Fusobacterium necrophorum was tested for the ability to survive and multiply in mouse peritoneal macrophages. F. necrophorum (5 x 108) was injected intraperitoneally into mice and following an infection period of 4 hours, the peritoneal macrophages were harvested, exposed to gentamycin, and maintained in tissue culture. Macrophages were lysed at 4, 24, 48, and 72 hours postinfection, and lysates were plated on agar medium and incubated anaerobically to recover viable bacteria. Escherichia coli LE392, a nonvirulent laboratory strain, served as a control in these experiments. F. necrophorum survived and multiplied in mouse peritoneal macrophages for at least 72 hours at a level of 105 fold higher than the control. E. coli LE392 survived for 24 hours in the mouse peritoneal macrophages but the number of bacteria dropped precipitously by 48 hours. Electron micrographs of the F. necrophorum- containing macrophages revealed that F. necrophorum were initially enclosed within a membrane-bound phagolysosome, but by 24 hours postinfection, the phagolysosome membrane was dissolved, and by 48 hours the macrophages were being destroyed and F. necrophorum was released. A gene library of F. necrophorum DNA was constructed in E. coli LE392 in order to identify and characterize the gene(s) involved in survival of F. necrophorum in mouse peritoneal macrophages. One thousand recombinant E. coli clones were tested for their ability to survive in mouse peritoneal macrophages. Two E. coli clones were identified which were able to survive for 72 hours in mouse peritoneal macrophages These clones contained over 20kb inserts. Subcloning revealed that a smaller 9kb fragment could cause E. coli to survive in macrophages. Sequence analysis is currently under way to further characterize the biological determinant for survival.
