Thus, it is possible that the immunization regimen induced an immune response that prevented a systemic dissemination of the bacteria. Studies by Sharma et al. provide support for this possibility. It is also possible that high titers of antigen-specific IgG, as well as cellmediated immune responses in the systemic compartment helped control bacterial growth in the spleen and liver. In the case of the lungs, it might be more difficult to clear the infection, especially considering the high number of bacteria that are rapidly lodged in the lungs after i.n. infection. Since the subunit vaccine used in this current study was tested for its efficacy in inducing a protective response against FT LVS and not against the Schu S4 strain, the question of the 4-(Benzyloxy)phenol degree of similarity between FT LVS and Schu S4-derived DnaK and Tul4 needs to be addressed. Analysis of the outer membrane proteins of FT LVS and Schu S4 has revealed almost identical bioinformatics findings and a shared sequence homology of 96 to 100%, which was confirmed with antiserum reactivity to homologous outer membrane proteins, such as Tul4, found in FT LVS and Schu S4. In a different study, DnaK was among the 3,4,5-Trimethoxyphenylacetic acid identified proteins to which T cell hybridomas showed select reactivity upon exposure to FT LVS and Schu S4 lysates. Moreover, the protein sequences encoding the DnaK and Tul4 T cell epitopes are conserved in FT LVS and Schu S4. Hence, the degree of similarity between the DnaK and Tul4 derived from FT LVS and Schu S4 is extremely high. Based upon these observations, we predict immune reactivity to DnaK and Tul4 in mice immunized with both DnaK+Tul4 will contribute towards a host defense against FT Schu S4 infection. However, it should be kept in mind that the levels of protein expression by a bacterium could influence the efficacy of a vaccine. Studies have shown that the expression of proteins by strains of FT can vary based upon growth conditions, e.g., laboratory media, host macrophages or murine spleens. Therefore, if FT Schu S4 exhibits low levels of DnaK and/or Tul4 expression, especially during the in vivo infection process, this could potentially limit any protective effects of a subunit vaccine. Future studies will be required to investigate this possibility and to determine the effectiveness of our subunit vaccine in protecting against a lethal challenge with FT Schu S4. Studies have shown that a protective response against FT LVS challenge does not necessarily translate into an effective response against a Schu S4 challenge. For example, immunization with the outer membrane protein FopA was effective against FT LVS, but not a Schu S4 challenge. In another study native outer membrane proteins afforded 50%, but not complete protection against a Schu S4 challenge. Similarly, LPS has been extensively shown to protect mice against FT LVS infection, but a response to LPS alone is not sufficient to completely protect against a type A strain. These findings add to the growing consensus that in order to provide effective protection against more virulent FT subspecies, immune reactivity to several FT antigens will be necessary. Moreover, a combinatorial strategy, where LPS is chemically conjugated to a protein antigen such as bovine serum albumin, significantly increased protection against a type A strain. This highlights the need to identify a diverse set of FT-specific immunodominant antigens that can stimulate antibody and cell-mediated responses to combat FT infection.