Shell-vial culture and real-time PCR applied to Rickettsia typhi and Rickettsia felis detection.

Murine typhus is a zoonosis transmitted by fleas, whose etiological agent is Rickettsia typhi. Rickettsia felis infection can produces similar symptoms. Both are intracellular microorganisms. Therefore, their diagnosis is difficult and their infections can be misdiagnosed. Early diagnosis prevents severity and inappropriate treatment regimens. Serology can't be applied during the early stages of infection because it requires seroconversion. Shell-vial (SV) culture assay is a powerful tool to detect Rickettsia. The aim of the study was to optimize SV using a real-time PCR as monitoring method. Moreover, the study analyzes which antibiotics are useful to isolate these microorganisms from fleas avoiding contamination by other bacteria. For the first purpose, SVs were inoculated with each microorganism. They were incubated at different temperatures and monitored by real-time PCR and classical methods (Gimenez staining and indirect immunofluorescence assay). R. typhi grew at all temperatures. R. felis grew at 28 and 32 °C. Real-time PCR was more sensitive than classical methods and it detected microorganisms much earlier. Besides, the assay sensitivity was improved by increasing the number of SV. For the second purpose, microorganisms and fleas were incubated and monitored in different concentrations of antibiotics. Gentamicin, sufamethoxazole, trimethoprim were useful for R. typhi isolation. Gentamicin, streptomycin, penicillin, and amphotericin B were useful for R. felis isolation. Finally, the optimized conditions were used to isolate R. felis from fleas collected at a veterinary clinic. R. felis was isolated at 28 and 32 °C. However, successful establishment of cultures were not possible probably due to sub-optimal conditions of samples.

Growth of Rickettsia felis in Drosophila melanogaster S2 cells.

Rickettsia felis is an obligate, intracellular, Gram-negative bacterium and a member of the transitional group rickettsiae. This bacterium has been shown to grow in vitro in amphibian, tick, and mosquito cell lines. Here, we present data to show the growth of R. felis strain LSU in Drosophila melanogaster S2 cells, an embryonic, hemocytic cell line with phagocytic properties. R. felis LSU was isolated from Ixodes scapularis E6 (ISE6) cells and used to infect S2 cells, grown at 25°C. By 19 days postinfection, the S2 cells were 100% infected with R. felis as determined by Acridine Orange and Diff-Quik staining. A species-specific R. felis qPCR assay was used to demonstrate that the kinetics associated with the S2 cell culture infection involved a lag/adaptation phase, followed by continued growth to 20 days postinfection. Moreover, R. felis organisms were observed in the S2 cells using transmission electron microscopy and a polyclonal antibody against spotted fever rickettsiae. The ability to use D. melanogaster S2 cells for growing rickettsial agents is a useful tool owing to the ease of manipulation of the S2 cultures and the wide-ranging possibility of Drosophila resources available for future studies.

Tryptose phosphate broth improves Rickettsia felis replication in mammalian cells.

In cell culture, Rickettsia felis grows only at low temperatures (< 31 °C). Therefore, its ability to enter, survive and grow in cell lines has primarily been tested in cells derived from amphibians and arthropods, which naturally grow at low temperatures, and only infrequently in mammalian cells. We subcultured R. felis in mammalian cells for more than 10 passages using media supplemented with tryptose phosphate broth (TPB) and found that TPB is critical for optimal growth of R. felis in mammalian cells.

Acquisition of Rickettsia felis by cat fleas during feeding.

Evidence for horizontal routes of transmission for Rickettsia felis has come from detection of R. felis infection in vertebrates and multiple blood-feeding arthropods; however, infection of cat fleas, Ctenocephalides felis, during blood feeding has not been demonstrated. In this study, the ability of cat fleas to acquire R. felis through an infectious blood meal with subsequent vertical transmission was examined. Utilizing an artificial feeding system, Rickettsia-naive fleas were exposed to R. felis-infected blood meals and monitored for subsequent infection at weekly intervals for 4 weeks. At 7 days postexposure (dpe) ~52% of fleas successfully acquired rickettsiae and R. felis DNA; rickettsial transcript and DNA was detected in cat flea feces. Quantitative real-time polymerase chain reaction determined that both the R. felis infection load and R. felis infection density was significantly greater in fleas assessed at later time points. Although a persistent R. felis infection was detected in adult fleas, R. felis infection was not observed in F(1) progeny. This study demonstrates that cat fleas are able to acquire R. felis infection from an infectious blood meal and will serve as a model to examine R. felis transmission between arthropod and vertebrate hosts.

Characterization and growth of polymorphic Rickettsia felis in a tick cell line.

Morphological differentiation in some arthropod-borne bacteria is correlated with increased bacterial virulence, transmission potential, and/or as a response to environmental stress. In the current study, we utilized an in vitro model to examine Rickettsia felis morphology and growth under various culture conditions and bacterial densities to identify potential factors that contribute to polymorphism in rickettsiae. We utilized microscopy (electron microscopy and immunofluorescence), genomic (PCR amplification and DNA sequencing of rickettsial genes), and proteomic (Western blotting and liquid chromatography-tandem mass spectrometry) techniques to identify and characterize morphologically distinct, long-form R. felis. Without exchange of host cell growth medium, polymorphic R. felis was detected at 12 days postinoculation when rickettsiae were seeded at a multiplicity of infection (MOI) of 5 and 50. Compared to short-form R. felis organisms, no change in membrane ultrastructure in long-form polymorphic rickettsiae was observed, and rickettsiae were up to six times the length of typical short-form rickettsiae. In vitro assays demonstrated that short-form R. felis entered into and replicated in host cells faster than long-form R. felis. However, when both short- and long-form R. felis organisms were maintained in cell-free medium for 12 days, the infectivity of short-form R. felis was decreased compared to long-form R. felis organisms, which were capable of entering host cells, suggesting that long-form R. felis is more stable outside the host cell. The relationship between rickettsial polymorphism and rickettsial survivorship should be examined further as the yet undetermined route of horizontal transmission of R. felis may utilize metabolically and morphologically distinct forms for successful transmission.

Rickettsia felis from cat fleas: isolation and culture in a tick-derived cell line.

Rickettsia felis, the etiologic agent of spotted fever, is maintained in cat fleas by vertical transmission and resembles other tick-borne spotted fever group rickettsiae. In the present study, we utilized an Ixodes scapularis-derived tick cell line, ISE6, to achieve isolation and propagation of R. felis. A cytopathic effect of increased vacuolization was commonly observed in R. felis-infected cells, while lysis of host cells was not evident despite large numbers of rickettsiae. Electron microscopy identified rickettsia-like organisms in ISE6 cells, and sequence analyses of portions of the citrate synthase (gltA), 16S rRNA, Rickettsia genus-specific 17-kDa antigen, and spotted fever group-specific outer membrane protein A (ompA) genes and, notably, R. felis conjugative plasmids indicate that this cultivatable strain (LSU) was R. felis. Establishment of R. felis (LSU) in a tick-derived cell line provides an alternative and promising system for the expansion of studies investigating the interactions between R. felis and arthropod hosts.

Isolation of Rickettsia felis in the mosquito cell line C6/36.

We report the isolation and establishment of Rickettsia felis in the C6/36 cell line. Rickettsial growth was intense, always with 90 to 100% of cells being infected after few weeks. The rickettsial isolate was confirmed by testing infected cells by PCR and sequencing fragments of three major Rickettsia genes (gltA, ompB, and the 17-kDa protein gene).

Rickettsia felis, from culture to genome sequencing.

Rickettsia felis has been recently cultured in XTC2 cells. This allows production of enough bacteria to create a genomic bank and to sequence it. The chromosome of R. felis is longer than that of previously sequenced rickettsiae and it possess 2 plasmids. Microscopically, this bacterium exhibits two forms of pili: one resembles a conjugative pilus and another forms hair-like projections that may play a role in pathogenicity. R. felis also exhibits several copies of ankyrin-repeat genes and tetratricopeptide encoding gene that are specifically linked to pathogenic host-associated bacteria. It also contains toxin-antitoxin system encoding genes that are extremely rare in intracellular bacteria and may be linked to plasmid maintenance.

Emended description of Rickettsia felis (Bouyer et al. 2001), a temperature-dependent cultured bacterium.

On the basis of phenotypic data obtained on the strain Marseille-URRWFXCal2(T), isolated from the cat flea Ctenocephalides felis, the description of Rickettsia felis (Bouyer et al., 2001) is emended and Marseille-URRWFXCal2(T) is proposed as the type strain of the species. On the basis of polyphasic characterization, especially the inability to grow at temperatures higher than 32 degrees C on Vero cells that allow growth of other Rickettsia to at least 35 degrees C, it is confirmed that this agent, although different from other recognized rickettsial species, is genotypically indistinguishable from bacteria previously detected within cat fleas and provisionally named ELB. Comparison of the phenotypic characteristics previously described for R. felis and those observed for the isolate in this study indicated some differences, although concurrent analysis of the two was not possible as no extant isolates of the first isolate of R. felis exist.