Rickettsia felis: molecular characterization of a new member of the spotted fever group (original) (raw)
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International Journal of Systematic and Evolutionary Microbiology
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.
Antigenic classification of Rickettsia felis by using monoclonal and polyclonal antibodies
2003
Rickettsia felis is a flea-transmitted rickettsia. There is a discrepancy between its reported phylogenic and phenotypic identifications. Following the first report of R. felis, it was considered by tests with serologic reagents to be closely related to another recognized flea-transmitted rickettia, R. typhi. Subsequently, it appeared to be more closely related to spotted fever group (SFG) rickettsiae by genetic analysis. In the present work, R. felis was studied by microimmunofluorescence (MIF) serologic typing and with monoclonal antibodies (MAbs). Mouse polyclonal antisera to R. felis cross-reacted only with SFG rickettsiae. A neighbor-joining analysis based on MIF indicated that R. felis is actually related to SFG rickettsiae antigenically, clustering with R. australis, R. akari, and R. montanensis. A panel of 21 MAbs was raised against a 120-kDa protein antigen or a 17-kDa polypeptide of R. felis. They cross-reacted with most members of the SFG rickettsiae but not with R. prowazekii, R. typhi, or R. canadensis of the typhus group (TG) rickettsiae. Sixty-four MAbs previously generated to seven other ricketttsial species were tested with R. felis. Three MAbs reacted with the 120-kDa antigen and were generated by R. africae, R. conorii, and R. akari, respectively. They exhibited cross-reactivities with R. felis. All our data show that R. felis harbors the antigenic profile of an SFG rickettsia. Rickettsia spp. are gram-negative and obligate intracellular bacteria (24). They are bacilli of 0.3 to 0.5 m in diameter and 0.8 to 0.2 m in length which retain basic fuchsin when they are stained by the method of Gimenez. Pathogenic rickettsiae are transmitted to humans by arthropods and cause clinical diseases that manifest typically as fever, rash, and vasculitis. At present, rickettsiae are divided into two groups, the spotted fever group (SFG) and the typhus group (TG), on the basis of their intracellular positions, optimal growth temperatures, percent GϩC DNA contents, clinical features, epidemiological aspects, and antigenic characteristics (24, 30). Recently, a novel rickettsia-like organism was observed in the midgut epithelial cells of cat fleas (Ctenocephalides felis) in California by electron microscopy (1). It was described as the ELB agent, for the EL Laboratory (Soquel, Calif.), where it was originally described (1, 23). In 1996, this flea-borne bacterium was proposed as a distinct species, "Rickettsia felis" (12), and was characterized as a typhus-like rickettsia. Monoclonal antibodies (MAbs) specific for R. typhi reacted with this organism (6). Its ultrastructure and tissue distribution in fleas resembled those of R. typhi (1, 6). However, molecular data, which were obtained by sequencing and phylogenetic analysis of a 17-kDa protein-encoding gene, a citrate synthase-encoding gene, a 155-kDa protein-encoding gene, a 120-kDa protein-encoding gene, and the metK, ftsY, polA, and dnaE genes, classified R. felis into the SFG rickettsiae (2, 6, 9, 26, 27, 29). Although advanced genetic techniques have been extensively used to classify rickettsial species, serotyping by indirect microimmunofluorescence (MIF) with mouse antisera is still considered valuable for its general applicability (20).
Journal of Clinical Microbiology, 2003
A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of Rickettsia rickettsii and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of R. rickettsii can be detected. SQ-PCR is suitable for quantitation of R. rickettsii and 10 other genotypes of spotted fever group rickettsiae but not for R. akari, R. australis, R. bellii, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks. on July 12, 2015 by guest http://jcm.asm.org/ Downloaded from on July 12, 2015 by guest http://jcm.asm.org/ Downloaded from 5472 EREMEEVA ET AL.
Analysis of the Spotted Fever and Thyphus Groups of Rickettsia Using 16 S rRNA Gene Sequences
The bacterial genus Rickettsia is traditionally divided into three biotypes, the spotted fever group (SFG), the typhus group (TG), and the scrub typhus group (STG) based on vector host and antigenic crossreactivity. DNA sequence data were gathered from the 16S ribosomal RNA gene of several SFG and TG species. Comparative sequence analysis shows that: i) all species of Rickettsia are closely related, exhibiting 0.3-1.6% sequence divergence; ii) although there are identifiable clusters corresponding to the SFG and TG, species of Rickettsia fall into more than two distinct phylogenetic groups; iii) the tick-borne species Rickettsia bellii and Rickettsia canada diverged prior to the schism between the sported fever and typhus groups; iv) the newly described AB bacterium is clearly a member of Rickettsia, but its phylogenetic placement within the genus is problematic; v) the mite-borne Rickettsia akari, the tick-borne Rickettsia al/stralis and the recently described flea-borne ELB agent form a loose cluster that cannot be definitively associated with either the TG or the traditional SFG cluster. This latter clade may represent a unique group(s) distinct from the main cluster of spotted fever and typhus group species. The divergence of Rickettsia was an ancient event within the a-subclass of the proteobacteria. The sequence divergence between Rickettsia and Ehrlichia, the closest known genus to Rickettsia, is nearly equal to the sequence divergence between Rickettsia and all other a-subclass proteobacterial taxa included in the analysis. When Rickettsia was compared to a representative group of the a-subclass, twenty-eight nucleotide sites were identified which uniquely characterize the 16S rRNA sequences of all species of Rickettsia. The approxi• mate time of divergence between the various species of Rickettsia, estimated from the bacterial 16S rRNA molecular clock, coincides with the approximate divergence time of the hard body ticks which are the arthropod hosts of many Rickettsia. Thus, the possibility of coevolution between these intracellular bacteria and their tick hosts exists.
Identification of rickettsiae from wild rats and cat fleas in Malaysia
Rickettsioses are emerging zoonotic diseases reported worldwide. In spite of the serological evidence of spotted fever group rickettsioses in febrile patients in Malaysia, limited studies have been conducted to identify the animal reservoirs and vectors of rickettsioses. This study investigated the presence of rickettsiae in the tissue homogenates of 95 wild rats and 589 animal ectoparasites. Using PCR assays targeting the citrate synthase gene (gltA), rickettsial DNA was detected in the tissue homogenates of 13 (13.7%) wild rats. Sequence analysis of the gltA amplicons showed 98.6–100% similarity with those of Rickettsia honei/R. conorii/R. raoultii (Rickettsiales: Rickettsiaceae). Sequence analysis of outer membrane protein A gene (ompA) identified Rickettsia sp. TCM1 strain from two rats. No rickettsia was detected from Laelaps mites, Rhipicephalus sanguineus and Haemaphysalis bispinosa ticks, and Felicola subrostratus lice in this study. R. felis was identified from 32.2% of 177 Ctenocephalides felis fleas. Sequence analysis of the gltA amplicons revealed two genotypes of R. felis (Rf31 and RF2125) in the fleas. As wild rats and cat fleas play an important role in the enzoonotic maintenance of rickettsiae, control of rodent and flea populations may be able to reduce transmission of rickettsioses in the local setting.
Transcription of the Rickettsia Felis OmpA gene in naturally infected fleas
The American journal of tropical medicine and hygiene
Rickettsia felis is maintained transovarially in Ctenocephalides felis fleas in a widespread geographic distribution and is transmitted to humans and animals, including opossums. This rickettsia is phylogenetically a member of the spotted fever group, most closely related to Rickettsia akari and R. australis. An unusual feature of this rickettsia is that the gene for the outer membrane protein A (OmpA) is interrupted by stop codons. To determine if this putatively dying gene is expressed, mRNA was extracted from laboratory-maintained, R. felis-infected cat fleas. Reverse transcriptase-polymerase chain reaction amplification of three segments of the ompA gene indicated that mRNA of ompA is actively transcribed in fleas. The cDNA sequences expressed represented mRNA of the first 1860-basepair segment of ompA, which includes domains I and II, part of domain III, the region from site 1836 to site 2180, despite the presence of several stop codons, and the open reading frame from site 2788 to site 3837. The detected sequences showed several differences in the amino acid composition when compared with the previously reported sequence.
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...
Polygenic detection of Rickettsia felis in cat fleas (Ctenocephalides felis) from Israel
The American journal of tropical medicine and hygiene, 2006
The presence of Rickettsia felis, an emerging bacterial pathogen, was investigated in 79 cat flea (Cteno-cephalides felis) pools from Israel (5 to 20 fleas each) by polymerase chain reaction (PCR) and sequencing of 5 different genes. Amplified targets included both metabolic (gltA and fusA) and surface antigen (ompA, ompB, and the 17-kDa antigen) genes. R. felis DNA was detected in 7.6% of the flea pools. Two genotypes similar in their housekeeping gene sequences but markedly different in their surface antigenic genetic milieus were characterized. This is the first detection of this flea-transmitted rickettsia within its vector in Israel and the Middle East. Although no clinical case has been reported in human beings in Israel to date, these findings suggest that this infection is prevalent in Israel.
Comparative Immunology Microbiology and Infectious Diseases
Rickettsia spp. are zoonotic pathogens and mainly transmitted by various arthropod vectors, such as fleas, ticks, and lice. Previous epidemiological studies indicated that ectoparasites infested on dogs or cats may be infected by Rickettsia spp., and transmit them to human beings accidentally. In this study, the prevalence of Rickettsia infection was evaluated using fleas and ticks from stray dogs and cats in Taiwan. A total of 158 pools made by 451 cat fleas (Ctenocephalides felis) from 37 dogs and 4 cats were used for analysis. Besides, 386 Rhipicephalus ticks collected from the other 62 stray dogs were included in this study. Nymphal and adult ticks were individually analyzed but larvae were separated into 21 pools for molecular detection. Partial sequencing analysis of the gltA gene was applied for Rickettsia identification. The results showed that 44.3% (70/158) of the cat flea pools were harboring Rickettsia DNA. Although 6.9% (13/187) of adult ticks were infected with Rickettsia, neither larval pools nor nymphal ticks were found to contain Rickettsia DNA. According to the results of sequencing analyses, all Rickettsia PCR-positive cat flea pools were infected with R. felis, and all Rickettsia PCR-positive adult ticks were infected with R. rhipicephali. The results of this study demonstrated that C. felis but not Rhipicephlus sanguineus (the brown dog tick) and Rh. haemaphysaloides collected from stray animals in could be infected the zoonotic pathogen R. felis. Moreover, R. rhipicephali was only identified in adult stage of Rhipicephalus sanguineus and Rh. haemaphysaloides. (C.-C. Chang). pathogens. Based on serological studies, Rickettsia spp. are divided into three groups: typhus group (TG), spotted fever group (SFG), and scrub typhus group (STG) [1]. The TG includes lice-transmitted R. prowazekii causing epidemic typhus [2], and flea-transmitted R. typhi causing endemic typhus [3]. The SFG contains R. rickettsii causing Rocky mountain spotted fever (RMSF) [4], and R. conorii subsp. conorii causing Mediterranean spotted fever (MSF) [5]. The replication of Rickettsia spp. can only occur within the host cells [1]. Arthropod vectors (such as fleas, ticks, and 0147-9571/$ -see front matter
Rickettsia felis infection in cat fleas Ctenocephalides felis felis
Brazilian Journal of Microbiology, 2010
The present study evaluated the rickettsial infection in a laboratory colony of cat fleas, Ctenocephalides felis felis (Bouche) in Brazil. All flea samples (30 eggs, 30 larvae, 30 cocoons, 30 males, and 30 females) tested by polymerase chain reaction (PCR) were shown to contain rickettsial DNA. PCR products, corresponding to the rickettsial gltA, htrA, ompA and ompB gene partial sequences were sequenced and showed to correspond to Rickettsia felis, indicating that the flea colony was 100% infected by R. felis. The immunofluorescence assay (IFA) showed the presence of R. felis-reactive antibodies in blood sera of 7 (87.5%) out of 8 cats that were regularly used to feed the flea colony. From 15 humans that used to work with the flea colony in the laboratory, 6 (40.0%) reacted positively to R. felis by IFA. Reactive feline and human sera showed low endpoint titers against R. felis, varying from 64 to 256. With the exception of one human serum, all R. felis-reactive sera were also reactive to Rickettsia rickettsii and/or Rickettsia parkeri antigens at similar titers to R. felis. The single human serum that was reactive solely to R. felis had an endpoint titer of 256, indicating that this person was infected by R. felis.