Characterization of PM20C2, A TadD- Pasteurella muliocida Mutant
Cotey, Alicia D.
A mutant strain of Pasteurella multocida, PM20C2, which contains a transposon disruption in the TadD gene, was characterized using genetic complementation studies and an in vitro/in vivo competition assay to assess its potential for use in a live vaccine. Complementation was achieved by incorporating either the wildtype P. multocida TadCD genes or the wildtype Actinobacillus actinomycetemcomitans TadCD genes into the pNF2176 plasmid. These complemented mutants were then used to infect mice and the resultant LD50 values were compared to the LD50 values of mice infected with the uncomplemented PM20C2 mutant, the wildtype TF5 P. multocida strain,or the PM20C2 mutant complemented with an empty pNF2176 vector plasmid. Neither TadCD from P. multocida nor that from A. actinomycetemcomitans successfully restored virulence. It is likely that the transposon disrupted the expression of genes downstream from its insertion in TadD. The in Vitro/in vivo competition assay involved creating a mixture of equivalent concentrations (cfu/ml) of PM20C2 and TF5. This was then used to either infect a mouse model or a falcon tube of broth. The output ratio was determined and entered, along with the input ratio, into the Competitive Index equation. The Competitive Index equation gives a numerical value to the fitness of the mutant when placed in competition with the wildtype. When compared to the wildtype, the PM20C2 mutant was near equally fit in vivo and less fit in vitro. Possible complementation by the presence of the wildtype, TF5, may explain the discrepancy in viability of the PM20C2 mutant in the competition assays vs. Signature Tagged Mutagenesis. From the results obtained, it is still inconclusive as to whether PM20C2 would be a good candidate for a live vaccine·.
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