This could partially be explained by the fact

that aerosol delivery of brPEI-pcDNA1/MOMPopt was performed by use of the Cirrus™ nebulizer, designed for aerosol therapy in humans. This nebulizer creates small aerosol droplets (up to 5 μm) which most likely target the lower airways and especially the lungs of birds, bypassing most parts of the upper airways. Additionally, birds have a limited number of RAD001 resident macrophages in the normal respiratory tract that could act as antigen presenting cells. However, avian respiratory macrophages are predominantly located in atrial connective tissue compartments of the lungs [31], which might explain the observed local protection. Formulation of the vaccine as dispersible dry powder could circumvent this problem. Corbanie et al. [32] recently developed a method for administering dry powder vaccines as an alternative for liquid spray and aerosol vaccination in chickens. Dispersion of the powder vaccine in isolators with chickens resulted in a uniform targeting of the upper and lower respiratory tract due to a more optimal and narrow particle size distribution. According to them dispersible dry powder would also allow lowering the dose of current vaccines. Theoretically, spray drying

of brPEI-pcDNA1/MOMPopt into a dry dispersible powder would be possible. Nevertheless, further research is needed and a final vaccination experiment in SPF turkeys would be necessary to prove the efficacy of a brPEI-pcDNA1/MOMPopt dry powder vaccine and to determine the minimal vaccine dose to provide effective protection. Primo

vaccination did not result in detectable MOMP-specific serum antibody titres. However, Apoptosis inhibitor antibody titres, observed at 3 weeks post-primo vaccination with pcDNA1/MOMP were also low (±1/20) [2]. This might be normal as immunisation one out day after hatching does not effectively activate antibody production, probably due to incomplete structural organisation of the secondary lymphoid structures in neonates. However, at the age of one week, with the plasmid still present, effective humoral immune responses with specific antibody production could normally occur [33] as birds meanwhile became fully immunocompetent. The occurrence of low antibody titres following DNA vaccination is in accordance with other studies stating that antibody responses following DNA vaccination are generally modest [34]. Interestingly, a superior B-cell response upon immunisation in combination with an ‘early’ secondary serum antibody response upon challenge was correlated with the best protection. Thus, humoral immune responses, albeit not considered as crucial, seem to contribute to protection in this study. Mucosal immunisation resulted in higher mean OD405 values for total mucosal antibodies and the presence of serum IgA antibodies in one animal, while IgA was not detected in intramuscularly immunised turkeys. Furthermore, the mean OD405 values were extremely low as also observed in our previous study [2].