In this study, we demonstrate that genetic differences (clades A1a, A1b and A2) among F. tularensis subsp. tularensis strains, as identified by PFGE and SNP typing, correlate directly with differences in virulence as assessed using C57BL/6 mice. Among type A clades, A1b strains were shown to be the most virulent in mice, with A1b infected mice succumbing to infection at significantly earlier times as compared to A1a and A2 infected mice. Additionally, A1a infected mice died within a narrower time span than A1b or A2 infected mice, suggesting that A1a strains are either more clonal than A1b or A2 strains or utilize a different mechanism for infection. Importantly, virulence differences appeared to be conserved among clades, as two representative strains with slightly differing PFGE patterns from each type A clade, A1b, A1a and A2, were utilized in this study. Virulence differences were also demonstrated between all type A clades and type B.
Measurement of bacterial burden within the blood, spleen and liver at the time of death in mice infected with A1a, A1b, A2 and type B strains indicated a significantly higher bacterial load in type B-infected mice as compared to mice infected with any of the type A clades. Similarly, mice infected with A2 strains had significantly higher bacterial loads in the blood and liver as compared to A1a and A1b infected mice. A correlation between bacterial burden and time to death was observed, with mice succumbing to infection earlier (i.e. A1a and A1b infected mice) having lower bacterial burdens than mice dying at later time points (i.e. A2 and type B infected mice). This data demonstrates that higher bacterial loads are needed for type B and A2 infected mice to die from infection, as compared to A1a or A1b infected mice. It is unknown if differences in growth rates exist between type A clades (A1a, A1b and A2) or between type A clades and type B. In addition to type B infected mice having higher bacterial burdens, organs were larger in type B infected mice, indicative of a strong inflammatory response that is not as pronounced in type A infected mice.
Our findings are in agreement with a study performed by Twine et al.
, which demonstrated virulence differences between two type A strains, Schu S4 and FSC033, in BALB/c mice 
. Although significant differences in survival curves were found between these two strains, with FSC033 appearing more virulent than Schu S4, they were not attributed to genetic differences. PFGE typing demonstrates that Schu S4 is an A1a strain (), whereas analysis of the genome sequence for FSC033 shows it contains single nucleotide polymorphisms characteristic of A1b strains (C at position 93857 in the FSC033 genome using primer set 1574929 and a G at position 5357 in the FSC033 genome using primer set 518892) 
Due to the low infectious dose of F. tularensis
for both type A and type B in mice (LD50
<10 CFU for both subspecies) and the acute and rapid onset of infection that results, few studies have used a mouse model to demonstrate tangible virulence differences between F. tularensis
. Nonetheless, our results indicate that survival curves can be used to assay differences in virulence in mice among both type A clades and subspecies of F. tularensis
. Several factors are likely to be critical for identifying virulence differences among F. tularensis
strains using a mouse model. In this study, we used death as an endpoint to generate survival curves, thereby eliminating any bias in using signs of morbidity to euthanize mice and determine time of death. Towards this end we have defined a specific temperature during infection that is an ethical indicator of death (unpublished observation). In the future, the use of temperature as an experimental endpoint will minimize pain and suffering of infected animals and will also serve as a valuable tool for unbiased comparisons of F. tularensis
virulence. Monitoring mice every 2 hours over the time course of infection, rather than 24 hr periods, was also important in this study for ensuring accuracy. Finally, genotyped clinical strains were chosen for this study in order to yield information directly related to previous epidemiologic findings 
The mechanisms underlying virulence differences among type A clades and between type A clades and type B are not known. To date, numerous studies have compared genetic variations present between type A and type B strains at both the genome and protein level and a few studies have also compared differences between type A clades, A1 and A2 
. A previous study showed that FSC033 was better able to disseminate as compared to Schu S4 from the original inoculation site to the liver and spleen of infected mice 
. Consistent with A1b strains being better able to disseminate is the epidemiologic finding that the majority of isolates (67%) from patients infected with A1b strains were recovered from invasive sites (blood, lung, pleural fluid or CSF) as compared to patients infected with A1a (44%), A2 (10%) or type B (37%) strains 
. Rates of dissemination, immunological responses, and tissue tropism are areas for future analyses.
In conclusion, our results correlate with epidemiological data for human culture-confirmed tularemia cases in the United States, which demonstrated that A1b infections were associated with higher mortality as compared to A1a, A2 and type B infections 
. Here we showed that mice infected with A1b strains had a significantly shortened survival time as compared to mice infected with A1a, A2 or type B strains, demonstrating the importance of human epidemiological data and how it can yield hypotheses that can be studied in the laboratory. This finding will be important for raising awareness in states where human infections with A1b strains occur, which may aid in preventing mortality. The virulence differences identified among type A clades indicate we can no longer consider F. tularensis
to be biologically homogeneous and the need to shift our attention from the subspecies to the clade level. Previous studies also showed differences in geographic distributions among type A clades 
. The demonstration of genetic and geographic, as well as virulence, differences among the A1a, A1b and A2 clades brings into question their classification, given that F. tularensis
was originally classified as a subspecies based on biochemical, geographic and virulence differences