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PubMed1999

Seroepidemiologic survey of Orientia tsutsugamushi, Rickettsia typhi, and TT118 spotted fever group rickettsiae in rubber estate workers in Malaysia.

The seroprevalence of Orientia tsutsugamushi, Rickettsia typhi, and TT118 spotted fever group (SFG) rickettsiae in 300 rubber estate workers in Slim River, Malaysia was determined in December 1996 and March 1997. In December, which was the wet season, 23.3%, 3.0%, and 57.3% of the population had antibodies detected against the three rickettsiae, respectively. The highest seropositive rate of 40% was detected for single infection with SFG rickettsiae, followed by a rate of 15.3% for both O. tsutsugamushi and SFG rickettsiae among the rubber estate workers. Subjects less than 21 years old had a lower seroprevalence of SFG rickettsiae compared with the other age groups. Indians had a higher seroprevalence of O. tsutsugamushi compared with other ethnic groups. Rubber tappers had a higher seroprevalence of SFG rickettsiae compared with other occupational groups. During the dry season in March 1997, there was a significant increase in the seroprevalence of R. typhi. The seroconversion rates for IgM against O. tsutsugamushi, R. typhi, and SFG rickettsiae were 5.7%, 12.3%, and 15.1%, respectively, during the four-month period. Significant variations of antibody titers towards the three rickettsiae was noted among subjects who were bled twice. This suggests a significant and continual exposure of rubber estate workers to the three rickettsiae.

Adolescent↗
PubMed2003

Isolation of a rickettsia related to Astrakhan fever rickettsia from a patient in Chad.

We isolated a novel spotted fever group rickettsia from a patient coming back from Chad with fever and a maculopapulous rash. In Africa, only six pathogenic spotted fever group rickettsiae have been identified, R. conorii, R. africae, R. akari, R. aeschlimannii, "R. mongolotimonae," and R. felis. Our isolate was identified by PCR amplification and sequencing of the 16S rRNA (16S rDNA), citrate synthase (gltA), and rOmpA (ompA) encoding genes. The 16S rDNA, gltA, and ompA sequences of the isolate were found to be 99.7, 99.6, and 99.5% identical with that of Astrakhan fever rickettsia, respectively. This rickettsia is endemic in the Caspian sea area and has also recently been identified in Kosovo. Using mouse serotyping, the currently accepted method for the identification of spotted fever group rickettsiae, the Chad isolate exhibited a specificity difference of 2 when compared to Astrakhan fever rickettsia and at least 4 when compared with other members of the R. conorii complex. The Chad isolate should be considered a variant of Astrakhan fever rickettsia. This is the first description of Astrakhan fever rickettsia outside Europe and the bacterium may be responsible for cases of spotted fever in Chad. Although Astrakhan fever rickettsia is transmitted by Rhipicephalus ticks in Europe, further studies are indicated to identify its vector in Africa where these ticks are also prevalent.

Adult↗
PubMed1997

Distribution of immunogenic epitopes on the two major immunodominant proteins (rOmpA and rOmpB) of Rickettsia conorii among the other rickettsiae of the spotted fever group.

Forty-four monoclonal antibodies were raised against strain Seven, the type strain of Rickettsia conorii. Of these 44 monoclonal antibodies, 13, 27, and 4 were demonstrated to be directed against the 116-kDa protein (rOmpA), the 124-kDa protein (rOmpB), and lipopolysaccharide-like antigen, respectively. The antiprotein monoclonal antibodies were found to be directed against 29 distinct epitopes, which were located on the two major immunodominant proteins discussed above. Further analysis showed that strain-specific epitopes were located on the rOmpA protein and species- and subgroup-specific epitopes were located on the rOmpB protein. R. conorii Manuel, Indian tick typhus rickettsia, and Kenya tick typhus rickettsia also possessed all 29 epitopes, whereas the other rickettsiae of the spotted fever group (SFG) expressed between 3 and 25 epitopes, with the exception of Rickettsia helvetica, R. akari, and R. australis which did not possess any epitopes. Additional analyses by Western immunoblotting confirmed that the epitopes shared among the SFG rickettsiae were located on the same two high-molecular-mass proteins as on R. conorii. However, although epitopes on the R. conorii rOmpB protein were expressed on the rOmpB proteins of most other SFG rickettsiae, some were found on the rOmpA proteins of R. aeschlimannii, R. rickettsii, and R. rhipicephali. Both proteins possessing the common epitopes were found to have different sizes in the SFG rickettsial species. The different distributions of common epitopes in the SFG rickettsiae were also used to build a taxonomic dendrogram, which demonstrated that all the R. conorii strains formed a relatively independent cluster within the SFG rickettsiae and was generally consistent with previously proposed taxonomies.

Antibodies, Monoclonal↗
PubMed1990

Species-specific monoclonal antibodies to Rickettsia japonica, a newly identified spotted fever group rickettsia.

A total of 192 hybridomas were developed from mice immunized with Rickettsia japonica, a newly identified spotted fever group rickettsia pathogenic for humans. Of these hybridomas, 101 were species specific, 37 were spotted fever group reactive, and the other 54 were also reactive with one or more of the other pathogenic species of spotted fever group rickettsiae, Rickettsia akari, Rickettsia australis, Rickettsia conorii, Rickettsia rickettsii, and Rickettsia sibrica. Seven of the species-specific monoclonal antibodies were characterized. These monoclonal antibodies all belong to the immunoglobulin G class and react with all five strains of R. japonica at the same immunofluorescence titers, indicating that the five strains all belong to a single species. The species-specific epitopes reactive with these monoclonal antibodies are located on the surface proteins of the organisms demonstrated as 145- and 120-kilodalton bands on Western immunoblots. These two antigenic bands were shown to be proteins, because treatment with proteinase K completely destroyed the reactivity of the bands with the monoclonal antibodies.

Animals↗
PubMed2006

Spotted fever group rickettsiae in ticks feeding on humans in northwestern Spain: is Rickettsia conorii vanishing?

During a 7-year study, we identified and analyzed by PCR 4,049 ticks removed from 3,685 asymptomatic patients in Castilla y León (northwestern Spain). A total of 320 ticks (belonging to 10 species) were PCR-positive for rickettsiae. Comparison of amplicon sequences in databases enabled us to identify eight different spotted fever group (SFG) rickettsiae: Rickettsia slovaca, Rickettsia sp. IRS3/IRS4, R. massiliae/Bar29, R. aeschlimannii, Rickettsia sp. RpA4/DnS14, R. helvetica, Rickettsia sp. DmS1, and R. conorii. Although Mediterranean spotted fever (MSF) is an endemic disease in Castilla y León, R. conorii was found in only one Rhipicephalus sanguineus tick, whereas other pathogenic SFG rickettsiae were much more prevalent in the same area. Our data suggest that in Castilla y León, many MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsiae present in ticks biting people in this region of Spain.

Animals↗
PubMed1991

Effects of typhus rickettsiae on peritoneal and alveolar macrophages: rickettsiae stimulate leukotriene and prostaglandin secretion.

Monolayers of mouse resident peritoneal macrophages were incubated with typhus rickettsiae, and macrophage secretion of leukotriene B4 (LTB4) was assessed. Macrophages incubated with native rickettsiae, but not those incubated with hemolytically inactivated rickettsiae, secreted significantly more LTB4 than did sham-treated macrophages. Antirickettsial antiserum was opsonic and blocked hemolysis, and macrophages incubated with rickettsiae in the presence of antiserum did not secrete more LTB4 than those incubated with buffer alone. Native rickettsiae also stimulated alveolar macrophages to secrete LTB4 and prostaglandin E2 (PGE2), but inactivated rickettsiae had no effect. Finally, because trifluoperazine did not alter macrophage LTB4 secretion in the presence of rickettsiae, but inhibited PGE2 secretion, it was suggested that the mechanisms by which rickettsiae stimulated production of these autacoids may have differed.

Analysis of Variance↗
PubMed2006

Isolation of Rickettsia rhipicephali and Rickettsia bellii from Haemaphysalis juxtakochi ticks in the state of São Paulo, Brazil.

In the present study, attempts to isolate Rickettsia in cell culture were performed individually in seven specimens of Haemaphysalis juxtakochi ticks collected in the state of São Paulo (southeastern Brazil). Rickettsia was successfully isolated by the shell vial technique and established in Vero cell culture from six ticks (six isolates). DNA extracted from infected cells of these isolates was tested by PCR and DNA sequencing, using genus-specific Rickettsia primers targeting the genes gltA, htrA, ompA, and ompB. After the generated sequences were compared with available sequences in GenBank, five out of the six isolates were identified as Rickettsia bellii (isolates HJ#1, HJ#2, HJ#3, HJ#4, and HJ#7). The sixth isolate (HJ#5) was closest to Rickettsia sp. strain R300, previously detected in H. juxtakochi in northern Brazil, and to Rickettsia rhipicephali, isolated from ticks in the United States. Following recent gene sequence-based criteria proposed for the identification of Rickettsia isolates, both isolate HJ#5 and strain R300 were identified as South American strains of R. rhipicephali, which was confirmed in this continent for the first time. Isolation of R. bellii from H. juxtakochi ticks, added to eight other tick species that have been reported to be infected with this bacterium in Brazil, indicates that R. bellii is indeed the most frequent Rickettsia species infecting ticks in Brazil. Currently, the role of both R. rhipicephali and R. bellii as human pathogens is regarded as unknown.

Animals↗
PubMed2002

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.

Actins↗
PubMed2005

Phylogenetic analysis of a novel molecular isolate of spotted fever group Rickettsiae from northern Peru: Candidatus Rickettsia andeanae.

Phylogenetic analysis of five rickettsial genes (17-kDa gene, gltA, ompB, ompA, and sca4) from two molecular isolates of Candidatus Rickettsia andeanae from two ticks (Amblyomma maculatum and Ixodes boliviensis) collected from two domestic horses living in two separate locations in northern Peru (Coletas and Naranjo) was conducted to more clearly characterize this recently reported novel spotted fever group (SFG) rickettsia. Following nested polymerase chain reaction (PCR) amplification of the 17-kDa gene, gltA, ompB, ompA, and sca4, amplicons were purified, sequenced, and compared to those downloaded from GenBank. Phylogenetic analyses of the Candidatus Rickettsia andeanae sequences generated from 17-kDa gene (483 bp), gltA (1185 bp), ompA (1598 bp), ompB (4839 bp), and sca4 (2634 bp) demonstrated that they aligned strongly with those of SFG rickettsiae. Moreover, the sequences of these five genes most closely aligned with the following rickettsiae: ompA: Rickettsia sp RpA4 (98.03%), R. sp DnS28 (97.90%), and R. rhipicephali and R. massiliae (97.11%); ompB: R. aeschlimannii (97.22%), R. rhipicephali (97.20%), and R. sp Bar 29 (97.10%); and sca4: R. massiliae (97.8%), R. rhipicephali, and R. slovaca (97.7%). These results from the additional phylogenetic analyses of Candidatus Rickettsia andeanae confirm its inclusion within, and distance and uniqueness from, other known SFG rickettsiae.

Animals↗
PubMed2003

Gene sequence-based criteria for identification of new rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov.

We propose genetic guidelines for the classification of rickettsial isolates at the genus, group, and species levels by using sequences of the 16S rRNA (rrs) gene and four protein-coding genes, the gltA, ompA, and ompB genes and gene D. To be classified as a member of the genus Rickettsia, an isolate should exhibit degrees of rrs and gltA homology with any of the 20 Rickettsia species studied of >/=98.1 and >/=86.5%, respectively. A member of the typhus group should fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of either Rickettsia typhi or Rickettsia prowazekii of >/=99.4, >/=96.6, >/=92.4, and >/=91.6%, respectively. A member of the spotted fever group should either possess the ompA gene or fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of any member of this group of >/=98.8, >/=92.7, >/=85.8, and >/=82.2%, respectively. The existence of a distinct "ancestral" group should be questioned. To be classified as a new Rickettsia species, an isolate should not exhibit more than one of the following degrees of nucleotide similarity with the most homologous validated species: >/=99.8 and >/=99.9% for the rrs and gltA genes, respectively, and, when amplifiable, >/=98.8, >/=99.2, and >/=99.3% for the ompA and ompB genes and gene D, respectively. By use of our classification scheme, "Rickettsia heilongjiangii" belongs to a new species for which we officially propose the name Rickettsia heilongjiangensis sp. nov.

DNA, Ribosomal↗
PubMed2005

Proposal to create subspecies of Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii.

BACKGROUND: Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness. RESULTS: Among the 39 isolates or tick amplicons studied, four MLST genotypes were identified: i) the Malish type; ii) the ITTR type; iii) the AFR type; and iv) the ISFR type. Among these four MLST genotypes, the pairwise similarity in nucleotide sequence varied from 99.8 to 100%, 99.4 to 100%, 98.2 to 99.8%, 98.4 to 99.8%, and 99.2 to 99.9% for 16S rDNA, gltA, ompA, ompB, and sca4 genes, respectively. Representatives of the 4 MLST types were also classified within four types using MST genotyping as well as mouse serotyping. CONCLUSION: Although homogeneous genotypically, strains within the R. conorii species show MST genotypic, serotypic, and epidemio-clinical dissimilarities. We, therefore, propose to modify the nomenclature of the R. conorii species through the creation of subspecies. We propose the names R. conorii subsp. conorii subsp. nov. (type strain = Malish, ATCC VR-613), R. conorii subspecies indica subsp. nov. (type strain = ATCC VR-597), R. conorii subspecies caspia subsp. nov. (type strain = A-167), and R. conorii subspecies israelensis subsp. nov. (type strain = ISTT CDC1). The description of R. conorii is emended to accomodate the four subspecies.

Gene Expression Regulation, Bacterial↗
PubMed1980

In vitro studies of Rickettsia-host cell interactions: ultrastructural study of Rickettsia prowazekii-infected chicken embryo fibroblasts.

Secondary chicken embryo fibroblasts infected in suspension with the Breinl strain of Rickettsia prowazekii and grown in monolayer culture were examined by both transmission and scanning electron microscopy at specific intervals after infection to study the effects of prolonged intracellular growth on the fine structure of the host cell and the rickettsiae. Cytopathological changes in the infected host cells were not apparent until late in the intracellular growth cycle when the cells began to rupture as a result of a large rickettsial burden. The only recognizable changes in heavily infected cells before lysis were the condensation of the intercristal matrix of some mitochondria and the apparent dissociation of ribosomes from the rough-surfaced endoplasmic reticulum. Although the effects of intracellular growth of rickettsiae on the fine structure of the host cell were rather unremarkable when compared with those imposed by Rickettsia rickettsii in a similar cell system, noticeable morphological changes in the rickettsiae were recognized during the intracellular growth cycle. These changes first became apparent about 40 h postinfection and consisted primarily of an increased electron density of the rickettsiae, the appearance of numerous vacuoles in the rickettsial cytoplasm, and a slight reduction in size of the rickettsiae. Changes of this nature may reflect transitional phases of growth characteristically seen in free-living bacterial cell systems.

Animals↗
PubMed2000

Phylogenetic analysis of the rompB genes of Rickettsia felis and Rickettsia prowazekii European-human and North American flying-squirrel strains.

The rickettsial outer membrane protein B (rompB) gene encodes the major surface antigens of Rickettsia species. We undertook sequencing and molecular analysis of the rompB gene of Rickettsia felis and a comparison with its homologs in spotted fever group (SFG) and typhus group (TG) rickettsiae, including the complete sequences of two North American flying squirrel strains and two European human strains of Rickettsia prowazekii. We sequenced 5,226 base pairs (bp) of the R. felis rompB, encoding a protein of 1,654 amino acids. We also sequenced 5,015 bp of rompB of the flying squirrel strains, encoding a protein of 1,643 amino acids. Analysis of the R. felis rompB gene sequence showed 10-13% divergence from SFG rickettsiae and 18% divergence from the TG rickettsiae. The rompB of all sequenced strains of R. prowazekii showed an overall similarity of 99.7-99.9%.

Animals↗
PubMed1993

Rickettsia massiliae sp. nov., a new spotted fever group Rickettsia.

We propose the name Rickettsia massiliae sp. nov. (type strain, Mtu1 in the Collection of the World Health Organization Collaborative Center for Rickettsial Reference, Marseille, France) for a spotted fever group rickettsia determined to be distinct from previously recognized species by the serotyping method (L. Beati, J.-P. Finidori, B. Gilot, and D. Raoult, J. Clin. Microbiol. 30:1922-1930, 1992). This rickettsia has biological characteristics similar to those of the other spotted fever group rickettsiae. In addition, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, a polymerase chain reaction followed by a restriction fragment length polymorphism analysis of DNA fragments, and pulsed-field electrophoresis of the genome of R. massiliae revealed unique migration patterns distinct from those of all previously recognized spotted fever group rickettsiae. These additional characteristics (Beati et al., J. Clin. Microbiol. 30:1922-1930, 1992), together with the data usually considered sufficient for description of rickettsiae, are crucial to the proposal of this new species and should be helpful in species identification.

Animals↗
PubMed1995

Prevalence of rickettsia-like organisms and spotted fever group rickettsiae in ticks (Acari: Ixodidae) from Zimbabwe.

The prevalence of rickettsia-like organisms in ticks from Zimbabwe was determined using the hemolymph test. Amblyomma hebraeum had the highest prevalence of rickettsia-like organisms. Other species with rickettsia-like organisms included Amblyomma sparsum, Amblyomma variegatum, Hyalomma marginatum rufipes, Ripicephalus simus, Haemaphysalis leachi, Amblyomma rhinocerotis, and Hyalomma truncatum. Ticks with no demonstrable rickettsia-like organisms infection were Boophilus decoloratus, Haemaphysalis spinulosa, Rhipicephalus appendiculatus, Rhipicephalus evertsi evertsi, and Rhipicephalus sanguineus. Polymerase chain reaction followed by restriction fragment-length polymorphism analysis on samples of hemolymph-positive ticks showed the agent of African tick-bite fever to be present in A. hebraeum, Rickettsia conorii to be present in Rhipicephalus simus and Haemaphysalis leachi, and a spotted fever group rickettsia similar to that in Hyalomma marginatum marginatum ticks from Morocco and Portugal to be present in Hyalomma marginatum rufipes.

Animals↗
PubMed1975

In vitro studies on rickettsia-host cell interactions: intracellular growth cycle of virulent and attenuated Rickettsia prowazeki in chicken embryo cells in slide chamber cultures.

Upon entry into the cytoplasm of irradiated chicken embryo cells in slide chamber cultures infected over a 2-h period, yolk sac-grown virulent (Breinl strain) and attenuated (E strain) Rickettsia prowazeki underwent indistinguishable reproducible intracellular growth cycles. They promptly entered an exponential growth phase, without detectable lag and without microscopic evidence for any unusual early replicative phase. The generation time for both strains was 8.8 to 8.9 h at 34 C. During most of this period, the state of the organisms and growth were very similar from one cell to another. The exponential-growth phase continued for at least 36 to 48 h, when the rickettsiae became too numerous to count by microscopic examination. Between about 36 and 48 h, cells packed with rickettsiae began irregularly to break down and release organisms. These began to initiate new infection cycles in previously uninfected cells over many hours, as demonstrated by the rise in percentage of cells infected, yielding a highly disordered infected culture with different cells containing rickettsiae in diverse stages of growth. The organisms underwent regular minor changes in morphology, similar to those seen in bacterial cultures, in the first infection cycle. As the cells became packed with rickettsiae, the microorganisms regularly diminished in size to become minute coccobacillary to coccoid forms. However, the rickettsiae in the second and subsequent infection cycles in aging cultures often assumed filamentous or swollen bizarre forms. Only the first infection cycle conformed closely to the concept of a one-step growth cycle. A set of terms is proposed and defined for the infection cycle.

Animals↗
PubMed2003

Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae.

A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of Rickettsia rickettsii and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of R. rickettsii can be detected. SQ-PCR is suitable for quantitation of R. rickettsii and 10 other genotypes of spotted fever group rickettsiae but not for R. akari, R. australis, R. bellii, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks.

Animals↗