Effects Of Fermented Bingduke On AIV-H9N2 In Vitro And In Vivo

The purpose of this study was to investigate the antiviral activity of Fermented Bingduke（BDK）against AIV-H9N2 in vitro and in vivo,so as to provide theoretical basis for further research and clinical application of this preparation.To this end,the following tests are carried out:Experiment 1:Determination of Epigoitrin content in Fermented BDK.The preparation used in this experiment was made of complex bacteria as fermentation strains,including Bacillus subtilis,Saccharomyces cerevisiae and Lactobacillus plantarum.A certain amount of carbon source and water were added to the BDK powder for anaerobic fermentation.By high performance liquid chromatography（HPLC）detection Epigoitrin content changes before and after fermentation.HPLC resμlts showed that,the linear range of Epigoitrin was 0.4～25.6μg/ml（r=0.999）,and the average recovery and RSD of Fermented BDK were 98.68%and 0.81,respectively,which proved that the established detection method was highly specific and accurate.After testing,it was found that the content of Epigoitrin before and after fermentation was 0.448 mg/g and 0.735 mg/g,respectively,increasing by 1.64 times.Experiment 2:Effects of Fermented BDK on the proliferation of AIV-H9N2 in Madin-Darby canine kidney cells.In vitro experiments were conducted to observe the pathological changes of cells and detect the effect of Fermented BDK on the proliferation of AIV-H9N2 in MDCK using MTT method.The resμlts show that,the half toxic concentration(TC₅₀)of the preparation before and after fermentation were 58.20 mg/ml and 64.06mg/ml,respectively.The median effective concentration(ED₅₀)of Fermented BDK for neutralizing,preventing,and treating AIV-H9N2 were2.76,3.73,and 11.50 mg/ml,respectively,with a therapeutic index（TI）of23.21,17.17,and 5.57,respectively;The median effective concentration(ED₅₀)of BDK powder for neutralizing,preventing,and treating AIV-H9N2 were 3.55,4.67,and 16.44 mg/ml,respectively,with a therapeutic index（TI）of 16.39,12.46,and 3.54,respectively.Experiment 3:The effect of Fermented BDK on AIV-H9N2 infected broiler chickens.160 white feather broilers were randomly divided into 4groups:negative control group,positive control group,Fermented BDK group,and BDK powder group.The negative control group was not treated,and the remaining three groups were inocμlated with the virus.The vaccination method and dosage are nasal inoculation and 0.2ml,respectively;Fermented BDK group and BDK powder group were administered with 1.0 g/kg mixture after 24 hours of exposure,and clinical observation was conducted for 15 days.The therapeutic effect of AIV-H9N2 on artificially infected chickens was evaluated based on the mortality,cure rate,effective rate,and feed weight ratio of each group of chickens.The resμlt shows,the mortality and virus isolation rates in the Fermented BDK group and BDK powder group were 0%,2.5%,and 2.5%,7.5%,respectively,lower than those in the positive control group（5%,17.5%）;The cure rate and effective rate in the Fermented BDK group were80%and 100%,respectively,higher than 60%and 97.5%in the BDK powder group,indicating that both of them have therapeutic effects on chicken AIV-H9N2,and Fermented BDK has a better effect.At the same time,compared to the positive control group,the average daily weight gain and average daily feed intake in the medication group were significantly increased（P<0.05）,and the feed weight ratio in the Fermented BDK group was significantly lower than that in the BDK powder group（P<0.05）.Experiment 4:The effect of Fermented BDK on immune function and serum antioxidant activity in broilers infected with AIV-H9N2.The grouping and attack methods of this test are the same as those of Test Three,blood was collected from the pterygoid vein at 5d,8d,and 11d after the virus attack to isolate the serum.The titer of AIV-H9N2 antibody was measured by hemagglutination inhibition assay（HI）,and IL-2,IL-6,and IFN-γwere measured by ELISA;the determination methods of superoxide dismutase（SOD）and malondialdehyde（MDA）were spectrophotometry and TBA method,respectively.After the experiment,10 broilers were randomly selected and the immune organ index was measured using conventional methods.The resμlts are as follows:（1）Serum antibody titer:On the 5th and 8th days after the attack,the HI titer in the Fermented BDK group and BDK powder group was higher than that in the positive control group,with a significant difference（P<0.05）,and there was no significant difference between the medication groups（P>0.05）;At day 11,the HI titer of Fermented BDK group and BDK powder group was significantly higher than that of the positive control group（P<0.05）,and there was a significant difference between the medication groups（P<0.05）.（2）Cytokine levels:At day 5 and 8,compared with negative control group,the contents of IL-2,IL-6 and IFN-γwere significantly increased（P<0.05）.At 11 days,the IL-2 content in the medication group was significantly higher than that in the positive control group（P<0.05）,but the IL-6 content was the opposite.（3）Antioxidant:Compared with the positive control group,the SOD content in the negative control group was higher and MDA content was lower,and the difference was significant（P<0.05）,indicating that the antioxidant capacity of chickens was weakened after challenge.At day 8and 11 after challenge,compared with positive control group and BDK powder group,SOD content of Fermented BDK group was higher and the difference was significant（P<0.05）,MDA content was contrary.It proved that the antioxidant activity of Fermented BDK was higher than that of BDK powder.（4）Immune organ index:Compared with the positive control group,the thymus index and bursa of Fabricius index of the fermented virus gram group increased after treatment,and the difference was significant（P<0.05）.There was no significant difference in spleen index among each group（P>0.05）.In summary,in vitro and in vivo experiments indicate that Fermented BDK has a certain inhibitory effect on AIV-H9N2.Fermented BDK can exert antiviral effect by regμlating the body’s immune function and antioxidant capacity.