|STALLKNECHT, DAVID - University Of Georgia|
Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2009
Publication Date: 3/1/2010
Citation: Lira, J., Moresco, K.A., Stallknecht, D., Swayne, D.E., Fisher, D.S. 2010. Single and combination diagnostic test efficiency and cost analysis for detection and isolation of avian influenza virus from wild bird cloacal swabs. Avian Diseases. 54:606-612.
Interpretive Summary: Some wild waterfowl can be infected with avian influenza viruses (AIV), but identifying these viruses is dependent upon the laboratory tests used. The purpose of this study was to develop a decision tree as to which diagnostic tests to use will maximize AIV isolations from wild bird surveillance samples. In a 50 sample set, AIV was isolated from 36% of samples by chorioallantoic sac (CAS) method and 46% samples by yolk-sac (YS) inoculation using embryonating chicken eggs (ECE), isolated from 20% of samples in Madin-Darby canine kidney cell culture, and detected in 54% of the samples by molecular gene test. Cost analysis for our laboratory indicates that molecular tests are an economical choice for screening samples before doing virus isolation in ECE if the AIV frequency is low in the samples. In contrast, isolation in ECE via CAS and YS inoculation routes without pre-screening by reverse transcriptase polymerase chain reaction (RRT-PCR) was most efficient and cost effective if the samples had an expected high frequency of AIV.
Technical Abstract: Effective laboratory methods for identifying avian influenza virus (AIV) in wild bird populations are crucial to understanding the ecology of this pathogen. The gold standard method has been AIV isolation in chorioallantoic sac (CAS) of specific-pathogen-free (SPF) embryonating chicken eggs (ECE), but in one study, combined use of yolk-sac (YS) and chorioallantoic membrane inoculation routes increased the number of virus isolations. In addition, cell culture for AIV isolation has been used. Most recently, real-time reverse transcriptase polymerase chain reaction (RRT-PCR) has been used to detect AIV genome in surveillance samples. The purpose of this study was to develop a diagnostic decision tree that would increase AIV isolations from wild bird surveillance samples when using combinations of laboratory detection and isolation methods under our laboratory conditions. Attempts to identify AIV was accomplished for 50 wild bird surveillance samples by isolation in ECE using CAS and YS routes of inoculation, and in Madin-Darby canine kidney (MDCK) cells, and by AIV matrix gene detection using RRT-PCR. AIV was isolated from 36% of samples by CAS inoculation and 46% samples by YS inoculation using ECE, isolated from 20% of samples in MDCK cells, and detected in 54% of the samples by RRT-PCR. AIV was isolated in ECE in 13 samples by both inoculation routes, 5 additional samples by allantoic, and 10 additional samples by yolk-sac inoculation, increasing the positive isolation of AIV in ECE to 56%. Allantoic inoculation and RRT-PCR detected AIV in 14 samples, with 4 additional by allantoic and 13 additional by RRT-PCR. Our data indicates that addition of YS inoculation of ECE will increase isolation of AIV from wild bird surveillance samples. Cost analysis for our laboratory indicates that RRT-PCR is an economical choice for screening samples before doing virus isolation in ECE if the AIV frequency is low in the samples. In contrast, isolation in ECE via CAS and YS inoculation routes without pre-screening by RRT-PCR was most efficient and cost effective if the samples had an expected high frequency of AIV.