069 mol/l, Acros BYL719 Organics, Geel, Belgium) and loaded onto a 0.8% agarose gel supplemented with ethidium bromide. The gel was run at 100 V for 30 min in 0.5 × TAE (Tris–acetate–EDTA). Smeared DNA bands indicate DNA degradation. Complex stability before and after nebulisation was examined by measuring particle size and zeta potential as described in Section 2.2, and by agarose gel electrophoresis as described above. The absence of visible pDNA bands indicates pDNA condensation and therefore complex stability. Results in BGM cells allowed the selection of a candidate formulation of the DNA vaccine (brPEI polyplexes with an N/P ratio of for subsequent in vivo studies. However, before starting in vivo

studies, we decided to check the cytotoxicity and transfection efficiency of brPEI polyplexes once again on an avian cell line, namely chicken embryo fibroblasts (DF-1 cells). Materials and methods are the same as described in Sections 2.3 and 2.4. The effect of parenteral (intramuscular injection; IM, m quadriceps) and mucosal (aerosol, AE) DNA vaccination

of turkeys was compared. For aerosol delivery Selleck CDK inhibitor we used the Cirrus™ Nebulizer (Intersurgical), designed to give tracheo-bronchial deposition of particles (up to 5 μm) in humans. Twenty-one SPF turkeys (AFSSA, Ploufragan, France) were divided into four groups and reared in negative pressure isolation units (IM1500, Montair, Sevenum, The Netherlands). Three groups received a primary DNA inoculation at 1 day of age and one booster inoculation 3 weeks later. Groups 1 and 2 were twice immunised intramuscularly others with respectively naked plasmid DNA or brPEI-pcDNA/MOMPopt, while group 3 was vaccinated at both time points through nebulisation of brPEI-pcDNA/MOMPopt. The control group (4) was left unvaccinated. Turkeys were challenged by aerosol infection at the age of 5.5 weeks using the Cirrus™ nebulizer. The challenge infection consisted of 108 TCID50 of Cp. psittaci strain 92/1293 (avian genotype D strain). All turkeys were euthanized at 25 days post-challenge (PC). The vaccination scheme and the experimental set-up

are presented in Table 1 and Table 2. The experimental design of the animal experiment was evaluated and approved by the Ethical Committee for Animal Experiments of Ghent University (Reference number: EC 2006/049). All turkeys were monitored daily for clinical signs. Pharyngeal and cloacal swabs were taken at day 1 of the experiment and subsequently every other day starting at 5 days PC until euthanasia. Swabs were stored at −80 °C in Cp. psittaci transport medium prior to isolation. Blood samples (v. ulnaris) for the detection of MOMP-specific serum antibody titres were taken immediately prior to each DNA vaccination, 1.5 weeks following booster vaccination, immediately prior to the challenge and at 2 and 3.5 weeks post-challenge.