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.     

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

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.     

Rickettsia monacensis sp. nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes ricinus) Collected in a European City Park

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.     

Propagation of arthropod-borne Rickettsia spp. in two mosquito cell lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Isolation of a rickettsial pathogen from a non-hematophagous arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod.     

Ultrastructure of a Japanese Rickettsial strain genetically identified as Rickettsia helvetica which was originally found in Europe

A rickettsial strain IO-1 has been isolated from a tick, Ixodes ovatus, in Japan and genetically identified as Rickettsia helvetica, a member of the spotted fever group rickettsiae. Ultrastructural observations were made on the microorganism. The ultrastructure of R. helvetica IO-1 appeared to be generally the same as that previously shown for other rickettsiae of the spotted fever and typhus groups. The rickettsiae were primarily found free in the cytoplasm of L929 cultured cells. Occasionally, the rickettsiae may also invade the host cell nucleus; however, the frequency of the nuclear localization was very low.     

Isolation of a spotted fever group Rickettsia, Rickettsia peacockii, in a Rocky Mountain wood tick, Dermacentor andersoni, cell line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

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.     

Phylogenetic analysis of spotted fever group Rickettsiae isolated from ticks in Japan

Eight spotted fever group (SFG) rickettsiae isolated from ticks in Japan were classified by phylogenetic analysis based on the nucleotide sequences of both the citrate synthase-encoding gene (gltA) and 190-kDa antigen-encoding gene (rOmpA). In the phylogenetic tree of gltA, strains DT-1 and FLA-1 isolated from the Dermacentor taiwanensis and Haemaphysalis frava ticks, respectively, were placed as Rickettsia japonica, and strains IO-1, IO-2, IO-25, IM-1 and IP-2 from genus Ixodes ticks were placed as Rickettsia helvetica. Strain AT-1 isolated from the Amblyomma testudinarium belonged to the cluster including Rickettsia akari, Rickettsia australis and Rickettsia felis. In the phylogenetic tree of the rOmpA, strains DT-1 and FLA-1 were placed as R. japonica, and strain AT-1 belonged to the cluster including Rickettsia cooleyi and the symbiont of Ixodes scapularis. The rOmpA fragments of 5 Ixodes isolates could not be amplified by PCR. The present study showed that strains DT-1 and FLA-1 were genotypically identical to R. japonica, and 5 Ixodes isolates were associated with the R. helvetica. Based on previous genotypic and antigenic data, and the phylogenetic analysis presented here, strain AT-1 should be considered as a new species among SFG rickettsiae.     

In vitro propagation of Candidatus Rickettsia andeanae isolated from Amblyomma maculatum

Candidatus Rickettsia andeanae was identified during an investigation of a febrile outbreak in northwestern Peru (2002). DNA sequencing from two ticks (Amblyomma maculatum, Ixodes boliviensis) collected during the investigation revealed a novel Rickettsia agent with similarity to the spotted fever group rickettsiae. Since then, Candidatus R.?andeanae has been detected in A.?maculatum ticks collected in the southeastern and southcentral United States, Argentina, and Peru. To date, Candidatus R.?andeanae has not been successfully cultivated in the laboratory. We present evidence for the continuous cultivation in three cell lines of Candidatus R.?andeanae isolated from an A.?maculatum tick (Portsmouth, Virginia).     

Restriction of the growth of a nonpathogenic spotted fever group rickettsia

The growth kinetics of pathogenic and nonpathogenic rickettsiae were compared to elucidate the mechanism responsible for the pathogenicity of rickettsiae. Vero and HeLa cells derived from mammals were inoculated with a nonpathogenic species of spotted fever group rickettsia, Rickettsia montanensis, before being infected with the pathogenic species Rickettsia japonica. The mammalian cells became persistently infected with R. montanensis and produced low levels of rickettsiae. On the other hand, superinfection of the R. montanensis-infected cells with R. japonica resulted in increased yields of R. montanensis accompanied by R. japonica growth. Both rickettsiae also grew well in the R. japonica-infected cells subjected to superinfection with R. montanensis. Western blotting with an antibody to the autophagy-related protein LC3B found that autophagy was induced in the cells infected with R. montanensis alone. On the contrary, autophagy was restricted in the cells that were co-infected with R. japonica. Electron microscopy of the cells infected with R. montanensis alone demonstrated rickettsia particles being digested in intracytoplasmic vacuoles. Conversely, many freely growing rickettsiae were detected in the co-infected cells.     

Electron microscopic studies on the in vitro proliferation of spotted fever group rickettsia isolated in Japan.

Rickettsia was isolated from a patient with Japanese spotted fever, and its proliferation in cultured green monkey kidney cells was observed by electron microscopy. In the course of this study, we observed fusion of infected cells to uninfected cells which may be a way of spreading the rickettsiae from a cell to another. On the other hand, whirlpool-like, multilayer membranous structures, similar to the mesosomes of gram-negative bacteria, were sometimes seen in the rickettsial cells. The other profiles common to the other rickettsiae in spotted fever group were observed, such as the electron-lucent halo zone around the rickettsiae, and external fibrous materials on their surface, but intranuclear multiplication was rarely observed.     

Rickettsia japonica sp. nov., the etiological agent of spotted fever group rickettsiosis in Japan.

We propose the name Rickettsia japonica sp. nov. (with type strain YH [= ATCC VR-1363]) for a serologically specific species of spotted fever group rickettsiae that are pathogenic for humans (J. Infect. Dis. 159:1122-1126, 1989; J. Clin. Microbiol. 28:1177-1180, 1990). The biologic and genomic characteristics of the organism (G+C content, 31.2 +/- 0.7 mol%) are essentially the same as those of other pathogenic spotted fever group rickettsiae, although the R. japonica isolates cause a persistent infection in Vero cells for many subcultures.     

Molecular detection of Candidatus Rickettsia asembonensis in fleas collected from pets and domestic animals in Puducherry,India

Rickettsia are obligate intracellular pathogens transmitted by arthropod vectors. The re-emergence of several rickettsioses imposes severe global health burden. In addition to the well-established rickettsial pathogens, newer rickettsial species and their pathogenic potentials are being uncovered. There are many reports of spotted and typhus fever caused by rickettsiae in India. Hence, in this study we screened the ectoparasites of pet and domestic animals for the presence of rickettsia using polymerase chain reaction. Nine cat flea samples (Ctenocephalides felis felis), that tested positive for the presence of rickettsia were subjected to Multi Locus Sequence Typing. Nucleotide sequencing and Phylogenetic analysis of gltA, ompB and 16rrs genes revealed that the rickettsiae detected in cat fleas was Rickettsia asembonensis. Further studies are required to assess Rickettsia asembonensis pathogenic potential to human and its enzootic maintenance of in various hosts and vectors.     

Rickettsia parkeri invasion of diverse host cells involves an Arp2/3 complex, WAVE complex and Rho-family GTPase-dependent pathway

Rickettsiae are obligate intracellular pathogens that are transmitted to humans by arthropod vectors and cause diseases such as spotted fever and typhus. Although rickettsiae require the host cell actin cytoskeleton for invasion, the cytoskeletal proteins that mediate this process have not been completely described. To identify the host factors important during cell invasion by Rickettsia parkeri, a member of the spotted fever group (SFG), we performed an RNAi screen targeting 105 proteins in Drosophila melanogaster S2R+ cells. The screen identified 21 core proteins important for invasion, including the GTPases Rac1 and Rac2, the WAVE nucleation-promoting factor complex and the Arp2/3 complex. In mammalian cells, including endothelial cells, the natural targets of R. parkeri, the Arp2/3 complex was also crucial for invasion, while requirements for WAVE2 as well as Rho GTPases depended on the particular cell type. We propose that R. parkeri invades S2R+ arthropod cells through a primary pathway leading to actin nucleation, whereas invasion of mammalian endothelial cells occurs via redundant pathways that converge on the host Arp2/3 complex. Our results reveal a key role for the WAVE and Arp2/3 complexes, as well as a higher degree of variation than previously appreciated in actin nucleation pathways activated during Rickettsia invasion.     

Identification of the spotted fever group rickettsiae detected from Haemaphysalis longicornis in Korea

Seven Haemaphysalis ticks were found positive in PCR assay of gltA gene to detect the spotted fever group (SFG) rickettsiae DNA from 100 ticks. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks and compared to those of other Rickettsia strains. The nucleotide sequence from 4 ticks showed high homologies (99.7 to 100%) with that of R. japonica YH, and that from 1 tick (tick no. 48) was identical with that of R. rickettsii R, suggesting that SFG rickettsiae exists in Korea. This is the first documentation of SFG rickettsiae in Korea.     

Isolation of a Spotted Fever Group Rickettsia, Rickettsia peacockii, in a Rocky Mountain Wood Tick, Dermacentor andersoni, Cell Line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona

Twenty Rhipicephalus sanguineus ticks collected in eastern Arizona were tested by PCR assay to establish their infection rate with spotted fever group rickettsiae. With a nested PCR assay which detects a fragment of the Rickettsia genus-specific 17-kDa antigen gene (htrA), five ticks (25%) were found to contain rickettsial DNA. One rickettsial isolate was obtained from these ticks by inoculating a suspension of a triturated tick into monolayers of Vero E6 monkey kidney cells and XTC-2 clawed toad cells, and its cell culture and genotypic characteristics were determined. Fragments of the 16S rRNA, GltA, rOmpA, rOmpB, and Sca4 genes had 100%, 100%, 99%, 99%, and 99%, respectively, nucleotide similarity to Rickettsia massiliae strain Bar29, previously isolated from R. sanguineus in Catalonia, Spain (L. Beati et al., J. Clin. Microbiol. 34:2688-2694, 1996). The new isolate, AZT80, does not elicit cytotoxic effects in Vero cells and causes a persistent infection in XTC-2 cells. The AZT80 strain is susceptible to doxycycline but resistant to rifampin and erythromycin. Whether R. massiliae AZT80 is pathogenic or infectious for dogs and humans or can cause seroconversion to spotted fever group antigens in the United States is unknown.     

Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Spotted fever rickettsioses in southern and eastern Europe

Mediterranean spotted fever due to Rickettsia conorii conorii was thought, for many years, to be the only tick-borne rickettsial disease prevalent in southern and eastern Europe. However, in recent years, six more species or subspecies within the spotted fever group of the genus Rickettsia have been described as emerging pathogens in this part of the world. Tick-borne agents include Rickettsia conorii israelensis, Rickettsia conorii caspia, Rickettsia aeschlimannii, Rickettsia slovaca, Rickettsia sibirica mongolitimonae and Rickettsia massiliae. Many Rickettsia of unknown pathogenicity have also been detected from ticks and could represent potential emerging pathogens to be discovered in the future. Furthermore, a new spotted fever rickettsia, Rickettsia felis, was found to be associated with cat fleas and is an emerging human pathogen. Finally, the mite-transmitted Rickettsia akari, the agent of rickettsialpox, is also known to be prevalent in Europe. We present here an overview of these rickettsioses, focusing on emerging diseases.