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.     

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.     

Horizontal transmission of Rickettsia felis between cat fleas, Ctenocephalides felis

Rickettsia felis is a rickettsial pathogen primarily associated with the cat flea, Ctenocephalides felis. Although laboratory studies have confirmed that R. felis is maintained by transstadial and transovarial transmission in C. felis, distinct mechanisms of horizontal transmission of R. felis among cat fleas are undefined. Based on the inefficient vertical transmission of R. felis by cat fleas and the detection of R. felis in a variety of haematophagous arthropods, we hypothesize that R. felis is horizontally transmitted between cat fleas. Towards testing this hypothesis, flea transmission of R. felis via a bloodmeal was assessed weekly for 4 weeks. Rhodamine B was used to distinguish uninfected recipient and R. felis-infected donor fleas in a rickettsial horizontal transmission bioassay, and quantitative real-time PCR assay was used to measure transmission frequency; immunofluorescence assay also confirmed transmission. Female fleas acquired R. felis infection more readily than male fleas after feeding on a R. felis-infected bloodmeal for 24 h (69.3% and 43.3%, respectively) and both Rickettsia-uninfected recipient male and female fleas became infected with R. felis after cofeeding with R. felis-infected donor fleas (3.3-40.0%). Distinct bioassays were developed to further determine that R. felis was transmitted from R. felis-infected to uninfected fleas during cofeeding and copulation. Vertical transmission of R. felis by infected fleas was not demonstrated in this study. The demonstration of horizontal transmission of R. felis between cat fleas has broad implications for the ecology of R. felis rickettsiosis.     

Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture

Anderson, Douglas R. (National Cancer Institute, Bethesda, Md.), Hope E. Hopps, Michael F. Barile, and Barbara C. Bernheim. Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture. J. Bacteriol. 90:1387-1404. 1965.-In an effort to make a valid comparison of the ultrastructure of several intracellular parasites, selected agents were propagated under identical conditions in a single type of tissue culture cell; such infected preparations were processed for examination by electron microscopy by use of a standardized procedure for fixation and embedding. The organisms studied were: the Breinl and E strains of epidemic typhus, Rickettsia prowazeki; the Bitterroot strain of R. rickettsii; the Karp strain of R. tsutsugamushi (R. orientalis); R. sennetsu; the P-4 strain of ornithosis virus; and the HEp-2 strain of Mycoplasma hominis type I. Each of the rickettsial species examined had a cell wall and a plasma membrane, and contained ribosomes and deoxyribonucleic acid (DNA) in a ground substance. However, certain differences were noted. Both strains of R. prowazeki contained numerous intracytoplasmic electron-lucent spherical structures (4 to 10 mmu), not previously described. R. sennetsu, unlike the other rickettsiae, was not free in the host cytoplasm but was always enclosed in a vacuole. R. rickettsii was observed intranuclearly and in digestive organelles of the host cell as well as in the cytoplasm. Cells infected with ornithosis virus contained several forms representing the stages in its life cycle. The "initial bodies," made up of ribosomes and DNA strands, were morphologically similar to the rickettsiae. In cultures infected with M. hominis, most of the cells became large and multinucleate. Although the Mycoplasma organisms were readily cultivated from these cultures, only a few could be found in the electron microscope preparations. These organisms were extracellular and lacked a cell wall, being bound only by a unit membrane. Again, the internal components were ribosomes and DNA strands. Under the uniform preparative conditions employed here, the three groups of organisms were morphologically distinguishable from one another.     

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.     

Molecular detection of various rickettsiae in mites (acari: trombiculidae) in southern Jeolla Province, Korea

This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission.     

Characteristics of rickettsial morphology during intracellular development

The normal anatomy of rickettsiae has been characterized with the use of R. prowazekii, R. conorii and R. akari in continuous cell cultures L-929, Al, FL and in primary chick embryo fibroblast culture. Rickettsiae are short rod-shaped cells with the dense cytoplasm and the regular structure of the cell wall--cytoplasmic membrane complex. The study has shown the absence of polymorphism in rickettsiae growing under permissive conditions, but at the same time these organisms easily develop into pathological forms. Pathological forms can be detected alongside normal rickettsiae in the same cells. The classification of the pathological forms of rickettsiae is presented. In this classification the compensating (reversible) and destructive (irreversible) forms of alterations, as well as hypertrophic and dystrophic processes on the level of the whole rickettsial cell or its organelles, are pointed out.     

S-adenosylmethionine transport in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.     

Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.     

Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis

Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.     

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.     

Differential proteomic analysis of Rickettsia prowazekii propagated in diverse host backgrounds

The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.     

Detection of Rickettsia spp. and host and habitat associations of fleas (Siphonaptera) in eastern Taiwan

Rickettsia typhi and Rickettsia felis (Rickettsiales: Rickettsiaceae) are two rickettsiae principally transmitted by fleas, but the detection of either pathogen has rarely been attempted in Taiwan. Of 2048 small mammals trapped in eastern Taiwan, Apodemus agrarius Pallas (24.5%) and Mus caroli Bonhote (24.4%) (both: Rodentia: Muridae) were the most abundant, and M. caroli hosted the highest proportion of fleas (63.9% of 330 fleas). Two flea species were identified: Stivalius aporus Jordan and Rothschild (Siphonaptera: Stivaliidae), and Acropsylla episema Rothschild (Siphonaptera: Leptopsyllidae). Nested polymerase chain reaction targeting parts of the ompB and gltA genes showed six fleas to be positive for Rickettsia spp. (3.8% of 160 samples), which showed the greatest similarity to R. felis, Rickettsia japonica, Rickettsia conorii or Rickettsia sp. TwKM01. Rickettsia typhi was not detected in the fleas and Rickettsia co-infection did not occur. Both flea species were more abundant during months with lower temperatures and less rainfall, and flea abundance on M. caroli was not related to soil hardness, vegetative height, ground cover by litter or by understory layer, or the abundance of M. caroli. Our study reveals the potential circulation of R. felis and other rickettsiae in eastern Taiwan, necessitating further surveillance of rickettsial diseases in this region. This is especially important because many novel rickettsioses are emerging worldwide.     

The rhizome of life: the sympatric Rickettsia felis paradigm demonstrates the random transfer of DNA sequences

The intracellular flea symbiont, Rickettsia felis, may meet other organisms intracellularly such as R. typhi. We used a single-gene phylogenetic approach of the 1375 R. felis genes to look for horizontal transfers that occurred as a result of the bacterial promiscuity with other organisms. Our results showed that besides genes that are linked to the Spotted Fever Group, 165 genes have a different history and are linked to other Rickettsia such as R. bellii (107 genes), R. typhi (15 genes), or to other bacteria such as Legionella sp. and Francisella sp. or to eukaryotes. Among these genes, we identified 73 individual genes and 34 spatial clusters containing 2-4 adjacent genes, a total of 79 genes, with evidence of en bloc transfer. We described 13 chimeric genes resulting from gene recombination with sympatric R. typhi. The transferred DNA sequences present different sizes and functions, suggesting that the horizontal transfer in R. felis is random and neutral within its specific host. Our study shows that the strict intracellular bacteria R. felis exhibits a mosaic genome. We therefore developed a new representation for the evolutionary history of R. felis showing its different putative ancestors in the form of a rhizome.     

Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.     

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.     

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.     

Interaction of Rickettsia akari with the host cell in vitro: multiplication, formation of spheroplast-like forms and its destruction in phagolyosomes

The electron microscopic study of the interaction of R. akari, strain CK, with the monolayer culture of L-cells was made 4 days after inoculation. Rickettsiae multiplied by transverse binary fission immediately in the cytoplasm of the cells and left the cells by gemmation, surrounded with plasmolemma and a fragment of the host cytoplasm. Alongside with multiplying rickettsiae, spheroplast-like rickettsiae and rickettsiae at the stage of destruction were regularly observed in phagolysosomes. The authors suggest that the normal interaction of rickettsiae with the host cell may be realized by three following routes (1) reproduction, (2) destruction in phagolysosomes and (3) formation of altered (anomalous) forms. The ability of the vegetative forms of rickettsiae and chlamydiae to yield spheroplast-like forms (the initial phase of bacterial L-transformation) indicates that these organisms are similar to bacteria and cannot be themselves regarded as L-forms.