Molecular Characterization of Pasteurella multocidaVaccine Strains
Molecular Characterization of Pasteurella multocidaVaccine Strains
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Date
2015-04-22
Authors
Badawi, Hajir
Journal Title
Journal ISSN
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Publisher
UOFK
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.
Description
82page
Keywords
Molecular Characterization,Pasteurella,multocidaVaccine, Strains