DRP000208
Francisella tularensis subsp. tularensis multi-isolate project
F. tularensis SCHU cultured confluently for 3 days on Chocolate II agar (BD) from glycerol stock was suspended with saline. The SCHU passage 0 (P0) clone 3 was isolated from this bacterial suspension. Three C57BL/6J mice were inoculated with 10^6 CFU/0.1 ml of above bacterial suspension. When mice showed the severe clinical signs or alive at 4 days after inoculation, three mice were sacrificed and their spleens were collected. The pool of spleen homogenate was plated on Chocolate II agar (BD) and then first passage F. tularensis SCHU were grown for 3days. The bacterial suspension was prepared with saline and then mice inoculated with this suspension were sacrificed. The nine times in vivo passages were carried out. Mice were inoculated with 10^8 to 10^9 CFU of SCHU in passage 2 to 5 because low amount of SCHU cannot produce the successive generations of SCHU in early passages. In contrast, mice were inoculated with 10^5 CFU of SCHU in passage 6 to 9. Since the increasing pathogenicities were observed during serial in vivo passage, SCHU P5 clone 3 and P9 clone 1 were isolated from pools of spleen homogenate at five and nine passages, respectively. In this study, SCHU P0 clone 3, SCHU P5 clone 3 and SCHU P9 clone 1 were named as SCHU P0, P5 and P9, respectively.