Objective: To determine whether characteristic changes in neutrophil morphology caused in vitro by Pasteurella haemolytica leukotoxin (LKT) can be observed in vivo by electron microscopic examination of infected tissue chamber fluids and pneumonic lungs.
Animals: 7 mixed-breed beef calves.
Procedure: Tissue chambers were implanted subcutaneously in 3 calves and were inoculated with P haemolytica or phosphate-buffered saline solution. Chamber fluid samples, obtained at 8 and 32 hours after inoculation, were examined, using electron microscopy. Experimental pneumonia was induced in an additional 4 calves by transthoracic inoculation with P haemolytica. These calves were euthanatized at 6, 12, 24, and 36 hours after inoculation and lung sections were examined, using transmission electron microscopy.
Results: On examination, using transmission electron microscopy, neutrophils in lung sections and tissue chamber fluids had cytoplasmic and nuclear changes indicative of irreversible cell injury, including cell swelling, loss of plasma membrane ruffling, mitochondrial swelling, autolytic vacuolation, disruption of plasma membrane, nuclear pyknosis, karyolysis, and karyorrhexis. On examination, using scanning electron microscopy, leukocytes obtained from tissue chambers did not have their typical convoluted surfaces, but appeared rounded and swollen or shrunken with pitted surfaces.
Conclusions: Pasteurella haemolytica-induced changes in neutrophil morphology in vivo were similar to those previously induced by in vitro exposure of neutrophils to LKT. Changes were suggestive of injury initiated by damage to the plasma membrane, which is consistent with the mechanism of action of pore-forming cytolysins.
Clinical relevance: Pasteurella haemolytica LKT appears to be an important virulence factor in vivo; a fact that should be addressed in the development of vaccines.