:

% Reduction = (1 - Viable parasites in Control / Viable parasites in Treaties) x

100

The concentrations of the spores of the different fungi isolated and used in the present invention, are from here and throughout the present document D1: 0.025x10 ⁶ ; D2: 0.05x10 ⁶ ; D3: 0.1x10 ⁶ ; D4: 0.2x10 ⁶ and D5: 0.4x10 ⁶ / L culture medium. Table 2. Ovicidal activity of different fungal isolates on eggs of the Calicophoron daubneyi parasite, according to the criteria described in Lysek (1982). Spore concentration

0.025x10 ^B 0.05x10 ^B 0.1x10 ^B 0.2x10 ^B 0.4x10 ^B Control

CECT 20824 2 2 3 4 3 0

Trichoderma 2 2 2 2 2 0

Fusarium 1 1 2 1 2 0

Table 2 shows the ovicidal activity of spores of fungi CECT 20824 (M. circinelloides), Trichoderma and Fusarium against the Calicophoron daubneyi parasite present in feces of grazing cattle.

Table 3 shows the ovicidal activity of fungi CECT 20824 (M. circinelloides), Trichoderma and Fusarium against eggs of the parasitic nematode Parascaris equorum obtained from feces of grazing foals. The spore concentrations of the different fungi used are the same as described above.

Table 3. Ovicidal activity of different fungal isolates on eggs of the parasitic nematode Parascaris equorum, according to the criteria described in Lysek (1982).

Table 4 shows the ovicidal activity of fungi CECT 20824 (M. circinelloides), Trichoderma and Fusarium against eggs of the parasite Trichuris spp. collected from dromedary feces (Camelus dromedarius) in "Marcelle Natureza". The spore concentrations of the different fungi used are the same as described above. Table 4. Ovicidal activity of different fungal isolates on eggs of the parasite Trichuris spp. according to the criteria described in Lysek (1982).

These results show that the greater ovicidal activity is shown in the presence of fungi of the genus Mucor, specifically in the presence of strain CECT 20824, corresponding to M. circinelloides, compared to the ovicidal activity presented by fungi of the genus Trichoderma , especially on the eggs of ascarids and tricurids. On the contrary, fungi of the genus Fusarium barely showed ovicidal activity on the eggs of the ascarids and tricurids analyzed in the present example. Example 4. Analysis of parasiticidal-ovicidal activity of different species of nematophagous fungi on coprocultures infected with parasites.

Five grams of feces from different species of animals infected with parasites and eliminating eggs from these parasites in their feces were placed on plastic boxes. Different concentrations of spores (see Example 3) of the fungal isolates described in Example 1 were added below. To simulate the actual conditions to which both fungi and parasites are exposed, plastic boxes with feces, Eggs from parasites and fungi were kept outdoors in grazing meadows. As control boxes were used with feces from infected animals to which fungal isolates were not added. The efficacy of the parasiticidal activity of the fungi analyzed was determined 30 days after the addition of the spores, by the formula described above.

Figure 2 shows the ovicidal activity of M. circinelloides fungi CECT 20824 and Trichoderma on eggs of the parasitic nematode Parascaris equorum. For This was prepared 60 boxes with five grams each of feces of zebras that eliminated 800 eggs of P. equorum (per gram of feces, hpg), which were divided into 3 groups:

• 25 boxes received spores of M. circinelloides CECT 20824,

· 25 boxes received Trichoderma spores

• 10 boxes were kept without spores-Control group.

The spore concentrations used were the same as previously used (see Example 3). As shown in Figure 2, the concentrations of spores, D2-D5, of M. circinelloides CECT 20824 show a percentage of reduction of ≥50% of eggs of the P. equorum parasite, while with the Trichoderma spores, values of 20% -42% reduction. The highest percentage of the ovicidal effect was reached when a spore concentration of 0.2x10 ⁶ spores (D4) of M. circinelloides CECT 20824 was used, obtaining a percentage of parasite egg reduction of 72%. In the control group no ovicidal activity was observed.

The ovicidal activity of the spores of these nematophagous fungi on feces cultures containing eggs of the parasitic nematode Trichuris spp (Figure 3) and of the parasitic trematode Calicophoron daubneyi (Table 5) was also analyzed. Figure 3 shows the ovicidal activity of fungi M. circinelloides CECT 20824 and Trichoderma on eggs of the parasitic nematode Trichuris spp. present in dromedary feces that eliminated 2,500 eggs of Trichuris spp. (per gram of feces). The results show that the percentages of reduction of viable eggs were higher when the feces were treated with spores of M. circinelloides CECT 20824, especially when spore concentrations greater than 0.1x10 ⁶ spores (D3) were used, observing a reduction of the number of eggs of the parasite that varied between 46-56%. Table 5 shows the ovicidal activity of the Mucor and Trichoderma fungi on eggs of the parasitic trematode Calicophoron daubneyi. The same experimental development as explained above was carried out, but using feces cultures of cows that removed different amounts of Calicophoron daubneyi eggs in feces (50, 100, 150 and 200), and a mixture of 2x10 was added ⁶ spores of Duddingtonia and Mucor, obtaining the following results: Table 5. Ovicidal activity of Mucor and Trichoderma spores on eggs of the Calicophoron daubneyi parasite. The table shows the percentage of egg reduction shown by spores of the nematophage fungi used.

Contrary to what happens with some nematodes (ascarids and tricurids), in which the infective phase (larva) does not leave the egg, in the tremátodos, once in the ground, inside the eggs the miracidio is formed, that leaves the egg, infects a snail (Galba truncatula), which emits cercarías, which eventually become the infectious phases, the metacercariae. The formation of miracidia depends on the temperature, ranging from 20-60 days at temperatures between 10 and 20 ° C. For this reason, it is necessary to take into account the time that elapses for the nematophago-ovicidal fungi to destroy the eggs of the trematodes. In this regard, an assay was designed in which the different spore concentrations of M. circinelloides CECT 20824, described throughout the present invention on five grams of cow feces that removed 250 eggs from the Calicophoron daubneyi trematode were added.

As can be seen in Figure 4, the earliest results of ovicidal activity shown by the spores of M. circinelloides CECT 20824 on the eggs of C. daubneyi, were obtained with the concentrations of D3-D5, proving that the viability of the eggs were significantly reduced by ≥50% after 17 days of being in contact with these spore concentrations of M. circinelloides CECT 20824. These results show that with the use of spore amounts of M. circinelloides CECT 20824 greater than 0.05 x10 ⁶ (D2) reduces the probability of infection by trematodes by at least 50%.

Example 5. Analysis of parasiticidal-ovicidal activity of different species of fungi on parasites in sandy environment.

As previously mentioned, among the most dangerous parasitic zoonoses for human health are ascariosis, caused mainly by Toxocara canis parasitic nematode. It is a helmintozoonosis in which pets, mainly dogs and cats, eliminate eggs that go outside with feces. In the soil, the development of these eggs is completed until larva 2 (L2), which does not leave the egg. Accidentally, for example in public parks, sandboxes, etc., it can take place the ingestion of eggs with L2 inside, which are released in the intestines of people, and perform migrations through the body, placing themselves in the eyeball , musculature, etc. The main risk group is found in children.

In order to simulate these conditions, five grams of coatis feces (Nasua narica) from the "Marcelle Natureza Zoological Park" (Outeiro de Rei, Lugo) were placed, eliminating 850 ascarid eggs per gram of feces in different plastic boxes. A total of 18 boxes were prepared, which were divided into 3 groups:

• 7 boxes containing spores of M. circinelloides CECT 20824

• 7 boxes containing spores of M. circinelloides CECT 20824 + sand

· 4 boxes that did not contain spores (Control group)

The boxes were kept in a meadow to simulate the real conditions to which they are exposed. The efficacy of the mushroom M. circinelloides CECT 20824 was determined with the formula described above, obtaining higher values of reduction of the number of viable eggs when M. circinelloides CECT 20824 was in a sandy environment, indicating that it is a very fungus useful in the prevention of parasitic infections of people by ingestion of eggs eliminated by domestic animals or cattle (zoonosis). The results obtained showed that the ovicidal activity of M. circinelloides CECT 20824 on parasites in sandy soil was a 72% reduction of said parasites versus a 61% reduction when M. circinelloides CECT 20824 is not found in sandy soil .

Example 6. Analysis of parasiticidal-larvicidal activity in spore culture plates of Duddingtonia flagrans CECT 20823 on plaque-grown parasites.

40 plates were prepared with enriched medium (agar, wheat flour, chloramphenicol, water), in which an inoculum of D. flagrans CECT 20823 was seeded. After 8-10 days, they were placed in 30 larval plates 3 (L3) of parasitic striated nematodes (ciatostominos) obtained after the incubation of horse feces at 22-25 ° C for 20 days. 15-17 days after the addition of the larvae, the larvicidal efficacy of the fungus was analyzed, using the formula:

% Reduction = 1 - (Parasites plates with CECT 20823 / Parasites control plates) x

100

The percentage of reduction of L3 larvae of the strungilated nematodes was 80-90%. Example 7. Analysis of parasiticidal-larvicidal activity of spores of D. flagrans CECT 20823 on coprocultures infected with parasites.

Four different concentrations of chlamydospores CECT 20823 D. flagrans 5x10 ^6, 10x10 ^6, 20x10 ⁶ and 40x10 ^6/100 g was used. feces, which were added to plastic boxes with 10 grams of horse feces that removed eggs from parasitic striated nematodes. Depending on the elimination of eggs, 3 groups were established: low (≤300), medium (800) and high (1200), observing a 89-99% reduction percentage according to the initial parasite load (Table 6). The highest parasiticidal activity of D. flagrans CECT 20823 was observed in the groups that had a high egg elimination level (> 1200) (Table 6).

Table 6. Parasiticidal activity of D. flagrans CECT 20823 against strongylated nematodes present in horse feces.

The results obtained based on the concentration of added D. flagrans CECT 20823 spores showed that the highest parasiticidal-larvicidal activity was observed when spore doses greater than 10x10 ^{6 were used} (Table 7). Table 7. Parasiticidal-larvicidal activity of different spore concentrations of D. flagrans CECT 20823 against strongylated nematodes present in horse feces.

Concentration of

N ° boxes% Reduction L3

spores

5x10 ⁶ 60 91

10x1 O ⁶ 40 92

20x1 O ⁶ 60 95

40x1 O ⁶ 36 99

Total 196 94

The parasiticidal-larvicidal activity of D. flagrans CECT 20823 on parasites present in piglet feces was also analyzed. In the farm of the association PRODEME (Pro Mentally Deficient) located in Monforte de Lemos (Lugo), feces were collected directly from the rectum of 20 piglets, which eliminated in the feces eggs of ascarid (Ascaris suum) and strungilated (Oesophagostomum) nematodes. Different groups were established based on the number of parasite eggs in the feces, placing 15 grams of feces in plastic boxes, preparing a total number of 66 boxes. 20x10 ⁶ clamidospores of D. flagrans CECT 20823 were added in 44 boxes, keeping 22 fungus-free boxes as controls. The efficacy of the parasiticidal activity of D. flagrans on this type of parasites was evaluated at 21 days using the formula:

% Reduction = 1 - (Box parasites with CECT 20823 / Control box parasites) x 100 As can be seen in Table 8, the percentage of reduction of strungilated nematodes (L3) was equal to or greater than 95%, regardless of the load initial (Eggs per gram of feces, hpg).

Table 8. Parasiticidal-larvicidal activity of D. flagrans CECT 20823 against L3 striated nematodes present in piglet stool coprocultures. Initial hpg 950-

0-300 350-600 650-900> 1200 strung 1200

% Reduction 95 96 95 97 99

No effect of D. flagrans CECT 20823 was observed on coccidia or ascarid parasites of piglets, which seems to be due to the fact that in this case the larval forms do not leave the egg until they are ingested again by a host, so the traps elaborated by the fungus D. flagrans CECT 20823, they are not useful in this case. On the contrary, the larvae L2 and L3 of the strungilated nematodes constitute the main target of this fungus. The parasiticidal-larvicidal activity of D. flagrans CECT 20823 on parasites present in calf feces was also analyzed. In the farm of the association PRODEME (Pro Mentally Deficient) located in Monforte de Lemos (Lugo), feces were collected directly from the rectum of 20 calves that eliminated eggs of strungilates (Ostertagia, Oesophagostomum and Trichostrongylus) in the feces. 70 plastic boxes with 15 grams of feces were prepared, which were grouped according to the number of eggs removed. 30x10 ⁶ clamidospores of D. flagrans CECT 20823 were added in 56 boxes, keeping 24 boxes without fungi as controls. The efficacy of fungal therapy was evaluated at 21 days with the formula described above, showing that the percentage of L3 larvae that remained viable after the addition of the spores was ≥5% (Table 9). These results corroborate the high efficacy of D. flagrans CECT 20823 against parasitic striated nematodes.

Table 9. Parasiticidal-larvicidal activity of D. flagrans CECT 20823 against L3 striated nematodes present in calf feces.

Example 8. Analysis of parasiticidal-larvicidal activity of spores of D. flagrans CECT 20823 on animals infected with parasites. Two animal tests were carried out. In the first place, spores of D. flagrans CECT 20823 were distributed directly over meadows that served as food for grazing horses (Farm "Gaioso-Castro", Castro Riberas de Lea, Provincial Council of Lugo). In the second place, the spore of D. flagrans CECT 20823 was orally administered to different species of equidae kept in the "Marcelle Natureza Zoological Park" (Outeiro de Rei, Lugo), by mixing with food concentrate (feed) .

Farm "Gaioso-Castro"

16 adult Purebred Galician mares were used, kept in three plots of two Ha, which eliminated eggs from parasitic strungy nematodes. The equines were divided into 3 groups:

• Group I (IVM): 6 animals treated with ivermectin (1 mg / kg pv Noromectin ^® , Norbrook, Ireland), a conventional antiparasitic,

• Group II (IVM + CECT 20823): 6 animals treated with ivermectin and held in grazing on a monthly where poured on the floor chlamydospores D. flagrans CECT 20823 of (2x10 ^6/100 m ²⁾

• Control Group: 4 animals that remained untreated.

Figure 5 shows that the administration of ivermectin suppressed the elimination of nematode eggs in the feces of animals of the IVM group and of the IVM + CECT 20823 group for 8 weeks. With the distribution of spores of D. flagrans CECT 20823 in the plot of horses of the group IVM + CECT 20823, figures for elimination of strungy eggs that varied between 50 and 150 hpg were maintained in that group, while These figures are much higher when spores are not administered (IVM group: between 50 and 400 hpg).

In order to reduce the indiscriminate and repeated use of anthelmintics in horses, the term selective therapy has been introduced, which consists of administering antiparasitics to animals that eliminate in the feces quantities greater than 300-400 eggs per gram, based on that this parasitic load begins to be pathogenic for horses, and therefore an anti-parasitic treatment is required (Uhlinger CA. Vet Clin North Am Equine Pract 2007; 23: 509-17 Francisco R, Paz-Silva A, Francisco I, Cortiñas FJ, Miguélez S, Suárez J, Cazapal-Monteiro CF, Suárez JL, Arias MS, Sánchez-Andrade R. J Eq Vet Sci 2012. 32: 274-280.). This demonstrates the usefulness of the addition of spores of D. flagrans to reduce parasitic load in the animal feces and therefore to reduce their chances of infection, since the numbers of strongy eggs present in the feces of the horses are kept below 300 hpg when spores are administered in the plots where they are found.

Marcelle Natureza

The efficacy of spore treatment of D. flagrans CECT 20823 in the prevention of reinfection of different equine species was tested: zebras (Equus quagga), domestic donkey (E. asinus) and African donkey (E. africanus asinus). The test consisted of administering every 2 days 2x10 ⁶ spores / animal, mixed with the food concentrate (feed).

Figure 7 shows that after the administration of antiparasitic (ivermectin + praziquantel (Equimax ^® , Virbac, Spain), 1, 07 g gel / 100 kg pv) in September 2010, the elimination of eggs was suppressed of strungillae in the 3 equine species. In November they reappeared and increased until March 201 1, at which time the animals were treated again with the anti parasitic. From May 2011 there was a new increase, and antiparasitic was applied in September of that year. From September 201 1, 2x10 ⁶ spores / animal of D. flagrans CECT 20823 were provided, every 15 days, mixed with the food concentrate, being able to maintain the elimination of strungy eggs between 50 and 400. No treatment was administered with antiparasitic from September 2011 to the current date (November 2012), maintaining the levels of elimination of strungy eggs among the above-mentioned values, without the help of commercial antiparasitic treatment.

Example 9. Parasiticidal efficacy of the spore combination of M. circinelloides CECT 20824 and D. flagrans CECT 20823.

As shown throughout the present invention, M. circinelloides CECT 20824 has the highest ovicidal activity and D. flagrans CECT 20823 has the highest larvicidal activity, of all the fungi analyzed. Therefore, its use has been considered in combination with the aim of expanding the range of action of these fungi on different infectious forms of parasites, eggs and larvae. Since it is known that some strains of nematophagous fungi, such as Trichoderma strains inhibit the growth of other nematophage strains, such as Fusarium (Hernández Mansilla AA, Sierra Peña A, Carr Pérez A. Phytosanity (Cuba) 10: 105- 108), it was verified that both strains of fungi, M. circinelloides CECT 20824 and D. flagrans CECT 20823, could grow together, observing that there was no problem, as can be seen in Figure 8. Next, we proceeded to the determination of the joint parasiticidal activity of both species of fungi on different parasitic stages that can be found in the soil.

Parasiticidal activity of M. circinelloides CECT 20824 + D. flagrans CECT 20823 on parasite eggs. Plate tests.

Twenty plates with wheat-agar were prepared, and each one was seeded simultaneously with an inoculum of D. flagrans CECT 20823 and another of M. circinelloides CECT 20824. At 10 days, once growth of both fungi was observed, they were added in each plate 200 eggs of Parascaris equorum and 500 L3 of strungilated nematodes. The effectiveness of parasiticidal activity was established 17 days later. The results obtained showed that the reduction percentage for ascarids (Parascaris equorum) was approximately 86% (72-96%) and for the L3 larvae of the strungilated nematodes of approximately 98% (96-99%) . These results show that the combination of the two fungi does not diminish their activity, since M. circinelloides CECT 20824 exerts an alteration on the eggs of type III ascarids (breakage of the cover and colonization of the interior), and an activity 4 on its viability (destruction of eggs greater than 80%), as demonstrated in Examples 2 and 3. On the other hand, the efficacy of D. flagrans CECT 20823 on strungilated larvae also reached the values observed previously in the Examples 6 and 7. Parasiticidal activity of M. circinelloides CECT 20824 + D. flaarans CECT 20823 on animals infected with parasites.

Taking into account that the association of M. circinelloides CECT 20824 + D. flagrans CECT 20823_ is capable of reducing the presence of eggs and larvae of parasites, it was considered that a very suitable mechanism for distribution in animals is their incorporation into the food concentrate ( I think) that animals receive. Of this In this way it is possible that the fungi act both in the animal itself and, mainly in their fecal matter, which also acts as a vehicle for parasitic forms to the ground, thus preventing the infection of grazing animals.

First, the heat resistance of a spore mixture of the two fungi (M. circinelloides CECT 20824 + D. flagrans CECT 20823) was analyzed, taking into account that during the feed manufacturing process a temperature of 70 ° C is reached for 1-2 min (drying phase), and that both factory raw materials and the final product (concentrate) may be exposed to temperatures close to 4 ° C. There are some studies that indicate that the growth of fungi of the genus Mucor occurs at low temperatures, indicating 30 ° C as the limit to observe the development of this fungus (de Hoog et al., 2000. Atlas of Clinical Fungí, 2nd ed, vol 1. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands). In the present invention it is shown, Tables 10 and 1 1, that the spores of M. circinelloides CECT 20824 and those of D. flagrans CECT 20823, show resistance to the heat treatment experienced during the manufacture of the food concentrate (70 ° C) , as well as its conservation in the factory (4 ° C). Table 10. Resistance of spores of M. circinelloides CECT 20824 and Duddingtonia flagrans CECT 20823 against treatments with high temperatures.

Temperature: 70 ° C

Reduction

L3 reduction

Egg growth time

(%) Parascaris (%)

0 ++++ 0 0

10 "++++ 95-99 56-78

30 "++++ 93-99 64-81

r ++++ 96-98 73-82

2 '++++ 91-97 69-77

5 '++++ 90-96 72-80

10 '+ 10 12 Table 11. Resistance of spores of M. circinelloides CECT 20824 against treatments with low temperatures.

Once the resistance of the spores to the heat treatment experienced during the manufacturing of the food concentrate, as well as to its conservation in the factory, was demonstrated, it was added to the Pro-Horse Club ^® (Nanfor) product at the NANTA (Nanfor) facilities , Nutreco) in Outeiro de Rei (Lugo). 72x10 ⁶ spores of each fungal species were added per 1200 kg feed. Samples (1 g) were taken from the concentrate thus prepared and dissolved in water (1 ml_), and deposited in Petri dishes with culture medium consisting of agar and wheat. At 7 days, growth of the two fungal species was observed, demonstrating that the production of the feed under the factory conditions does not alter its development capacity.

Next, 200 eggs of the parasitic nematode Parascaris equorum + 500 L3 of strongilated nematodes (ciatostominos) of horses were added on each plate. At 14 days it was determined that the percentage of egg reduction ranged from 72 to 82%, and that of larvae between 95-99%, confirming that the spores remain viable after the preparation of the food concentrate.

Subsequently, in order to determine if the mixture of spores crossed the digestive tract and left unscathed in the feces, a group of 8 horses, 2.5 kg feed / horse / day, was administered to ensure that 15x10 were provided ⁶ spores of each fungus / animal / day. The animals were kept for 6 days. The method described in Ojeda-Robertos et al., 2008 was followed to determine the percentage of spore recovery in feces. Ojeda-Robertos NF, Torres-Acosta JF, Aguilar-Caballero AJ, Ayala-Burgos A, Cob-Galera LA, Sandoval-Castro CA, Barrientos-Medina RC, from Gives PM. Vet Parasitol. 2008; 158: 329-335. The results obtained (Table 12) revealed that between 5 and 20% of the spores left with the feces, which coincides with results of previous investigations in which the spores of D. flagrans were administered in aqueous solution by oral route ( Chandrawathani et al., 2003) (Chandrawathani P, Jamnah O, Waller PJ, Larsen M, Gillespie AT, Zaharí WM. Vet Parasitol. 2003; 1 17: 173-83).

Table 12. Spore recovery percentage of M. circinelloides CECT 20824 and D. flagrans CECT 20823 in horse feces.

Subsequently, it was administered to two groups of think horses, which contained the mixture of spores of both fungal species and feed without spores (control group). Purebred Galician horses kept in grazing were used, which are supplemented with 2.5 Kg feed / horse / day. The food concentrate is Pro-Horse Club ^® . As seen in Figure 9, 15 days after the administration of an anthelmintic (ivermectin; 1 mg / Kg pv Noromectin ^® , Norbrook, Ireland) to both groups of animals, the horses of the control group again eliminated eggs from nematode parasites (strung) in feces, while those who received feed with spores did so after 45 days. It can also be seen that the addition of the spores to the feed reduced the elimination rates of eggs in the horses, keeping values below 200, while the control group reached figures of 600.

Example 10. Efficacy M. circinelloides CECT 20824 for the prevention of zoonoses caused by ascarids.

Zoonoses are those diseases that can be transmitted between vertebrate animals and man or vice versa. Helmintozoonosis are infections caused by helminth parasites, and among them are ascariosis, which appear in urban, peri-urban and rural settings (Bojar, H., Klapec, T. Ann Agrie Environ Med. 2012, 19: 267-270). They are very prevalent chronic infectious diseases in people, with a current estimate of 2 billion cases worldwide, which are encompassed within soil-transmitted zoonoses (soil-transmitted helminthic zoonoses), since people can become infected by accidental ingestion of embryonated eggs (they contain 2) larvae eliminated in animal feces. Toxocara spp. (7 canis and 7 cati) and Toxascaris leonina are species that affect pets (dogs and cats) as well as wild carnivores (lynx, etc.). People dedicated to the rearing of animals for rent are also exposed to infection by ascarids when they perform tasks such as cleaning stables or boxes without gloves. Ascaris suum is described in suidae, and in Parascaris spp equidae. (P. equorum and P. univalens). At present, it has gained much relevance, due to its wide distribution and severity of the conditions it causes, zoonosis caused by the ingestion of eggs from Baylisascaris procyonis, ascarid that mainly affects raccoons, and to a lesser extent other mammals and even birds. In some areas of the United States it is described that 68-82% of raccoons are parasitized (Kahn, LH Emerg Infecí Dis. 2006, 12: 556-561). In Europe they constitute a serious problem in Germany, Poland, or France. In Spain, stable colonies of raccoons have already been denounced in Madrid, Valencia, Extremadura and Galicia.

Certain species of fungi (Pochonia chlamydosporía, Verticillium sp.), In the presence of parasitic forms (eggs) develop the mycelium and are able to adhere to the cover of the eggs, penetrate and eliminate the embryo that is inside. In this way they manage to meet their energy and nitrogen needs. In the absence of parasitic forms, they remain in a state of latency. In order to establish the ability to reduce the number of viable ascarid eggs in the feces of parasitized animals, different tests were performed that consisted of adding a quantity of spores of M. circinelloides CECT 20824 directly to the feces, and after 30 days calculate the effect by comparing the egg count in this stool and in which it remained without spores (controls). All tests were performed under natural conditions.

10. 1. Efficacy on Toxocara canis in feces of Doberman puppies

20 boxes were prepared with 5 grams of feces of 4-month-old Doberman puppies, which initially removed 708 Toxocara canis eggs per gram of feces (HPG), and were kept for 30 days in field conditions. A lot of 10 boxes received 3 mL of COPFr culture medium that transported 0.1-0.2x106 Mucor spores and another of the same number of boxes served as a control group (without spores). With the addition of Mucor spores, the viability of the eggs was reduced by 53% (Table 13). Table 13. Reduction of the viability of Toxocara eggs with M. circinelloides CECT 20824.

10.2. Efficacy on Toxascaris leonina in feces of boreal lynx

Also, feces of boreal lynx containing 3161 HPG of Toxascaris leonina were placed in 20 plastic boxes (5 g / box); 0.1-0.2x106 Mucor spores were added to 10 boxes, and the other 10 remained fungus-free as controls (Table 14). The same scheme was repeated with the feces of other boreal lynxes that removed 5316 HPG from Toxascaris. As can be seen in Table 14, the percentage of reduction in the number of viable eggs decreased between 64 and 67%.

Table 14.- Reduction of the viability of Toxascaris eggs with M. circinelloides CECT 20824.

10.3 Efficacy on Ascaris suum and Parascaris equorum in feces of Porch echones

Celtic To determine the degree of prevention of infection by ascarids that affect animals for rent, 5 grams of feces of piglets of native Porco Celta breed that removed 960 HPG from Ascaris suum were placed. 18 boxes were prepared, which were divided into 3 lots, with Mucor spores (12) and without spores (controls) (6). Likewise, the feces of Pura Raza Galega (PRG) foals containing 300 HPG of P. equorum were placed in boxes (5 g), and the Witness groups (12) and Mucor (12) were established.

A 53% reduction percentage was estimated on viable A. suum eggs through the Mucor action (Table 15), much higher than that indicated in previous investigations with other ovicidal fungi (Araújo JV, Braga FR, Silva AR, Araujo JM , Tavela AO. Parasitol Res, 2008, 102: 787-790).

Table 15. Reduction of the viability of saccharide eggs in animals for rent.

Table 15 shows that the action of the 2 ovicidal fungi reduced the presence of viable Parascaris eggs to 1/3, and no significant differences were observed between them (P> 0.05). In tests carried out on Petri dishes, with Pochonia an ovicidal effect of type III was reached on Parascaris eggs, and the percentage of reduction of its viability was 45% (De Carvalho LM, Braga FR, Domingues RR, Araujo JM, Lelis RT, by Paula AT, da Silveira WF, by Araújo JV. J. Basic Microbiol, 2013; doi: 10.1002 / jobm. 201300586).

10.4 Efficacy on Baylisascaris in raccoon and coati feces

In a total of 12 plastic boxes, 5 grams of raccoon feces (Procyon lotor) were disposed of that eliminated 216 HPG from Baylisascaris. 2 groups of 6 boxes each were used, with Mucor spores and without spores (controls). The same was applied design with raccoon feces with 2700 HPG, and coatis (Nasua nanea) with 1 HPG from Baylisascarís.

Table 16. Reduction percentage of viable Baylisascarís eggs with CECT 20824 circinelloides.

As summarized in Table 16, the Mucor effect meant a reduction in the viability of Baylisascarís proeyonis eggs by 52-74%. The absence of biological control data on Baylisascarís eggs limits the possibilities for discussion of the results obtained. However, it adds significant value to them, since it is currently a serious Public Health problem in forested or peri-urban areas of countries such as the US. Nor should we forget the severity of the measures used to reduce the presence of eggs of this ascarid on the ground, based on the flaming of any surface that has been in contact (or suspicious) with raccoon feces (Page LK, Beasley JC , Olson ZH, Smyser TJ, Downey M, Kellner KF, McCord SE, Egan TS 2nd, Rhodes OE Jr. Emerg Infected Dis. 201 1, 17: 90-93.). Although it sounds a bit distant, it should also be taken into account that stable populations of raccoons have been detected in Spain, specifically in Madrid, Valencia and Extremadura, and even in Galicia.

10.5. Efficacy on Toxocara canis and Baylisascarís proeyonis in sandy areas contaminated with feces of dog puppies and coatis respectively Occasionally, the transmission of helmintozoonosis takes place during the enjoyment of recreational areas, sandboxes, parks ..., places to that also access pets or wild animals, and when defecating they can be contaminating them. For this reason, 2 trials were carried out to simulate the conditions of sandy areas. Be they used 6 boxes with 5 grams of feces of dog puppies that removed 708 HPG from Toxocara canis, to which Mucor spores were added, and 5 g of absorbent cat litter. In addition, 5 grams of coatis feces with 2700 HPG of Baylisascaris procyonis were also placed in 6 boxes, and Mucor and sand spores (5 g / box) were added.

Table 17. Reduction of the viability of ascarid eggs in feces and sand.

With the incorporation of Mucor spores, a 58% reduction in the viability of Toxocara eggs was obtained, and 81% in Baylisascaris eggs. Comparing these results with those of Tables 13 and 16, it can be observed that in the presence of sand the reduction values of viable eggs did not decrease, increasing slightly (P> 0.05). These data represent a very important contribution to the possibilities of control of helmintozoonosis by ascites, since it is concluded that the use of Mucor reduces the amount of viable eggs to less than half.

Fungi Parasi

PCT

Printing (original in electronic format)

(This sheet is not part of the international application and does not count as one of its sheets)

1 The indications made to

then refer to (fos)

microorganism (s) or other material

biological deposited mentioned in

The description in:

-1 page 4

-2 line 24

-3 Identification of) deposit

-3-1 Name of the CECT deposit institution Spanish Type Crop Collection Type -3-2 Address of the depository institution Building 3 CUE. Stop Scientific

University of Valencia, Professor Agustín Escardino, 9, 46980 Paterna (Valencia), Spain

-3-3 Presentation date 18 December 2012 (18.12.2012)

-3-4 Order number CECT 20824

-5 Indications are given for All designations

following designated states

-6 Separate supply of indications Certificate of strain deposit included as accompanying element

Indications lists will be presented to

a International office later

Fungi Parasi

PCT

Printing (original in electronic format)

2 Indications made to

then refer to

microorganism (s) or other material

biological deposited mentioned in

The description in:

2-1 page 4

2-2 line 28

2-3 Deposit Identification

2-3-1 Name of the CECT deposit institution Spanish Type Crop Collection

2-3-2 Address of the depository institution Building 3 CUE, Pare Cientific

2-3-3 Presentation date December 18, 2012 (18.12.2012)

2-3-4 Order number CECT 20823

2-5 Indications are given for All designations

following designated states

2-6 Separate supply of indications Certificate of strain deposit included as accompanying element

These indications will be presented to

the International Bureau later

FOR USE OF THE RECEIVING OFFICE ONLY

-4-1 Authorized Officer

FOR USE OF THE INTERNATIONAL OFFICE ONLY -5 This form was received by the

International Office on:

-5-1 Authorized Officer

Claims

47 CLAIMS

1. Biological control agent comprising at least one fungus or a reproductive form thereof, belonging to the species M. circinelloides.

2. Biological control agent according to claim 1 characterized in that the fungus or a reproductive form thereof is M. circinelloides CECT 20824.

3. Biological control agent according to any of claims 1-2 characterized in that it also comprises at least one other fungus or a reproductive form thereof, belonging to the species D. flagrans.

4. Biological control agent according to claim 3 characterized in that the fungus or a reproductive form thereof is D. flagrans CECT 20823.

5. Biological control agent according to any of claims 1-4 characterized in that it also comprises at least one other fungus or a reproductive form thereof, belonging to a genus selected from any of the following: Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or any of its combinations.

6. Biological control agent according to any of claims 1-5 characterized in that it is an additive for animal feed.

7. Biological control agent according to any of claims 1-5 characterized in that it is a parasiticidal composition.

8. Biological control agent according to any of claims 6 or 7 characterized in that it is administered orally.

9. Biological control agent according to claim 7 characterized in that the parasiticidal composition is administered additionally or alternatively to oral administration, on the feces of the animals and / or on the ground.

10. Biological control agent according to any of claims 1-9 characterized in that it comprises reproductive forms of fungi.

1 1. Biological control agent according to claim 10 characterized in that the reproductive forms of the fungi are live spores.

12. Biological control agent according to any of claims 1-1 1 characterized in that the biological control is exercised against the viable forms of the helminth parasites selected from any of the following: nematodes, trematodes, cestodes and / or any of your combinations

13. Biological control agent according to any of claims 1-12, characterized in that the biological control is exercised in animals selected from any of the list: dogs, cats, bovids, ovids, sweats and equidae. 48

14. Use of at least one fungus or a reproductive form thereof belonging to the species Mucor circinelloides for the preparation of a biological control agent.

15. Use according to claim 14 characterized in that the fungus or a reproductive form thereof is M. circinelloides CECT 20824.

16. Use according to any of claims 14-15 characterized in that it further comprises the use of at least one other fungus or a reproductive form thereof belonging to the species Duddingtonia flagrans.

17. Use according to claim 16 characterized in that the fungus or a reproductive form thereof is D. flagrans CECT 20823.

18. Use according to any of claims 14-17 characterized in that it further comprises at least one other fungus or a reproductive form thereof belonging to a genus selected from any of the following: Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or Any of your combinations.

19. Use according to any of claims 14-18 characterized in that the biological control agent is an additive for animal feed.

20. Use according to any of claims 14-18 characterized in that the biological control agent is a parasiticidal composition.

21. Use according to any of claims 19 or 20 characterized in that the biological control agent is administered orally.

22. Use according to claim 20 characterized in that the parasiticidal composition is additionally or alternatively administered to oral administration, on the feces of the animals and / or on the ground.

23. Use according to any of claims 14-22 characterized in that it comprises reproductive forms of fungi.

24. Use according to claim 23 characterized in that the reproductive forms of fungi are live spores.

25. Use according to any of claims 14-24 characterized in that the biological control is exercised against the viable forms of the parasites selected from any of the following: nematodes, trematodes, cestodes and / or any combination thereof.

26. Use according to any of claims 14-25 characterized in that the biological control is exercised in animals selected from any of the list: dogs, cats, bovids, eggs, suidae and equidae.

27. Cultivable strain CECT 20824 cultivable characterized by belonging to the species M. circinelloides.

28. Biologically pure culture of the fungal strain M. circinelloides CECT 20824. 49

29. Method of reducing the parasitic load present in animal feces and in the environments where they are found by administering, either to feces and / or the environment, an effective amount of at least one fungus or a reproductive form thereof, of the species M. circinelloides CECT 20824 alone or in combination with at least one other fungus or a reproductive form thereof, selected from any of the following: D. flagrans CECT 20823, Trichoderma, Fusarium, Verticillum, Arthrobotrys, Pochonia and / or any of its combinations; or of a biological control agent according to any of claims 1-13.

30. A method of reducing the parasitic load according to claim 29, characterized in that the reduction in the amount of parasites is at least 50%.

31. Method of reducing the parasitic load according to any of claims 29-30 characterized in that the reduction in the amount of parasites is at least 75%.

32. Method of reducing the parasitic load according to any of claims 29-31 characterized in that the reduction of the parasitic load is carried out on the viable forms of the helminth parasites selected from any of the following: nematodes, trematodes, cestodes and / or any of their combinations.

33. Method of reducing the parasitic load according to any one of claims 29-32, characterized in that the animals are selected from any of the list: dogs, cats, bovids, ovids, suidae and equidae.

34. Food concentrate for animal feed comprising the biological control agent according to claim 6.