The Research Of National Reference Standard And Evaluation Of New Castle Disease Low Virulent Attnuate Vaccine (La Sota)

Newcastle disease (ND) that caused by Newcastle disease virus (NDV) is a highly contagious virus disease of birds. NDV can infect many species of domestic and wild birds, resulting in great harm on the poultry breeding industry, thus, ND has been considered one of the most important poultry diseases worldwide. Vaccine is one of the most effective methods of Newcastle disease control in China, even in the world. The vaccine containing the La Sota strain of NDV are wildly used, since they are characterized by virulence stability and good immune response. However, in present the determination of the quality of Newcastle disease vaccine (La Sota) just depends on relative written standards, lacking of physical standards. In these cases, random errors are beyond control in the effectiveness tests which cause the calibration values of effectiveness of Newcastle disease vaccine (La Sota) are not accurate, and the vaccines are uneven in quality. All of these problems result in the inaccurate effects that differ with the expected results, and these problems have become the obstacles of the development of poultry breeding industry.The reference standard of Newcastle disease can better control the quality of vaccine, achieving the standardization of the calibration value of effectiveness. The national standard materials of Newcastle disease were established through the selection, analysis and the determination of its standard features of semi-liquid chicken. This research primarily finished the establishment of the preparation and selection methods of mild living Newcastle disease vaccine (La Sota) through the studies of candidate semi-liquid chicken. According to the test results, the batches of vaccine candidate were selected and the final national standard material candidates were produced. After the basic quality inspection and effectiveness calibration, these national standard material candidates can be preserved.Three candidate product samples were done by the studies of three batches candidate vaccine through series of tests, such as exogenous virus test, aseptic test, specificity test and homogeneity test. Through strict statistic methods, No.3 candidate material which were qualified, homogenous and had relatively high effectiveness were determined to be used for the preparation of standard material. The initial effectiveness values (lgEID50=7.586±0.0857) of No.3 candidate material were also determined.3023 No.3 candidate standard Newcastle disease vaccines (La Sota) were produced, all of which meet the standard quality criteria after initial inspection procedures. The ampoule vacuum sealing preservation act was improved to ampoule filling nitrogen preservation act for the storage of standard material. The stability tests proved that the two methods had no significant differences in the preservation stability of low virulent Newcastle disease living vaccine, realizing the mass production of standard material.During the research of the effectiveness of vaccine, the quantitative RT-PCR method for the measurement of viral load in vaccine was established. This method was validated to be stable, reproducible, sensitive and specific. Vaccine contained virus copies 750×10^[(46.76-Ct)/3.29]/bottle, and the amplification efficiency of virus was 1.013 when the DNA copy number was between 5.0×102～5.0×109/ul. Using this method, it is verified that when lgEID50 is 7.5, the virus load of low virulent Newcastle living vaccine is 1.46×1011～2.78×1011 copy, and when the lgEID50 is 6.5, the virus load is 2.45×1010～6.0×1010 copy. There are about one magnitude difference of virus load between the low virulent Newcastle living vaccines whose lgEID50 are 7.5 and 6.5 respectively. When lgEID50 is 6.0 (the minimum lgEID50 of qualified vaccines), the virus load is 8.51×109～2.30×1010 copy. The virus load test results of No.3 Newcastle disease vaccine (La Sota) samples indicated that there had significant relationship between the Ct value get through this method and 50% egg infective dose, proving this method can be carried out for measuring the number of virus in Newcastle disease vaccine (La Sota). The number of virus load was 1.21×1011～3.85×1011 copy/bottle. Meanwhile, immunofluorescence technique for measuring the effectiveness of virus of low virulent Newcastle disease living vaccine (La Sota) was established. This method can determine the 1025EID50 unit NDV, depending on the significant relationship between 50% cell infective dose (lgTCID50) and 50% egg infective dose(lgEID50).