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In vitro activities of telithromycin compared to those of erythromycin against Rickettsia spp., Bartonella spp., Coxiella burnetii, and Ehrlichia chaffeensis were determined. Telithromycin was more active than erythromycin against Rickettsia, Bartonella, and Coxiella burnetii, with MICs of 0.5 microg/ml, 0.003 to 0.015 microg/ml, and 1 microg/ml, respectively, but was inactive against Ehrlichia chaffeensis.
Using PCR, we screened 411 ticks from four genera collected in Russia and Kazakhstan for the presence of rickettsiae and ehrlichiae. In Russia, we detected "Rickettsia heilongjiangensis," Rickettsia sp. strain RpA4, and Ehrlichia muris. In Kazakhstan, we detected Rickettsia sp. strain RpA4 and a rickettsia closely related to Rickettsia aeschlimannii. These agents should be considered in a differential diagnosis of tick-borne infections in these areas.
Rickettsia species phylotype G022 (G022) and Rickettsia tillamookensis both maintain a low infection prevalence in Ixodes pacificus ticks and share a close phylogenetic history with pathogenic Rickettsia species. The transmission mechanisms and pathogenic potential of I. pacificus-borne Rickettsia are poorly understood. The low prevalence of G022 and R. tillamookensis suggests that vertebrate species are utilized for maintenance, though identification of vertebrate amplifier and reservoir hosts utilized by Rickettsia is limited. To evaluate the molecular occurrence of G022 and R. tillamookensis and the sentinel utility of domestic dogs, a molecular survey was conducted. Buffy coat and blood plasma samples from 175 domestic dogs collected from Humboldt and Sonoma counties in northern California were screened for rickettsial DNA and anti-rickettsial antibodies, respectively. Dogs were screened for antibodies against Rickettsia spp. and tested for seroreactivity against R. tillamookensis, Rickettsia rickettsii subsp. californica, and Rickettsia rhipicephali. Because G022 has not yet been isolated, this Rickettsia species was omitted from the serological survey. Of the 175 dogs tested, three (1.71%; 95% CI: 0.58%-4.90%) were seropositive for Rickettsia spp. by indirect immunofluorescence antibody assay (IFA); two dogs (1.14%; 95% CI: 0.31%-4.10%) demonstrated seroreactivity by enzyme-linked immunosorbent assay (ELISA), suggesting previous exposure to Rickettsia spp. Among dogs from Sonoma County, 2.88% (3/104; 95% CI: 1.0%-8.14%) of plasma samples were seropositive for Rickettsia spp. by IFA, and 1.92% (2/104; 95% CI: 0.53%-6.74%) demonstrated seroreactivity by ELISA, while all the dogs from Humboldt County were seronegative for both methods. Although the ELISA results were not considered species-specific, two plasma samples demonstrated the greatest seroreactivity against R. tillamookensis and R. rickettsii subsp. californica, respectively, suggesting previous exposure to Rickettsia spp. with epitope similarity to R. tillamookensis and R. rickettsii subsp. californica, respectively. No DNA was detected for G022 or R. tillamookensis by quantitative real-time PCR, with a limit of detection of approximately 3.6 target copies per reaction for G022 and 1 genomic copy per 2 μl of template DNA per reaction for R. tillamookensis.
The eubacterial genus Rickettsia belongs to the alpha subgroup of the phylum Proteobacteria. This genus is usually divided into three biotypes on the basis of vector host and antigenic cross-reactivity characteristics. However, the species Rickettsia bellii does not fit into this classification scheme; this organism has characteristics common to both the spotted fever group and the typhus group biotypes and also exhibits some unique features. Sequences of the 16S rRNA and 23S rRNA genes from Rickettsia rickettsii (spotted fever group), Rickettsia prowazekii (typhus group), and R. bellii were studied to determine the position of R. bellii in the rickettsial classification scheme. The 23S rRNA gene sequences described in this paper are the first 23S rRNA sequences reported for any member of the Rickettsiaceae. The 23S rRNA gene contains substantially more phylogenetic information than is contained in the 16S rRNA sequences, and the 23S rRNA gene sequence has diverged about 1.9 times faster in the three Rickettsia species which we studied. Taken together, the molecular data obtained from the two genes indicate that R. bellii is not a member of either the spotted fever group or the typhus group; rather, this organism appears to be the product of a divergence which predates the separation of the genus into the spotted fever group and the typhus group. Consequently, different combinations of the ancestral characteristics retained by R. bellii have been retained in the more derived lineages of the genus.(ABSTRACT TRUNCATED AT 250 WORDS)
The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-beta and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-gamma), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.
To determine the phylogenetic position of two new rickettsial strains isolated from ticks in China, 16S ribosomal DNA, gltA, and ompA (apart from the tandem repeat units) genes were amplified by PCR and sequenced. The phylogenetic relationships between these strains and other rickettsiae were inferred from the comparison of sequences of the three genes by the parsimony, neighbor-joining, and maximum-likelihood methods. The results demonstrated that the 054 strain, a rickettsia pathogenic in humans, and the HL-93 strain were related and clustered together with Rickettsia japonica. Significant statistical bootstrap values (100 and 92%) supported the nodes in this cluster. Based on previous genotypic and antigenic data and the phylogenetic analysis presented here, the 054 and HL-93 strains should be considered as new species, and we formally propose that they be named "Rickettsia heilongjiangii" and "Rickettsia hulinii," respectively.
Levofloxacin, the L-isomer of ofloxacin, is approximately twice as active as ofloxacin against most Gram-positive and Gram-negative bacteria, and has improved intracellular pharmacokinetic and pharmacodynamic properties. The present work deals with the in-vitro activity of levofloxacin against the obligate intracellular bacteria Rickettsia rickettsii, Rickettsia conorii, 'Israeli spotted fever group rickettsia' (Israeli SFGR) and Coxiella burnetii. Fluoroquinolones, including ofloxacin, have previously been shown to be bacteriostatic against Rickettsia spp. and C. burnetii in vitro. They are reliable alternatives to tetracycline therapy for Mediterranean spotted fever, scrub typhus and acute Q fever. Levofloxacin was bacteriostatic against R. rickettsii, R. conorii and the Israeli SFGR at concentrations of 0.5-1 mg/L, as determined by both a plaque assay and a dye uptake assay. It was also bacteriostatic against C. burnetii isolates, including the Nine Mile, Priscilla and Q212 strains, at concentrations of 0.5-2 mg/L, as determined using the shell vial assay. Overall, levofloxacin could inhibit rickettsial growth at concentrations equal to or half of those necessary for growth inhibition by ofloxacin. Levofloxacin was not bactericidal against C. burnetii at concentrations up to 4 mg/L.
An enzyme-linked immunosorbent assay (ELISA) has been developed for the titration of rickettsial antibodies in human and animal sera. Two preparations of soluble typhus-group antigens were obtained from Rickettsia typhi and Rickettsia prowazekii by ether extraction: a standard antigen from infected yolk sacs (YS antigen) and one free of yolk sac contaminants from Renografin-purified rickettsiae (PR antigen). Rabbit, mouse, and guinea pig sera were obtained by immunization with viable purified R. typhi or R. prowazekii. Human sera were obtained from individuals who had recovered from laboratory infections with either typhus rickettsia months or years previously. Goat-derived anti-immunoglobulins were conjugated to alkaline phosphatase with glutaraldehyde. Although the PR and YS antigens gave equivalent antibody titers in the complement fixation test, the PR antigen was clearly superior in the ELISA. With this antigen, the titration curves of all antisera were linear over a wider range of serum concentrations and the titers were higher than with the YS antigen. With YS and PR antigens, ELISA titers were higher than those obtained by complement fixation by one and two orders of magnitude, respectively. In human sera, immunoglobulin G and immunoglobulin M antibodies were demonstrated by their respective anti-immunoglobulins and by differential susceptibility to ethanethiol. ELISA titers showed some type specificity, whereas none was observed in complement fixation tests. The ELISA is highly sensitive, reproducible, and easily adaptable to the various requirements of clinical and research laboratories.
Rickettsia typhi and Rickettsia conorii, the etiologic agents of, respectively, murine typhus and Mediterranean spotted fever, are recognized as frequent causes of fever of intermediate duration in southern Spain; in addition, in recent years Rickettsia felis has been detected in potential vectors in this area. Nevertheless, limited data exist regarding the actual prevalence of past infection due to these three pathogens. In the present study, the prevalence of past infection due to R. felis, R. typhi, and R. conorii was determined in a representative population of southern Spain during 2002. In addition, the possible risk factors associated with exposure to these pathogens were investigated. An epidemiological survey was completed by all subjects included in the study. Serum samples were tested by indirect immunofluorescence assay. The prevalence of past infection due to R. felis, R. typhi, and R. conorii among the 504 total subjects was 6.5, 3.8 and 8.7%, respectively. In multivariate analysis, infection due to R. felis was independently associated with a high-risk occupation (one that required working outdoors in nature, close contact with domestic animals, or potential contact with rodents) (OR=5.8; 95%CI 2.1-15.6), while infection due to R. typhi was associated with older age (factor of 1.04 [95%CI 1.008-1.068]) and frequent insect bites (OR=10.3; 95%CI 2.3-45.5). Two factors were associated with infection due to R. conorii: a high-risk occupation (OR=9.3; 95%CI 3.7-23.2), and participation in outdoor activities (OR=7.2; 95%CI 1.4-38.5). The results confirm the widespread prevalence of past infection due to R. felis, R. typhi, and R. conorii in the population of southern Spain.
The present study was undertaken to investigate the ability of members of two different groups of Rickettsia to stimulate macrophages or immune lymphocytes to produce TNF. It was found that R. conorii, a spotted fever group rickettsia, readily induced murine peritoneal macrophages or the macrophage-like cell line P388D1 to produce relatively high levels of TNF. The interaction of macrophages with viable organisms or heat-killed organisms resulted in TNF production. In contrast, viable or killed R. tsutsugamushi did not stimulate the production of detectable TNF even though viable organisms grew to high numbers in both cell types. It was found that the appropriate immune spleen cells stimulated with heat-killed R. tsutsugamushi or R. conorii produced TNF, and TNF activity was found in the sera of immune mice after injection with rickettsial antigen. Infection of naive mice with viable R. tsutsugamushi resulted in high TNF levels in ascites, but TNF was not found in ascites obtained from infected athymic (nu/nu) mice. These data support the suggestion that spotted fever group rickettsiae, such as R. conorii, possess components perhaps on the surface that interact with macrophages to induce TNF production and this component is lacking in R. tsutsugamushi. Antigens of R. tsutsugamushi and R. conorii will stimulate immune cells to produce TNF activity. These data are compatible with the suggestion that the TH-1 subset of T cells is predominant in immunity to R. tsutsugamushi.
Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy.
In serum samples obtained from all the healthy humans, horses, dogs, and donkeys present on three farms in the Pedreira Municipality, an endemic area for Brazilian spotted fever, an indirect immunofluorescence assay (IFA) detected antibodies against Rickettsia rickettsii in 17 (77.3%) horses, 5 (31.3%) dogs (titers ranging from 64 to 4,048), and none of 4 donkeys or 50 humans. Five canine and eight equine sera with high antibody titers to R. rickettsii were also tested by IFA against R. bellii, R. akari, and R. africae antigens. Sera from two horses and two dogs that showed similar high antibody titers against two rickettsial antigens were evaluated after cross-absorption. Sera from seven horses and two dogs contained antibodies specific for R. rickettsii, and one dog serum had antibodies against a Rickettsia species very closely related to R. africae. The latter may have been caused by infection with the recently identified COOPERI strain.
The Barbash strain of spotted fever group rickettsia was reexamined in this study by the microimmunofluorescence test with mouse antisera and with monoclonal antibodies. Protein immunoblotting was performed for comparison of purified antigens of R. rickettsii, R. sibirica, R. conorii and Barbash strain. Comparison of Barbash strain, R. rickettsii (Sheila Smith strain), R. conorii (Malish 7 strain), and R. sibirica (strains 232, 246 and Jinghe-74) of the spotted fever group in the microimmunofluorescence test of Philip et al. revealed that Barbash strain has antigens that yield homologous titers with the R. conorii strains and differ from R. sibirica and R. rickettsii. Monoclonal antibodies specific for R. conorii react at identical titres with the Barbash strain, and a monoclonal antibody specific for R. sibirica does not react with the Barbash strain. Likewise, T-cell hybridomas reactive with R. conorii but not R. sibirica yield a strong response when stimulated by Barbash strain antigens. Western immunoblotting with the same polyclonal and monoclonal antibodies confirmed the presence of specific protein antigens of R. conorii and different protein antigenic composition of R. sibirica when compared with Barbash strain. Thus, Barbash strain is a strain of R. conorii.
The antigens of Rickettsia sibirica, Rickettsia rickettsii, and Rickettsia conorii were examined by Western immunoblots and the standard immunofluorescent serotyping assay with mouse anti-Rickettsia sibirica serum that is used for determining the species identification of spotted fever group rickettsiae. Serum was employed prior to absorption and after absorption with purified native or heated Rickettsia sibirica. The unabsorbed antiserum, the antiserum absorbed with heated Rickettsia sibirica and the antibodies eluted from native Rickettsia sibirica recognized two antigenic polypeptides of Rickettsia sibirica (130 and 118 kDa), Rickettsia rickettsii (151 and 133 kDa), and Rickettsia conorii (136 and 113 kDa), respectively when examined in native state, not denatured by heat. The antiserum absorbed with native Rickettsia sibirica reacted most strongly with one polypeptide of Rickettsia sibirica (118 kDa), Rickettsia rickettsii (133 kDa), and Rickettsia conorii (136 kDa); at the endpoint of the immunofluorescence assay there was minimal reactivity with the 130 kDa polypeptide by immunoblotting. Both Western immunoblots and immunofluorescence assay showed that the antiserum absorbed with native Rickettsia sibirica reacted with homologous and heterologous antigens at a much lower titer than did the antiserum that was not absorbed. However, the titers of reaction with Rickettsia sibirica, Rickettsia rickettsii, or Rickettsia conorii were not diminished by absorption of the native antiserum with heated Rickettsia sibirica. The antibody eluted from native Rickettsia sibirica reacted by immunofluorescence with all the rickettsiae at a higher dilution than the antibody eluted from heated Rickettsia sibirica.(ABSTRACT TRUNCATED AT 250 WORDS)
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Murine typhus (MT) is a cause of fever of intermediate duration in the south of Spain. Rickettsia typhi has been described as the MT etiological agent. Rickettsia felis produces an infection similar to MT. The aim of the study is to determine their seroprevalence in humans in Catalonia. Antibodies to Rickettsia typhi and Rickettsia felis from 217 serum samples were examined by indirect immunofluorescence assay (IFA). Age, gender, residence area, contact with animals, and occupation were surveyed. Rickettsia typhi and Rickettsia felis seroprevalences were 8.8% and 3.2%, respectively. Rickettsia typhi was present in 7.6% of the samples in urban, 8.5% in semirural, and in 21.4% in rural areas, whereas Rickettsia felis was present in 3.5% in urban, 1.7% in semirural, and 7.1% in rural area. The only statistically significant association observed was that between Rickettsia felis seropositivity and age. Our data seem to indicate the presence of Rickettsia typhi and Rickettsia felis in humans in Catalonia.
Three common European 'anthrophilic' ticks, Ixodes ricinus, Haemaphysalis concinna and Dermacentor reticulatus, were collected in Hungary and tested, in assays based on nested PCR, for rickettsiae of the spotted-fever group. Low percentages of I. ricinus (2.7%) and H. concinna (1.0%) and a high percentage of D. reticulatus (26.8%) were found to be infected. The rickettsiae in the ticks were then identified, by sequencing of the genes coding for 16S ribosomal RNA (16S rDNA), citrate synthase (gltA) and the rOmpA outer-membrane protein (ompA), as Rickettsia helvetica, Rickettsia monacensis, Rickettsia sp. RpA4, or what is probably a newly recognized Rickettsia species ('Candidatus Rickettsia kotlanii'). These results raise the possibility that rickettsiae other than Rickettsia slovaca are involved in human disease in Hungary. Current knowledge on the distributions of the rickettsiae of the spotted-fever group that are emerging in Europe is also summarized.