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Abstract: The type A and B subspecies of Francisella tularensis cause severe disease, tularemia, in humans. However, only the former can be lethal especially if inhaled. It is likely that non-lethal infection is due at least in part to the ability of innate host defenses to control pathogen growth whilst acquired immunity develops. Most common small laboratory animals rapidly succumb to clinical strains of F. tularensis and are, therefore, poor models with which to study innate immunity. In an attempt to improve upon this situation in the present study, Sprague–Dawley and Fischer 344 rats were examined for their ability to combat challenge with type A and B strains of the pathogen. Sprague–Dawley rats were significantly more resistant than Fischer rats to infection with either subspecies. This correlated with the ability of Sprague–Dawley rats to arrest the growth of the pathogen at both the site of challenge and at sites of disseminated infection. The rapidity with which F. tularensis kills susceptible rats and the early onset of control of infection in resistant rats suggests that differences in innate immunity account for these disparate outcomes. Thus, the rat might be a more useful model for studying innate immunity to virulent F. tularensis than other small mammals. [Copyright &y& Elsevier]

BALB/c and C57BL/6 mice were challenged by aerosol or intradermally with low doses (∼10–20 colony forming units) of virulent type A and type B strains of the facultative intracellular pathogen, Francisella tularensis, and the course of infection was monitored. Both mouse strains were equally susceptible to infection, but type A strains reached lethal numbers a few days earlier than type B strains regardless of challenge route. BALB/c mice showed overt signs of infection for several days, whereas C57BL/6 mice remained asymptomatic until a few hours before death. Histological changes were extensive and severe in the liver and spleen, but much more limited in the lungs, even in mice challenged by aerosol. Thus, it appears that regardless of the route of infection, systemic rather than pulmonary infection was the likely cause of death following low dose challenge with virulent F. tularensis. [Copyright &y& Elsevier]

The present study examines the relationship between the structure and important biological effects of the lipopolysaccharide (LPS) of the intracellular bacterial pathogen, Francisella tularensis LVS. It shows that treating mice with sub-immunogenic amounts of intact F. tularensis LPS rapidly induces an enhanced resistance to intradermal or aerogenic challenge with strains of the pathogen of varying virulence. However, neither the free Lipid A nor core-O-chain produced by mild acid hydrolysis of LPS appeared able to elicit this host defense mechanism. [Copyright &y& Elsevier]

Francisella tularensis is a zoonotic, facultative intracellular bacterial pathogen capable of initiating infection, tularemia, via multiple routes including dermal micro-abrasions and inhalation. Mouse models of systemically-initiated infection with F. tularensis LVS have been used extensively to reveal potential host defence mechanisms against the pathogen. Such studies have demonstrated the critical need for neutrophils and interferon-gamma (IFN-γ) to combat the early stages of primary experimental tularaemia initiated by systemic routes. Surprisingly, however, the present study shows that these defences appear not to combat early pulmonary tularaemia initiated by inhalation of the pathogen into the lower airways. The results imply that the effectiveness of particular anti-bacterial host defences vary with invasion site. Thus, it is impossible to predict effective host defence mechanisms against inhalation-initiated tularaemia from current knowledge of anti- Francisella defences that have been shown to combat systemically-initiated infection. [Copyright &y& Elsevier]

The current study determined the ability of Francisella novicida to act as a live vaccine against the much more virulent, but closely related pathogen, Francisella tularensis. Live attenuated strains of the latter are effective vaccines against human tularemia. However, the molecular cause of their attenuation remains unknown, and this is a regulatory barrier for licensing such vaccines. Moreover, F. tularensis is exceptionally difficult to manipulate genetically. This is hampering the development of rationally attenuated vaccine strains. F. novicida shares a lot of genetic homology with F. tularensis and is more amenable to genetic manipulation. If the former naturally expresses the protective antigens of the latter, it could be used to develop a defined tularemia vaccine. However, the results presented herein show that wild-type F. novicida elicits almost no protection in mice against challenge with virulent F. tularensis. [Copyright &y& Elsevier]

Abstract: Francisella tularensis subspecies tularensis is one of the most virulent of bacterial pathogens for humans. Protective immunity against the pathogen can be induced in humans and some, but not all, mouse strains by vaccination with live, but not killed, vaccines. In mice, this protection is mediated predominantly by CD4+ and CD8+ T cells. This is thought to be the case too for humans. Nevertheless, it is possible that successful vaccination elicits antigen-specific antibodies that can serve as correlates of protection. To test this hypothesis we examined the repertoire of antibodies induced following successful immunization of BALB/c and CH3/HeN mice versus unsuccessful vaccination of C57BL/6 and DBA⧹2 mice with F. tularensis Live Vaccine Strain or following unsuccessful vaccination of BALB/c mice with highly related subspecies, F. novicida. The results showed that successful vaccination elicited antibodies to at least six proteins that were not recognized by antisera from vaccinated but unprotected mice. [Copyright &y& Elsevier]

Abstract: Understanding the pathogenesis of infectious diseases requires comprehensive knowledge of the proteins expressed by the pathogen during in vivo growth in the host. Proteomics provides the tools for such analyses but the protocols required to purify sufficient quantities of the pathogen from the host organism are currently lacking. Here, we present a rapid immunomagnetic protocol for the separation of Francisella tularensis, a highly virulent bacterium and potential biowarfare agent, from the spleens of infected mice. In less than one hour, bacteria can be isolated in quantities sufficient to carry out meaningful proteomic comparisons with in vitro grown bacteria. Furthermore, the isolates are virtually free from contaminating host proteins. Two-dimensional gel analysis revealed a host induced proteome in which 78 proteins were differentially expressed in comparison to in vitro grown controls. The results obtained clearly demonstrate the complexity of the adaptive response of F. tularensis to the host environment, and the difficulty of mimicking such behavior in vitro. [Copyright &y& Elsevier]

Abstract: Francisella tularensis subspecies tularensis (type A F. tularensis) is considered to be one of the most virulent of all bacterial pathogens. Mice are extremely susceptible to infection with this subspecies (LD100 via various inoculation routes is <10cfu). However, it has not been established whether overt virulence differences exist amongst type A strains of F. tularensis. To this end, the present study compared the virulence of two distinct type A strains, FSC033 and SCHU S4, for naïve mice and mice immunized with the live vaccine strain of the pathogen, F. tularensis LVS. One nominal isolate of SCHU S4 was found to be completely avirulent. Another isolate was highly virulent, but all examined cases appeared somewhat less virulent than FSC033. The implication of these findings for future infection and immunity studies is discussed. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a gram-negative facultative intracellular bacterium and the causative agent of tularemia. Two subspecies (type A and B strains) of the pathogen exist, the former being much more virulent than the latter for humans and other higher mammals. In this study, we examined the effect of virulent strains of F. tularensis infection on the thymus and thymocytes and the potential mechanisms involved. Low-dose aerosol exposure of C57BL/6 mice with type A, but not type B, F. tularensis caused severe reduction in thymus weight and destruction of thymocytes, particularly CD4+CD8+ thymocytes, by day 4 after infection. The depletion of thymocytes was accompanied by a significant increase in circulating cortisone levels and could be partially prevented by adrenalectomy. Moreover, thymus atrophy and thymocyte depletion following infection were abolished in mice deficient in tumor necrosis factor receptors 1 and 2, but not in FasL-deficient mice. The severe destruction of the thymus and selective depletion of immature thymocytes during type A F. tularensis infection may represent a key pathogenic mechanism in tularemia and could hinder the development of an effective primary immune response against this highly virulent pathogen. [Copyright &y& Elsevier]

Francisella tularensis, the causative agent of tularemia, is a gram-negative facultative intracellular bacterium. Toll-like receptor (TLR) 4 is considered to be critical for inducing host innate immunity against many gram-negative bacteria including many respiratory pathogens. To determine the role of TLR4 in host defense against airborne F. tularensis infection, TLR4-defective C3H/HeJ (TLR4d) or wild-type C3H/HeOuJ (WT) mice were challenged by low-dose aerosol with type A F. tularensis, and the course of the infection and host responses were compared at day 2 and 4 post-inoculation (dpi). At dpi 2, bacterial burdens in the lungs were similar between TLR4d and WT mice, but TLR4d mice surprisingly harbored approximately 10-fold fewer bacteria in their spleens and livers. However, the bacterial burdens at dpi 4, the mortality and median time to irreversible moribundity were indistinguishable between the two mouse strains. In addition, the inflammatory responses to the infection, as reflected by the cytokine levels and leukocyte influx in the bronchoalveolar lavage fluid and histopathological analysis, were similar between both mouse strains. Additionally, as with C3H mice, we found no difference in either the median time to death or the survival rate between TLR4-deleted C57BL/10ScNJ mice and WT C57BL/10 mice. Combined, these data suggest that TLR4 does not contribute to resistance of mice to airborne type A F. tularensis infection. [Copyright &y& Elsevier]

Previous studies have shown that IFN-γ, TNF-α and NOS-2, but not B cells, are crucial for host defense against primary systemic infection with the attenuated live vaccine strain (LVS) of Francisella tularensis. In this study, we examined the importance of these and additional immune components in host resistance against infection with virulent strains of F. tularensis initiated by systemic and airborne routes. Wild-type (WT) mice and mice deficient in IFN-γ, TNFR1R2, NOS-2, or B cells were equally susceptible to low dose (∼10 colony forming units) aerosol or intradermal challenge with virulent type B F. tularensis, and succumbed to the infection between days 6 and 8 post-inoculation. Quantitative bacteriology showed that IFN-γ−/− and B cell−/− mice consistently harbored up to one log10 more bacteria in their lungs, spleens and livers than WT mice at day 5 post aerosol exposure. Surprisingly, however, compared to other strains of KO mice and WT control mice, IFN-γ−/− mice showed only mild liver damage as assessed by histopathology and liver function tests. Additional experiments established that even mice with broad immunodeficiency (SCID, neutropenic, splenectomized or thymectomized mice and mice treated with corticosteroid) were no more susceptible to aerosol-initiated infection with virulent type B or type A F. tularensis than immunosufficient control mice. Combined, our results indicate that, unlike LVS, normal type A and type B F. tularensis strains are so extremely virulent that even immunocompetent mice are virtually defenseless to low dose aerosol and intradermal challenges with them. [Copyright &y& Elsevier]