Molecular Characterization of Pasteurella multocidaVaccine Strains

Abstract

The present study was carried out tostudy the national haemorrhagic septicaemia vaccine strains at their molecular level. The vaccine is bivalent contain Pasteurella multocidaserotype E:2 and B:2. The vaccine strains were obtained from Central Veterinary Research Laboratory, Department of Biological Product, Soba. Culture characteristic and colonial morphology was ascertained in common laboratory media. The bacterial strains were characterized by using sodium-dodecyl sulphate polyacrylamide gel electrophoresis SDSPAGE, western blotting and by using PCR for capsular serotyping. Firstly, the two strains were characterized by SDS-PAGE technique. The bacteria were cultured in liquid media, and then the bacterial whole cell lysates were prepared. SDS-PAGE was carried out for both strains and the proteins bands werestained with coomassie brilliant blue. Then the molecular weights of the proteins bands were determined; they were 175 kDa, 165 kDa, 150 kDa, 123 kDa, 102kDa, 90 kDa, 85 kDa, 70 kDa, 64 kDa, 60 kDa, 51 kDa, 42 kDa, 37 kDa, 22 kDa and16 kDa. The protein profiles of both vaccine strains were similar. P. multocidastrains protein profiles were investigated by immunoblotting using specific hyperimmune sera prepared in rabbits. Immunostaining with enzyme conjugate was done after transfer of proteins in nitrocellulose acetate paper. Eight proteins in whole cell lysate, of approximately 175 kDa, 102 kDa, 90 kDa, 85 kDa, 70 kDa, 42kDa, 37 kDa, and 16 kDa, were recognized by the sera.

The PCR assay was performedfor strain B and E of P. multocidaby using primers that amplify capsulegene. Two extraction methods for DNA were used. There were the Boiling method and the kits method in which a lysis buffer is used. The two methods of DNA extraction gave good DNA yield. However the kit method was better in this respect than the boiling method. Primers of strain E gave an amplification product of 511 bp for vaccine strain E. In contrast primers of strain B did not amplify strain B DNA vaccine strain but amplified strain B field strain which were obtained from Department of Microbiology Faculty of Veterinary Medicine, University of Khartoum stock culture and the amplicon was 760 bp.