A previously described plaque reduction assay was modified and used to evaluate the virus neutralization activity of collected sera after vaccination [25] . Briefly, serum samples from each treatment group were pooled. An equal volume of a 1 : 10 dilution of pooled serum and LPAI subtype H5N2 was mixed and incubated for 30 min at 37 • C. A commercially available anti-M2 antibody (ProSci-Inc, Poway, CA) was used in a 1 : 1000 dilution as a control for antibody activity. Chicken embryo kidney cell (CEKC) monolayers in 6-well plates were washed twice with prewarmed PBS and 400 μL of the above mixture was added to the CEKC monolayer. The plates were incubated for 60 min at 37 • C. Then, the inoculums were removed and after 2 washes with prewarmed PBS, they were overlaid with 0.8% agar (University of Connecticut Cell Culture Facility) in Minimum Essential Medium Eagle (MEM). After 72 h, the plates were checked for plaque formation and for further evaluation were fixed with 99% methanol and stained with crystal violet for plaque counting.