Molecular investigation of the role of Apxl and Apxll in the virulence of Actinobacillus pleuropneumoniae serotype 5

Abstract

The extracellular hemolytic toxins (ApxI and ApxII) of Actinobacillus pleuropneumoniae are thought to be important factors in this microorganism's virulence and the pathogenesis of swine pleuropneumonia. Using the polymerase chain reaction, the apxI locus of a non-hemolytic, avirulent mutant of A. pleuropneumoniae serotype 5 (mIT4-H) generated by chemical mutagenesis (Inzana T. J., Todd J., Veit H. P. Microb Pathog 1991; 10: 281-96) was found to contain deletions that affected major parts of the entire apxICABD operon, thus inactivating each gene in the operon. The apxII locus was not affected. Monoclonal antibodies to ApxI and ApxII were used to confirm that ApxI was not synthesized, and that ApxII was synthesized but not secreted from the cell. The apxICABD genes and apxIBD genes were cloned into a broad host range vector to obtain plasmids pJFF800 and pJFF801, respectively. Each recombinant plasmid was electroporated into strain mIT4-H to obtain strain mIT4-H/pJFF800 and strain mIT4-H/pJFF801, respectively. Strain mIT4-H/pJFF800 exported ApxI and ApxII, and produced hemolytic activity comparable to or exceeding that of wild type strain J45. Strain mIT4-H/pJFF801 exported only ApxII and produced weak hemolytic activity. Strain mIT4-H/pJFF800 was virulent in mice, and had an LD50 of about 2 x 10(6) colony forming units. In contrast, mIT4-H/pJFF801 and mIT4-H were essentially avirulent in mice, and LD50s for these strains could not be calculated. Strain mIT4-H/pJFF800 was virulent in pigs and caused lethal pleuropneumonia, whereas parent strain mIT4-H was avirulent. Strain mIT4-H/pJFF801 was also able to induce pleuropneumonia in pigs, although a higher dose was required to induce lesions similar to those caused by mIT4-H/pJFF800. Thus, A. pleuropneumoniae strains that produce ApxI and ApxII require ApxI for full virulence and toxic activity in pigs. However, other factors including ApxII contribute to the virulence of A. pleuropneumoniae in pigs.