Isolation And Identification Of NDV And Detection Of NDV By Immunohistochemical Method

Newcastle disease(ND) is one of the infectious diseases with high contiguity and truculence;which induces big economic loss of poultry production in our country,and as one of the official reported infectious diseases.listed by Office International desEpizooties. In Anhui province,along with the generally use of vaccine,the cosmically break out of ND has been obviously controlled.However,due to the problem of the quality of vaccine,the use method and the process of immunity,the non-typical ND was induced in a mass of occurrence.Along with the appearance and popularity of enlarge of animal species which are infected and virus variance,the prevention and diagnoses of ND become more difficulty,this is induced high attention to detect and prevent NDV in every walk of life.Firstly,twenty materials with typical and non-typical ND clinical symptom are collected,the brain tissue were sampled.The NDV are isolated and purified by SPF embryo of eggs.The primary identification of allantois of eggs is carded out by HA and HI. The results identify three kinds of NDV,named NDV-AH1,NDV-AH2 and NDV-AH3.Secondly,the NDV-AH1,NDV-AH2 and NDV-AH3 were made for immunogenic antigen,the non-immune chicken are injected vaccine for seven days.The blood is collected after injected for three times,and then serum is separated.The mutual test of HI were done on the same condition between different separated viruses and Lasota which are used for antigen by minimal method,the HI antibody of serum are measured to detect the relativity between different separated viruses and Lasota,and the R value is caculated to compare the difference of their relativity in antigen.The results show that:the R value of three different separated viruses is 0.90 between NDV-AH1 and Lasota,0.95 between NDV-AH2 and Lasota,0.91 between NDV-AH3 and Lasota.The immunity was no obvious differences when the value of R:0.67≤R≤1.5,so the differences between NDV-AH1 and Lasota,NDV-AH2 and Lasota,NDV-AH3 and Lasota were not obvious.It can provide theory of inoculating ND vaccine in these areas.Furthermore,F48E9 virus with strong virulence has built in the test to infect PCEF, and then the pathological changes cells are observed,the pathological changes cells are used as material to establish immunohistochemical method,the best test consistency in conditions of the first antibody and second antibody are built,the results indicate:the first antibody is diluted 10 times,the condition of the reaction is 37℃for one hour,the second antibody which is signed by enzyme is diluted 400 times,the condition of the reaction is 37℃for forty minute,under these conditions,the effect of test reach high-point,the slices of cell are fine dyed,the background is clear.Finally,the non-immune chicken which are infected by F48E9 and NDV-AH1 for seven days are used as material to establish immunohistochemical method,the chicken are killed after infected for three or four days,then the brain,lung,liver,pancreas,duodenum and trachea are collected to observe the distribution of F48E9 and NDV-AH1,to find out the distribution of virus in each tissues when the animal are infected by two kinds of ND. The results indicate:the viruses distribute in lung,liver and brain when infected by F48E9, the virus distribute in lung,liver and brain when infected by NDV-AH1.Above all,three kinds NDV were collected;separated and identified in twenty materials with typical and non-typical ND clinical symptom.The mutual test of immunity indicates that the differences between separate NDV and Lasota aren't obvious.Chicken which are infected by F48E9 and NDV-AH1 are used as material to establish immunohistochemical method to find out the distribution of virus in each tissues.