The mouse was found to be natively susceptible to Listeria monocytogenes. Its susceptibility was attributed to the capacity of the organism to survive and multiplying in host macrophages. During the first 3 days of a primary infection the bacterial populations of spleen and liver were found to increase at a constant rate. On the 4th day of infection the host became hypersensitive to Listeria antigens and at the same time bacterial growth ceased. A rapid inactivation of the organism ensued. Convalescent mice were resistant to challenge, but no protective factor could be found in their serum. Histological evidence suggested that acquired resistance was the result of a change occurring in the host's mononuclear phagocytes. When challenged in vitro, the macrophages of convalescent mice were found to resist infection with Listeria monocytogenes. Listeria-resistant cells appeared during the course of infection at a time which corresponded with the development of the antibacterial mechanism in the spleen. They persisted for as long as the antibacterial mechanism remained intact in this organ. This period of absolute resistance to Listeria lasted about 3 weeks. Thereafter, the host remained hypersensitive but unable to inactivate a challenge inoculum of Listeria. However, it remained capable of producing an accelerated response to reinfection. This was thought to depend upon an ability to generate a new population of resistant cells from a residuum of specifically sensitized macrophages or macrophage precursors still surviving in the tissues as a result of the immunological activation which occurred during the primary infection.
Objective: To determine the LD50 of Listeria monocytogenes in mice through serial passage via intraperitoneal or intravenous injection, and to assess changes in bacterial virulence across successive passages
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Prepare serial half-log dilutions of sonicated Listeria monocytogenes suspension from appropriate source (either macrophage culture outgrowth or 16-hour BHI culture)
“Groups of 10 mice were infected by the intraperitoneal or intravenous injection of 0.2 ml of serial half-log dilutions of a sonicated suspension of L. monocytogenes”
If using BHI culture source, centrifuge 16-hour culture and resuspend in BSS containing 1 percent calf serum
“Bacterial suspensions were prepared from the outgrowth of infected macrophage cultures or from a 16 hour culture in BHI medium. The latter was centrifuged and resuspended in BSS containing 1 per cent calf serum”
Disperse bacterial suspension for 5 seconds in a Mullard-M.S.E. magneto-strictive ultrasonicator
“Suspensions were dispersed for 5 seconds in a Mullard-M.S.E. magneto-strictive ultrasonicator”
Adjust bacterial suspension to appropriate concentration by dilution in culture medium
“adjusted to an appropriate concentration by dilution in culture medium”
Inject groups of 10 mice with 0.2 ml of serial half-log dilutions via intraperitoneal or intravenous route
“Groups of 10 mice were infected by the intraperitoneal or intravenous injection of 0.2 ml of serial half-log dilutions”
Observe mice for signs of infection and identify moribund mice receiving minimal lethal dose
“The organism was recovered from the spleen of a moribund mouse of the group receiving a minimal lethal dose”
Remove spleen from moribund mouse aseptically and recover Listeria organism
“The organism was recovered from the spleen of a moribund mouse of the group receiving a minimal lethal dose”
Grow recovered organism in BHI medium for 16 hours
“After 16 hours growth in BHI it was used to infect a further series of mice”
Use 16-hour BHI culture of recovered organism to infect a further series of mice via intraperitoneal or intravenous injection
“After 16 hours growth in BHI it was used to infect a further series of mice”
Calculate LD50 using the method of Reed and Muench for each passage
“The LD50 was calculated by the method of Reed and Muench”
Remove spleen and liver aseptically from infected mice at specified time intervals and place in homogeniser tubes containing 5.0 ml BHI medium
“Organs were removed aseptically to homogeniser tubes containing 5.0 ml of BHI medium”
Disperse organ tissue for 1 minute with motorised teflon pestle
“They were dispersed for 1 minute with a motorised teflon pestle”
Serially dilute homogenised organ samples
“were then serially diluted and plated on well dried BHI-agar in quadrant plates”
Plate serial dilutions on well-dried BHI-agar in quadrant plates
“serially diluted and plated on well dried BHI-agar in quadrant plates”
Incubate plates for 24 hours and make colony counts
“Colony counts were made after incubation for 24 hours”
Animals fed balanced diet in cubed form with no antibiotic additives