Growth conditions and environmental factors impact aerosolization but not virulence of Francisella tularensis infection in mice

Seth A Faith,Le'Kneitah P Smith,Angela S Swatland,Douglas S Reed

doi:10.3389/fcimb.2012.00126

Abstract

In refining methodology to develop a mouse model for inhalation of Francisella tularensis, it was noted that both relative humidity and growth media impacted the aerosol concentration of the live vaccine strain (LVS) of F. tularensis. A relative humidity of less than 55% had a negative impact on the spray factor, the ratio between the concentration of LVS in the aerosol and the nebulizer. The spray factor was significantly higher for LVS grown in brain heart infusion (BHI) broth than LVS grown in Mueller–Hinton broth (MHb) or Chamberlain's chemically defined medium (CCDM). The variability between aerosol exposures was also considerably less with BHI. LVS grown in BHI survived desiccation far longer than MHb-grown or CCDM-grown LVS (~70% at 20 min for BHI compared to <50% for MHb and CCDM). Removal of the capsule by hypertonic treatment impacted the spray factor for CCDM-grown LVS or MHb-grown LVS but not BHI-grown LVS, suggesting the choice of culture media altered the adherence of the capsule to the cell membrane. The choice of growth media did not impact the LD50 of LVS but the LD99 of BHI-grown LVS was 1 log lower than that for MHb-grown LVS or CCDM-grown LVS. Splenomegaly was prominent in mice that succumbed to MHb- and BHI-grown LVS but not CCDM-grown LVS. Environmental factors and growth conditions should be evaluated when developing new animal models for aerosol infection, particularly for vegetative bacterial pathogens.

Highlights

Tularemia is a disease found throughout North America, Europe, and Asia that can be contracted by contact, arthropod vectors, or inhalation (Dennis et al, 2001; Pechous et al, 2009)
We report here our efforts to establish a system for reproducibly infecting animals with F. tularensis and infect mice with small particle aerosols containing live vaccine strain (LVS) as a surrogate model of human disease
Spray factor is the ratio of the aerosol concentration to the nebulizer concentration; it can be used as both a means to determine the nebulizer concentration needed to achieve a desired presented dose and the performance of an aerosol relative to other aerosols of the same agent (Roy and Pitt, 2005)

Summary

Introduction

Tularemia is a disease found throughout North America, Europe, and Asia that can be contracted by contact, arthropod vectors, or inhalation (Dennis et al, 2001; Pechous et al, 2009). The causative agent, Francisella tularensis, was first identified in 1912. F. tularensis can infect a significant number of mammals, arthropods, and even fresh-water amoebae. Four subspecies have been identified: tularensis, holarctica, mediasiactica, and novicida (Ellis et al, 2002). Subspecies tularensis strains, designated as type A, are the most virulent and are most frequently found in North America (type A can be further divided into A.1 and A.2) (Gurycova, 1998; Staples et al, 2006). Subspecies holarctica, or type B strains, are not as virulent as type A but they can cause human disease and are found throughout North America and Europe

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