Deletion of the znuA virulence factor attenuates Actinobacillus pleuropneumoniae and confers protection against homologous or heterologous strain challenge

•ΔznuA was generated by site-directed mutagenesis of A. pleuropneumoniae WT SLW01.•The ΔznuA deletion mutant was found to be lethal in low-Zn2+ medium.•ΔznuA virulence was attenuated at least 22-fold in Balb/C mice.•ΔznuA reduce mortality and morbidity in a murine model of infection.•ΔznuA immunisation induced homologous or heterologous challenge-protection in pigs.

The znuA gene is known to be important for growth and survival in Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida under low Zn2+ conditions. This gene is also present in Actinobacillus pleuropneumoniae serotype 1; therefore, the aim of this study was to investigate the existence of a similar role for the znuA gene in the growth and virulence of this organism. A precisely defined ΔznuA deletion mutant of A. pleuropneumoniae was constructed based on the sequence of the wild-type SLW01 using transconjugation and counterselection. This mutation was found to be lethal in low-Zn2+ medium. Furthermore, the ΔznuA mutant strain exhibited attenuated virulence (≥22-fold) as well as reduced mortality and morbidity in a murine (Balb/C) model of infection. The majority of the bacteria were cleared from the lungs within 2 weeks. The ΔznuA mutant strain caused no adverse effects in pigs at doses of up to 1.0 × 109 CFU/mL. The ΔznuA mutant strain induced a significant immune response and conferred 80% and 100% protection on immunised pigs against challenge with A. pleuropneumoniae strains belonging to homologous or heterologous serovars, respectively, compared to the blank controls. The data obtained in this study indicate the potential of the mutant ΔznuA strain for development as a live vaccine capable of inducing reliable cross-serovar protection following intratracheal immunisation.

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