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WO2015185779A1 - Method for treating coccidiosis - Google Patents

Method for treating coccidiosis Download PDF

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Publication number
WO2015185779A1
WO2015185779A1 PCT/ES2015/070435 ES2015070435W WO2015185779A1 WO 2015185779 A1 WO2015185779 A1 WO 2015185779A1 ES 2015070435 W ES2015070435 W ES 2015070435W WO 2015185779 A1 WO2015185779 A1 WO 2015185779A1
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Prior art keywords
seq
gene
sequence shown
carotene
transgene
Prior art date
Application number
PCT/ES2015/070435
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Spanish (es)
French (fr)
Inventor
Carmen Nogareda Burch
José Antonio MORENO MARTÍNEZ
Eduardo Angulo Asensio
Paul Christou
Original Assignee
Universitat De Lleida
Centre De Recerca En Agrotecnologia-Agrotecnio
Institució Catalana De Recerca I Estudis Avançats (Icrea)
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Publication of WO2015185779A1 publication Critical patent/WO2015185779A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/179—Colouring agents, e.g. pigmenting or dyeing agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • the present invention falls within the methods of treatment or prevention of diseases, specifically coccidiosis, pododermatitis and digital ulcers in a subject.
  • Coccidiosis is a disease caused by protozoan parasites of microscopic size, known as coccidia, belonging to the phylum Apicomplexa; a group of protists characterized by the presence of a unique organelle called apical complex, which includes different families and genera of parasites of great economic and sanitary importance (for example, Coccidia, Piroplasma, and Plasmodium).
  • the family Eimieridae causes diseases that affect most commercially raised animals for consumption, for example, poultry (eg, chickens, roosters, chickens, turkeys, ducks, geese, etc.), domestic mammals (eg, sheep, cows, pigs, horses, water buffalo, etc.), etc.
  • poultry eg, chickens, roosters, chickens, turkeys, ducks, geese, etc.
  • domestic mammals eg, sheep, cows, pigs, horses, water buffalo, etc.
  • the life cycle of these parasites depends on the genus and the species, and most of them complete the cycle in a single host.
  • the cycle develops in a single host;
  • Each species of Eimeria has a specific period of preparation (time between infection and elimination of oocysts) and patent (duration of oocyst excretion) (in general, between 2 and 12 days depending on the species).
  • the dissemination of the oocysts, inside and outside the farm, is done by feces, bed, dust, arthropods, tools, etc.
  • Coccidiosis is characterized by its high morbidity and can lead to clinical or sub-clinical infections.
  • the clinical picture is characterized by diarrhea that can be hemorrhagic, fever, anorexia, asthenia and, sometimes, anemia, causing a decrease in growth and production rates with great economic losses.
  • the different measures applied in the fight against coccidiosis can be grouped into two groups, therapeutic measures based on the use of anticoccidials, such as coccidiostats (preventive treatment) and coccidicides (curative treatment); and preventive measures based on immunizations.
  • Coccidiostats are products that inhibit or stop the development of parasites
  • coccidicides are products that destroy such parasites.
  • Coccidiostats by not completely eliminating oocysts, allow the development of immunity in birds, especially being used in broilers.
  • Coccidicides by destroying parasites, inhibit the development of immunity so they are currently used when clinical problems appear in lots of birds.
  • the extract of an antioxidant compound, grape seed proanthocyanidin, incorporated into the chicken diet has beneficial effects in the treatment of coccidiosis caused by E. tenella (Wang ML. Et al., Poult. Sci. 2008; 87: 2273- 80).
  • Other studies such as Alien PC. et al., (Clin. Microbiol. Rev., 2002, 15: 58-65) show that antioxidants in the diet of birds are not effective in the treatment against E. tenella, but of infections caused by E. acervulina and E. maximum. Therefore, the control of coccidiosis requires better and more effective alternatives, avoiding economic losses triggered by the parasite and, on the other hand, allowing to produce high quality meat without pharmacological residues.
  • the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof, for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of coccidiosis in a subject.
  • the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of pododermatitis or ulcers in a subject.
  • the invention in a third aspect, relates to a composition
  • a composition comprising a part of an antioxidant-enriched corn plant or a derivative thereof and an anticoccidic agent.
  • the invention relates to the use of the composition of the invention for the prevention or treatment of coccidiosis in a subject or for the prevention or treatment of pododermatitis or ulcers associated with coccidiosis in a subject.
  • the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for obtaining a nutritive or nutraceutical composition.
  • the invention relates to a nutritive or nutraceutical composition
  • a nutritive or nutraceutical composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof.
  • Figures 1 and 2 Pododermatitis and number of ulcers according to Welfare Quality ® (2009) Welfare Quality® assessment protocol for poultry (broilers, laying hens).
  • Welfare Quality® Consortium Lelystad, Netherlands. Data were obtained from 14 animals / group.
  • T1 control diet in uninfected chickens
  • T2 control diet in chickens infected with E. tenella
  • T3 diet of corn rich in carotenoids in uninfected chickens
  • T4 diet of corn rich in carotenoids in chickens infected with E. tenella.
  • T2 control diet in chickens infected with E. tenella
  • T4 diet of corn rich in carotenoids in chickens infected with E. tenella. (The uninfected groups T1 and T3 were negative).
  • FIG. 4 From left to right: two legs of each group: T1: control diet in uninfected chickens, T2: control diet in chickens challenged with E. tenella, T3: diet of carotenoid-rich corn in uninfected chickens, T4: Carotenoid-rich corn diet in chickens infected with E. tenella.
  • FIGS. 5 and 6 show histopathology of the Fabrizio pouch of the T2 group (control diet in chickens infected with E. tenella) with medullary and cortical vacuolization.
  • FIG. 7 Histopathology of the Fabrizio pouch of group T4 (diet with corn rich in antioxidants in chickens infected with E. tenella) with little medullary and cortical vacuolization.
  • the authors of the present invention have observed that birds raised with a diet of corn enriched in antioxidants exhibit a reduction in pododermatitis and finger ulcers. In addition, these birds showed a reduction of about four times in the amount of Eimeria oocysts excreted by feces, indicating that the diet of antioxidant-enriched corn is effective against coccidiosis.
  • the histopathology of the Fabrizio pouch allowed the quantification of medullary and cortical vacuolization in the follicles and it was observed that infected animals fed with antioxidant-enriched corn showed a significant reduction of vacuoles.
  • carotenoids play a protective role against oxidative stress due to coccidia infection and allow an increase in immunity.
  • antioxidant-enriched corn is a surprisingly effective vehicle to maintain poultry health and immunity, to reduce dermatitis in the legs and foot ulcers.
  • corn plant refers to a photosynthetic living being belonging to the Zea mays species. Said term also includes the various subspecies.
  • a "part of a plant”, as used in the present invention, refers to a specific part of a plant, such as stem, leaf, root, flower, seed, endosperm, grain, fruit or yolk.
  • “Derived from a part of the plant” refers to any product obtained after processing a part of the plant of the invention from physical and / or chemical modification, by way of illustration, not limitation, flour, semolina, oil, syrup, starch or starch.
  • the corn plant is "enriched in antioxidants," that is, it has higher levels of molecules capable of retarding or preventing the oxidation of other molecules, compared to a reference level.
  • Antioxidants are responsible for capturing free radicals produced in the oxidation reactions of the metabolism.
  • Illustrative non-limiting examples of antioxidants are carotenoids, lutein, lycopene, selenium, vitamin A, vitamin C and vitamin E.
  • the term "nutritional composition" of the present invention refers to that food that, regardless of providing nutrients to the subject who takes it, beneficially affects one or more functions of the organism, so as to provide a better state of health and well-being.
  • said nutritional composition may be intended for the prevention and / or treatment of a disease or the causative factor of a disease. Therefore, the term “nutritional composition” of the present invention can be used as a synonym for functional food or food for specific nutritional purposes or medicinal food.
  • a nutritional composition has an appearance similar to that of a conventional food and is consumed as part of a normal diet.
  • coccidiosis refers to a disease caused by protozoan parasites of microscopic size, known as "coccidia.” They are included in the phylum Apicomplexa, including the family Eimeriidae and are considered several genera, eg, Eimeria, Isospora, etc.
  • This disease affects most commercially raised animals for food purposes, for example, birds (eg, chickens, roosters, chickens, turkeys, ducks, geese, etc.), mammals (eg, sheep, cows, pigs, rabbits , horses, water buffalo, etc.), fish, etc., or for other commercial purposes (eg, dogs, cats, marsupials, etc.).
  • the disease is transmitted by contact with feces or by ingestion of infected tissues.
  • the primary symptom is diarrhea, which can become bloody in severe cases.
  • Most animals infected by coccidia are asymptomatic; however, young or immunodeficient can suffer severe symptoms, including death.
  • subject refers to a member of an animal species, preferably a bird, a mammal or a fish, of any sex, race or age.
  • bird includes chickens, water birds and game birds.
  • Pododermatitis refers to lesions in the pads of the legs and fingers of the legs and in the skin located in the tibiotarsian joint. They can vary in size and depth. At first it is characterized by hyperkeratosis (thickening of the skin). In a second stage, a brown coloration of the skin that corresponds to tissue necrosis appears. In the most severe cases, erosion and ulceration of the tissues occurs accompanied by an inflammation of the subcutaneous tissue. In the central zone the complete destruction of the epidermis originates. The ulcer produces an exudate that forms a black-looking scab. The main cause of pododermatitis is moisture.
  • Anticoccidic agent includes coccidiostats and coccidiocides, thus classified according to their effects on parasites.
  • Eimeria includes any species of the genus Eimeria, including, among others, Eimeria species that infect birds (eg, chickens, roosters, chickens, capons, graffiti (Guinea fowl), puladas, turkeys, ducks, geese, pheasants, partridges, quails, ostriches, etc.), Eimeria species that infect mammals (eg, sheep, cows, pigs, rabbits, horses, dogs, cats, etc.), Eimeria species that infect fish (eg, eels, conger, anchovies or anchovies, herring, sardines, carp, cod, hake, sea bass, horse mackerel, monkfish, etc.
  • Eimeria species that infect birds eg, chickens, roosters, chickens, capons, graffiti (Guinea fowl), puladas, turkeys, ducks, geese, pheasants, part
  • Illustrative, non-limiting examples of such species of Eimeria include species that infect chickens, such as E .tenella, E. acervulina, E. brunetti, E. Hagai, E. maxim, E. mitis, E. mivati, E. necatrix, E. praecox, etc., species that infect turkeys, such as E. adenoeides, E. gallopavonis, E. innocua, E. meleagr ⁇ dis, E. meleagrimitis, E. subrotunda, etc., species that infect partridges, t ales such as E. alectoreae, E. caucasian, E. gonzalezcastroi, E.
  • species that infect chickens such as E .tenella, E. acervulina, E. brunetti, E. Hagai, E. maxim, E. mitis, E. mivati, E. necatrix, E. praecox, etc.
  • E. phasiani species that infect quail, such as E. bater ⁇ , E. coturnicis, E. lettyae, etc., species that infect pheasants, such as E. colchici, E. duodenalis, E. langeroni, E. megalostomata, E. pacifica, E. phasiani, E. paisana, etc., painted infecting species, such as E. gorakhpur ⁇ , E. grenieri, E. numidae, etc., species that infect ducks, such as E. anatis, E. aythyae, E.battakhi, E.danailovi, E.
  • kr ⁇ lovi, etc. species that infect geese, such as E. stigmosa, E. truncata, etc., species that infect cows, such as E. bovis, E. zuernii, E. ellipsoidalis, E. cylindr ⁇ ca, E. auburnensis, E. alabamensis, E. subspher ⁇ ca, E. wyomingensis, etc., species that infect goats, such as E. ar ⁇ oingi, E. chr ⁇ stenseni, E. ninakohlyakimovae, E. alijevi, E. hirci, E. caprina, E. caprovina, E.
  • E. jolchijevi E. apsheronica, etc.
  • species that infect sheep such as E. a hsata, E. bakuensis, E.crandallis, E. faurei, E. intricata, E. marsica, E. ovinoidalis, E. pallida, E. parva, E. weybridgensis, E. gilruthi, E. gonzalezi, E. granulosa, E. punctata, etc.
  • species that infect pigs such as E. scabra, E.porci, E. polita, E.suis, etc., species that infect rabbits, such as E.
  • Eimeria tenella refers to the stenoxen parasite of the Eimeria species that causes intestinal bleeding and primarily affects the blind of chickens and chickens.
  • Body refers to the vertebrate animal having the modified forelimbs as wings, with the body covered with feathers and a corneal beak without teeth.
  • poultry includes domestic birds raised by man to obtain eggs, meat and / or feathers.
  • poultry include roosters, chickens, turkeys, ducks, geese, quails, pheasants, pigeons, pigeons, emus, ostriches and rheas.
  • cken refers to a brood, especially chicken.
  • transgenic corn refers to that corn plant whose genome has been modified by genetic engineering in order to achieve different and / or improved biological characteristics with respect to those of the wild isogenic plant.
  • Transgene of the carotenoid biosynthesis pathway refers to a gene that encodes an enzyme that participates in the carotenoid synthesis pathway.
  • carotene biosynthesis occurs in the membranes of chromoplasts.
  • Carotenoids refers to organic pigments found naturally in plants and other photosynthetic organisms. Most carotenoids are tetraterpenoids, composed of 40 carbon atoms formed by eight isoprenoid units and contain rings at one or both ends of the molecule. They can function as antioxidants, protecting against self-oxidation.
  • genes that participate in carotenoid biosynthesis are not limited to phytoeno synthase, carotene desaturase, lycopene ⁇ -cyclase, ⁇ -carotene hydroxylase and carotenoid ketolase.
  • phytoeno synthase or psyl as used in the present invention it refers to the gene that codes for the enzyme that catalyzes the reaction that converts two GGDP molecules into phytoeno, the first step in the synthesis of provitamin A.
  • Carotene desaturase or crt ⁇ , as used in the present invention, refers to the gene that codes for the enzyme responsible for the production of lycopene from three substrates, neurosporene, an electron acceptor and oxygen.
  • Lycb refers to the gene that codes for the enzyme that transforms lycopene into provitamin A.
  • 3-carotene hydroxylase or bch, as used in the present invention, refers to the gene that codes for the enzyme responsible for the conversion of beta-carotene to xanthophylls.
  • “Carotenoid ketolase” or crtw refers to the gene that codes for the enzyme responsible for adding a carbonyl group with carbon 4 and 4 'of the beta carotene molecule.
  • Vitamin C biosynthesis transgene refers to a gene that encodes an enzyme that participates in the biosynthesis of vitamin C.
  • An example is dehydroascorbate reductase.
  • Vitamin C refers to the compound with CAS number 50-81-7, is an antioxidant and cofactor in various enzymatic reactions.
  • “Dhar dehydroascorbate reductase”, as used in the present invention, refers to the gene encoding the enzyme that catalyzes the reduction of dehydroascorbate to ascorbate in a glutathione-dependent reaction.
  • Falate biosynthesis transgene refers to a gene encoding an enzyme that participates in the folate biosynthesis pathway, for example GTP cyclohydrolase.
  • “Folate”, as used in the present invention, refers to a set of substances derived from folic acid itself, (pteroyl-L-glutamic acid), which can be easily transformed into it.
  • GTP cyclohydrolase or GCH1 refers to the enzyme encoded by the folE gene and which catalyzes the hydrolysis reaction of GTP.
  • Specific endosperm promoter refers to a nucleic acid molecule that allows to direct or regulate the expression of a gene in the endosperm.
  • the "endosperm” is a characteristic formation of Angiosperm seeds, it is a nutritious tissue for the embryo.
  • the corn endosperm originates from a series of free nuclear divisions, followed by cellization and subsequent formation of a range of functional cell domains.
  • the endosperm is the main storage organ of corn seeds; It nourishes the embryo while the seed develops and provides nutrients to the germinating seedling.
  • Barley D-hordein refers to the protein with accession number Q40054 from the Uniprot database as of March 17, 2014.
  • the promoter of D-hordein has the sequence shown in SEQ ID NO: 10
  • LMW wheat glutenin refers to protein with accession number Q41603 from the Uniprot database as of March 17, 2014.
  • the LMW promoter of glutenin has the sequence shown in SEQ ID NO: 11
  • Seed refers to each of the bodies that are part of the fruit that gives rise to a new plant.
  • endosperm refers to the main organ of storage in corn seeds, nourishing the embryo while the seed develops and providing nutrients to the young germinating plant.
  • seed derivative or endosperm refers to any product obtained from the physical and / or chemical modification of the seed or endosperm.
  • frlour as understood in the present invention, is the product obtained from the milling of any seed of plants of the Zea genus that may be stripped to a greater or lesser extent of the bran or seed husk.
  • seed refers to coarse flour (poorly ground corn seeds), that is, endosperm fragments with a variable amount of seed husk.
  • Corn oil refers to the oil obtained after drying the germinated corn grain.
  • corn starch refers to a finally ground white silky powder made from the endosperm of dried corn.
  • Corn starch is the polysaccharide obtained through wet milling.
  • Corn syrup also known as corn syrup, is a liquid sweetener made from starch or cornstarch after a heating and hydrolyzing process.
  • the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof, for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of coccidiosis in a subject.
  • the invention relates to a part of an antioxidant-enriched corn plant or a derivative thereof for use in the prevention or treatment of coccidiosis in a subject.
  • the invention relates to a part of a corn plant enriched in antioxidants or a derivative thereof for use in medicine.
  • the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of pododermatitis or ulcers in a subject.
  • the invention relates to a part of a corn plant enriched in antioxidants or a derivative thereof for use in the prevention or treatment of pododermatitis or ulcers in a subject.
  • the ulcers are found in the legs or feet.
  • a part of a corn plant or a derivative thereof is enriched in antioxidants, when it has antioxidant levels higher than the levels of a reference sample or control sample.
  • a part of a plant or derivative of it presents higher levels of antioxidants when the levels are at least 1 time, 1, 5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more with respect to the reference sample or control sample.
  • the person skilled in the art will understand that it is not necessary for an antioxidant-enriched plant to contain high levels of said antioxidants in all tissues of said plant, but it is sufficient that at least one plant tissue contains high levels of antioxidants so that The plant can be considered as "enriched in antioxidants".
  • the reference sample to determine the degree of antioxidant enrichment is determined by comparison with antioxidant levels is a sample of the same tissue or part of a control plant.
  • a control or reference sample, as used in the present invention may be the value that corresponds to the levels of antioxidants present in a part of a corn plant or derived therefrom that is in the wild, it is say not genetically modified.
  • the antioxidant capacity can be determined by quantifying the ability of antioxidant compounds to react with a given free radical, or by determining the potential such compounds would have to reduce the complex formed between Fe ions (lll) and the TPTZ reagent (2, 4,6-tripyridyl-s-triazine).
  • the corn plant is white corn or yellow corn.
  • White corn refers to a variety of corn in which the endosperm has a white color, as a result of the absence of carotenoid pigments that confer yellow color to the endosperm of corn known as yellow corn.
  • the antioxidants are carotenoids.
  • the carotenoids are selected from the group consisting of ⁇ -carotene, ⁇ -carotene, ⁇ -carotene, a-cryptoxanthin, ⁇ -cryptoxanthin, lycopene, lutein and zeaxanthin.
  • the part of the antioxidant-enriched corn plant is the endosperm or the seed or derivatives thereof.
  • the derivative of the seed or endosperm can be a flour, semolina, corn oil, corn starch, cornstarch or corn syrup among others.
  • treatment refers to any type of therapy, which aims at termination, prevention, improvement or reduction of the susceptibility to suffer from a clinical condition, as described herein.
  • the term “treatment” refers to prophylactic treatment (ie, a therapy to reduce the susceptibility to suffer a clinical condition) of a disorder or condition.
  • prophylactic treatment ie, a therapy to reduce the susceptibility to suffer a clinical condition
  • treatment refers to obtaining a desired pharmacologically or physiologically desired effect, which covers any treatment of a pathological condition or disorder in a mammal, including humans.
  • the effect can be prophylactic in terms of providing total or partial prevention of a disorder and / or adverse effect attributable to it.
  • treatment includes (1) preventing the disease from occurring or recurring in a subject, (2) inhibiting the disease, for example by stopping its development, (3) interrupting or ending the disorder or at least the associated symptoms at the same time, so that the patient would no longer suffer the disease or its symptoms, for example cause the regression of the disease or its symptoms by restoring or repairing a lost, absent or defective function, or stimulating an inefficient process, or ( 4), reduce, relieve or improve the disease, or the symptoms associated with it, where reducing is used in a broad sense to refer to at least one reduction in the magnitude of a parameter, such as inflammation, pain, or immune deficiency
  • coccidiosis is caused by Eimera.
  • said Eimeria species are selected from E. acervulina, E. brunetti, E. hagai, E. maxim, E. mitis, E. mivati, E. necatrix, E. praecox, E.tenella, E. adenoeides, E. gallopavonis, E. innocua, E. meleagridis, E. meleagrimitis, E. subrotunda, E. scabra, E.porci, E. polita, E.suis, E. bovis, E. zuernii, E. ellipsoidalis, E. cylindrica, E. auburnensis, E. alabamensis, E.
  • Eimeria includes any strain of any species of Eimeria, including attenuated and virulent strains.
  • the species of Eimeria is E. tenella.
  • Techniques that allow the diagnosis of a subject suffering from coccidiosis.
  • Techniques include the detection of oocysts in feces by a conventional flotation technique with subsequent counting, or the detection of oocysts in the intestine, such as the quantitative McMaster technique. Additionally, it is possible to observe the parasite in serial scrapes of the intestinal mucosa (MRSMI) in a simple way using current staining methods.
  • MRSMI serial scrapes of the intestinal mucosa
  • pododermatitis in a subject In relation to the diagnosis of pododermatitis in a subject, whether associated or not with coccidiosis, it is usually done by visual and radiological inspection, to assess the state of soft tissues and bone. Thermography allows detecting pododermatitis before the appearance of lesions, due to the greater difference between the maximum and minimum temperatures of the plantar pad and the thermal pattern. There are several criteria to determine the severity of the lesions, including the 3-point scale or the 1-1-point scale. Other methods are based on the severity or area of the absolute or relative injury. In other cases, the severity and the surface are combined.
  • the severity of pododermatitis can be classified into three types.
  • Type I It is the beginning of an injury in the palm of the claw. It is usually located on a single finger. It is inflammatory and although very weakened, there is still a cover epithelium. The lesion may be proliferative or degenerative with a tendency to ulceration. Within this type there are no clinical signs of pain or discomfort at first. It is usually diagnosed by routine inspection. There is no apparent clinical infection Type II It is one more degree of the previous type. Signs of infection begin to appear. In cultures, Staphilococcus aureus, Proteus sp and E. Coli are normally isolated. These laboratory findings confirm the epithelial and fecal origin of these infections. A marked pain begins to appear, with withdrawal of the support in the affected leg and load in the other, (situation that eventually deteriorates both limbs). Suppurating abscesses and marked fibrous reaction of the tissues appear.
  • Type III It is the most advanced degree of pododermatitis. Deformations and an abundant fibrous reaction are distinguished. Osteomyelitis with severe joint involvement is very common. In these cases, medical treatments are usually attempted, but they inevitably end with the death of the animal, either due to progressive deterioration or euthanasia. Serious complications: osteomyelitis, valvular endocarditis.
  • the part of an antioxidant-enriched corn plant or a derivative thereof or the compositions of the invention are used according to the present invention for the treatment of type I pododermatitis, of type II or type III.
  • the subject to be treated is a bird, more particularly a poultry and more particularly a chicken.
  • the antioxidant-enriched corn plant is a transgenic corn that expresses at least one gene from the carotenoid biosynthesis pathway.
  • the gene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene ⁇ -cyclase, a gene that encodes a ⁇ - carotene hydroxylase, and a gene that encodes a carotenoid ketolase.
  • the antioxidant-enriched corn plant is a transgenic corn that expresses at least 2 genes from the carotenoid biosynthesis pathway, more particularly at least one gene encoding a phytoen synthase and a gene that encodes a carotene desaturase; a gene that encodes a phytoen synthase and a gene that encodes a lycopene ⁇ -cyclase; a gene that encodes a phytoeno synthase and a gene that encodes a ⁇ -carotene hydroxylase; a gene that encodes a phytoeno synthase and a gene that encodes a carotenoid ketolase; a gene that encodes a carotene desaturase and a gene that encodes a lycopene ⁇ -cyclase; a gene that encodes a carotene desaturase and a gene that encodes a ⁇ -carotene hydroxylase, a gene that encodes a caro
  • the antioxidant-enriched corn plant is a transgenic corn that expresses at least 3 genes of the carotenoid biosynthesis pathway, more particularly at least one gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase and a gene encoding a lycopene ⁇ -cyclase; a gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase and a gene that encodes a ⁇ -carotene hydroxylase; a gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase and a gene that encodes a carotenoid ketolase; a gene that encodes a phytoeno synthase, a gene that encodes a lycopene ⁇ -cyclase and a gene that encodes a ⁇ -carotene hydroxylase; a gene that encodes a gene that encode
  • the antioxidant-enriched corn plant is a transgenic corn that expresses at least 4 genes of the carotenoid biosynthesis pathway, more particularly at least one gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase, a gene encoding a lycopene ⁇ -cyclase and a gene encoding a ⁇ -carotene hydroxylase; a gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene ⁇ -cyclase and a gene that encodes a carotenoid ketolase; a gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a ⁇ -carotene hydroxylase and a gene that encodes a carotenoid ketolase; a gene that encodes a phytoeno synth
  • the antioxidant-enriched corn plant is a transgenic corn that expresses at least 5 genes of the carotenoid biosynthesis pathway, more particularly a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene which encodes a lycopene ⁇ -cyclase, a gene that encodes a ⁇ -carotene hydroxylase and a gene that encodes a carotenoid ketolase.
  • the gene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1, of Zea mays.
  • the gene encoding a carotene desaturase has the sequence shown in SEQ ID NO: 2 of Pantoea ananatis, formally Erwinia uredovora.
  • the gene encoding a lycopene ⁇ -cyclase has the sequence shown in SEQ ID NO: 3 of Gentiana luteal
  • the gene encoding a ⁇ -carotene hydroxylase has the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12 of Zea mays or the gene with sequence shown in SEQ ID NO: 5 of Gentiana lutea.
  • the gene encoding a carotenoid ketolase has the sequence shown in SEQ ID NO: 6 of Paracoccus sp N81106.
  • the antioxidant-enriched corn plant can express other additional transgenes such as a transgene the vitamin C biosynthesis pathway, in particular a gene encoding dehydroascorbate reductase, more particularly the gene with the sequence shown in SEQ ID NO: 7 of E. coli. Additionally, the antioxidant-enriched corn plant can express other additional transgenes such as a transgene folate biosynthesis pathway, more particularly the gene with sequence shown in SEQ ID NO: 8 of Orazy sativa.
  • a transgene the vitamin C biosynthesis pathway in particular a gene encoding dehydroascorbate reductase, more particularly the gene with the sequence shown in SEQ ID NO: 7 of E. coli.
  • the antioxidant-enriched corn plant can express other additional transgenes such as a transgene folate biosynthesis pathway, more particularly the gene with sequence shown in SEQ ID NO: 8 of Orazy sativa.
  • such methods may include: 1) use of a live vector that carries the genetic material to the white cell by means of genetically modified viruses or the natural mechanism of infection of the soil bacterium Agrobacterium tumefaciens 2) The use of protoplasts, which are plant cells to which they have been freed from the cell wall, or 3) the biolistics that consists of bombarding the cells with microscopic metal particles coated with the DNA that they wish to introduce.
  • transgenic corn plants the introduction of DNA into corn cells has been proposed including electroporation, microinjection, microprojectile bombardment, liposome fusion, Agrobacterium-mediated transfer, macroinjection and exposure to naked DNA in solution. .
  • the gene contained in the recombinant DNA molecule is expressed at the appropriate levels and tissues, it is desirable, but not necessary for genes of plant origin, that it is contained in an expression cassette that includes a promoter sequence of the functional transcription in plants, the coding part of the gene of interest and a terminator of functional transcription in plants. Additionally, a sequence encoding a transit peptide that directs the enzyme to a specific cell compartment can be included.
  • the transit peptide and the corresponding processing signals can be derived from any plant protein that is synthesized in the cytoplasm and is translocated to the subcellular compartment of interest, be it the plastid or the mitochondria.
  • sequences derived from genes encoding the small subunit of the ribulose bisphosphate carboxylase or CAB proteins which are CAB proteins can be used to locate plastids those that bind with chlorophylls a and b to formalize light harvesting antennas (Van den Broek et al Nature (London) 313, 358-363 (1985)).
  • a promoter In the case of genes that are not of plant origin, a promoter must be included in the transcription initiation sequence and an optimal translation start signal for plants or one of the so-called translation enhancers can also be used.
  • transgenes that can be used in the present invention can be placed under the control of regulatory elements that ensure expression in plant cells.
  • regulatory elements comprise an active promoter in plant cells.
  • promoters that can be used include promoters of the plant to be transformed, promoters of other plants or of any origin that are functional in the white plant and direct a specific constitutive, inducible or tissue expression.
  • promoters derived from the Ti plasmid of Agrobactertum tumefaciens can be used such as the promoters of octopine synthetase, nopahna synthetase or sgropin synthetase.
  • promoters such as the 35S promoter of cauliflower mosaic virus or those derived from geminivirus may be included.
  • promoters see Peremarti A. et al, Plant Mol Biol 73: 363-378, 2010.
  • the transcription terminator sequencer can be derived from the same gene from which the transcription promoter sequence was obtained or from a different gene.
  • the terminator sequence may be derived from genes contained in the T-DNA of the Ti plasmid from Agrobacterium, from the cauliflower mosaic virus or from genes of plant origin.
  • the terminator sequence of the nopaline synthetase gene or the terminator sequence of the ADPGP gene is frequently used in Plant Genetic Engineering.
  • the nopaline synthase gene transcription terminator sequence has the sequence shown in SEQ ID NO: 14, and the ADPGPP gene terminator sequence has the sequence shown in SEQ ID NO: 15.
  • the different nucleic acid sequences that are part of the recombinant DNA molecule can be linked by conventional methods described in the literature.
  • the sequences must be cloned and linked in the correct orientation and order to achieve functional expression in plant cells.
  • the plants of interest are transformed with a recombinant DNA molecule that encodes at least one enzyme that participates in the biosynthesis of carotenoids.
  • the different nucleotide sequences that compose it and that comprise the regulatory sequences of the transcription and the coding sequence of interest may have been subjected to different types of processing, such as ligaments, digestion with restriction enzymes , in vitro mutagenesis, oligonucleotide addition or modifications by means of the chain polymerization reaction (PCR). Therefore, the components of the recombinant molecule before being bound could be subject to deletions, insertions or internal modifications. Since the recombinant molecule is derived from components that originate from different organisms and that have been isolated, purified or synthesized, it is not a molecule that exists as such in nature.
  • the inclusion of other DNA sequences may be necessary.
  • a molecular cloning vector that allows replication in E. coli and, for some cases, that allows replication in A. tumefaciens.
  • the Agrobacterium Ti plasmid-mediated genetic transformation system to introduce the recombinant molecule into the genome of the plant cell, it is necessary to include the T-DNA border sequences, so that they are present at both ends of the gene which encodes the enzyme that synthesizes organic acids and in some cases the dominant selection gene.
  • the use of unarmed Agrobacterium strains that is, those whose genes responsible for tumor formation have been removed, but maintain the ability to transfer DNA to plant cells, allows the regeneration of transgenic plants that contain the recombinant molecule of interest.
  • a dominant selection gene is also generally included to identify and select the cells that stably incorporated the recombinant molecule into their genome.
  • the marker genes can be the rrn16 mutation (16S rRNA) that confers resistance to spectinomycin and streptinomycin, the rps12 mutation (ribosome) that confers streptinomycin resistance, aadA gene (E.
  • coli that encodes the 3 'adenyltransferase aminoglycoside enzyme that confers resistance to the antibiotics spectinomycin and streptinomycin
  • nptll gene from the bacterial transposon Tn5 encodes the enzyme neomycin phosphotransferase that confers resistance to the antibiotic kanamycin
  • the aphA-6 gene of Acinetobacter baumannii that encodes the enzyme aminoglycoside phosphotransferase which confers resistance to the antibiotic kanamycin
  • coli cat gene confers resistance to chloramphenicol
  • ASA gene responsible of resistance to tryptophan analogs
  • Streptomyces hygroscopicus bar gene that encodes the phosphinothricin acetyl transferase enzyme (PAT) that confers phosphinothricin resistance
  • PAT phosphinothricin acetyl transferase enzyme
  • Streptomyces viridochromogenes pat gene converts ammonium glufosinate into nacetyl glufosinate which is a non-compound phytotoxic for the plant.
  • the selection gene is the bar gene.
  • the gene has the sequence shown in SEQ ID NO: 9
  • the plant selection marker gene is eliminated.
  • Temporary, inducible or specific tissue expression can be achieved through the use of promoters or regulatory sequences that have the desired expression specificity.
  • tissue specific promoters see Stockhaus, EMBO J. 8 (1989), 2245-2251). Promoters that are specifically active in seeds of different plant species, such as corn, are also known.
  • transgene or transgenes is under the control of a specific endosperm promoter.
  • Specific endosperm promoters useful in the present invention are, among others, gene promoters that code for seed storage proteins, Galili et al., 1993, Trends Cell Biol 3: 437-442.
  • Prolamines are the most abundant cereal storage proteins, in corn the zeins ( ⁇ , ⁇ , ⁇ and ⁇ -zein zeins, in gluten glutenins, in hordein barley, rye secalin and oats acenin.
  • Illustrative, non-limiting examples of specific endosperm promoters that may be employed in the present invention are among others the LMW wheat gluten, barley hordein, rice prolamine, rice glutelin 1 promoters, corn zein, zeina 27 Kda, rice ADP-glucose pyrophosphorylase, rice glutelin I as described in Russel DA. et al., Transgenic research 6, 157-168 (1997). Also useful are the promoters of MEG-1 as described in EP1680506B1.
  • the promoter is selected from the barley D-hordein promoter with sequence shown in SEQ ID NO: 10 or from the LMW wheat glutenin promoter with sequence shown in SEQ ID NO: 1 1.
  • the phytoeno synthase gene is under the control of the LMW glutenin promoter, preferably of the LMW wheat glutenin promoter.
  • the carotene desaturase gene is under the control of the D-hordein promoter, preferably of the barley D-hordein promoter.
  • the regulatory elements may further comprise functional transcription and / or translation enhancers in plant cells. Additionally, the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly-A tail to the transcript that can improve its stability.
  • transcription termination signals such as a poly-A signal
  • the enzyme encoded by said transgene forms a fusion protein comprising a sequence for the targeting of proteins to the N-terminal position chloroplast. regarding the sequence of the transgene.
  • the sequence for the targeting of proteins to the chloroplast is the small subunit sequence of ribulose 1, 5 carboxylase / oxygenase diphosphate has the sequence shown in SEQ ID NO: 13.
  • compositions of the invention and uses thereof
  • the invention in another aspect, relates to a composition
  • a composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof and an anticoccidic agent.
  • the antioxidants are carotenoids.
  • the carotenoids are selected from the group. formed by ⁇ -carotene, ⁇ -carotene, ⁇ -carotene, ⁇ -cryptoxanthin, ⁇ -cryptoxanthin, lycopene, lutein and zeaxanthin.
  • the antioxidant enriched corn is a transgenic corn that expresses at least one transgene of the carotenoid biosynthesis pathway.
  • the transgene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene ⁇ -cyclase, a gene that encodes a ⁇ -carotene hydroxylase, and a gene that encodes a carotenoid ketolase or a combination thereof.
  • the transgene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1
  • the transgene encoding carotene desaturase has the sequence shown in SEQ ID NO: 2
  • the transgene encoding for Lycopene ⁇ -cyclase has the sequence shown in SEQ ID NO: 3
  • the transgene encoding ⁇ -carotene hydroxylase has the sequence shown in SEQ ID NO: 4
  • the transgene coding for the carotenoid ketolase has the sequence shown in SEQ ID NO: 6.
  • the corn plant contains the transgene encoding phytoen synthase and the gene encoding carotene desaturase.
  • Other combinations, such as those mentioned in the context of the therapeutic use of the invention are equally applicable.
  • the transgene or transgenes is or is under the control of a specific endosperm promoter.
  • the specific endosperm promoter is the D-hordein promoter or the LMW glutenin promoter.
  • the D-hordein promoter has the sequence shown in SEQ ID NO: 10 and the LMW glutenin promoter has the sequence shown in SEQ ID NO: 1 1.
  • the corn plant contains the transgene that encodes the phytoen synthase under the control of the LMW glutenin promoter and the gene that encodes the carotene desaturase under the control of the barley D-hordein.
  • the part of the antioxidant-enriched corn plant is selected from the endosperm and seed group.
  • the anticoccidic agent can be selected from coccytostatics and coccidiocides. With coccidiostats it is observed that intracellular growth of coccidia stops, but their development can continue after the withdrawal of the drug; if Coccidiocides coccidia die during their development when exposed to the drug. Some anticoccidic drugs may be coccidiostatic when administered in the short term, but coccidicidal when administered in the long term. Another classification of anticoccidics is by their origin: ionophores, which are fermentation products, and chemical-synthetics. Among the ionophores are monensin, salinomycin, lasalocide, narasin, maturemicin and semduramycin.
  • Monensin, narasin and salinomycin are more effective against E.acervulina, but ineffective in the control of E. tenella and E. maxim.
  • Lasolacida and Maduramycin are effective against E. tenella, but low effectiveness against E. acervulina.
  • the dose of the ionophores is within 25-100 ppm, with the exception of mature microwaves that are dosed at 5 ppm.
  • the chemical-synthetic compounds are amprolium, clopidol and quinolines (for example decoquinate, methylbenzocuate), folic acid antagonists, including sulfonamides, 2,4-diaminopyrimidines and ethopabate, halofuginone hydrobromide, nicarbazine, nitrobenzamides (for example dinitolmide), robenidine, roxarsona, diclazuril and toltrazuril.
  • amprolium for example decoquinate, methylbenzocuate
  • folic acid antagonists including sulfonamides, 2,4-diaminopyrimidines and ethopabate, halofuginone hydrobromide, nicarbazine, nitrobenzamides (for example dinitolmide), robenidine, roxarsona, diclazuril and toltrazuril.
  • anticoccidic agents also include oral vaccines.
  • the anticoccidic vaccine may be not attenuated (virulent) or attenuated.
  • Non-attenuated vaccines contain oocysts, live virulent coccidia, that is, chickens are fully exposed to the pathogenic parasites of the vaccine, in addition to the virulent spread of coccidia in the poultry yard. Therefore, when using these vaccines, there is a risk of strong reaction after vaccination and clinical coccidiosis outbreak especially in birds with a weakened immune system. Therefore, after the administration of virulent vaccines, preventive treatment with anticoccidials is often necessary and recommended to be applied even by the manufacturers of these vaccines.
  • the attenuated vaccines that is, the second generation vaccines, contain live oocysts of attenuated lines of Eimerian species. These vaccines are much safer due to the biologically attenuated pathogenicity of vaccine parasites, and the challenge of controlled infection that therefore presents no risk of clinical coccidiosis outbreak. In the event that attenuated vaccines are used, it is important that coccidiostats are not used to avoid reducing the effectiveness of the vaccine.
  • An example of an oral anticoccidic vaccine is IMMUCOXOT based on live Eimeria spp oocysts, or PARACOXTM containing live sporulated oocysts derived from eight early coccidial lines or HIPRACOX® based on live sporulated oocysts derived from five attenuated lines.
  • the invention relates to the use of a composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof and an anticoccidic agent for the manufacture of a medicament, nutritive or nutraceutical composition for prevention or treatment of pododermatitis or ulcers associated with coccidiosis in a subject.
  • the invention relates to a composition
  • a composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof and an anticoccidic agent for use in the prevention or treatment of pododermatitis or ulcers associated with coccidiosis in a subject .
  • the anticoccidic agent is administered at a subtherapeutic dose.
  • Subtherapeutic doses refers to the dose below the amount required for a therapeutic effect. Particularly, the subtherapeutic dose is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, or at least 90% lower or more than the minimum dose required for a therapeutic effect.
  • the minimum dose of several anticoccidic drugs are shown in Table 1.
  • the invention in another aspect relates to a nutritive or nutraceutical composition
  • a nutritive or nutraceutical composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof.
  • nutritive or nutraceutical composition is used herein in a broad sense and covers food for humans as well as food for production animals (ie, a feed).
  • the nutritive or nutraceutical composition is for animal consumption.
  • the nutritive or nutraceutical composition may be in the form of a solution or as a solid, depending on the use and / or the mode of application and / or the mode of administration.
  • the antioxidants are carotenoids.
  • the carotenoids are selected from the group consisting of ⁇ -carotene, ⁇ -carotene, ⁇ -carotene, a-cryptoxanthin, ⁇ -cryptoxanthin, lycopene, lutein and zeaxanthin.
  • the antioxidant enriched corn is a transgenic corn that expresses at least one transgene of the carotenoid biosynthesis pathway.
  • the transgene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene ⁇ -cyclase, a gene that encodes a ⁇ -carotene hydroxylase, and a gene that encodes a carotenoid ketolase or a combination thereof.
  • the transgene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1
  • the transgene encoding carotene desaturase has the sequence shown in SEQ ID NO: 2
  • the transgene encoding lycopene ⁇ -cyclase has the sequence shown in SEQ ID NO: 3
  • the transgene encoding ⁇ -carotene hydroxylase has the sequence shown in SEQ ID NO: 4
  • the transgene encoding for Carotenoid ketolase has the sequence shown in SEQ ID NO: 6.
  • the corn plant contains the transgene encoding phytoen synthase and the gene encoding carotene desaturase.
  • Other combinations, such as those mentioned in the context of the therapeutic use of the invention are equally applicable.
  • the transgene or transgenes is or is under the control of a specific endosperm promoter.
  • the specific endosperm promoter is the D-hordein promoter or the LMW glutenin promoter.
  • the D-hordein promoter has the sequence shown in SEQ ID NO: 10 and the LMW glutenin promoter has the sequence shown in SEQ ID NO: 1 1.
  • the corn plant contains the transgene that encodes the phytoen synthase under the control of the LMW glutenin promoter and the gene that encodes the carotene desaturase under the control of the barley D-hordein.
  • the part of the antioxidant-enriched corn plant is selected from the endosperm and seed group.
  • compositions of the invention or nutraceuticals may be in a liquid, semi-solid or solid form.
  • compositions or nutraceuticals of the invention are in solid form and more particularly it is a feed.
  • the nutritive or nutraceutical compositions of the invention can be formulated with the usual excipients and adjuvants for oral compositions or food supplements, such as and without limitation, fatty components, aqueous components, humectants, preservatives, texturizing agents, flavors, aromas, antioxidants and dyes common in the food sector.
  • excipients and adjuvants for oral compositions or food supplements such as and without limitation, fatty components, aqueous components, humectants, preservatives, texturizing agents, flavors, aromas, antioxidants and dyes common in the food sector.
  • the nutritive or nutraceutical compositions of the invention comprise at least 0.01% of the part of a part of a plant enriched in antioxidants or derived therefrom, more preferably at least 0.10%, 0.50%, 1 %, 2. 3. 4. 5%, 6%, 7%, 8%, 9%, 10%, 15% 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95 %.
  • Nutritive compositions useful for feeding poultry, and in particular fattening for meat chickens or broilers, chick rearing (weeks 0 to 8), breeding for chicks (week 9 -20) or starting from week 21 a are known in the state of the art, for example in ES2051706T3.
  • compositions or nutraceuticals of the invention are useful for the treatment and prevention of coccidiosis, pododermatitis and ulcers. Therefore the part of a corn plant enriched in antioxidants or a derivative thereof must be in a therapeutically effective amount.
  • the nutritive or nutraceutical composition of the invention comprises corn grains in at least 20%, preferably in at least 30%, more preferably at least 40%, preferably more than 50%, more preferably more of 60%.
  • the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for obtaining a nutritive or nutraceutical composition.
  • the nutritive or nutraceutical composition according to the present invention can be prepared directly from the part of the plant enriched in carotenoids.
  • the part of the plant is the endosperm or corn kernels, in which case said composition contains at least 40%, preferably more than 50% or more preferably more than 60%.
  • the part of the plant that is used to prepare the nutritive or nutraceutical composition according to the present invention can be subjected to a partial purification to obtain a derivative enriched in the antioxidant product.
  • the antioxidant compounds are carotenoids and that the part of the plant that is used is the endosperm
  • the nutritional composition or the nutraceutical they contain an extract of corn kernels or endosperm that can be found partially- or fully purified so that the carotenoid content is higher than what appears in the part of the enriched corn plant.
  • Corn derivatives that can be used in the present invention include corn flour, corn bran, corn oil, corn starch, corn syrup, and the like.
  • Carotenoids have been shown to potentiate both specific and nonspecific immunological functions.
  • Various immunological modulation reactions can increase the tumoricidal activity of cytotoxic T cells, macrophages and / or natural killer cells and promote immunological antimicrobial functions.
  • beta-carotene can reduce the incidence of cardiovascular events, having been demonstrated in clinical studies (Gazianio JM. Et al., Circulation 82: 201-204, 1990). Additionally, population groups that consume foods rich in beta-carotene are more protected against skin and lung cancers, particularly smokers.
  • Several mechanisms have been proposed to explain the physiological activity of carotenoids. The most plausible is based on the strong antioxidant character and its ability to deactivate harmful chemicals such as singlet oxygen, free radicals and lipid peroxide radicals. There is also evidence that several carotenoids produce a direct stimulation of the immune system and the immune response.
  • the nutritive or nutraceutical composition of the invention must comply with the country's food regulations and legislation.
  • the color of the meat and skin of the breast, thigh and leg in the chicken carcasses was measured by a compact portable CM-700d spectrophotometer, Konica Minolta (Japan). The color of each sample was measured by the trichromatic CIELAB system using the luminosity (L), red (a) and yellow (b) values. The classic fan of the Roche scale has also been used for the color of the leg.
  • lyophilized samples were extracted in 20 ml of 50/50 (v / v) tetrahydrofuran and methanol at 60 ° C for 15-20 min and quantified by measuring the absorbance at 450 nm.
  • the solvent was evaporated under a stream of nitrogen gas at 37 ° C, re-dissolved in 50 L of acetone, and an aliquot of 20 was immediately injected.
  • the samples were separated on a 3- ⁇ Nucleosil C18 column with acetonitrile / methanol / 2-propanol (85: 15: 5, v / v / v) as a mobile phase at 25 0 C.
  • the color of the leg of the leg was also determined as described above.
  • the oocyst counts were determined in stool samples collected daily from each group from day 6 to day 9 after infection. Stool collection for oocyst analysis was done twice a day. A saturated solution of NaCI and McMaster chambers was used for oocyst counting and the results are presented as the number of oocysts per gram of fresh feces. See Table 2 and Figure 3.
  • the Fabrizio bag, liver and blind were collected and fixed in 4% buffered formaldehyde and processed in paraffin for histopathological study.
  • antioxidant-rich corn reduces the excretion of coccidia oocysts in infected animals, reduces pododermatitis and digital ulcers in the legs of chickens.
  • Reduction of pododermatitis and leg ulcers can occur not only in animals affected by coccidiosis, but also among diseases or conditions that can cause diarrhea in domestic birds such as cold stress or heat, bacteria or viruses (colibacillosis, lymphoid leukosis, Marek's disease), parasites (Ascardiasis or Capilariasis) etc.
  • the reduction of pododermatitis, digital ulcers and excretion of coccidia oocysts can also be expected in other animal species and possibly in humans.
  • the histopathology of the Fabrizio bag indicates that animals infected with Eimeria and fed the corn diet rich in antioxidants had better protection and immunity than animals fed with the control diet, which may indicate that the same may occur with others diseases.
  • T1 non-infected control diet
  • T2 control diet challenged with E. tenella
  • T3 non-carotenoid-rich corn diet
  • T4 diet of corn rich in carotenoids challenged with E. tenella.
  • T2 control diet challenged with E. tenella
  • T4 control diet challenged with E. tenella
  • the decrease in oocysts on days 8 and 9 after infection may be related to the parasite cycle.
  • the cycle of these protozoa begins with the asexual phase (schizogonia) followed by the sexual phase (gametogonia) that ends with the expulsion of oocysts through feces.
  • the oocysts sporulate (sporogonia) in a few days outside between the feces and the bed depending on the temperature and humidity. Ingestion of a large number of new sporulated oocysts begins a massive new cycle and a new wave of oocysts that are excreted in the feces again.
  • the control diet group may have started a new wave of oocyst excretion on day 9 post infection, but the antioxidant-rich diet group may have improved their immunity and this new wave may have been delayed or reduced.
  • the specific immunity to coccidia is similar in both treatments, but in the second cycle the group with corn-based diet rich in antioxidants may have improved humoral immunity.
  • the retinol levels in the serum of animals fed the carotenoid-rich diet were almost double that of the control diet, confirming the effective conversion of the carotenoids of provitamin A into vitamin A. See Table 4.
  • T1 non-infected control diet
  • T2 control diet challenged with E. tenella
  • T3 carotenoid-rich corn diet uninfected
  • T4 carotenoid-rich corn diet challenged with E. tenella.
  • a, b, c The values in the same column with one letter in common do not differ significantly. The significance of the data is as follows: *: 0.05> P> 0.01.
  • Sequence listing in the sequence list refers to "Sequence list", “Artificial Sequence” to “Artificial sequence” and the term “DNA” refers to “DNA” and “mise feature” refers to characteristic miscellany.

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Abstract

The invention relates to the use of a corn plant enriched in anti-oxidants or a derivative of same, to produce a medication, nutritional composition or nutraceutical for the prevention or treatment of coccidiosis, pododermatitis or ulcers in a subject. The invention also relates to a composition comprising a part of a corn plant enriched in anti-oxidants or a derivative of same and an anticoccidial agent, and to the use of said composition for the prevention or treatment of pododermatitis or ulcers associated with coccidiosis in a subject. The invention further relates to the use of a part of a corn plant enriched in anti-oxidants or a derivative of same to produce a nutritional composition or nutraceutical, and to a nutritional composition or nutraceutical comprising a part of a corn plant enriched in anti-oxidants or a derivative of same.

Description

MÉTODO DE TRATAMIENTO DE COCCIDIOSIS DESCRIPCIÓN  COCCIDIOSIS TREATMENT METHOD DESCRIPTION
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se encuadra dentro de los métodos de tratamiento o prevención de enfermedades, concretamente de coccidiosis, pododermatitis y úlceras digitales en un sujeto. The present invention falls within the methods of treatment or prevention of diseases, specifically coccidiosis, pododermatitis and digital ulcers in a subject.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
La coccidiosis es una enfermedad producida por parásitos protozoarios de tamaño microscópico, conocidos como coccidios, pertenecientes al phylum Apicomplexa; un grupo de protistas caracterizado por la presencia de un orgánulo único denominado complejo apical, que incluye diferentes familias y géneros de parásitos de gran importancia económica y sanitaria (por ejemplo, Coccidia, Piroplasma, y Plasmodium). Coccidiosis is a disease caused by protozoan parasites of microscopic size, known as coccidia, belonging to the phylum Apicomplexa; a group of protists characterized by the presence of a unique organelle called apical complex, which includes different families and genera of parasites of great economic and sanitary importance (for example, Coccidia, Piroplasma, and Plasmodium).
Dentro del phylum Apicomplexa, la familia Eimeriidae provoca enfermedades que afectan a la mayoría de los animales criados comercialmente para consumo, por ejemplo, aves de corral (e.g., gallinas, gallos, pollos, pavos, patos, ocas, etc.), mamíferos domésticos (e.g., ovejas, vacas, cerdos, caballos, búfalos de agua, etc.), etc. Within the phylum Apicomplexa, the family Eimeriidae causes diseases that affect most commercially raised animals for consumption, for example, poultry (eg, chickens, roosters, chickens, turkeys, ducks, geese, etc.), domestic mammals (eg, sheep, cows, pigs, horses, water buffalo, etc.), etc.
El ciclo de vida de estos parásitos depende del género y de la especie, y la mayoría de ellos completan el ciclo en un único hospedador. En el caso del género Eimeria que afectan a Gallus gallus, el ciclo se desarrolla en un único hospedador; cada especie de Eimeria presenta un periodo concreto de de prepatencia (tiempo entre la infección y la eliminación de ooquistes) y patencia (duración de la excreción de ooquistes) (en general, entre 2 y 12 días dependiendo de la especie). La diseminación de los ooquistes, dentro y fuera de la granja, se realiza mediante heces, cama, polvo, artrópodos, herramientas, etc. The life cycle of these parasites depends on the genus and the species, and most of them complete the cycle in a single host. In the case of the Eimeria genus that affect Gallus gallus, the cycle develops in a single host; Each species of Eimeria has a specific period of preparation (time between infection and elimination of oocysts) and patent (duration of oocyst excretion) (in general, between 2 and 12 days depending on the species). The dissemination of the oocysts, inside and outside the farm, is done by feces, bed, dust, arthropods, tools, etc.
La coccidiosis se caracteriza por su gran morbilidad y puede dar lugar a infecciones clínicas o sub-clínicas. El cuadro clínico se caracteriza por diarrea que puede ser hemorrágica, fiebre, anorexia, astenia y, a veces, anemia, ocasionando una disminución de las tasas de crecimiento y producción con grandes pérdidas económicas. La infección por algunas de las especies más patógenas, como E. tenella, desencadena un alto índice de mortalidad. Se han realizado numerosos esfuerzos para frenar y controlar los riesgos de la coccidiosis a la vista de la extraordinaria resistencia de sus agentes etiológicos a los procedimientos aplicados contra ellos. En general, las distintas medidas aplicadas en la lucha frente a la coccidiosis pueden agruparse en dos grupos, medidas de orden terapéutico basadas en el empleo de anticoccidiales, tales como coccidiostáticos (tratamiento preventivo) y coccidicidas (tratamiento curativo); y medidas de orden preventivo basadas en inmunizaciones. Los coccidiostáticos son productos que inhiben o detienen el desarrollo de los parásitos, mientras que los coccidicidas son productos que destruyen dichos parásitos. Los coccidiostáticos, al no eliminar totalmente los ooquistes, permiten el desarrollo de inmunidad en las aves, utilizándose especialmente en pollos de engorde. Los coccidicidas, al destruir los parásitos, inhiben el desarrollo de la inmunidad por lo que actualmente se usan cuando aparecen problemas clínicos en los lotes de aves. Además, se cree que los productos anticoccidiales no actúan sobre todas las especies de Eimeria sp. y que pueden inducir resistencia después de un uso continuado. A raíz de la introducción de los coccidiostáticos en la alimentación de las aves, disminuyeron los casos severos de coccidiosis; no obstante, la aparición de resistencias frente a los coccidiostáticos y la presión ejercida por los consumidores en demanda de carne sin residuos farmacológicos han limitado el uso de estos fármacos en alimentación animal. En el marco legal europeo, el Reglamento (CE) No. 1831/2003, establece la supresión progresiva de la utilización de los coccidiostáticos y los histomonóstatos como aditivos para alimentación animal. Por otra parte, a falta de desarrollo de nuevas moléculas, la coccidiosis vuelve a generar graves problemas económicos. Coccidiosis is characterized by its high morbidity and can lead to clinical or sub-clinical infections. The clinical picture is characterized by diarrhea that can be hemorrhagic, fever, anorexia, asthenia and, sometimes, anemia, causing a decrease in growth and production rates with great economic losses. Infection by some of the most pathogenic species, such as E. tenella, triggers a high mortality rate. Numerous efforts have been made to curb and control the risks of coccidiosis in view of the extraordinary resistance of its etiological agents to the procedures applied against them. In general, the different measures applied in the fight against coccidiosis can be grouped into two groups, therapeutic measures based on the use of anticoccidials, such as coccidiostats (preventive treatment) and coccidicides (curative treatment); and preventive measures based on immunizations. Coccidiostats are products that inhibit or stop the development of parasites, while coccidicides are products that destroy such parasites. Coccidiostats, by not completely eliminating oocysts, allow the development of immunity in birds, especially being used in broilers. Coccidicides, by destroying parasites, inhibit the development of immunity so they are currently used when clinical problems appear in lots of birds. In addition, it is believed that anticoccidial products do not act on all species of Eimeria sp. and that can induce resistance after continued use. Following the introduction of coccidiostats in bird feeding, severe cases of coccidiosis decreased; However, the emergence of resistance to coccidiostats and the pressure exerted by consumers in demand for meat without pharmacological residues have limited the use of these drugs in animal feed. In the European legal framework, Regulation (EC) No. 1831/2003, establishes the progressive suppression of the use of coccidiostats and histomonostats as additives for animal feed. On the other hand, in the absence of the development of new molecules, coccidiosis once again generates serious economic problems.
Hoy en día existe un creciente interés por el desarrollo de alternativas no farmacológicas para un control saludable, efectivo y barato de la coccidiosis aviar, entre ellas la modificación de la dieta de los animales. Today there is a growing interest in the development of non-pharmacological alternatives for a healthy, effective and cheap control of avian coccidiosis, including the modification of the diet of animals.
El extracto de un compuesto antioxidante, proantocianidina de semilla de uva, incorporado en la dieta de pollos tiene efectos beneficiosos en el tratamiento de coccidiosis causada por E. tenella (Wang ML. et al., Poult. Sci. 2008; 87: 2273-80). Sin embargo otros estudios como el de Alien PC. et al., (Clin. Microbiol. Rev., 2002, 15: 58-65) demuestran que los antioxidantes en la dieta de las aves no son efectivos en el tratamiento frente a E. tenella, pero sí de infecciones causadas por E. acervulina y E. máxima. Por lo tanto, el control de la coccidiosis requiere de mejores alternativas y más eficaces, evitando las pérdidas económicas desencadenadas por el parásito y, por otra parte, permitir producir carne de alta calidad sin residuos farmacológicos. The extract of an antioxidant compound, grape seed proanthocyanidin, incorporated into the chicken diet has beneficial effects in the treatment of coccidiosis caused by E. tenella (Wang ML. Et al., Poult. Sci. 2008; 87: 2273- 80). However other studies such as Alien PC. et al., (Clin. Microbiol. Rev., 2002, 15: 58-65) show that antioxidants in the diet of birds are not effective in the treatment against E. tenella, but of infections caused by E. acervulina and E. maximum. Therefore, the control of coccidiosis requires better and more effective alternatives, avoiding economic losses triggered by the parasite and, on the other hand, allowing to produce high quality meat without pharmacological residues.
COMPENDIO DE LA INVENCIÓN SUMMARY OF THE INVENTION
En un primer aspecto, la invención se relaciona con el uso de una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma, para la fabricación de un medicamento, composición nutritiva o nutracéutico para la prevención o tratamiento de coccidiosis en un sujeto. In a first aspect, the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof, for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of coccidiosis in a subject.
En un segundo aspecto, la invención se relaciona con el uso de una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma para la fabricación de un medicamento, composición nutritiva o nutracéutico para la prevención o tratamiento de pododermatitis o úlceras en un sujeto. In a second aspect, the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of pododermatitis or ulcers in a subject.
En un tercer aspecto, la invención se relaciona con una composición que comprende una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma y un agente anticoccídico. In a third aspect, the invention relates to a composition comprising a part of an antioxidant-enriched corn plant or a derivative thereof and an anticoccidic agent.
En un cuarto aspecto, la invención se relaciona con el uso de la composición de la invención para la prevención o tratamiento de coccidiosis en un sujeto o para la prevención o tratamiento de pododermatitis o úlceras asociadas a coccidiosis en un sujeto. In a fourth aspect, the invention relates to the use of the composition of the invention for the prevention or treatment of coccidiosis in a subject or for the prevention or treatment of pododermatitis or ulcers associated with coccidiosis in a subject.
En un quinto aspecto, la invención se relaciona con el uso de una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma para la obtención de una composición nutritiva o nutracéutico. In a fifth aspect, the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for obtaining a nutritive or nutraceutical composition.
En un sexto aspecto, la invención se relaciona con una composición nutritiva o nutracéutico que comprende una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma. In a sixth aspect, the invention relates to a nutritive or nutraceutical composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figuras 1 y 2: Pododermatitis y cantidad de úlceras de acuerdo a Welfare Quality ® (2009) Welfare Quality® assessment protocol for poultry (broilers, laying hens). Welfare Quality® Consortium, Lelystad, Netherlands. Los datos se han obtenido de 14 animales / grupo. T1 : dieta control en pollos no infectados, T2: dieta control en pollos infectados con E. tenella, T3: dieta de maíz rico en carotenoides en pollos no infectados, T4: dieta de maíz rico en carotenoides en pollos infectados con E. tenella. Figures 1 and 2: Pododermatitis and number of ulcers according to Welfare Quality ® (2009) Welfare Quality® assessment protocol for poultry (broilers, laying hens). Welfare Quality® Consortium, Lelystad, Netherlands. Data were obtained from 14 animals / group. T1: control diet in uninfected chickens, T2: control diet in chickens infected with E. tenella, T3: diet of corn rich in carotenoids in uninfected chickens, T4: diet of corn rich in carotenoids in chickens infected with E. tenella.
Figura 3. Excreción de ooquistes de 6 a 9 días post infección. T2: dieta control en pollos infectados con E. tenella, T4: dieta de maíz rico en carotenoides en pollos infectados con E. tenella. (Los grupos no infectados T1 y T3 fueron negativos). Figure 3. Excretion of oocysts 6 to 9 days post infection. T2: control diet in chickens infected with E. tenella, T4: diet of corn rich in carotenoids in chickens infected with E. tenella. (The uninfected groups T1 and T3 were negative).
Figura 4. De izquierda a derecha: dos patas de cada grupo: T1 : dieta control en pollos no infectados, T2: dieta control en pollos desafiados con E. tenella, T3: dieta de maíz rico en carotenoides en pollos no infectados, T4: dieta de maíz rico en carotenoides en pollos infectados con E. tenella. Figure 4. From left to right: two legs of each group: T1: control diet in uninfected chickens, T2: control diet in chickens challenged with E. tenella, T3: diet of carotenoid-rich corn in uninfected chickens, T4: Carotenoid-rich corn diet in chickens infected with E. tenella.
Figuras 5 y 6 (mayor aumento). Histopatología de la bolsa de Fabricio del grupo T2 (dieta control en pollos infectados con E. tenella) con vacuolización medular y cortical. Figures 5 and 6 (largest increase). Histopathology of the Fabrizio pouch of the T2 group (control diet in chickens infected with E. tenella) with medullary and cortical vacuolization.
Figura 7. Histopatología de la bolsa de Fabricio del grupo T4 (dieta con maíz rico en antioxidantes en pollos infectados con E. tenella) con poca vacuolización medular y cortical. Figure 7. Histopathology of the Fabrizio pouch of group T4 (diet with corn rich in antioxidants in chickens infected with E. tenella) with little medullary and cortical vacuolization.
DESCRIPCIÓN DETALLADA DETAILED DESCRIPTION
Los autores de la presente invención han observado que las aves criadas con una dieta de maíz enriquecido en antioxidantes exhiben una reducción de pododermatitis y úlceras en los dedos. Además, dichas aves mostraron una reducción de cerca de cuatro veces en la cantidad de ooquistes de Eimeria excretados por las heces, lo que indica que la dieta de maíz enriquecido en antioxidantes es efectiva frente a la coccidiosis. La histopatología de la bolsa de Fabricio permitió la cuantificación de la vacuolización medular y cortical en los folículos y se observó que los animales infectados alimentados con maíz enriquecido en antioxidantes mostraban una reducción significativa de vacuolas. Esta observación demuestra que los carotenoides ejercen un papel protector contra el estrés oxidativo debido a la infección por coccidios y permiten un aumento de la inmunidad. Los resultados demuestran que el maíz enriquecido en antioxidantes es un vehículo sorprendentemente eficaz para mantener la salud y la inmunidad de aves de corral, para reducir la dermatitis en las patas y las úlceras en los pies. The authors of the present invention have observed that birds raised with a diet of corn enriched in antioxidants exhibit a reduction in pododermatitis and finger ulcers. In addition, these birds showed a reduction of about four times in the amount of Eimeria oocysts excreted by feces, indicating that the diet of antioxidant-enriched corn is effective against coccidiosis. The histopathology of the Fabrizio pouch allowed the quantification of medullary and cortical vacuolization in the follicles and it was observed that infected animals fed with antioxidant-enriched corn showed a significant reduction of vacuoles. This observation demonstrates that carotenoids play a protective role against oxidative stress due to coccidia infection and allow an increase in immunity. The results show that antioxidant-enriched corn is a surprisingly effective vehicle to maintain poultry health and immunity, to reduce dermatitis in the legs and foot ulcers.
Definiciones Por "planta de maíz", según se emplea en la presente invención, se refiere a un ser vivo fotosintético perteneciente a la especie Zea mays. Dicho término incluye también las diversas subespecies. Definitions By "corn plant", as used in the present invention, it refers to a photosynthetic living being belonging to the Zea mays species. Said term also includes the various subspecies.
Una "parte de una planta", tal y como se usa en la presente invención, se refiere a una parte específica de una planta, tal como tallo, hoja, raíz, flor, semilla, endospermo, grano, fruto o yema. A "part of a plant", as used in the present invention, refers to a specific part of a plant, such as stem, leaf, root, flower, seed, endosperm, grain, fruit or yolk.
"Derivado de una parte de la planta" se refiere a cualquier producto obtenido tras el procesamiento de una parte de la planta de la invención a partir de la modificación física y/o química, a modo ilustrativo no limitativo, harina, sémola, aceite, sirope, almidón o fécula. "Derived from a part of the plant" refers to any product obtained after processing a part of the plant of the invention from physical and / or chemical modification, by way of illustration, not limitation, flour, semolina, oil, syrup, starch or starch.
Según la invención, la planta de maíz se encuentra "enriquecida en antioxidantes", es decir, presenta niveles superiores de moléculas capaces de retardar o prevenir la oxidación de otras moléculas, comparado con un nivel de referencia. Los antioxidantes se encargan de capturar los radicales libres producidos en las reacciones de oxidación propias del metabolismo. Ejemplos ilustrativos no limitativos de antioxidantes son carotenoides, luteína, licopeno, selenio, vitamina A, vitamina C y vitamina E. According to the invention, the corn plant is "enriched in antioxidants," that is, it has higher levels of molecules capable of retarding or preventing the oxidation of other molecules, compared to a reference level. Antioxidants are responsible for capturing free radicals produced in the oxidation reactions of the metabolism. Illustrative non-limiting examples of antioxidants are carotenoids, lutein, lycopene, selenium, vitamin A, vitamin C and vitamin E.
El término "composición nutritiva" de la presente invención se refiere a aquel alimento que, con independencia de aportar nutrientes al sujeto que lo toma, afecta beneficiosamente a una o varias funciones del organismo, de manera que proporciona un mejor estado de salud y bienestar. Como consecuencia, dicha composición nutritiva puede ser destinada a la prevención y/o tratamiento de una enfermedad o del factor causante de una enfermedad. Por tanto, el término "composición nutritiva" de la presente invención se puede emplear como sinónimo de alimento funcional o alimento para fines nutricionales específicos o alimento medicinal. Una composición nutritiva tiene una apariencia similar a la de un alimento convencional y se consume como parte de una dieta normal. The term "nutritional composition" of the present invention refers to that food that, regardless of providing nutrients to the subject who takes it, beneficially affects one or more functions of the organism, so as to provide a better state of health and well-being. As a consequence, said nutritional composition may be intended for the prevention and / or treatment of a disease or the causative factor of a disease. Therefore, the term "nutritional composition" of the present invention can be used as a synonym for functional food or food for specific nutritional purposes or medicinal food. A nutritional composition has an appearance similar to that of a conventional food and is consumed as part of a normal diet.
Por "nutracéutico", palabra derivada de nutrición y farmacéutico, se entiende un producto elaborado a partir de un alimento, pero que se encuentra en forma de pildora, polvos y otras presentaciones farmacéuticas no asociadas generalmente con los alimentos y que presenta propiedades beneficiosas para el tratamiento y/o prevención de enfermedades. El término "coccidiosis", tal como aquí se utiliza, se refiere a una enfermedad producida por parásitos protozoarios de tamaño microscópico, conocidos como "coccidios". Se incluyen en el phylum Apicomplexa, entre ellos a la familia Eimeriidae y se consideran varios géneros, e.g., Eimeria, Isospora, etc. Invaden células epiteliales del intestino, o de otros órganos, de los animales infectados, produciendo la destrucción de las células hospedadoras, e.g., enterocitos, etc. Esta enfermedad afecta a la mayoría de los animales criados comercialmente bien con fines alimenticios, por ejemplo, aves (e.g., gallinas, gallos, pollos, pavos, patos, ocas, etc.), mamíferos (e.g., ovejas, vacas, cerdos, conejos, caballos, búfalos de agua, etc.), peces, etc., o bien con otros fines comerciales (e.g., perros, gatos, marsupiales, etc.). La enfermedad se transmite por el contacto con heces o por ingestión de tejidos infectados. El síntoma primario es la diarrea, que pueden llegar a ser sangrienta en los casos severos. La mayoría de los animales infectados por coccidios son asintomáticos; sin embargo, los jóvenes o inmunodeficientes pueden sufrir síntomas severos, incluyendo muerte. By "nutraceutical", a word derived from nutrition and pharmaceutical, is meant a product made from a food, but which is in the form of a pill, powders and other pharmaceutical presentations not generally associated with food and that has beneficial properties for treatment and / or prevention of diseases. The term "coccidiosis," as used herein, refers to a disease caused by protozoan parasites of microscopic size, known as "coccidia." They are included in the phylum Apicomplexa, including the family Eimeriidae and are considered several genera, eg, Eimeria, Isospora, etc. They invade epithelial cells of the intestine, or other organs, of infected animals, causing the destruction of host cells, eg, enterocytes, etc. This disease affects most commercially raised animals for food purposes, for example, birds (eg, chickens, roosters, chickens, turkeys, ducks, geese, etc.), mammals (eg, sheep, cows, pigs, rabbits , horses, water buffalo, etc.), fish, etc., or for other commercial purposes (eg, dogs, cats, marsupials, etc.). The disease is transmitted by contact with feces or by ingestion of infected tissues. The primary symptom is diarrhea, which can become bloody in severe cases. Most animals infected by coccidia are asymptomatic; however, young or immunodeficient can suffer severe symptoms, including death.
El término "sujeto", tal como aquí se utiliza, se refiere a un miembro de una especie de animal, preferentemente un ave, un mamífero o un pez, de cualquier sexo, raza o edad. A modo de ejemplo ilustrativo no limitativo el término ave incluye pollos, aves acuáticas y las aves de caza. The term "subject", as used herein, refers to a member of an animal species, preferably a bird, a mammal or a fish, of any sex, race or age. By way of illustrative, non-limiting example, the term bird includes chickens, water birds and game birds.
"Pododermatitis", tal y como se usa en la presente invención, se refiere a lesiones en las almohadillas de las patas y los dedos de las patas y en la piel situada en la articulación tibiotarsiana. Pueden variar en tamaño y en profundidad. Al principio se caracteriza por una hiperqueratosis (engrosamiento de la piel). En una segunda etapa, aparece una coloración marrón de la piel que se corresponde con la necrosis de los tejidos. En los casos más graves se produce la erosión y ulceración de los tejidos acompañados de una inflamación del tejido subcutáneo. En la zona central se origina la destrucción completa de la epidermis. La úlcera produce un exudado que forma una costra de aspecto negro. La causa principal de la pododermatitis es la humedad. Todos los factores que pueden favorecer la humedad del suelo, tales como el tipo de cama, el sistema de abastecimiento de agua, la digestibilidad de los alimentos o la presencia de diarrea, también pueden contribuir a su aparición. Por tanto, la pododermatitis o úlceras digitales pueden aparecer de forma secundaria a la coccidiosis por el aumento en la humedad del suelo como consecuencia de las diarreas que sufren los animales. "Agente anticoccídico", tal y como se usa en la presente invención, incluye los coccidiostáticos y los coccidiocidas, clasificados así según sus efectos sobre los parásitos. "Pododermatitis", as used in the present invention, refers to lesions in the pads of the legs and fingers of the legs and in the skin located in the tibiotarsian joint. They can vary in size and depth. At first it is characterized by hyperkeratosis (thickening of the skin). In a second stage, a brown coloration of the skin that corresponds to tissue necrosis appears. In the most severe cases, erosion and ulceration of the tissues occurs accompanied by an inflammation of the subcutaneous tissue. In the central zone the complete destruction of the epidermis originates. The ulcer produces an exudate that forms a black-looking scab. The main cause of pododermatitis is moisture. All the factors that can favor soil moisture, such as the type of bed, the water supply system, the digestibility of food or the presence of diarrhea, can also contribute to its appearance. Therefore, pododermatitis or digital ulcers may appear secondary to coccidiosis due to the increase in soil moisture as a result of diarrhea suffered by animals. "Anticoccidic agent", as used in the present invention, includes coccidiostats and coccidiocides, thus classified according to their effects on parasites.
El término "Eimeria", tal como aquí se utiliza, incluye cualquier especie del género Eimeria, incluyendo, entre otras, especies de Eimería que infectan aves (e.g., gallinas, gallos, pollos, capones, pintadas (gallinas de Guinea), pulardas, pavos, patos, ocas, faisanes, perdices, codornices, avestruces, etc.), especies de Eimería que infectan mamíferos (e.g., ovejas, vacas, cerdos, conejos, caballos, perros, gatos, etc.), especies de Eimeria que infectan peces (e.g., anguilas, congrios, anchoas o boquerones, arenques, sardinas, carpas, bacalaos, merluzas, lubinas, jureles, rapes, etc. Ejemplos ilustrativos, no limitativos, de dichas especies de Eimería incluyen especies que infectan pollos, tales como E.tenella, E. acervulina, E. brunetti, E. hagani, E. máxima, E. mitis, E. mivati, E. necatrix, E. praecox, , etc., especies que infectan pavos, tales como E. adenoeides, E. gallopavonis, E. innocua, E. meleagrídis, E. meleagrimitis, E. subrotunda, etc., especies que infectan perdices, tales como E. alectoreae, E. caucásica, E. gonzalezcastroi, E. kofoidi, E. phasiani, etc., especies que infectan codornices, tales como E. baterí, E. coturnicis, E. lettyae, etc., especies que infectan faisanes, tales como E. colchici, E. duodenalis, E. langeroni, E. megalostomata, E. pacifica, E. phasiani, E. paisana, etc., especies que infectan pintadas, tales como E. gorakhpurí, E. grenieri, E. numidae, etc., especies que infectan patos, tales como E. anatis, E. aythyae, E.battakhi, E.danailovi, E.krílovi, etc., especies que infectan ocas, tales como E. stigmosa, E. truncata, etc., especies que infectan vacas, tales como E. bovis, E. zuernii, E. ellipsoidalis, E. cylindríca, E. auburnensis, E. alabamensis, E. subspheríca, E. wyomingensis, etc., especies que infectan cabras, tales como E. aríoingi, E. chrístenseni, E. ninakohlyakimovae, E. alijevi, E. hirci, E. caprina, E. caprovina, E. pallida, E. jolchijevi, E. apsheronica, etc., especies que infectan ovejas, tales como E. ahsata, E. bakuensis, E.crandallis, E. faurei, E. intricata, E. marsica, E. ovinoidalis, E. pallida, E. parva, E. weybridgensis, E. gilruthi, E. gonzalezi, E. granulosa, E. punctata, etc., especies que infectan cerdos, tales como E. scabra, E.porci, E. polita, E.suis, etc., especies que infectan conejos, tales como E. stiedae, E. irresidua, E. magna, E. media, E. períorans, etc., especies que infectan caballos, tales como E. leuckarti, E. solipedum, E. iniungulsti, etc. "Eimeria tenella", tal y como se usa en la presente invención, se refiere al parásito estenoxeno de la especie de Eimeria que causa hemorragias intestinales y afecta principalmente a los ciegos de gallinas y pollos. The term "Eimeria", as used herein, includes any species of the genus Eimeria, including, among others, Eimeria species that infect birds (eg, chickens, roosters, chickens, capons, graffiti (Guinea fowl), puladas, turkeys, ducks, geese, pheasants, partridges, quails, ostriches, etc.), Eimeria species that infect mammals (eg, sheep, cows, pigs, rabbits, horses, dogs, cats, etc.), Eimeria species that infect fish (eg, eels, conger, anchovies or anchovies, herring, sardines, carp, cod, hake, sea bass, horse mackerel, monkfish, etc. Illustrative, non-limiting examples of such species of Eimeria include species that infect chickens, such as E .tenella, E. acervulina, E. brunetti, E. Hagai, E. maxim, E. mitis, E. mivati, E. necatrix, E. praecox, etc., species that infect turkeys, such as E. adenoeides, E. gallopavonis, E. innocua, E. meleagrídis, E. meleagrimitis, E. subrotunda, etc., species that infect partridges, t ales such as E. alectoreae, E. caucasian, E. gonzalezcastroi, E. kofoidi, E. phasiani, etc., species that infect quail, such as E. baterí, E. coturnicis, E. lettyae, etc., species that infect pheasants, such as E. colchici, E. duodenalis, E. langeroni, E. megalostomata, E. pacifica, E. phasiani, E. paisana, etc., painted infecting species, such as E. gorakhpurí, E. grenieri, E. numidae, etc., species that infect ducks, such as E. anatis, E. aythyae, E.battakhi, E.danailovi, E. krílovi, etc., species that infect geese, such as E. stigmosa, E. truncata, etc., species that infect cows, such as E. bovis, E. zuernii, E. ellipsoidalis, E. cylindríca, E. auburnensis, E. alabamensis, E. subspheríca, E. wyomingensis, etc., species that infect goats, such as E. aríoingi, E. chrístenseni, E. ninakohlyakimovae, E. alijevi, E. hirci, E. caprina, E. caprovina, E. pallida, E. jolchijevi, E. apsheronica, etc., species that infect sheep, such as E. a hsata, E. bakuensis, E.crandallis, E. faurei, E. intricata, E. marsica, E. ovinoidalis, E. pallida, E. parva, E. weybridgensis, E. gilruthi, E. gonzalezi, E. granulosa, E. punctata, etc., species that infect pigs, such as E. scabra, E.porci, E. polita, E.suis, etc., species that infect rabbits, such as E. stiedae, E. irresidua, E. magna, E. media, E. períorans, etc., species that infect horses, such as E. leuckarti, E. solipedum, E. iniungulsti, etc. "Eimeria tenella", as used in the present invention, refers to the stenoxen parasite of the Eimeria species that causes intestinal bleeding and primarily affects the blind of chickens and chickens.
"Ave" tal y como se usa en la presente invención, se refiere al animal vertebrado que presentan las extremidades anteriores modificadas como alas, con el cuerpo recubierto de plumas y un pico córneo sin dientes. "Bird" as used in the present invention, refers to the vertebrate animal having the modified forelimbs as wings, with the body covered with feathers and a corneal beak without teeth.
"Ave de corral", incluye aves domésticas criadas por el hombre para la obtención de huevos, carne y/o plumas. Los ejemplos de las aves de corral incluyen gallos, gallinas, pavos, patos, gansos, codornices, faisanes, palomas, palomas, emúes, avestruces y ñandúes. "Poultry" includes domestic birds raised by man to obtain eggs, meat and / or feathers. Examples of poultry include roosters, chickens, turkeys, ducks, geese, quails, pheasants, pigeons, pigeons, emus, ostriches and rheas.
Por "pollo" tal y como se usa en la presente invención, se refiere a una cría de ave especialmente de gallina. By "chicken" as used in the present invention, it refers to a brood, especially chicken.
Por "maíz transgénico", según se usa en la presente invención, se refiere a aquella planta de maíz cuyo genoma ha sido modificado mediante ingeniería genética con el objetivo de conseguir características biológicas diferentes y/o mejoradas respecto a las de la planta isogénica silvestre. By "transgenic corn", as used in the present invention, it refers to that corn plant whose genome has been modified by genetic engineering in order to achieve different and / or improved biological characteristics with respect to those of the wild isogenic plant.
"Transgén de la ruta de biosíntesis de carotenoides", tal y como se usa en la presente invención se refiere a un gen que codifica para una enzima que participa en la ruta de síntesis de carotenoides. En el grano, la biosíntesis de carotenos ocurre en las membranas de los cromoplastos. "Transgene of the carotenoid biosynthesis pathway", as used in the present invention refers to a gene that encodes an enzyme that participates in the carotenoid synthesis pathway. In the grain, carotene biosynthesis occurs in the membranes of chromoplasts.
"Carotenoides", tal y como se usa en la presente invención, se refiere a pigmentos orgánicos que se encuentran de forma natural en plantas y otros organismos fotosíntéticos. La mayoría de los carotenoides son tetraterpenoides, compuestos de 40 átomos de carbono formados por ocho unidades isoprenoides y contienen anillos a uno o ambos extremos de la molécula. Pueden funcionar como antioxidantes, protegiendo frente a la auto-oxidación. "Carotenoids," as used in the present invention, refers to organic pigments found naturally in plants and other photosynthetic organisms. Most carotenoids are tetraterpenoids, composed of 40 carbon atoms formed by eight isoprenoid units and contain rings at one or both ends of the molecule. They can function as antioxidants, protecting against self-oxidation.
Ejemplos de genes que participan en la biosíntesis de carotenoides, a modo ilustrativo, no limitativo son fitoeno sintasa, caroteno desaturasa, licopeno β-ciclasa, β-caroteno hidroxilasa y carotenoide cetolasa. Por "Fitoeno sintasa" o psyl tal y como se usa en la presente invención se refiere al gen que codifica para la enzima que cataliza la reacción que convierte dos moléculas de GGDP en fitoeno, el primer paso en la síntesis de la provitamina A. Examples of genes that participate in carotenoid biosynthesis, by way of illustration, are not limited to phytoeno synthase, carotene desaturase, lycopene β-cyclase, β-carotene hydroxylase and carotenoid ketolase. By "Phytoeno synthase" or psyl as used in the present invention it refers to the gene that codes for the enzyme that catalyzes the reaction that converts two GGDP molecules into phytoeno, the first step in the synthesis of provitamin A.
"Caroteno desaturasa" o crt\, tal y como se usa en la presente invención, se refiere al gen que codifica para la enzima encargada de la producción de licopeno a partir de tres substratos, neurosporeno, un aceptor de electrones y oxígeno. "Carotene desaturase" or crt \, as used in the present invention, refers to the gene that codes for the enzyme responsible for the production of lycopene from three substrates, neurosporene, an electron acceptor and oxygen.
"Licopeno 3-ciclasa" o Lycb, tal y como se usa en la presente invención, se refiere al gen que codifica para la enzima que transforma el licopeno en provitamina A. "Lycopene 3-cyclase" or Lycb, as used in the present invention, refers to the gene that codes for the enzyme that transforms lycopene into provitamin A.
"3-caroteno hidroxilasa" o bch, tal y como se usa en la presente invención, se refiere al gen que codifica para la enzima encargada de la conversión del beta-caroteno a xantofilas. "3-carotene hydroxylase" or bch, as used in the present invention, refers to the gene that codes for the enzyme responsible for the conversion of beta-carotene to xanthophylls.
"Carotenoide cetolasa" o crtw tal y como se usa en la presente invención, se refiere al gen que codifica para la enzima encargada de añadir un grupo carbonilo con el carbono 4 y 4 'de la molécula de beta caroteno. "Carotenoid ketolase" or crtw, as used in the present invention, refers to the gene that codes for the enzyme responsible for adding a carbonyl group with carbon 4 and 4 'of the beta carotene molecule.
"Transgén de la biosíntesis de vitamina C" tal y como se usa en la presente invención, se refiere a un gen que codifica para una enzima que participa en la biosíntesis de la vitamina C. Un ejemplo es la dehidroascorbato reductasa. "Vitamin C biosynthesis transgene" as used in the present invention, refers to a gene that encodes an enzyme that participates in the biosynthesis of vitamin C. An example is dehydroascorbate reductase.
"Vitamina C", tal y como se usa en la presente invención, se refiere al compuesto con número CAS 50-81-7, es antioxidante y cofactor en varias reacciones enzimáticas. "Vitamin C", as used in the present invention, refers to the compound with CAS number 50-81-7, is an antioxidant and cofactor in various enzymatic reactions.
"Dehidroascorbato reductasa" dhar, tal y como se usa en la presente invención, se refiere al gen que codifica para la enzima que cataliza la reducción del dehidroascorbato a ascorbato en una reacción dependiente de glutatión. "Dhar dehydroascorbate reductase", as used in the present invention, refers to the gene encoding the enzyme that catalyzes the reduction of dehydroascorbate to ascorbate in a glutathione-dependent reaction.
"Transgén de la biosíntesis de folato", tal y como se usa en la presente invención, se refiere a un gen que codifica para una enzima que participa en la ruta de biosíntesis de folato, por ejemplo GTP ciclohidrolasa. "Folate biosynthesis transgene", as used in the present invention, refers to a gene encoding an enzyme that participates in the folate biosynthesis pathway, for example GTP cyclohydrolase.
"Folato", tal y como se usa en la presente invención, se refiere a un conjunto de substancias derivadas del ácido fólico propiamente dicho, (el ácido pteroil-L- glutámico), que pueden transformarse en él fácilmente. "Folate", as used in the present invention, refers to a set of substances derived from folic acid itself, (pteroyl-L-glutamic acid), which can be easily transformed into it.
"GTP ciclohidrolasa" o GCH1, tal y como se usa en la presente invención, se refiere a la enzima codificada por el gen folE y que cataliza la reacción de hidrólisis del GTP. "Promotor específico de endospermo", tal y como se usa en la presente invención, se refiere a una molécula de ácido nucleico que permite dirigir o regular la expresión de un gen en el endospermo. El "endospermo" es una formación característica de las semillas de Angiospermas, es un tejido nutritivo para el embrión. El endospermo de maíz se origina a partir de una serie de divisiones nucleares libres, seguidas de la celularización y posterior formación de un rango de dominios celulares funcionales. El endospermo es el órgano de almacenamiento principal de las semillas de maíz; nutre al embrión mientras la semilla se desarrolla y proporciona nutrientes a la plántula en germinación. "GTP cyclohydrolase" or GCH1, as used in the present invention, refers to the enzyme encoded by the folE gene and which catalyzes the hydrolysis reaction of GTP. "Specific endosperm promoter", as used in the present invention, refers to a nucleic acid molecule that allows to direct or regulate the expression of a gene in the endosperm. The "endosperm" is a characteristic formation of Angiosperm seeds, it is a nutritious tissue for the embryo. The corn endosperm originates from a series of free nuclear divisions, followed by cellization and subsequent formation of a range of functional cell domains. The endosperm is the main storage organ of corn seeds; It nourishes the embryo while the seed develops and provides nutrients to the germinating seedling.
"D-hordeína de cebada", tal y como se emplea en la presente invención, se refiere a la proteína con el número de acceso Q40054 de la base de datos Uniprot a fecha 17 de marzo de 2014. En una realización particular, el promotor de D-hordeína tiene la secuencia mostrada en SEQ ID NO: 10 "Barley D-hordein," as used in the present invention, refers to the protein with accession number Q40054 from the Uniprot database as of March 17, 2014. In a particular embodiment, the promoter of D-hordein has the sequence shown in SEQ ID NO: 10
"LMW glutenina de trigo", tal y como se emplea en la presente invención, se refiere a proteína con el número de acceso Q41603 de la base de datos Uniprot a fecha 17 de marzo de 2014. En una realización particular, el promotor de LMW de glutenina tiene la secuencia mostrada en SEQ ID NO: 11 "LMW wheat glutenin", as used in the present invention, refers to protein with accession number Q41603 from the Uniprot database as of March 17, 2014. In a particular embodiment, the LMW promoter of glutenin has the sequence shown in SEQ ID NO: 11
"Semilla" tal y como se usa en la presente invención, se refiere a cada uno de los cuerpos que forman parte del fruto que da origen a una nueva planta. "Seed" as used in the present invention, refers to each of the bodies that are part of the fruit that gives rise to a new plant.
El término "endospermo", según se usa en la presente invención, se refiere al órgano principal de almacenamiento en las semillas de maíz, nutriendo el embrión mientras la semilla se desarrolla y proporcionando nutrientes a la planta joven en germinación. The term "endosperm", as used in the present invention, refers to the main organ of storage in corn seeds, nourishing the embryo while the seed develops and providing nutrients to the young germinating plant.
Por "derivado de semilla o endospermo", tal y como se usa en la presente invención se refiere a cualquier producto obtenido a partir de la modificación física y/o química de la semilla o endospermo By "seed derivative or endosperm", as used in the present invention it refers to any product obtained from the physical and / or chemical modification of the seed or endosperm.
El término "harina" según se entiende en la presente invención, es el producto obtenido de la molturacion de cualquier semilla de plantas del género Zea que puede estar despojado en mayor o menor grado del salvado o la cascarilla de la semilla. The term "flour" as understood in the present invention, is the product obtained from the milling of any seed of plants of the Zea genus that may be stripped to a greater or lesser extent of the bran or seed husk.
El término "sémola" hace referencia a harina gruesa (semillas de maíz poco molidas), es decir, fragmentos de endospermo con una cantidad variable de cascarilla de semilla. "Aceite de maíz", tal y como se usa en la presente invención, se refiere al aceite obtenido tras el secado del grano de maíz germinado. The term "semolina" refers to coarse flour (poorly ground corn seeds), that is, endosperm fragments with a variable amount of seed husk. "Corn oil", as used in the present invention, refers to the oil obtained after drying the germinated corn grain.
Por "almidón de maíz", tal y como se emplea, se refiere a un polvo sedoso finalmente molido blanco hecho del endospermo del maíz seco. By "corn starch," as used, it refers to a finally ground white silky powder made from the endosperm of dried corn.
"Fécula de maíz" es el polisacárido obtenido a través de la molienda húmeda. "Corn starch" is the polysaccharide obtained through wet milling.
"Sirope de maíz", también conocido como jarabe de maíz, es un edulcorante liquido elaborado a partir del almidón o la fécula de maíz tras un proceso de calentamiento e hidrolización. "Corn syrup", also known as corn syrup, is a liquid sweetener made from starch or cornstarch after a heating and hydrolyzing process.
Usos médicos de la invención Medical uses of the invention
En un primer aspecto, la invención se relaciona con el uso de una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma, para la fabricación de un medicamento, composición nutritiva o nutracéutico para la prevención o tratamiento de coccidiosis en un sujeto. In a first aspect, the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof, for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of coccidiosis in a subject.
Alternativamente, la invención se relaciona con una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma para su uso en la prevención o tratamiento de coccidiosis en un sujeto. Alternatively, the invention relates to a part of an antioxidant-enriched corn plant or a derivative thereof for use in the prevention or treatment of coccidiosis in a subject.
Asimismo, la invención se relaciona con una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma para su uso en medicina. Also, the invention relates to a part of a corn plant enriched in antioxidants or a derivative thereof for use in medicine.
En un segundo aspecto, la invención se relaciona con el uso de una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma para la fabricación de un medicamento, composición nutritiva o nutracéutico para la prevención o tratamiento de pododermatitis o úlceras en un sujeto. In a second aspect, the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of pododermatitis or ulcers in a subject.
Alternativamente, la invención se relaciona con una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma para su uso en la prevención o tratamiento de pododermatitis o úlceras en un sujeto. Alternatively, the invention relates to a part of a corn plant enriched in antioxidants or a derivative thereof for use in the prevention or treatment of pododermatitis or ulcers in a subject.
En una realización particular las úlceras se encuentran en las patas o pies. In a particular embodiment the ulcers are found in the legs or feet.
Se entiende que una parte de una planta de maíz o un derivado de la misma se encuentra enriquecida en antioxidantes, cuando presenta niveles de antioxidantes superiores a los niveles de una muestra de referencia o muestra control. En particular, se puede considerar que una parte de una planta o derivado de la misma presenta niveles superiores de antioxidantes cuando los niveles son de al menos 1 vez, 1 ,5 veces, 5 veces, 10 veces, 20 veces, 30 veces, 40 veces, 50 veces, 60 veces, 70 veces, 80 veces, 90 veces, 100 veces o incluso más con respecto a la muestra de referencia o muestra control. El experto en la materia entenderá que no es necesario que una planta enriquecida en antioxidantes contenga niveles elevados de dichos antioxidantes en todos los tejidos de dicha planta, sino que es suficiente con que al menos un tejido de la planta contenga niveles elevados de antioxidantes para que la planta pueda ser considerada como "enriquecida en antioxidantes". En ese caso, la muestra de referencia para determinar el grado de enriquecimiento en antioxidantes se determina mediante comparación con los niveles de antioxidantes es una muestra del mismo tejido o parte de una planta control. It is understood that a part of a corn plant or a derivative thereof is enriched in antioxidants, when it has antioxidant levels higher than the levels of a reference sample or control sample. In particular, It can be considered that a part of a plant or derivative of it presents higher levels of antioxidants when the levels are at least 1 time, 1, 5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more with respect to the reference sample or control sample. The person skilled in the art will understand that it is not necessary for an antioxidant-enriched plant to contain high levels of said antioxidants in all tissues of said plant, but it is sufficient that at least one plant tissue contains high levels of antioxidants so that The plant can be considered as "enriched in antioxidants". In that case, the reference sample to determine the degree of antioxidant enrichment is determined by comparison with antioxidant levels is a sample of the same tissue or part of a control plant.
Una muestra control o de referencia, tal y como se usa en la presente invención, puede ser el valor que corresponde a los niveles de antioxidantes presentes en una parte de una planta de maíz o derivado de la misma que se encuentra en estado salvaje, es decir no modificada genéticamente. A control or reference sample, as used in the present invention, may be the value that corresponds to the levels of antioxidants present in a part of a corn plant or derived therefrom that is in the wild, it is say not genetically modified.
El experto en la materia conoce diversos métodos para determinar si una planta de maíz o derivado de la misma se encuentra enriquecida en antioxidantes, por ejemplo determinando los niveles de antioxidantes, particularmente carotenoides mediante HPLC (cromatografía líquida de alta resolución) o determinando el incremento de la capacidad antioxidante. The person skilled in the art knows various methods to determine if a corn plant or derivative thereof is enriched in antioxidants, for example by determining antioxidant levels, particularly carotenoids by HPLC (high performance liquid chromatography) or determining the increase in antioxidant capacity
La capacidad antioxidante puede determinarse mediante la cuantificación de la capacidad que tienen los compuestos antioxidantes para reaccionar con un radical libre determinado, o determinando el potencial que tales compuestos tendrían para reducir el complejo formado entre iones Fe(lll) y el reactivo TPTZ (2,4,6-tripiridil-s- triazina). The antioxidant capacity can be determined by quantifying the ability of antioxidant compounds to react with a given free radical, or by determining the potential such compounds would have to reduce the complex formed between Fe ions (lll) and the TPTZ reagent (2, 4,6-tripyridyl-s-triazine).
Entre aquellos ensayos que se basan en la medición de la capacidad de los antioxidantes para reaccionar con un radical libre, caben destacar los siguientes, ensayo ORAC (del inglés Oxygen Radical Absorbance Capacity o capacidad de absorción de radicales de oxigeno), ensayo TEAC (del inglés Trolox Equivalent Antioxidant Capacity o Capacidad Antioxidante como Equivalentes Trolox) y Ensayo DPPH (2,2-Difenil-1-picrilhidrazil). En una realización particular, la planta de maíz es maíz blanco o maíz amarillo. Maíz blanco se refiere a una variedad de maíz en el que el endospermo tiene color blanco, como consecuencia de la ausencia de los pigmentos carotenoides que confieren el color amarillo al endospermo del maíz conocido como maíz amarillo. Among those tests that are based on the measurement of the ability of antioxidants to react with a free radical, the following should be noted: ORAC test (Oxygen Radical Absorbance Capacity or oxygen radical absorption capacity), TEAC test (from English Trolox Equivalent Antioxidant Capacity or Antioxidant Capacity as Trolox Equivalents) and DPPH Test (2,2-Diphenyl-1-picrilhidrazil). In a particular embodiment, the corn plant is white corn or yellow corn. White corn refers to a variety of corn in which the endosperm has a white color, as a result of the absence of carotenoid pigments that confer yellow color to the endosperm of corn known as yellow corn.
En una forma preferida de realización, los antioxidantes son carotenoides. En una forma de realización aún más preferida, los carotenoides se seleccionan del grupo formado por β-caroteno, γ-caroteno, α-caroteno, a-criptoxantina, β-criptoxantina, licopeno, luteína y zeaxantina. In a preferred embodiment, the antioxidants are carotenoids. In an even more preferred embodiment, the carotenoids are selected from the group consisting of β-carotene, γ-carotene, α-carotene, a-cryptoxanthin, β-cryptoxanthin, lycopene, lutein and zeaxanthin.
En una realización particular, la parte de la planta de maíz enriquecida en antioxidantes es el endospermo o la semilla o derivados de la misma. In a particular embodiment, the part of the antioxidant-enriched corn plant is the endosperm or the seed or derivatives thereof.
A modo de ejemplo ilustrativo no limitativo, el derivado de la semilla o endospermo puede ser una harina, sémola, aceite de maíz, almidón de maíz, fécula de maíz o sirope de maíz entre otros. As an illustrative, non-limiting example, the derivative of the seed or endosperm can be a flour, semolina, corn oil, corn starch, cornstarch or corn syrup among others.
El término "tratamiento", tal como se utiliza aquí, se refiere a cualquier tipo de terapia, que tenga como objetivo la terminación, la prevención, la mejora o la reducción de la susceptibilidad a padecer una condición clínica, como se describe aquí. En una forma de realización preferida, el término tratamiento se refiere al tratamiento profiláctico (es decir, una terapia para reducir la susceptibilidad a padecer una condición clínica) de un trastorno o condición. Así, "tratamiento", "tratar", y sus términos equivalentes se refieren a la obtención de un efecto deseado farmacológica o fisiológicamente, que cubre cualquier tratamiento de una afección patológica o trastorno en un mamífero, incluyendo el ser humano. El efecto puede ser profiláctico en términos de proporcionar prevención total o parcial de un trastorno y/o efecto adverso atribuible al mismo. Es decir, "tratamiento" incluye (1) prevenir que la enfermedad ocurra o se repita en un sujeto, (2) inhibir la enfermedad, por ejemplo deteniendo su desarrollo, (3) interrumpir o finalizar el desorden o por lo menos los síntomas asociados al mismo, por lo que el paciente ya no sufriría la enfermedad o sus síntomas, por ejemplo provocar la regresión de la enfermedad o sus síntomas mediante la restauración o reparación de una función perdida, ausente o defectuosa, o estimular un proceso ineficiente, o (4), aminorar, aliviar o mejorar la enfermedad, o los síntomas asociados a la misma, donde aminorar se utiliza en un sentido amplio para referirse a, al menos, una reducción en la magnitud de un parámetro, tal como inflamación, dolor, o deficiencia inmune. En otra realización particular, la coccidiosis está causada por Eimera. The term "treatment", as used herein, refers to any type of therapy, which aims at termination, prevention, improvement or reduction of the susceptibility to suffer from a clinical condition, as described herein. In a preferred embodiment, the term "treatment" refers to prophylactic treatment (ie, a therapy to reduce the susceptibility to suffer a clinical condition) of a disorder or condition. Thus, "treatment", "treat", and its equivalent terms refer to obtaining a desired pharmacologically or physiologically desired effect, which covers any treatment of a pathological condition or disorder in a mammal, including humans. The effect can be prophylactic in terms of providing total or partial prevention of a disorder and / or adverse effect attributable to it. That is, "treatment" includes (1) preventing the disease from occurring or recurring in a subject, (2) inhibiting the disease, for example by stopping its development, (3) interrupting or ending the disorder or at least the associated symptoms at the same time, so that the patient would no longer suffer the disease or its symptoms, for example cause the regression of the disease or its symptoms by restoring or repairing a lost, absent or defective function, or stimulating an inefficient process, or ( 4), reduce, relieve or improve the disease, or the symptoms associated with it, where reducing is used in a broad sense to refer to at least one reduction in the magnitude of a parameter, such as inflammation, pain, or immune deficiency In another particular embodiment, coccidiosis is caused by Eimera.
En una realización particular, dichas especies de Eimeria se seleccionan entre E. acervulina, E. brunetti, E. hagani, E. máxima, E. mitis, E. mivati, E. necatrix, E. praecox, E.tenella, E. adenoeides, E. gallopavonis, E. innocua, E. meleagridis, E. meleagrimitis, E. subrotunda, E. scabra, E.porci, E. polita, E.suis, E. bovis, E. zuernii, E. ellipsoidalis, E. cylindrica, E. auburnensis, E. alabamensis, E. subspherica, E. wyomingensis, E. ahsata, E. bakuensis, E.crandallis, E. faurei, E. intricata, E. marsica, E. ovinoidalis, E. pallida, E. parva, E. weybridgensis, E. gilruthi, E. gonzalezi, E. granulosa, E. punctata, E. arloingi, E. christenseni, E. ninakohlyakimovae, E. alijevi, E. hirci, E. caprina, E. caprovina, E. pallida, E. jolchijevi, E. apsheronica, E. stiedae, E. irresidua, E. magna, E. media y E. perforans. Asimismo, el término "Eimeria" incluye cualquier cepa de cualquier especie de Eimeria, incluyendo las cepas atenuadas y las virulentas. In a particular embodiment, said Eimeria species are selected from E. acervulina, E. brunetti, E. hagai, E. maxim, E. mitis, E. mivati, E. necatrix, E. praecox, E.tenella, E. adenoeides, E. gallopavonis, E. innocua, E. meleagridis, E. meleagrimitis, E. subrotunda, E. scabra, E.porci, E. polita, E.suis, E. bovis, E. zuernii, E. ellipsoidalis, E. cylindrica, E. auburnensis, E. alabamensis, E. subspherica, E. wyomingensis, E. ahsata, E. bakuensis, E.crandallis, E. faurei, E. intricata, E. marsica, E. ovinoidalis, E. pallida, E. parva, E. weybridgensis, E. gilruthi, E. gonzalezi, E. granulosa, E. punctata, E. arloingi, E. christenseni, E. ninakohlyakimovae, E. alijevi, E. hirci, E. caprina, E. caprovina, E. pallida, E. jolchijevi, E. apsheronica, E. stiedae, E. irresidua, E. magna, E. media and E. perforans. Also, the term "Eimeria" includes any strain of any species of Eimeria, including attenuated and virulent strains.
En una realización más particular, la especie de Eimeria es E. tenella. In a more particular embodiment, the species of Eimeria is E. tenella.
Un experto en la materia conoce técnicas que permiten diagnosticar a un sujeto que padece coccidiosis. Entre las técnicas destaca la detección de ooquistes en heces mediante una técnica de flotación convencional con recuento posterior, o la detección de ooquistes en el intestino, como la técnica cuantitativa de McMaster. Adicionalmente es posible observar el parásito en raspados seriados de la mucosa intestinal (MRSMI) de manera sencilla empleando métodos de tinción corriente. A person skilled in the art knows techniques that allow the diagnosis of a subject suffering from coccidiosis. Techniques include the detection of oocysts in feces by a conventional flotation technique with subsequent counting, or the detection of oocysts in the intestine, such as the quantitative McMaster technique. Additionally, it is possible to observe the parasite in serial scrapes of the intestinal mucosa (MRSMI) in a simple way using current staining methods.
En relación con el diagnóstico de pododermatitis en un sujeto, ya sea asociada o no a coccidiosis, suele realizarse por inspección visual y radiológica, para valorar el estado de los tejidos blandos y el hueso. La termografía permite detectar la pododermatitis antes de la aparición de lesiones, por la mayor diferencia entre las temperaturas máxima y mínima de la almohadilla plantar y por el patrón térmico. Existen diversos criterios para determinar la gravedad de las lesiones, entre ellos destacan las escala de 3 puntos o la escala de 1 1 puntos. Otros métodos están basados en la severidad o en el área de la lesión absoluta o relativa. En otros casos, la severidad y la superficie están combinados. In relation to the diagnosis of pododermatitis in a subject, whether associated or not with coccidiosis, it is usually done by visual and radiological inspection, to assess the state of soft tissues and bone. Thermography allows detecting pododermatitis before the appearance of lesions, due to the greater difference between the maximum and minimum temperatures of the plantar pad and the thermal pattern. There are several criteria to determine the severity of the lesions, including the 3-point scale or the 1-1-point scale. Other methods are based on the severity or area of the absolute or relative injury. In other cases, the severity and the surface are combined.
A modo de ejemplo, la severidad de la pododermatitis puede clasificarse en tres tipos. As an example, the severity of pododermatitis can be classified into three types.
Tipo I: Es el inicio de una lesión en la palma de la garra. Normalmente está localizada en un solo dedo. Es de características inflamatorias y aunque muy debilitado, todavía existe un epitelio de cubierta. La lesión puede ser proliferativa o degenerativa con tendencia a la ulceración. Dentro de este tipo no existen signos clínicos de dolor o molestias al principio. Normalmente se diagnostica por inspección rutinaria. No existe una infección clínica aparente Tipo II Es un grado más del tipo anterior. Comienza a aparecer signos de infección. En los cultivos normalmente se aisla Staphilococcus aureus, Proteus sp y E. Coli. Estos hallazgos de laboratorio confirman el origen epitelial y fecal de estas infecciones. Comienza a aparecer un dolor marcado, con retirada del apoyo en la pata afectada y carga en la otra, (situación que con el tiempo termina deteriorando ambas extremidades). Aparecen abscesos supurantes y marcada reacción fibrosa de los tejidos. Type I: It is the beginning of an injury in the palm of the claw. It is usually located on a single finger. It is inflammatory and although very weakened, there is still a cover epithelium. The lesion may be proliferative or degenerative with a tendency to ulceration. Within this type there are no clinical signs of pain or discomfort at first. It is usually diagnosed by routine inspection. There is no apparent clinical infection Type II It is one more degree of the previous type. Signs of infection begin to appear. In cultures, Staphilococcus aureus, Proteus sp and E. Coli are normally isolated. These laboratory findings confirm the epithelial and fecal origin of these infections. A marked pain begins to appear, with withdrawal of the support in the affected leg and load in the other, (situation that eventually deteriorates both limbs). Suppurating abscesses and marked fibrous reaction of the tissues appear.
Tipo III Es el grado de pododermatitis más avanzado. Se distinguen deformaciones y una reacción fibrosa abundante. Es muy común la aparición de osteomielitis con compromiso articular grave. En estos casos se suelen intentar tratamientos médicos, pero indefectiblemente terminan con la muerte del animal, ya sea por un deterioro progresivo o por eutanasia. Complicaciones graves: osteomielitis, endocarditis valvular.  Type III It is the most advanced degree of pododermatitis. Deformations and an abundant fibrous reaction are distinguished. Osteomyelitis with severe joint involvement is very common. In these cases, medical treatments are usually attempted, but they inevitably end with the death of the animal, either due to progressive deterioration or euthanasia. Serious complications: osteomyelitis, valvular endocarditis.
Así, en formas preferidas de realización, la parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma o las composiciones de la invención se emplean de acuerdo a la presente invención para el tratamiento de la pododermatitis de tipo I, de tipo II o de tipo III. Thus, in preferred embodiments, the part of an antioxidant-enriched corn plant or a derivative thereof or the compositions of the invention are used according to the present invention for the treatment of type I pododermatitis, of type II or type III.
En una realización particular, el sujeto a ser tratado es un ave, más particularmente un ave de corral y más particularmente un pollo. In a particular embodiment, the subject to be treated is a bird, more particularly a poultry and more particularly a chicken.
En otra realización particular, la planta de maíz enriquecida en antioxidantes es un maíz transgénico que expresa al menos un gen de la ruta de biosíntesis de carotenoides. En una realización más particular el gen de la ruta de biosíntesis de carotenoides se selecciona entre un gen que codifica la fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa, y un gen que codifica una carotenoide cetolasa. In another particular embodiment, the antioxidant-enriched corn plant is a transgenic corn that expresses at least one gene from the carotenoid biosynthesis pathway. In a more particular embodiment the gene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase, a gene that encodes a β- carotene hydroxylase, and a gene that encodes a carotenoid ketolase.
En otra realización la planta de maíz enriquecida en antioxidantes es un maíz transgénico que expresa al menos 2 genes de la ruta de biosíntesis de carotenoides, más particularmente al menos un gen que codifica una fitoeno sintasa y un gen que codifica una caroteno desaturasa; un gen que codifica una fitoeno sintasa y un gen que codifica una licopeno β-ciclasa; un gen que codifica una fitoeno sintasa y un gen que codifica una β-caroteno hidroxilasa; un gen que codifica una fitoeno sintasa y un gen que codifica una carotenoide cetolasa; un gen que codifica una caroteno desaturasa y un gen que codifica una licopeno β-ciclasa; un gen que codifica una caroteno desaturasa y un gen que codifica una β-caroteno hidroxilasa, un gen que codifica una caroteno desaturasa y un gen que codifica una carotenoide cetolasa; un gen que codifica una licopeno β-ciclasa y un gen que codifica una β-caroteno hidroxilasa; un gen que codifica una licopeno β-ciclasa y un gen que codifica una carotenoide cetolasa o al menos un gen que codifica una β-caroteno hidroxilasa y un gen que codifica una carotenoide cetolasa. In another embodiment, the antioxidant-enriched corn plant is a transgenic corn that expresses at least 2 genes from the carotenoid biosynthesis pathway, more particularly at least one gene encoding a phytoen synthase and a gene that encodes a carotene desaturase; a gene that encodes a phytoen synthase and a gene that encodes a lycopene β-cyclase; a gene that encodes a phytoeno synthase and a gene that encodes a β-carotene hydroxylase; a gene that encodes a phytoeno synthase and a gene that encodes a carotenoid ketolase; a gene that encodes a carotene desaturase and a gene that encodes a lycopene β-cyclase; a gene that encodes a carotene desaturase and a gene that encodes a β-carotene hydroxylase, a gene that encodes a carotene desaturase and a gene that encodes a carotenoid ketolase; a gene that encodes a lycopene β-cyclase and a gene that encodes a β-carotene hydroxylase; a gene encoding a lycopene β-cyclase and a gene encoding a carotenoid ketolase or at least one gene encoding a β-carotene hydroxylase and a gene encoding a carotenoid ketolase.
En otra realización la planta de maíz enriquecida en antioxidantes es un maíz transgénico que expresa al menos 3 genes de la ruta de biosíntesis de carotenoides, más particularmente al menos un gen que codifica una fitoeno sintasa, un gen que codifica una caroteno desaturasa y un gen que codifica una licopeno β-ciclasa; un gen que codifica una fitoeno sintasa, un gen que codifica una caroteno desaturasa y un gen que codifica una β-caroteno hidroxilasa; un gen que codifica una fitoeno sintasa, un gen que codifica una caroteno desaturasa y un gen que codifica una carotenoide cetolasa; un gen que codifica una fitoeno sintasa, un gen que codifica una licopeno β- ciclasa y un gen que codifica una β-caroteno hidroxilasa; un gen que codifica una fitoeno sintasa, un gen que codifica una licopeno β-ciclasa y un gen que codifica una carotenoide cetolasa; un gen que codifica una fitoeno sintasa, un gen que codifica una β-caroteno hidroxilasa y un gen que codifica una carotenoide cetolasa; un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa y un gen que codifica una β-caroteno hidroxilasa; un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa y un gen que codifica una carotenoide cetolasa; un gen que codifica una caroteno desaturasa, un gen que codifica una β- caroteno hidroxilasa y un gen que codifica una carotenoide cetolasa o al menos un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa y un gen que codifica una carotenoide cetolasa. In another embodiment, the antioxidant-enriched corn plant is a transgenic corn that expresses at least 3 genes of the carotenoid biosynthesis pathway, more particularly at least one gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase and a gene encoding a lycopene β-cyclase; a gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase and a gene that encodes a β-carotene hydroxylase; a gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase and a gene that encodes a carotenoid ketolase; a gene that encodes a phytoeno synthase, a gene that encodes a lycopene β-cyclase and a gene that encodes a β-carotene hydroxylase; a gene that encodes a phytoeno synthase, a gene that encodes a lycopene β-cyclase and a gene that encodes a carotenoid ketolase; a gene that encodes a phytoeno synthase, a gene that encodes a β-carotene hydroxylase and a gene that encodes a carotenoid ketolase; a gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase and a gene that encodes a β-carotene hydroxylase; a gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase and a gene that encodes a carotenoid ketolase; a gene that encodes a carotene desaturase, a gene that encodes a β-carotene hydroxylase and a gene that encodes a carotenoid ketolase or at least one gene that encodes a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase and a gene which encodes a carotenoid ketolase.
En otra realización la planta de maíz enriquecida en antioxidantes es un maíz transgénico que expresa al menos 4 genes de la ruta de biosíntesis de carotenoides, más particularmente al menos un gen que codifica una fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa y un gen que codifica una β-caroteno hidroxilasa; un gen que codifica una fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa y un gen que codifica una carotenoide cetolasa; un gen que codifica una fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una β- caroteno hidroxilasa y un gen que codifica una carotenoide cetolasa; un gen que codifica una fitoeno sintasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa y un gen que codifica una carotenoide cetolasa o al menos un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa y un gen que codifica una carotenoide cetolasa. In another embodiment, the antioxidant-enriched corn plant is a transgenic corn that expresses at least 4 genes of the carotenoid biosynthesis pathway, more particularly at least one gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase, a gene encoding a lycopene β-cyclase and a gene encoding a β-carotene hydroxylase; a gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase and a gene that encodes a carotenoid ketolase; a gene that encodes a phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a β-carotene hydroxylase and a gene that encodes a carotenoid ketolase; a gene that encodes a phytoeno synthase, a gene that encodes a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase and a gene that encodes a carotenoid ketolase or at least one gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase and a gene that encodes a carotenoid ketolase.
En otra realización particular, la planta de maíz enriquecida en antioxidantes es un maíz transgénico que expresa al menos 5 genes de la ruta de biosíntesis de carotenoides, más particularmente un gen que codifica la fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa y un gen que codifica una carotenoide cetolasa. In another particular embodiment, the antioxidant-enriched corn plant is a transgenic corn that expresses at least 5 genes of the carotenoid biosynthesis pathway, more particularly a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene which encodes a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase and a gene that encodes a carotenoid ketolase.
En otra realización particular, el gen que codifica para la fitoeno sintasa tiene la secuencia mostrada en SEQ ID NO: 1 , de Zea mays. In another particular embodiment, the gene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1, of Zea mays.
En otra realización particular, el gen que codifica una caroteno desaturasa tiene la secuencia mostrada en SEQ ID NO:2 de Pantoea ananatis, formalmente Erwinia uredovora. In another particular embodiment, the gene encoding a carotene desaturase has the sequence shown in SEQ ID NO: 2 of Pantoea ananatis, formally Erwinia uredovora.
En otra realización particular, el gen que codifica una licopeno β-ciclasa tiene la secuencia mostrada en SEQ ID NO:3 de Gentiana lútea In another particular embodiment, the gene encoding a lycopene β-cyclase has the sequence shown in SEQ ID NO: 3 of Gentiana luteal
En otra realización particular, el gen que codifica una β-caroteno hidroxilasa tiene la secuencia mostrada en SEQ ID NO: 4 o SEQ ID NO: 12 de Zea mays o el gen con secuencia mostrada en SEQ ID NO: 5 de Gentiana lútea. In another particular embodiment, the gene encoding a β-carotene hydroxylase has the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12 of Zea mays or the gene with sequence shown in SEQ ID NO: 5 of Gentiana lutea.
En otra realización particular, el gen que codifica una carotenoide cetolasa tiene la secuencia mostrada en SEQ ID NO: 6 de Paracoccus sp N81106. In another particular embodiment, the gene encoding a carotenoid ketolase has the sequence shown in SEQ ID NO: 6 of Paracoccus sp N81106.
Adicionalmente la planta de maíz enriquecida en antioxidantes puede expresar otros transgenes adicionales tales como un transgén la ruta de biosíntesis de vitamina C, particulamente un gen que codifica para la dehidroascorbato reductasa, más particularmente el gen con la secuencia mostrada en SEQ ID NO: 7 de E. coli. Adicionalmente la planta de maíz enriquecida en antioxidantes puede expresar otros transgenes adicionales tales como un transgén la ruta de biosíntesis de folato, más particularmente el gen con secuencia mostrada en SEQ ID NO: 8 de Orazy sativa. Additionally, the antioxidant-enriched corn plant can express other additional transgenes such as a transgene the vitamin C biosynthesis pathway, in particular a gene encoding dehydroascorbate reductase, more particularly the gene with the sequence shown in SEQ ID NO: 7 of E. coli. Additionally, the antioxidant-enriched corn plant can express other additional transgenes such as a transgene folate biosynthesis pathway, more particularly the gene with sequence shown in SEQ ID NO: 8 of Orazy sativa.
Un experto en la materia conoce las técnicas adecuadas para la generación de plantas transgénicas. Para una revisión de la transferencia génica a plantas, incluyendo vectores, métodos de transferencia de ADN, etc, véase, por ejemplo, Twyman RM.2004, Handbook of Plant Biotechnology. John Wiley & Sons Inc., NY, pp 263-289, o Komari et al. , 2004 Handbook of Plant Biotechnology. John Wiley & Sons Inc., NY, pp 233-262. One skilled in the art knows the appropriate techniques for the generation of transgenic plants. For a review of gene transfer to plants, including vectors, DNA transfer methods, etc., see, for example, Twyman RM.2004, Handbook of Plant Biotechnology. John Wiley & Sons Inc., NY, pp 263-289, or Komari et al. , 2004 Handbook of Plant Biotechnology. John Wiley & Sons Inc., NY, pp 233-262.
Brevemente dichos métodos pueden incluir: 1) empleo de un vector vivo que lleve el material genético a la célula blanco mediante virus genéticamente modificados o el mecanismo natural de infección de la bacteria del suelo Agrobacterium tumefaciens 2) El uso de protoplastos, que son células vegetales a las que se les ha liberado de la pared celular, o 3) la biolística que consiste en bombardear las células con partículas metálicas microscópicas recubiertas del ADN que se desea introducir. Briefly, such methods may include: 1) use of a live vector that carries the genetic material to the white cell by means of genetically modified viruses or the natural mechanism of infection of the soil bacterium Agrobacterium tumefaciens 2) The use of protoplasts, which are plant cells to which they have been freed from the cell wall, or 3) the biolistics that consists of bombarding the cells with microscopic metal particles coated with the DNA that they wish to introduce.
En concreto, para la generación de plantas de maíz transgénico se ha propuesto la introducción de ADN en células de maíz incluyendo la electroporación, microinyección, bombardeo con microproyectiles, fusión de liposomas, transferencia mediada por Agrobacterium, macroinyección y la exposición a ADN desnudo en solución. Specifically, for the generation of transgenic corn plants, the introduction of DNA into corn cells has been proposed including electroporation, microinjection, microprojectile bombardment, liposome fusion, Agrobacterium-mediated transfer, macroinjection and exposure to naked DNA in solution. .
Para lograr que el gen contenido en la molécula de ADN recombinante se exprese en los niveles y en los tejidos adecuados, es deseable, pero no necesario para genes de origen vegetal, que esté contenido en un casette de expresión que incluya una secuencia promotora de la transcripción funcional en plantas, la parte codificante del gen de interés y un terminador de la transcripción funcional en plantas. Adicionalmente se puede incluir una secuencia que codifique para un péptido de tránsito que dirija la enzima a un compartimiento específico de la célula. El péptido de tránsito y las señales de procesamiento correspondientes puede ser derivado de cualquier proteína vegetal que se sintetiza en el citoplasma y es translocada al compartimiento subcelular de interés, ya sea este el plástido o la mitocondria. Por ejemplo, para localizar en los plástidos pueden ser usadas secuencias derivadas de genes que codifican para la subunidad pequeña de la ribulosa bisfosfato carboxilasa o las proteínas CAB que son aquellas que se unen a las clorofilas a y b para formal las antenas cosechadoras de luz (Van den Broek et al Nature (London) 313, 358-363 (1985)). To ensure that the gene contained in the recombinant DNA molecule is expressed at the appropriate levels and tissues, it is desirable, but not necessary for genes of plant origin, that it is contained in an expression cassette that includes a promoter sequence of the functional transcription in plants, the coding part of the gene of interest and a terminator of functional transcription in plants. Additionally, a sequence encoding a transit peptide that directs the enzyme to a specific cell compartment can be included. The transit peptide and the corresponding processing signals can be derived from any plant protein that is synthesized in the cytoplasm and is translocated to the subcellular compartment of interest, be it the plastid or the mitochondria. For example, sequences derived from genes encoding the small subunit of the ribulose bisphosphate carboxylase or CAB proteins which are CAB proteins can be used to locate plastids those that bind with chlorophylls a and b to formalize light harvesting antennas (Van den Broek et al Nature (London) 313, 358-363 (1985)).
Para el caso de genes que no son de origen vegetal, en la secuencia iniciadora de la transcripción se debe incluir un promotor y se puede emplear también una señal de inicio de la traducción óptima para plantas o uno de los llamados potenciadores de la traducción. In the case of genes that are not of plant origin, a promoter must be included in the transcription initiation sequence and an optimal translation start signal for plants or one of the so-called translation enhancers can also be used.
Los transgenes que pueden emplearse en la presente invención pueden ponerse bajo el control de elementos reguladores que aseguran la expresión en las células de las plantas. En general, tales elementos reguladores comprenden un promotor activo en las células de las plantas. The transgenes that can be used in the present invention can be placed under the control of regulatory elements that ensure expression in plant cells. In general, such regulatory elements comprise an active promoter in plant cells.
Por tanto, los promotores que pueden ser usados incluyen promotores de la planta que se desea transformar, promotores de otras plantas o de cualquier origen que sean funcionales en la planta blanco y dirijan una expresión constitutiva, inducible o tejido específica. Por ejemplo, se pueden utilizar promotores derivados del plásmido Ti de Agrobactertum tumefaciens como son los promotores de la octopina sintetasa, la nopahna sintetasa o la sgropina Sintetasa. Además, se pueden incluir otros promotores como el promotor 35S del virus del mosaico de la coliflor o aquellos derivados de geminivirus. Para una revisión de promotores útiles puede verse Peremarti A. et al, Plant Mol Biol 73:363-378, 2010. Therefore, promoters that can be used include promoters of the plant to be transformed, promoters of other plants or of any origin that are functional in the white plant and direct a specific constitutive, inducible or tissue expression. For example, promoters derived from the Ti plasmid of Agrobactertum tumefaciens can be used such as the promoters of octopine synthetase, nopahna synthetase or sgropin synthetase. In addition, other promoters such as the 35S promoter of cauliflower mosaic virus or those derived from geminivirus may be included. For a review of useful promoters, see Peremarti A. et al, Plant Mol Biol 73: 363-378, 2010.
La secuenciadora terminadora de la transcripción puede ser derivada del mismo gen de donde se obtuvo la secuencia promotora de la transcripción o de un gen distinto. La secuencia terminadora puede ser derivada de genes contenidos en el T-DNA del plásmido Ti de Agrobacterium, del virus del mosaico de la coliflor o de genes de origen vegetal. Por ejemplo, la secuencia terminadora del gen de la nopalina sintetasa o la secuencia terminadora del gen ADPGP (ADP glucosa pilrofosforilasa de arroz) es frecuentemente usado en la Ingeniería Genética de plantas. En una realización particular, la secuencia terminadora de la transcripción del gen de a nopalina sintasa tiene la secuencia mostrada en SEQ ID NO: 14, y la secuencia terminadora del gen ADPGPP tiene la secuencia mostrada en SEQ ID NO: 15. The transcription terminator sequencer can be derived from the same gene from which the transcription promoter sequence was obtained or from a different gene. The terminator sequence may be derived from genes contained in the T-DNA of the Ti plasmid from Agrobacterium, from the cauliflower mosaic virus or from genes of plant origin. For example, the terminator sequence of the nopaline synthetase gene or the terminator sequence of the ADPGP gene (ADP glucose rice pyrophosphorylase) is frequently used in Plant Genetic Engineering. In a particular embodiment, the nopaline synthase gene transcription terminator sequence has the sequence shown in SEQ ID NO: 14, and the ADPGPP gene terminator sequence has the sequence shown in SEQ ID NO: 15.
La diferentes secuencias de ácidos nucleicos que forman parte de la molécula de ADN recombinante pueden ser unidas por métodos convencionales descritos en la literatura. Las secuencias deben ser clonadas y unidas en la orientación y orden correcto para lograr la expresión funcional en células vegetales. De acuerdo con uno de los aspectos de esta invención, las plantas de interés son transformadas con una molécula de ADN recombinante que codifica al menos una enzima que participa en la biosíntesis de carotenoides. En el desarrollo de la molécula recombinante, las diferentes secuencias nucleotídicas que la componen y que comprenden las secuencias regulatorias de la transcripción y la secuencia codificante de interés, pueden haber sido sujetas a diferentes tipos de procesamiento, como son ligaciones, digestiones con enzimas de restricción, mutagénesis in vitro, adición de oligonucleótidos o modificaciones por medio de la reacción de polimerización en cadena (PCR). Por lo tanto, los componentes de la molécula recombinante antes de ser unidos pudieron estar sujetos a deleciones, inserciones o modificaciones internas. Como la molécula recombinante es derivada de componentes que se originan de diferentes organismos y que han sido aislados, purificados o sintetizados, no es una molécula que existe como tal en la naturaleza. The different nucleic acid sequences that are part of the recombinant DNA molecule can be linked by conventional methods described in the literature. The sequences must be cloned and linked in the correct orientation and order to achieve functional expression in plant cells. According to one of the aspects of this invention, the plants of interest are transformed with a recombinant DNA molecule that encodes at least one enzyme that participates in the biosynthesis of carotenoids. In the development of the recombinant molecule, the different nucleotide sequences that compose it and that comprise the regulatory sequences of the transcription and the coding sequence of interest, may have been subjected to different types of processing, such as ligaments, digestion with restriction enzymes , in vitro mutagenesis, oligonucleotide addition or modifications by means of the chain polymerization reaction (PCR). Therefore, the components of the recombinant molecule before being bound could be subject to deletions, insertions or internal modifications. Since the recombinant molecule is derived from components that originate from different organisms and that have been isolated, purified or synthesized, it is not a molecule that exists as such in nature.
Dependiendo del método de transformación utilizado para introducir la molécula recombinante a la célula vegetal de interés, puede ser necesario la inclusión de otras secuencias de ADN. Por ejemplo, es necesario incluir un vector de clonación molecular que permita su replicación en E. coli y, para algunos casos, que permita la replicación en A. tumefaciens. Para el uso del sistema de transformación genética mediada por el plásmido Ti de Agrobacterium para introducir la molécula recombinante en el genoma de la célula vegetal, es necesario incluir las secuencias bordes del T- DNA, de manera tal que estén presentes a ambos extremos del gen que codifica la enzima que sintetiza ácidos orgánicos y en algunos casos el gen de selección dominante. El uso de cepas de Agrobacterium desarmadas, es decir aquellas a las que se les han removido los genes responsables de la formación de tumores, pero que mantienen la capacidad de transferir ADN a células vegetales, permite la regeneración de plantas transgénicas que contienen la molécula recombinante de interés. Depending on the transformation method used to introduce the recombinant molecule to the plant cell of interest, the inclusion of other DNA sequences may be necessary. For example, it is necessary to include a molecular cloning vector that allows replication in E. coli and, for some cases, that allows replication in A. tumefaciens. For the use of the Agrobacterium Ti plasmid-mediated genetic transformation system to introduce the recombinant molecule into the genome of the plant cell, it is necessary to include the T-DNA border sequences, so that they are present at both ends of the gene which encodes the enzyme that synthesizes organic acids and in some cases the dominant selection gene. The use of unarmed Agrobacterium strains, that is, those whose genes responsible for tumor formation have been removed, but maintain the ability to transfer DNA to plant cells, allows the regeneration of transgenic plants that contain the recombinant molecule of interest.
También generalmente se incluye un gen de selección dominante que permita identificar y seleccionar las células que incorporaron de manera estable la molécula recombinante en su genoma. A modo de ejemplo ilustrativo no limitativo, los genes marcadores pueden ser la mutación rrn16 (16S rRNA) que confiere resistencia a espectinomicina y estreptinomicina, la mutación rps12 (ribosoma) que confiere resistencia a estreptinomicina, gen aadA (E. coli) que codifica la enzima aminoglucósido 3' adeniltransferasa que confiere resistencia a los antibióticos espectinomicina y estreptinomicina, gen nptll procedente del transposón bacteriano Tn5 codifica la enzima neomicina fosfotransferasa que confiere resistencia al antibiótico kanamicina, el gen aphA-6 de Acinetobacter baumannii que codifica la enzima aminoglucósido fosfotransferasa la cual le confiere resistencia al antibiótico kanamicina, gen cat de E. coli confiere resistencia a cloranfenicol, gen ASA2 responsable de la resistencia a análogos del triptófano, el gen bar de Streptomyces hygroscopicus que codifica para la enzima fosfinotricina acetil transferasa (PAT) que confiere resistencia a fosfinotricina y el gen pat de Streptomyces viridochromogenes convierten el glufosinato de amonio en nacetil glufosinato que es un compuesto no fitotóxico para la planta. A dominant selection gene is also generally included to identify and select the cells that stably incorporated the recombinant molecule into their genome. As an illustrative non-limiting example, the marker genes can be the rrn16 mutation (16S rRNA) that confers resistance to spectinomycin and streptinomycin, the rps12 mutation (ribosome) that confers streptinomycin resistance, aadA gene (E. coli) that encodes the 3 'adenyltransferase aminoglycoside enzyme that confers resistance to the antibiotics spectinomycin and streptinomycin, nptll gene from the bacterial transposon Tn5 encodes the enzyme neomycin phosphotransferase that confers resistance to the antibiotic kanamycin, the aphA-6 gene of Acinetobacter baumannii that encodes the enzyme aminoglycoside phosphotransferase which confers resistance to the antibiotic kanamycin, E. coli cat gene confers resistance to chloramphenicol, ASA gene responsible of resistance to tryptophan analogs, the Streptomyces hygroscopicus bar gene that encodes the phosphinothricin acetyl transferase enzyme (PAT) that confers phosphinothricin resistance and the Streptomyces viridochromogenes pat gene converts ammonium glufosinate into nacetyl glufosinate which is a non-compound phytotoxic for the plant.
En una realización preferida el gen de selección es el gen bar. En una realización más particular, el gen óartiene la secuencia mostrada en SEQ ID NO: 9 In a preferred embodiment the selection gene is the bar gene. In a more particular embodiment, the gene has the sequence shown in SEQ ID NO: 9
Dependiendo del tipo de gen de selección que se incluya, puede ser aconsejable que una vez se ha identificado la planta transgénica, se elimine el gen marcador de selección de la planta. Depending on the type of selection gene that is included, it may be advisable that once the transgenic plant has been identified, the plant selection marker gene is eliminated.
La expresión temporal, inducible o tejido específica puede ser lograda mediante el uso de promotores o secuencias reguladoras que tienen la especificidad de expresión deseada. Con objeto de conseguir la expresión en tejidos específicos de una planta transgénica es posible utilizar promotores específicos de tejido (véase Stockhaus, EMBO J. 8 (1989), 2245-2251). Se conocen también promotores que son específicamente activos en semillas de diferentes especies de plantas, tales como maíz. Temporary, inducible or specific tissue expression can be achieved through the use of promoters or regulatory sequences that have the desired expression specificity. In order to achieve expression in specific tissues of a transgenic plant it is possible to use tissue specific promoters (see Stockhaus, EMBO J. 8 (1989), 2245-2251). Promoters that are specifically active in seeds of different plant species, such as corn, are also known.
En una realización particular el transgén o transgenes se encuentra bajo el control de un promotor específico de endospermo. In a particular embodiment the transgene or transgenes is under the control of a specific endosperm promoter.
Promotores específicos de endospermo útiles en la presente invención, son entre otros los promotores de genes que codifican para proteínas de almacenamiento en semillas, Galili y col., 1993, Trends Cell Biol 3:437-442. Las prolaminas, son las proteínas de almacenamiento de cereales más abundantes, en maíz las zeínas (zeínas α, β, γ y δ- zeínas, en trigo las gluteninas, en cebada hordeína, secalina en centeno y acenina en avena. Specific endosperm promoters useful in the present invention are, among others, gene promoters that code for seed storage proteins, Galili et al., 1993, Trends Cell Biol 3: 437-442. Prolamines are the most abundant cereal storage proteins, in corn the zeins (α, β, γ and δ-zein zeins, in gluten glutenins, in hordein barley, rye secalin and oats acenin.
Ejemplos ilustrativos, no limitativos, de promotores específicos de endospermo que pueden emplearse en la presente invención son entre otros los promotores de glutenina LMW de trigo, hordeína de cebada, prolamina de arroz, glutelina 1 de arroz, zeína de maíz, de zeina 27 Kda, ADP-glucosa pirofosforilasa de arroz, glutelina I de arroz tal y como se describe en Russel DA. et al., Transgenic research 6, 157-168 (1997). También son útiles, los promotores de MEG-1 tal y como se describe en EP1680506B1. Illustrative, non-limiting examples of specific endosperm promoters that may be employed in the present invention are among others the LMW wheat gluten, barley hordein, rice prolamine, rice glutelin 1 promoters, corn zein, zeina 27 Kda, rice ADP-glucose pyrophosphorylase, rice glutelin I as described in Russel DA. et al., Transgenic research 6, 157-168 (1997). Also useful are the promoters of MEG-1 as described in EP1680506B1.
En una realización particular el promotor se selecciona del promotor de D-hordeína de cebada con secuencia mostrada en SEQ ID NO: 10 o del promotor de LMW glutenina de trigo con secuencia mostrada en SEQ ID NO: 1 1 . In a particular embodiment the promoter is selected from the barley D-hordein promoter with sequence shown in SEQ ID NO: 10 or from the LMW wheat glutenin promoter with sequence shown in SEQ ID NO: 1 1.
En una forma preferida de realización, el gen de la fitoeno sintasa se encuentra bajo control del promotor de la LMW glutenina, preferiblemente del promotor de la LMW glutenina de trigo. En otra forma preferida de realización, el gen de la carotena desaturasa se encuentra bajo control del promotor de la D-hordeina, preferiblemente del promotor de la D-hordeina de cebada. In a preferred embodiment, the phytoeno synthase gene is under the control of the LMW glutenin promoter, preferably of the LMW wheat glutenin promoter. In another preferred embodiment, the carotene desaturase gene is under the control of the D-hordein promoter, preferably of the barley D-hordein promoter.
Los elementos reguladores pueden comprender adicionalmente intensificadores de la transcripción y/o traducción funcionales en las células de las plantas. Adicionalmente, los elementos reguladores pueden incluir señales de terminación de la transcripción, tales como una señal poli-A, que conducen a la adición de una cola poli-A al transcrito que puede mejorar su estabilidad. The regulatory elements may further comprise functional transcription and / or translation enhancers in plant cells. Additionally, the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly-A tail to the transcript that can improve its stability.
En otro aspecto preferido, si el transgén no es de origen vegetal, por ejemplo el transgén es de bacteria, entonces la enzima codificada por dicho transgen forma una proteína de fusión que comprende una secuencia para el direccionamiento de proteínas al cloroplasto en posición N-terminal respecto a la secuencia del transgén. In another preferred aspect, if the transgene is not of plant origin, for example the transgene is bacterial, then the enzyme encoded by said transgene forms a fusion protein comprising a sequence for the targeting of proteins to the N-terminal position chloroplast. regarding the sequence of the transgene.
En una realización preferida, la secuencia para el direccionamiento de proteínas al cloroplasto, es la secuencia de la subunidad pequeñas de la ribulosa 1 , 5.bifosfato carboxilasa/oxigenasa tiene la secuencia mostrada en SEQ ID NO: 13. In a preferred embodiment, the sequence for the targeting of proteins to the chloroplast is the small subunit sequence of ribulose 1, 5 carboxylase / oxygenase diphosphate has the sequence shown in SEQ ID NO: 13.
Composiciones de la invención y usos de las mismas Compositions of the invention and uses thereof
En otro aspecto, la invención se relaciona con una composición que comprende una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma y un agente anticoccídico. In another aspect, the invention relates to a composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof and an anticoccidic agent.
En una forma preferida de realización, los antioxidantes son carotenoides. En una forma de realización aún más preferida, los carotenoides se seleccionan del grupo formado por β-caroteno, γ-caroteno, α-caroteno, α-criptoxantina, β-criptoxantina, licopeno, luteína y zeaxantina. In a preferred embodiment, the antioxidants are carotenoids. In an even more preferred embodiment, the carotenoids are selected from the group. formed by β-carotene, γ-carotene, α-carotene, α-cryptoxanthin, β-cryptoxanthin, lycopene, lutein and zeaxanthin.
En una forma preferida de realización, el maíz enriquecido en antioxidantes es un maíz transgénico que expresa al menos un transgén de la ruta de biosíntesis de carotenoides. En una forma de realización más preferida, el transgén de la ruta de biosíntesis de carotenoides se selecciona de entre un gen que codifica la fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa, y un gen que codifica una carotenoide cetolasa o una combinación de los mismos. En otra forma de realización, el transgén que codifica para la fitoeno sintasa tiene la secuencia mostrada en SEQ ID NO: 1 , el transgen que codifica para la caroteno desaturasa tiene la secuencia mostrada en SEQ ID NO: 2, el transgén que codifica para la lycopeno β- ciclasa tiene la secuencia mostrada en SEQ ID NO:3, el transgén que codifica para la β-caroteno hidroxilasa tiene la secuencia mostrada en SEQ ID NO:4, SEQ ID NO:5 o SEQ ID NO: 12, el transgén que codifica para la carotenoide cetolasa tiene la secuencia mostrada en SEQ ID NO:6. En una forma de realización aún más preferida, la planta de maíz contiene el transgén de que codifica la fitoeno sintasa y el gen que codifica la caroteno desaturasa. Otras combinaciones, tales como las mencionadas en el contexto del uso terapéutico de la invención son igualmente aplicables. In a preferred embodiment, the antioxidant enriched corn is a transgenic corn that expresses at least one transgene of the carotenoid biosynthesis pathway. In a more preferred embodiment, the transgene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase, and a gene that encodes a carotenoid ketolase or a combination thereof. In another embodiment, the transgene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1, the transgene encoding carotene desaturase has the sequence shown in SEQ ID NO: 2, the transgene encoding for Lycopene β-cyclase has the sequence shown in SEQ ID NO: 3, the transgene encoding β-carotene hydroxylase has the sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12, the transgene coding for the carotenoid ketolase has the sequence shown in SEQ ID NO: 6. In an even more preferred embodiment, the corn plant contains the transgene encoding phytoen synthase and the gene encoding carotene desaturase. Other combinations, such as those mentioned in the context of the therapeutic use of the invention are equally applicable.
En otra forma preferida de realización, el transgén o transgenes se encuentra o encuentran bajo el control de un promotor específico de endospermo. En una forma de realización más preferida, el promotor específico de endospermo es el promotor de D- hordeína o el promotor de LMW glutenina. En una forma de realización aún más preferida, el promotor de D-hordeína tiene la secuencia mostrada en SEQ ID NO: 10 y el promotor de LMW glutenina tiene la secuencia mostrada en SEQ ID NO: 1 1. En una forma de realización aún más preferida, la planta de maíz contiene el transgén de que codifica la fitoeno sintasa bajo el control del promotor de la glutenina LMW y el gen que codifica la caroteno desaturasa bajo el control de la D-hordeina de cebada. In another preferred embodiment, the transgene or transgenes is or is under the control of a specific endosperm promoter. In a more preferred embodiment, the specific endosperm promoter is the D-hordein promoter or the LMW glutenin promoter. In an even more preferred embodiment, the D-hordein promoter has the sequence shown in SEQ ID NO: 10 and the LMW glutenin promoter has the sequence shown in SEQ ID NO: 1 1. In an even more embodiment Preferably, the corn plant contains the transgene that encodes the phytoen synthase under the control of the LMW glutenin promoter and the gene that encodes the carotene desaturase under the control of the barley D-hordein.
En una forma preferida de realización, la parte de la planta de maíz enriquecida en antioxidantes se selecciona del grupo de endospermo y la semilla. In a preferred embodiment, the part of the antioxidant-enriched corn plant is selected from the endosperm and seed group.
El agente anticoccídico puede seleccionarse entre cocciostáticos y coccidiocidas. Con los coccidiostáticos se observa que el crecimiento intracelular de coccidios se detiene, pero su desarrollo puede continuar después de la retirada del medicamento; en el caso de coccidiocidas los coccidios mueren durante su desarrollo al exponerse al fármaco. Algunos fármacos anticoccidicos pueden ser coccidiostáticos cuando se administran a corto plazo, pero coccidicidas cuando se administran a largo plazo. Otra clasificación de los anticoccidicos es por su origen: ionóforos, que son productos de fermentación, y químicos-sintéticos. Entre los ionóforos se encuentran monensina, salinomicina, lasalocida, narasina, maduramicina y semduramicina. La monensina, narasina y salinomicina son más eficaces en contra de E.acervulina, pero poco eficaces en el control de E. tenella y E. máxima. La lasolacida y maduramicina son efectivos contra E. tenella, pero baja efectividad contra E. acervulina. La dosis de los ionóforos va dentro del 25-100 ppm, con excepción de la maduramicina que se dosifica a 5 ppm. Entre los compuestos químicos-sintéticos se encuentran amprolio, clopidol y quinolinas (por ejemplo decoquinato, metilbenzocuato), antagonistas del ácido fólico, entre los que se encuentran las sulfonamidas, las 2,4- diaminopirimidinas y el etopabato, hidrobromuro de halofuginona, nicarbazina, nitrobenzamidas (por ejemplo dinitolmida), robenidina, roxarsona, diclazurilo y toltrazurilo. The anticoccidic agent can be selected from coccytostatics and coccidiocides. With coccidiostats it is observed that intracellular growth of coccidia stops, but their development can continue after the withdrawal of the drug; if Coccidiocides coccidia die during their development when exposed to the drug. Some anticoccidic drugs may be coccidiostatic when administered in the short term, but coccidicidal when administered in the long term. Another classification of anticoccidics is by their origin: ionophores, which are fermentation products, and chemical-synthetics. Among the ionophores are monensin, salinomycin, lasalocide, narasin, maturemicin and semduramycin. Monensin, narasin and salinomycin are more effective against E.acervulina, but ineffective in the control of E. tenella and E. maxim. Lasolacida and Maduramycin are effective against E. tenella, but low effectiveness against E. acervulina. The dose of the ionophores is within 25-100 ppm, with the exception of mature microwaves that are dosed at 5 ppm. Among the chemical-synthetic compounds are amprolium, clopidol and quinolines (for example decoquinate, methylbenzocuate), folic acid antagonists, including sulfonamides, 2,4-diaminopyrimidines and ethopabate, halofuginone hydrobromide, nicarbazine, nitrobenzamides (for example dinitolmide), robenidine, roxarsona, diclazuril and toltrazuril.
Adicionalmente, los agentes anticoccidicos también incluyen vacunas orales. Additionally, anticoccidic agents also include oral vaccines.
A modo de ejemplos ilustrativos, no limitativos, la vacuna anticoccídica puede ser no atenuada (virulentas) o atenuada. By way of illustrative, non-limiting examples, the anticoccidic vaccine may be not attenuated (virulent) or attenuated.
Vacunas no atenuadas contienen ooquistes, coccidios virulentos vivos, es decir, los pollos están totalmente expuestos a los parásitos patógenos de la vacuna, además de la propagación virulenta de coccidia en el corral de aves. Por lo tanto, al utilizar estas vacunas, existe un riesgo de fuerte reacción después de la vacunación y de brote de coccidiosis clínica especialmente en las aves con un sistema inmunitario debilitado. Por lo tanto después de la administración de las vacunas virulentas es a menudo necesario y recomendado para ser aplicado incluso por los fabricantes de estas vacunas el tratamiento preventivo con anticoccidiales. Non-attenuated vaccines contain oocysts, live virulent coccidia, that is, chickens are fully exposed to the pathogenic parasites of the vaccine, in addition to the virulent spread of coccidia in the poultry yard. Therefore, when using these vaccines, there is a risk of strong reaction after vaccination and clinical coccidiosis outbreak especially in birds with a weakened immune system. Therefore, after the administration of virulent vaccines, preventive treatment with anticoccidials is often necessary and recommended to be applied even by the manufacturers of these vaccines.
Las vacunas atenuadas, es decir, las vacunas de la segunda generación, contienen ooquistes vivos de líneas atenuadas de especies de Eimerian. Estas vacunas son mucho más seguras debido a la patogenicidad biológicamente atenuada de los parásitos vacunales, y el desafío de infección controlado que presenta por lo tanto no hay riesgo de brote de coccidiosis clínica. En el caso de que se utilicen las vacunas atenuadas, es importante que no se utilicen coccidiostáticos para evitar la reducción de la eficacia de la vacuna. Ejemplo de vacuna anticoccídica por vía oral es IMMUCOXOT basada en ooquistes de Eimeria spp vivo, o PARACOX TM que contiene ooquistes esporulados en vivo derivadas de ocho líneas precoces de coccidios o HIPRACOX® basado en ooquistes esporulados en vivo derivados de cinco líneas atenuadas. The attenuated vaccines, that is, the second generation vaccines, contain live oocysts of attenuated lines of Eimerian species. These vaccines are much safer due to the biologically attenuated pathogenicity of vaccine parasites, and the challenge of controlled infection that therefore presents no risk of clinical coccidiosis outbreak. In the event that attenuated vaccines are used, it is important that coccidiostats are not used to avoid reducing the effectiveness of the vaccine. An example of an oral anticoccidic vaccine is IMMUCOXOT based on live Eimeria spp oocysts, or PARACOXTM containing live sporulated oocysts derived from eight early coccidial lines or HIPRACOX® based on live sporulated oocysts derived from five attenuated lines.
En otro aspecto la invención se relaciona con el uso de una composición que comprende una parte de una planta de maíz enriquecida en antioxidantes o de un derivado de la misma y un agente anticoccídico para la fabricación de un medicamento, composición nutritiva o nutraceutico para la prevención o tratamiento de pododermatitis o úlceras asociadas a coccidiosis en un sujeto. In another aspect the invention relates to the use of a composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof and an anticoccidic agent for the manufacture of a medicament, nutritive or nutraceutical composition for prevention or treatment of pododermatitis or ulcers associated with coccidiosis in a subject.
Alternativamente, la invención se relaciona con una composición que comprende una parte de una planta de maíz enriquecida en antioxidantes o de un derivado de la misma y un agente anticoccídico para su uso en la prevención o tratamiento de pododermatitis o úlceras asociadas a coccidiosis en un sujeto. Alternatively, the invention relates to a composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof and an anticoccidic agent for use in the prevention or treatment of pododermatitis or ulcers associated with coccidiosis in a subject .
En una realización particular de la composición que comprende una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma y un agente anticoccídico para su uso en la prevención o tratamiento de coccidiosis en un sujeto o para la revención de úlceras asociadas con pododermatitis asociadas a coccidiosis en un sujeto, el agente anticoccídico se administra a una dosis subterapéutica. In a particular embodiment of the composition comprising a part of an antioxidant-enriched corn plant or a derivative thereof and an anticoccidic agent for use in the prevention or treatment of coccidiosis in a subject or for the prevention of ulcers associated with pododermatitis associated with coccidiosis in a subject, the anticoccidic agent is administered at a subtherapeutic dose.
Dosis subterapéuticas, como se usa aquí, se refiere a la dosis por debajo de la cantidad requerida para un efecto terapéutico. Particularmente, la dosis subterapéuticas es al menos 10 % inferior, al menos 20 % inferior, al menos 30 % inferior, al menos 40 % inferior, al menos 50 % inferior, al menos 60 % inferior, al menos 70 % inferior, al menos 80 % menor , o al menos 90 % más bajo o más de la dosis mínima requerida para un efecto terapéutico. A modo de ejemplos ilustrativos, no limitativos , la dosis mínima de varios fármacos anticoccidicos se muestran en la Tabla 1. Subtherapeutic doses, as used herein, refers to the dose below the amount required for a therapeutic effect. Particularly, the subtherapeutic dose is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, or at least 90% lower or more than the minimum dose required for a therapeutic effect. By way of illustrative, non-limiting examples, the minimum dose of several anticoccidic drugs are shown in Table 1.
Fármaco Dosis mínima Drug Minimum dose
Narasina 40 ppm  Narasin 40 ppm
Nicarbazina 40 ppm  Nicarbazine 40 ppm
Monensina 100 ppm  Monensin 100 ppm
Narasina 60 ppm  Narasin 60 ppm
Diclazurilo 50 ppm  Diclazuril 50 ppm
Semduramicina 25 ppm  Semduramycin 25 ppm
Maduramicina 5 ppm Halofuginono 2 ppm obenidino 50 ppm Maduramycin 5 ppm Halofuginono 2 ppm obenidino 50 ppm
Lasalocida 75 ppm  Lasalocide 75 ppm
Amprolium 125 ppm  Amprolium 125 ppm
Sulfaquinoxalina 50 mg/L Sulfaquinoxaline 50 mg / L
Nicarbazina 125 ppm Nicarbazine 125 ppm
Furazolidono 75 ppm  Furazolidone 75 ppm
Decoquinato 20 ppm  20 ppm decocinate
Salinomicina 50 ppm  Salinomycin 50 ppm
Toltrazuriol 0,175 mg/Kg peso vivo  Toltrazuriol 0.175 mg / kg live weight
Tabla 1. Dosis mínimas de fármacos anticoccídicos (dosis en ppm se refieren a la concentración del fármaco en el pienso y las dosis en mg/l se refieren a la concentración de fármaco por litro de agua de bebida)  Table 1. Minimum doses of anticoccidic drugs (doses in ppm refer to the concentration of the drug in the feed and the doses in mg / l refer to the concentration of the drug per liter of drinking water)
Composiciones nutracéuticas de la invención, métodos para su preparación y usos de las mismas  Nutraceutical compositions of the invention, methods for their preparation and uses thereof
En otro aspecto la invención se relaciona con una composición nutritiva o nutracéutica que comprende una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma. In another aspect the invention relates to a nutritive or nutraceutical composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof.
La expresión "composición nutritiva o nutracéutico" se usa en el presente documento en un sentido amplio y cubre alimentos para seres humanos así como alimentos para animales de producción (es decir, un pienso). En un aspecto preferido, la composición nutritiva o nutracéutico es para consumo animal. La composición nutritiva o nutracéutico puede estar en forma de una disolución o como un sólido, dependiendo del uso y/o el modo de aplicación y/o el modo de administración. The term "nutritive or nutraceutical composition" is used herein in a broad sense and covers food for humans as well as food for production animals (ie, a feed). In a preferred aspect, the nutritive or nutraceutical composition is for animal consumption. The nutritive or nutraceutical composition may be in the form of a solution or as a solid, depending on the use and / or the mode of application and / or the mode of administration.
En una forma preferida de realización, los antioxidantes son carotenoides. En una forma de realización aún más preferida, los carotenoides se seleccionan del grupo formado por β-caroteno, γ-caroteno, α-caroteno, a-criptoxantina, β-criptoxantina, licopeno, luteína y zeaxantina. In a preferred embodiment, the antioxidants are carotenoids. In an even more preferred embodiment, the carotenoids are selected from the group consisting of β-carotene, γ-carotene, α-carotene, a-cryptoxanthin, β-cryptoxanthin, lycopene, lutein and zeaxanthin.
En una forma preferida de realización, el maíz enriquecido en antioxidantes es un maíz transgénico que expresa al menos un transgén de la ruta de biosíntesis de carotenoides. En una forma de realización más preferida, el transgén de la ruta de biosíntesis de carotenoides se selecciona de entre un gen que codifica la fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa, y un gen que codifica una carotenoide cetolasa o una combinación de los mismos. En otra forma de realización, el transgén que codifica para la fitoeno sintasa tiene la secuencia mostrada en SEQ ID NO: 1 , el transgen que codifica para la caroteno desaturasa tiene la secuencia mostrada en SEQ ID NO: 2, el transgén que codifica para la lycopeno β- ciclasa tiene la secuencia mostrada en SEQ ID NO:3, el transgén que codifica para la β-caroteno hidroxilasa tiene la secuencia mostrada en SEQ ID NO:4, SEQ I D NO: 5 o SEQ ID NO: 12, el transgén que codifica para la carotenoide cetolasa tiene la secuencia mostrada en SEQ ID NO:6. En una forma de realización aún más preferida, la planta de maíz contiene el transgén de que codifica la fitoeno sintasa y el gen que codifica la caroteno desaturasa. Otras combinaciones, tales como las mencionadas en el contexto del uso terapéutico de la invención son igualmente aplicables. In a preferred embodiment, the antioxidant enriched corn is a transgenic corn that expresses at least one transgene of the carotenoid biosynthesis pathway. In a more preferred embodiment, the transgene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase, and a gene that encodes a carotenoid ketolase or a combination thereof. In another form of embodiment, the transgene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1, the transgene encoding carotene desaturase has the sequence shown in SEQ ID NO: 2, the transgene encoding lycopene β-cyclase has the sequence shown in SEQ ID NO: 3, the transgene encoding β-carotene hydroxylase has the sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12, the transgene encoding for Carotenoid ketolase has the sequence shown in SEQ ID NO: 6. In an even more preferred embodiment, the corn plant contains the transgene encoding phytoen synthase and the gene encoding carotene desaturase. Other combinations, such as those mentioned in the context of the therapeutic use of the invention are equally applicable.
En otra forma preferida de realización, el transgén o transgenes se encuentra o encuentran bajo el control de un promotor específico de endospermo. En una forma de realización más preferida, el promotor específico de endospermo es el promotor de D- hordeína o el promotor de LMW glutenina. En una forma de realización aún más preferida, el promotor de D-hordeína tiene la secuencia mostrada en SEQ ID NO: 10 y el promotor de LMW glutenina tiene la secuencia mostrada en SEQ ID NO: 1 1. En una forma de realización aún más preferida, la planta de maíz contiene el transgén de que codifica la fitoeno sintasa bajo el control del promotor de la glutenina LMW y el gen que codifica la caroteno desaturasa bajo el control de la D-hordeina de cebada. In another preferred embodiment, the transgene or transgenes is or is under the control of a specific endosperm promoter. In a more preferred embodiment, the specific endosperm promoter is the D-hordein promoter or the LMW glutenin promoter. In an even more preferred embodiment, the D-hordein promoter has the sequence shown in SEQ ID NO: 10 and the LMW glutenin promoter has the sequence shown in SEQ ID NO: 1 1. In an even more embodiment Preferably, the corn plant contains the transgene that encodes the phytoen synthase under the control of the LMW glutenin promoter and the gene that encodes the carotene desaturase under the control of the barley D-hordein.
En una forma preferida de realización, la parte de la planta de maíz enriquecida en antioxidantes se selecciona del grupo de endospermo y la semilla. In a preferred embodiment, the part of the antioxidant-enriched corn plant is selected from the endosperm and seed group.
Las composiciones de la invención o nutracéuticos pueden estar en una forma líquida, semisólida o sólida. En una realización particular las composiciones o nutracéutico de la invención se encuentran en forma sólida y más particularmente es un pienso. The compositions of the invention or nutraceuticals may be in a liquid, semi-solid or solid form. In a particular embodiment the compositions or nutraceuticals of the invention are in solid form and more particularly it is a feed.
Las composiciones nutritivas o nutracéuticas de la invención pueden formularse con los excipientes y adyuvantes usuales para las composiciones orales o suplementos alimentarios, como por ejemplo y sin sentido limitativo, componentes grasos, componentes acuosos, humectantes, conservantes, agentes textu rizantes, sabores, aromas, antioxidantes y colorantes comunes en el sector alimentario. The nutritive or nutraceutical compositions of the invention can be formulated with the usual excipients and adjuvants for oral compositions or food supplements, such as and without limitation, fatty components, aqueous components, humectants, preservatives, texturizing agents, flavors, aromas, antioxidants and dyes common in the food sector.
Las composiciones nutritivas o nutracéuticas de la invención comprenden al menos un 0,01 % de la parte de una parte de una planta enriquecida en antioxidantes o derivado de la misma, más preferiblemente al menos un 0,10%, 0,50%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, o, al menos un 95%. The nutritive or nutraceutical compositions of the invention comprise at least 0.01% of the part of a part of a plant enriched in antioxidants or derived therefrom, more preferably at least 0.10%, 0.50%, 1 %, 2. 3. 4. 5%, 6%, 7%, 8%, 9%, 10%, 15% 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95 %.
Las necesidades nutritivas de los animales son bien conocidas en el estado de la técnica. Por ejemplo, las necesidades nutritivas de las aves de corral son descritas en Gail Damerow The Chicken Encyclopedia: An lllustrated Reference. Storey Publishing. pp. 118-1 19, 135-136 y en el libro Animal Nutrition handbook, sección 12 páginas 316-331 , 2009. The nutritional needs of animals are well known in the state of the art. For example, the nutritional needs of poultry are described in Gail Damerow The Chicken Encyclopedia: An Illustrated Reference. Storey Publishing. pp. 118-1 19, 135-136 and in the book Animal Nutrition handbook, section 12 pages 316-331, 2009.
Composiciones nutritivas útiles para la alimentación de aves de corral, y en concreto de engorde para pollos de carne o broilers, de cría de pollitas (semanas 0 a 8), de recría para pollitas (semana 9 -20) o puesta a partir de la semana 21a son conocidas en el estado de la técnica, por ejemplo en ES2051706T3. Nutritive compositions useful for feeding poultry, and in particular fattening for meat chickens or broilers, chick rearing (weeks 0 to 8), breeding for chicks (week 9 -20) or starting from week 21 a are known in the state of the art, for example in ES2051706T3.
Según la invención, las composiciones o nutracéutico de la invención son útiles para el tratamiento y prevención de coccidiosis, pododermatitis y úlceras. Por tanto la parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma debe encontrarse en una cantidad terapéuticamente eficaz. According to the invention, the compositions or nutraceuticals of the invention are useful for the treatment and prevention of coccidiosis, pododermatitis and ulcers. Therefore the part of a corn plant enriched in antioxidants or a derivative thereof must be in a therapeutically effective amount.
En una realización particular, la composición nutritiva o nutracéutico de la invención comprende granos de maíz en al menos un 20%, preferiblemente en al menos un 30%, más preferiblemente al menos un 40%, preferiblemente más de un 50%, más preferiblemente más de un 60%. In a particular embodiment, the nutritive or nutraceutical composition of the invention comprises corn grains in at least 20%, preferably in at least 30%, more preferably at least 40%, preferably more than 50%, more preferably more of 60%.
En otro aspecto, la invención se relaciona con el uso de una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma para la obtención de una composición nutritiva o nutracéutico. In another aspect, the invention relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for obtaining a nutritive or nutraceutical composition.
La composición nutritiva o nutracéutico de acuerdo a la presente invención se puede preparar a partir directamente de la parte de la planta enriquecida en carotenoides. En una forma preferida de realización, la parte de la planta es el endospermo o granos de maíz, en cuyo caso dicha composición contiene al menos un 40%, preferiblemente más de un 50% o más preferiblemente más de un 60%. The nutritive or nutraceutical composition according to the present invention can be prepared directly from the part of the plant enriched in carotenoids. In a preferred embodiment, the part of the plant is the endosperm or corn kernels, in which case said composition contains at least 40%, preferably more than 50% or more preferably more than 60%.
Alternativamente, la parte de la planta que se usa para preparar la composición nutritiva o el nutracéutico de acuerdo a la presente invención puede someterse a una purificación parcial para obtener un derivado enriquecido en el producto antioxidante. Así, en el caso de que los compuestos antioxidantes sean carotenoides y que la parte de la planta que se usa sea el endospermo, la composición nutritiva o el nutracéutico contienen un extracto de granos de maíz o de endospermo que puede encontrarse parcial- o totalmente purificado de forma que el contenido en carotenoides sea superior al que aparece en la parte de la planta de maíz enriquecida. Derivados de maíz que se pueden usar en la presente invención incluyen harina de maíz, salvado de maíz, aceite de maíz, almidón de maíz, sirope de maíz, y similares. Alternatively, the part of the plant that is used to prepare the nutritive or nutraceutical composition according to the present invention can be subjected to a partial purification to obtain a derivative enriched in the antioxidant product. Thus, in the case that the antioxidant compounds are carotenoids and that the part of the plant that is used is the endosperm, the nutritional composition or the nutraceutical they contain an extract of corn kernels or endosperm that can be found partially- or fully purified so that the carotenoid content is higher than what appears in the part of the enriched corn plant. Corn derivatives that can be used in the present invention include corn flour, corn bran, corn oil, corn starch, corn syrup, and the like.
Así, por ejemplo, de acuerdo a la presente invención, es posible extraer los carotenoides presentes en la parte de las plantas de maíz o derivados de la misma para obtener una composición nutritiva o nutracéutico. Thus, for example, according to the present invention, it is possible to extract the carotenoids present in the part of the corn plants or derivatives thereof to obtain a nutritive or nutraceutical composition.
El experto en la materia conoce diversos métodos para extraer carotenoides de una parte de una planta de maíz, tal y como extracción a partir de endospermos congelados y HPLC, tal y como se describe en Naqvi S. et al., PNAS 2009, vol. 106, no 19. The person skilled in the art knows various methods for extracting carotenoids from a part of a corn plant, such as extraction from frozen endosperms and HPLC, as described in Naqvi S. et al., PNAS 2009, vol. 106, not 19.
Se ha comprobado que los carotenoides potencian tanto las funciones inmunologicas específicas como las no específicas. Diversas reacciones de modulación inmunologicas pueden aumentar la actividad tumoricida de las células T citotóxicas, los macrófagos y/o las células asesinas naturales y fomentar las funciones antimicrobianas inmunologicas. Por ejemplo, el beta-caroteno puede reducir la incidencia de sucesos cardiovasculares, habiéndose demostrado en estudios clínicos (Gazianio JM. et al., Circulation 82:201-204, 1990). Adicionalmente los grupos de población que consumen alimentos ricos en beta-caroteno están más protegidos contra los cánceres de piel y pulmón, particularmente los fumadores. Se han propuesto varios mecanismos para explicar la actividad fisiológica de los carotenoides. La más plausible se basa en el fuerte carácter antioxidante y en su capacidad para desactivar sustancias químicas perjudiciales como el oxígeno singlete, los radicales libres y los radicales peróxido lipidíeos. También hay evidencia de que varios carotenoides producen una estimulación directa del sistema inmunológico y de la respuesta inmunológica. Carotenoids have been shown to potentiate both specific and nonspecific immunological functions. Various immunological modulation reactions can increase the tumoricidal activity of cytotoxic T cells, macrophages and / or natural killer cells and promote immunological antimicrobial functions. For example, beta-carotene can reduce the incidence of cardiovascular events, having been demonstrated in clinical studies (Gazianio JM. Et al., Circulation 82: 201-204, 1990). Additionally, population groups that consume foods rich in beta-carotene are more protected against skin and lung cancers, particularly smokers. Several mechanisms have been proposed to explain the physiological activity of carotenoids. The most plausible is based on the strong antioxidant character and its ability to deactivate harmful chemicals such as singlet oxygen, free radicals and lipid peroxide radicals. There is also evidence that several carotenoids produce a direct stimulation of the immune system and the immune response.
Como entenderá un experto en la materia, la composición nutritiva o nutracéutico de la invención deben cumplir las normativas y legislación alimentaria del país. As one skilled in the art will understand, the nutritive or nutraceutical composition of the invention must comply with the country's food regulations and legislation.
Existen diversos métodos conocidos para la elaboración de composiciones nutritivas y nutracéuticos ampliamente conocidas por el experto en la materia y que pueden emplearse en la presente invención. La invención se describe ahora en detalle por medio de los siguientes ejemplos que se deben considerar como meramente ilustrativos y no limitantes del ámbito de la invención. There are various known methods for the preparation of nutritive and nutraceutical compositions widely known to those skilled in the art and which can be used in the present invention. The invention is now described in detail by means of the following examples that should be considered as merely illustrative and not limiting the scope of the invention.
Materiales y métodos Alimentación y animales Materials and methods Food and animals
Pollos comerciales de tipo broilers se criaron en las instalaciones de la Universidad de Lleida (España). Se utilizaron piensos a base de maíz rico en carotenoides y antioxidantes, según se describe en Naqvi S. et al, PNAS, 2009 vol,. 106, no. 19 páginas 7762-7767) y su isogénico control (wild type). A los 35 días de edad, los animales fueron sacrificados siguiendo las normas de bienestar animal y se necropsiaron. Se recogieron muestras de sangre para análisis bioquímicos y estudios inmunológicos. Se recogieron muestras de carne y piel de la pechuga, carne y piel del muslo, piel de las patas, la cresta y la grasa abdominal, que se congelaron y liofilizaron para el análisis de carotenoides. Commercial broiler-type chickens were raised in the facilities of the University of Lleida (Spain). Corn-based feedstuffs rich in carotenoids and antioxidants were used, as described in Naqvi S. et al, PNAS, 2009 vol ,. 106, no. 19 pages 7762-7767) and its isogenic control (wild type). At 35 days of age, the animals were slaughtered according to animal welfare standards and necropsied. Blood samples were collected for biochemical analysis and immunological studies. Samples of meat and skin were collected from the breast, flesh and skin of the thigh, skin of the legs, crest and abdominal fat, which were frozen and lyophilized for carotenoid analysis.
Mediciones Measurements
Se midió el color de la carne y la piel de la pechuga, muslo y pata en las canales de pollo mediante un espectrofotómetro CM-700d portátil compacto, Konica Minolta (Japón). El color de cada muestra se midió por el sistema CIELAB tricromático mediante los valores de luminosidad (L), rojos (a) y la amarillos (b). Para el color de la pata también se ha utilizado el clásico abanico de la escala Roche. The color of the meat and skin of the breast, thigh and leg in the chicken carcasses was measured by a compact portable CM-700d spectrophotometer, Konica Minolta (Japan). The color of each sample was measured by the trichromatic CIELAB system using the luminosity (L), red (a) and yellow (b) values. The classic fan of the Roche scale has also been used for the color of the leg.
Para el análisis de los carotenoides totales, las muestras liofilizadas se extrajeron en 20 mi de 50/50 (v/v) de tetrahidrofurano y metanol a 60 °C durante 15-20 min y se cuantificaron midiendo la absorbancia a 450 nm. Para la separación por HPLC, el disolvente se evaporó bajo una corriente de gas nitrógeno a 37 °C, se volvió a disolver en 50 L de acetona, y una alícuota de 20 se inyectó inmediatamente. Las muestras se separaron en una columna Nucleosil C18 de 3-μ con acetonitrilo/ metanol/ 2 - propanol (85: 15:5 , v/v/v) como fase móvil a 25 0 C. En cada caso, la zeaxantina y la luteína se separaron en tramos paralelos en una columna C18 Vydac 218TP54, con metanol que contiene 2 % de agua como fase móvil Romer et al., 2002 Metab. Eng. 4:263-272). Las muestras fueron monitorizadas con una Kontron DAD detector de matriz de fotodiodos 440 con el registro en línea de las medidas. Todos los carotenoides se identificaron por la co-cromatografía con compuestos auténticos de referencia, que fueron biosintetizados en E. coli como se describe en Sandmann G.et.al. 2002 Chem. Bio.Chem. 3, 629-635. y la comparación de sus medidas. Estas muestras de referencia también se utilizaron para la cuantificación en combinación con los coeficientes de extinción de Davies B.H. 1976, Chemistry and biochemistry of plant pigments, vol. 2. Academic Press, Inc. (London), Ltd., London. For the analysis of the total carotenoids, lyophilized samples were extracted in 20 ml of 50/50 (v / v) tetrahydrofuran and methanol at 60 ° C for 15-20 min and quantified by measuring the absorbance at 450 nm. For separation by HPLC, the solvent was evaporated under a stream of nitrogen gas at 37 ° C, re-dissolved in 50 L of acetone, and an aliquot of 20 was immediately injected. The samples were separated on a 3-μ Nucleosil C18 column with acetonitrile / methanol / 2-propanol (85: 15: 5, v / v / v) as a mobile phase at 25 0 C. In each case, zeaxanthin and Lutein was separated in parallel sections on a C18 Vydac 218TP54 column, with methanol containing 2% water as mobile phase Romer et al., 2002 Metab. Eng. 4: 263-272). The samples were monitored with a Kontron DAD 440 photodiode array detector with online recording of the measurements. All carotenoids were identified by co-chromatography with authentic compounds of reference, which were biosynthesized in E. coli as described in Sandmann G.et.al. 2002 Chem. Bio.Chem. 3, 629-635. and the comparison of their measurements. These reference samples were also used for quantification in combination with the extinction coefficients of Davies BH 1976, Chemistry and biochemistry of plant pigments, vol. 2. Academic Press, Inc. (London), Ltd., London.
Desafío con E. tenella Challenge with E. tenella
Se criaron 4 grupos de pollos con 14 animales por grupo, y dos grupos se alimentaron con cada una de las dietas(control y dieta de maíz enriquecida en carotenoides) y un grupo de cada dieta fue desafiado con E. tenella. El desafío con E. tenella se llevó a cabo a los 13 días de edad con 3 χ 104 ooquistes esporulados de E. tenella Houghton, cepa de HIPRA, España. Todos los pollos se pesaron individualmente en el momento de llegada y luego semanalmente. El color de las patas de todos los animales (en el momento de su sacrificio: 8 animales a los 19 días de edad y 6 animales a los 22 días de edad) de todos los grupos se registraron regularmente y las lesiones (pododermatitis) y las úlceras se contabilizaron el día de su sacrificio (Welfare Quality ® 2009). Ver Tabla 1 y Figuras 1 y 2. Four groups of chickens were raised with 14 animals per group, and two groups were fed each of the diets (control and diet of corn enriched in carotenoids) and one group of each diet was challenged with E. tenella. The challenge with E. tenella was carried out at 13 days of age with 3 χ 10 4 sporulated oocysts of E. tenella Houghton, strain of HIPRA, Spain. All chickens were weighed individually at the time of arrival and then weekly. The color of the legs of all animals (at the time of slaughter: 8 animals at 19 days of age and 6 animals at 22 days of age) of all groups were recorded regularly and lesions (pododermatitis) and Ulcers were counted on the day of their sacrifice (Welfare Quality ® 2009). See Table 1 and Figures 1 and 2.
El color de la caña de la pata también se determinó como se describe anteriormente. Los recuentos de ooquistes se determinaron en muestras de heces recogidas diariamente de cada grupo desde el día 6 al día 9 después de la infección. La recogida de heces para su análisis de ooquistes se hizo dos veces al día. Para el recuento de ooquistes se utilizó una solución saturada de NaCI y cámaras de McMaster y los resultados se presentan como el número de ooquistes por gramo de heces frescas. Ver Tabla 2 y Figura 3. The color of the leg of the leg was also determined as described above. The oocyst counts were determined in stool samples collected daily from each group from day 6 to day 9 after infection. Stool collection for oocyst analysis was done twice a day. A saturated solution of NaCI and McMaster chambers was used for oocyst counting and the results are presented as the number of oocysts per gram of fresh feces. See Table 2 and Figure 3.
La bolsa de Fabricio, el hígado y el ciego se recogieron y se fijaron en formaldehído al 4% tamponado y se procesaron en parafina para su estudio histopatológico. The Fabrizio bag, liver and blind were collected and fixed in 4% buffered formaldehyde and processed in paraffin for histopathological study.
EJEMPLO 1 EXAMPLE 1
Los resultados demuestran que el maíz rico en antioxidantes reduce la excreción de ooquistes de coccidios en animales infectados, reduce la pododermatitis y las úlceras digitales en las patas de los pollos. The results show that antioxidant-rich corn reduces the excretion of coccidia oocysts in infected animals, reduces pododermatitis and digital ulcers in the legs of chickens.
La reducción de la pododermatitis y las úlceras en las patas se puede producir no sólo en animales afectados de coccidiosis, sino también entre las enfermedades o condiciones que pueden causar diarrea en aves domésticas como el estrés por frío o calor, bacterias o virus (colibacilosis, leucosis linfoide, enfermedad de Marek), parásitos (Ascardiasis o Capilariasis) etc. La reducción de pododermatitis, las úlceras digitales y la excreción de ooquistes de coccidios también se puede esperar en otras especies animales y posiblemente en humanos. Reduction of pododermatitis and leg ulcers can occur not only in animals affected by coccidiosis, but also among diseases or conditions that can cause diarrhea in domestic birds such as cold stress or heat, bacteria or viruses (colibacillosis, lymphoid leukosis, Marek's disease), parasites (Ascardiasis or Capilariasis) etc. The reduction of pododermatitis, digital ulcers and excretion of coccidia oocysts can also be expected in other animal species and possibly in humans.
La histopatología de la bolsa de Fabricio indica que los animales infectados con Eimeria y alimentados con la dieta de maíz rico en antioxidantes tuvieron una mejor protección e inmunidad que los animales alimentados con la dieta control, lo cual puede indicar que lo mismo puede ocurrir con otras enfermedades. The histopathology of the Fabrizio bag indicates that animals infected with Eimeria and fed the corn diet rich in antioxidants had better protection and immunity than animals fed with the control diet, which may indicate that the same may occur with others diseases.
Los resultados también demuestran que, incluso en ausencia de infección, los animales del grupo alimentado con la dieta de maíz rico en antioxidantes presentaban una significativa reducción de pododermatitis y úlceras digitales en comparación con el grupo de animales alimentados con la dieta de control (véase T1 y T3 de la Tabla 2). The results also demonstrate that, even in the absence of infection, the animals in the group fed the antioxidant-rich corn diet had a significant reduction in pododermatitis and digital ulcers compared to the group of animals fed the control diet (see T1 and T3 of Table 2).
Figure imgf000033_0001
Figure imgf000033_0001
Tabla 2. Media±ES de la pododermatitis y número de úlceras plantares de  Table 2. Mean ± ES of pododermatitis and number of plantar ulcers of
acuerdo a: Welfare Quality ® (2009). Protocolo de evaluación para aves  according to: Welfare Quality ® (2009). Evaluation protocol for birds
(pollos de engorde, gallinas ponedoras) Welfare Quality ®. Welfare Quality  (broilers, laying hens) Welfare Quality ®. Welfare Quality
® Consorcio, Lelystad, Países Bajos. Los datos obtenidos son de 14  ® Consortium, Lelystad, The Netherlands. The data obtained are 14
animales/grupo. T1 : dieta control no infectados, T2: dieta control desafiados con E. tenella, T3: dieta de maíz rico en carotenoides no  Animals / group T1: non-infected control diet, T2: control diet challenged with E. tenella, T3: non-carotenoid-rich corn diet
infectado, T4: dieta de maíz rico en carotenoides desafiado con E. tenella.  infected, T4: diet of corn rich in carotenoids challenged with E. tenella.
a, b, c: Los valores en la misma columna con una letra en común no  a, b, c: The values in the same column with one letter in common do not
difieren significativamente. La significación de los datos es la siguiente: **:  They differ significantly. The significance of the data is as follows: **:
0,01 > P> 0,001. Prueba de Duncan.  0.01> P> 0.001. Duncan test.
Según esta Tabla 2, se observan diferencias significativas en el nivel de pododermatitis y la cantidad de úlceras entre los grupos no infectados (T1 y T3) lo que indica que la dieta antioxidante incluso en animales no infectados (T3) mejora su estado sanitario y su bienestar. Comparando los grupos infectados entre sí, se observan más diferencias significativas importantes con menos pododermatitis y lesiones en el grupo de dieta antioxidante (T2 yT4). According to this Table 2, significant differences are observed in the level of pododermatitis and the number of ulcers between the uninfected groups (T1 and T3), which indicates that the antioxidant diet even in non-infected animals (T3) improves their health status and their wellness. Comparing the infected groups with each other, it They observe more significant significant differences with less pododermatitis and lesions in the antioxidant diet group (T2 and T4).
Figure imgf000034_0001
Figure imgf000034_0001
Tabla 3. Recuento de ooquistes utilizando solución  Table 3. Oocyst count using solution
saturada de NaCI en cámaras de McMaster. Se  saturated of NaCI in McMaster chambers. Be
presentan como el número de ooquistes por gramo de  presented as the number of oocysts per gram of
heces. Los datos se obtienen de la mezcla de las  stool The data is obtained from the mixture of
heces de todos los animales del grupo y repetido 4  feces of all animals in the group and repeated 4
veces. T2: dieta control desafiados con E. tenella, T4:  times. T2: control diet challenged with E. tenella, T4:
dieta de maíz rico en carotenoides desafiado con E.  Carotenoid-rich corn diet challenged with E.
tenella. (Los grupos no infectados T1 y T3 fueron  tenella. (The uninfected groups T1 and T3 were
negativos y no se presentan en la tabla). *: T4 difiere  negative and are not presented in the table). *: T4 differs
significativamente de T2 (P <0,05).  significantly from T2 (P <0.05).
El descenso de ooquistes en los días 8 y 9 después de la infección puede estar relacionado con el ciclo del parásito. Después de la infección, el ciclo de estos protozoos se inicia con la fase asexual (esquizogonia) seguido de la fase sexual (gametogonia) que termina con la expulsión de los ooquistes a través de las heces. Los ooquistes esporulan (esporogonia) en pocos días en el exterior entre las heces y la cama dependiendo de la temperatura y la humedad. La ingestión de un elevado número de nuevos ooquistes esporulados inicia un ciclo nuevo masivo y una nueva ola de ooquistes que se excretan por las heces de nuevo. El grupo con dieta control puede haber iniciado una nueva ola de excreción de ooquistes en el día 9 post infección, pero el grupo de la dieta rica en antioxidantes puede haber mejorado su inmunidad y esta nueva ola puede haberse retrasado o reducido. Podemos especular que en el primer ciclo, la inmunidad específica a los coccidios es similar en ambos tratamientos, pero en el segundo ciclo el grupo con dieta a base de maíz rico en antioxidantes puede haber mejorado la inmunidad humoral. Los niveles de retinol en el suero de los animales alimentados con la dieta rica en carotenoides eran casi el doble que los de dieta control, lo que confirma la conversión efectiva de los carotenoides de la provitamina A en vitamina A. Ver Tabla 4. The decrease in oocysts on days 8 and 9 after infection may be related to the parasite cycle. After infection, the cycle of these protozoa begins with the asexual phase (schizogonia) followed by the sexual phase (gametogonia) that ends with the expulsion of oocysts through feces. The oocysts sporulate (sporogonia) in a few days outside between the feces and the bed depending on the temperature and humidity. Ingestion of a large number of new sporulated oocysts begins a massive new cycle and a new wave of oocysts that are excreted in the feces again. The control diet group may have started a new wave of oocyst excretion on day 9 post infection, but the antioxidant-rich diet group may have improved their immunity and this new wave may have been delayed or reduced. We can speculate that in the first cycle, the specific immunity to coccidia is similar in both treatments, but in the second cycle the group with corn-based diet rich in antioxidants may have improved humoral immunity. The retinol levels in the serum of animals fed the carotenoid-rich diet were almost double that of the control diet, confirming the effective conversion of the carotenoids of provitamin A into vitamin A. See Table 4.
Figure imgf000035_0001
Figure imgf000035_0001
alimentados con dieta control y con el maíz rico en antioxidantes.  fed diet control and corn rich in antioxidants.
***: 0.001 > P> 0,0001 carotenoides de sangre (g / mi de suero)  ***: 0.001> P> 0.0001 blood carotenoids (g / mi serum)
Figure imgf000035_0002
Figure imgf000035_0002
la media del número de vacuolas en 10 campos de microscopía 40x de 5 animales / grupo. T1 : dieta control no infectados, T2: dieta control desafiados con E. tenella, T3: dieta de maíz rico en carotenoides no infectado, T4: dieta de maíz rico en carotenoides desafiado con E. tenella. a, b, c: Los valores en la misma columna con una letra en común no difieren significativamente. La significación de los datos es la siguiente: *: 0,05>P>0,01.  The average number of vacuoles in 10 40x microscopy fields of 5 animals / group. T1: non-infected control diet, T2: control diet challenged with E. tenella, T3: carotenoid-rich corn diet uninfected, T4: carotenoid-rich corn diet challenged with E. tenella. a, b, c: The values in the same column with one letter in common do not differ significantly. The significance of the data is as follows: *: 0.05> P> 0.01.
El término "Sequence listing" en la lista de secuencias se refiere a "Lista de secuencias", "Artificial Sequence" a "Secuencia artificial" y el término "DNA" se refiere a "ADN" y "mise feature" se refiere a característica miscelánea. The term "Sequence listing" in the sequence list refers to "Sequence list", "Artificial Sequence" to "Artificial sequence" and the term "DNA" refers to "DNA" and "mise feature" refers to characteristic miscellany.

Claims

REIVINDICACIONES
1. Uso de una parte de una planta de maíz enriquecida en antioxidantes o de un derivado de la misma, para la fabricación de un medicamento, composición nutritiva o nutracéutico para la prevención o tratamiento de coccidiosis en un sujeto. 1. Use of a part of a corn plant enriched in antioxidants or a derivative thereof, for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of coccidiosis in a subject.
2. Uso de una parte de una planta de maíz enriquecida en antioxidantes o de un derivado de la misma para la fabricación de un medicamento, composición nutritiva o nutracéutico para la prevención o tratamiento de pododermatitis o úlceras en un sujeto. 2. Use of a part of an antioxidant-enriched corn plant or a derivative thereof for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of pododermatitis or ulcers in a subject.
3. Uso según las reivindicaciones 1 o 2 en donde los antioxidantes son carotenoides. 3. Use according to claims 1 or 2 wherein the antioxidants are carotenoids.
4. Uso según la reivindicación 3 en donde los carotenoides se seleccionan del grupo formado por β-caroteno, γ-caroteno, α-caroteno, a-criptoxantina, β-criptoxantina, licopeno, luteína y zeaxantina. 4. Use according to claim 3 wherein the carotenoids are selected from the group consisting of β-carotene, γ-carotene, α-carotene, a-cryptoxanthin, β-cryptoxanthin, lycopene, lutein and zeaxanthin.
5. Uso según la reivindicación 4 en donde la planta de maíz enriquecida en antioxidantes es una planta de maíz transgénica que expresa al menos un transgén de la ruta de biosíntesis de carotenoides. 5. Use according to claim 4 wherein the antioxidant enriched corn plant is a transgenic corn plant that expresses at least one transgene of the carotenoid biosynthesis pathway.
6. Uso según la reivindicación 5 en donde el transgén de la ruta de biosíntesis de carotenoides se selecciona de entre un gen que codifica la fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa, y un gen que codifica una carotenoide cetolasa. 6. Use according to claim 5 wherein the transgene of the carotenoid biosynthesis pathway is selected from a gene encoding phytoeno synthase, a gene encoding a carotene desaturase, a gene encoding a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase, and a gene that encodes a carotenoid ketolase.
7. Uso según la reivindicación 6 en donde el transgén que codifica para la fitoeno sintasa tiene la secuencia mostrada en SEQ ID NO: 1 , el transgen que codifica para la caroteno desaturasa tiene la secuencia mostrada en SEQ ID NO: 2, el transgén que codifica para la lycopeno β-ciclasa tiene la secuencia mostrada en SEQ ID NO:3, el transgén que codifica para la β-caroteno hidroxilasa tiene la secuencia mostrada en SEQ ID NO:4, SE ID NO:5 o SEQ ID NO: 12 ,el transgén que codifica para la carotenoide cetolasa tiene la secuencia mostrada en SEQ ID NO:6. 7. Use according to claim 6 wherein the transgene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1, the transgene encoding carotene desaturase has the sequence shown in SEQ ID NO: 2, the transgene that coding for lycopene β-cyclase has the sequence shown in SEQ ID NO: 3, the transgene encoding β-carotene hydroxylase has the sequence shown in SEQ ID NO: 4, SE ID NO: 5 or SEQ ID NO: 12 , the transgene encoding the carotenoid ketolase has the sequence shown in SEQ ID NO: 6.
8. Uso según cualquiera de las reivindiciones 5 a 7 en donde el transgén se encuentra bajo el control de un promotor específico de endospermo. 8. Use according to any of claims 5 to 7 wherein the transgene is under the control of a specific endosperm promoter.
9. Uso según la reivindicación 8 en donde el promotor específico de endospermo es el promotor de D-hordeína o el promotor de LMW glutenina. 9. Use according to claim 8 wherein the endosperm specific promoter is the D-hordein promoter or the LMW glutenin promoter.
10. Uso según la reivindicación 9 en donde el promotor de D-hordeína tiene la secuencia mostrada en SEQ ID NO: 10 y el promotor de LMW glutenina tiene la secuencia mostrada en SEQ ID NO: 1 1. 10. Use according to claim 9 wherein the D-hordein promoter has the sequence shown in SEQ ID NO: 10 and the LMW glutenin promoter has the sequence shown in SEQ ID NO: 1 1.
1 1. Uso según cualquiera de las reivindicaciones 1 a 10 en donde la parte de la planta de maíz enriquecida en antioxidantes se selecciona del grupo de endospermo y semilla. 1 1. Use according to any one of claims 1 to 10 wherein the part of the antioxidant enriched corn plant is selected from the endosperm and seed group.
12. Uso según cualquiera de las reivindicaciones 1 a 1 1 en donde la coccidiosis es causada por Eimeria. 12. Use according to any of claims 1 to 1 1 wherein coccidiosis is caused by Eimeria.
13. Uso según la reivindicación 12 en donde la coccidiosis es causada por E. tenella. 13. Use according to claim 12 wherein coccidiosis is caused by E. tenella.
14. Uso según cualquiera de las reivindicaciones 1 a 13 en donde el sujeto es un ave. 14. Use according to any of claims 1 to 13 wherein the subject is a bird.
15. Uso según la reivindicación 14 en donde el ave es un ave de corral. 15. Use according to claim 14 wherein the bird is a poultry.
16. Uso según la reivindicación 15 en donde el ave de corral es pollo. 16. Use according to claim 15 wherein the poultry is chicken.
17. Composición que comprende una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma y un agente anticoccídico. 17. Composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof and an anticoccidic agent.
18. Composición según la reivindicación 17 en donde los antioxidantes son carotenoides. 18. Composition according to claim 17 wherein the antioxidants are carotenoids.
19. Composición según la reivindicación 18 en donde los carotenoides se seleccionan del grupo formado por β-caroteno, γ-caroteno, a-caroteno, α-criptoxantina, β- criptoxantina, licopeno, luteína y zeaxantina. 19. Composition according to claim 18 wherein the carotenoids are selected from the group consisting of β-carotene, γ-carotene, a-carotene, α-cryptoxanthin, β-cryptoxanthin, lycopene, lutein and zeaxanthin.
20. Composición según la reivindicación 19 en donde la planta de maíz enriquecida en antioxidantes es una planta de maíz transgénica que expresa al menos un transgén de la ruta de biosíntesis de carotenoides. 20. Composition according to claim 19 wherein the antioxidant-enriched corn plant is a transgenic corn plant that expresses at least one transgene of the carotenoid biosynthesis pathway.
21. Composición según la reivindicación 20 en donde el transgén de la ruta de biosíntesis de carotenoides se selecciona de entre un gen que codifica la fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa, y un gen que codifica una carotenoide cetolasa. 21. Composition according to claim 20 wherein the transgene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase, and a gene that encodes a carotenoid ketolase.
22. Composición según la reivindicación 21 en donde el transgén que codifica para la fitoeno sintasa tiene la secuencia mostrada en SEQ ID NO: 1 , el transgen que codifica para la caroteno desaturasa tiene la secuencia mostrada en SEQ ID NO: 2, el transgén que codifica para la lycopeno β-ciclasa tiene la secuencia mostrada en SEQ ID NO:3, el transgén que codifica para la β-caroteno hidroxilasa tiene la secuencia mostrada en SEQ ID NO:4, SEQ I D NO:5 o SEQ ID NO: 12 ,el transgén que codifica para la carotenoide cetolasa tiene la secuencia mostrada en SEQ ID NO:6. 22. Composition according to claim 21 wherein the transgene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1, the transgene encoding carotene desaturase has the sequence shown in SEQ ID NO: 2, the transgene that encoding for lycopene β-cyclase has the sequence shown in SEQ ID NO: 3, the transgene encoding β-carotene hydroxylase has the sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12 , the transgene encoding the carotenoid ketolase has the sequence shown in SEQ ID NO: 6.
23. Composición según cualquiera de las reivindicaciones 20 a 22 en donde el transgén se encuentra bajo el control de un promotor específico de endospermo. 23. Composition according to any of claims 20 to 22 wherein the transgene is under the control of a specific endosperm promoter.
24. Composición según la reivindicación 23 en donde el promotor específico de endospermo es el promotor de D-hordeína o el promotor de LMW glutenina. 24. Composition according to claim 23 wherein the endosperm specific promoter is the D-hordein promoter or the LMW glutenin promoter.
25. Composición según la reivindicación 24 en donde el promotor de D-hordeína tiene la secuencia mostrada en SEQ ID NO:10 y el promotor de LMW glutenina tiene la secuencia mostrada en SEQ ID NO: 1 1. 25. Composition according to claim 24 wherein the D-hordein promoter has the sequence shown in SEQ ID NO: 10 and the LMW glutenin promoter has the sequence shown in SEQ ID NO: 1 1.
26. Composición según cualquiera de las reivindicaciones 17 a 25 en donde la parte de la planta de maíz enriquecida en antioxidantes se selecciona del grupo de endospermo y la semilla. 26. Composition according to any one of claims 17 to 25 wherein the part of the antioxidant enriched corn plant is selected from the endosperm and seed group.
27. Uso de la composición cualquiera de las reivindicaciones 17 a 26 para la fabricación de un medicamento, composición nutritiva o nutracéutico para la prevención o el tratamiento de coccidiosis en un sujeto o para la prevención o tratamiento de pododermatitis o úlceras asociadas a coccidiosis en un sujeto. 27. Use of the composition any of claims 17 to 26 for the manufacture of a medicament, nutritive or nutraceutical composition for the prevention or treatment of coccidiosis in a subject or for the prevention or treatment of pododermatitis or ulcers associated with coccidiosis in a subject.
28. Uso de la composición según la reivindicación 27 en donde el agente anticoccídico se administra a una dosis subterapeútica. 28. Use of the composition according to claim 27 wherein the anticoccidic agent is administered at a subtherapeutic dose.
29. Uso de una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma para la obtención de una composición nutritiva o nutracéutico. 29. Use of a part of a corn plant enriched in antioxidants or a derivative thereof to obtain a nutritive or nutraceutical composition.
30. Uso según la reivindicación 29 en donde los antioxidantes son carotenoides. 30. Use according to claim 29 wherein the antioxidants are carotenoids.
31. Uso o composición según la reivindicación 30 en donde los carotenoides se seleccionan del grupo formado por β-caroteno, γ-caroteno, α-caroteno, a- criptoxantina, β-criptoxantina, licopeno, luteína y zeaxantina. 31. Use or composition according to claim 30 wherein the carotenoids are selected from the group consisting of β-carotene, γ-carotene, α-carotene, acryptoxanthin, β-cryptoxanthin, lycopene, lutein and zeaxanthin.
32. Uso según cualquiera de las reivindicaciones 30 a 31 en donde el maíz enriquecido en antioxidantes es un maíz transgénico que expresa al menos un transgén de la ruta de biosíntesis de carotenoides. 32. Use according to any of claims 30 to 31 wherein the antioxidant enriched corn is a transgenic corn that expresses at least one transgene of the carotenoid biosynthesis pathway.
33. Uso según la reivindicación 32 en donde el transgén de la ruta de biosíntesis de carotenoides se selecciona de entre un gen que codifica la fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa, y un gen que codifica una carotenoide cetolasa. 33. Use according to claim 32 wherein the transgene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase, and a gene that encodes a carotenoid ketolase.
34. Uso según la reivindicación 33 en donde el transgén que codifica para la fitoeno sintasa tiene la secuencia mostrada en SEQ ID NO: 1 , el transgén que codifica para la caroteno desaturasa tiene la secuencia mostrada en SEQ ID NO: 2, el transgén que codifica para la lycopeno β-ciclasa tiene la secuencia mostrada en SEQ ID NO:3, el transgén que codifica para la β-caroteno hidroxilasa tiene la secuencia mostrada en SEQ ID NO:4, SEQ I D NO:5 o SEQ ID NO: 12 ,el transgén que codifica para la carotenoide cetolasa tiene la secuencia mostrada en SEQ ID NO:6. 34. Use according to claim 33 wherein the transgene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1, the transgene encoding carotene desaturase has the sequence shown in SEQ ID NO: 2, the transgene which encoding for lycopene β-cyclase has the sequence shown in SEQ ID NO: 3, the transgene encoding β-carotene hydroxylase has the sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12 the transgene coding for the carotenoid ketolase has the sequence shown in SEQ ID NO: 6.
35. Uso según cualquiera de las reivindicaciones 32 a 34 en donde el transgén se encuentra bajo el control de un promotor específico de endospermo. 35. Use according to any of claims 32 to 34 wherein the transgene is under the control of a specific endosperm promoter.
36. Uso según la reivindicación 35 en donde el promotor específico de endospermo es el promotor de D-hordeína o el promotor de LMW glutenina. 36. Use according to claim 35 wherein the endosperm specific promoter is the D-hordein promoter or the LMW glutenin promoter.
37. Uso según la reivindicación 36 en donde el promotor de D-hordeína tiene la secuencia mostrada en SEQ ID NO: 10 y el promotor de LMW glutenina tiene la secuencia mostrada en SEQ ID NO: 1 1. 37. Use according to claim 36 wherein the D-hordein promoter has the sequence shown in SEQ ID NO: 10 and the LMW glutenin promoter has the sequence shown in SEQ ID NO: 1 1.
38. Uso según cualquiera de las reivindicaciones 29 a 37 en donde la parte de la planta de maíz enriquecida en antioxidantes es el endospermo o la semilla o derivados de los mismos. 38. Use according to any of claims 29 to 37 wherein the part of the corn plant enriched in antioxidants is the endosperm or the seed or derivatives thereof.
39. Composición nutritiva o nutracéutico que comprende una parte de una planta de maíz enriquecida en antioxidantes o un derivado de la misma. 39. Nutritious or nutraceutical composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof.
40. Composición según la reivindicación 39 en donde los antioxidantes son carotenoides. 40. Composition according to claim 39 wherein the antioxidants are carotenoids.
41. Composición según la reivindicación 409 en donde los carotenoides se seleccionan del grupo formado por β-caroteno, γ-caroteno, a-caroteno, α-criptoxantina, β- criptoxantina, licopeno, luteína y zeaxantina. 41. Composition according to claim 409 wherein the carotenoids are selected from the group consisting of β-carotene, γ-carotene, a-carotene, α-cryptoxanthin, β-cryptoxanthin, lycopene, lutein and zeaxanthin.
42. Composición según cualquiera de las reivindicaciones 39 a 41 en donde el maíz enriquecido en antioxidantes es un maíz transgénico que expresa al menos un transgén de la ruta de biosíntesis de carotenoides. 42. Composition according to any of claims 39 to 41 wherein the antioxidant enriched corn is a transgenic corn that expresses at least one transgene of the carotenoid biosynthesis pathway.
43. Composición según la reivindicación 42 en donde el transgén de la ruta de biosíntesis de carotenoides se selecciona de entre un gen que codifica la fitoeno sintasa, un gen que codifica una caroteno desaturasa, un gen que codifica una licopeno β-ciclasa, un gen que codifica una β-caroteno hidroxilasa, y un gen que codifica una carotenoide cetolasa. 43. Composition according to claim 42 wherein the transgene of the carotenoid biosynthesis pathway is selected from a gene that encodes phytoeno synthase, a gene that encodes a carotene desaturase, a gene that encodes a Lycopene β-cyclase, a gene that encodes a β-carotene hydroxylase, and a gene that encodes a carotenoid ketolase.
44. Composición según la reivindicación 43 en donde el transgén que codifica para la fitoeno sintasa tiene la secuencia mostrada en SEQ ID NO: 1 , el transgen que codifica para la caroteno desaturasa tiene la secuencia mostrada en SEQ ID NO: 2, el transgén que codifica para la lycopeno β-ciclasa tiene la secuencia mostrada en SEQ ID NO:3, el transgén que codifica para la β-caroteno hidroxilasa tiene la secuencia mostrada en SEQ ID NO:4 , SEQ ID NO:5 o SEQ I D NO: 12,el transgén que codifica para la carotenoide cetolasa tiene la secuencia mostrada en SEQ ID NO:6. 44. Composition according to claim 43 wherein the transgene encoding phytoen synthase has the sequence shown in SEQ ID NO: 1, the transgene encoding carotene desaturase has the sequence shown in SEQ ID NO: 2, the transgene that encoding for lycopene β-cyclase has the sequence shown in SEQ ID NO: 3, the transgene encoding β-carotene hydroxylase has the sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12 , the transgene encoding the carotenoid ketolase has the sequence shown in SEQ ID NO: 6.
45. Composición según de las reivindicaciones 42 a 44 en donde el transgén se encuentra bajo el control de un promotor específico de endospermo. 45. Composition according to claims 42 to 44 wherein the transgene is under the control of a specific endosperm promoter.
46. Composición según la reivindicación 45 en donde el promotor específico de endospermo es el promotor de D-hordeína o el promotor de LMW glutenina. 46. Composition according to claim 45 wherein the endosperm specific promoter is the D-hordein promoter or the LMW glutenin promoter.
47. Composición según la reivindicación 46 en donde el promotor de D-hordeína tiene la secuencia mostrada en SEQ ID NO:10 y el promotor de LMW glutenina tiene la secuencia mostrada en SEQ ID NO: 1 1. 47. Composition according to claim 46 wherein the D-hordein promoter has the sequence shown in SEQ ID NO: 10 and the LMW glutenin promoter has the sequence shown in SEQ ID NO: 1 1.
48. Composición según cualquiera de las reivindicaciones 39 a 47 en donde la parte de la planta de maíz enriquecida en antioxidantes es el endospermo o la semilla o derivados de los mismos. 48. Composition according to any one of claims 39 to 47 wherein the part of the corn plant enriched in antioxidants is the endosperm or the seed or derivatives thereof.
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