A genetically-defined riboflavin auxotroph of Actinobacillus pleuropneumoniae as a live attenuated vaccine.

Actinobacillus pleuropneumoniae is a gram negative pleiomorphic rod that is the causative agent of a severe, highly infectious and often fatal pleuropneumonia in swine. We have previously reported the construction of genetically-defined stable riboflavin auxotrophs by replacement of a portion of the APP riboflavin biosynthetic operon (ribGBAH) with an antibiotic cassette encoding resistance to kanamycin, and have demonstrated that such riboflavin auxotrophs are avirulent. In this study, we evaluated riboflavin auxotrophs of A. pleuropneumoniae for their ability to stimulate protective immunity against pleuropneumonia. An initial challenge experiment demonstrated that intramuscular vaccination with a live attenuated serotype 1A rib mutant, in a vaccine formulation that included a limiting amount of exogenous riboflavin, provided better protection against challenge with virulent A. pleuropneumoniae than either intratracheal immunization or intramuscular immunization with live bacteria in the absence of exogenous riboflavin. Subsequent studies in which the vaccine inoculating dose, concentration of exogenous riboflavin, and serotype of the vaccine strain were varied demonstrated that immunization with live avirulent riboflavin auxotrophs could elicit significant protection against experimental challenge with both homologous and heterologous virulent serotypes of A. pleuropneumoniae.