Culture Isolate of Rickettsia felis from a Tick

Abstract

Although the cat flea, Ctenocephalides felis, has been identified as the primary vector of Rickettsia felis, additional flea, tick, mite, and louse species have also been associated with this bacterium by molecular means; however, the role of these arthropods in the transmission of R. felis has not been clarified. Here, we succeeded in culture isolation of R. felis from a host-seeking castor bean tick, Ixodes ricinus, the most common tick in Slovakia. The bacterial isolation was performed on XTC-2 cells at 28 °C using the shell-vial technique. An evaluation of the growth properties was performed for both the XTC-2 and Vero cell lines. We observed R. felis in the infected host cells microscopically by Gimenez staining and immunofluorescence assay. The R. felis isolate was purified by gradient ultracentrifugation and visualized by electron microscopy. Fragments of the genes gltA, ompA, ompB, htrA, rpoB, sca4, rffE, and rrs were amplified and compared with the corresponding sequences of the type strain URRWXCal2 and other R. felis culture -isolated strains. We did not detect any nucleotide polymorphisms; however, plasmid pRFδ, characteristic of the standard strain, was absent in our isolate. Herein, we describe the first successful isolation and characterization of a tick-derived R. felis strain "Danube", obtained from an I. ricinus nymph.

Keywords: Ixodes ricinus; Rickettsia felis; cell culture; shell-vial technique; vector-borne bacteria.

Figure 1

Tick sampling location in Slovakia. On the left: a political map of Europe indicating the location of Slovakia (in light green). Ticks were collected in the Bratislava district (marked with a triangle) by flagging method (maps from www.vidiani.com, accessed on 27 January 2022, licensed under CC-BY 3.0, desaturated from the original). On the right: I. ricinus nymph collected from the local vegetation (SMZ1500 Stereomicroscope).

Figure 2

Identification of the isolate Danube by PCR. Agarose gel electrophoresis of PCR products of the genes gltA (a) and rffE (b), amplified with primers CS-78 and CS-323, MQ32 and MQ33, respectively. M: molecular marker; 1: R. helvetica C9P9; 2: R. helvetica IR16; 3: R. felis Danube; 4: negative control. Note: primer set MQ32, MQ33 does not amplify R. helvetica.

Figure 3

Growth of R. felis Danube in XTC-2 and Vero cells. Rickettsiae were propagated in XTC-2 and Vero cells at 28 °C and 32 °C, respectively. We observed an apparent cytopathic effect (plaque formation, black arrows) in the infected host cells at 6 dpi in XTC-2 and 14 dpi in Vero cells (Zeiss Axiovert 40 CFL trinocular inverted phase-contrast microscope; scale bar: 100 µm). Rickettsia felis stained magenta in contrast to host cells in blue by Gimenez technique (Leica DM 4500B microscope; scale bar 10 µm).

Figure 4

Graphical representation of the rickettsial rate of intracellular growth in XTC-2 cells. Amphibian host cells incubated in 12-well plates and approaching confluence were infected with MOI 10 rickettsiae. Infected cells from culture wells were harvested in 24 h intervals. To demonstrate the growth kinetics of R. felis Danube, the approximate mean numbers of bacterial genome equivalents (GE) were calculated based on the amplification of the rpsL gene by qPCR. Standard errors of the mean were calculated from two biological replicates. For statistical analysis, one-way ANOVA was performed, followed by Dunnett’s multiple comparison test (** p < 0.01, **** p < 0.0001) (a). To confirm viability of rickettsiae, the estimated transcript numbers of the same gene were evaluated per well, as described in Materials and Methods. The red line represents nonlinear regression, Beta growth then decay (b).

Figure 5

Agarose gel electrophoresis of PCR amplicons of the ompA gene and plasmids from R. felis Danube. The partial sequence of the gene ompA was amplified using R. felis-specific primers Rf190.1790fw and Rf190.2857rev (a). The pRF plasmid was detected using the primer pairs pRFa-pRFb (expected size 159 bp) and pRFc-pRFd (expected size 1342 bp), but not pRFδ (primers pRFa and pRFd; expected size 1168 bp) (b). M: molecular marker; 1: uninfected XTC-2 cells; 2: infected XTC-2 cells; 3: purified R. felis Danube; 4: negative control; 5: infected XTC-2 cells; 6: purified R. felis Danube; 7: extracted plasmid DNA from purified R. felis Danube; 8: negative control.

Figure 6

Detection of R. felis Danube by immunofluorescence assay. Infected XTC-2 and Vero cells were fixed with 4% paraformaldehyde in PHEM buffer, and rickettsiae were labeled with rabbit polyclonal antibody against R. conorii, followed by rhodamine-conjugated goat anti-rabbit antibody (red signal). Actin filaments of host cells were labeled with conjugated phalloidin (green signal), and cell nuclei were stained with DAPI (blue signal). Uninfected cells treated with both primary and secondary antibodies were used as negative controls (scale bar 10 µm).

Figure 7

Visualization of purified R. felis Danube by electron microscopy. Negative staining of rickettsial binary fission captured by transmission electron microscopy (a). Scanning electron microscopy of purified and glutaraldehyde-fixed R. felis sedimented onto circular coverslips and silicon wafer (b).