Title: Avian embryos, real-time polymerase chain reaction, and cell culture for detection of avian influenza and Newcastle disease virus in wild bird surveillance samples Authors
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 10, 2012
Publication Date: April 23, 2012
Citation: Moresco, K.A., Stallknecht, D.E., Swayne, D.E. 2012. Avian embryos, real-time polymerase chain reaction, and cell culture for detection of avian influenza and Newcastle disease virus in wild bird surveillance samples. Journal of Veterinary Diagnostic Investigation. 24(3):563-567. Interpretive Summary: Wild bird surveillance studies for avian influenza virus and avian paramyxovirus involve detection of viral genes in samples using molecular methods as well as demonstration of virus growth in fertile chicken eggs. The percentage of samples positive by molecular methods is often higher than the percentage of virus isolates grown in chicken eggs. This study was conducted to explore the possibility of higher rates of virus growth using fertile duck and turkey eggs as well as two widely available cell lines. Results indicate marginal differences in the virus growth rate of avian influenza virus and avian paramyxovirus when comparing the three types of avian embryos and significant differences in detection when comparing virus growth in avian embryos to molecular methods. Results for cell culture indicate significant reduction of virus growth and detection when compared to virus growth using avian embryos and detection using molecular methods.
Technical Abstract: Two hundred samples collected from Anseriformes, Charadriiformes, Gruiformes, and Galliformes were assayed using real-time reverse transcriptase polymerase chain reaction (RRT-PCR) for presence of avian influenza virus and avian paramyxovirus-1. Virus isolation using embryonating chicken eggs, embryonating turkey eggs, embryonating duck eggs, Madin-Darby canine kidney cell, and African green monkey kidney cells was performed on RRT-PCR positive samples. Three virus isolation attempts consisted of two chorioallantoic sac inoculations and one yolk sac inoculation were performed. Agent identification was achieved by confirmation of avian influenza virus by a commercial antigen capture enzyme-linked immunosorbent assay and avian paramyxovirus-1 by hemagglutination inhibition assay. Virus detection by real-time polymerase chain reaction (RT-PCR) was significantly greater than virus isolation for avian influenza virus and avian paramyxovirus. There were no significant differences in avian influenza virus and avian paramyxovirus virus isolation methods during pairwise comparisons but small differences in number of isolate were observed. Virus isolation rate for avian influenza virus in embryonating chicken eggs was 62.5%, 50% in turkey eggs, and 43.8% in duck eggs for samples with cycle threshold less than or equal to 37 (n=16). Virus isolation rate for avian paramyxovirus in embryonating chicken eggs was 75%, turkey and duck eggs both had 100% virus isolation rate for samples with cycle threholds of less than or equal to 36 (n=8). Results indicate no real predictive quality for virus isolation using the cycle threshold values obtained from RT-PCR, as well as, comparable virus isolation efficiency for all three types of avian embryos.