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

Abstract

14Inhalation of Francisella tularensis (Ft) causes pneumonic tularemia in humans, a severe disease 15 with a 30-60% mortality rate. Reproducible delivery of aerosolized virulent bacteria in relevant 16 animal models is essential for evaluating medical countermeasures. Here we developed 17 optimized protocols for infecting New Zealand White (NZW) rabbits with aerosols containing 18 Ft. We evaluated relative humidity, aerosol exposure technique, and bacterial culture conditions 19 to optimize spray factor (SF), a central metric of aerosolization. This optimization reduced both 20 inter-and intra-daily variability and were applicable to multiple isolates of Ft. Further 21 improvements in the accuracy and precision of the inhaled pathogen dose were achieved through 22 enhanced correlation of bacterial culture OD and CFU. Plethysmograph data collected during 23 exposures found that respiratory function varied considerably between rabbits, was not a 24 function of weight, and did not improve with acclimation to the system. Vaccine Strain (LVS)-25 vaccinated rabbits were challenged via aerosol with human-virulent Ft SCHU S4 that had been 26 cultivated in either Mueller Hinton Broth (MHB) or Brain Heart Infusion (BHI) broth. LVS-27 vaccinated animals challenged with MHB-SCHU S4 experienced short febrile periods (median: 28 3.2 days), limited weight loss (< 5%), and longer median survival times (~18 d) that were 29 significantly different than unvaccinated controls. In contrast, LVS-vaccinated rabbits 30 challenged with BHI SCHU S4 experienced longer febrile periods (median: 5.5 days), greater 31 weight loss (> 10%), and median survival times that were not significantly different than 32 unvaccinated controls. These studies highlight the importance of careful characterization and 33 optimization of protocols for aerosol challenge with pathogenic agents. 34 35 36 Francisella tularensis (Ft) is a gram-negative coccobacillus that causes a severe zoonotic 37 disease in humans known as tularemia (a.k.a. rabbit fever). Transmission of Ft is by direct 38 contact with infected animals or tissues, arthropods, ingestion, or inhalation; tularemia is not 39 typically spread person-to-person. Inhalation of as few as 15 organisms is sufficient to cause 40 disease; Ft is readily grown to high concentration in culture and relatively stable in aerosol, 41 properties considered 'ideal' in a biological weapon. For these reasons, both the former Soviet 42 Union and United States of America (prior to 1969) included Ft in their offensive biological 43 weapons programs (1). There are no licensed vaccines for tularemia; antibiotics are typically 44 given and show good efficacy against most isolates of Ft. Considering the high 45 morbidity/mortality and infectivity by aerosol, as well as the possibility of antibiotic resistant 46strains, Ft is a tier 1 select agent, those for which there is the most concern about malicious use 47 (2). 48 We have previously shown that rabbits are a relevant model of the human disease caused 49 by inhal...