Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
• • 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
• • 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
  • A23K20/111—Aromatic compounds
• • 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

Definitions

• the invention relates to the use of a food additive comprising at least one phytonutrient for stimulating vaccine immunity against apicomplexes in a monogastric animal such as poultry or pork, or in a ruminant such as a cattle, sheep or goat.
• Apicomplexes or sporozoans are unicellular organisms that are parasites of vertebrates or invertebrates. These parasites can cause various pathologies.
• coccidiosis which is an intestinal disease caused by several distinct species of protozoa Eimeria, is an economically important parasitic infection for the poultry industry around the world.
• This vaccination technique has the major disadvantage that live oocysts can only be produced by chickens, which requires a lot of work and involves many manipulations.
• coccidiosis an immunogenic protein of Eimeria.
• profilin an immunogenic protein of Eimeria.
• Recombinant vaccinations using profilin by subcutaneous immunization in young chickens or by immunization of embryos have provided some protection against avian coccidiosis.
• protein vaccines have limited immunogenicity and their efficacy as a vaccine for reducing production losses due to coccidiosis is less than that of traditional anti-coccidiostats and live oocysts vaccine.
• various strategies have been examined including adjuvants and cytokines.
• carvacrol is one of the most common components of essential oils, has extensive antimicrobial activity, and has been approved as a safe food additive in the United States of America and Europe;
• cinnamaldehyde showed antifungal, anti-pyretic, antioxidant, antimicrobial and larvicidal activity, in addition to modulating T-cell differentiation;
• Capsicum spp reduces intestinal pro-inflammatory cytokine levels, increases weight gain and reduces parasite fecundity in E. acervulina-infected poultry;
• Curcuma plants including C. longa, have anti-oxidant and anti-inflammatory properties, and chicken macrophages treated with turmeric extracts have shown increased levels of IL-6 and IFN-gamma mRNA. .
• One of the new drug-free approaches that has not been tested so far is the combined strategy of using a vaccine with a nutrition-mediated dietary immuno-modulation, particularly by phytonutrients as nutritional supplements.
• the main object of the invention is to combat apicomplexes present in the body of animals, such as poultry or ruminants, such as cattle or goats.
• the inventors have surprisingly discovered that it is possible to better control apicomplexes by stimulating the immunity induced in the host after vaccination. against the infectious agent through the use of food additives.
• these food additives have the advantage of being known phytonutrients and generally easy to find commercially, such as those belonging to the class of terpenoid.
• phytonutrients have not shown toxicity or adverse effects as is often the case for conventional antiparasitic drugs.
• FIG. 1 shows the effects of dietary phytonutrient supplements, VAC and MC, on body weight and excretion of oocysts in farmed chickens after E. tenella infection. Columns that do not have the same letters in common are significantly different (P ⁇ 0.05) according to Duncan's test. ;
• FIG. 2 presents the effects of dietary phytonutrient supplements, VAC and MC, on serum antibody responses to profilin in E. tenella infected farmed chickens;
• FIG. 3 presents the effects of dietary phytonutrient supplements, VAC and MC, on the proliferation of splenic lymphocytes in farmed chicken infected with E. tenella;
• FIG. 4 shows the effects of dietary phytonutrient supplements, VAC and MC, on intestinal cytokine levels produced.
• the mRNA level is quantified for each of the following cytokines: IFN- ⁇ in Fig.4A, IL-6 in Fig.4B, IL-17F in Fig.4C, and TNSF15 in Fig.4D, and these levels are standardized to the level of mRNA encoding GAPDH.
• the present invention relates to a food additive for stimulating immunity immunity in non-human animals infected with an Apicomplexa sporozoan, comprising at least one phytonutrient.
• boosting immunity immunity means that the animals are vaccinated against the infectious agent for the prevention of disease related to an apicomplex pathogen.
• phytonutrient any bioreactive compound derived from plants that has positive effects on the health of animals.
• phytonutrients targeted by the present invention there are in particular those of the plant terpenoid group, some of which are known and used because of their aromatic qualities (such as essential oils), but also for their various antiseptic, antioxidant properties. , anti-fungal, anti-microbial and / or larvicidal.
• the inventors have shown with their work carried out on several of them that they could surprisingly enhance the immune protection against infectious agents when they were used together with a vaccine immunization.
• the supplementation is carried out by adding at least one phytonutrient chosen from carvacrol, cinnamaldehyde, capsicum and curcumin. These phytonutrients can be used alone or in combination.
• phytonutrients there may be mentioned in particular the carvacrol / cinnamaldehyde combination, as well as the combination turmeric / caspicum.
• chilli Capsicum annuum or Capsicum frutescens
• oleoresin a capsicum
• the inventors of the present invention have shown in in vivo tests their synergistic effects by stimulation of the protective immunity after vaccination against the infectious agent, and this surprisingly given their completely different previously recognized properties (mentioned in FIG. part "background of the invention").
• the result was that in hosts who received supplementation compared to those who did not, including good weight gain higher, a reduction in excreted oocysts, an increase in the antibody response and significantly reduced levels of several proinflammatory cytokines, which had not been shown in the prior art, nor even suggested.
• the invention also relates to a non-human animal feed intended to prevent infectious diseases related to the sporozoan Apicomplexa comprising, in addition to a preferably balanced diet, supplementation with at least one phytonutrient.
• a balanced diet consists of providing all the nutrients that birds (chicks, young or adults) need during their growth and fattening, in terms of proteins, carbohydrates, fats, but also vitamins and minerals. Generally, it consists of cereal, oilseed and protein meal, to which supplements such as vitamins and trace elements can be added.
• the supplementation of the food ration is carried out by at least one phytonutrient chosen from carvacrol, cinnamaldehyde, capsicum and curcumin, used alone or in combination.
• the phytonutrients will be used in a total phytonutrient concentration (s) of between 1 and 50 mg per kilogram of food intake or between 0.5 and 20 mg per liter of drinking water.
• the supplementation may be in solid form, that is to say by adding to the diet, or in liquid form by adding to the drinking water.
• the supplemented food for non-human animals of the invention is in the form of powder, granules or tablets.
• Vaccination is conducted as early as possible, preferably as of 1 day old animals.
• the immunization of the hosts, in particular the poultry, is carried out by vaccination.
• the vaccine associated with phytonutrient supplementation may be derived from recombinant protein.
• the recombinant protein vaccine comes from profilin which is an immunogenic protein of Eimeria and which has been shown beforehand to induce significant protection against avian coccidiosis and to reduce the fecundity of the parasite.
• the type of vaccine used in the examples served as a model. However, at present, this is not the main type of vaccine available. The majority of vaccines used are from attenuated oocysts.
• the vaccination of the hosts can be carried out by various ways of inoculation (subcutaneous, systemic etc ..) but the oral way is obviously that privileged since not only it allows good results in terms of immunogenicity but so for its side convenient ; it suffices to simply add the vaccine solution in the drinking water, as is commonly the case in farms.
• the vaccines can also be in the form of a gel which is sprayed directly on the animals, for example the chicks, which ingest it by contact.
• the food additive or nutritional supplement of the present invention as well as the food or the drinking water containing it is suitable for preventing, in non-human animals, infectious diseases related to sporozoites belonging to the genus Apicomplexa, among which find in particular the agents of coccidiosis (coccidia); toxoplasmosis (toxoplasm); cryptosporidiosis (cryptosporidia); malaria
• neosporosis (Neospora).
• the invention is particularly applicable to the control of coccidiosis, which can be caused by various species of Eimeria including those which affect the chicken / laying hen (E. tenella, E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox); those affecting turkey (E. meleagrimitis, E. adenoeides, E. dispersa, E. gallopavonis); those affecting goose (E. truncata, E. anseris); those that affect the duck (E. tyzzeria perniciosa, E. mulardi); those affecting the guinea fowl (E. numidia, E. grenieri); or that which affects the pigeon (E. labbeana).
• the invention applies to non-human animals that are concerned with these diseases related to apicomplexes and for which immunization immunization against these infectious agents exists.
• These animals may be monogastric such as poultry, pig or rabbit, or in a ruminant such as a cow, sheep or goat.
• the chicken / laying hen but also the turkey, goose, duck, guinea fowl, quail, doe and pigeon.
• They are farm animals intended for human consumption that can be kept in cages arranged in batteries or on the ground, in poultry houses or living in outdoors or at liberty. The important thing is to be able to provide them daily food supplemented with phytonutrients; these animals having received an immunization against the sporozoites mentioned above.
• VAC is composed of 5 mg carvacrol, 3 mg cinnamaldehyde and 2 mg oleoresin capsicum per kg feed ration
• MC is composed of 4 mg oleoresin turmeric and 4 mg oleoresin capsicum per kg diet.
• the synergistic effect of VAC and MC supplementation on profilin immunization against avian coccidiosis was assessed by examining body weight, excretion of fecal oocysts, splenocyte subpopulations and rates. in cytokines.
• CON control animals, that is to say uninfected, unvaccinated, not fed with plant extracts
• CON-MC-V infected with Eimeria tenella animals having received a vaccination, fed with oleoresins of turmeric and capsicum and infected with Eimeria tenella.
• ARS IACUC USDA's Agriculture Research Service Institutional Animal Care and the Utilization Committee
• the chickens were kept in a thermally controlled environment (29 ° C). All diets contained 24.2% crude protein, 54.0% carbohydrate, 15% vitamin / mineral blend, 4.7% fat, and 2.4% fiber (USDA / FeedMill, Beltsville, MD).
• the chickens in the vaccinated groups were immunized with profilin (CON-V, VAC-V, MC-V) at 7 days of age, the animals were infected with coccidia at 17 days of age by inoculation. orally 2.0 x 10 4 spore oocysts of Eimeria tenella as previously described in the literature.
• the chickens were previously raised in facilities without Eimeria for 2 weeks and then transferred to cages (2 chickens / cage) and maintained until the end of the period. experimental in places separate from those in which they had been infected.
• Example 1 Effect of supplementation with VAC and MC on body weight
• Body weight was measured at Day 0 and 9 after infection (DPI).
• DPI body weight
• the chickens were placed in special cages to collect oocysts (2 chickens / cage, 12 chickens / group) and faecal samples were collected from day 5 to day 9 after infection.
• Feeding of chickens reared with a supplemented VAC and MC diet showed no toxic effect in the host based on changes in body weight and other physical characteristics at the doses used in this test. During the first 17 days after hatching, the chickens who received the supplemented diets did not show any change in their body weight, compared to those who received the standard diet alone. After infection with E. tenella (Day 10 after infection), however, the chickens immunized with the profilin vaccine (CON-V, VAC-V, MC-V) had higher body weights of 8 to 21% compared to the infected controls who received the unsupplemented diet (CON, Figure 1A).
• total oocysts / chicken [number of oocysts x dilution factor x (volume of fecal sample / volume of the counting cell)] / 2.
• Blood samples (4 chickens / group) were collected by cardiac puncture immediately after euthanasia on Day 10 after infection, the sera were obtained by centrifugation and analyzed by ELISA to measure profilin specific antibody responses as described. previously in the literature. Briefly, 96-well microtiter plates were overlaid overnight with 1.0 g / well of a purified recombinant profilin. The plates were rinsed with phosphate buffered saline containing 0.05% Tween® (PBS-T) and blocked with PBS containing 1% BSA. The diluted sera (1:50) were added (100 ⁇ l / well), incubated with stirring for 2 hours at room temperature and rinsed with PBS-T.
• PBS-T phosphate buffered saline containing 0.05% Tween®
• Bound antibodies were detected using peroxidase-conjugated rabbit anti-chicken IgGs and the substrate 3,3 ', 5,5'-tetramethylbenzidine (Sigma®, St. Louis, MO). Optical density (OD) was measured at 450 nm (OD 450) by an automated microplate reader (Bio-Rad®, Richmond, CA). All samples were analyzed in four copies.
• the chickens (4 chickens / group) were euthanized by cervical dislocation, the spleens were removed and placed in petri dishes with 10 ml of balanced Hank's saline solution supplemented with 100 U / ml penicillin. and 100 ⁇ / ⁇ of spectromycin (Sigma®, St. Louis, MO). Cell suspensions were prepared by delicate cell-cell rinsing and the lymphocytes were purified by density gradient centrifugation on a Histopaque-1077 (Sigma®).
• the number of cells was adjusted to a concentration of 5.0 x 10 6 cells / ml; these were placed in RPMI medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin and incubated in 20 ⁇ / ⁇ of profilin in 96-well plates in a humidified incubator (Forma, Marietta, OH) at 41 ° C and 5% CO 2 for 24 hours.
• Cell proliferation was measured using the WST-8® cell counting kit (Dojindo Molecular Technologies, Gaithersburg, MD) at 450 nm using a microplate spectrophotometer (BioRad®, Hercules, CA) as previously described in Literature. Lymphocyte proliferation was expressed as a stimulation index which is the ratio of the mean OD value of the profilin-stimulated group divided by the mean value of the OD of the stimulated group only by the CON medium.
• the proliferative lymphocyte response of splenocytes caused by the antigen as expressed by the stimulation index was increased in the groups vaccinated with profilin (from 1.05 ⁇ 0.05 to 1.21 ⁇ 0.11) compared with to the non-immunized CON group (0.87 ⁇ 0.01) at Day 10 post infection ( Figure 3).
• the MC-V group had the highest value.
• Caecal tonsils were obtained from uninfected and infected chickens that were fed with control (CON-V) and phytonutrient supplemented (VAC and MC-V) diets on Days 0 and 6 after infection (4 chickens). / group), and the expression of their genes encoding cytokines was determined using RTQ-PCR as previously described in the literature.
• the intestines were removed, cut lengthwise, and washed three times with ice-cold Hank's Balanced Saline Solution (HBSS) containing 100 U / ml penicillin and 100 ⁇ g / ml streptomycin.
• HBSS Hank's Balanced Saline Solution
• the mucosal layer was removed by thorough scraping using a surgical scalpel and the tissue was washed with HBSS.
• RNAs were extracted using TRIzol® (Invitrogen®, Carlsbad, CA). Five micrograms of total RNAs were treated with 1.0 U of DNAse I and 1.0 ⁇ l of 10X reaction buffer (Sigma®) and incubated for 15 minutes at room temperature. After incubation, 1.0 ⁇ g stop solution was added to inactivate DNAse I and the mixture was heated at 70 ° C for 10 minutes. RNA has undergone a reverse transcribed using the synthesis system 1 strand StrataScript® (Statagene®, La Jolla, CA) according to manufacturer's recommendations. The RT-PCT oligonucleotide primers for chicken cytokines and the internal control of GAPDH are shown in the table below.
• Figure 4 demonstrates the effect of food supplementation with VAC and MC on the production of caecal cytokines after immunization with the profilin vaccine.
• the profilin-vaccinated groups fed with VAC and MC showed overall a reduction of the pro-inflammatory cytokines IFN- ⁇ , IL-6, IL-17F, and TNFSF15 in the caeca compared to the CON group.
• unvaccinated P ⁇ 0.05.
• Cytokine IFN- ⁇ , IL-17F, and TNFSF15 caecal levels increased after oral challenge infection with E. coli. tenella in relation to their pre-infection values.
• IFN- ⁇ and IL-6 levels in the profilin-vaccinated group fed MC supplemented diet were significantly increased compared to the CON-V group at Day 6 post-infection.
• mice monoclonal antibodies specific for the major histocompatibility complex (MHC) class II, CD4, CD8, K1, T cell receptor 1 (TCR1), or chicken T cell receptor 2 (TCR2).
• MHC major histocompatibility complex
• TCR1 T cell receptor 1
• TCR2 chicken T cell receptor 2
• HB2 a human T-cell specific antibody (American Type Culture Collection, ARS-USDA) was included and an antibody against C6B12, a class 1 chicken MHC antigen, was used as a positive control .
• the cells were rinsed three times with FCA buffer and incubated with 100 ⁇ l of fluorescein isothiocyanate labeled IgG anti-mouse IgG secondary antibody (Sigma®) for 45 minutes on ice. Cells were rinsed three times with FCA buffer and fluorescence was then analyzed on 1 x 10 4 viable cells using FACSCalibur® (BD Science, Boston). Results
• peripheral blood lymphocyte subpopulations in farmed chickens are shown in the table below.
• the percentages of T cells expressing MHC class II, CD4, CD8, TCR1 and TCR2 were significantly increased compared to those in CON groups.
• -V and CON-VAC-V on Day 10 after infection; most of the values being twice that of the CON-V and CON-MC-V groups.
• the percentage of cells expressing a macrophage Klde marker was significantly increased in the CON-VAC-V group compared to that of the CON-V and CON-VAC-V groups at Day 10 post infection.

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