Characterization and Different Antigenic Preparations of P. multocida Along with their Quantitative and Qualitative Analysis

Pasteurella multocida is a causative agent of a number of economically important diseases in livestock. Due to the fatal nature of the disease, there is urgent need for rapid diagnosis so that appropriate therapeutic and preventive measures could be undertaken. A study was designed to extract different antigens of P. multocida capsular type A and B which included whole bacterium, Capsular antigen, Outer membrane proteins (OMPs) and Lipopolysaccharides (LPS). Bacteria were grown on brain heart infusion broth and capsular antigen was separated by fractional precipitation with addition of polar organic solvents yielding capsular polysaccharide. OMPs were extracted by ultracentrifugation of the supernatant obtained from bacterial broth by addition of HEPES Buffer containing Sodium Lauryl Sarcosinate detergent and the detergent insoluble OMP enriched fractions were obtained. Whole cell antigen was obtained by centrifugation of sonicated P. multocida suspended in HEPES buffer. LPS was extracted by formalinized saline killing of bacteria followed by ultracentrifugation. Total protein concentration was found to be 8.97 mg/ml and 5.67 mg/ml for OMP capsular type A & OMP type B respectively, while for WCL it was 22.38 mg/ml& 26.89 mg/ml. Carbohydrate estimation of capsular type A & type B of LPS and capsular polysaccharide concentrations were estimated to be 188.32 μg/ml, 330.71 μg/ml and 4.08 mg/ml, 2.38 mg/ml respectively. OMPs and whole cell lysate extracted were subjected to discontinuous SDS-PAGE. Nine polypeptides of MW ranging from 15–100 kDa from OMPs preparation & fifteen polypeptides of MW ranging from 25–98 kDa from whole cell lysate were visualised.