Development, characterization and standardization of a nose-only inhalation exposure system for exposure of rabbits to small particle aerosols containing Francisella tularensis.

Abstract

Inhalation of Francisella tularensis (Ft) causes pneumonic tularemia in humans, a severe disease with a 30-60% mortality rate. Reproducible delivery of aerosolized virulent bacteria in relevant animal models is essential for evaluating medical countermeasures. Here we developed optimized protocols for infecting New Zealand White (NZW) rabbits with aerosols containing Ft. We evaluated relative humidity, aerosol exposure technique, and bacterial culture conditions to optimize spray factor (SF), a central metric of aerosolization. This optimization reduced both inter-and intra-daily variability and were applicable to multiple isolates of Ft. Further improvements in the accuracy and precision of the inhaled pathogen dose were achieved through enhanced correlation of bacterial culture OD and CFU. Plethysmograph data collected during exposures found that respiratory function varied considerably between rabbits, was not a function of weight, and did not improve with acclimation to the system. Vaccine Strain (LVS)-vaccinated rabbits were challenged via aerosol with human-virulent Ft SCHU S4 that had been cultivated in either Mueller Hinton Broth (MHB) or Brain Heart Infusion (BHI) broth. LVS-vaccinated animals challenged with MHB-SCHU S4 experienced short febrile periods (median: 3.2 days), limited weight loss (< 5%), and longer median survival times (~18 d) that were significantly different than unvaccinated controls. In contrast, LVS-vaccinated rabbits challenged with BHI SCHU S4 experienced longer febrile periods (median: 5.5 days), greater weight loss (> 10%), and median survival times that were not significantly different than unvaccinated controls. These studies highlight the importance of careful characterization and optimization of protocols for aerosol challenge with pathogenic agents.