10th International Aerosol Conference September 2 - September 7, 2018 America's Center Convention Complex St. Louis, Missouri, USA
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Development of an Inhalational Francisella Tularensis Exposure Therapeutic Model in New Zealand White Rabbits
ROY BARNEWALL, Chrs Cirimotich, Susan Reed, Kristin Patton, Heather Northup, Amber Lindsay, Phyllis Herr-Calomeni, Brian Miller, Gloria Sivko, Battelle, Columbus Ohio
Abstract Number: 250 Working Group: Infectious Bioaerosol
Abstract Introduction Tularemia, caused by the Gram-negative bacterium Francisella tularensis, is a zoonotic disease that causes a debilitating illness in humans. Human cases of tularemia are typically sporadic, with a primary concern around intentional release of the bacteria for biological warfare or bioterrorism purposes. Such an event would likely be attempted through aerosolization of the bacteria and result in primarily pneumonic tularemia, which is characterized by pneumonia, fever, chills, and other nonspecific symptoms. The natural incidence of F. tularensis in humans is too low to support clinical testing of new therapeutic countermeasures against the bacterium. Therefore, therapeutics will need to be tested under the US FDA Animal Rule and likely require two well-characterized animal models of pneumonic tularemia. To satisfy this requirement, a New Zealand White (NZW) rabbit inhalational tularemia therapeutic model was developed.
Materials and Methods This model development was performed using NZW Rabbits in two phases: Phase I was performed to determine an inhaled median lethal dose (LD50) for F. tularensis Schu S4 and Phase II used the data generated under Phase I to develop an therapeutic model of lethal inhalation F. tularensis Schu S4 infection evaluating the efficacy of two classes of antibiotics, doxycycline (2.5 mg/kg, twice daily) and levofloxacin (50 mg/kg, once daily), when administered orally for 10 days after the onset of clinical illness in this model.
Phase I. 54 rabbits were challenged with varying doses of aerosolized F. tularensis (target 1 to 10,000 colony forming units (CFU)) and clinical observations were performed twice daily for 14 days for mortality. Blood samples for bacteremia analysis were collected from sedated animals (acepromazine; 1-6 mg/kg intramuscular) on days 3, 4, 5, 6, 7, and 14 post-challenge.
Phase II. 54 rabbits were randomized into 3 groups and challenged with a lethal dose of aerosolized F. tularensis to evaluate disease progression, including body weight and temperature, hematologic and clinical chemistry status, bacteremia status, mortality, and bacterial tissue burden for 14 days. It was determined a significant increase in body temperature (SIBT; two consecutive readings greater than two standard deviations above the average baseline temperature for an individual animal) was a suitable trigger for antibiotic treatment in this model. Thus, doxycycline (group 2) or levofloxacin (group 3) were administered after two consecutive SIBTs were detected for 10 consecutive days. Doxycycline was administered twice daily and levofloxacin once daily. All rabbits that died or were euthanized had necropsy and histology performed on select tissues to confirm death or illness due to tularemia. Samples of lung, liver, and spleen were collected for quantitative analysis of bacterial burden.
Results Phase I Inhaled LD50 Determination. The calculated inhaled doses ranged from 4 to 24,168 CFU. The inhaled LD50 was calculated using a logistic model generated from the rabbit mortality data and was determined to be 266 CFU (95% Fieller interval: 120, 797).
Phase II. Rabbits were challenged with a mean inhaled dose of 4568 CFU of F. tularensis and were provided with antibiotics (doxycycline – Group 2; levofloxacin – Group 3) or no treatment (Group 1) following two consecutive SIBTs. Levofloxacin was completely protective against F. tularensis bacteremia, bacterial tissue burden, and tularemia-associated death, doxycycline was only partially protective with all but one animal succumbing and an average time to death of 266.4 ± 134.16 hours (11.1 ± 5.59 days). There were no survivors in the untreated controls with an average time to death of 138.41 ± 18.29 hours (5.76 ± 0.76 days).
Conclusion: The results support the use of NZW rabbits as an animal model for evaluating therapeutic efficacy of candidate inhalational tularemia post-exposure countermeasures.