Improved stability of live attenuated vaccine gdhA derivative Pasteurella multocida B:2 by freeze drying method for use as animal vaccine

The efficacy of attenuated strain of gdhA derivative Pasteurella multocida B:2 mutant as a live vaccine to control haemorrhagic septicaemia (HS) disease in cattle and buffaloes has been demonstrated. In order to use P. multocida B:2 mutant as a commercial product, it is essential to optimise its formulation for high viability and stability of the live cells. The effectiveness of freeze-drying process using different protective agent formulations for improving cells viability was explored. Sugar and nitrogen compounds were used as protective agents in freeze-drying and the capability of these compounds in maintaining the viability of mutant P. multocida B:2 during subsequent storage was investigated. A complete loss in viability of freeze-dried mutant P. multocida B:2 was monthly observed until 6–12 months of storage at −30 °C, 4 °C and 27 °C when nitrogen compound or no protective agent was added. Trehalose and sucrose showed significantly high survival rate of 93–95% immediately after freeze-drying and the viability was retained during the subsequent storage at −30 °C and 4 °C. A smooth cell surface without any cell-wall damage was observed for the cells formulated with trehalose under scanning electron micrograph. This study presented a freeze-drying process generating a dried live attenuated vaccine formulation with high stability for commercial applications. •Conditions applied during freeze drying significantly influence cells viability.•Trehalose and sucrose are the best cryoprotectants for live mutant P. multocida B:2.•Nitrogen compounds are not suitable cryoprotectant for live mutant P. multocida B:2.•-30 °C is the best storage temperature for freeze-dried mutant P. multocida B:2.