Studies On The Detetion Of Antibodies Against New Duck Plague Virus By An Indirect ELISA And The Detection Of New Duck Plague Virus By An Indirect Immunofluorescent Assay

In this study,the purifying method of the new duck plague virus(NDPV,named tentatively),the detecting of the serum against the NDPV by indirect ELISA,the locatilation of virus by IFA,and the pathogenicity of the different generation's virus to duck and the detecting of virus were studied.We confirmed the best purifying method of the virus,the best confiditions of the indirect ELISA and its sensitivity,specificity and repetivity,locating the virus,confirming the relation between the virus'pathogenicity to duck and the detective rate.We can obtain pure virus by extracting the virus with 50% ammonium sulfate salting-out or 10% PEG6000 and then purifying the virus by means of Sephadex G-200 chromatography;the indirect ELISA is very sensitive,specificand and repetitive;the IFA confirmed that the most of viruses are in liver and spleen,and the others are in blood,brain,rhinal secretion;the pathogenicity of old virus is attenuated to duck,S20 virus can't do harm to duck,but the virus can be detected.This research consists of four parts: PartⅠ:The purification of the NDPV A viral strain was obtained from the natural infectious ducks with clinical sympotom,which was inoculated muscovy duck embryo at 12-day-old,we collected the allantoid liquid that the embryo died in 48 hours to 120 hours after inoculation and had typical pathological changes,then extrating crudly the virus with 50% ammonium sulfate salting-out,60% ammonium sulfate salting-out,8%PEG6000,10% PEG6000 and then purifying the virus by means of Sephadex G-200 chromatography with different velocity of flow.The result confirmed that it has same effect with 50% ammonium sulfate salting-out and 10% PEG6000,the velocity of flow with 0.5ml/min is good,it is diffcult to detach the virus and the effect is bad when the velocity of flow under 0.5ml/min because the peak value becomes broad and protracted.PartⅡ:Detecting the serum antibody against NDPV by indirect ELISA The polystyrene microtitration plates was coated with the purified virus,and the concentration of coating was 1.17ul/cave.The plates should be kept a whole evening at 4℃ after an hour at 37℃.Then we blocked the plates,joining the sera and the antibody conjugated with HRP,and all the steps above were done at 37℃ for an hour.It indicated that the critical number was 0.20 by detecting 30 negative sera,and the serum was positive if the number exceeded 0.20;the result of detecting the serum antibody of ducks infected experimentall were positive expect the ducks which died in one to three days after coming to disease,and the detective rate was 80%;all the immune young ducks were positive .By detecting the standard positive sera and the positive sera made by oneself,it confirmed that the serum against NDPV is partly correlative with the serum against DPV,but having nothing with others.The results of detecting 6 sera under the same conditions but at different time were same.All the studies above indicated that the indirect ELISA is very sensitive,specificand and repetitive.ParyⅢ:Detecting the virus by IFA We infected experimently 10-day-old ducks and 50-day-old ducks with NDPV and DPV,collecting organs in different time after infection,then we detected the virus in the organs by IFA.The result demonstrated that NDPV infects young ducks easily and DPV infects old ducks;we should collect the samples of young ducks at 72th hour after infection with NDPV and at 48th hour after infection with DPV.By detecting different organs,it indicated NDPV and DPV can multiply in the same organs,and the most of viruses are in liver and spleen,and the others are in blood,brain,rhinal secretion.The detective rate of iced slice is high obviously than pressed slice.Part Ⅳ:The relation between the virus'infectious ability to ducks and its detective rate We infected young muscovy at 1-day-old,infecting 5 ducks with every generation of virus.With increasing of the generation,the virus is attenuated obviously;2th generation,5th generation,10th generation can infect all young duck...