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PubMed1998

Rickettsia slovaca sp. nov., a member of the spotted fever group rickettsiae.

The name Rickettsia slovaca sp. nov. (type strain is strain B) is proposed for a member of the spotted fever group (SFG) rickettsiae which was isolated from Dermacentor marginatus ticks in Slovakia in 1968, and was recently implicated in human febrile illness. This rickettsia can be phenotypically distinguished from other SFG rickettsiae by microimmunofluorescence serotyping, SDS-PAGE, Western blotting and mAbs. Genotypic differences between R. slovaca and the other SFG representatives can be demonstrated by PCR-RFLP analysis, pulsed-field gel electrophoresis and sequencing of 16S rRNA, gltA and ompA genes.

Animals↗
PubMed1972

Metabolism of Rickettsia typhi and Rickettsia akari in irradiated L cells.

L cells that had been exposed to 3,000 r of (60)Co the previous day were used to study the growth and metabolism of Rickettsia typhi and R. akari. Viable (unirradiated) L cells were used to study the effect of rickettsial infection on host-cell metabolism. Monolayers were infected with a rickettsial multiplicity of 1.2 and given Eagle's minimal essential medium containing 25 mmN-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid buffer and 10% calf serum. At various intervals, cycloheximide (2 mug/ml) was added to one set of cultures, to inhibit eukaryotic protein and deoxyribonucleic acid (DNA) metabolism; phosphate-buffered saline (PBS) was added to another set. After 1 hr, the cultures received a mixture of 15 (14)C-labeled amino acids or adenine-8-(14)C. The cultures were harvested 16 hr later and were tested for incorporation of labeled carbon into the fraction precipitated by cold trichloroacetic acid. Viable cells were exposed to thymidine-2-(14)C for 2-hr periods. Infectivity of R. typhi increased to a peak of 150 to 400 hemolytic units/culture on day 4; the titer remained approximately the same on days 5 and 6, and declined rapidly on day 7. Total amino acid incorporation was about the same in infected and uninfected cultures up to day 6, but metabolic activity was reduced to a negligible level on day 7 in infected cells. Cycloheximide-resistant activity was higher in the infected cultures, with a peak equivalent to one-half the total activity at day 4 to 5. Total as well as cycloheximide-resistant adenine incorporation was higher in the infected cells between days 3 and 5 after infection, with a peak at day 3 to 4. Somewhat similar results were obtained with R. akari, except that the cycle of infection and of cycloheximide-resistant activity proceeded and was completed more rapidly. (14)C-DNA of both rickettsiae was isolated from infected cultures that had received labeled adenine. With labeled thymidine, which was not incorporated by the rickettsiae, it was shown that R. typhi and R. akari differ considerably in their effects on the host cell. R. typhi elicited moderate inhibition, whereas R. akari infection led to a complete inhibition of thymidine incorporation by the third day, at the time of highest rickettsial activity. It is concluded that rickettsiae have the necessary enzymes for protein and nucleic acid synthesis, but, thus far, these enzymes have been activated or induced only in an intracellular environment.

Adenine↗
PubMed1981

Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis.

A simple procedure for the selective isolation of the protective species-specific protein antigens (SPAs) of Rickettsia typhi and Rickettsia prowazekii was developed to permit use of the SPAs in the immunodiagnosis and immunoprophylaxis of typhus infections. Although the SPAs were readily extracted from lysozyme- or detergent-treated rickettsiae, as measured by rocket immunoelectrophoresis, other polypeptides were also present, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, both water and seven buffers, each at a 10 mM concentration and pH 7.6, were nearly equally effective in the selective release of the SPAs from whole cells by extraction for 30 min at 45 degrees C. High-ionic-strength buffers and MgCl2 abolished this SPA release, thus suggesting that divalent cations were important in the binding of the SPAs to the cell envelope. The efficacy of the dilute buffer extraction procedure for isolation of large amounts of SPAs was tested by further characterization of the supernatants obtained by centrifugation (200,000 x g) of two successive tris-(hydroxymethyl)aminomethane-hydrochloride buffer (Tris) extracts. With this procedure, between 10 and 15 mg of SPA was obtained from 100 mg of purified rickettsiae. Although low-molecular-weight ribonucleic acid fragments were released into the Tris extracts in significant amounts, only the SPAs were detected, in significant quantities, as measured by polyacrylamide gel electrophoresis and rocket immunoelectrophoresis. The Tris extracts contained the same major and minor SPA polypeptides as those observed previously in SPA preparations obtained by extensive diethylaminoethyl-cellulose column chromatography, but the Tris SPAs were more satisfactory antigens in an enzyme-linked immunosorbent assay.

Antigens, Bacterial↗
PubMed2006

Isolation of Rickettsia rickettsii and Rickettsia bellii in cell culture from the tick Amblyomma aureolatum in Brazil.

Brazilian spotted fever (BSF) is a highly lethal disease caused by Rickettsia rickettsii. In the present study, rickettsial infection was evaluated in 669 Amblyomma aureolatum adult ticks collected from naturally infested dogs in Taiaçupeba, a BSF-endemic area in the state of São Paulo. Ten (1.49%) ticks were infected with Rickettsia bellii, and 6 (0.89%) ticks were infected with R. rickettsii. Both Rickettsia species were isolated and established in Vero cell cultures. The Rickettsia isolates were characterized by molecular analyses, sequencing fragments of different rickettsial genes. Our results suggest that A. aureolatum is an important vector of R. rickettsii in Brazil.

Animals↗
PubMed2005

Susceptibility of Rickettsia monacensis and Rickettsia peacockii to Cecropin A, Ceratotoxin A, and lysozyme.

Ticks host obligate intracellular bacteria that range from benign symbiotes to virulent human pathogens. The effects on those bacteria of antimicrobial peptides (AMPs) involved in arthropod innate immunity to microbial infections are largely unknown. We evaluated effects of AMPs and a c-type lysozyme on host cell-free suspensions of the tick symbiotes Rickettsia monacensis and Rickettsia peacockii with stain-based infectivity and viability assays. Cecropin A at a concentration of 8 muM: had a lethal effect on both rickettsiae while ceratotoxin A was approximately 20-fold less effective. Toxicity of both AMPs was synergized by lysozyme, an enzyme expressed by ticks. Lactoferrin, a transferrin, had no effect on R. monacensis at up to 110 microM. The rickettsiae were less sensitive to the AMPs than is typical of bacteria that grow extracellularly. Our assays may be useful in the study of AMP activity against other obligate intracellular bacteria.

Antimicrobial Cationic Peptides↗
PubMed1997

Rickettsia aeschlimannii sp. nov., a new spotted fever group rickettsia associated with Hyalomma marginatum ticks.

We formally propose the name Rickettsia aeschlimannii sp. nov. for a new spotted fever group (SFG) rickettsia, strain MC16T, isolated from Hyalomma marginatum marginatum ticks collected in Morocco. This organism shows a typical rickettsial morphology when analyzed by electron microscopy. After characterization by serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblotting, PCR-restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis, and 16S rDNA sequencing, this organism was found to be different from all of the recognized SFG rickettsiae. Identical PCR-RFLP profiles have, however, been found in H. marginatum marginatum from Portugal and H. marginatum rufipes from Zimbabwe, which suggests that the distribution of this rickettsia reaches from the Mediterranean to southern Africa.

Animals↗
PubMed1979

In vitro studies of rickettsia-host cell interactions: ultrastructural changes induced by Rickettsia rickettsii infection of chicken embryo fibroblasts.

Secondary chicken embryo fibroblasts infected with the Sheila Smith strain of Rickettsia rickettsii and grown in monolayer culture undergo rapid morphological alterations. Transmission electron microscopic examination of cells at intervals after infection showed several progressive host cell lesions, including widespread dilatation of the rough endoplasmic reticulum and outer nuclear envelope and the accumulation of electron-dense material within the cisternae of intracellular membranes. Dilatation of the rough endoplasmic reticulum is a common, early reversible manifestation of other forms of cell injury. However, the severity of the damage to the host cell resulting from the progressive distention of intracellular membranes and the subsequent formation of small segments of membrane-bound host cytoplasm within the cisternae of these membranes is unknown. Early in the infection cycle, the rickettsiae were found free in the host cell cytoplasm, within invaginations of the nuclear envelope, occasionally free in the space between the outer and inner nuclear membranes, and in the host nucleoplasm, but not within cisternae formed by swollen endoplasmic reticulum. As a consequence of intracisternal swelling and fusion of intracellular membranes later in the infection cycle, the majority of the rickettsiae were found surrounded by host cytoplasm bound by host-derived internal membranes and appeared to persist in this state until cell lysis. The overall cytopathological changes in cells infected with R. richettsii appear dramatic and, from other studies in our laboratory, are significantly different from those observed in cells infected with Rickettsia prowazekii.

Animals↗
PubMed1983

Role of T-lymphocytes in production of antibody to antigens of Rickettsia tsutsugamushi and other Rickettsia species.

The requirement of thymus-dependent lymphocytes for antibody production to Rickettsia tsutsugamushi, Rickettsia akari, Rickettsia conorii, and Rickettsia typhi was investigated by comparing antibody production in athymic (nu/nu) or thymus-bearing BALB/c mice. Athymic BALB/c mice produced antibody after infection with R. akari, R. conorii, and R. typhi as measured by indirect fluorescent antibody titration or radioimmunoassay. Antibody production in these mice was a great or greater than in the thymus-bearing mice and demonstrated similar kinetics. In contrast, athymic BALB/c mice infected either intraperitoneally or subcutaneously with the Gilliam strain of R. tsutsugamushi failed to produce demonstrable antibody. The requirement of thymus-dependent lymphocytes for antibody production to R. tsutsugamushi was further suggested by the demonstration of antibody production after transfer of immune thymus-dependent lymphocytes to athymic mice and the demonstration of R. tsutsugamushi-specific T helper cells in immune thymus-bearing mice. The antibody produced in athymic mice after infection with R. akari, R. conorii, and R. typhi was predominantly immunoglobulin M, based on isotype-specific radioimmunoassays and sucrose gradient fractionation. Furthermore, the antibody produced by athymic mice in response to R. akari infection reacted with a carbohydrate-containing outer membrane component.

Animals↗
PubMed1979

Ultrastructure of Rickettsia rhipicephali, a new member of the spotted fever group rickettsiae in tissues of the host vector Rhipicephalus sanguineus.

Rickettsia rhipicephali is similar in ultrastructure to R. rickettsii while differing from other rickettsiae of the typhus group and of Q fever and others by its lack of a prominently reticulated cytoplasmic matrix and in the thickness of the inner osmophilic layer of the cell wall. In tissues of the tick vector Rhipicephalus sanguineus, R. rhipicephali had a mean length and width of 1.2 and 0.46 micrometer, respectively. It possessed a trilaminar cell wall with an adhering capsule-like layer. The trilaminar cell wall was approximately 12 to 18 nm thick; its inner osmophilic layer was thicker than that previously reported for other rickettsiae. The capsule-like layer varied from 7 to 18 nm thick. The plasma membrane was similar in structure, measurement, and appearance to that of other reported rickettsiae. The cytoplasm appeared to be composed of a finely granular, amorphous, ground substance and randomly dispersed ribosomes and lacked a reticular matrix or nuclear fibrils. In massively infected salivary glands and ovarial tissues of its tick vector, R. rhipicephali produced a low degree of histopathology which does not appear to affect the engorgement and egg-laying process of the ticks.

Animals↗
PubMed2006

Prevalence data of Rickettsia slovaca and other SFG Rickettsiae species in Dermacentor marginatus in the southeastern Iberian peninsula.

In southern Spain, Dermacentor marginatus ticks can be infected with several genospecies of spotted fever Group (SFG) Rickettsia. We developed a nested polymerase chain reaction assay by using a species-specific probe targeting the ompA gene to detect and differentiate between the two groups of rickettsiae previously described in D. marginatus. SFG rickettsia has been detected in 85.15% of ticks studied (26.7% of positives have been to R. slovaca, the causative agent of TIBOLA-DEBONEL, and 73.3% to SFG rickettsia closely related to strains RpA4-JL-02-DnS14-DnS28).

Animals↗
PubMed2004

Rickettsia bellii and Rickettsia amblyommii in Amblyomma ticks from the State of Rondônia, Western Amazon, Brazil.

This study evaluates the rickettsial presence in Amblyomma ticks from eight areas of the Amazon forest in Rondônia, Brazil. The following tick species (number in parentheses) were examined: Amblyomma ovale Koch (121), Amblyomma cajennense (F.) (41), Amblyomma naponense (Packard) (36), Amblyomma scalpturatum Neumann (35), Amblyomma oblongoguttatum Koch (30), Amblyomma incisum Neumann (27), Amblyomma rotundatum Koch (16), Amblyomma coelebs Neumann (10), and Amblyomma humerale Koch (6). Ticks were examined individually or in pools (2-10 ticks) by polymerase chain reaction (PCR) targeting the gltA gene. The PCR-determined minimal infection rate for each tick species was A. ovale 28%, A. cajennense 27%, A. naponense 0%, A. scalpturatum 11%, A. oblongoguttatum 3%, A. incisum 0%, A. rotundatum 87%, A. coelebs 10%, and A. humerale 50%. Partial sequences of the gltA gene of Rickettsia from A. ovale, A. scalpturatum, A. oblongoguttatum, A. rotundatum, and A. humerale were 99.9% (349/350) identical to Rickettsia bellii. DNA sequences of PCR products from A. cajennense and A. coelebs were 100% (350/350) identical to Rickettsia amblyommii. R. bellii organisms were isolated in Vero cells from A. scalpturatum, A. ovale, A. rotundatum, and A. oblongoguttatum, but only one of the isolates, cultured from A. scalpturatum, was established in continuous cell culture passage. R. amblyommii was isolated from A. cajennense and was successfully established in continuous passage in cell culture. R. amblyommii infection of Vero cells was analyzed by transmission electron microscopy. This study adds South America to the known geographic distribution of R. amblyommii and reports rickettsiae in six Amblyomma species for the first time.

Animals↗
PubMed2001

Short report: Thai tick typhus, Rickettsia honei, and a unique rickettsia detected in Ixodes granulatus (Ixodidae: Acari) from Thailand.

A unique Rickettsia species (Rickettsia thailandii sp. nov.) was identified in an Ixodes granulatus by use of polymerase chain reaction heteroduplex mobility assay by use of segments of the citrate synthase gene. This tick was collected from Rattus rattus from Nakhon Ratchasima province in 1970. Another I. granulatus was infected with Thai tick typhus strain TT-118, Rickettsia honei sp. nov. Stenos, Roux, Walker & Raoult; this tick was removed from a R. rattus collected 4 years later from the same province. Ixodes granulatus is the first tick species in Australasia shown to be infected with R. honei and the unique Rickettsia species.

Animals↗
PubMed2002

Prevalence of Rickettsia conorii and Rickettsia typhi infections in the population of northern Greece.

Seroepidemical surveys concerning the prevalence of Rickettsia conorii and Rickettsia typhi have never been studied in northern Greece. We examined 1,584 sera samples from residents of northern Greece for the detection of antibodies to R. conorii and to R. typhi by means of immunofluorescence assay. In addition, we compared the prevalence of rickettsial infections among the demographic variables of sex, age, occupation, and area of residence. Antibodies to R. conorii and R. typhi were found in 125 (7.9%) and 31 (2.0%) of the examined subjects, respectively. The prevalence of antibodies to R. conorii correlated with increasing age and was statistically higher in men. Farmers had significantly higher prevalence of antibodies to both species of rickettsiae studied versus other professions. Residents of rural areas showed a statistically higher prevalence for R. conorii versus urban residents, although this difference was not demonstrated for R. typhi. We also detected differences in the prevalence of rickettsial infections among the different prefectures. Our data show the wide distribution of R. conorii in northern Greece and indicate the presence of R. typhi.

Adolescent↗
PubMed2006

Molecular detection of Rickettsia felis, Rickettsia typhi and two genotypes closely related to Bartonella elizabethae.

A total of 56 fleas were collected from mice, rats, and one hedgehog in national parks of mainland Portugal and the Madeira Island. All fleas were tested for the presence of bacteria of the genera Rickettsia and Bartonella using PCR assays. In fleas from mainland Portugal, we detected Rickettsia felis in one Archaeopsylla erinacei maura flea and in one Ctenophtalmus sp. In five Leptopsylla segnis fleas taken from rats in the Madeira Island, we identified Rickettsia typhi. In addition, in four fleas from the genera Ornithophaga and Stenoponia collect from mice and a rat in mainland Portugal, we detected the presence of two new Bartonella genotypes closely related to Bartonella elizabethae. Our findings emphasize the potential risk of flea-transmitted infections in mainland Portugal and the Madeira archipelago, and extend our knowledge of the potential flea vectors of human pathogens.

Animals↗
PubMed2001

Lack of association between Kawasaki syndrome and infection with Rickettsia conorii, Rickettsia typhi, Coxiella burnetii or Ehrlichia phagocytophila group.

BACKGROUND: The etiology of Kawasaki syndrome (KS) is unknown. Rickettsiae, intracellular microorganisms that invade the vascular endothelium, might cause KS. OBJECTIVES: To investigate whether there is an association between KS and infection with Rickettsia conorii, Rickettsia typhi, Coxiella burnetii or Ehrlichia phagocytophila group. METHODS: All children who were diagnosed with KS at the University of Athens Second Department of Pediatrics from December, 1999, through November, 2000, were prospectively studied. Paired serum specimens were obtained from all patients and antibody titers against R. conorii, R. typhi, C. burnetii and E. phagocytophila group were assessed by microimmunofluorescence assay. RESULTS: Eleven children with a median age of 2.5 years were included in the study. A 15-month-old child had a 4-fold rise of antibody titers against C. burnetii, which is indicative of acute Q fever. The patient had a history of recent exposure to possible sources of C. burnetii. The remaining patients tested negative for the presence of antibodies against R. conorii, R. typhi, C. burnetii and E. phagocytophila group. CONCLUSIONS: Our study does not provide serologic evidence that KS is the result of infection with R. conorii, R. typhi, C. burnetii or E. phagocytophila group. It is suggested that C. burnetii may cause a KS-like illness in young children.

Boutonneuse Fever↗
PubMed1993

Confirmation that Rickettsia helvetica sp. nov. is a distinct species of the spotted fever group of rickettsiae.

We propose the name Rickettsia helvetica sp. nov. for a rickettsial serotype of unknown pathogenicity isolated in 1979 in Switzerland from Ixodes ricinus ticks and designated the Swiss agent. The growth characteristics and the results of microimmunofluorescence serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting (immunoblotting) with specific mice sera, and a polymerase chain reaction followed by restriction fragment length polymorphism analysis confirmed previously reported preliminary findings which suggested that this rickettsia, to which a name was given provisionally, does represent a new member of the spotted fever group of rickettsiae. The type strain is C3 (Reference Center for Rickettsioses, Marseille, France).

Animals↗
PubMed1978

External layers of Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer.

Using a simple specific-antibody stabilization procedure on organisms gently liberated from their host cells, we have demonstrated by electron microscopy that Rickettsia prowazekii and Rickettsia rickettsii possess a coat of variable thickness, external to the outer leaflet of the cell wall and the structure designated by others as a "microcapsule," which corresponds most closely to the slime layer of certain other bacteria. Reactions in the methenamine silver and ruthenium red staining procedures and the failure to be visualized by standard procedures suggest that the slime layer is largely polysaccharide in nature. It is postulated that this slime layer accounts in large part for the large, electron-lucent, halo-like zone which is found by electron microscopy to surround organisms of the typhus and spotted fever groups in the cytoplasm of their host cells, that it may be the locus of some major group-specific antigens, and that it may function as an antiphagocytic mechanism, as an aid for attachment of rickettsiae to potential host cells, or both. Moreover, because the attenuated E strain of R. prowazekii has been shown to possess a substantial slime layer, the basis for attenuation is not likely to be a simple smooth-to-rough variation.

Antigens, Bacterial↗
PubMed1981

Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis.

Species-specific antigens from Rickettsia typhi and Rickettsia prowazekii were readily solubilized by French pressure cell extraction or sonication of Renografin density gradient-purified rickettsiae and were identified by rocket immunoelectrophoresis. As measured by quantitative rocket immunoelectrophoresis, the species-specific typhus rocket antigens (STRAs) appeared to be proteins; they were denatured by heating at 56 degrees C for 30 min but not by 50 degrees C treatment, and they were sensitive to pronase and trypsin but were not affected by periodate oxidation, glycosidases of various specificities, phospholipase A, or lipase. STRAs from both R. typhi and R. prowazekii were separated from common antigens by DE52 column chromatography of 100,000-X-g supernatant fractions of rickettsial extracts. The purified STRAs were characterized by crossed immunoelectrophoresis, by polyacrylamide gel electrophoresis on Davis and sodium dodecyl sulfate gels, and by an enzyme-linked immunosorbent assay. The two purified STRAs were proteins with similar native electrophoretic mobilities in agarose and polyacrylamide gels, and these proteins had similar polypeptide patterns on sodium dodecyl sulfate gels. Most of the STRA activity migrated as a single protein band on sodium dodecyl sulfate-polyacrylamide and Davis polyacrylamide gels, although minor protein bands with STRA activity were also detected. The major STRA proteins constituted 10 to 15% of the total cellular protein of R. typhi and R. prowazekii. According to sensitive enzyme-linked immunosorbent assay titrations, the STRA of R. prowazekii had substantial cross-reactivity with rabbit antiserum prepared against R. typhi, as shown also by rocket immunoelectrophoresis, whereas the STRA of R. typhi reacted only very weakly with antiserum prepared against R. prowazekii according to the enzyme-linked immunosorbent assay and not at all according to rocket immunoelectrophoresis.

Antigens, Bacterial↗