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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: Evaluation and attempted optimization of avian embryos and cell culture methods for efficient isolation and propagation of low pathogenicity avian influenza viruses

Authors
item Moresco, Kira
item Stallknecht, David -
item Swayne, David

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 1, 2009
Publication Date: March 1, 2010
Citation: Moresco, K.A., Stallknecht, D., Swayne, D.E. 2010. Evaluation and attempted optimization of avian embryos and cell culture methods for efficient isolation and propagation of low pathogenicity avian influenza viruses. Avian Diseases. 54:622-626.

Interpretive Summary: Some wild waterfowl can be infected with avian influenza viruses (AIV), but identifying these viruses is dependent upon the laboratory tests used. In this study, we compared duck embryos, turkey embryos, and multiple cell cultures for the ability to grow AIV compared to chicken embryos. There were no differences in AIV growth in duck or turkey compared to chicken embryos. The cell cultures were less efficient than chicken embryos for AIV growth, but the best cell lines were Madin-Darby canine kidney (MDCK), primary chicken embryo kidney (CEK), and primary chicken embryo fibroblast (CEF) cell cultures. Overall, chicken embryos were the most efficient system for growth of AIV. However, the savings in time and resources incurred with the use of the cell cultures, would allow a higher volume of samples to be processed with the same fiscal and financial resources, thus being potentially advantageous despite the lower growth rates.

Technical Abstract: Surveillance of wild bird populations for avian influenza viruses (AIV) contributes to our understanding of AIV evolution and ecology. Both real-time reverse transcriptase polymerase chain reaction (RRT-PCR) and virus isolation in embryonating chicken eggs (ECE) are standard methods for detecting AIV in swab samples from wild birds, but AIV detection rates are higher with RRT-PCR than isolation in ECE. In this study we tested duck embryos, turkey embryos, and multiple cell lines for AIV growth as compared to ECE for improved isolation and propagation of AIV 16 hemagglutinin subtypes. There were no differences in low pathogenicity avian influenza virus (LPAIV) propagation titers in duck or turkey embryos compared to ECE. The replication efficiency of LPAIV was lower in each of the cell lines tested compared to ECE. LPAIV titers were 1-3 log mean tissue culture infective doses (TCID50) lower in Madin-Darby canine kidney (MDCK), primary chicken embryo kidney (CEK), and primary chicken embryo fibroblast (CEF) cell cultures and 3-5 log TCID50 lower in chicken bone marrow macrophage (HD11), chicken fibroblast (DF-1), and mink lung epithelial (Mv1Lu) cells than the corresponding mean embryo infective doses (EID50) in ECE. The quail fibroblast (QT-35), baby hamster kidney (BHK-21), cell lines produced titers 5-7 log TCID50 less than EID50 in ECE. Overall, ECE were the most efficient system for growth of LPAIV. However, the savings in time and resources incurred, with the use of the MDCK, CEK, and CEF cultures, would allow a higher volume of samples to be processed with the same fiscal and financial resources, thus being potentially advantageous despite the lower replication efficiency and lower isolation rates.

Last Modified: 12/17/2014