Isolation, Quantification and Characterisation of Pasteurella multocida Pm52-Derived Lipopolysaccharide and its Effect on Nitric Oxide Production in Mice

Lipopolysaccharide (LPS) of Pasteurella multocida is regarded as one of the most important virulence factors associated with this organism. It also helps in its classification, serodiagnosis and in the induction of experimental inflammatory disease models like septic shock and general immune stimulation. This paper describes the standardisation and modification of existing protocols for LPS extraction (hot water phenol method), purification and characterisation from the bacterium in a much simplified, efficient and economical manner. During isolation, an additional step of phenol treatment was used to avoid cumbersome steps like freeze drying and ultracentrifugation. Higher (600 μg) quantities of LPS were obtained compared with earlier protocols. Overdevelopment of the gel was avoided using a stopping solution instead of distilled water. Quantification of LPS measured indirectly by determining the carbohydrate content was found to be a highly efficient, inexpensive and reliable method (R2>0.98). The in vitro and in vivo studies confirmed that the extracted LPS retained its biological properties and stimulated significantly higher levels of nitric oxide production (p<0.05). The present modifications can be thus used for extraction, characterisation and quantification of LPS from P. multocida in a much simplified, efficient, economical and reliable manner.