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United States Department of Agriculture

Agricultural Research Service

Research Project: APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS Title: Improved avian influenza virus isolation rates from wild waterfowl cloacal swabs using yolk sac inoculation of embryonating chicken egg

item Lira, Jennifer
item Moresco, Kira
item Swayne, David

Submitted to: International Symposium on Avian Influenza
Publication Type: Abstract Only
Publication Acceptance Date: January 20, 2009
Publication Date: April 5, 2009
Citation: Lira, J., Moresco, K.A., Swayne, D.E. 2009. Improved avian influenza virus isolation rates from wild waterfowl cloacal swabs using yolk sac inoculation of embryonating chicken egg [abstract]. Abstracts of the 7th International Symposium on Avian Influenza, April 5-8, 2009, Athens, Georgia. p. 48.

Technical Abstract: Avian influenza virus (AIV) remains of interest to researchers as a pathogen that infects many economically important bird species. Asymptomatic wild birds, such as waterfowl species, can shed virus and spread it to domestic poultry, where it can cause severe damage. Effective laboratory methods to isolate AIVs from wild bird populations are crucial to understanding the ecology of this pathogen. The standard method for AIV isolation is inoculation of sample into the chorioallantoic cavity of specific-pathogen-free (SPF) embryonating chicken eggs (ECE). In this study, AIV isolation was attempted for 50 wild bird surveillance samples in ECE using allantoic cavity and yolk-sac routes of inoculation and in Madin-Darby canine kidney (MDCK) cells, and the results were compared with AIV matrix gene detection by real-time reverse transcriptase polymerase chain reaction (RRT-PCR). Presumptive virus isolation was based on hemagglutination test and the presence of AIV was confirmed with AIV specific antigen-capture test (BinaxNow®). AIV was isolated from 34% of samples by allantoic inoculation, 54% samples by yolk-sac inoculation, and detected in 36% of the samples by RRT-PCR. AIV was isolated in ECE in 14 samples by both inoculation routes, 3 additional samples by allantoic, and 13 additional samples by yolk-sac inoculation, increasing the positive isolation of AIV in ECE to 60%. Allantoic inoculation and RRT-PCR detected AIV in 10 samples, with 7 additional by allantoic and 8 additional by RRT-PCR. Samples positive for AIV by RRT-PCR with a Ct-value greater than or equal to 38 were confirmed using RT-PCR for the NS1 gene. Investigation regarding the positive isolation rate of AIV in MDCK cells is ongoing. To date, our data indicates that addition of yolk sac inoculation of ECE may increase isolation of AIV from wild bird surveillance samples.

Last Modified: 8/24/2016