Optimization of Environmental Sampling Methods for the Detection of Virus Contamination in a Layer Chicken Farm: A...

Environmental testing of poultry farms after an outbreak of infectious viral disease such as infectious bronchitis or avian influenza is essential for verifying viral clearance and supporting the safe resumption of operations. To find optimal sampling locations inside and outside of chicken farms, a laboratory study simulating a respiratory virus-infected poultry house with wire layer cages was conducted using a commercial live attenuated infectious bronchitis virus vaccine as a surrogate virus administered to 21-day-old layer chickens. Oropharyngeal swab samples of chickens, air, and four locations within the poultry house, including manure conveyor belt, floor surface, environmental dust, and on-farm boots, were evaluated on the day before and 1, 3, 5, and 8 days after spray vaccination. Tools that were used outside the poultry house, such as off-farm shoes, farm work clothes, protective face masks, cotton gloves, egg transport vehicles, and egg transport pallets, were also evaluated. Due to inherent differences in environmental conditions and facility structures across farms, environmental sampling was performed following a standardized protocol designed in this study to ensure consistency across all collection sites. Virus loads at each sampling site were determined by quantitative real-time reverse transcription PCR and expressed as 50% egg infectious dose (EID₅₀) equivalents. In addition, a subset of samples was further subjected to virus re-isolation through egg inoculation. The highest viral RNA loads inside and outside of the farm were observed from the manure conveyer belts and off-farm shoes, respectively. Samples taken from floor surfaces and on-farm boots also consistently showed high amounts of viral RNA. While infectious viruses were successfully re-isolated from several internal samples, no infectious virus was detected in samples collected outside the farm, suggesting that no viral spillover occurred under the experimental conditions. Our findings identified high-risk virus accumulation sites within poultry farms and highlight the utility of optimized, standardized environmental sampling protocols in enhancing detection sensitivity and supporting effective risk-based surveillance.