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Lysates of turkey-grown Pasteurella multocida: examination of vaccine preparations by electron microscopy
Abstract:
The effects of differential centrifugation, density gradient centrifugation, freeze-thawing, and chemical lysis on the morphology of Pasteurella multocida from the blood of infected turkeys were examined by electron microscopy. Morphologic differences were not seen between thin-section preparations of P multocida after differential and density gradient centrifugation procedures. The internal ultrastructure of in vivo-grown cells was different from that observed previously for P multocida grown in vitro. Large membranous blebs were also observed on the surface of negative-stained preparations of organisms from the plasma, but not in thin sections of cells from the same preparation. Freeze-thaw of bacterial suspensions in sucrose resulted in partial lysis, revealing bacteria in different phases of degradation. Complete lysis (but not solubilization) was enhanced by treatment with EDTA, lysozyme, and Triton X-100. Centrifuged lysate pellets were thin-sectioned or negative-stained. Pellets consisted of vesicles, ranging in size from 0.05 to 1.0 micrometer, that had a characteristic trilaminar membranous appearance similar to those reported from other gram-negative bacteria grown in vitro and treated similarly.
Insights
Electron microscopy revealed distinct Pasteurella multocida ultrastructure in vivo. Chemical lysis and freeze-thawing induced partial or complete bacterial degradation, forming vesicles.
Area of Science:
- Veterinary Microbiology
- Cell Biology
- Electron Microscopy
Background:
- Pasteurella multocida is an important avian pathogen.
- Understanding bacterial morphology is crucial for diagnostics and treatment.
- Previous studies on P. multocida morphology primarily focused on in vitro-grown cells.
Purpose of the Study:
- To investigate the effects of various preparation methods on Pasteurella multocida morphology.
- To compare the ultrastructure of in vivo-grown P. multocida with in vitro-grown cells.
- To characterize the morphological changes induced by differential centrifugation, density gradient centrifugation, freeze-thawing, and chemical lysis.
Main Methods:
- Electron microscopy (thin-sectioning and negative staining) was used.
- Morphological analysis of Pasteurella multocida isolated from infected turkey blood.
- Comparison of cells processed by differential centrifugation, density gradient centrifugation, freeze-thawing, and chemical lysis (EDTA, lysozyme, Triton X-100).
Main Results:
- No significant morphologic differences were observed between P. multocida processed by differential and density gradient centrifugation.
- In vivo-grown P. multocida exhibited different internal ultrastructure compared to previously reported in vitro-grown cells.
- Freeze-thawing in sucrose caused partial lysis, while EDTA, lysozyme, and Triton X-100 enhanced complete lysis.
- Vesicles with a trilaminar membranous appearance were observed in centrifuged lysate pellets.
Conclusions:
- Differential and density gradient centrifugation preserve P. multocida morphology.
- In vivo-grown P. multocida possesses unique ultrastructural characteristics.
- Chemical lysis and freeze-thawing effectively degrade P. multocida, yielding characteristic vesicles.