Factors influencing in vitro infectivity and growth of Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni (Acari, Ixodidae)

Abstract

Rickettsia peacockii, a spotted fever group rickettsia, is a transovarially transmitted endosymbiont of Rocky Mountain wood ticks, Dermacentor andersoni. This rickettsia, formerly known as the East Side Agent and restricted to female ticks, was detected in a chronically infected embryonic cell line, DAE100, from D. andersoni. We examined infectivity, ability to induce cytopathic effect (CPE) and host cell specificity of R. peacockii using cultured arthropod and mammalian cells. Aposymbiotic DAE100 cells were obtained using oxytetracycline or incubation at 37 degrees C. Uninfected DAE100 sublines grew faster than the parent line, indicating R. peacockii regulation of host cell growth. Nevertheless, DAE100 cellular defenses exerted partial control over R. peacockii growth. Rickettsiae existed free in the cytosol of DAE100 cells or within autophagolysosomes. Exocytosed rickettsiae accumulated in the medium and were occasionally contained within host membranes. R. peacockii multiplied in other cell lines from the hard ticks D. andersoni, Dermacentor albipictus, Ixodes scapularis, and Ixodes ricinus; the soft tick Carios capensis; and the lepidopteran Trichoplusia ni. Lines from the tick Amblyomma americanum, the mosquito Aedes albopictus, and two mammalian cell lines were non-permissive to R. peacockii. High cell densities facilitated rickettsial spread within permissive cell cultures, and an inoculum of one infected to nine uninfected cells resulted in the greatest yield of infected tick cells. Cell-free R. peacockii also were infectious for tick cells and centrifugation onto cell layers enhanced infectivity approximately 100-fold. The ability of R. peacockii to cause mild CPE suggests that its pathogenicity is not completely muted. An analysis of R. peacockii-cell interactions in comparison to pathogenic rickettsiae will provide insights into host cell colonization mechanisms.

Fig. 1

Comparison of growth (transfer frequency) of uninfected D. andersoni, DAE100 cultures with cultures of DAE100 cells persistently infected with R. peacockii. The slower growing persistently infected line is shown with the plot having open circles. During the 22nd passage, selected cultures were cured of the endosymbiont by incubation at 37 °C or by the addition of oxytetracycline to the medium. The faster growing aposymbiotic oxytetracycline-treated subline is depicted in the plot having closed circles.

Fig. 2

Transmission electron photomicrograph (TEM) of a D. andersoni DAE100 cell infected with R. peacockii. (A) Note cluster of rickettsiae in the cytoplasm (small arrowheads) and degenerating rickettsiae within putative lysosomes (large arrowheads). The cell contains numerous vacuoles filled with residual bodies. Some of the vacuoles in the cell periphery appear to be releasing their contents (large arrows). Bar=2μm. (B) TEM of R. peacockii in direct contact with host cell cytoplasm (arrows) and within a putative lysosome (arrowhead). The area shown here is from the rectangle outlined in (A). Bar=0.2μm.

Fig. 3

Rickettsiae and cellular debris released by DAE100 cells persistently infected with R. peacockii. (A) Phase contrast photomicrograph of cells in culture. Note abundance of extracellular particles in the spaces between the cells. Bar=50 μm. (B) TEM micrograph of extracellular rickettsiae and cellular debris released from persistently infected DAE100 cells. Arrowheads point to rickettsiae that are free of host provided membranes. Arrow points to a rickettsia within an extracellular multivesicular body. Bar=0.2 μm.

Fig. 4

Aposymbiotic DAE100A line cleared of R. peacockii by addition of oxytetracycline to the medium. (A) Phase contrast photomicrograph of uninfected DAE100 cells. Note absence of extracellular debris and R. peacockii. (B) TEM of a cell from the subline DAE100A. Note numerous vacuoles with residual bodies and overall similarity to cell shown in Fig. 2A. Bar = 3 μm.

Fig. 5

TEM of R. peacockii in the I. scapularis embryonic cell line IDE2. (A) Photomicrograph of cell heavily infected with R. peacockii. Cell was infected using cell-free rickettsiae released from DAE100 cells. Arrowheads point to rickettsiae free within the cytoplasm and arrows to rickettsiae within putative autophagolysomes. Bar = 2 μm. (B) TEM of R. peacockii in direct contact with host cell cytoplasm (arrowheads) and within a putative lysosome (arrows). The area shown here is from the rectangle outlined in (A). Bar = 0.2 μm.

Fig. 6

Infectivity of cell-free R. peacockii for I. scapularis embryonic cell line ISE6. Rickettsiae were released from infected cells, separated from host cell debris, and 10-fold serial dilutions were made of the resultant rickettsial suspension. ISE6 cells in multiwell plates were inoculated with the suspensions. In one set of plates, the rickettsial suspension was centrifuged onto the cell layer (open squares) and with the other sets the rickettsiae were allowed to settle onto the cell layer by gravity (solid circles). Wells were harvested after 15 (solid lines) or 27 (dotted lines) days of incubation, prepared for Giemsa staining, and the proportion of cell infected with rickettsiae was determined by light microscopy. Values shown are means ± SD.