Virulence and genome analysis of three historical Francisella tularensis ssp. holarctica isolates for development of a Type B test panel

<i>Francisella tularensis</i> ssp<i>. tularensis</i> (Type A) and ssp. <i>holarctica</i> (Type B) are the etiological agent of tularemia. Most studies on <i>F. tularensis</i> pathogenesis and vaccine development have focused on the Type A Schu S4 strain. However, Type B isolates remain less understood, emphasizing the need for further research. To address this concern and provide well characterized Type B isolates to test future vaccine efficacy, we selected three Type B isolates available in the USAMRIID repository (VT68, strain 425, and strain 503). These strains were chosen based on the original isolation source, the availability of historical <i>in vivo</i> data, and genomic sequence data. Strains were characterized for extracellular and intracellular growth, lipopolysaccharide profile via western blot analysis, and LD₅₀ values were determined by both murine intranasal challenge and aerosolization of Fischer 344 rats. Strain 425 displayed several attenuation indicators and was completely attenuated in a rat aerosol challenge model. In contrast, VT68 and 503 remained highly virulent in rodent models but displayed some differences in lesion severity. Although <i>F. tularensis</i> genomes are known to be highly conserved, genomic analysis revealed multiple inversions and 68 unique genetic differences among these three Type B isolates. From this study, we were able to provide well-characterized Type B <i>F. tularensis</i> strains to test future vaccines and therapeutics.