Barbash strain spotted fever group rickettsia is a strain of Rickettsia conorii and differs from Rickettsia sibirica

The Barbash strain of spotted fever group rickettsia was reexamined in this study by the microimmunofluorescence test with mouse antisera and with monoclonal antibodies. Protein immunoblotting was performed for comparison of purified antigens of R. rickettsii, R. sibirica, R. conorii and Barbash strain. Comparison of Barbash strain, R. rickettsii (Sheila Smith strain), R. conorii (Malish 7 strain), and R. sibirica (strains 232, 246 and Jinghe-74) of the spotted fever group in the microimmunofluorescence test of Philip et al. revealed that Barbash strain has antigens that yield homologous titers with the R. conorii strains and differ from R. sibirica and R. rickettsii. Monoclonal antibodies specific for R. conorii react at identical titres with the Barbash strain, and a monoclonal antibody specific for R. sibirica does not react with the Barbash strain. Likewise, T-cell hybridomas reactive with R. conorii but not R. sibirica yield a strong response when stimulated by Barbash strain antigens. Western immunoblotting with the same polyclonal and monoclonal antibodies confirmed the presence of specific protein antigens of R. conorii and different protein antigenic composition of R. sibirica when compared with Barbash strain. Thus, Barbash strain is a strain of R. conorii.     

Molecular detection of Rickettsia massiliae,Rickettsia sibirica mongolitimonae and Rickettsia conorii israelensis in ticks from Israel

Rickettsioses are recognized as important emerging vector-borne infections of humans worldwide. Previous reports documented the presence of two spotted fever group rickettsiae in Israel, Rickettsia conorii israelensis and Rickettsia felis. The aim of this study was to characterize the diversity of rickettsiae in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 131 tick pools, 83 of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (each with 2–10 ticks per pool), were included in this study. In addition, 13 Hyalomma sp. ticks were collected. The ticks were molecularly screened for rickettsiae, targeting the citrate synthase (gltA) and the outer membrane protein A (ompA) gene loci. Rickettsia massiliae ompA DNA (100% sequence identity; 180 bp) was detected in 32 Rh. turanicus and 12 Rh. sanguineus tick pools. R. conorii israelensis was detected in three Rh. sanguineus pools. Rickettsia sibirica mongolitimonae ompA DNA (100% sequence identity; 182 bp) was found in one Hyalomma tick. This study reports the first detection of R. massiliae and R. sibirica mongolitimonae in ticks from Israel. This is the first report describing the presence of these human pathogens in the Middle East.     

Detection of the Rocky Mountain spotted fever agent, Rickettsia rickettsii, in dead ticks, Dermacentor andersoni.

Rickettsia rickettsii remained viable and retained chromophilic properties for not more than 24 h after Dermacentor andersoni were killed by freezing. Antigenic reactivity was detected for at least 71 days by direct immunofluorescence. However, rickettsiae in ticks suffocated with mineral oil remained pathogenic for at least 14 days. Accordingly, ticks removed from a host by mineral oil or dying from desiccation in transit are still suitable for rickettsial examination.     

Cross-reactive lymphocyte responses and protective immunity against other spotted fever group rickettsiae in mice immunized with Rickettsia conorii.

Lymphocyte proliferation in response to antigens on spotted fever group rickettsiae was used as a method to investigate the group-specific protective immunity to rechallenge characteristic of this group of rickettsiae at the T-cell receptor level. Spleen cells from Rickettsia conorii-immune C3H/HeJ mice proliferated in response to R. rickettsii Sheila Smith, R. sibirica 246, R. australis, and all tested strains of R. conorii (Casablanca, Moroccan, and Malish). Spleen cells from these mice, however, responded poorly or not at all to antigens prepared from the Kaplan or Hartford strain of R. akari. Proliferation of immune T cells maintained as in vitro cell lines showed a similar pattern of reactivity to these antigens; however, response to R. akari was consistently demonstrable. Spleen cells from C3H/HeJ mice immunized with R. akari responded to R. akari and R. conorii antigens as well as antigens from the other spotted fever group rickettsiae. Lymphocytes obtained from lymph nodes draining foot pads infected with R. conorii or R. akari demonstrated cross-reactivity similar to that found with immune spleen cells. If immunization was accomplished with R. conorii antigen emulsified in Freund complete adjuvant, the resulting lymph node cells were able to respond to R. akari antigens. These data suggest that infection with R. conorii induces a population of T lymphocytes that recognize an antigen(s) that also is found on other spotted fever rickettsiae and that may be responsible for cross-protective immunity. This antigen probably is not a major antigen on R. akari.     

Ultrastructural study of the response of cells infected in vitro with causative agent of spotted fever group rickettsiosis in Japan.

The response of host cells L929 infected with causative agent of spotted fever group (SFG) rickettsiosis in Japan, the Katayama strain, was studied by electron microscopy. The rickettsiae penetrated the cytoplasm and multiplied here and after prolonged incubation progressed into the dilated cisternae of rough endoplasmic reticulum (rER), the perinuclear space, the deep invaginated nuclear membrane, and then the nucleoplasm of the host cells. The intranuclear rickettsiae showed different states: one type was enclosed by the double membrane of the host cell and the other type was free in the nucleoplasm. In addition to these double membrane-bound and membrane-free intranuclear rickettsiae, various membrane structures, including rER-like structures, were also found in the nucleus. The cells infected with the rickettsiae underwent distinctive morphological alterations which occurred mainly within intracellular membranes of the host cells. These findings indicate the possibility that the intracellular membranes are characteristic cytopathological sites in rickettsia-host cell interaction, and that these alterations may be related to a possible route of rickettsial penetration into the nucleus: passage through vesicles formed from invaginations in the nuclear membrane.     

Detection of a novel spotted fever group Rickettsia in the gophertortoise tick

The gophertortoise tick, Amblyomma tuberculatum (Marx), is distributed throughout the southeastern United States, and its immature life stages have been reported to occasionally bite humans. Here we report detection of a novel spotted fever group (SFG) Rickettsia in A. tuberculatum ticks collected in the southern United States. Among questing ticks collected in Georgia, 10 pools of larvae were identified as gophertortoise ticks, A. tuberculatum. Each of these samples was positive for SFG Rickettsiae. The restriction fragment-length polymorphism profiles were identical to each other, but distinct from those of other rickettsiae previously found in Amblyomma spp. ticks. Partial genetic characterization of the novel agent was achieved by sequencing the 17 kDa, gltA, ompB, ompA, rpoB, and sca4 genes. Analysis of a concatenated tree of four genes (gltA, ompB, ompA, and sca4) demonstrates close relatedness of the detected Rickettsia to several SFG Rickettsia spp. The identical rickettsial DNA was detected in 50 and 70% of adult A. tuberculatum ticks from Mississippi and Florida, respectively. The results indicate wide distribution of a novel Rickettsia, capability for transovarial transmission, and high prevalence in tested tick populations.     

Seroepidemiological study of Rickettsia felis, Rickettsia typhi, and Rickettsia conorii infection among the population of southern Spain

Rickettsia typhi and Rickettsia conorii, the etiologic agents of, respectively, murine typhus and Mediterranean spotted fever, are recognized as frequent causes of fever of intermediate duration in southern Spain; in addition, in recent years Rickettsia felis has been detected in potential vectors in this area. Nevertheless, limited data exist regarding the actual prevalence of past infection due to these three pathogens. In the present study, the prevalence of past infection due to R. felis, R. typhi, and R. conorii was determined in a representative population of southern Spain during 2002. In addition, the possible risk factors associated with exposure to these pathogens were investigated. An epidemiological survey was completed by all subjects included in the study. Serum samples were tested by indirect immunofluorescence assay. The prevalence of past infection due to R. felis, R. typhi, and R. conorii among the 504 total subjects was 6.5, 3.8 and 8.7%, respectively. In multivariate analysis, infection due to R. felis was independently associated with a high-risk occupation (one that required working outdoors in nature, close contact with domestic animals, or potential contact with rodents) (OR=5.8; 95%CI 2.1–15.6), while infection due to R. typhi was associated with older age (factor of 1.04 [95%CI 1.008–1.068]) and frequent insect bites (OR=10.3; 95%CI 2.3–45.5). Two factors were associated with infection due to R. conorii: a high-risk occupation (OR=9.3; 95%CI 3.7–23.2), and participation in outdoor activities (OR=7.2; 95%CI 1.4–38.5). The results confirm the widespread prevalence of past infection due to R. felis, R. typhi, and R. conorii in the population of southern Spain.     

Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden.

A spotted fever group rickettsia isolated from the common tick, Ixodes ricinus, was genetically characterized by PCR and genomic sequencing. This study was performed with nymphal and adult ticks collected in southern and central Sweden. I. ricinus is the only North European tick species of medical importance which is regularly collected from humans. No species of the genus Rickettsia has previously been found in Scandinavian ticks, nor has any case of domestic rickettsial infection in humans or animals been reported. According to the nucleotide sequencing, the present Rickettsia sp. belongs to the spotted fever group of rickettsiae. Ticks are the most common arthropod reservoirs and vectors of the rickettsiae of this group. Among 748 ticks investigated, 13 (1.7%) were positive for a Rickettsia sp. Borrelia burgdorferi was detected in 52 (7%) of the ticks, a prevalence similar to or somewhat lower than that previously been recorded in other Swedish studies. There was no evidence of ehrlichial or chlamydial DNA in these ticks. The Rickettsia sp. was further characterized by 16S ribosomal DNA (rDNA) sequencing and restriction fragment length polymorphism (RFLP). The 16S rDNA sequencing resulted in a sequence identical to that described for Rickettsia helvetica, but the pattern obtained with RFLP of the citrate synthetase gene diverged from previously known patterns. The rickettsial agent of one tick which was positive by PCR was confirmed by transmission electron microscopy. The morphology of this rickettsia was similar to that of the spotted fever and typhus group rickettsiae. This represents the first documented isolate of a Rickettsia sp. from Swedish ticks.     

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.     

Diagnosis of Mediterranean spotted fever by cultivation of Rickettsia conorii from blood and skin samples using the centrifugation-shell vial technique and by detection of R. conorii in circulating endothelial cells: a 6-year follow-up.

Rickettsia conorii, an obligate intracellular bacterium that infects vascular endothelial cells, is the etiologic agent of Mediterranean spotted fever. We correlated the results of 205 R. conorii blood and skin cultures for 157 patients and the results of 48 detections of R. conorii in circulating endothelial cells (CEC) for 41 patients with relevant serological, clinical, and therapeutic data. R. conorii was cultured from 40% of patients and 29.8% of samples. R. conorii was detected in CEC in 50% of samples, representing 46.2% of patients. When these calculations were limited to the samples from untreated patients prior to their seroconversion to R. conorii, the sensitivity of culture was 59%, whereas it remained at 50% for detection in CEC. We also performed PCRs for the detection of R. conorii on eight shell vial supernatants from positive cultures and on 43 blood samples. Only nonfrozen supernatants from fresh cultures were positive. The methods described in this report are suitable for use in all laboratories. Our findings suggest that for samples to be suitable for culture they must be collected prior to the initiation of an antibiotic regimen, as early as possible in the course of the disease, and be inoculated onto shell vials with minimal delay, if R. conorii is to be successfully isolated. For patients who have been treated or who have a delayed diagnosis, detection of R. conorii in CEC remains helpful.     

Causative agent of spotted fever group rickettsiosis in Japan.

Since 1984, it has been known that spotted fever group rickettsiosis exists in Japan. We isolated three strains of the causative rickettsiae, designated Katayama, Misaka, and Abe, from patients with the disease and studied the characteristics of the isolates. Nude mice and cyclophosphamide-treated mice died after infection with the isolates. However, infected normal mice recovered and acquired immunity. Infected adult male guinea pigs had fever, a scrotal reaction, and seroconversion. The isolates propagated well in tissue-cultured Vero cells. Analysis by the cross-immunofluorescence antibody method showed that these isolates were closely related serologically. To reveal their immunological properties in detail, we produced 21 anti-Katayama monoclonal antibodies. Seven of these antibodies reacted with all representative strains of spotted fever group rickettsiae used in this study, and five others reacted only with the homologous strain, revealing that the Katayama strain has a strain-specific antigen(s) different from those of other spotted fever group rickettsiae. Moreover, these strain-specific antibodies also reacted with the Misaka and Abe strains. These results demonstrate that the causative agent of spotted fever group rickettsiosis in Japan is a new serotype of spotted fever group rickettsiae.     

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.     

Molecular evidence for a spotted fever group Rickettsia species in the tick Amblyomma longirostre in Brazil

Two Amblyomma longirostre adult male ticks were collected from a Brazilian porcupine Coendou prehensilis L. in the state of Rondonia, Western Amazon, Brazil. The two ticks were pooled for DNA extraction and tested for the presence of rickettsial DNA by amplifying portions of the gltA, 17-kDa, ompA, and ompB rickettsial genes by polymerase chain reaction (PCR). Portions of the four genes were amplified from the sample and subsequently sequenced. These results indicated the presence of a Rickettsia strain infecting A. longirostre, which was designated as strain Aranha. Compared with homologous ompA rickettsial sequences, "Rickettsia amblyommii" ompA seemed to be the closest relative to Aranha (similarity values: 99.0-99.3%). Phylogenetic analyses of more conserved genes including 17-kDa and gtlA partial sequences indicated that this Rickettsia sp. is a spotted fever group rickettsia. The partial ompB sequence of strain Aranha was distinct from all homologous sequences available in GenBank. Although our ompA analysis suggested a very close molecular phylogenetic relationship of Aranha with "R. amblyommii," we cannot at this time determine if Aranha is a new strain of "R. amblyommii" or a new Rickettsia species in South America.     

Primary humoral antibody response to Coxiella burnetii, the causative agent of Q fever.

Of 147 patients with acute Q fever diagnosed during a major outbreak in Birmingham, England, in early summer 1989, 41 provided sets of sera which allowed us to make a detailed analysis of the primary humoral immune response. Antibody titers specific for Coxiella burnetii were measured by the complement fixation test and by an immunoglobulin M (IgM)- and IgG-specific indirect immunofluorescence test. The relative avidity of specific IgGs was determined by the indirect immunofluorescence test with and without treatment of antigen-antibody complexes with 8 M urea. The IgG subclass responses after primary infection and their avidities were also determined for a limited number of paired serum specimens. Specific IgM titers persisted for more than 6 months in the majority of cases and were therefore not a sufficient criterion for the diagnosis of recent infection. However, for serial samples the antibody titer ratios (IgG/IgM) and the ratios (IgG titer with treatment/IgG titer without treatment) that indicated relative avidity changed significantly, depending on the time postinfection. Within the IgG class, the C. burnetii-specific antibody response over time was almost exclusively represented by subclass 1 molecules, which thus showed affinity maturation.     

Detection of a spotted fever group Rickettsia in the tick Ixodes tasmani collected from koalas in Port Macquarie, Australia

Four species of Rickettsia are recognized as endemic to Australia. This study reports the detection of a new spotted fever group Rickettsia in the common marsupial tick Ixodes tasmani Neumann collected from koalas (Phascolarctos cinereus) in Port Macquarie, NSW, Australia. Based on the results of polymerase chain reaction (PCR) amplification of extracted tick DNA with primers targeting the citrate synthase gene (gltA) and the outer membrane proteins A and B (ompA. ompB), Rickettsiae were detected in 22 of 78 I. tasmani tick samples (28.2%). Sequence data obtained for the three genes displayed the closest degree of similarity to Rickettsia heilongjiangiensiss for gltA (99.4%; 331/333 bp), Rickettsia amblyommii for the ompA gene (94.8%; 417/440 bp), and both Rickettsia massiliae and Rickettsia rhipicephali for the ompB gene (97%; 770/803 bp). BLAST and phylogenetic analysis of partial sequences obtained for the three genes were found to have sufficient nucleotide variation from the current recognized Australian species to be considered a distinct spotted fever group Rickettsia.     

Purification of Cytoecetes phagocytophila, the causative agent of tick-borne fever, from infected ovine blood.

Cytoecetes phagocytophila, the causative agent of tick-borne fever, was successfully separated by Percoll and Renografin density gradient centrifugation and by cellular affinity chromatography, from the peripheral blood leucocytes of experimentally infected sheep. After centrifugation on Renografin or Percoll density gradients, infectious particles of C. phagocytophila banded at buoyant densities which ranged between 1.063 to 1.140. Rickettsiae separated by wheat germ lectin cellular affinity chromatography retained their morphology but often lost their infectivity. Cell-free C. phagocytophila remained infective to susceptible sheep for 6 months when kept at -114 degrees C in sucrose phosphate buffer with 10 per cent dimethylsulphoxide as a cryopreservative.     

Isolation of the causative agent of Karelian fever from Aedes sp. mosquitoes

The results of isolation of an Alphavirus strain from Aedes sp. mosquitoes collected in the focus of Karelian fever are presented. According to the results of serological studies, the isolated strain LEIV-9298 Karelia belongs to the antigenic complex Sindbis-Western equine encephalomyelitis. The appurtenance of the isolated agent to the Alphavirus genus has been confirmed by electron microscopic examinations. A rise of antibody titres to LEIV-9298 Karelia virus in paired sera of subjects with a history of Karelian fever allows it to be considered the causative agent of this disease.     

Laboratory diagnosis of Rocky Mountain spotted fever by immunofluorescent demonstration of Rickettsia in Cutaneous lesions.

Direct immunofluorescent staining for Rickettsia rickettsii was performed on cryostat sections of skin biopsies from 27 patients suspected of having Rocky Mountain spotted fever. In nine of the 17 patients whose final diagnosis was Rocky Mountain spotted fever, coccobacillary forms of R. rickettsii were identified in endothelium and vascular walls within the dermis. Facotrs recognized as contributing to false-negative results were prior treatment with tetracycline or chloramphenicol for 24--48 hours or longer and failure to obtain a section through the focus of vasculitis. No false-positive result was obtained in the ten patients whose final diagnoses were not Rocky Mountain spotted fever. The laboratory test offers an immediate, positive laboratory diagnosis for this treatable, life-threatening disease.     

Characterization of and application of monoclonal antibodies against Rickettsia africae, a newly recognized species of spotted fever group rickettsia.

Rickettsia africae is a newly described species which causes African tick bite fever. Mediterranean spotted fever caused by R. conorii is endemic in the same regions of Africa as tick bite fever, and differentiation of the two syndromes by characterization of their etiological agents is important for epidemiological studies. R. africae and R. conorii are, however, difficult to distinguish, and therefore, our aim was to produce monoclonal antibodies to address this problem. Monoclonal antibodies were produced against R. africae by fusing splenocytes from BALB/C mice immunized with purified rickettsial organisms and SP2/0-Ag14 myeloma cells. A total of 355 hybridomas producing monoclonal antibodies to R. africae were identified by initial screening with six different antigens by microimmunofluorescence assay. A panel of 23 representative monoclonal antibodies were selected and subcloned. This panel was screened with a further 17 different spotted fever group (SFG) rickettsial reference antigens. Of these 23 monoclonal antibodies, 1 cross-reacted with only R. parkeri, whereas the others cross-reacted with more than two different antigens. Immunoblotting indicated that all the monoclonal antibodies were directed against the epitopes on two major high-molecular-mass heat-labile proteins, of which the molecular masses were 128 and 135 kDa, respectively. This monoclonal antibody panel was used successfully to identify R. africae in the blood culture of an infected patient, in infected cells within shell vials, and in infected ticks collected from Africa. Furthermore, the cross-reactivity of each SFG rickettsia with each of these 23 monoclonal antibodies was scored and was used to build a dendrogram of taxonomic relatedness between R. africae and the other SFG rickettsiae on the basis of Jaccard coefficients and unweighted pair group method with arithmetic mean analysis. The relatedness was generally consistent with that obtained by other methods of comparison.     

Molecular detection of spotted fever group Rickettsia in Dermacentor silvarum from a forest area of northeastern China

In total, 676 Dermacentor silvarum Olenev (Acari: Ixodidae) from a forest area of Jilin Province in northeastern China were examined by polymerase chain reaction for the presence of spotted fever group (SFG) Rickettsia. The overall positive rate was 10.7%, with a 95% confidence interval from 8.3 to 13.0%. The SFG Rickettsia infection was more prevalent in adults than in nymphs, and in fed ticks obtained from domestic animals than in those collected on vegetation. Sequence analysis of the partial outer membrane protein A gene confirmed the existence of R. sibirica and discovered a novel rickettsial agent in this area, the sequence of which was identical to that of DnS14 genotype Rickettsia previously reported in the former Soviet Union.