Construction and characterisation of attenuated derivatives of Pasteurella multocida : serotype B:2 strains
The project was concerned with the construction of defined attenuated derivatives of P. multocida serotype B:2 strains, causative agents of haemorrhagic septicaemia, and attempts were made to construct defined mutations in genes such as aroA, cya, and galE loci that have been used to induce attenuation in other bacterial strains. Mutants defective in the aroA gene were constructed by allelic exchange of the locus in the chromosome of the wild-type strains with a cloned aroA gene interrupted with a cassette encoding kanamycin resistance (KmR). The aroA defective strains were confirmed by PCR, Southern blotting, lack of growth on minimal medium and by enzyme assay. KmR inactivated aroA mutants JRMT1 and JRMT2 strains derived from P. multocida 85020 and Quetta strains, respectively, were highly attenuated in a mouse model, with an LD50 108 C.F.U./mouse after injection intraperitoneally (i.p.). In contrast, the wild-type strains had LD50 <50 C.F.U./mouse by this route. Vaccination once by the i.p. route or twice by the i.n. route with these aroA mutants gave complete protection to the mice against subsequent challenge i.p. with 10,000 LD50 of the homologous wild-type strain or 1000 LD50 of the heterologous wild-type strain. Vaccination with these by the s.c. route was not protective. When high doses of the attenuated strains were inoculated by the i.p. or i.n. routes, there was some spread to the internal organs but the organisms were cleared by 24 and 72 hrs respectively. In contrast, the wild-type parent strains spread rapidly and multiplied in high numbers and killed the mice by 24 and 96 hrs respectively.