The 2015 outbreak of Highly Pathogenic Avian Influenza (HPAI) took a toll on the United States poultry industry. This epidemic displayed a need for improvements in monitoring and detecting Avian Influenza Virus (AIV). Traditional testing for HPAI virus has been voluntary and through serology using serum and commercial ELISA tests or oropharyngeal swabs using Polymerase Chain Reaction (PCR) analysis. Since the industry uses environmental sampling for detection of other pathogens such as Salmonella enteritidis, one question was if similar methods could provide AIV monitoring and surveillance activities such as collection of environmental specimens from various locations in the poultry house. This study was conducted to evaluate different environmental sampling locations, sample matrices, collection materials and their utilization for simple and accurate detection of AIV as part of routine surveillance.
This project was co-led by three primary investigators located at Iowa State University : Dr. Phillip Gauger, associate professor, Department of Veterinary Diagnostic and Production Animal Medicine; Dr. Yuko Sato, assistant professor and extension poultry veterinarian for the state of Iowa and Dr. Karen Harmon, clinical associate professor in the Department of Veterinary Diagnostic and Production Animal Medicine. Additional research team members included Dr. Kyoung-Jin Yoon, Dr. Jianqiang Zhang and Shahan Azeem.
It was concluded that environmental samples can be successfully utilized to monitor the presence of pathogens such as HPAI virus. The first phase of the study collected environmental samples from five different poultry houses representing 12.9 million layers. Samples were collected ranging from 99 to 148 days after the house tested positive for HPAI virus during the epidemic. Sampling occurred in various areas of the houses including floors, manure belts and pits, egg belts, water troughs, and ventilation equipment using Swiffer® Sweeper™ dry pads and drag swabs.
The second phase of the study included evaluating various sample matrices (Swiffer, Drag and Water) for detection of AIV by PCR. Overall, the limit of detection and estimated titers by PCR indicated that the three sample matrices were comparable for AIV detection.
The third phase of the study included feces and water spiked with various concentrations of H5N2 Low Pathogenic Avian Influenza (LPAI) virus and a production environment simulation. Swiffer® Sweeper™ dry pads, drag swabs and bootie swabs were evaluated in this simulated environment using feces and water spiked with LPAI. In this environmental simulation, the team found that overall, the three collection materials yielded similar PCR results. Additionally, a comparison of floor and non-floor samples indicated that floor samples resulted in lower virus detection, suggesting environmental degradation of the virus.
Other results demonstrated that samples should be taken and immediately placed in a chilled cooler for transport to the testing laboratory since virus detection dropped when samples were stored at or above 98.6 degrees Fahrenheit (37 degrees Celsius) for 48 hours or longer.
Additional research is needed using similar sample types, locations and methods to evaluate the ability of environmental samples to detect the presence of a pathogen prior to an outbreak. This would require ongoing monitoring through sample collection in poultry houses to detect the presence of an influenza A virus of swine-origin that often infect turkeys, or detect a LPAI virus as a surrogate for HPAI virus. The industry could also evaluate similar practices for other high-impact poultry diseases such as Newcastle.
An issue with using PCR-based testing of environmental samples after cleaning and disinfection of poultry houses previously infected with HPAI is the detection of residual viral genetic material in the environment, requiring further evaluation by virus isolation to ascertain the presence or absence of viable AIV.