2.1. Collection and processing of samples

The present study was performed by using 480 Archival blood samples that collected in the spring (240) and the fall (240) of 2018 from randomly selected backyard hens located in six different provinces (Mugla, Izmir, Aydin, Afyonkarahisar, Kutahya and Manisa) of the Western Anatolia region with 80 samples from each province (40 in spring and 40 in fall seasons), this region geographically lies in the west of Turkey as depicted in Figure 1. The sample size and random sampling procedure were determined by using the described method of Houe et al. (2004), in which a selection was made without regard to exposure or disease status.

Map of Turkish provinces where hens sera were collected. ^●Provinces where serum was collected. ^▲Provinces where positive serum was found

After taking blood samples from the hens wing veins (<1 year), collected from each province, it was then transported to the laboratory under the cold chain. Blood samples were centrifuged at 1500 rpm for 10 min. After that serum samples were aliquoted into sterile tubes then inactivated for 30 min at 56°C and stored until investigation at –20°C.