Evaluation of a PCR Assay for Quantitation of Rickettsia rickettsii and Closely Related Spotted Fever Group Rickettsiae (original) (raw)
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Journal of Clinical Microbiology, 1994
Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified genes was used to study spotted fever group (SFG) rickettsiae, extending the previous work of Regnery et al. (R.L. Regnery, C.L. Spruill, and B.D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991). Twenty-six strains of SFG rickettsia were studied, including several recognized species which have never been studied (R. parkeri, R. helvetica, and R. japonica) as well as strains which are not currently classified. Two previously used primer pairs derived from the R. prowazekii citrate syntase gene and the R. rickettsii 190-kDa protein antigen gene were studied, as were primer pairs obtained from the R. rickettsii 120-kDa protein antigen gene. By using three amplifications and three enzyme digestions, it was possible to differentiate between almost all of the known SFG rickettsia species and to differentiate between several strains of the R. conorii complex. Two human pathogens, "R. africae" and the Israeli ...
The American journal of tropical medicine and hygiene, 2005
A highly specific real-time polymerase chain reaction (PCR) assay was developed to detect spotted fever and typhus group rickettsiae using the citrate synthase gene as the target. The assay amplified rickettsial members of the spotted fever and typhus group including Rickettsia akari, R. australis, R. conorii, R. honei, "R. marmionii," R. sibirica, R. rickettsii, R. typhi, and R. prowazekii. The ancestral group rickettsia, R. bellii, did not produce a positive reaction, nor did other members of the order Rickettsiales or any non-rickettsial bacteria. The assay had a sensitivity of one target copy number per reaction as determined by serial dilutions of a plasmid containing a spotted fever group target sequence. This quantitative assay is useful for the enumeration of rickettsiae in clinical specimens and the diagnosis of rickettsial illnesses, when rickettsial numbers are very low.
Rickettsia felis: molecular characterization of a new member of the spotted fever group
International Journal of Systematic and Evolutionary Microbiology, 2001
In this report, placement of Rickettsia felis in the spotted fever group (SFG) rather than the typhus group (TG) of Rickettsia is proposed. The organism, which was first observed in cat fleas (Ctenocephalides felis) by electron microscopy, has not yet been reported to have been cultivated reproducibly, thereby limiting the standard rickettsial typing by serological means. To overcome this challenge, several genes were selected as targets to be utilized for the classification of R. felis. DNA from cat fleas naturally infected with R. felis was amplified by PCR utilizing primer sets specific for the 190 kDa surface antigen (rOmpA) and 17 kDa antigen genes. The entire 5513 bp rompA gene was sequenced, characterized and found to have several unique features when compared to the rompA genes of other Rickettsia. Phylogenetic analysis of the partial sequence of the 17 kDa antigen gene indicated that R. felis is less divergent from the SFG rickettsiae than from the TG rickettsiae. The data corroborate results from previous reports that analysed the citrate synthase, 16S rRNA, rompB (135 kDa surface antigen), metK, ftsY, polA and dnaE genes that placed R. felis as a member of the SFG. The organism is passed transstadially and transovarially, and infection in the cat flea has been observed in the midgut, tracheal matrix, muscle, hypodermis, ovaries and testes.
Characterization and comparison of Australian human spotted fever group rickettsiae
Journal of Clinical Microbiology, 1992
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 n...
Proteinic and genomic identification of spotted fever group rickettsiae isolated in the former USSR
Journal of Clinical Microbiology, 1993
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 Ricketts...
Pathogens, 2019
The clinical features of spotted fever group (SFG) Rickettsia induced disease range from a mild to severe illness. The clinical complexity is even greater due to the fact that the disease can be caused by different species with varying degrees of virulence. Current knowledge asserts that the Israeli SFG (ISF) strain Rickettsia conorii israelensis is the only human pathogenic SFG member in Israel. Current diagnostic procedures distinguish between SFG and the typhus group rickettsiosis, assuming all SFG-positive clinical samples positive for ISF. Molecular studies on questing ticks over the past decade have uncovered the existence of other SFG strains besides ISF in Israel and the region. This study describes the first documented analysis of SFG-positive samples from Israeli patients with the goal of distinguishing between ISF and non-ISF SFG strains. We managed to identify a new Rickettsia isolate from three independent clinical samples in Israel which was shown to be an as-yet unkno...
Microorganisms
The spotted fever group of Rickettsiae is a heterogeneous group of Rickettsiae transmitted by ticks, causing similar diseases in humans (spotted fever). Until recently, it was supposed that a single pathogenic tick-borne SFG Rickettsia circulated in each different geographic area and that R. conorii subsp. conorii was the SFG Rickettsiae circulating in Italy, but in the last decade, thanks to molecular diagnostic, several different Rickettsia species, previously not considered pathogenic for decades, have been isolated from ticks and definitively associated to human disease, also in Italy. The present survey was carried out with the aim of investigating the presence of different SFG Rickettsia species in a geographic area where no information was available. Ticks collected from animals submitted to necropsy, removed from humans in local hospitals and collected from the environment were identified and tested by PCR for Rickettsia spp. based on the gltA gene, and positive PCR products...
Journal of clinical microbiology, 1997
Rickettsial proteins rOmp A and rOmp B exist in both Rickettsia australis and Rickettsia honei but differ in molecular weight and antigenicity; in addition, they produce distinct immunogenic responses and appear to be to conformationally dependent antigens. Species-specific monoclonal antibodies for other spotted fever group rickettsial species did not react with R. honei. A PCR product of the repeat region of the rOmp A gene from R. honei was amplified and calculated to contain 11 repeat units.