Restriction of the growth of typhus group rickettsiae in tick cells

Abstract

The vectors of typhus group rickettsiae (TGR) are lice andfleas that belong to Insecta. Those of spotted fever group rickettsiae (SFGR), however, are ticks classified as Arachnida. Until recently, the host dependency of the growth of TGR and SFGR in cells derived from arthropods had not been well documented. As for insect cells, it was reported by Rovery et al. that the transcription of spoT gene paralogues was suppressed during the infection of Rickettsia conorii in Aedes albopictus (C6 ⁄ 36) cells at 10 C for 38 days. Shifting the temperature to 37 C was followedby a rapidupregulationof spoT1 gene expression [1]. Uchiyama reported that some SFGR (Rickettsia japonica and Rickettsia montanensis) do not grow in the AeAl2 cell line from A. albopictus, even though the rickettsiae achieved adherence and invasion [2]. On the contrary, Sakamoto et al. demonstrated that some non-pathogenic SFGR, R. montanensis and Rickettsia peacockii successfully grew in two mosquito cell lines (A. albopictus cell line Aa23 and Anopheles gambie cell line Sua5B) [3]. Concerning tick cell lines, several reports regarding the growth of SFGR in the cells have been published [4,5]. In this study, the growth of TGR in tick cells was examined. The DALBE3 cell line from Dermacentor albipictus (a generous gift of U.G. Munderloh) and the ISE6 cell line from Ixodes scapuralis (generously donated by I. Takashima under agreement of U.G. Munderloh) were used as tick cells. As mammalian cells, Vero and ECV304 cells were used. These cells were inoculated with R. japonica, strain YH and R. conorii, strain Malish 7 as SFGR and Rickettsia prowazekii, strain Breinl and Rickettsia typhi, strain Wilmington as TGR at a multiplicity of infection (MOI) of 0.1 PFU ⁄ cell. The infected cells were cultured at 34 C in a medium described previously [4,5]. The medium was changed every 3 days and the yields of rickettsiae were plaqueassayed on Vero cell monolayers. As shown in Fig. 1(a), SFGR grew well in DALBE3 and ISE6 cells as well as in Vero and ECV304 cells (data from ISE6, Vero and ECV304 cells are not shown). However, growth of TGR was restricted in these tick cells contrary to their growth in Vero and ECV304 mammalian cells (data from ISE6, Vero and ECV304 cells are not shown). Rickettsial infectivity decreased to an undetectable level up to 2 weeks after infection. Because the growth of TGR in these cells was restricted, the adherence step was analysed to determine if it was completed. We inoculated the tick cells with rickettsiae at an MOI of 1.0 PFU ⁄ cell and incubated these cells for 5, 10, 60 or 180 min at 25 C. As shown in Fig. 1b, the adherence of these cells by TGR was successfully completed as were those in other combinations of rickettsiae and host cells, although the extent of adherence differed among the combinations tested. We also performed Gimenez staining of the tick cells inoculated with either group of rickettsiae at an MOI of 50 PFU ⁄mL and found that the cells showed adherence and ⁄ or invasion by rickettsiae (data not shown). Transmission electron microscopy was performed to confirm these findings and examine morphological changes. DALBE3 cells were inoculated with TGR and SFGR at an MOI of 50 PFU ⁄ cell and incubated at 25 C for 10 min, 60 min, and 180 min, and then cultured at 34 C for 24 h and 72 h until fixed. Thin sections of the samples were observed under a Hitachi H-7650 transmission electron microscope. DALBE3 cells demonstrated adherence and invasion by these rickettsiae independent of their group (data not shown). Rickettsiae under the process of escaping from phagosomes to the cytoplasm after being engulfed by DALBE3 cells were also shown. Thus, it is suggested that restriction occurred after these steps. Further analyses of the restrictive steps Corresponding author and reprint requests: T. Uchiyama, Department of Virology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770–8503, Japan E-mail: uchiyama@basic.med.tokushima-u.ac.jp