Rickettsia felis: Molecular characterization of a new member of the spotted fever group

In this report, placement of Rickettsia felis in the spotted fever group (SFG) rather than the typhus group (TG) of Rickettsia is proposed. The organism, which was first observed in cat fleas (Ctenocephalides felis) by electron microscopy, has not yet been reported to have been cultivated reproducibly, thereby limiting the standard rickettsial typing by serological means. To overcome this challenge, several genes were selected as targets to be utilized for the classification of R. felis. DNA from cat fleas naturally infected with R. felis was amplified by PCR utilizing primer sets specific for the 190 kDa surface antigen (rOmpA) and 17 kDa antigen genes. The entire 5513 bp rompA gene was sequenced, characterized and found to have several unique features when compared to the rompA genes of other Rickettsia. Phylogenetic analysis of the partial sequence of the 17 kDa antigen gene indicated that R. felis is less divergent from the SFG rickettsiae than from the TG rickettsiae. The data corroborate results from previous reports that analysed the citrate synthase, 16S rRNA, rompB (135 kDa surface antigen), metK, ftsY, poIA and dnaE genes that placed R. felis as a member of the SFG. The organism is passed transstadially and transovarially, and infection in the cat flea has been observed in the midgut, tracheal matrix, muscle, hypodermis, ovaries and testes

Immunization with Ehrlichia P28 Outer Membrane Proteins Confers Protection in a Mouse Model of Ehrlichiosis▿

The obligately intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the etiologic agent of human monocytotropic ehrlichiosis (HME). HME is an emerging and often life-threatening, tick-transmitted infectious disease in the United States. Effective primary immune responses against Ehrlichia infection involve generation of Ehrlichia-specific gamma interferon (IFN-γ)-producing CD4+ T cells and cytotoxic CD8+ T cells, activation of macrophages by IFN-γ, and production of Ehrlichia-specific antibodies of the Th1 isotype. Currently, there are no vaccines available against HME. We evaluated the ability of 28-kDa outer membrane proteins (P28-OMP-1) of the closely related Ehrlichia muris to stimulate long-term protective memory T and B cell responses and confer protection in mice. The spleens of mice vaccinated with E. muris P28-9, P28-12, P28-19, or a mixture of these three P28 proteins (P28s) using a DNA prime-protein boost regimen and challenged with E. muris had significantly lower bacterial loads than the spleens of mock-vaccinated mice. Mice immunized with P28-9, P28-12, P28-19, or the mixture induced Ehrlichia-specific CD4+ Th1 cells. Interestingly, mice immunized with P28-14, orthologs of which in E. chaffeensis and E. canis are primarily expressed in tick cells, failed to lower the ehrlichial burden in the spleen. Immunization with the recombinant P28-19 protein alone also significantly decreased the bacterial load in the spleen and liver compared to those of the controls. Our study reports, for the first time, the protective roles of the Ehrlichia P28-9 and P28-12 proteins in addition to confirming previous reports of the protective ability of P28-19. Partial protection induced by immunization with P28-9, P28-12, and P28-19 against Ehrlichia was associated with the generation of Ehrlichia-specific cell-mediated and humoral immune responses