Identification of Rickettsia rickettsii in formalin-fixed, paraffin-embedded tissues by immunofluorescence.
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With slight modification of a trypsin digestion technique, Rickettsia rickettsii were demonstrated specifically by immunofluorescence staining in Formalin-fixed, paraffin-embedded tissue sections from a human, rhesus monkey, and guinea pig with Rocky Mountain spotted fever and in infected membranes from a chicken embryo. Tissues were cut at 4 micron and, using geltain as a tissue adhesive, were hydrated in a routine manner. Sections were then digested in refrigerated 0.1% trypsin for 16 h, washed, and stained specifically for R. rickettsii by direct or indirect immunofluorescence. Rickettsial organisms were localized in affected vessels of the mammalian species and within the yolk sac epithelium of the chicken embryo. Specificity was confirmed by adsorbing antibody conjugates with R. rickettsii organisms. Trypsin digestion probably decreased tissue proteins which interfered with immunochemical attachment of antibody to the rickettsiae. The technique is valuable in that a diagnosis of Rocky Mountain spotted fever can be confirmed from Formalin-fixed tissues processed in a routine manner. (+info)
Efficacy of doxycycline, azithromycin, or trovafloxacin for treatment of experimental Rocky Mountain spotted fever in dogs.
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Dogs were experimentally inoculated with Rickettsia rickettsii (canine origin) in order to compare the efficacies of azithromycin and trovafloxacin to that of the current antibiotic standard, doxycycline, for the treatment of Rocky Mountain spotted fever. Clinicopathologic parameters, isolation of rickettsiae in tissue culture, and PCR amplification of rickettsial DNA were used to evaluate the response to therapy or duration of illness (untreated infection control group) in the four groups. Concentrations of the three antibiotics in plasma and blood cells were measured by high-performance liquid chromatography. Doxycycline and trovafloxacin treatments resulted in more-rapid defervescence, whereas all three antibiotics caused rapid improvement in attitudinal scores, blood platelet numbers, and the albumin/total-protein ratio. Based upon detection of retinal vascular lesions by fluorescein angiography, trovafloxacin and doxycycline substantially decreased rickettsia-induced vascular injury to the eye, whereas the number of ocular lesions in the azithromycin group did not differ from that in the infection control group. As assessed by tissue culture isolation, doxycycline resulted in the earliest apparent clearance of viable circulating rickettsiae; however, rickettsial DNA could still be detected in the blood of some dogs from all four groups on day 21 postinfection, despite our inability to isolate viable rickettsiae at that point. As administered in this study, trovafloxacin was as efficacious as doxycycline but azithromycin proved less efficacious, possibly due to the short duration of administration. (+info)
Surface proteins of typhus and spotted fever group rickettsiae.
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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. (+info)
Hidden mortality attributable to Rocky Mountain spotted fever: immunohistochemical detection of fatal, serologically unconfirmed disease.
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Rocky Mountain spotted fever (RMSF) is the most severe tickborne infection in the United States and is a nationally notifiable disease. Since 1981, the annual case-fatality ratio for RMSF has been determined from laboratory-confirmed cases reported to the Centers for Disease Control and Prevention (CDC). Herein, a description is given of patients with fatal, serologically unconfirmed RMSF for whom a diagnosis of RMSF was established by immunohistochemical (IHC) staining of tissues obtained at autopsy. During 1996-1997, acute-phase serum and tissue samples from patients with fatal disease compatible with RMSF were tested at the CDC. As determined by indirect immunofluorescence assay, no patient serum demonstrated IgG or IgM antibodies reactive with Rickettsia rickettsii at a diagnostic titer (i.e., >/=64); however, IHC staining confirmed diagnosis of RMSF in all patients. Polymerase chain reaction validated the IHC findings for 2 patients for whom appropriate samples were available for testing. These findings suggest that dependence on serologic assays and limited use of IHC staining for confirmation of fatal RMSF results in underestimates of mortality and of case-fatality ratios for this disease. (+info)
Evidence of rickettsial spotted fever and ehrlichial infections in a subtropical territory of Jujuy, Argentina.
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Between November 1993 and March 1994, a cluster 6 pediatric patients with acute febrile illnesses associated with rashes was identified in Jujuy Province, Argentina. Immunohistochemical staining of tissues confirmed spotted fever group rickettsial infection in a patient with fatal disease, and testing of serum of a patient convalescing from the illness by using an indirect immunofluorescence assay (IFA) demonstrated antibodies reactive with spotted fever group rickettsiae. A serosurvey was conducted among 16 households in proximity to the index case. Of 105 healthy subjects evaluated by IFA, 19 (18%) demonstrated antibodies reactive with rickettsiae or ehrlichiae: 4 had antibodies reactive with Rickettsia rickettsii, 15 with Ehrlichia chaffeensis, and 1 with R. typhi. Amblyomma cajennense, a known vector of R. rickettsii in South America, was collected from pets and horses in the area. These results are the first to document rickettsial spotted fever and ehrlichial infections in Argentina. (+info)
Serologic study of the prevalence of rickettsiosis in Yucatan: evidence for a prevalent spotted fever group rickettsiosis.
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Because of the discovery of a spotted fever group rickettsiosis with signs and symptoms similar to dengue fever in Yucatan, Mexico, immunofluorescence assay (IFA) serology was performed on sera from 390 persons selected from a representative geographic distribution of rural Yucatan to detect antibodies reactive with Rickettsia rickettsii, R. akari, a Thai strain (TT-118) that is most closely related to a rickettsia identified in Amblyomma cajennense ticks in southern Texas, and R. typhi. The IFA antibodies at titers > or = 1:64 against R. akari were detected in 22 (5.6%) of the samples with the expected cross-reactivity against the other antigens of the spotted fever group. Immunoblotting with antigens of R. akari identified antibodies against antigens of spotted fever group lipopolysaccharides and not against rickettsial outer membrane proteins A and B, which contain the species-specific epitopes. A rickettsiosis most likely caused by a relative of R. akari appears to be both prevalent and widely distributed geographically in Yucatan. (+info)
Characterization of mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species.
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Rickettsiae are gram-negative, obligately intracellular bacteria responsible for arthropod-borne spotted fevers and typhus. Experimental studies have delineated a cluster of naturally rifampin-resistant spotted fever group species. We sequenced the 4, 122- to 4,125-bp RNA polymerase beta-subunit-encoding gene (rpoB) from typhus and spotted fever group representatives and obtained partial sequences for all naturally rifampin-resistant species. A single point mutation resulting in a phenylalanine-to-leucine change at position 973 of the Rickettsia conorii rpoB sequence and present in all the rifampin-resistant species was absent in all the rifampin-susceptible species. rpoB-based phylogenetic relationships among these rickettsial species yielded topologies which were in accordance with previously published phylogenies. (+info)
Discrepancies in Weil-Felix and microimmunofluorescence test results for Rocky Mountain spotted fever.
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Only 4.2% of 284 single specimens and 17.6% of 51 pairs of sera reactive in Weil-Felix agglutination tests for Rocky Mountain spotted fever were confirmed by a specific Rickettsia rickettsii microimmunofluorescence test. (+info)