Optimizing sample collection methods for detection of respiratory viruses in poultry housing environments.

Viral respiratory diseases, such as avian influenza, Newcastle disease, infectious bronchitis and infectious laryngotracheitis, have considerable negative economic implications for poultry. Ensuring the virus-free status of premises by environmental sampling after cleaning and disinfection is essential for lifting a quarantine and/or safely restocking the premises following an outbreak. The objectives of this study were to identify optimal sample collection devices and to determine the locations in poultry housing which are best for poultry respiratory virus sample collection. Chickens exposed to infectious bronchitis virus, which was used as a representative virus for enveloped poultry respiratory viruses, were housed in floor-pens in either a curtain-sided wood framed house or a cement block house. Foam swabs, cellulose sponges, polyester swabs, dry cotton gauze and pre-moistened cotton gauze were evaluated for comparative efficiency in recovering viral RNA. Cotton gauze pre-moistened with the viral transport media had the highest sensitivity among the devices (wood-framed house: 78% positive, geometric mean titre [GMT] of 2.6 log10 50% egg infectious doses [EID50 ] equivalents/ml; cement block houses: 55% positive, GMT of 1.7 log10 EID50 equivalents/ml). Targeting virus deposition sites is also crucial for efficient virus elimination procedures and subsequent testing; therefore, 10 locations within the houses were compared for virus detection. In both housing types, the highest viral RNA loads were recovered from the tops of drinker lines within the pen. Places the chickens could contact directly (e.g., feeder rim) or were contacted by caretaker feet (hallway floor) also yielded higher levels of viral RNA more consistently. These results will facilitate the establishment of efficient environmental sampling procedures for respiratory viruses of poultry.

Validation of specific quantitative real-time RT-PCR assay panel for infectious bronchitis using synthetic DNA standards and clinical specimens

Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan sup TM /sup -based quantitative real-time RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan sup TM /sup -based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log sub 10 /sub dynamic range with a reproducible limit of detection of =10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%-115%. Further validation of specificity using clinical and biological specimens was also successful.