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At least 19 recordsLinked to original sources
PubMed2026

Detection of Rickettsia species with seroreactivity to Rickettsia tillamookensis and Rickettsia rickettsii subsp. californica in domestic dogs in Northern California.

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.

Animals↗
PubMed1977

Sensitive enzyme-linked immunosorbent assay for detection of antibodies against typhus rickettsiae, Rickettsia prowazekii and Rickettsia typhi.

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.

Animals↗
PubMed1979

In vitro studies of rickettsia-host cell interactions: ultrastructural changes induced by Rickettsia rickettsii infection of chicken embryo fibroblasts.

Secondary chicken embryo fibroblasts infected with the Sheila Smith strain of Rickettsia rickettsii and grown in monolayer culture undergo rapid morphological alterations. Transmission electron microscopic examination of cells at intervals after infection showed several progressive host cell lesions, including widespread dilatation of the rough endoplasmic reticulum and outer nuclear envelope and the accumulation of electron-dense material within the cisternae of intracellular membranes. Dilatation of the rough endoplasmic reticulum is a common, early reversible manifestation of other forms of cell injury. However, the severity of the damage to the host cell resulting from the progressive distention of intracellular membranes and the subsequent formation of small segments of membrane-bound host cytoplasm within the cisternae of these membranes is unknown. Early in the infection cycle, the rickettsiae were found free in the host cell cytoplasm, within invaginations of the nuclear envelope, occasionally free in the space between the outer and inner nuclear membranes, and in the host nucleoplasm, but not within cisternae formed by swollen endoplasmic reticulum. As a consequence of intracisternal swelling and fusion of intracellular membranes later in the infection cycle, the majority of the rickettsiae were found surrounded by host cytoplasm bound by host-derived internal membranes and appeared to persist in this state until cell lysis. The overall cytopathological changes in cells infected with R. richettsii appear dramatic and, from other studies in our laboratory, are significantly different from those observed in cells infected with Rickettsia prowazekii.

Animals↗
PubMed1978

External layers of Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer.

Using a simple specific-antibody stabilization procedure on organisms gently liberated from their host cells, we have demonstrated by electron microscopy that Rickettsia prowazekii and Rickettsia rickettsii possess a coat of variable thickness, external to the outer leaflet of the cell wall and the structure designated by others as a "microcapsule," which corresponds most closely to the slime layer of certain other bacteria. Reactions in the methenamine silver and ruthenium red staining procedures and the failure to be visualized by standard procedures suggest that the slime layer is largely polysaccharide in nature. It is postulated that this slime layer accounts in large part for the large, electron-lucent, halo-like zone which is found by electron microscopy to surround organisms of the typhus and spotted fever groups in the cytoplasm of their host cells, that it may be the locus of some major group-specific antigens, and that it may function as an antiphagocytic mechanism, as an aid for attachment of rickettsiae to potential host cells, or both. Moreover, because the attenuated E strain of R. prowazekii has been shown to possess a substantial slime layer, the basis for attenuation is not likely to be a simple smooth-to-rough variation.

Antigens, Bacterial↗
PubMed1978

Biochemical characteristics of typhus group rickettsiae with special attention to the Rickettsia prowazekii strains isolated from flying squirrels.

Six strains of Rickettsia prowazekii, two derived from human infections and four isolated from flying squirrels, two strains of R. typhi, and the single available strain of R. canada, were characterized by several biochemical procedures. The electrophoretic patterns on polyacrylamide gels of rickettsial proteins solubilized by sodium dodecyl sulfate revealed several species differences, but strains of the same species appeared to have identical patterns. Cytoplasmic fractions of the rickettsiae were examined for enzymatic activities and for polyacrylamide gel isoelectric focusing patterns. Some species differences were encountered in the activities or ratios of activities of glutamate-oxaloacetate transaminase, glutamate dehydrogenase, and malate dehydrogenase. When polyacrylamide gels were stained for malate dehydrogenase after electrophoresis, a single band became apparent with single extracts or mixtures of two strains of R. prowazekii, but two bands were seen with mixtures of a strain of R. prowazekii and one of R. typhi. The isoelectric focusing patterns of the soluble proteins revealed numerous species differences, especially between R. canada and the other two species, and a few differences among the strains of R. prowazekii. The patterns of the two human strains, Breinl and E(R), differed in at least one location, and both differed from the flying squirrel strains in the displacement of one band. One of the flying squirrel strains, GvF-16, contained a protein band not seen in the other five strains. Despite these minor differences, a striking similarity was revealed by all the biochemical tests performed between the R. prowazekii strains of human and flying squirrel origin.

Animals↗
PubMed1977

Antigenic relations of Rickettsia prowazekii and Rickettsia canada, established in the study of sera of patients with Brill's disease.

Sera of patients with Brill's disease and of healthy persons with spotted fever in their past history were examined in the complement fixation reaction (CFR) to determine antigenic relations between R. prowazekii and R. canada. R. canada was found to have common antigenic determinants with R. prowazekii and R. mooseri. However, the antigenic determinants of R. canada differed from those of the mentioned rickettsiae. The titres of complement-fixing antibodies in the sera of patients with Brill's disease with the antigen of R. mooseri were lower than the titres with the homologous antigen within the range of 1-2 twofold dilutions of the serum. However, the oscillations of the titres with the antigen of R. canada in the study of the same sera were expressed in 1-5 twofold dilutions. In serological identification of canada rickettsiosis, antigens of rickettsiae of the spotted fever group should invariably be included in the investigation of the sera.

Antigens↗
PubMed1979

Sensitive microplate enzyme-linked immunosorbent assay for detection of antibodies against the scrub typhus rickettsia, Rickettsia tsutsugamushi.

A microtiter enzyme-linked immunosorbent assay (ELISA) has been developed for the titration of antibodies against scrub typhus in human and animal sera. Scrub typhus rickettsiae were grown in monolayers of irradiated mouse LM3 cells and separated from host cell materials by differential centrifugation, filtration through a glass filter (AP-20, Millipore Corp.), and isopycnic banding in Renografin density gradients. The scrub typhus ELISA antigens were obtained from the purified viable rickettsiae by French pressure cell disruption and addition of 0.2% Formalin to the soluble extract. Antisera prepared in rabbits against the prototype Karp, the Kato, and the Gilliam strains of scrub typhus were used to standardize the ELISA and to compare its sensitivity and specificity to that of the indirect fluorescent antibody test (IFA). ELISA titers were measured as the greatest serum dilution showing an optical density 0.25 above controls or by the optical density achieved at a fixed serum dilution. The IFA and ELISA end point titers were quite similar, and all three measures of titer had comparable specificity for the strains of scrub typhus. No cross-reactions between the typhus and scrub typhus wera were observed by ELISA. Both the immunoglobulin M (IgM) and IgG antibody titers of 12 sequential sera from four patients with scrub typhus were obtained by IFA and ELISA. The IFA and ELISA end point titers for IgM and IgG had correlation coefficients of 0.91 and 0.97, respectively, whereas the ELISA optical density values at a serum dilution of 1:100 had slightly lower correlations with IFA titers (0.80 and 0.94). Early rising IgM titers followed by rising IgG titers were demonstrated by ELISA in three patients with primary scrub typhus infections, whereas the IgG response predominated in a patient with a reinfection. It is concluded that the ELISA for scrub typhus is a very satisfactory alternative to the IFA test.

Animals↗
PubMed1976

In vitro studies on Rickettsia-host cell interactions: lag phase in intracellular growth cycle as a function of stage of growth of infecting Rickettsia prowazeki, with preliminary observations on inhibition of rickettsial uptake by host cell fragments.

Two Rickettsia prowazeki seeds, an "early" seed in the logarithmic or exponential growth phase and a "late" seed in the stationary or possibly early decline phase, were prepared in chicken embryo (CE) cell cultures and compared with respect to morphology and infection cycle in CE cells in culture. Differences in size and ultrastructure of the organisms in the two seeds were similar to those seen in other gram-negative bacteria at comparable stages to growth. Vacuolar structures, rare in log-phase organisms, were common in stationary-phase organisms. Minute spherical forms reminiscent of minicells were seen in the stationary-phase preparations. In quantitative uptake experiments, organisms, typical in size and morphology of each preparation, had comparable capacity per plaque-forming unit to penetrate into CE cells in suspension when the seeds had been depleted of host cell membrane fragments and other debris. This suggests that host cell fragments, presumably of membrane origin, competitively inhibit rickettsial uptake by intact CE cells. Organisms of the log-phase organisms displayed a lag phase of about 7.5 h, during which they enlarged and increased in intensity of staining, before entering the log phase of growth.

Cells, Cultured↗
PubMed1979

Changes in immunoferritin labeling of Rickettsia tsutsugamushi after serial cultivation in 60Co-irradiated BHK cells.

The immunolabeling characteristics of Rickettsia tsutsugamushi (Gilliam strain) were examined by using a purified immunoglobulin G fraction of antibody to R. tsutsugamushi raised in rabbits. Formalin-fixed rickettsiae were reacted with this antibody and then with ferritin-conjugated goat anti-rabbit Fc antibody. R. tsutsugamushi cultivated in yolk sacs was used to raise antibody for this study. When rickettsiae in BHK-21 cells infected from yolk sac seed material were immunoferritin labeled, the binding of ferritin was found to be dense and uniform on the outer surface of the rickettsiae in disrupted host cells. Immunolabeling of purified suspensions of extracellular rickettsiae resulted in the uniform ferritin labeling of the microorganism. Aggregation of these rickettsiae by antibody appeared to depend upon the purity of the pellets. Immunoferritin labeling examined at high magnification revealed ferritin very close to the outer dense leaflet of the outer membrane. On some rickettsiae or on focal sites of others, the labelin; was several ferritin particles thick, suggesting the presence of a thick coating. The immunoferritin labeling of R. tsutsugamushi during successive serial passages in BHK-21 cells revealed decreased labeling with each passage, and by the 10th passage there was no detectable labeling. However, these rickettsiae inoculated back into yolk sacs regained their immunoferritin labeling. R. tsutsugamushi passed back into yolk sacs after four serial propagations in BHK-21 cells regained their labeling on the first passage in yolk sacs. However, rickettsiae from the 20th serial passage in BHK-21 cells required five passages in yolk sacs to reestablish their previous labeling affinity. Rickettsiae which did not label after 20 passages in BHK cells regained some of their labeling characteristics when sonicated. Antibody against rickettsiae cultivated in BHK-21 cells continued labeling rickettsiae even after 9 serial passages in BHK-21 cells.

Animals↗
PubMed1978

Penetration of cultured mouse fibroblasts (L cells) by Rickettsia prowazeki.

The association of Rickettsia prowazeki with L cells was examined by using a novel radioactive assay in which [alpha-(32)P]ATP-labeled rickettsiae were incubated with L-cell monolayers. Rickettsial association with the monolayer involved adherence and internalization steps that could be experimentally distinguished. Since R. prowazeki but not L cells possess an ATP-ADP obligate exchange transport system, addition of excess unlabeled ATP resulted in exchange of the labeled ATP from external, adherent rickettsiae but not from internalized rickettsiae. Rickettsial association was temperature dependent and was a linear function of both time and concentration. More than 90% of the biologically active rickettsiae associated with L cells was internalized. Rickettsial internalization required active participation of both rickettsiae and L cells; inactivation of either greatly reduced internalization. Rickettsial adherence to poisoned L cells was a saturable function of time and concentration. Adherence showed less temperature dependence than did internalization, but like rickettsial internalization, the extent of adherence was extremely low at 0 degrees C. The rate and extent of adherence by inactivated and native rickettsiae to inactivated L cells were similar. Although inactive rickettsiae adhered to active and inactive L cells to a similar extent, inactive rickettsiae were internalized poorly by active L cells. These data form the basis for the hypothesis that R. prowazeki are internalized by the host cell through a process of "induced phagocytosis" and that inactivated rickettsiae adhere to the host cell differently from native rickettsiae, failing to trigger the endocytosis mechanism.

4-Chloromercuribenzenesulfonate↗
PubMed2025

Polysaccharide synthesis operon modulates Rickettsia-endothelial cell interactions.

Pathogenic Rickettsia species target vascular endothelial cells and cause systemic vasculitis. As obligate intracellular bacterial pathogens, Rickettsia must secure nutritional resources within the cytoplasm of endothelial cells while simultaneously subverting the innate immune defense system. With advances in rickettsial and host genetics, recent studies have identified novel molecular mechanisms involved in the complex interactions between Rickettsia and endothelial cells. However, it remains unclear how Rickettsia shields pathogen-derived immune stimulants, such as lipopolysaccharides (LPS) and peptidoglycan fragments, from immune recognition during intracellular replication. Prior work described two Rickettsia conorii variants with kkaebi transposon insertions in the polysaccharide synthesis operon (pso). Biochemical and immunological analyses revealed that pso is responsible for the biosynthesis of O-antigen (O-Ag) and the proper assembly of surface proteins. In the present work, we document that pso variant HK2 exhibits reduced capacities to adhere to and invade microvascular endothelial cells. Despite the low intracellular abundance, HK2 induced significantly higher levels of proinflammatory cytokines and chemokines, leading to premature cell death. Notably, HK2 exhibited defective intracellular survival in bone marrow-derived macrophages. This inability to dampen endothelial cell-mediated immune stimulation and resist macrophage-induced bactericidal activities resulted in the rapid elimination of viable Rickettsia in the mouse model of spotted fever. Further, when tested as a live-attenuated vaccine, HK2 elicited robust protective immunity against lethal spotted fever pathogenesis. Our work highlights the crucial role of pso in enabling Rickettsia to evade immune surveillance during intracellular replication within endothelial cells, ultimately delaying pathogen-induced programmed cell death and escaping immune defense mechanisms.

Operon↗
PubMed1978

Antigenic relationships between the typhus and spotted fever groups of rickettsiae.

Paired sera from cases of epidemic typhus in Ethiopia and from probable cases of Rocky Mountain spotted fever (RMSF) in the United States were examined by microagglutination (MA) and microimmunofluorescence (micro-IF) tests for antibodies against Rickettsia prowazekii, Rickettsia typhi, Rickettsia canada, Rickettsia rickettsii, Rickettsia conorii and Ricksettsia akari. IgG and IgM antibodies against the various rickettsiae were titrated with specific fluorescein-conjugated anti-IgG and anti-IgM sera. Purified, particulate rickettsial antigens were employed in all tests. A majority of patients acutely ill with epidemic typhus produced both IgG and IgM antibodies against R. prowazekii, R. typhi and R. canada. Concurrently they produced IgG (but seldom IgM) antibodies against members of the spotted fever group. In contrast, patients ill with probably spotted fever, while producing IgG and IgM antibodies against R. rickettsii, R. conorii and R. akari, also produced both IgG and IgM antibodies with about equal frequency against members of the typhus group. It was concluded that a relatively broad antigenic relationship exists between rickettsiae of the typhus and spotted fever groups.

Agglutination Tests↗