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F. tularensis LVS ΔiglC 8.08 x 105 CFU
F. tularensis LVS ΔdeoB 5.57 x 105 CFU
In Vivo Growth of F. tularensis
An in vivo growth curve was characterized within B. dubia roaches where both intracellular and extracellular growth was observed. After inoculation of 106 CFU of F. tularensis LVS, total growth to 109 cells was observed after 96 hours. Two hours after treatment with gentamicin revealed a small fraction of in vivo growth was intracellular. After 96 hours post initial inoculation of B. dubia roaches with F. tularensis LVS, 108 cells were classified as intracellular. A steady increase in CFU is observed in both total and intracellular growth, as show in figure 2.
Antibiotic Rescue
To explore the drug treatment potential of Blaptica dubia cockroaches, antibiotics were used in aims of curing roaches post inoculation of Francisella tularensis LVS. After infection of 106 CFU of F. tularensis LVS, antibiotics were administered both systemically through hemocel injections, and orally through a sucrose solution. 100\% survival was observed after treatment with systemic doxycycline, and 90\% survival was observed with oral administration of doxycycline. Streptomycin prolonged survival of B. dubia roaches when administered orally, but 0\% survival was observed after 7 days of initial F. tularensis LVS inoculation. In contrast, 80\% survival was observed when streptomycin was observed when streptomycin was delivered through an intrahemoceol injection.
Figure 3. Rescue of B. dubia roaches with antibiotics after lethal dose of F. tularensis LVS.
Survival graph shows effectiveness of antibiotics after 106 CFU inoculation of F. tularensis LVS via systemic injection into B. dubia roaches. Antibiotics were administered 2, 48, and 96 hours post-infection either via systemic injection or oral administration. Roaches with no antibiotic treatment resulted in 0\% survival after 4 days PI, while prolonged survival was observed in roaches treated with streptomycin delivered orally. Survival of over 80\% was observed in both groups treated with doxycycline and systemic injection of streptomycin.
Temperature Dependent Pathogenesis of F. tularensis
The virulent activity of F. tularensis LVS at different temperatures was also determined. B. dubia roaches were injected with F. tularensis LVS as described previously, and groups of roaches were incubated at different temperatures (40°C, 37°C, 30°C, and 22°C). An increase in temperature correlated to a decrease in LD50 value. The 40C LD50 of F. tularensis LVS was calculated to be 1.24E+04, while the 22°C group had an LD50 value of 2.00+06. Survival of B. dubia roaches also reflected the decrease in virulence, with prolonged survival in the groups of roaches incubated at 22°C and 30°C compared to those incubated at 37°C and 40°C.
Table 2. Lethal dose 50\% values of F. tularensis LVS at variable temperatures.
Values calculated by the survival of roaches seven days post-infection of F. tularensis LVS using the Reed-Muench method. Increasing LD50 values reflect decreasing incubation temperatures.
Incubation Temperature
LD50 Value
40°C 1.24 x 104 CFU
37°C 2.86 x 104 CFU
30°C 1.93 x 105 CFU
22°C 2.00 x 106 CFU
Figure 4. Temperature dependent virulence of F. tularensis LVS.
The above graph shows survival of B.dubia roaches after >3x106 CFU inoculation of F. tularensis LVS via systemic injection. 0\% survival was observed in groups incubated at 40°C and 37°C after 5 days post infection. Prolonged survival was observed in groups of roaches incubated at 30°C and 22°C, with 20\% and 30\% survival after seven days, respectively.