J Vet Sci.  2020 Mar;21(2):e20. 10.4142/jvs.2020.21.e20.

Construction and immunization with double mutant ΔapxIBD Δpnp forms of Oryctolagus cuniculus serotypes 1 and 5

  • 1College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 24341, Korea
  • 2Key Laboratory of Veterinary Medicine, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi 100000, Vietnam


Actinobacillus pleuropneumoniae (APP) causes a form of porcine pleuropneumonia that leads to significant economic losses in the swine industry worldwide. The apxIBD gene is responsible for the secretion of the ApxI and ApxII toxins and the pnp gene is responsible for the adaptation of bacteria to cold temperature and a virulence factor. The apxIBDand pnp genes were deleted successfully from APP serotype 1 and 5 by transconjugation and sucrose counter-selection. The APP1ΔapxIBD Δpnp and APP5ΔapxIBD Δpnp mutants lost hemolytic activity and could not secrete ApxI and ApxII toxins outside the bacteria because both mutants lost the ApxI- and ApxII-secreting proteins by deletion of the apxIBD gene. Besides, the growth of these mutants was defective at low temperatures resulting from the deletion of pnp. The APP1ΔapxIBD Δpnp and APP5ΔapxIBD Δpnp mutants were significantly attenuated compared with wild-type ones. However, mice vaccinated intraperitoneally with APP5ΔapxIBD Δpnpdid not provide any protection when challenged with a 10-times 50% lethal dose of virulent homologous (APP5) and heterologous (APP1) bacterial strains, while mice vaccinated with APP1ΔapxIBD Δpnp offered 75% protection against a homologous challenge. The ΔapxIBD Δpnp mutants were significantly attenuated and gave different protection rate against homologous virulent wild-type APP challenging.


Actinobacillus pleuropneumoniae; deletion mutation; apxIBD gene; pnp gene; virulence


  • Fig. 1. PCR analysis of the wild-type and apxIBD, pnp-deleted mutant of APP1 (A) and APP5 (B) using primer pairs P5–P6 and P11–P12. Lane M, DNA molecular weight ladder 1 kb; lane 1, negative control; lane 2, apxIBD gene amplified from genomic DNA of wild-type APP; lane 3, apxIBD gene amplified from genomic DNA of the Δ apxIBDΔ pnp mutant; lane 4, pnp gene amplified from genomic DNA of the wild-type APP; lane 5, pnp gene amplified from genomic DNA of Δ apxIBDΔ pnp mutant. PCR, polymerase chain reaction; APP, Actinobacillus pleuropneumoniae.

  • Fig. 2. Phenotypes of the APP mutants. Hemolytic activity of wild-type and mutants of APP1 (A) and APP5 (B). The black arrow indicates the clear zone caused by the hemolytic activity surrounding bacteria on blood agar. Cold shock adaptive ability of wild-type and mutant APP1 (C) and APP5 (D). Open arrow indicates colony growth on BHI agar containing a final amount of 10 µg/mL NAD. APP, Actinobacillus pleuropneumoniae; BHI, brain heart infusion; NAD, nicotinamide adenine dinucleotide.

  • Fig. 3. Growth curves of wild-type and mutants of APP1 (A) and APP5 (B). Symbols: ο, wild-type APP; ▲, mutant APP Δ apxIBDΔ pnp. APP, Actinobacillus pleuropneumoniae; OD600, optical density at 600 nm.

  • Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Apx toxins in wild-type and the mutants of APP1 (A) and APP5 (B). Lane M, Xpert Prestained Protein marker; lane 1, culture supernatant of the wild-type APP before precipitating; lane 2, culture supernatant of the mutants before precipitating; lane 3, RTX toxins of wild-type after precipitating with ammonium sulfate; lane 4, RTX toxins of the mutants after precipitating with ammonium sulfate. APP, Actinobacillus pleuropneumoniae; RTX, repeats in toxin.

  • Fig. 5. Evaluation of the stability deletion gene in the genome of APP1Δ apxIBDΔ pnp and APP5Δ apxIBDΔ pnp over 10 passages by PCR using primer pairs P5–P6 and P11–P12 for amplifying apxIBD and pnp genes, respectively. Amplified apxIBD gene (A) and pnp gene (B) from APP1Δ apxIBDΔ pnp. Amplified apxIBD gene (C) and pnp gene (D) from APP5Δ apxIBDΔ pnp. Lane M, DNA Ladder 1kb; lane 1, negative control; lane 2, target gene amplified from genomic DNA of wild-type APP, lanes 3–12, deletion gene amplified from genome DNA of mutant through 10 continuous passages. APP, Actinobacillus pleuropneumoniae; PCR, polymerase chain reaction.



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