Figure 1.
Detection of Rickettsia in booklice.
(A) Rickettsia on formalin-fixed paraffin-embedded booklice sections were labeled with mouse polyclonal antibody against R. felis followed by FITC-conjugated goat anti-mouse IgG. Whole booklice tissues were counterstained with Evan's blue and nuclei were stained with DAPI as shown in red and blue, respectively. (B) Negative control staining using non-infected mouse serum. (C)–(F) Variation of mycetomes located in booklice abdomen. High magnification view of mycetomes shows densely packed rickettsiae. Bar = 100 µm.
Figure 2.
Electron micrographs of Rickettsia in booklice.
(A) Rickettsiae free in the cytosol of gut epithelial cells. MV represents microvilli. (B) Higher magnification of Fig. 2A shows typical rickettsial ultrastructure: rickettsial cell wall including trilaminar cell wall membrane associated with the external surface microcapsule layer and an internal trilaminar cytoplasmic membrane (arrowheads), surrounded by an outermost “halo” zone (h). Solid arrow indicates a small vacuole inside rickettsial cytoplasm. (C) Rickettsiae (R) in ovary. Inset: higher magnification view of the boxed rickettsiae. (D) Ultrathin section of a mycetome-like, several cells of which contain large vacuoles tightly packed with rickettsiae of irregular shape with dense cytoplasm. Bar = 500 nm.
Figure 3.
Propagation of Rickettsia isolated from booklice in ISE6 cells.
(A) ISE6 cells infected with Rickettsia isolated from booklice were Cytospin centrifuged and rickettsiae were visualized by Diff-Quik staining. (B) The infected cells on coverslips were fixed and permeabilized prior to stain with mouse polyclonal antibody against R. felis and FITC-conjugated goat anti-mouse IgG. Host cell actin and nuclei were stained with with Rhodamine-Phalloidin and DAPI as shown in red and blue, respectively. Bar = 5 µm.
Figure 4.
Electron micrographs of Rickettsia isolated from booklice in ISE6 cells.
(A) Typical infected cells containing rickettsiae in cytosol. (B) Typical ultrastructure of Rickettsia. Inset: higher magnification view of rickettsial cell wall including trilaminar cell wall associated with the external surface microcapsule layer (solid arrows) and an internal trilaminar cytoplasmic membrane (arrowheads). m represents mitochondria. (C) Rickettsiae surrounded by a halo zone (h). (D) Rickettsiae being destroyed in a phagolysosome. Long arrows indicate phagolysosomal membrane. Bar = 500 nm.
Table 1.
Identity of sequenced R. felis LSU-Lb isolate genes compared to R. felis URRWXcal2 Accession numbers CP000053 (genome) and CP000054 (plasmid pRF).
Figure 5.
Rickettsial OmpB antigen detected in the L. bostrychophila isolate.
Rickettsial proteins were extracted from partially purified LSU or LSU-Lb R. felis isolates. Rickettsial antigen was detected by Western immunoblotting using an anti-rickettsial OmpB monoclonal antibody. The reactive bands represent the R. felis OmpB protein. Protein extracted from non-infected ISE6 cells was served as a negative control.
Figure 6.
Rickettsial load in individual L. bostrychophila and C. felis (LSU colony).
Twelve individual booklice and cat fleas were collected at two time points (June 2010 and October 2010). Rickettsial loads were determined by qPCR. Scatter plot shows significantly greater rickettsial loads in L. bostrychophila, compared to C. felis. Bars represent medians of the data set. The medians with the same symbol are not significantly different (p≤0.05).
Table 2.
Primers used for PCR amplification and sequencing of R. felis (LSU-Lb) and primer/probe combination used for quantitative real-time PCR for Rickettsia.