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.     

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.     

Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone

Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.     

Taxonomy and classification of Rickettsiae

During the recent 20 years the improvement of the methodological research basis including the molecular and genetic analysis of microorganisms parasites of eukaryotic cells, led to considerable changes in the taxonomy and classification of rickettsiae. This problem was not discussed in the Russian literature. At present the term "rickettsiae" conventionally embraces the group of microorganisms of the class Proteobacteria, nominated as species belonging to the genera Rickettsia, Orientia, Ehrlichia, Coxiella and Bartonella. An original scheme of the modern taxonomy of rickettsiae, pathogenic for man, is presented. The scheme is made with due regard to their phenotypic, clinico-epidemiological and genetic features.     

Genotype characterization of the bacterium expressing the male-killing trait in the ladybird beetle Adalia bipunctata with specific rickettsial molecular tools.

The male-killing ladybird beetle (LB) bacterium (AB bacterium) was analyzed with specific rickettsial molecular biology tools in the LB Adalia bipunctata strains. Eight phenotype-positive LB strains showing mortality of male embryos were amplified with rickettsial genus-specific primers from the gene for citrate synthase (CS) and the gene for a 17-kDa protein and spotted fever group-specific primers from the gene for the 120-kDa outer membrane protein (ompB). The specificity of amplification was confirmed by Southern hybridization and the absence of the above-listed gene products in three phenotype-negative LB strains. Restriction polymorphism patterns of three examined amplicons from the CS gene, 17-kDa-protein gene, and ompB gene were identical among the eight phenotype-positive LB strains and were unique among all known rickettsiae of the spotted fever and typhus groups. Amplified fragments of the CS genes of the AB bacterium, Rickettsia prowazekii Breinl, Rickettsia typhi Wilmington, Rickettsia canada 2678, and Rickettsia conorii 7 (Malish) were sequenced. The greatest differences among the above-listed rickettsial and AB bacterium CS gene sequences were between bp 1078 and 1110. Numerical analysis based on CS gene fragment sequences shows the close relationships of the AB bacterium to the genus Rickettsia. Expanding of knowledge about rickettsial arthropod vectors and participation of rickettsiae in the cytoplasmic maternal inheritance of arthropods is discussed.     

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.     

Species-specific BALB/c mouse antibodies to rickettsiae studied by Western blotting

Abstract BALB/c mice were inoculated intraperitoneally either once only, or up to four times at weekly intervals, with viable Rickettsia rickettsii, Rickettsia conorii or the Israeli spotted fever group rickettsia. Sera collected one week after the last inoculation were tested for the presence of antibodies reactive with the above organisms by indirect fluorescent antibody testing and Western blot. With repeated inoculations there was a general progressive rise in homologous and heterologous immunofluorescence titers although the increase after the first inoculation was always the greatest. For each rickettsia, the homologous titers were higher than the heterologous titers. Western blots showed that the reactive antibodies were against rickettsial high molecular mass species specific protein antigens and homologous species-specific antibody reactions were detectable earlier than heterologous cross-reacting antibody reactions. Antibodies in mice sera did not react with the group specific lipopolysaccharide-like antigens of the rickettsiae although such reactivity was strong in Western blots with sera from patients suffering from acute Rickettsia conorii infections. Our findings suggest that the intraperitoneal route of inoculation of BALB/c mice can be used for the differentiation of spotted fever group rickettsiae.     

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.     

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.     

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.     

Experimental forms of infection and serological analysis of the antigenic structure of Rickettsia canada

Experimental forms of Rickettsia canada infection were characterized and serological analysis of the antigenic structure of R. canada was carried out. According to its pathogenicity for experimental animals, R. canada can be characterized as a poorly virulent species of rickettsiae, similar to R. prowazekii (for guinea pigs). The complement-fixing, haemagglutinating and agglutinating antigens of R. canada are fairly similar to those of the typhus group rickettsiae. The region of antigenic activity common to or identical in R. canada and the typhus group rickettsiae, is larger in R. canada than in the typhus group rickettsiae. R. canada has common antigens with Proteus OX19. R. canada has active toxic substances similar to those of R. prowazekii which, however, are detectable only with sera of Brill's disease convalescents. The position of R. canada in the taxonomy of rickettsiae is discussed.     

Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes.

DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.     

Evaluation of clinical specimens for Rickettsia, Bartonella, Borrelia, Coxiella, Anaplasma, Franciscella and Diplorickettsia positivity using serological and molecular biology methods

We monitored clinical samples from patients of different age groups from selected regions in Slovakia. Overall seroprevalence evaluated by immunofluorescence (IFA) using nine Bartonella, two Borrelia, six rickettsial (spotted fever and typhus group), two Coxiella, and one human granulocytic ehrlichiosis Anaplasma, Franciscella tularensis and Diplorickettsia massiliensis antigens, in rural and city populations of Slovak Republic, was found to be 32% positive for spotted fever group rickettsiae. Only five (10%) of the rickettsia-positive cases evaluated by IFA were confirmed by polymerase chain reaction. Rickettsia helvetica, Rickettsia slovaca, and Rickettsia raoultii infection appear to be prevalent in Slovakia. Furthermore, Coxiella burnetii, Borrelia and, for the first time, Bartonella elisabethae were confirmed in Slovakia.     

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.     

A Rickettsia genome overrun by mobile genetic elements provides insight into the acquisition of genes characteristic of an obligate intracellular lifestyle

We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae (Orientia tsutsugamushi) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi, REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ~35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis, its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity.     

Analysis of the peptidoglycan of Rickettsia prowazekii.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycan-linked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.     

Detection of Rickettsia hulinii in ticks of the Haemaphysalis concinna species in Russia

The presence of Rickettsia hulinii was detected in ticks of the Haemaphysalis concinna species in the Asiatic part of Russia. It was the first detection of these rickettsiae outside the territory of the Chinese People's Republic. This tick is spread over a broken area and may occur in different regions of Eurasia. At present at least 3 rickettsial species of the tick-borne spotted fever group were found in Ixodes ticks on the territory of Eastern Siberia: R. sibirica, known to be the etiological agent of tick-borne rickettsiosis, R. hulinii with proved pathogenicity for laboratory animals and R.DnS14 with no data available on its pathogenicity.