Spotted Fever Group Rickettsia Species Research Articles

Bacterial Pathogens in Ixodid Ticks from a Piedmont County in North Carolina: Prevalence of Rickettsial Organisms

In North Carolina, reported human cases of tick-borne illness, specifically Rocky Mountain spotted fever, have escalated over the past decade. To determine the relative abundance of vectors and to estimate the risk of acquiring a tick-borne illness in peri-residential landscapes, ticks were collected in Chatham County, a typical Piedmont county and, samples of the ticks were tested for infection with selected bacterial pathogens using real-time quantitative polymerase chain reaction assays. Ticks (n = 3746) were collected by flagging vegetation at 26 sites from April to July 2006. The predominant questing tick was Amblyomma americanum (98.5%) with significantly fewer Dermacentor variabilis (1.0%) and Ixodes scapularis (0.5%) collected. Spotted fever group (SFG) rickettsiae were detected in 68.2% of 1590 A. americanum with 56.4% of the molecular isolates identified as Rickettsia amblyommii, an informally named member of the SFG rickettsiae. Comparatively, smaller numbers of A. americanum contained Ehrlichia chaffeensis (1.8%) and Borrelia lonestari (0.4%). Of 15 I. scapularis nymphs tested, 6 (40%) were positive for Borrelia burgdorferi sensu lato. Seven (19.4%) of 36 adult D. variabilis tested positive for Rickettsia montanensis, 4 (11.1%) were positive for R. amblyommii, and 5 (13.9%) were infected with unidentified species of SFG rickettsiae. The tick population in Chatham County contains a diverse array of microbes, some of which are known or potential pathogens. Highest attack rates would be expected from A. americanum ticks, and highest potential risk of infection with a tick-transmitted agent would be to rickettsial organisms, particularly R. amblyommii. Accordingly, longitudinal eco-epidemiology investigations are needed to determine the public health importance of A. americanum infected with rickettsial organisms.

TheRickettsia conoriiAutotransporter Protein Sca1 Promotes Adherence to Nonphagocytic Mammalian Cells

The pathogenesis of spotted fever group (SFG) Rickettsia species, including R. conorii and R. rickettsii, is acutely dependent on adherence to and invasion of host cells, including cells of the mammalian endothelial system. Bioinformatic analyses of several rickettsia genomes revealed the presence of a cohort of genes designated sca genes that are predicted to encode proteins with homology to autotransporter proteins of Gram-negative bacteria. Previous work demonstrated that three members of this family, rOmpA (Sca0), Sca2, and rOmpB (Sca5) are involved in the interaction with mammalian cells; however, very little was known about the function of other conserved rickettsial Sca proteins. Here we demonstrate that sca1, a gene present in nearly all SFG rickettsia genomes, is actively transcribed and expressed in R. conorii cells. Alignment of Sca1 sequences from geographically diverse SFG Rickettsia species showed that there are high degrees of sequence identity and conservation of these sequences, suggesting that Sca1 may have a conserved function. Using a heterologous expression system, we demonstrated that production of R. conorii Sca1 in the Escherichia coli outer membrane is sufficient to mediate attachment to but not invasion of a panel of cultured mammalian epithelial and endothelial cells. Furthermore, preincubation of a recombinant Sca1 peptide with host cells blocked R. conorii cell association. Together, these results demonstrate that attachment to mammalian cells can be uncoupled from the entry process and that Sca1 is involved in the adherence of R. conorii to host cells.

Adherence to and Invasion of Host Cells by Spotted Fever Group Rickettsia Species

The pathogenic lifecycle of obligate intracellular bacteria presents a superb opportunity to develop understanding of the interaction between the bacteria and host under the pretext that disruption of these processes will likely lead to death of the pathogen and prevention of associated disease. Species of the genus Rickettsia contain some of the most hazardous of the obligate intracellular bacteria, including Rickettsia rickettsii and R. conorii the causative agents of Rocky Mountain and Mediterranean spotted fevers, respectively. Spotted fever group Rickettsia species commonly invade and thrive within cells of the host circulatory system whereby the endothelial cells are severely perturbed. The subsequent disruption of circulatory continuity results in much of the severe morbidity and mortality associated with these diseases, including macropapular dermal rash, interstitial pneumonia, acute renal failure, pulmonary edema, and other multisystem manifestations. This review describes current knowledge of the essential pathogenic processes of adherence to and invasion of host cells, efforts to disrupt these processes, and potential for disease prevention through vaccination with recently identified bacterial adherence and invasion proteins. A more complete understanding of these bacterial proteins will provide an opportunity for prevention and treatment of spotted fever group Rickettsia infections.

Identification of Spotted Fever Group Rickettsia Species by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of the Sca4 Gene

DNA samples from four rickettsial species in Japan--R. japonica Aoki, R. asiatica IO-1, R. helvetica IP-1, and Rickettsia tamurae AT-1--were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in which a portion of the sca4 gene was amplified and restriction digested with the MspI enzyme. Each spotted fever group rickettsial species exhibited unique restriction profiles after MspI digestion. To examine the possibility of applying PCR-RFLP analysis to field samples, 36 Ixodes persulcatus ticks were collected from the Tokachi region (Hokkaido, Japan) by the flagging method and subjected to PCR-RFLP analysis. Of the ticks collected, five had identical restriction profiles to that of R. helvetica. Amplification and sequencing of the gltA gene for two of the five positive samples revealed that the sequences were identical to that of the Rickettsia sp. Tokachi-B-IP17m, which exhibits the highest similarity to R. helvetica. These results demonstrate that PCR-RFLP analysis is useful for identifying Rickettsia species in Japan and is applicable to epidemiological studies involving tick samples.

First description of Rickettsia monacensis in Ixodes ricinus in Algeria

Tick-borne spotted fever group (SFG) rickettsioses are caused by obligate intracellular Gram-negative bacteria belonging to the genus Rickettsia. Although some rickettsias have been known as human pathogens since the beginning of the century, tick-borne SFG rickettsioses are also recognized as important emerging worldwide zoonoses [1]. In Algeria, three species of SFG rickettsia have been detected in ticks, including Rickettsia conorii conorii (the agent of Mediterranean spotted fever) in Rhipicephalus sanguineus, and two emerging pathogens, i.e. Rickettsia aeschlimannii in Hyalomma marginatum marginatum, and Rickettsia massiliae in Rhipicephalus sanguineus and Rhipicephalus turanicus [2]. We present here the detection and characterization of rickettsiae in more ticks collected in Algeria using PCR and sequence analysis of amplified products.

First detection of Rickettsia aeschlimannii in Hyalomma aegyptium from Algeria

Tick-borne spotted fever group (SFG) rickettsioses are zoonoses caused by obligate intracellular Gram-negative bacteria belonging to the genus Rickettsia [1]. In Algeria, three species of SFG rickettsia transmitted by ticks are known to occur, including Rickettsia conorii conorii (the agent of Mediterranean spotted fever), transmitted by Rhipicephalus sanguineus, Rickettsia aeschlimannii, an emerging pathogen transmitted by Hyalomma marginatum marginatum, and Rickettsia massiliae, another emerging pathogen transmitted by Rh. sanguineus and Rhipicephalus turanicus [2]. We report here the detection of SFG rickettsiae in Hyalomma aegyptium ticks collected in Algeria.

HumanRickettsia sibirica mongolitimonaeInfection, Spain

To the Editor: Rickettsia sibirica mongolitimonae has been recently reported as a subspecies of R. sibirica (1). The first evidence of R. sibirica mongolitimonae pathogenicity in humans was documented in France in 1996 (2). Since then, 11 more cases in France, Algeria, South Africa, Greece, and Portugal have been reported (3–6). Because the main clinical manifestations include lymphangitis, the acronym LAR (lymphangitis-associated rickettsiosis) has been proposed (3). We report a case from Spain that confirms the broad distribution of this agent in southern Europe. A 41-year-old man was admitted on June 19, 2007, to the Hospital de Cruces (Baracaldo, Spain) with fever (39°C), malaise for a week, sweating, lumbar and knee pain, disseminated myalgias, and headache. He reported that 20 days before admission he had removed an engorged tick from his right leg while working as a topographer in the Balmaseda Mountains, 30 km from Bilbao. He had also removed several ticks from his body 4 days before the onset of symptoms. Physical examination did not demonstrate relevant findings. There was no inoculation eschar at the tick-bite sites. Rash, lymphadenopathies, and lymphangitis were not observed. Chest radiograph did not show consolidation or other abnormality. Initial laboratory examination, on June 21, 2007, showed a leukocyte count 5.2 × 103/μL, hemoglobin 14.1 g/dL, platelet count 190,000/μL, erythrocyte sedimentation speed 9 mm/h, urea 38 mg/dL, creatinine 0.9 mg/dL, aspartate aminotransferase 229 IU/L, alanine aminotransferase 170 IU/L, alkaline phosphatase 158 IU/L, gamma-glutamyl-transpeptidase 111 IU/L, total bilirubin 1.3 mg/dL, and C-reactive protein 4.3 mg/dL. Because the patient had been bitten by a tick, acute-phase serum and EDTA-treated blood samples were sent to the Special Pathogens Laboratory (Area de Enfermedades Infecciosas – Hospital San Pedro from La Rioja), where a presumptive diagnosis of rickettsiosis was made. On June 22, 2007, treatment with doxycycline was begun (100 mg/day for 12 days), and his condition rapidly improved. The early-phase serum yielded low immunoglobulin (Ig) G titer (<64) against Rickettsia conorii and Anaplasma phagocytophilum antigens, and results of ELISA and Western blotting for Lyme borreliosis were negative. A convalescent-phase serum sample collected 7 weeks later did not contain IgG antibodies against spotted fever group Rickettsia species when R. conorii antigen was used. DNA was extracted from the early whole-blood specimen by using QIAamp DNA Blood minikit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. This DNA extract was used as template in nested PCR assays targeting the spotted fever group rickettsial ompB (420 bp) and gltA (337 bp) genes (7). Quality control included both positive (with R. conorii Malish #7 grown in Vero cells) and negative controls that were extracted and PCR amplified in parallel with the specimens. Negative controls consisted of sterile water instead of template DNA. Amplification products of the expected size were obtained. The sequences of these amplicons allowed the identification of R. sibirica mongolitimonae with 99.5% and 100% similarity for ompB and gltA, respectively (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:DQ097083,term_id:73620233}}DQ097083 and {type:entrez-nucleotide,attrs:{text:DQ097081,term_id:73620146}}DQ097081). To our knowledge, Rickettsia species have never been detected in ticks or human specimens in Spain. The host ticks of this rickettsia are likely Hyalomma species, which are more prevalent in southern Spain. In our region in northern Spain, Hyalomma marginatum represented 8% of ticks that fed on humans during 2001–2005, although an increase in this number was recorded last year (data not shown). In our patient, Rickettsia’s pathogenic role was demonstrated by PCR, a technique that has previously enabled us to identify other arthropod-borne Rickettsia species (8,9). This case suggests that R. sibirica mongolitimonae infection should be considered in the differential diagnosis of rickettsiosis and tick-bite febrile patients in Spain and confirms the distribution of this rickettsia in southern Europe. According to the literature (3), some patients in whom R. sibirica mongolitimonae infection is diagnosed have >1 eschar, which raises the suspicion that some cases of Mediterranean spotted fever with multiple eschars reported in Spain could be caused by this rickettsial species. More studies about the vectors of this bacteria are needed because studies of Hyalomma and Rhipicephalus ticks (the suspected hosts) conducted in our area have not demonstrated the presence of this Rickettsia species.

Rickettsia felisin Chile

To the Editor: Rickettsiosis due to Rickettsia felis is an emerging disease that has been reported worldwide (1). Fever, headache, myalgia, and macular rash have been attributed to R. felis infection in humans (1). In South America, R. felis infection in fleas (mostly Ctenocephalides spp.) has been reported only in Brazil, Peru, and Uruguay (2–3). Although a growing number of articles have reported that R. felis is transmitted by fleas, the acquisition mechanism of R. felis by vertebrates or uninfected fleas in nature remains unknown (4). Cats experimentally exposed to R. felis–infected fleas have been shown to become seropositive (5). However, neither serologic nor molecular evidence of R. felis infection has been reported in cats under natural conditions, despite the fact that most C. felis fleas are infected by R. felis (6,7). In November 2006, we investigated the presence of rickettsial DNA in 30 C. felis fleas randomly collected from 22 domestic cats privately owned and housed indoors in a single household in Santiago, Chile. To detect rickettsial DNA in each individual flea, PCRs were performed that targeted a 398-nt fragment of the rickettsial gltA gene and an 856-nt fragment of the rickettsial ompB gene (7,8). A total of 21 individual fleas (70%) yielded expected PCR products for both gltA and ompB genes. PCR gltA products from the 21 fleas and ompB products from 5 fleas were subjected to DNA sequencing as described (7). The gltA partial sequences obtained from 21 fleas were identical, as were the ompB partial sequences from 5 fleas. These sequences were 100% identical to corresponding sequences in the R. felis genome (GenBank accession no. {type:entrez-nucleotide,attrs:{text:C53,term_id:67003925,term_text:C53}}C53). Blood serum samples were collected from the 22 cats and tested by indirect immunofluorescence assay (IFA) with crude antigens derived from 6 Rickettsia isolates from Brazil: R. bellii, R. amblyommii, R. rhipicephali, R. rickettsii, R. parkeri, and R. felis (7,9). Serum was considered to contain antibodies against rickettsiae if it displayed a reaction at 1:64 dilution. End-point titers against each Rickettsia species were determined by testing serial 2-fold serum dilutions. Reactive serum specimens were tested in 2 or 3 replications by 2 readers before the end-point titer was determined. Serum showing a Rickettsia species titer at least 4-fold higher than those observed for the other Ricketttsia species was considered homologous to the first Rickettsia species or to a very closely related genotype (7,9). In each slide, a nonreactive cat serum specimen (negative control) and a known reactive cat serum specimen (positive control) were tested at the 1:64 dilution (7). IFA detected antibodies reactive with R. felis (titer >64) in 16 (72.7%) of 22 cats. Among those, 5 (22.7%) also reacted with R. rhipicephali, 4 (18.2%) with R. bellii, 3 (13.6%) with R. parkeri, 2 (9.1%) with R. rickettsii, and 1 (4.5%) with R. amblyommii. No serum reacted with any other Rickettsia species without reacting with R. felis (Table). Four cat serum specimens (cats 1, 3, 8, and 11) showed titers to R. felis at least 4-fold higher than those to any of the other 5 antigens. The antibody titers in these 4 animals were considered to have been stimulated by R. felis infection. For the remaining 12 seropositive cats, we could not discern whether R. felis had been the infection agent because the results displayed a single titer of 64 for R. felis or showed similar titers for other Rickettsia species. Table End-point titers of indirect immunofluorescence assay (IFA) for 6 Rickettsia species in cats from a household in Santiago, Chile* We report 70% R. felis–infected fleas in this study on the basis of the concordant results of 2 PCR amplifications (gltA and ompB) and DNA sequencing. This infection rate is within the range (13.5%–90%) that has been reported for R. felis infecting Ctenocephalides fleas in Brazil and Uruguay (2,3,7). Sixteen (72.7%) cats contained R. felis–reactive antibodies; 4 of them showed titers to R. felis at least 4-fold higher than those to the other 5 rickettsial strains, findings that enabled us to technically conclude that these cats were exposed to R. felis or a closely related organism (1,7,9). Our finding of 70% R. felis infection in fleas infesting the cats indicates that cats acquired the infection through infected fleas. However, the mechanism of R. felis transmission by fleas is yet to be demonstrated under experimental conditions. To our knowledge, the presence of R. felis, or a spotted fever group Rickettsia species, has not been reported in Chile. Recent investigations have provided clinical and serologic evidence of canine (10) and human (K. Abarca and J. Lopez, unpub. data) infection by spotted fever rickettsia in Chile, confirmed by IFA that used R. conorii commercial antigen. Since substantial serologic cross-reaction occurs between R. conorii and R. felis antigens (1), R. felis could be causing infection in dogs or humans in Chile.

Human Infection withRickettsiasp. relatedto R. japonica, Thailand

Human Infection with<i>Rickettsia</i>sp. related<i>to R. japonica</i>, Thailand

Far Eastern Tick‐Borne Rickettsiosis

We recently reported the first documented cases of a new rickettsial disease caused by Rickettsia heilongjiangensis in the Russian Far East (Far Eastern tick-borne rickettsiosis). Here we report the amplification of DNA of R. heilongjiangensis from both the skin biopsy of an acutely ill patient and the tick removed from him prior to the disease development. The tick has been identified as Haemaphysalis spp. The clinical picture was that of a spotted fever group rickettsiosis and a seroconversion was noted with R. heilongjiangensis antigen. Screening testing of both species of Haemaphysalis ticks inhabiting Russian Far Eastern regions showed that up to 28.13% of H. concinnae and 4.48% of H. japonica douglasii ticks harbor R. heilongjiangensis. It has been concluded that H. concinnae may serve as the main vector for the transmission of R. heilongjiangensis. H. japonica douglasii ticks harbor several varieties of rickettsiae. DNA of "Candidatus Rickettsia tarasevichiae," previously found in Ixodes persulcatus ticks, was amplified from one male tick. Two sequenced complete gltA genes belong to the novel spotted fever group rickettsial species provisionally called here "Candidatus Rickettsia principis" variants Hjd54 and Hjd61. The rate of infection has been found to be not higher than 1.5%.

Serological evidence of human infection with rickettsial strain Bar29 in Catalonia, northeastern Spain

Mediterranean spotted fever (MSF) is an endemic disease in Catalonia, northeastern Spain. The causative agent is Rickettsia conorii, which is transmitted by the dog tick Rhipicephalus sanguineus in the Mediterranean area [1, 2]. However, it is strongly suspected that another spotted fever group (SFG) rickettsial strain could be implicated in some cases of MSF diagnosed in Catalonia [3, 4]. In 1990, Mtu1 and Mtu5 were isolated from Rhipicephalus turanicus ticks collected in the south of France. Both were new serotypes related to the SFG rickettsiae [5]. Mtu1 was named Rickettsia massiliae and proposed as a new SFG Rickettsia species [6]. Strains related to R. massiliae have been identified throughout the world [7]. In Catalonia, a new SFG rickettsial strain (Bar29) was isolated by our group in 1996 from R. sanguineus; it is identical to the Mtu5 strain and closely related to R. massiliae [3]. R. massiliae Bar29 belongs to the R. massiliae group of SFG that includes R. massiliae, Rickettsia aeschlimannii, Rickettsia montanensis and Rickettsia rhipicephali [7]. A previous study we conducted using sera from Catalonian patients suggested R. massiliae Bar29 could be implicated in cases of MSF diagnosed in our region [4]. Currently, R. massiliae and R. aeschlimannii are recognized as human pathogens [7, 8]. The aim of the study presented here was to determine the seroprevalence of R. massiliae Bar29 in a representative population of Catalonia and to identify possible risk factors for infection. The study was performed in a predominantly urban area near the coast in northeastern Spain. Between September 1993 and January 1994, 211 subjects who had received attention at Sabadell’s Hospital were chosen to participate in a study searching for antibodies to R. conorii and Borrelia burgdorferi. The sample size had been estimated taking into account overall prevalences of 11 and 3% of antibodies to R. conorii and B. burgdorferi, respectively (given an alpha error of 0.05). The sample included adults undergoing minor surgery and children who visited the Pediatric Emergency Service. Taking into account the actual population, the study population was stratified by age and residential area. After informed consent was obtained, a serum sample was collected. Age, sex, demographic area, occupation, contact with dogs, contact with wild animals, contact with farm animals, and history of MSF were surveyed. One milliliter of blood was collected in tubes containing heparin. The sample of heparinized blood was sedimented and the supernatant (0.5 ml) was collected and stored at −80°C. Human sera were evaluated using an indirect immunofluorescence assay (IFA) for IgG antibodies [9]. Antigen was obtained from the R. massiliae Bar29 strain previously isolated from ticks and cultured in our laboratory [3]. Titers ≥1/40 were considered positive. Statistical analysis was performed using the chi-square test, Fisher's Eur J Clin Microbiol Infect Dis (2006) 25:541–543 DOI 10.1007/s10096-006-0176-1

Potential Roles for Regulatory Oxygenases in Rickettsial Pathogenesis

Clinical and experimental evidence suggests an important role for oxidative stress and associated cellular defense mechanisms in the pathogenesis of vasculopathic rickettsioses. Our laboratory has reported that R. rickettsii infection of endothelial cells in vitro induces the expression of HO-1, the inducible isoform of the antioxidant defense enzyme heme oxygenase. HO-1 plays a critical role in maintaining the integrity of the vasculature and controls the functioning of the cyclooxygenase (COX) system. This study was undertaken to investigate the expression of COX and HO isozymes during in vitro infection of EC with two major representatives of spotted fever group Rickettsia species. The mRNA expression of COX-2 was significantly increased in endothelial cells infected with R. rickettsii and R. conorii, while that of COX-1 remained unaffected. Western blot analysis using total protein lysates from infected endothelial cells and corresponding uninfected controls further confirmed specific induction of COX-2 in response to infection. ELISA measurements on culture supernatants also suggested enhanced secretion of 6-keto PGF(1alpha) (stable hydrolysis product of PGI(2) and PGE(2). As a functional consequence of HO-1 upregulation, increased expression of the iron storage protein ferritin following R. rickettsii and R. conorii infection was also evident. Since products of HO-1 and COX-2 reactions govern a variety of physiologically important functions in the vasculature, further studies to define their regulation in the host cell should provide useful insights into the pathogenesis of rickettsial diseases.

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.

Rickettsia honei sp. nov., the aetiological agent of Flinders Island spotted fever in Australia.

The name Rickettsia honei, strain RBT, has been proposed for a unique spotted fever group (SFG) agent which is pathogenic for humans. This agent has previously been compared to the other SFG agents and was shown to be distinct in protein structure by SDS-PAGE and by immunoblotting. Genetic comparisons of the 16S rRNA, rompA, gltA and the 17 kDa antigen genes with the other SFG rickettsiae confirmed the phylogenetic distance between R. honei and the previously described species. Genetically, Rickettsia honei is more closely related to the Thai tick typhus (TT-118) rickettsia than to any other member of the SFG. Indeed, it is proposed that TT-118 is a strain of R. honei which was previously isolated in Thailand. These results elucidate the presence of a unique SFG rickettsial species in Australasia.

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.

Protein characterization of Australian spotted fever group rickettsiae and monoclonal antibody typing of Rickettsia honei.

Rickettsial proteins rOmp A and rOmp B exist in both Rickettsia australis and Rickettsia honei but differ in molecular weight and antigenicity; in addition, they produce distinct immunogenic responses and appear to be to conformationally dependent antigens. Species-specific monoclonal antibodies for other spotted fever group rickettsial species did not react with R. honei. A PCR product of the repeat region of the rOmp A gene from R. honei was amplified and calculated to contain 11 repeat units.

Antigenic diversity of Rickettsia conorii.

Analysis of seven strains designated as Rickettsia conorii for reactivity with a panel of 12 monoclonal antibodies to surface-protein epitopes of spotted fever group rickettsiae and by Western immunoblotting with standard serotyping sera revealed remarkable antigenic diversity. Rickettsial strains from France, Morocco, Ethiopia, Kenya, South Africa, India, and the USSR differed from one another in reactivity with at least one and as many as five monoclonal antibodies. Simko and Indian strains were similar to one another and differed substantially from other R. conorii strains. All seven strains reacted with three R. conorii-specific monoclonal antibodies. Western immunoblotting demonstrated a major 120-kD protein and a major 135-kD protein in all strains. The principal differences were the presence of a major undenatured 130-kD protein in all strains except Indian and Simko, which had an analogous protein of 124 kD. Immunodominant antigenically related, heat-denatured protein bands of 170 kD (Malish 7 strain), 175 kD (Manuel strain), and 190 kD (Kenya tick typhus, Indian, and Simko strains) were not detected in the M-1 and Moroccan strains. This antigenic diversity is greater than that previously reported for other spotted fever group rickettsial species, suggesting that R. conorrii is an older species than R. rickettsii with a longer period of time for evolutionary divergence.

Antigenic Analysis of Chinese Strains of Spotted Fever Group Rickettsiae by Protein Immunobiotting

Protein immunoblotting demonstrated that 6 Chinese strains of spotted fever group (SFG) rickettsiae from northern China had antigenic polypeptides identical to those of Rickettsia siberica (strains 246 and 232), and dissimilar to other SFG rickettsiae. The various species of other SFG rickettsiae exhibited serologically distinct epitopes as well as many cross-reactive epitopes in protein immunoblotting. All SFG rickettsiae examined in this study had major antigenic polypeptides of 113-160 kDa.