Development and optimization of virus neutralization test in chicken embryonated eggs for indirect identification of avian influenza and Newcastle disease virus

Abstract

Avian viral problems have been consistently reported in commercial poultry of Pakistan causing heavy economic losses to the poultry farmers. Authentic idenfication and confirmation of the causative agent is always been question mark for the selection of vaccinal strain in this regard. Current study was therefore undertaken to optimize the virus neutralization test for the serological survey of vaccinated poultry particularly for avian influenza virus’s subtypes and Newcastle disease virus. Various physiochemical factors such as concentration of antigen and antibody, Incubation temperature and incubation period for in vitro and in-vivo reaction of antigen and antibody were optimized in chicken embryonated eggs. Serum samples were obtained from vaccinated breeder birds of five commercial poultry breeder companies and subjected for VNT using different concentration of three antigen and their respective homologous antibodies under optimized conditions. AIV H9 (EID50-1×109.0/ml) and NDV (EID50-1×108.2/ml) having biological titer of 10-7 /50ul HA units were neutralize with 10-2/50ul HIU of antibody and incubated at 37°C for 30 minutes was injected subsequently into 10 day old chicken embryo followed by incubation at 37°C for 38 hours showed ≥90% neutralizing specificity. Furthermore, sera obtained from five AIV-H9, AIV-H5 and NDV exposed commercial poultry farms revealed that Big bird broiler, Big bird breeders and A&S chicks are 100% sensitive and specific whereas, Gateway chicks and Waqas poultry breeders showed 100% homology for AIV-H5 virus but do not confers similarity with prevailing AIV-H9 and NDV field strains. Therefore, high sensitivity, reproducibility and specificity VNT, it could be a tool for indirect detection of homology between vaccinal strain and wild virus antigen using known antisera. Particularly, for those organisms possess natural ability to mutate in the adverse climatic conditions.
 Keywords: Virus neutralization test, Avian Influenza Virus, Newcastle Disease Virus, Sensitivity, Specificity