Alternative method using Real Time PCR for evaluation of inactivated Newcastle disease viral vaccine

The present work aims to establish a new alternative protocol to evaluate in vitro potency of inactivated Newcastle disease virus vaccine using Real Time PCR. Aqueous phases of seven inactivated Newcastle disease virus vaccines batches of different manufacturers were extracted by isopropyl myristate. The Newcastle disease virus antigen of each vaccine sample was determined by a standard Real Time PCR assay. Vaccines were inoculated into separate groups of 3-week-old specific pathogen free chickens using the recommended dose of vaccine. The immunogenicity was assessed for each vaccine by the Newcastle disease virus hemagglutination inhibition antibody titers. Individual serum samples were collected 4 weeks post vaccination, then vaccine efficacy and protection rates were recorded after challenge test of birds vaccinated with the virulent Newcastle disease virus. There is the possibility of using the Real Time PCR as an in vitro assay for vaccine evaluation. The Cycle Threshold values were ranged between 21.17 and 25.23. On the other hand, the hemagglutination inhibition titers ranged between 7.1 log2 to 6.2. The comparison between the Cycle Threshold values of the antigen extracts and the corresponding results of challenge test and in vivo hemagglutination inhibition assays using sera of vaccinated birds proved a strong correspondence between the in vitro and in vivo results(AU)

El presente trabajo pretende establecer un nuevo protocolo alternativo para la evaluación in vitro de la potencia de la vacuna de virus inactivado contra la enfermedad de Newcastle mediante PCR en tiempo real. Las fases acuosas de siete lotes de vacunas inactivadas contra el virus de la enfermedad de Newcastle de distintos fabricantes se extrajeron mediante miristato de isopropilo. El antígeno del virus de la enfermedad de Newcastle de cada muestra de vacuna se determinó mediante un ensayo estándar de PCR en tiempo real. Las vacunas se inocularon en grupos separados de pollos libres de patógenos específicos de 3 semanas de edad utilizando la dosis recomendada de vacuna. La inmunogenicidad se evaluó para cada vacuna mediante los títulos de anticuerpos de inhibición de la hemaglutinación del virus de la enfermedad de Newcastle. Se recogieron muestras individuales de suero 4 semanas después de la vacunación y, a continuación, se registraron la eficacia de la vacuna y los índices de protección tras la prueba de reto de las aves vacunadas con el virus virulento de la enfermedad de Newcastle. Existe la posibilidad de utilizar la PCR en tiempo real como ensayo in vitro para la evaluación de vacunas. Los valores del umbral de ciclo oscilaron entre 21,17 y 25,23. Por otra parte, los títulos de anticuerpos inhibidores de la hemaglutinación oscilaron entre 7,1 log2 y 6,2. La comparación entre los valores del umbral de ciclo de los extractos de antígeno con los resultados correspondientes de la prueba de reto y los ensayos de inhibición de la hemaglutinación in vivo, utilizando sueros de aves vacunadas, demostró una fuerte correspondencia entre los resultados in vitro e in vivo(AU)

Primary survey of avian influenza virus and Newcastle disease virus infection in wild birds in some areas of Heilongjiang Province, China

Two hundred thirty specimens of wild birds were collected from some areas in Heilongjiang Province during the period of 2003~2004, including two batches of specimens collected randomly from a same flock of mallards in Zhalong Natural Reserve in August and December, 2004, respectively. Primary virus isolation and identification for avian influenza virus (AIV) and Newcastle disease virus (NDV) were performed. The results showed that only two specimens of young mallards collected from Zhalong Natural Reserve in August, 2004 were positive to AIV (isolation rate 0.9%), and one strain (D57) of these two virus isolates was identified to be H9 subtype by hemagglutination inhibition test. Meanwhile, the two batches of blood serum samples of mallards from Zhalong were also examined for antibodies against AIV and NDV. Among 38 blood serum samples collected in August, antibodies against the hemagglutinin of H1, H3, H5, H6 and H9 subtypes of AIV were found in 1, 0, 2, 0 and 8 samples, respectively; and 11 samples were found with antibody against NDV. Whereas the NDV isolation in both two batches of specimens of mallard was negative, all of the 32 blood serum samples collected in December were negative for antibodies against AIV and NDV.