WO2013124441A1 - Utilisation de thiosulfinate et/ou thiosulfonate de dialkyle destiné à améliorer la résistance d'un animal infecté par un agent pathogène - Google Patents

Utilisation de thiosulfinate et/ou thiosulfonate de dialkyle destiné à améliorer la résistance d'un animal infecté par un agent pathogène Download PDF

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WO2013124441A1
WO2013124441A1 PCT/EP2013/053603 EP2013053603W WO2013124441A1 WO 2013124441 A1 WO2013124441 A1 WO 2013124441A1 EP 2013053603 W EP2013053603 W EP 2013053603W WO 2013124441 A1 WO2013124441 A1 WO 2013124441A1
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animal
garlic
ptso
genes
compound
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PCT/EP2013/053603
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English (en)
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David Bravo
Hyun Lillehoj
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Pancosma S.A.
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    • 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
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • 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
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • 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
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents

Definitions

  • the invention concerns the use of a compound dialkyl-thiosulfinate and/or a compound dialkyl-thiosulfonate to improve the disease resistance of an animal infected with a pathogen, such as a virus, bacteria or apicomplexa.
  • a pathogen such as a virus, bacteria or apicomplexa.
  • AGPs antibiotics growth promoters
  • the invention concerns the use of a compound dialkyl- thiosulfinate (also called propy thiosulfinate, PTS) and/or a compound dialkyl- thiosulfonate (also called propy thiosulfonate, PTSO) to improve the disease resistance of an animal infected by a pathogen.
  • PTS propy thiosulfinate
  • PTSO propy thiosulfonate
  • the inventors discovered that these compounds were able to improve the resistance of the host animal to pathogenic challenges.
  • compound dialkyl-thiosulfinate or dialkyl-thiosulfonate has the effect of reducing the number of specific receptors intended to detect pathogenic bacteria and viruses in the gut. By reducing the number of these receptors, the animal is less sensitive to these organisms and its immune response against this infection is decreased. As a result, the animal has fewer symptoms due to the infection.
  • the compound is used in an amount of 1 g to 20 g per ton of feed or 0,5g to lOg per ton of drink.
  • the compound is used in an amount of 1 g to 10 g per ton of feed or 0,5 g to 5 g per ton of drink.
  • the compound can be used in an amount of 4g per ton of feed or 2g per ton of drink.
  • the animal is a monogastric (poultry, swine) or a pre-ruminant (calf, lamb, goat kid).
  • Figure 1 represents the effects of PTSO on the splenocyte proliferation
  • Figure 2 represents the effects of dietary PTSO on body weight (A), fecal oocyst shedding (B), and serum antibody responses against profilin (C) in broiler chickens post challenge infection with E. acervulina; a b Means lacking common letters differ in uninfected or infected chickens (P ⁇ 0.05);
  • Figure 3 represents the most significant network exhibiting >2.0-fold up- and down-regulated gene expression levels after dietary supplementation of Garlicon40 (Up- and down-regulated genes are illustrated with signs (+) or (-) respectively and the intensity of each gene indicates the expression level of the genes; the overlaid numbers are the fold changes of each gene expression)
  • Figure 4 represents a comparison between microarray analysis and qRT-PCR for levels of mRNAs corresponding to selected genes (each bar represents fold changes of mRNA levels in Garlicon 40-fed chickens vs. non-treated controls.
  • adiponectin receptor 2, ADIPOR2 aspartate beta-hydroxylase, ASPH; interleukin 8, IL8; tumor necrosis factor (ligand) superfamily, member 13b, TNFSF13B).
  • the invention is based on the discovery that feeding animals with at least one alliaceous extract, for example a garlic, onion or shallot extract, containing a dialkyl- thiosulfinate and/or a dialkyl-thiosulfonate like PTS (di(n-propyl) thiosulfinate) and PTSO (di(n-propyl) thiosulfonate) has a positive impact on immune parameters of animals, for example birds.
  • the dietary supplementation of, for example, 10 ppm of an alliaceous extract standardized with 40% PTS or PTSO (Garlicon 40) modifies the expression of an important number of genes (1227) in intra-epithelial lymphocytes.
  • TLR3 and TLR5 Toll Like Receptors 3 and 5
  • TLR3 and TLR5 Toll Like Receptors 3 and 5
  • TLR3 and TLR5 These receptors detect the presence of invading micro-organisms, and are specific.
  • TLR3 detects viruses and TLR5 detects bacteria.
  • a reduced number of TLR reduces the sensitivity of the host to the organism.
  • body weight gain of chickens infected by Eimeria acervulina and fed with 10 ppm Garlicon 40 are higher than infected not supplemented chickens similar to birds in untreated non infected group.
  • the use of the product Garlicon 40 in the animal feed in an amount of 10 ppm of Garlicon 40 that is standardized with 40% PTS or PTSO in the feed for the animal (this corresponds to an dose of 4ppm of PTS or PTSO in the feed), enhances the resistance of the animal as well as the oocyst excretion in case of coccidosis infection.
  • the compound can be added in the drink for the animal, instead of the feed. In this case, the compound would be added in an amount of 2 ppm in the drink (2 g per ton of drink).
  • Splenocytes were collected from each treatment group (4/group) following euthanasia at 14 dph as described (28).
  • the cells were adjusted to 5.0 x 10 6 cells/ml in RPMI medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 ⁇ g ml streptomycin and incubated in a humidified incubator at 41°C and 5% C0 2 for 48 hr with or w/o serial dilution of PTSO or 2.5 ⁇ g ml of concanavalin A (ConA) in 96-well plates.
  • Cell proliferation was measured using WST-8 (Cell-Counting Kit-8, Dojindo Molecular Technologies, Gaithersburg, MD) (29) and was defined as the optical density at 450 nm (OD 450 ).
  • Serum samples were obtained at 14 dph (4/group) and tested by ELISA to determine serum antibody levels against profiling, an immunogenic protein of Eimeria, as described (30). Briefly, 96-well plates were coated overnight with 10 ug/well of purified recombinant profiling protein. The plates were washed with PBS containing 0.05% Tween (PBS-T), and blocked with PBS containing 1% BSA. Diluted sera (1: 100) were added (100 ul/well), incubated with agitation for 1 hr at room temperature, washed with PBS-T, and bound antibody detected with peroxidase-conjugated rabbit anti-chicken IgG (Sigma, St.
  • PTSO induced significant lymphocyte proliferation response in dose-dependent manner when compared with medium control.
  • the level of PTSO-induced proliferation was comparable to that of Con A-treated group.
  • the optimal dose PTSO needed for the stimulation of chicken splenocyte proliferation was 10 ng/ml.
  • Body weights were taken from individual birds at 0 and 10 dpi, and mean weight gains of uninfected and E. acervulina infected birds were calculated over the 10-d infection period.
  • the dietary supplementation with PTSO significantly (P ⁇ 0.05) increased body weight gain in the E. acervulna infected bird compared with non-treated control.
  • PTSO-supplemented group shed significantly less oocysts per bird (P ⁇ 0.05) than non-treated control (Figure 2B).
  • Eimeria profilin protein which is a conserved Eimeria protein, was used in this study.
  • Figure 2C shows that E. acervulina-mfected chickens produced significantly higher profilin-specific serum antibodies when they were fed PTSO-supplemented diet compared to controls (P ⁇ 0.05).
  • PTSO splenocyte lymphoproliferation
  • Several immunomodulatory proteins including garlic extracts showed effects on lymphocytes, mast cells, and basophils in rat (49).
  • PTSO exerted direct killing effects on an invasive stage of Eimeria and enhanced serum antibody titers against coccidian.
  • PTSO a garlic-derived compound
  • PTSO- supplemented diet clearly improved disease susceptibility with improved body weight growth of garlic-fed chickens.
  • results of present study show clear interaction of garlic with host genes and suggest that dietary immunomodulation by garlic-derived compounds may represents a sound alternative to antibiotics for poultry production.
  • Broiler chickens (Ross/Ross, Longenecker's Hatchery, Elizabethtown, PA) were randomly housed in Petersime starter brooder units to four groups (control and treatment group with or w/o infection) at one day post hatch (dph).
  • Garlicon40 an extract of garlic fruits representing secondary metabolite of garlic, PTSO, was obtained from Pancosma S.A. (Geneva, Switzerland).
  • Garlicon40 contains about 40% of a mixture of PTS and PTSO.
  • the diets were formulated to meet the nutrient requirements for chickens as recommended by the National Research Council (1994). Chickens were fed with a standard diet alone (control) or with diets supplemented with 10.0 ppm of Garlicon40 during the experimental period. The Garlicon40 dose was chosen based on the optimal doses from our pretrial experiments (data not shown).
  • IELs Intestinal intraepithelial lymphocytes
  • Trizol Intestinal intraepithelial lymphocytes
  • RNAs were amplified using the Two-Color Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA) with cyanine 3 (Cy3)- or cyanine 5 (Cy5)-labeled CTP.
  • RNA probes were hybridized with a Chicken Gene Expression Microarray (Agilent Technologies, Santa Clara, CA) containing 43,803 genes. Two hybridization replications were performed for each sample with alternation of fluorescent dye to prevent data distortion from sample labeling (24). The microarray images were scanned, and data extraction and analysis were performed using Feature Extraction software version 10.7.3.1.
  • GeneSpring GX10 software (Silicon Genetics, Redwood, CA) was used to qualify and normalize image analysis data and to determine fold-changes in gene expression. Median signal intensities were qualified by subtraction of the median local background and normalized by block LOWESS (locally-weighted regression and smoothing scatter plots) methods. Flag information was applied to strain the spots with 100% valid values from each samples and asymptotic t-test analysis was performed to analyze the significance between Garlicon40-fed and control groups. To generate signal ratios, signal channel values from Garlicon40-fed birds were divided by values from controls. Modulated elements were defined as RNAs with > 2.0-fold increased or decreased levels with P ⁇ 0.05. The significantly differentially expressed genes were filtered using the Volcano Plot method (25) built by the t-test. All microarray information and data will be deposited in the Gene Expression Omnibus (GEO) database.
  • GEO Gene Expression Omnibus
  • IPA Ingenuity Pathway Analysis
  • RNA samples used for microarray hybridizations were reverse-transcribed using the StrataScript First Strand Synthesis System (Stratagene, La Jolla, CA). Oligonucleotide primers are listed in Table 1. Amplification and detection were carried out using the Mx3000P system and Brilliant SYBR Green qRT-PCR master mix (SABioscience, Stratagene, Frederick, MD). Standard curves were generated using log 10 diluted standard RNA and levels of individual transcripts were normalized to those of GAPDH by the Q-gene program (27). For the calculation of fold changes between GD and groups, Ct values of the target gene were normalized to GAPDH and calibrated to the relevant control line values. Each analysis was performed in triplicate. Lymphocyte proliferation and serum antibody levels
  • Splenocytes were collected from each treatment group (4/group) following euthanasia at 14 dph as described (28).
  • the cells were adjusted to 5.0 x 10 6 cells/ml in RPMI medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 ⁇ g ml streptomycin and incubated in a humidified incubator at 41°C and 5% C0 2 for 48 hr with or w/o serial dilution of PTSO or 2.5 ⁇ g ml of concanavalin A (ConA) in 96-well plates.
  • Cell proliferation was measured using WST-8 (Cell-Counting Kit-8, Dojindo Molecular Technologies, Gaithersburg, MD) (29) and was defined as the optical density at 450 nm (OD 450 ).
  • the analysis on Bio Function and Canonical Pathway classified the modified genes by PTSO feeding to the categories of biological functions.
  • the significance value associated with a function in this analysis is a measure for the likelihood that genes from the dataset file under investigation participate in that function.
  • the significance is expressed as a P value that is calculated using the right-tailed Fisher's exact test.
  • the expression patterns observed by microarray analysis were validated by qRT- PCR with 4 selected transcripts whose levels were significantly modulated when comparing PTSO-fed vs. non-treated chickens. These genes were adiponectin receptor 2 (ADIPOR2), aspartate beta-hydroxylase (ASPH), interleukin 8 (IL8), and tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B).
  • the levels of all 4 transcripts that were up-or down-regulated by microarray hybridization were also correspondingly up- or down-regulated when analyzed by qRT-PCR (Figure 5).
  • the differences in the magnitude of the changes observed by the two techniques might be due to differences in the normalization methods used and/or the different fluorescent dyes used (32).
  • PTSO induced significant lymphocyte proliferation response in dose-dependent manner when compared with medium control.
  • the level of PTSO-induced proliferation was comparable to that of Con A-treated group.
  • the optimal dose PTSO needed for the stimulation of chicken splenocyte proliferation was 10 ng/ml.
  • TLRs Tolllike receptors
  • IRF interferon-regulatory factor
  • PTSO a garlic-derived compound
  • PTSO- supplemented diet clearly improved disease susceptibility with improved body weight growth of garlic-fed chickens.
  • the results of present study shows clear interaction of garlic with host genes and suggest that dietary immunomodulation by garlic-derived compounds may represents a sound alternative to antibiotics for poultry production.
  • Tsao SM Yin MC. In-vitro antimicrobial activity of four diallyl sulphides occurring naturally in garlic and Chinese leek oils. J Med Microbiol. 2001 Jul;50:646-9. 12. Kyo E, Uda N, Kasuga S, Itakura Y. Immunomodulatory effects of aged garlic extract. J Nutr. 2001 Mar;131: 1075S-9S.
  • Garlic oil and DDB comprised in a pharmaceutical composition for the treatment of patients with viral hepatitis, prevents acute liver injuries potentiated by glutathione deficiency in rats. Chem Biol Interact. 2005 Jun 30;155:82-96.
  • TLRs differential adapter utilization by toll-like receptors mediates TLR-specific patterns of gene expression. Mol Interv. 2003 Dec;3:466-77.
  • Clement F Pramod SN, Venkatesh YP. Identity of the immunomodulatory proteins from garlic (Allium sativum) with the major garlic lectins or agglutinins. Int Immunopharmacol. Mar; 10:316-24.
  • MDK 23.142 growth factor promoting factor 2 A_ _87_ _P009319 Space
  • VAX1 ventral anterior homeobox 1 11.949 Nucleus
  • TLR5 Altered T Cell and B Cell Signaling TLR5, HLA-DRB1 , TLR3,

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Abstract

La présente invention concerne l'utilisation d'un composé de thiosulfinate de dialkyle et/ou un composé de thiosulfonate de dialkyle destiné à améliorer la résistance aux maladies d'un animal infecté par un agent pathogène. L'agent pathogène peut être un virus, une bactérie ou un apicomplexe. Le composé est utilisé en une quantité de 1 g à 20 g par tonne d'aliment (ou 0,5 g à 10 g par tonne de boisson). Le composé est utilisé de préférence, dans une quantité de 1 g à 10 g par tonne d'aliment (ou 0,5 g à 5 g par tonne de boisson).
PCT/EP2013/053603 2012-02-23 2013-02-22 Utilisation de thiosulfinate et/ou thiosulfonate de dialkyle destiné à améliorer la résistance d'un animal infecté par un agent pathogène WO2013124441A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015128516A1 (fr) * 2014-02-26 2015-09-03 Investfood, LLC Nouvelles méthodes d'utilisation d'un produit pour la modulation de la réponse immunologique chez des humains
US9163162B2 (en) 2012-08-30 2015-10-20 Hitachi Chemical Company, Ltd. Polishing agent, polishing agent set and method for polishing base
EP3338774A1 (fr) * 2016-12-23 2018-06-27 Mixscience Composition à base de composés thiosulfinate et/ou de thiosulfonate pour son utilisation dans la prévention des infections bactériennes chez les animaux aquatiques

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