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.     

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.     

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.     

Molecular evidence for novel tick-associated spotted fever group rickettsiae from Thailand

Ticks are of considerable medical and veterinary importance because they directly harm the host through their feeding action and indirectly through vectoring many bacterial pathogens. Despite many ticks being known from Thailand, very little is known about the bacteria they may harbor. We report here the results of a survey of tick-associated bacteria in Thailand. A total of 334 individuals representing 14 species of ticks in five genera were collected from 10 locations in Thailand and were examined for the human pathogens, Borrelia, Francisella, Rickettsia, and the common arthropod endosymbionts, Wolbachia, by polymerase chain reaction (PCR) assay using specific primers. Rickettsial DNA was detected in 30% (9/30) of Amblyomma testudinarium (Koch, 1844) collected from Khao Yai National Park, Nakhon Nayok Province and 16.84% (16/95) of Hemaphysalis ornithophila (Hoogstraal and Kohls, 1959) collected from Khao Yai National Park, Nakhon Nayok Province and Khao Ang Rue Nai Wildlife Sanctuary, Chachoengsao Province. Rickettsial DNA was not detected in any of the other tick species and no DNA of Borrelia, Francisella, or Wolbachia was detected in any of 14 tick species. Phylogenetic relationships among the rickettsiae detected in this study and those of other rickettsiae were inferred from comparison of sequences of the 17-kDa antigen gene, the citrate synthase gene (gltA), and the 190-kDa outer membrane protein gene (ompA). Results indicated that the three Thai rickettsiae detected in this study represent new rickettsial genotypes and form a separate cluster among the spotted fever group rickettsiae.     

Surface proteins of typhus and spotted fever group rickettsiae.

Six proteins, previously established as major constituents of intact organisms, were identified in cell envelopes obtained from intrinsically radiolabeled Rickettsia prowazekii. Extrinsic radioiodination of intact organisms conducted at 0.5 micronM iodide indicated that protein 4 was the most peripheral, although protein 1 also had reactive groups exposed on the surface of the organisms. A 10-fold increase in iodide concentration resulted in labeling of protein 2, and at 50 micronM iodide, all six major proteins were radiolabeled. Similar selective labeling was not achieved with R. conorii. Analysis of both typhus and spotted fever group organisms radiolabeled with galactose suggested that carbohydrate was associated with proteins 1, 3, and 4. Typhus soluble antigen included all major proteins except protein 2, which remained attached to particulate rickettsiae after ether extraction. Protein 4 appeared to be prominent in the surface topography of R. prowazekii, was a component of soluble antigen and may have an important role in rickettsiae-host interactions.     

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.     

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.     

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.     

福建省斑点热群立克次体的检测,分离及鉴定

作者用ＰＣＲ／ＲＦＬＰ分型系统首次对福建省宁化县和漳州市的蜱和啮齿动物进行了斑点热群立克次体的检测。并从宁化县收集的越原血蜱中分离到一株斑点热群立克次体，命名为ＮＨ－９５株，分别用ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎｂｌｏｔ及ＰＣＲ／ＲＦＬＰ进行了鉴定。结果显示：宁化县的越原血蜱、微小血蜱、金泽革蜱和漳州市的黄胸鼠可能感染西伯利亚立克次体；ＮＨ－９５株在蛋白多肽、抗原多肽和ＰＣＲ／ＲＦＬＰ图谱上均与西伯利亚立克次体一致，在中国南方地区首次从病原学证明存在北亚蜱传斑点热的自然疫源地。     

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.     

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

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

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.     

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.     

Indirect hemagglutination test for human antibody to typhus and spotted fever group rickettsiae.

An indirect hemagglutination (IHA) test is described that uses glutaraldehyde-stabilized erythrocytes treated with a rickettsial erythrocyte-sensitizing substance obtained from Rickettsia typhi or Rickettsia rickettsii. The serological reagent was stable for at least 3 months at room temperature and 6 months at 4 C. It exhibited group specificity and no group cross-reactivity. At a minimum dilution of 1:40, acute and early convalescent epidemic and murine typhus antisera showed 86% positive reactors, whereas similar spotted fever antisera had 74% positive reactors. In comparison with the indirect fluorescent antibody test, the IHA procedure gave lower titers but showed comparable detection of seroconversion with most paired sera. The IHA test demonstrated significantly higher titers than the complement fixation test and was more sensitive than either the complement fixation or Weil-Felix test in identifying seroconversion. No agglutination was observed when sensitized erythrocytes were tested with rodent sera known to contain rickettsial antibodies.     

Human and tick spotted fever group Rickettsia isolates from Israel: a genotypic analysis.

The genomes of spotted fever group rickettsiae isolated in different geographical areas of Israel (two from ticks and four from humans, obtained over a span of 20 years) were studied by polymerase chain reaction (PCR) and restriction endonuclease fragment length polymorphism (RFLP) analysis. The human isolates were obtained from patients suffering from rickettsial disease of different degrees of severity. The PCR products obtained with five pairs of oligonucleotide primers (two primer sets derived from the 190-kDa polypeptide gene and three from the 120-kDa polypeptide gene) and cleaved with restriction endonucleases were used to study the Israeli isolates and reference Rickettsia conorii isolates. Subtle differences between the PCR-RFLP patterns of Israeli isolates and the two R. conorii reference strains (Moroccan and no. 7) were seen when the PCR products derived from the 190-kDa gene-derived primer sets were digested. All of the Israeli isolates were identical by RFLP analysis using all of the primer sets. This study showed that the Israeli spotted fever group isolates (from both ticks and humans) were genetically homogeneous by the criteria used in this study, despite the time and location differences in their original isolation, and different as a group from R. conorii.     

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.     

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.