Protective immunity conferred by attenuated aroA derivatives of Pasteurella multocida B:2 strains in a mouse model of hemorrhagic septicemia

Hemorrhagic septicemia (HS) is a fatal systemic disease of cattle and buffaloes. In South Asia HS is caused by infection with Pasteurella multocida serotype 13:2, Some control is achieved with alum-precipitated or oil-adjuvanted killed whole-cell vaccines injected subcutaneously, but these vaccines provide only short-term immunity and require annual administration for effective use. Live attenuated vaccines have the advantage of a natural route of entry into the host, but for live strains to be used as vaccines, the mode of attenuation should be well defined. We constructed aroA attenuated derivatives of two P. multocida serotype 13:2 strains by allelic exchange of the native aroA sequence with aroA sequences disrupted with a kanamycin resistance cassette or with marker-free aroA sequences containing an internal deletion. These strains were confirmed to be aroA mutants by PCR and Southern blot analysis, enzyme assay, and lack of growth on minimal medium. The aroA derivatives were highly attenuated for virulence in a mouse model of HS. Mouse challenge experiments showed that intraperitoneal or intranasal vaccination of an aroA strain completely protected mice against challenge with a high dose (> 1,000 50% lethal doses) of either the parent strain or the other wild-type 13:2 strain. The spread of the parent and the aroA derivatives to different organs was compared when the organisms were inoculated by different routes.