Development Of "Guyuan Granule" Immunopotentiator And Its Applications In Chicken Vaccination

Traditional Chinese medicine?TCM?has an obvious effect of enhancing immunity,and has become an important research direction for the application of alternative antibiotics.The original formula of"Guyuan Granules"caming from the"New Drug Conversion Standard"formulated from astragalus and raw sun ginseng rutabaga is effective in benefiting Qi and strengthening the body.But due to the high cost of its original formula,it was difficult to be popularized in veterinary clinics and animal husbandry.However,some of the subsidiary extracts that have similar effects to the main ingredients usually produced during the processing of TCM are favored by new veterinary drug developers because of their low cost.Therefore,Astragalus Polysaccharides?APS?and Ginseng Stem-leaf Saponins?GSLS?were substituted for Astragalus and Ginseng in the formulation of"Guyuan granules"to produce soluble pellets called veterinary"Guyuan granules"for enhancing resistance to disease and vaccine immunity in animals.Next,the preparation processes and quality standards of Guyuan granules were studied.The safety,effectiveness and optimal addition of the preparation to chickens,as well as its immunomodulatory effect on chickens and mice that under immunosuppressive conditions were then evaluated.Moreover,the molecular mechanism of the granules in activating TLR4 signaling pathway and promoting related cytokine secretion in laying hen lymphocytes has been preliminary explored.Finally,in order to provide new methods for the prevention and treatment of viral diseases of poultry,the application experiments were conducted on chickens.The main results are summarized as follows:1.The formulation technology of Guyuan granules was optimized and determined.The optimal molding process was determined through pre-experiment,formulation selection,wetting agent screening and verification tests.The formula was:astragalus polysaccharide 110 g,ginseng stem-leaf saponins 100 g,sucrose 590 g and dextrin 200 g.Mix them evenly with 75%ethanol as a wetting agent,then make soft materials,granulate,dry at 60°C,and granulate.The obtained particles are easy to shape,the granules are complete,and the fine powder is less.The test results show that the quality of granules obtained from the three batches is consistent,indicating that the process is stable and feasible.2.High performance liquid chromatography was established and the content of ginsenosides Rg1,Re,and Rd in the granules was determined.It was confirmed by precision test,repeatability test,stability test and recovery test that RSD<2%.Three batches of solid element particles were measured.The total amount of Rg1+Re+Rd in each 1 g of particles was above 30 mg.The results show that the method is easy to operate and accurate in measurement,and can be included in the quality standard as the content of the quality control of the preparation.3.Guyuan granules have both safety and effectiveness.First,the chickens were orally administered with the granules at the recommended doses of 0,1,3,5,and 10 times?0,0.05,0.15,0.25,and 0.5 g/kg body weight?for 7 consecutive days and discontinued for 2 weeks.The behaviors and weight changes of chickens were observed,and the blood routine indexes,blood biochemical indexes and organ indexes were detected.The results obtained were not abnormal,indicating that the preparation has high safety for chicken clinical use.In addition,different dosages of Guyuan granules can effectively improve the antibody titer level,erythrocyte rosette rate and production performance indicators of laying hens,among which 50 mg/kg is the most suitable dosage.Guyuan granules have immune-enhancing effects on chickens that have been vaccinated with Newcastle Disease vaccine.50 mg/kg of the granules was used to perform immunomodulatory tests on the vaccinated chickens,the organ index,the Newcastle Disease antibody titer,and the spleen lymphocyte transformation index were measured.It was confirmed that the anti-NDV antibody titer,was significantly higher than that in the control group?P<0.05?after 2-3 weeks of administration.Guyuan granules have immunomodulatory effects on immunosuppressed chickens.After oral administration of the granules to immunosuppressed chickens induced by cyclophosphamide?CTX?,the immunosuppressive phenomena were significantly alleviated.The levels of Newcastle Disease antibody were significantly higher than those in the CTX group?P<0.05?on day 21 after immunization,and there was no significant difference from the vaccine control group?VC?.In addition,on 7th,14th,and 21 days after the vaccine immunization,the ConA and LPS-induced spleen lymphocyte stimulation of four drug-treated groups were significantly higher than those of the CTX group?P<0.05?.There was no significant difference between the middle dose group and the blank control group?BC??P>0.05?.Besides,Guyuan granules also have immunomodulatory effects on immunosuppressed mice.The medium and high dose groups of Guyuan granules can significantly increase the spleen index and thymus index of immunosuppressed mice?P<0.05?.The results showed that after 15 days of administration,the levels of IL-2 and IFN-?in immunosuppressed mice were close to those of normal mice,and the effect of the middle dose group?50 mg/kg?was the most obvious.In addition,the spleen lymphocyte conversion rate of each dose group was significantly different from that of the CTX group?P<0.01?.4.The immune response effects of Guyuan granules on chicken may be related to the activation of TLR4 signaling pathway and the secretion of related cytokines in laying hen lymphocytes.The peripheral blood lymphocytes of laying hens were isolated and cultured with different concentrations of Guyuan granules?final concentrations of 200,100,50,25,and 0?g/mL,and the positive control was astragalus polysaccharide with a concentration of 100?g/mL?for 16 h,24 h,32 h and 48 h,respectively.RT-PCR was used to detect the expression of TLR4,MyD88,TRAF-6,TRIF,IRF3,and IFN-?mRNA in lymphocytes.After that,the supernatants of lymphocytes cultured for 24 h were collected,and the contents of cGMP,cAMP,NO,iNOS,and Ca²⁺were measured by ELISA.The results showed that Guyuan granules activated MyD88-dependent and TRIF-dependent signaling pathways through TLR4 receptors,which could also reduce the content of cAMP in the culture supernatant of chicken peripheral lymphocytes and increase the content of cGMP,Ca²⁺,NO and iNOS,thereby regulating the immune function of laying hens.5.The application of"Guyuan granules"in chickens can promote the immune effects of vaccines.Two chicken houses were randomly selected and divided into the"Guyuan granule"+vaccine immunization group and the single vaccine immunization group?n=3100?,and clinical application trials were conducted at a dose of 50 mg/kg body weight.Compared with the vaccine control group,the immunization antibody titers of the"Guyuan Granule"test group increased significantly,the morbidity and mortality of the group administrated with Guyuan granules decreased by 0.7 and 0.8 percentage points respectively.It shows that"Guyuan granules"can effectively reduce the morbidity and mortality in large groups of clinical trials and increase the breeding income.In summary,the stable preparation process of"Guyuan Granules"and its quality standards have been established to confirm the safety and effectiveness of the formula,and preliminary confirmation that the granule preparation can activate MyD88 and TRIF pathways through TLR4receptors,reduce the content of cAMP in the culture supernatant of chicken peripheral lymphocytes,increase the content of cGMP,Ca²⁺,NO,and iNOS,thereby regulating the immune response of chickens.Guyuan granules have a good application prospect because they can significantly increase the antibody levels of laying hens and reduce morbidity and mortality.