Investigation of survival mechanisms of Pasteurella haemolytica and Pasteurella trehalosi in vivo and in vitro

Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10 were cultured in "in-vivo" fluids (ruminant tracheobronchial washings and serum), under defined iron-restrictive conditions, and compared for changes in cellular composition. In analyses by SDS PAGE and Western blotting, envelope and cell contents of the bacteria showed differences in protein content and changes in antigens recognised by convalescent antiserum. Capsule size was not influenced by growth medium. Lipopolysaccharide was detected by PAGE in all the fluids except bovine tracheobronchial washings. However, a Limulus amoebocyte lysate assay did detect trace amounts of endotoxin. Leukotoxin activity was only detected in broth and iron-restricted cultures and not in any "in vivo" fluids. Neutralisation of the toxin with homologous and heterologous convalescent antiserum showed little cross-reactivity in the neutralisation capacity against A1 leukotoxin but heterologous antiserum was effective with leukotoxin of the other serotypes. Using the same fluids to monitor long term culture, all serotypes showed the capacity for extended survival and resuscitation with the addition of nutrients. All three strains survived better in ruminant than in other species' fluids. In non-ruminant fluids and natural water viability was detected only at low temperatures. Morphological changes in both colonial and microscopic appearances were apparent during long term survival. These results imply that this organism possesses some starvation survival mechanisms. Immunomagnetic beads with bound antibody specific for the three serotypes proved a useful reagent for the isolation of target organisms from host tissues and fluids. Differing protein profiles from those seen in bacteria grown in-vitro were demonstrated. Western blotting unexpectedly failed to identify many antigens when recovered whole cells were compared to those grown in laboratory culture, indicating that perhaps mechanisms were present which helped to evade an immune response. Superoxide dismutases were detected in all three strains. Level of enzyme activity, electrophoretic mobility on native PAGE, and metal-active sites were shown to differ.