Pasteurella multocida P52 aroA mutant conferred protection to rabbits and mice against haemorrhagic septicaemia

Pasteurella multocida serotype B:2 is the etiological agent of haemorrhagic septicaemia (HS) in cattle and buffaloes, a disease that causes significant economic losses to the dairy industry in India. Several killed vaccines are in uses that provide protection to limited period of time. The aim of this study was to generate an attenuated strain of Pasteurella multocida P52 by targeted gene knock-out approach and to demonstrate its efficacy as a live vaccine to induce protective immunity in mouse and rabbit model of infection. We have made a defined, complete deletion of aroA in the P. multocida P52 vaccine strain, replacing the gene with a kanamycin resistance gene cassette. The developed mutant, P52Δaro, was highly attenuated in mice and rabbits. The mutant failed to grow in Pasteurella minimal medium without the aromix supplement. Wild-type and mutant strain showed similar morphological and biochemical properties. In addition, mutant strain was also confirmed as P. multocida serotype B:2 by genus and serotype specific PCR. In vivo studies showed that P52Δaro mutant was quickly removed from the system and no pathological lesions were observed in liver, spleen and lung. Immunization of mice and rabbits with P52Δaro conferred significant level of protection. Rabbits served as a better model than mice for immunogenicity studies. Immunization of rabbits with aroA mutant elicited high antibody titer which persisted for long duration of time. Upon immunization with P52Δaro, rabbits developed a solid immunity for prolonged period against virulent challenge with parental wild-type or a heterologous strain of P. multocida isolated from a field outbreak. The mutant, P52Δaro, may serve as a potent live vaccine candidate for control of HS in cattle and buffaloes.