Avian Flavivirus Surveillance Protocol

Blood was collected from the brachial vein in the wing from nearly 750 wild-caught adult birds using sterile syringes, into EDTA-microfil tubes. Plasma was separated from the cellular part by centrifugation of the blood samples. Resulting plasma was stored at − 70°C. Additionally, faecal samples were collected for screening of an active virus excretion. Flaviviruses have been detected in birds from cloacae swab samples in previous studies [25 (link), 26 (link)]. Faecal samples were taken from 300 birds, and placed in virus transport medium (Hanks balanced salt solution containing 0.5% lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 μg/ml streptomycin, 100 U/ml polymyxin B sulphate, and 250 μg/ml gentamycin, and 50 U/ml nystatin; Sigma) in 2011. Faecal samples were further collected from 202 birds in 2013 and placed into empty tubes and frozen immediately to − 70°C.