Isolation of Avian influenza virus from polymerase chain reaction-negative cloacal samples of waterfowl

Avian influenza virus (AIV) is one of the most important zoonotic pathogens because of its potential to cause severe disease outbreaks in avian and human hosts. Virus isolation in embryonated chicken eggs (ECEs) remains a gold standard technique for AIV detection. However, some laboratories prefer molecular methods, such as real-time quantitative reverse transcription polymerase chain reaction (qRTPCR), for initial sample screening because of their high throughput sample processing and rapid results. Samples found positive on real-time qRT-PCR are then inoculated in ECEs for virus isolation and characterization. This approach is based on the premise that real-time qRT-PCR will detect all AIV-positive samples. The current study aimed to determine if AIV can be isolated from cloacal samples of waterfowl that were initially found to be negative by real-time qRT-PCR screening. Quantitative RT-PCR-negative cloacal samples (1,369) were inoculated for virus isolation in commercial nonspecific pathogen-free ECEs. After 4 days of incubation, the allantoic fluids were harvested and inoculated in fresh ECEs for a second passage. Allantoic fluids from 147 samples were positive for hemagglutination with chicken erythrocytes. Of the 147 hemagglutination-positive allantoic fluids, 82 were AIV positive when confirmed with real-time qRT-PCR. Ten isolates were subtyped as H7N2 (n 5 7), H7N1, H1N2, and H2N2. In addition, N subtype could be determined for isolates from an additional 25 samples. These results highlight the fact that screening by realtime qRT-PCR may result in some false-negative cloacal samples for AIV.