Abstract:Objective To develop a DNA vaccine by loading avian pathogenic Escherichia coli ghosts with the fimbrium gene flfA of Gallibacterium anatis and evaluate the immune effect of this vaccine in chickens.Methods Taking flfA of G. anatis as the target gene, we constructed the eukaryotic expression plasmid pCAGGS-flfA from the plasmid pCAGGS-HA carrying the chicken β-actin promoter, which enhanced the efficacy of vaccine. The temperature-sensitive plasmid PBV-E-SN was then constructed and transformed into the ampicillin-sensitive avian pathogenic E. coli isolate to prepare a bacterial ghost. Finally, pCAGGS-flfA was loaded into the bacterial ghost to prepare a bacterial ghost vaccine. Seven-day-old chickens were assigned into five groups: bacterial ghost loading pCAGGS-flfA, pCAGGS-flfA, empty bacterial ghost, PBS, and normal control. The primary vaccination was carried out for 7-day-old chickens, and the booster immunization was performed two weeks after the primary immunization. The chickens were challenged with G. anatis four weeks after the primary immunization, and the immune effect of the vaccine was evaluated.Results The eukaryotic expression plasmid pCAGGS-flfA was successfully expressed in cells in vitro. The lysis rate of the constructed avian pathogenic E. coli ghosts heated at 42 °C for 210 min reached 99.94%. The specific IgG antibody titer measured by ELISA, the number of shedding bacteria in chicken cloacal swabs and throat swabs, and the bacterial loads in tissue and organs of challenged chickens showed that the immune effects in the bacterial ghost loading pCAGGS-flfA group and the pCAGGS-flfA group were much higher than those in other groups. Moreover, the bacterial ghost loading pCAGGS-flfA showed stronger immune effect than pCAGGS-flfA.Conclusion Bacterial ghosts as the carriers of pCAGGS-flfA significantly enhanced the immune effect of the DNA vaccine.
