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.     

Rocky Mountain spotted fever. Gastrointestinal and pancreatic lesions and rickettsial infection

Recent clinical studies have shown a high incidence of nausea, vomiting, diarrhea, and abdominal pain in Rocky Mountain spotted fever (RMSF), and case reports have documented rickettsial infection and vascular injury in the small intestine, appendix, and gallbladder. To determine the incidence and distribution of Rickettsia rickettsii and rickettsial lesions that might be the basis for these clinical manifestations of RMSF, tissues that were available from the stomach, small intestine, colon, and pancreas in fatal cases of RMSF were examined. Lesions were identified in pancreatic tissue in 91% of cases and in tissue obtained from the stomach, small intestine, and colon in all cases. Most tissues were judged to be only moderately injured. Organisms of R rickettsii were demonstrated by immunofluorescence in 14 (50%) of 28 cases and, when identified, correlated topographically with the location of vascular injury. These observations support the concept of rickettsial vascular injury of the gastrointestinal (GI) tract and pancreas leading to GI signs and symptoms in RMSF.     

Microimmunofluorescence test for the serological study of rocky mountain spotted fever and typhus.

A microimmunofluorescence test was used to study antibody responses to various spotted fever group and typhus group rickettsiae during Rocky Mountain spotted fever (RMSF) and epidemic typhus (ET). Patients with RMSF reacted most strongly to Rickettsia rickettsii; those with ET reacted predominantly to R. prowazekii. The degree of cross-reaction to other rickettsial strains varied from patient to patient, but a particular pattern of cross-reaction was consistently observed in serial sera from the same patient. Fresh isolates from three Montana RMSF cases were indistinguishable from each other and from strain R of R. rickettsii used as a standard antigen in all tests. Immunoglobulin M (IgM) antibodies were usually present in high titer in early-convalescent-phase sera from RMSF, as well as ET, patients. After RMSF, IgM antibodies persisted for a few months and, in one instance, for as long as 10 months. IgM responses to laboratory-acquired infections were infrequent in persons previously vaccinated with antigens related to the infecting strain. Previous antigenic conditioning from infection or vaccination may have accounted partly for the apparent lack of IgM response in a few study participants.     

Detection and identification of spotted fever group rickettsiae in Dermacentor species from southern California

Dermacentor occidentalis Marx and Dermacentor variabilis (Say) commonly bite humans in California. These Dermacentor species may play a role in transmitting spotted fever group (SFG) rickettsiae to humans in many parts of the state where Dermacentor andersoni Stiles, a known vector for the etiologic agent of Rocky Mountain spotted fever, Rickettsia rickettsii, is absent. However, the specific rickettsial agents present in these ticks and their current prevalence are poorly understood. In total, 365 D. occidentalis and 10 D. variabilis were collected by flagging vegetation at 16 sites in five counties of southern California. The presence of SFG rickettsial DNA in these ticks was detected with rOmpA and GltA gene polymerase chain reaction (PCR) assays. The rickettsial species were identified by sequencing PCR amplicons. Of 365 D. occidentalis, 90 (24.7%) contained R. rhipicephali DNA, 28 (7.7%) contained DNA of unclassified genotype 364D, two (0.55%) contained R. bellii DNA, and one (0.3%) contained R. rickettsii DNA. Of 10 D. variabilis, four (40%) contained only R. rhipicephali. Four new genotypes of R. rhipicephali were discovered. For the first time, we detected R. rickettsii in D. occidentalis. Our study provides the first molecular data on the prevalence and species identification of SFG rickettsiae circulating in populations of these California ticks. Because neither D. variabilis nor R. rickettsii were abundant, 364D should be evaluated further as a potential cause of human SFG rickettsioses in southern California.     

Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) from Kansas

The role of lone star ticks as vectors for Rocky Mountain spotted fever (RMSF) remains poorly described. We compared the entomological inoculation rates (EIRs) for Rickettsia spp. for representative sites in Missouri and Kansas, states that frequently report RMSF each year. Host-seeking ticks were collected during 2006 and pooled tick homogenates analyzed by polymerase chain reaction to detect probable R. rickettsii, with confirmation for multiple gene targets performed on individual ticks from pools that screened positive. Of 870 adult and nymphal lone star ticks, Amblyomma americanum (L.), 0.46% contained DNA of Rickettsia rickettsii. Interestingly, two of these positive ticks were concurrently infected by R. amblyommii. More than 90% of lone star tick pools contained R. amblyommii DNA. Of 169 dog ticks that were analyzed, none were infected by R. rickettsii. The entomological inoculation rate for spotted fever group (SFG) rickettsiae within lone star ticks was an order of magnitude greater than that for dog ticks. We conclude that lone star ticks may be epidemiologically significant vectors of Rocky Mountain spotted fever and of spotted fever group rickettsiae.     

Genetic variation in Australian spotted fever group rickettsiae.

Rickettsiae were isolated by cell culture of buffy coat blood from six patients with spotted fever from southeastern Australia and Flinders Island in Bass Strait. The isolates were genetically compared with two previous Rickettsia australis patient isolates. The genus-specific 17-kDA genes from the isolates were compared after DNA amplification and restriction fragment analysis of the amplified DNA. This comparison revealed that mainland rickettsial isolates from southeastern Australia were identical to two previous isolates of R. australis from northeastern Australia. Rickettsial isolates from Flinders Island were distinct from the mainland isolates. The 16S rRNA gene sequences from the isolates were determined and compared. The Flinders Island rickettsial agent was most closely related (0.3% structural divergence) to Rickettsia rickettsii, Rickettsia conorii, and Rickettsia slovaca. The Flinders Island rickettsial agent was 1.3 and 2.1% structurally divergent from R. australis and Rickettsia akari, respectively. The 16S rRNA gene sequence from the Flinders Island agent shows that this rickettsia is more closely related to the rickettsial spotted fever group than is R. australis. We conclude that there are two populations of spotted fever group rickettsiae in Australia and propose that the genetically distinct causative organism of Flinders Island spotted fever be designated Rickettsia honei. The extent of distribution and animal host reservoirs remain to be elucidated.     

Cloning, sequencing, and expression of the gene coding for an antigenic 120-kilodalton protein of Rickettsia conorii.

Several high-molecular-mass (above 100 kDa) antigens are recognized by sera from humans infected with spotted fever group rickettsiae and may be important stimulators of the host immune response. Molecular cloning techniques were used to make genomic Rickettsia conorii (Malish 7 strain) libraries in expression vector lambda gt11. The 120-kDa R. conorii antigen was identified by monospecific antibodies to the recombinant protein expressed on construct lambda 4-7. The entire gene DNA sequence was obtained by using this construct and two other overlapping constructs. An open reading frame of 3,068 bp with a calculated molecular mass of approximately 112 kDa was identified. Promoters and a ribosome-binding site were identified on the basis of their DNA sequence homology to other rickettsial genes and their relative positions in the sequence. The DNA coding region shares no significant homology with other spotted fever group rickettsial antigen genes (i.e., the R. rickettsii 190-, 135-, and 17-kDa antigen-encoding genes). The PCR technique was used to amplify the gene from eight species of spotted fever group rickettsiae. A 75-kDa portion of the 120-kDa antigen was overexpressed in and purified from Escherichia coli. This polypeptide was recognized by antirickettsial antibodies and may be a useful diagnostic reagent for spotted fever group rickettsioses.     

A recombinant Rickettsia conorii vaccine protects guinea pigs from experimental boutonneuse fever and Rocky Mountain spotted fever.

There are no vaccines against boutonneuse fever and Rocky Mountain spotted fever. Previous studies have identified a Rickettsia rickettsii surface protein as a vaccine candidate and shown that an antigenically related protein is present in R. conorii, which causes boutonneuse fever. The gene encoding the R. rickettsii protein has been cloned and expressed in Escherichia coli. We confirmed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rickettsial lysates followed by immunoblotting with a monoclonal antibody raised against the R. rickettsii protein that an analogous protein exists in R. conorii. Although these proteins were previously called 155-kilodalton (kDa) proteins, we found that their apparent molecular masses were 198 kDa for R. conorii Kenya tick typhus and 190 kDa for R. rickettsii R. Using the R. rickettsii gene probe, we cloned and expressed a 5.5-kilobase HindIII fragment from R. conorii Kenya tick typhus genomic DNA in E. coli JM107. The expressed recombinant product was recognized by a monospecific polyclonal rabbit antiserum prepared against the 198-kDa protein. Guinea pigs immunized with sonic lysates of the E. coli strain expressing the recombinant gene product developed antibodies recognizing R. conorii when tested by a microimmunofluorescence antibody assay. Upon immunoblotting of rickettsial lysates, those antisera specifically recognized the 198-kDa R. conorii protein and its 190-kDa analog in R. rickettsii. Guinea pigs immunized with sonic lysates of the recombinant E. coli expressing the 198-kDa protein were protected from experimental infections with the homologous R. conorii strain and partially protected from experimental infections with a strain of the heterologous species R. rickettsii. These findings show that the 198-kDa R. conorii protein is a candidate for a vaccine against boutonneuse fever.     

Computer simulation of Rocky Mountain spotted fever transmission by the American dog tick (Acari: Ixodidae)

A computer model was developed for simulation of the transmission of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), by the American dog tick, Dermacentor variabilis (Say). The model of RMSF was combined with a model for population dynamics of the American dog tick and included simulation of infection and transmission of rickettsiae between ticks and host mammals and transmission of RMSF to humans. The model simulated the effects of biotic and environmental variables such as weather, host density, habitat, transovarial transmission, fecundity of infected ticks, and infectivity level of ticks and mammals. Some parameters in the model were fitted by iterative simulations to produce realistic rates of R. rickettsii infection in adult ticks and small and medium-sized mammal hosts. Parameters also were fitted to yield the historical average number of RMSF cases for Virginia. Comparisons of the simulated and actual number of cases for nine other states indicated a reasonable level of validity for the model. A theoretical tick density threshold of 252 unfed adult ticks/ha for transmission of RMSF was determined from a relationship between rate of transmission to humans and density of ticks. The transmission threshold can be used for additional modeling efforts to study the effects of management technologies on tick densities and RMSF human cases. The model can serve as a framework for modeling other tick-borne diseases such as Lyme disease, babesiosis, and heartwater.     

Biological properties of rabbit antibodies to a surface antigen of Rickettsia rickettsii

Because of the potential significance of external components of the obligate intracellular parasite Rickettsia rickettsii in host-parasite interactions, we have begun the first phase of a study to isolate and characterize surface antigens of this organism. An antiserum to a rickettsial surface component was obtained from rabbits inoculated with immune precipitates prepared by crossed immunoelectrophoresis of Triton X-100 extracts of R. rickettsii strain R. This antiserum (i) protected guinea pigs inoculated with 10,000 guinea pig 50% infectious doses of R. rickettsii against fever, (ii) prevented death of mice challenged with 2 50% lethal doses of R. rickettsii, and (iii) reacted in the microimmunofluorescence test with 9 of 13 spotted fever group serotypes tested. The location of this antigen on the rickettsial surface was demonstrated by immunoelectron microscopy with ferritin-labeled antibodies.     

Fulminant Rocky Mountain spotted fever. Its pathologic characteristics associated with glucose-6-phosphate dehydrogenase deficiency

Three patients with documented fulminant Rocky Mountain spotted fever (RMSF) (death on or before day 5 of illness) had severe multisystemic injury as shown by clinical signs and laboratory data, but on microscopic examination showed minimal evidence of the typical mononuclear leukocytic response to rickettsial vascular infection and injury. Thrombosis was more extensive than in classic RMSF, with fibrin thrombi located in foci of rickettsial infection. These patients had a rash either preterminally or not at all, particularly severe Rickettsia-associated pulmonary lesions, and other shock-related lesions, eg, centrilobular hepatic necrosis. All three patients were male blacks with glucose-6-phosphate dehydrogenase deficiency, a condition recently associated with severity of RMSF. Diagnosis of fulminant RMSF requires awareness of its pathologic and epidemiologic aspects, and use of rickettsial isolation or specific immunofluorescence.     

Detection of Rickettsia tsutsugamushi in Experimentally infected mice by PCR.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.     

Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteins

Actin-based motility (ABM) is a mechanism for intercellular spread that is utilized by vaccinia virus and the invasive bacteria within the genera Rickettsia, Listeria, and Shigella. Within the Rickettsia, ABM is confined to members of the spotted fever group (SFG), such as Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Infection by each agent induces the polymerization of host cell actin to form the typical F (filamentous)-actin comet tail. Assembly of the actin tail propels the pathogen through the host cytosol and into cell membrane protrusions that can be engulfed by neighboring cells, initiating a new infectious cycle. Little is known about the structure and morphogenesis of the Rickettsia rickettsii actin tail relative to Shigella and Listeria actin tails. In this study we examined the ultrastructure of the rickettsial actin tail by confocal, scanning electron, and transmission electron microscopy. Confocal microscopy of rhodamine phalloidin-stained infected Vero cells revealed the typhus group rickettsiae, Rickettsia prowazekii and Rickettsia typhi, to have no actin tails and short (approximately 1- to 3-micrometer) straight or hooked actin tails, respectively. The SFG rickettsia, R. rickettsii, displayed long actin tails (>10 micrometer) that were frequently comprised of multiple, distinct actin bundles, wrapping around each other in a helical fashion. Transmission electron microscopy, in conjunction with myosin S1 subfragment decoration, revealed that the individual actin filaments of R. rickettsii tails are >1 micrometer long, arranged roughly parallel to one another, and oriented with the fast-growing barbed end towards the rickettsial pole. Scanning electron microscopy of intracellular rickettsiae demonstrated R. rickettsii to have polar associations of cytoskeletal material and R. prowazekii to be devoid of cytoskeletal interactions. By indirect immunofluorescence, both R. rickettsii and Listeria monocytogenes actin tails were shown to contain the cytoskeletal proteins vasodilator-stimulated phosphoprotein profilin, vinculin, and filamin. However, rickettsial tails lacked ezrin, paxillin, and tropomyosin, proteins that were associated with actin tails of cytosolic or protrusion-bound Listeria. The unique ultrastructural and compositional characteristics of the R. rickettsii actin tail suggest that rickettsial ABM is mechanistically different from previously described microbial ABM systems.     

Detection of Rickettsia rickettsii and Bartonella henselae in Rhipicephalus sanguineus ticks from California

Sixty-two questing adult Rhipicephalus sanguineus (Latreille) ticks were collected by direct removal from blades of turfgrass and adjacent concrete walkways at a suburban home in Riverside County, CA, and tested for the presence of Rickettsia, Bartonella, and Ehrlichia DNA. Polymerase chain reaction (PCR) was used to amplify fragments of the 17-kDa antigen gene and the rOmpA gene of the spotted fever group rickettsiae. One male tick contained R. rickettsii DNA; its genotype differed from R. rickettsii isolates found in Montana and Arizona that cause Rocky Mountain spotted fever and from Hlp#2 and 364D serotypes. One male tick and one female tick contained B. henselae DNA. No Ehrlichia platys or Ehrlichia canis DNAs were detected using nested PCR for their 16S rRNA genes. These findings extend the area where Rickettsia rickettsii may be vectored by Rh. sanguineus. Rh. sanguineus also may be infected with Bartonella henselae, a human pathogen that is typically associated with fleas and causes cat scratch disease.     

Evaluation of PCR-based assay for diagnosis of spotted fever group rickettsiosis in human serum samples

A nested PCR assay was developed for the detection of spotted fever group (SFG) rickettsiae in serum samples. The assay was based on specific primers derived from the rickettsial outer membrane protein B gene (rompB) of Rickettsia conorii. An SFG rickettsia-specific signal is obtained from R. akari, R. japonica, R. sibirica, and R. conorii. Other bacterial species tested did not generate any signal, attesting to the specificity of the assay. As few as seven copies of the rompB gene of R. conorii could be detected in 200 microl of serum sample. The assay was evaluated with a panel of sera obtained from patients with acute-phase febrile disease tested by immunofluorescent antibody assay (IFA). The SFG rickettsia-specific DNA fragment was detected in 71 out of 100 sera, which were proven to have immunoglobulin M antibodies against SFG rickettsial antigen by IFA. The results were further confirmed by restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated that this PCR assay is suitable for the diagnosis of spotted fever group rickettsiosis in Korea.     

Identification of a novel rickettsial infection in a patient diagnosed with murine typhus.

Identification of ELB agent-infected fleas and rodents within several foci of murine typhus in the United States has prompted a retrospective investigation for this agent among human murine typhus patients. This agent is a recently described rickettsia which is indistinguishable from Rickettsia typhi with currently available serologic reagents. Molecular analysis of the 17-kDa antigen gene and the citrate synthase gene has discriminated this bacterium from other typhus group and spotted fever group rickettsiae. Current sequencing of its 16S ribosomal DNA gene indicates a homology of 98.5% with R. typhi and 99.5% with R. rickettsii. Through a combination of restriction fragment length polymorphism and Southern hybridization analysis of rickettsia-specific PCR products, one of five tested patient blood samples was shown to be infected with ELB while R. typhi infections were confirmed in the remaining samples. This is the first reported observation of a human infection by the ELB agent and underscores the utility of PCR-facilitated diagnosis and discrimination of these closely related rickettsial infections.     

Effect of immunosuppression on Rickettsia rickettsii infection in guinea pigs.

The role of the immune response in the pathogenesis of Rickettsia rickettsii infection in guinea pigs was investigated by immunosuppression, using antilymphocyte serum. Twenty guinea pigs were inoculated with R. rickettsii, Sheila Smith strain, on day 0. Fifteen animals received antilymphocyte serum on days --1, 0, 2, 4, and 6. Five animals received normal rabbit serum on the same schedule. At necropsy, specimens were collected for histological examination, rickettsial immunofluorescence, rickettsial titration, and antirickettsial antibody titration. All normal rabbit serum recipients and 12 of 15 antilymphocyte serum recipients developed typical disease. Comparison of animals in terminal stages of disease revealed the same clinical course and gross lesions, but differing rickettsial burden and cellular response. Immunosuppressed animals had higher titers of splenic rickettsiae and greater numbers of immunofluorescent rickettsiae. Thus, although antibody was undetectable in both groups, there appeared to be an inhibition of antirickettsial immunity. Microscopic vasculitis was similar quantitatively, but differed qualitatively, with immunocompetent animals having the typical monouclear/lymphocytic inflammation and immunosuppressed animals having neutrophilic predominance. This study demonstrates that immunopathological mechanisms are not necessary for the pathogenesis of experimental Rocky Mountain spotted fever. The rickettsiae themselves seem capable of causing cellular and tissue damage.     

Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where Brazilian spotted fever is endemic

Owing to the potential role of the tick Amblyomma cooperi in the enzootic cycle of Rickettsia rickettsii, the etiologic agent of Brazilian spotted fever (BSF), this study evaluated infection by Rickettsia species in A. cooperi ticks collected from an area in Brazil where BSF is endemic. Among a total of 40 A. cooperi adult ticks collected in an area of BSF endemicity in the state of S?o Paulo, PCR analysis detected DNA of Rickettsia bellii in 16 ticks (40%), and 3 other ticks (7.5%) were positive for a previously unidentified spotted-fever-group (SFG) rickettsia. Cultivation in Vero cell cultures by the shell vial technique with individual A. cooperi ticks resulted in two isolates of R. bellii and one isolate genotypically characterized as an SFG rickettsia. The two R. bellii isolates were established in Vero cell cultures in the laboratory and were confirmed to be R. bellii by molecular analysis of the gltA and 17-kDa protein-encoding genes and by electron microscopic analysis. The SFG rickettsial isolate could not be stably passaged in cell culture in the laboratory, but molecular analysis of early passages suggested that it was closely related to Rickettsia parkeri, Rickettsia africae, and Rickettsia sibirica. These results do not support the role of A. cooperi in the ecology of R. rickettsii in the area studied, but they add two more species of rickettsiae to the poorly developed list of species occurring in ticks in South America.     

Demonstration and partial characterization of antigens of Rickettsia rhipicephali that induce cross-reactive cellular and humoral immune responses to Rickettsia rickettsii.

The relatively unrelated spotted fever group rickettsia Rickettsia rhipicephali conferred on guinea pigs protective immunity against challenge with virulent R. rickettsii. Immunity was conferred at all doses of R. rhipicephali used in the study. Because of the serologic unrelatedness of these two rickettsiae, determined by the use of microimmunofluorescence and other serological assays, further studies were performed to define the nature of the immune response elicited by R. rhipicephali and the characteristics of the rickettsial antigens that evoke cross-reactive antibody responses. Animals immune to R. rhipicephali tested at the time of challenge showed a complete cross-reactive lymphocyte proliferative response to rickettsial antigens prepared from each species. In fact, spleen cells from R. rhipicephali-immune animals responded better to R. rickettsii antigens than to homologous immunizing antigens. Serum samples were obtained from R. rhipicephali-infected animals at various times after infection and tested by the use of Western immunoblot assay for antibodies that were cross-reactive with antigens of R. rickettsii. By 10 days after infection with R. rhipicephali, antibodies to antigens of both species were noted, and by 37 days after infection, sera from immune animals showed strong reactivity to antigens of R. rhipicephali with apparent molecular masses of 107 and 151 kDa. The cross-reactive antibody response to antigens of R. rickettsii was relatively strong and involved predominantly the rOmpB protein and the rickettsial lipopolysaccharide. These findings establish the presence of T-cell-dependent epitopes associated with antigens of R. rhipicephali, which confer protective immunity against challenge with R. rickettsii. Results of Western immunoblot assays support the contention that the R. rickettsii rOmpB surface antigen contains important protective epitopes.     

Characterization and comparison of Australian human spotted fever group rickettsiae.

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus (QTT) caused by Rickettsia australis and the recently described Flinders Island spotted fever (FISF) were compared. Clinically and serologically, the diseases are similar. Cell culture reveals differences in the plaque-forming abilities of the isolates. Characterization of the gene encoding the genus-specific 17-kDa antigen of R. australis revealed a unique nucleotide sequence unlike those of the FISF isolate and Rickettsia rickettsii. Southern blot analysis of rickettsial DNA from the isolates with a 17-kDa-antigen gene probe revealed the presence of this gene in all isolates but no difference in banding patterns. When a probe for the rRNA genes was used, clear differences in banding patterns of isolates from patients with QTT and FISF were revealed. Thus, the rickettsiae isolated from patients with FISF differ from those from patients with QTT and may represent a new rickettsial species.