All samples were centrifugated at 2500 rpm for 5 minutes (mini centrifuge with A12-2P rotor “Dlab: D2012 plus”). The biological samples were pooled by species and collection date (3 to 4 samples/pool). Subsequently, automated RNA extraction was performed from 200μL of each pooled supernatant using the Cador® Pathogen 96 QIAcube HT Kit (Qiagen) following the manufacturer’s instructions. The qRT-PCR was carried out with the VetMAX™-Gold SIV Detection Kit (Life Technologies) following the manufacturer’s instructions (Ma et al., 2014). Negative and positive controls were included in each run. Samples with a cycle threshold value (Ct) <38 were considered positive as described by the manufacturer. Positive pools were tested again individually. Positive IAV samples were inoculated into the allantoic cavity of 9-to-11-day-old specific-pathogen-free (SPF) chicken embryos as described before (Spackman & Killian, 2014). The allantoic fluid was harvested, and a haemagglutination assay was performed with 0.5% chicken red blood cells (Killian, 2014).