Aim:This study aimed to create rapid characterization and genotyping of Pasteurella multocida (PM) protocol using modern molecular biology techniques.Materials and Methods:Thirty bacterial isolates were characterized by capsular and somatic identification using conventional procedure followed by multiplex polymerase chain reaction (PCR), restriction endonucleases analysis (REA), and finally confirmed by sequence analysis. Two local vaccine strains and two field isolates were identified as PM Type A and B.Results:A total of 30 isolates were found positive for PM either morphologically and biochemically; however, multiplex PCR technique identified only 22 isolates as Pasteurella species using universal primers while 8 isolates were found negative for PM. 12 of 22 isolates (54%) were characterized at the same reaction into PM Type A, five isolates (23%) were Type B and the rest five isolates (23%) of tested isolates were negative for Types A, B, and D. Hemorrhagic septicemia Type B: 2 or B: 5 could be identified somatically within PM capsular serogroup B using PCR technique. Somatic characterization of PM was done using REA that could identify all PM Type A into A:1 and all PM Type B into B: 2. These protocols were verified for its accuracy and reliability by sequence analysis of two vaccine strains of PM Type A and B that were characterized previously by biochemical and serological methods as well as two selected isolates from the 22 positive isolates representing PM Type A and B.Conclusion:PCR and REA could confirm the identity of PM and provide a rapid and reliable characterization in comparison with biochemical analysis and conventional serotyping that may take up to 2 weeks. Hence, they can reduce the time needed for polyvalent vaccine production and when the reference antisera are unavailable. Moreover, the identity of Omp-H for vaccine and field strains may provide better data to control Pasteurellosis in Egypt.

No abstract

Corynebacterium pseudotuberculosis is a small Gram-positive bacillus containing mycolic acid in the structure of the cell wall. The bacterium is responsible for Caseous Lymphadenitis (CLA) in small ruminants (sheep and goats). The bacteria are also responsible for Ulcerative Lymphangitis in equines. The disease causes great economic loss in the animal industry. This work aimed to check the ability of Quadruplex PCR (Q-PCR) for genotyping and identification of Egyptian isolates of C. pseudotuberculosis and sequence analysis of phospholipase D (PLD) gene of local isolates. Four of C. pseudotuberculosis local isolates previously biochemically identified were tested for narG gene (nitrate reductase gene). Both nitrate negative biovar (ovis) and nitrate positive biovar (equi) showed a positive result for 16S rRNA, rpoB and PLD genes of C. pseudotuberculosis species. The sequence analysis of our local isolates' PLD gene revealed minor changes in PLD proteins between ovine and equine strains compared with other published PLD sequences in GenBank. It was concluded that the Q-PCR method is able to differentiate between C. pseudotuberculosis equi and ovis biovars. Also, the sequence of PLD gene of local isolates representing the two biovars revealed some variation, which leads to an accurate diagnosis of C. pseudotuberculosis biovars and generates a mapping of immerged local isolates. Further, the PCR and sequence of these isolates provide rapid and accurate genotyping, especially with hyperimmune serum's unavailability.