Immunoregulating Effects Of Two Chinese Herbal Polysaccharides On Muscovy Duck Infecting Of MDRV

Although some herbal polysaccharides have been applied clinically for the immunoregulation of the immunosuppressive animals in recent years, the mechanism of immunosuppression caused by infectious diseases and the mechanism of immunoregulation of herbal polysaccharide have not been fully known now. It was reported that the muscovy duck reovirous(MDRV) could lead to immunosuppression in infected muscovy ducklings. In this study, the experiments were conducted to study the mechanism of immunoregulation of astragalus polysaccharides(APS) and hericium erinaceus polysaccharide (HPS) on the immunosuppression muscovy ducks infected with MDRV through establishing MDRV natural infection models, and to analyse the influences of herbal polysaccharide on partial cytokines, neurotransmitters, hormones in serum and the expression level of interleukin 2(IL-2) mRNA and interleukin 6(IL-6) mRNA in slpeens. The results showed as follows:1. Three specific primers were designed by gene sequences ofβ-actin, IL-2 and IL-6 of relevant species. The gene fragments of Mdβ-actin, MdIL-2 and MdIL-6 were cloned from the spleens of muscovy ducks at the age of 35 days which were induced by ConA, and then tested by gene sequenceing. The results showed that the sizes of Mdβ-actin, MdIL-2 and MdIL-6 were 786bp,423bp and 322bp respectively, which were accorded with the primers disigned. The homology of nucleic acid sequences was 98.7% between Mdβ-actin andβ-actin of anas platyrhynchos (EF667345),97.3% between Mdβ-actin andβ-actin of gallus,100% between MdIL-2 and MdIL-2(AY193713),98.35% between MdIL-2 and IL-2 of anas platyrhynchos (AF294323), and 99.7% between MdIL-6 and IL-6 of anas platyrhynchos (AB191038).2. The muscovy ducklings infected artificially with MDRV-YB4 strains at the dose of 2000TCID50 were mixedly raised three days later with the health muscovy ducks which were uninoculated MDRV at the proportion of 2:5. The results showed that the latter group had the pathogenic process, antopsy changes and clinical symptoms of muscovy duck reovirus disease under natural condition. The MDRV natural infection models were established for further researchs.3. The MDRV natural infection models were used to study the protective effects of the infected muscovy ducks fed with polysaccharides at different drinking doses, The results showed that APS and HPS at the dose of 0.6 g/L and 0.2 g/L respectively had better protective effects. 4. The one-day-old muscovy duckling were randomly separated into seven groups which were artificial infected MDRV group(AIG), no-treatment control group(NCG), cohabited MDRV infection control group(CICG), hericium polysaccharide control group(HCG), hericium polysaccharide prevention group(HPG), astragalus polysaccharide control group(ACG) and astragalus polysaccharide prevention group(APG), and the muscovy duckling of AIG, CICG, HPG and APG were infected by MDRV as the MDRV natural infection models. After divided into groups at random, the muscovy duckling of HCG and HPG were given hericium erinaceus polysaccharide (0.2 g/L) orally in drinking water, and the muscovy duckling of ACG and APG were given astragalus polysaccharides (0.6 g/L) orally in drinking water to observ the protective effects. The results were as follows:The mortality of HPG and APG groups was 12% and 4% respectively, significantly lower than the CICG group(24%). The clinical symptoms of the HGP and APG groups were changed to be moderate, such as mental state, appetite and soft limbs. The necropsy changes were lighter particularly at anaphase. The dissection analysis showed that there were no obvious MDRV infected lesions in the APG group.5. The content of interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4(IL-4), interleukin 6 (IL-6), adrenal cortical hormone(ACTH), glucocorticoid(GC), growth hormone(GH) andβ-endorphins(β-EP) in muscovy duck serum by Radioimmunoassay (RIA). The results showed as followed:(1) After MDRV infection, the secretion of IL-1, IL-2, IL-4, IL-6, GH and ACTH was inhibited; The level of GC was high at the midanaphase of MDRV infection; The level ofβ-EP was fluctuated, which was negatively associated with stress.(2) HPS and APS could promote muscovy ducks to secrete IL-1, IL-2 and IL-4, maintain the concentration of IL-6, ACTH andβ-EP to be relatively steady, restrain GC of muscovy ducks infected with MDRV to increase rapidly and made it maintain at the normal range, and promote the muscovy ducks of low GH to produce GH. The reasults also showed that the regulation capability of APS was better than HPS.6. The relative expression level of IL-2mRNA and IL-6mRNA from the muscovy Duck spleens was detected by real-time RT-PCR, usingβ-actin mRNA as reference genes. The research indicated as followed:(1) The apoptosis of the Th cells caused by MDRV had leaded to immunodepression, which was directly reflected through the decline of the expression level of IL-2 mRNA and IL-6 mRNA. (2) HPS and APS were helpful for the stable expression level of IL-2 mRNA and IL-6 mRNA of muscovy ducks infected with MDRV.7. Plasmid of pGEX6p-1/MdIL-2, was restructured by MdIL-2 ORF (open reading box) cDNA sequences after clone selection and sequencing, and then was transformed into E. coli DH5a to be cultured at 37℃by IPYG, The Sds-page and Western blotting analysis showed that the molecular of MdIL-2 fusion protein was about 40 kD. The cloning and the expression of original nuclear of MdIL-2 genes laid a good foundation for the further activity detection and application research of MdIL-2.In brief, the study showed that:①MDRV could inhibit neuroendocrine-immune system to secrete IL-1, IL-2, IL-4, IL-6, GH and ACTH, promote the secretion of GC, and lead to immunosuppression.②APS and HPS could promote the muscovy ducks to secrete IL-1, IL-2 and IL-4, inhibit cell apoptosis, remain the content of IL-6, ACTH andβ-EP comparatively steady, inhibit the content of GC of muscovy ducks infected with MDRV to increase rapidly, regulate the neuroendocrine-immune system to be steady, and promote the muscovy ducks of immunosuppression caused by MDRV to rehabilitation. The research provide a theoretical basis for prevention and treatment of animal diseases with HPS and APS as immune modulators.