A riboflavin auxotroph of Actinobacillus pleuropneumoniae is attenuated in swine

Abstract

Actinobacillus pleuropneumoniae is the etiological agent of a highly contagious and often fatal pleuropneumonia in swine. A riboflavin-requiring mutant of A. pleuropneumoniae serotype 1, designated AP233, was constructed by deleting a portion of the riboflavin biosynthetic operon (ribGBAH) and replacing it with a gene cassette encoding kanamycin resistance. The genes affected included both the alpha- and beta-subunits of riboflavin synthase as well as a bifunctional enzyme containing GTP cyclohydrase and 3,4-dihydroxy-2-butanone-4-phosphate synthase activities. AP233 was unable to grow in the absence of exogenous riboflavin but otherwise was phenotypically identical to the parent wild-type strain. Experimental infection studies with pigs demonstrated that the riboflavin-requiring mutant was unable to cause disease, on the basis of mortality, lung pathology, and clinical signs, at dosages as high as 500 times the normal 50% lethal dose for the wild-type parent. This is the first demonstration of the attenuation of A. pleuropneumoniae by introduction of a defined mutation in a metabolic gene and the first demonstration that mutations in the genes required for riboflavin biosynthesis can lead to attenuation in a bacterial pathogen.