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.     

Chemical properties of lipopolysaccharides from spotted fever group rickettsiae and their common antigenicity with lipopolysaccharides from Proteus species.

The lipopolysaccharides (LPS) isolated from spotted fever group (SFG) rickettsia strains Thai tick typhus TT-118 and Katayama were characterized by chemical analyses, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. These LPS did not contain heptose, but they contained 3-deoxy-D-manno-octulosonic acid (KDO), glucosamine, quinovosamine, phosphate, ribose, an unknown neutral sugar, and palmitic acid. Resolution of the apparent molecular masses of these LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with silver showed ladder-like bands. In an ELISA, convalescent-phase sera from 10 patients with Japanese spotted fever reacted with LPS from the Katayama strain, and 90% (9 of 10) of these sera also reacted with LPS isolated from Proteus vulgaris OX2. Immunoblotting revealed that the sera reacted with the high-molecular-mass bands of LPS from SFG rickettsiae, in addition to those of OX2 LPS. In an ELISA, immunoglobulin M antibodies from these sera reacted with the O-polysaccharide and lipid A portions of LPS from P. vulgaris OX2. The epitopes common to LPS of SFG rickettsiae and P. vulgaris OX2 may be in the O-polysaccharide and lipid A portions.     

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.     

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.     

Genotypic and antigenic identification of two new strains of spotted fever group rickettsiae isolated from China.

Four isolates of spotted fever group rickettsiae isolated from ticks in China were compared with all known species and strains of spotted fever group rickettsiae by immunofluorescence assay, DNA polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblot. The Chinese isolates belonged to three types, including a novel serotype which has not been described before. One isolate obtained from tick ova of Dermacentor nuttallii in Inner Mongolia was antigenically and genotypically identical to Rickettsia sibirica. Two isolates obtained from Dermacentor sinicus collected from Beijing were identical, different from other members of spotted fever group rickettsiae but apparently closely related to R. sibirica. HA-91, a strain isolated from Hyalomma asiaticum bv. kozlovi olenew, was antigenically and genotypically unique among spotted fever group rickettsiae, and we feel that data presented here should prompt consideration of it as a new species on the basis of current rickettsial taxonomy.     

Isolation of a spotted fever group rickettsia from a patient and related ecologic investigations in Xinjiang Uygur Autonomous Region of China.

Investigation of patients, healthy persons, and ticks in Jinghe County, Xinjiang Uygur Autonomous Region, People's Republic of China, for evidence of spotted fever group (SFG) rickettsiosis demonstrated strong evidence for a high prevalence of pathogenic SFG rickettsiae. Antibodies to SFG rickettsiae were detected in 62.5% of healthy subjects tested by enzyme-linked immunosorbent assay and 20% tested by complement fixation test. Two febrile patients were documented as having acute spotted fever rickettsiosis by complement fixation seroconversion. One, and 11-year-old Kazakh boy with eschar and regional lymphadenopathy, had an SFG rickettsia (An strain) isolated from his blood. A hemolymph test revealed that 20% of ticks contained rickettsiae. Two strains of SFG rickettsiae were isolated from male and female Dermacentor nuttalli ticks. The human SFG rickettsial isolate is the first to be obtained in the People's Republic of China.     

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.     

Characterization of Sicilian strains of spotted fever group rickettsiae by using monoclonal antibodies.

Twenty-two hybridomas producing anti-Rickettsia conorii monoclonal antibodies were obtained by nine fusion experiments. The strain chosen for immunization of mice was MAVI, an R. conorii strain isolated from a Sicilian patient with Boutonneuse fever. When tested for immunoglobulin isotype by an indirect immunofluorescence (IIF) assay, 46.6% of supernatants from the 22 hybridomas were immunoglobulin M. The supernatants were tested in the IIF assay for binding to the MAVI strain and four spotted fever group rickettsia strains isolated from Sicilian ticks (two virulent and two nonpathogenic when inoculated intraperitoneally in male guinea pigs). Only five of the supernatants showed a positive IIF result on all tested strains, although they produced different titers to the various strains, possibly an indication that they recognized an antigen common to spotted fever group rickettsiae. Immunodominant epitopes for humans were determined by using patient sera to analyze inhibition of binding to the MAVI strain. Although a limited number of serum samples were screened, a high percentage of Boutonneuse fever patients produced antibodies recognizing the same epitopes as were recognized by the mouse monoclonal antibodies. A striking heterogeneity was found both in the expression of mouse-recognized epitopes on the five rickettsial strains and in the serum antibody responses of Boutonneuse fever patients to these epitopes.     

Protective monoclonal antibodies recognize heat-labile epitopes on surface proteins of spotted fever group rickettsiae.

Thirty-eight monoclonal antibodies that have not been reported previously were developed from mice immunized with Rickettsia rickettsii, R. conorii, and R. sibirica. Western immunoblotting showed that these monoclonal antibodies are directed against heat-sensitive epitopes which are located on two major surface polypeptides with molecular sizes ranging from 115 to 150 kilodaltons. The detection of the two bands did not depend on the presence of 2-mercaptoethanol. Both bands were destroyed by treatment with proteinase K. Monoclonal antibodies examined by immunofluorescence assay reacted with epitopes that are species specific, group reactive, or shared among a smaller subset of species of spotted fever group rickettsiae. Nine of the monoclonal antibodies were evaluated for their ability to neutralize rickettsial infection and thus protect animals against disease caused by homologous species of rickettsiae. Treatment of rickettsiae with monoclonal antibodies F3-12, F3-14, and F3-36 completely protected guinea pigs against illness caused by the homologous organism R. rickettsii. Monoclonal antibodies F9-5G11 and F15-5B12, derived from mice immunized with R. sibirica, conferred partial protection by delaying the onset and shortening the duration of fever in guinea pigs inoculated with R. sibirica. Monoclonal antibodies F2-15, F2-31, F2-53, and F3-12 protected mice from a lethal infection with R. conorii. Heat-labile epitopes of spotted fever group rickettsial surface proteins are important candidate antigens for development of vaccines to confer protective immunity.     

Natural infection of dogs on Cape Cod with Rickettsia rickettsii.

Four isolates of rickettsiae from sick dogs on Cape Cod, Mass., were serologically identical to isolates of Rickettsia rickettsii from human patients with Rocky Mountain spotted fever. The antigenic analysis used the indirect fluorescent-antibody test and antisera prepared in mice to each of the isolates and to reference strains of R. rickettsii and Rickettsia montana. Serological responses of infected dogs were specific for R. rickettsii, although antibodies to R. montana were also detected in the sera of most of the canines.     

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.     

Epidemiology of Tsutsugamushi disease in relation to the serotypes of Rickettsia tsutsugamushi isolated from patients, field mice, and unfed chiggers on the eastern slope of Mount Fuji, Shizuoka Prefecture, Japan.

A total of 59 strains of Rickettsia tsutsugamushi were isolated from patients (24 isolates), Apodemus speciosus mice (30 isolates), and unfed larvae of Leptotrombidium scutellare (2 isolates) and Leptotrombidium pallidum (3 isolates) in the Gotenba-Oyama District, Shizuoka Prefecture, Japan. All these isolates were classified into the three serotypes Karp, Kawasaki, and Kuroki based on reactivity with strain-specific monoclonal antibodies. Kawasaki- and Karp-type rickettsiae were isolated from L. scutellare and L. pallidum, respectively, and the geographic distribution of patients and rodents infected with these two types of rickettsiae coincided with the areas densely populated by the respective chiggers. From these results, we conclude that Kawasaki-type rickettsiae are transmitted by L. scutellare and Karp-type ones are transmitted by L. pallidum. Kawasaki-type rickettsial infections were prevalent in early autumn, and Karp-type infections showed a peak of occurrence in the late autumn, reflecting the seasonal fluctuations of L. scutellare and L. pallidum. Isolates of Kuroki-type rickettsiae were obtained only from four patients in October and November, and the relationship between this type of rickettsia and its vector species could not be fully defined.     

Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens

The genetic and antigenic relatedness of eight antigens in three strains of Rickettsia tsutsugamushi has been studied by using recombinant organisms expressing epitopes of the 150-, 110-, 72-, 58-, 56-, 49-, 47-, and 20-kilodalton (kDa) polypeptide antigens of the Karp strain. Southern blot analysis of Karp, Kato, and Gilliam strain genomic DNA by using probes specific for each antigen class indicated that while strong homology exists between each of the corresponding antigen genes in these three strains, some restriction fragment length polymorphism exists. Antibodies affinity purified against each recombinant antigen class reacted with a comparably sized polypeptide in the Karp, Kato, and Gilliam strains in Western blots (immunoblots). Against more recent human isolates of R. tsutsugamushi, the affinity-purified antibodies against the 58-kDa recombinant antigen (anti-58-kDa) reacted with all nine isolates, anti-56-kDa reacted with eight of nine isolates, anti-47-kDa reacted with eight of nine isolates, anti-72-kDa reacted with eight of nine isolates, and anti-110-kDa reacted with four of nine isolates. Additional analysis indicated that the 110-kDa antigen may contain strain-specific epitopes similar to those previously reported for the 56-kDa polypeptide. Evidently, the strain heterogeneity among scrub typhus rickettsiae is a result of multiple components that exhibit variability in a background of strong homology.     

Serological studies of antigenic similarity between Japanese spotted fever rickettsiae and Weil-Felix test antigens.

Acute and convalescent-phase sera obtained from 10 patients infected with a Japanese strain of spotted fever group (SFG) rickettsia were tested by the indirect immunoperoxidase test, the Weil-Felix test, an enzyme-linked immunosorbent assay (ELISA), and immunoblotting. By the Weil-Felix test, the reactivity of these sera to the OX2 antigen was higher than those to the OX19 antigen, as is the case with sera from persons infected with other SFG rickettsiae. By ELISA, the titers of immunoglobulin M (IgM) antibodies against OX2 corresponded to the Weil-Felix test titers of these sera against OX2 but not to the titers obtained with IgG antibodies. The reactivity of the patient sera with the OX2 antigen in the Weil-Felix test was probably due to IgM antibodies against antigens which OX2 and SFG rickettsiae have in common. By immunoblotting tests, both IgG and IgM antibodies from the patient sera reacted with lipopolysaccharides from SFG rickettsiae and Proteus strain OX2. These results may show that these lipopolysaccharides contain similar epitopes.     

Reactivity of monoclonal antibodies to Rickettsia rickettsii with spotted fever and typhus group rickettsiae.

Analysis of 15 spotted fever group (SFG) and 2 typhus group strains of rickettsiae with a panel of monoclonal antibodies revealed a number of shared and unique epitopes of the 120- and 155-kilodalton surface proteins. All of the SFG strains but neither of the typhus group strains reacted with antibody to the lipopolysaccharidelike antigen of Rickettsia rickettsii; possibly the lipopolysaccharidelike antigen is the common antigen which defines the SFG. North Carolina and Montana strains of R. rickettsii known to differ slightly in virulence for guinea pigs differed in at least one epitope of the 120-kilodalton protein.     

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.     

Selective modulation of antioxidant enzyme activities in host tissues during Rickettsia conorii infection

The involvement of oxidative mechanisms in the pathogenesis of rickettsiosis was investigated using infection of C3H/HeN mice with sub-lethal and lethal infectious doses of Rickettsia conorii, the causative agent of Mediterranean spotted fever. Microscopic examination of tissues at 48 and 96 h post-infection revealed characteristic pathologic features and the presence of rickettsiae in the endothelium of infected tissues. Activities of key antioxidant enzymes, namely glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and superoxide dismutase, at these times exhibited a pattern of differential and selective modulation in brain, lungs, and testes of mice infected with viable organisms, whereas heat-inactivated or sonically disrupted rickettsiae had no effect. Of these, most significant changes were evident in the lungs of infected animals. Adaptive alterations in oxidant-scavenging enzymes occurred in apparent correlation with the dose and duration of infection. Treatment with an antioxidant, alpha-lipoic acid, protected against infection-induced oxidative injury via regulation of antioxidant enzyme activities and maintenance of reduced glutathione levels. These results suggest the involvement of regulatory enzymes of glutathione redox and superoxide scavenging systems in the antioxidant response during in vivo infection, the extent of which varies with the titer of viable rickettsiae in different organs of the host.     

Susceptibility of inbred mice to rickettsiae of the spotted fever group.

A mouse strain susceptible to lethal infection with Rickettsia conorii was required for testing vaccine efficacy and for studying the immunology and pathogenesis of infection. Among 20 strains of inbred mice inoculated intraperitoneally with the Malish strain of R. conorii, the C3H/HeJ mouse strain was the most susceptible, with a 50% lethal dose of approximately 10 PFU. Infection of all mouse strains resulted in a measurable antibody response; the highest titers correlated with the greatest degree of rickettsial replication as measured by plaque assay of infected spleen homogenates. Inoculation of C3H/HeJ mice with 5.0 log10 organisms of strain Malish by the subcutaneous route did not result in lethal infection. The Casablanca and Moroccan strains of R. conorii were not lethal for C3H/HeJ mice and, in addition, produced plaques in L-929 cells morphologically distinct from those produced by the Malish strain. The only other spotted fever group rickettsia tested which produced a lethal infection in C3H/HeJ mice was Rickettsia sibirica. Sublethal infection with any of the spotted fever rickettsiae tested protected against lethal infection with R. conorii. These data established a lethal challenge system for examining the protective efficacy of spotted fever immunogens and presented evidence of biological variation among strains of R. conorii.     

Properties of selected rickettsiae of the spotted fever group.

Eight strains of spotted fever group rickettsiae were studied to gain insight into the extent of variation of their properties. Two standard strains of Rickettsia rickettsii and one strain of Rickettsia conorii were included among the eight for comparison. The molar percentage of guanine plus cytosine for each strain did not differ significantly from that for R. rickettsii, 32.6 +/- 0.7%. Two strains caused extended fever in guinea pigs, one strain caused fever of short duration, and the other strains induced little or no fever. Polyacrylamide gel electrophoresis of the detergent-solubilized rickettsial proteins indicated that the protein content of all strains, except the two strains of R. rickettsii, were different, particularly in the molecular weight range of 40,000 to 60,000. Virulent strains produced large clear plaques in Vero cells monolayers; the strains of low virulence generally produced smaller or more turbid, or both, plaques. On the basis of agglutination reactions with rabbit antisera, the eight strains were placed into five serotypes. These results indicate considerable heterogeneity in properties of spotted fever group rickettsiae in the United States.     

First isolation ofRickettsia helvetica fromIxodes ricinus ticks in France

Two rickettsial isolates recovered fromIxodes ricinus ticks in Puy-de-Dôme (Central France) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot immunoassay, restriction fragment length polymorphism analysis, and sequencing of a portion of the citrate-synthase gene. By these methods, the isolates appeared to be identical to a member of the spotted fever group rickettsiae,Rickettsia helvetica. This first isolation ofRickettsia helvetica in France has epidemiologic importance; a serosurvey on Mediterranean spotted fever conducted previously in Puy-de-Dôme, where the infection is not endemic, demonstrated a high seroprevalence of nonspecific antibodies directed against spotted fever group rickettsiae lipopolysaccharides, and thus the possibility of infection due to a rickettsia different fromRickettsia conorii was suggested. The isolation ofRickettsia helvetica in anthropophilic ticks in the same area further supports this hypothesis.