Addition of monoclonal antibodies specific for Rickettsia akari to the rickettsial diagnostic panel.

Monoclonal antibodies were produced from mice infected with Rickettsia akari (the etiologic agent of rickettsialpox) and evaluated for specificity in indirect fluorescent-antibody tests with 23 different rickettsial antigens. Of the nine antibodies that were evaluated, two were specific for R. akari and four reacted with R. akari and all other spotted fever group rickettsiae. The remaining three antibodies reacted with some, but not all, members of the spotted fever group. None of the antibodies reacted with typhus, scrub typhus, trench fever, or Q fever rickettsiae. Adding these antibodies to the list of available diagnostic reagents will facilitate identification of rickettsial diseases, particularly those caused by members of the spotted fever group, where the clinical presentations are similar and the etiologic agents are closely related antigenically.     

Serological approach to the occurrence of rickettsioses in the Central African Republic

A serosurvey for evidence of human rickettsial infections was carried out in the Republic of Central Africa on 144 sera by indirect immunofluorescence (IIF) and microagglutination tests (MA). There was no serological evidence of epidemic typhus and only two sera were positive for murine typhus. Approximately 15% of the surveyed population was serologically positive by MA for R. conorii antibodies. However, 48% of this population had spotted fever group antibodies as detected by IIF but were negative in MA for R. conorii, R. rickettsii and R. akari antibodies. These sera with high titers in IIF and negative in MA lead us to believe that in Central Africa there are rickettsiae pathogenic for man that are related to the Spotted Fever group and are yet to be identified.     

A rickettsia-like organism showing positive immunofluorescence with antisera to Coxiella burnetii in Haemaphysalis inermis ticks

A rickettsia-like organism (RLO) was detected in the oocytes of Haemaphysalis inermis ticks. The RLOs were about 5 microns long bent rods with a definite inner structure. They were Gram-negative and could be visualized by Giemsa but not by Gimenez staining. Attempts to cultivate the RLO in chick embryo yolk sacs, various types of cell culture and tick body cavities were unsuccessful. The RLO displayed a bright immunofluorescence with antisera to Coxiella burnetii, but no immunofluorescence was obtained with antisera to representatives of typhus and spotted fever group rickettsiae, with the exception of very weak fluorescence with serum from a rabbit immunized with Rickettsia akari and one serum from Apodemus flavicollis immunized with Rickettsia conorii. These findings should be taken into consideration when studying the infestation of ticks with rickettsiae.     

Proteins of typhus and spotted fever group rickettsiae.

Purified radioactive rickettsiae were obtained from irradiated and cycloheximide-inhibited L cells, and their proteins were analyzed by polyacrylamide gel electrophoresis. Rickettsial species could be distinguished by comparing the relative mobilities of constituent proteins after migration of two differentially labeled preparations in a single gel. Distinct differences were observed in gel patterns of rickettsiae from the typhus and spotted fever groups, as well as with different species within a group. Rickettsial organisms causing murine and epidemic typhus were clearly distinguished, as were the causative agentsof boutonneuse fever and rickettsialpox. The use of both internal and external molecular weight standards allowed molecular weight estimates for 19 proteins from both Rickettsia prowazekii and Rickettsia conorii. A flexible system for designating rickettsial proteins is proposed that lends itself to modification as more detailed analysis progresses.     

First serologic evidence of human spotted fever group rickettsiosis in Korea

To investigate the prevalence of spotted fever group rickettsioses in Korea, a serosurvey of Japanese spotted fever rickettsiosis in patients with acute febrile illness was conducted with an indirect immunofluorescence assay. Overall, 19.88% of the patients were found to have polyvalent antibody against Rickettsia japonica. This study is the first documentation of spotted fever group rickettsiosis in Korea.     

Development and characterization of high-titered, group-specific fluorescent-antibody reagents for direct identification of rickettsiae in clinical specimens.

Rabbits were inoculated with purified antigen preparations of Coxiella burnetii and representative species of the spotted fever and typhus groups of rickettsiae. Their antibody responses were monitored by complement fixation tests; high-titered antisera were fractionated with ammonium sulfate and then labeled with fluorescein isothiocyanate by the dialysis method. The conjugates had homologous 3+ staining titers of 1:256 to 1:2,048 and did not exhibit nonspecific staining. The Rickettsia rickettsii, R. conorii, and R. akari conjugates reacted only with rickettsiae of the spotted fever group; the R. canada, R. prowazekii, and R. typhi conjugates were specific for the typhus group rickettsiae; and the C. burnetii conjugate stained only homologous organisms. One of these conjugates (R. rickettsii) is currently being used to identify rickettsiae in clinical specimens and has already proven its value as a diagnostic tool.     

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.     

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.     

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.     

Production and characterization of monoclonal antibodies to Rickettsia rickettsii

Five mouse ascitic fluids (MAFs) containing monoclonal antibody to Rickettsia rickettsii were produced from three original fusions by murine hybridoma technology. The five MAFs were fractionated and purified; each contained monoclonal antibody of the immunoglobulin G2a subclass. Each monoclonal antibody-containing MAF was titrated by indirect immunofluorescence against three R. rickettsii isolates from humans and four other spotted fever group rickettsiae. Each MAF was also titrated in the complement fixation, latex agglutination, microagglutination, and indirect hemagglutination tests. Two of the MAFs were examined for their ability to prevent fever and rickettsemia in susceptible guinea pigs after a 1:100 dilution of each was mixed with viable R. rickettsii, and all five MAFs were titrated in the mouse toxicity phenomenon assay. All MAFs had high indirect immunofluorescence titers to the three strains of R. rickettsii (1:200,000 to 1:800,000), reduced indirect immunofluorescence titers to R. montana, and were nonreactive with R. akari, R. sibirica, and R. conorii. Each MAF was able to fix complement in the presence of spotted fever group antigen reagent and agglutinate a suspension of purified R. rickettsii, and each was negative in both the latex agglutination and the indirect hemagglutination tests. The two MAFs which were tested proved to be capable of preventing rickettsemia and death in guinea pigs, and each MAF was able to prevent death in mice at dilutions ranging from 1:40 to 1:80.     

Biological Properties of Rickettsia prowazekii Strains Isolated from Flying Squirrels

Four strains of Rickettsia prowazekii, isolated from flying squirrels (Glaucomys volans volans) from Florida and Virginia, were compared with other strains of the typhus biotype, two previously established strains each of R. prowazekii and R. typhi and one strain of R. canada, for similarities in a number of unrelated phenotypic characteristics. R. akari served as a spotted fever biotype control. All strains produced small plaques on chicken embryo cell monolayers that were clearly recognized only after 10 days of incubation at 32 degrees C. All strains were highly susceptible to erythromycin. The Renografin density gradient centrifugation procedure of separating rickettsiae from the infected yolk sacs of surviving chicken embryos was equally satisfactory in all cases and resulted in moderate to large yields of purified rickettsiae. There was relatively small variation in specific hemolytic activity or specific CO(2) formation from glutamate. None of the strains catabolized glucose. There was some strain variation in virulence for the chicken embryo, but none of the above tests separated the three species of the typhus biotype. On the other hand, R. akari was clearly distinguished by its more rapid plaque formation and by higher resistance to erythromycin. It is concluded that by the tests conducted thus far, the biological properties of the flying squirrel strains do not differ substantially from those of other strains of the typhus biotype.     

Primary surveillance of spotted fever group antibodies on rats in the Kinmen area

The positive rate of rickettsial antibodies of 107 rats in the Kinmen area by indirect immunofluorescent antibody (IFA) technique was 0% (0/107) in typhus fever, 38.3% (41/107) in scrub typhus and 66.4% (71/107) in spotted fever group; the positive rate (42.9%) of spotted fever group of 21 rats in Taiwan island also higher than scrub typhus (19.0). It suggests that spotted fever group patients may be present in our country but have not been discovered.     

Proteinic and genomic identification of spotted fever group rickettsiae isolated in the former USSR.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), restriction fragment length polymorphism of polymerase chain reaction-amplified genes (RFLP-PCR), and pulsed-field gel electrophoresis (PFGE) were used to identify 25 isolates of spotted fever group rickettsia collected in the former USSR. Six Rickettsia akari isolates which were identical to the MK reference strain from the American Type Culture Collection were found. Also, 14 isolates were found to be Rickettsia sibirica and identical to reference strain 246. Two of three isolates previously considered as atypical, low-pathogenic strains of R. sibirica, were found to be strains of Rickettsia slovaca. The third, strain S, was similar in its RFLP-PCR profile to "R. africae" sp. nov. (proposed name for a rickettsia pathogenic for human beings in southern Africa) but in its SDS-PAGE and PFGE profiles was unique among spotted fever group rickettsiae. Strain M-1 was confirmed as a genetic variant of Rickettsia conorii. The Astrachan isolate, the causative agent of a tick-bite rickettsiosis at the North of the Caspian Sea, showed a previously described RFLP-PCR profile identical to that of the Israeli tick typhus rickettsia, but its SDS-PAGE and PFGE profiles different from those of the other strains tested.     

Evidence of Rickettsia spp. infection in Sweden: a clinical, ultrastructural and serological study

Sweden is an area potentially endemic for spotted fever rickettsioses. Rickettsia helvetica has been isolated from its tick vector Ixodes ricinus, and in a handful of cases linked to human disease. This study demonstrates for the first time in Sweden the transmission of rickettsial infection after a tick bite and the attack rate in an endemic area. We present three cases of documented rickettsial infection and a prospective serological study of Swedish recruits who were trained in the area where the patients lived and showed seroconversion to spotted fever rickettsiae. All patients showed a four-fold increase in antibody titer to the spotted fever rickettsia, R. helvetica, and immunohistochemical examination revealed rickettsia-like organisms in the walls of skin capillaries and veins. Electron microscopy showed organisms resembling R. helvetica and immunogold labeling with two anti-rickettsial antibodies demonstrated specific labeling of the rickettsial organisms in the skin biopsy specimens. Eight of the thirty-five recruits showed a four-fold increase in IgG titer reflecting a high rate of exposure. The results of this study demonstrate that spotted fever rickettsioses should be taken into consideration in the diagnosis of tick-transmitted infections in Sweden.     

Prevalence of vectors of the spotted fever group Rickettsiae and murine typhus in a Bedouin town in Israel

A survey of the vectors of spotted fever group Rickettsiae and of murine typhus was carried out in Rahat, a Bedouin town in the Negev Desert, where the diseases are endemic. Houses with known cases of spotted fever group Rickettsiae or murine typhus were compared with those without reported clinical cases. A neighboring Jewish community, Lehavim, where no cases of spotted fever group Rickettsiae and murine typhus were reported in recent years, was used as a control. In the houses of patients with spotted fever group Rickettsiae in Rahat, an average of 7.4 times more ticks were found than in control houses. Out of 190 ticks isolated from sheep and goats or caught by flagging in Rahat, 90% were Rhipicephalus sanguineus (Latreille), 7.9% Rhipicephalus turanicus Pomerantzev, and 2.1% were Hyalomma sp. In the houses of patients with murine typhus, three times more rats were caught and, on the average, each rat was infested with 2.2 times more fleas than rats in the control houses. Out of 323 fleas collected from 35 Norwegian rats (Rattus norvegicus Berkenhout), 191 were Xenopsylla cheopis Rothschild and 132 Echidnophaga murina Tiraboschi. Thus, there was a six to seven times higher probability of encountering a tick or flea vector where infections had occurred than in control houses in Rahat. The percentage of rats seropositive to Rickettsia typhi was similar in study and control households (78.3 and 76.2, respectively). In the control settlement, Lehavim, only three Mus musculus L. were caught, which were not infested with ectoparasites and their sera were negative for murine typhus. Out of 10 dogs examined in this settlement, 15 R. sanguineus and eight specimens of the cat flea (Ctenocephalides felis felis Bouché) were isolated. No rats were caught in this settlement. These data indicate that there is a correlation among the density of domestic animals, their ectoparasites, and the incidence of spotted fever group Rickettsiae and murine typhus in Rahat.     

Immune responses to Rickettsia akari infection in congenitally athymic nude mice.

Athymic BALB/c nude mice and euthymic BALB/c mice were infected with Rickettsia akari by the intraperitoneal route. The rickettsialpox infection was terminated in euthymic mice with only two intraperitoneal injections of the antibiotic oxytetracycline, whereas prolonged treatment was necessary to terminate the infection in athymic mice. Both athymic and euthymic mice produced specific antibody, but athymic mice were still susceptible to reinfection. Killed R. akari served as a protective immunogen in euthymic, but no in athymic, mice. When spleen cells from convalescent euthymic mice were transferred to syngeneic athymic mice, recipients showed protection against challenge. This suggests that a T-cell-dependent step is generally necessary to terminate the rickettsialpox infection.     

Rhipicephalus sanguineus: vector of a new spotted fever group rickettsia in the United States.

A rickettsia related to but distinct from the spotted fever agent, Rickettsia rickettsii, has been detected in 167 (18.9%) of 884 Rhipicephalus sanguineus taken off dogs in central and northern Mississippi. The organisms could readily be isolated in male meadow voles (Microtus pennsylvanicus), where it produced massive infections in the tissues of tunica vaginalis. It was practically nonpathogenic for male guinea pigs, although inoculation of these animals with infected tunica vaginalis of voles afforded in 30 of 38 instances solid immunity to challenge with virulent R. rickettsii. The Rhipicephalus rickettsia grew well in monolayers of chicken embryo fibroblast, Vero, mouse L, and HeLa cells. Cytopathogenic effects were minimal unless large concentrations of rickettsiae were used as inocula. It also could be established in embryonated hen eggs but only after injection of massive doses of L cell-propagated organisms. Serological tests (complement fixation, microagglutination and/or micro immunofluorescence) indicated that the newly described Rickettsia belongs to the spotted fever group but differs from R. rickettsii, R. akari, and R. conorii. Antigenic differences were also demonstrated by direct fluorescence microscopy as well as by vaccine potency and mouse-toxin neutralization tests.     

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.