On days 1, 2, 3 and 6 and prior to euthanasia, swabs were taken from each ferret from nasal, throat and rectal regions. Following scheduled euthanasia, the nasal turbinates and the right caudal lobe of the lung from each ferret, which was divided laterally into four segments, were isolated. All swab and tissue samples were snap-frozen in liquid nitrogen and stored at 280uC until analyzed for virus titer by TCID 50 . Frozen tissues were weighed and diluted 10% weight per volume into cold DMEM with 1% penicillin/ streptomycin and 0.2% BSA before being homogenized and centrifuged to remove debris. Tissue homogenate and swabs were serially diluted 10-fold in DMEM with 2 mg/mL TPCK-Trypsin, 0.2% BSA, 4.5 g/L glucose, 1% penicillin/streptomycin, 2 mM L-glutamine, and 25 mM HEPES. Each sample was analyzed in quadruplicate following incubation in 96-well plates with Madin-Darby canine kidney (MDCK) cell monolayers at 37uC in 5% CO 2 for three days. Supernates were collected from each well were assayed for hemagglutination activity using 0.5% turkey red blood cells as an indicator of infection. Viral titers were expressed as log 10 TCID 50 /mL and were calculated using the Reed-Muench method [48] .