Tadpoles were euthanased by bathing for $5 minutes in 0.2% MS-222 (see Preparation of immunogen and inoculation trials). Tadpoles for real time PCR and for detection of viral adult globin were then frozen at 280uC until further processing. Tadpoles for blood sampling were euthanased as described then decapitated and blood was collected using a micropipette containing 0.2 M EDTA to prevent clotting. Red blood cells (RBCs) were pelleted at 13,0006g for 5 min, washed with PBS then lysed by resuspending in an equal volume of distilled water. Following centrifugation at 13,0006 g for 5 min the supernatant containing globin was removed and stored at 280uC.