Sensitive, Semi-nested RT-PCR Amplification Of F Sequences For The Rapid Detection And Differentiation Of Newcastle Disease Virus And Protective Effect Of Different Genotypic Inactivated NDV Vaccines

Chicken newcastle disease (ND) is one of urgent,highly contagious,violent avian infectious disease which had done great harm to poultry enterprise. It was listed by Office International des Epizooties (OIE). Every year it cause thousands of millions yuan economic loss. In recent years, there is occurrence continuously in the ND LaSota vaccinated flocks and high antibody chickens. For this reason semi-nested RT-PCR amplification was performed to discriminate virulent NDVs from live attenuated vaccine strains and protective effect of different genotypes of oil-adjuvant inactivated NDV vaccine was also been studied in high antibody chickens.1,Sensitive, semi-nested RT-PCR amplification of F sequences for the rapid detection and differentiation of Newcastle disease virusHere we described a sensitive and specific semi-nested RT-PCR approach for the detection and differentiation of virulent Newcastle disease viruses (NDV) from avirulent viruses. Based on the previously published GenBank sequences of the fusion protein (F) genes of NDVs, we designed a pair of outer primers targeting the F genes of both virulent and avirulent strains and an inner primer specific for the F0 cleavage site of virulent strain. With these primers, we developed the semi-nested RT-PCR and characterized the pathotypies of 62 NDV strains, including 52 field strains isolated in 2006 in a blind study. We found that determination of the pathotype of NDVs with the semi-nested RT-PCR approach was fast. The pathotype results obtained from semi-nested RT-PCR were consistent with those from the analysis of nucleotide sequences at cleavage sites of F genes and those from the assays of plaque forming units, mean death time (MDT) and intracerebral pathogenicity index (ICPI). Furthermore, with semi-nested RT-PCR approach, we also characterized 13 Classâ… type NDV strains, which were rarely characterized by the conventional methods. In the optimization of semi-nested RT-PCR, we found that both addition of 2% dimethyl sulfoxide (DMSO) and addition of 0.06 mg/ml acetylated bovine serum albumin (BSA) improved the efficiency of the one step RT-PCR. However, no synergistic effects were observed between DMSO and BSA. The detection threshold for the newly-developed semi-nested RT-PCR was 2 PFU of F48E9 NDV strain in allantoic fluid and the same level of prevalent virulentâ…¦d isolate (APMV1/ch/China4031) in oral or cloacal swab specimens collected from chickens vaccinated with live attenuated vaccines. Taken together, our results suggested that the semi-nested RT-PCR approach represents a feasible strategy for the rapid detection and differentiation of virulence NDVs.2,Protective effect of different genotypic inactivated NDV vaccinesIn order to realize the antigenic variation of epidemic strain and to develop more effective novel vaccine, the representative NDV genotypeâ…¦andâ…¨epidemic strain (NDV4222 and NDV251), combined with the traditional LaSota strain (geneâ…¡type) were chosen to prepare five different monovalent and polyvalent oil-adjuvant inactivated vaccines, the immune effect of each vaccine was evaluated. After inactivation, the HA titer ofâ…¦-type strains NDV4222 dramatically dropped to 0, but was still immunogenic , which suggested that theâ…¦-type strain NDV4222 is antigenically different from other genotype strains.First of all, we used the live vaccine strain LaSota as basis of immunization in 10-day-old chicken, and then in the fourth week, each group was injected with different inactivated oil vaccine to enhance the immunity. It was found that single and composite genotype immunization both can elicit a strong immune response and the level of antibody changes in each group was very similar. NDV antibody was generated 4 days after immunized with live vaccine although its HI titer is low. Two weeks after the second immunization, antibody titer increased steeply and then slightly in the third week. The HI titer of each group using F48E9 as testing antigen was 29-210 before challenge.Immunization protective rate of all vaccine groups was 100%. Despite similar antibody titers in each group, we found that Group E, vaccination with the three genotype composite vaccine, has the lightest tissue lesions after challenge in histopathologic examination. Only in the virus entry site - the trachea (challenge through intranasal and eye), there was some epithelial cell necrosis, or loss. In addition, virus shedding had not been detected in all immunized groups, demonstrated that such immunization procedures had significance of biological safety in laying hens.