Cross protective immunity conferred by a marker-free aroA mutant of Pasteurella multocida

The aroA gene from Pasteurella multocida serotype A:1 (X-73) was cloned by complementation of the Escherichia coli aroA mutant AB2829 with a DNA library constructed in pUC18. The cloned aroA gene was inactivated by deletion of a 300 bp internal sequence and reintroduced by homologous recombination into the chromosome of X-73 and P-1059 (serotype A:3) using a Pasteurella-E. coli shuttle vector pPBA1100. By subjecting the transformed cells to repeated subculturing in the presence of antibiotic selection coupled with auxotrophic enrichment, marker-free aroA mutants of X-73 and of P-1059 were isolated and designated PMP1 and PMP3, respectively. PMP1 and PMP3 were highly attenuated and capable of conferring complete protection against subsequent lethal challenge infection in a mouse model. Moreover, PMP3-immunized mice were protected against heterologous challenge infection with serotype A:1 or A:4.