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4). Open in a separate window Figure 4. Pathological lesion in the organs of immunized mice after challenge. of Rocky Asaraldehyde (Asaronaldehyde) Mountain spotted fever (RMSF), a life-threatening tick-transmitted contamination.1 Contamination with damages blood vessels throughout the body, causing increased vascular permeability, diminished serum oncotic pressure, and reduced perfusion of various organs.2 The case fatality rate of RMSF in untreated patients is as high as 60%, with a range of 5C10% in treated patients. Fatality rates tend to rise if treatment with antibiotics (tetracycline or chloramphenicol) is usually delayed.3 There have been concerted efforts to develop an effective vaccine against RMSF. Three different vaccines made up of nonviable have been prepared from cells, which is usually difficult and hazardous because is usually highly infectious. A good alternative would therefore be subunit vaccines against RMSF comprising protective antigens that can be efficiently and safely produced using modern techniques. In recent years, many surface proteins have been acknowledged in the spotted fever group (SFG) rickettsiae. The Sca0 (OmpA) and Sca1 proteins are involved in the attachment of rickettsiae to host cells,9,10 while Sca5 (OmpB) is usually associated with the rickettsial invasion of host cells.4 Sca2 serves as a formin mimic that is associated with actin-based motility of rickettsiae in host cells.4 Sca4 can activate vinculin and interacts with the actin cytoskeleton of host cells. 11 Both OmpA and OmpB have the ability to elicit efficient protection against contamination.12,13 Other Sca proteins have not been demonstrated to be protective antigens of infection in a murine model.14 TolC, a bacterial membrane-associated protein, is recognized as a major SEP through the proteomic analysis of biotinylated cell surface proteins of infection. Results Immunoblotting Purified rYbgF (44?kDa) and rTolC (63?kDa) were recognized by immunoblotting of sera collected from cell lysates were separated by 12% SDS-PAGE and stained with G-250 Coomassie brilliant blue (A). Immunoblotting analysis of rYbgF and rTolC. Lane M, protein molecular mass markers; lane 1, rYbgF; lane 2, rTolC (B). Microscopy detection of YbgF and TolC The presence of YbgF or TolC was determined by indirect immunofluorescence assays (IFA) and transmission Asaraldehyde (Asaronaldehyde) electron microscopy (TEM). Distinct fluorescent spots were observed in rickettsial cells stained with sera collected from mice immunized with rYbgF (Fig. 2A) or rTolC (Fig. 2B). Fluorescence was not observed in rickettsial cells stained with sera from mock-immunized mice (Fig. 2C). From our TEM results, the outer membrane (OM) and inner membrane (IM) of cells were covered with colloidal gold particles when sera from mice immunized with rYbgF (Fig. 2D) or rTolC (Fig. 2E) were used. Open in a separate window Physique 2. Microscopy analysis of YbgF and TolC in cells. were cultured in Vero cells and incubated with antibodies against rYbgF (A) or rTolC Asaraldehyde (Asaronaldehyde) (B), or with na?ve (C) serum. Secondary antibodies conjugated to fluorescein isothiocyanate (FITC) were then applied. The p18 white arrows indicate YbgF or TolC in cells. TEM analysis of in Vero cells involved staining with antibodies against rYbgF (D), rTolC (E), or with na?ve (F) serum. A goat anti-mouse IgG labeled with colloidal gold particles was then added to samples. The black arrows indicate the locations of YbgF or TolC in the inner membrane (IM) and outer membrane (OM) of cells. Immune protection against challenge Using quantitative polymerase chain reaction (qPCR) assays, the loads of rickettsia in the spleens (Fig. 3A), livers (Fig. 3B), or lungs (Fig. 3C) of mice immunized with rYbgF or whole cell antigen (WCA) were significantly lower than those in mock-immunized mice. The rickettsial loads in the spleens or lungs of mice immunized with rTolC were not significantly different from those in PBS-immunized mice. Open in a separate window Physique 3. Immune protection against contamination. C3H/HeN mice were immunized 3?occasions with rYbgF, rTolC, WCA, or PBS. At day 14 after the last immunization, mice were challenged with test or Wilcoxon Two-Sample Test based on their normality and equality of variances and are indicated as follows: *, 0.05; ***, 0.001; ns, no significance. * 0.05; ** 0.01; ns, no significance, respectively. Serious pathological damages were observed in rTolC- and PBS-immunized mice (Fig. 4). In liver tissues, we observed hydropic degeneration, nuclear pyknosis, inflammatory infiltrates consisting of mononuclear cells and polymorphonuclear leukocytes that were focused on the portal area of the liver. In spleen tissues, large numbers of macrophages were observed. For lung lesions, interstitial pneumonia was characterized by numerous inflammatory infiltrates of mononuclear cells, polymorphonuclear leukocytes, alveolar interstitial thickening, and alveolar.