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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: Comparison of pooling eleven and five oropharyngeal swabbings for detecting avian influenza virus by real-time RT-PCR in broiler chickens

Authors
item Ladman, Brian -
item Spackman, Erica
item Gelb, Jack -

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 15, 2011
Publication Date: April 1, 2012
Citation: Ladman, B., Spackman, E., Gelb, J. 2012. Comparison of pooling eleven and five oropharyngeal swabbings for detecting avian influenza virus by real-time RT-PCR in broiler chickens. Avian Diseases. 56(1):227-229.

Interpretive Summary: Currently all commercial chicken flocks in the U.S. are tested for avian influenza virus (AIV) prior to being sent to slaughter. It has been statistically determined that testing 11 birds is sufficient to detect the virus. These 11 samples are currently split between 2 sample vials, one with 5 and one with 6 swabs because this was the maximum number of samples recommended per vial by the National Veterinary Services Laboratories. Combining all 11 swabs into one vial would drastically reduce testing costs for the poultry industry by essentially cutting the sample number in half. A study was conducted to determine if combining all 11 samples in one vial will effect virus detection. Detection of virus in pools of 5 swabs versus 11 swabs was very close. Therefore combining all 11 swabs from a single flock may be a viable method to improve the practicality of AIV detection in broiler chickens prior to slaughter.

Technical Abstract: The effect of pooling five or 11 orophyarngeal (O/P) swabbings on detecting avian influenza virus (AIV) by real-time reverse transcription-polymerase chain reaction (RRT-PCR) was evaluated. The model used for the evaluation was designed to minimize viral load and thus assess the effect of the pooling on detection. Two-week-old broiler chickens were inoculated via the intranasal route with the low pathogenicity (LP) chicken/Maryland/Minh Ma/04 H7N2 strain or remained uninoculated. On day 2, 3, 4, 5, 7, 9, 11, and 14 post-inoculation (PI), O/P swabbings were collected from individual infected birds and pooled with either four or ten O/P swabs from uninfected broilers to produce ten replicate pools of five and 11 swabbings, respectively. AIV was readily detected (80-100%) by RRT-PCR in the pools of five and pools of eleven swabbings from day 2 through day 5 PI. Detection in pools of both types decreased to similar levels on Day 7 (50% for the pools of 5 and 40% for the pools of 11). AIV was not detected on day 9, 11, and 14 PI. On a given sample day PI, mean cycle threshold (Ct) values were consistently lower (higher genome levels) in the pools of five compared to the pools of 11. These differences were statistically significant only on day 3 and 5 PI, yet Ct values were both types of pools were clearly interpretable as AIV positive.

Last Modified: 11/23/2014
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