Rickettsia monacensis sp. nov., a spotted fever group Rickettsia, from ticks (Ixodes ricinus) collected in a European city park

Abstract

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich(T)) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.

FIG. 1.

Transmission electron photomicrographs of R. monacensis IrR/Munich^T within cultured tick and mammalian cells. (A) Infected I. scapularis (ISE6) cell with rickettsiae in the cytoplasm (arrow), as well as being digested in vacuoles (arrowhead). Bar = 2 μm. N, host cell nucleus. (B) Vero cell filled with cytoplasmic rickettsiae (arrows). N, host cell nucleus. Bar = 2 μm. (C) R. monacensis within the nucleus (N, arrowheads) and cytoplasm (arrow) of an ISE6 cell. Bar = 0.5 μm. (D) Rickettsia (arrow) within a pseudopodial extension of an ISE6 cell. Bar = 0.5 μm. (E) High magnification of R. monacensis showing the typical rickettsial morphology of the organism. Bar = 0.2 μm. Shown are the inner periplasmic membrane (white arrowhead), the electron-lucent periplasmic space, and the cell wall (arrow). The insert shows an enlargement of the outer membrane. Bars delineate the trilaminar cell wall; small arrowheads indicate the faint microcapsular layer.

FIG. 2.

RFLP analyses of R. monacensis IrR/Munich^T, I. ricinus tick no. 5 salivary glands (sg.) and gut tissues, and R. helvetica C9P9 PCR products. (A) 16S rRNA gene PCR products from the fD1-Rc16S.452n primer set uncut or digested with restriction endonuclease RsaI or BstXI. (B) Citrate synthase gene (gltA) sequences corresponding to the RpCS.877p-RpCS.1258n primer set, uncut or digested with restriction endonuclease AluI or Sau3AI. The molecular sizes indicated on the left correspond to φX174 replicative-form DNA digested with HaeIII (Life Technologies).

FIG. 3.

Neighbor-joining phylogram based on partial rompA sequences showing the phylogenetic placement of R. monacensis sp. nov. IrR/Munich^T among the validated SFG rickettsial species. Bootstrap support (>50%) for phylogenetic groupings and the scale of percent difference between taxa are indicated. R. australis PHS was used as the outgroup (see Materials and Methods).

FIG. 4.

Western blot analyses of R. monacensis IrR/Munich^T, R. helvetica C9P9, Rickettsia strain MOAa, and R. rickettsii Hlp#2 sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated antigens reacted with pooled polyclonal sera from two hamsters inoculated with IrR/Munich^T (A) and MAb 13-5 to rOmpA (B). Arrows indicate the approximate positions of rOmpA, as determined by MAb 13-5. The arrowhead indicates the approximate position of rOmpB.

FIG. 5.

Immunofluorescence photomicrographs of R. monacensis IrR/Munich^T-infected eukaryotic cells as visualized by simultaneous excitation of the appropriate wavelengths for the Texas red and FITC fluorochromes. Rickettsiae were labeled with a hamster anti-IrR/Munich^T polyclonal serum reacted with a secondary anti-hamster IgG antibody conjugated to FITC, and host cell actin was labeled with Texas red phalloidin. The arrows indicate actin tail structures associated with rickettsiae. The arrowheads indicate clustering of rickettsiae at the poles of cells. Panels: A, tick (D. andersoni) DAE100 cells; B, mouse (Mus musculus) L-929 cells. Bars = 10 μm.