Effect Of Enteromorpha Polysaccharide And Propolis On Mucosal Immunity Of Inactivated H9N2 Subtype Avian Influenza Virus

As the most prevalent subtype of influenza viruses in chickens in China,H9N2 not only causes a great economic loss for the poultry industry,but also poses a great threat to public health.Recent research has shown that Specific Medicinal Plant Polysaccharides and propolis could improve the effects of different vaccines.The present work summarizes the preparation,extraction,purification,and characterization along with quantitative analysis of polysaccharides from Enteromorpha prolifera(PEP).In addition,co-adjuvant effects of Enteromorpha polysaccharide-propolis flavone oral liguid(PPO)on chickens immunized orally with H9N2 avian influenza inactivated vaccine is discussed.This study would clarify the adjuvant activity of PPO and provide the theoretical basis for the development of new mucosal vaccine adjuvants.The main results were as follows:1.Isolation,purification and identification of PEP:The crud polysaccharides(PEP)were extracted by decoction and alcohol sedimentation technique.Purification of the PEP by a gel filtration using Sephadex G-75 column and DEAE-cellulose column chromatography.The carbohydrate,protein,uronic acid,and acidophobe contents of PEP were determinated recpectively by phenol-sulfuric acid,Bradford protein assay,carbazole-sulfuric acid,Oxygen Flask Combustion methods.Identification of the polysaccharides structural by ultraviolet(UV)spectra,fourier transforms infrared spectroscopy(FTIR),high performance liquid chromatography(HPLC),Gel size exclusion chromatography(SEC),scanning electron microscope(SEM),and nuclear magnetic resonance spectroscopy(NMR)analysis.Thermodynamic Properties of PEP were analyzed by differential scanning calorimeter(DSC)and thermo-gravimetric(TG).Our results show that the extraction rate of PEP was 1.926%.The carbohydrate content of PEP was the highest up to 90.21%w/w.The uronic acid units of PEP was found to be 11.37%w/w.The protein and Sulfate Groups contents of PEP were lower than1%.PEP were polysaccharide substances and contained no proteins and a little of nucleic acids from UV scanning spectrum.Polysaccharides had characteristic absorption peak in infrared spectrum;molecular weight distribution showed that PEP had two different retention time,indicating the existence of a narrow symmetrical peak and the polysaccharides were homogeneous with higher purity.The average molecular weight of PEP were 6.3×10~3-1.77×10~5Da;monosaccharide composition analysis showed that PEP was mainly composed of glucose(58.23%),galacturonic acid(11.04%)arabia(9.34%),galactose(15.013%),xylose(8.01%);1 H-NMR and 13 C-NMR showed that PEP linked mainly by(1→3)-β-D-Glcp residues.In addition,there may exist a small amount ofα-L-1,6-Arap residues too.From the thermogravimetric analysis(TGA)data,the second mass loss(268.37~318.82℃)attributed to polysaccharide degradation.The results of SEM showed that schistose structures were observed in PEP.The DSC thermograms of polysaccharides revealed endothermic peaks at around 84.61℃for PEP,which corresponded to the melting temperature of polysaccharides.In a word,PEP prepared by our method has high purity and can be used for further study.2.The Assessment of Enhancing Immune Effects of inactivated avian H9N2 influenza virus vaccine(IAIV)with PPO.14-day-old chickens were randomly assigned into 7 groups and,except for blank control(BC)group,orally immunized with inactivated avian H9N2 influenza virus vaccine,revaccination at 28 days old.At the same time of each vaccination,the chickens in high,middle,and low doses of PPO groups were taken orally respectively with PPO of 4mg,2mg and 1 mg,and the chickens in drug control group were taken orally respectively with PEP and PF 2mg,once a day for three consecutive days.In vaccination control(VC)group and blank control(BC)group,no administration.(1)The detection results of immune indexes in serum:AIV-specific HI antibody,AlV-specific lgA and IgG antibody,IL-4 and IFN-γwere tested on 7,14,21,28 and 35 days after the first vaccination.The results showed that antibody titer at every time point and IL-4 and IFN-γcontent at most of the points in high and middle doses of PPO groups were higher or significantly higher than VC group.The levels of the IgA and IgG antibody on 7d~21d increased significantly(P<0.05)with IAIV in combination with PPO after oral immunization compared with VC group.These results suggested that PPO could significantly enhance the systemic immune response,and middle and high dosage of PPO had a better effect.(2)The detection results of immune indexes in small intestine:On 7d,21d and 35d after the first vaccination,six chickens were selected randomly from each group to measure slgA and IL-17 content in washing liquor of duodenum,the number of lymphocyte in duodenal endothelium,IgA~+cell and mast cells in jejunal endothelium.The results showed in every time points of the first vaccination slgA and IL-17 content in washing liquor of duodenum,lymphocyte in duodenal endothelium,the number of IgA~+cell and mast cells in jejunal endothelium were significantly higher than that of VC group and were higher or significant higher than PEP and PF groups.These results indicate that PPO could enhance the intestinal mucosal immunity.The effects of the middle and high doses of PPO were the best.