Genotypic identification and phylogenetic analysis of the spotted fever group rickettsiae by pulsed-field gel electrophoresis

157

We formally propose the name Rickettsia aeschlimannii sp. nov. for a new spotted fever group (SFG) rickettsia, strain MCMT, isolated from Hyalumma marginatum marginatum ticks collected in Morocco. This organism shows a typical rickettsial morphology when analyzed by electron microscopy. After characterization by serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblotting, PCR-restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis, and 16s rDNA sequencing, this organism was found to be different from all of the recognized SFG rickettsiae. Identical PCR-FWLP profiles have, however, been found in H. marginatum marginatum from Portugal and H. marginatum rujipes from Zimbabwe, which suggests that the distribution of this rickettsia reaches from the Mediterranean to southern Africa.Although the first descriptions of Mediterranean spotted fever and its ecology were made in northern Africa (13) and one of the reference strains of Rickettsia conorii is called the Moroccan strain, there have been few recent reports about the ecology and epidemiology of rickettsioses in this area (31). In early studies, all three African spotted fever group (SFG) rickettsioses, Mediterranean spotted fever, South African tick bite fever, and Kenyan tick typhus (25), were attributed to infection with R. conorii. The recognized vectors were dog ticks: Rhipicephalus sanguineus in the Mediterranean area (11) and Rhipicephalus simus or Haemaphysalis leachi in the eastern and southern regions of Africa (22,27). When other tick species were found to be infected with rickettsia-like organisms (RLO), they were considered to be "secondary" vectors of R. conorii (21). More recently, however, it has been shown that other SFG rickettsiae occur throughout Africa and that several tick species carry rickettsiae different from R. conorii (7,41).The presence of RLO in Hyalomma ticks of Morocco and Sudan was first reported in the 1950s (21). RLO have been detected by PCR and direct immunofluorescence in H. impeltatum, H. dromedari, and H. anatolicum in Egypt; however, they have not been identified (30). During a large-scale field survey in Zimbabwe, hemolymph tests showed that 11% of Hyalomma marginatum rufipes ticks were infected with RLO (7). Although isolation attempts were unsuccessful, PCR-restriction fragment length polymorphism (RFLP) analysis showed that these ticks contained SFG rickettsiae genotypically similar to a strain (PoTiR8) isolated from H. marginatum marginaturn ticks from Portugal (2). Strain PoTiR8 has, however, not been further characterized.In Morocco, H. marginatum marginaturn is one of the most widely distributed tick species, possibly representing up to 42% of the tick burden of cattle (3, 32). Although in its juvenile stages it usually bites birds, under particular circumstances it may also infest humans (23). We present here the first description of an SFG rickettsial strain (MC16T) isolated from Moroccan H. marginaturn marginaturn. This isolate was compared to the recogn...

Section: Methodsmentioning

confidence: 99%

Section: Sds-page and Western Blot Immunoassaymentioning

confidence: 99%

Rickettsia aeschlimannii sp. nov., a New Spotted Fever Group Rickettsia Associated with Hyalomma marginatum Ticks

Béati

Meskini

Thiers

et al. 1997

157

“…When the citrate synthase gene amplified material was digested by AluI, it produced a unique profile. The migration patterns for the genome of strain MtulT digested with different enzymes (SmaI, EagI, and BssHII) and analyzed by pulsed-field electrophoresis were clearly different from the profiles obtained for the reference SFG rickettsiae Therefore, strain MtulT can be considered a new species distinct from all previously recognized SFG rickettsiae, not only on the basis of the usual serological criteria, but also on the basis of the results of two different genetic analyses (3,4,8). The polymerase chain reaction-restriction fragment length polymorphism profile of MtulT, obtained after amplification of the citrate synthase gene and digestion with AluI, is unique and distinct from the profile found for all previously described SFG rickettsiae.…”

mentioning

confidence: 99%

“…Gimenez-stained infected cells contain small, intracellular, rod-shaped or diplobacillary bacteria that are slightly shorter (0.3 to 0.4 by 0.6 to 1 pm) than the other SFG rickettsiae (3). This organism can also be detected in infected cells by indirect immunofluorescence by using human immune sera reactive with the lipopolysaccharide of Rickettsia cunurii, a cross- (8).…”

mentioning

confidence: 99%

NOTES: Rickettsia massiliae sp. nov., a New Spotted Fever Group Rickettsia

Béati

Raoult²

1993

113

We propose the name Rickettsia lttaSSiliae sp. nov. (type strain, Mtul in the Collection of the World Health Organization Collaborative Center for Rickettsia1 Reference, Marseille, France) for a spotted fever group rickettsia determined to be distinct from previously recognized species by the serotyping method (L. Beati, J.-P. Finidori, B. Gilot, and D. Raoult, J. Clin, Microbiol, 30:1922Microbiol, 30: -1930Microbiol, 30: ,1992)-This rickettsia has biological characteristics similar to those of the other spotted fever group rickettsiae. In addition, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, a polymerase chain reaction followed by a restriction fragment length polymorphism analysis of DNA fragments, and pulsed-field electrophoresis of the genome of R. massiliae revealed unique migration patterns distinct from those of all previously recognized spotted fever group rickettsiae. These additional characteristics (Beati et al., J. Clin. Microbiol. 30:1922Microbiol. 30: -1930Microbiol. 30: , 1992, together with the data usually considered sufficient for description of rickettsiae, are crucial to the proposal of this new species and should be helpful in species identification.In this paper, we formally name a rickettsia isolated from the hemolymph of ticks (3). The first isolate, strain MtulT (T = type strain) was isolated in 1990 from a Rhipicephalus turanicus tick in an area near Marseille, France. Since then, another strain has been isolated from a Rhipicephalus sanguineus tick (brown dog tick) collected in Sisteron, France, and other strains have been detected in a Rhipicephalus sanguineus tick from Portugal (1) and in Rhipicephalus mushamae, Rhipicephalus sulcatus, and Rhipicephalus lunulatus ticks collected in the Central African Republic (2).As determined by the serotyping method (9, we found that the new isolates were distinct from all previously described spotted fever group (SFG) rickettsiae (15 species).Cross-titrations of murine polyclonal sera with these new strains and with the other SFG rickettsiae (3,4) revealed 6 to 13 specific differences between the new organisms and the other SFG rickettsiae (3, 4).The results of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein analysis revealed different electrophoretic mobilities for the major immunodominant hi h-molecular-weight surface polypeptides of strain Mtul compared with the antigens of the standard SFG rickettsiae, including Rickettsia slovaca 13-B, Rickettsia sibirica 232, Rickettsia conorii Moroccan, Rickettsia rickettsii Sheila Smith, Rickettsia rhipicephali 2-7-6, Rickettsia montana ATCC VR-611, Rickettsia helvetica C3T, Rickettsia parkeri Maculatum 20, Israeli spotted fever rickettsia strain ISTT CDC-1, Rickettsia japonica YM, Thai tick typhus rickettsia strain TI'-118, Rickettsia akari MK-Kaplan, and Rickettsia australis Phillips (3,4).Genetic analyses of the citrate synthase and 190-kDa protein genes (7) of strain MtulT amplified by the polymerase chain reaction and digested with res...

“…Using the 16s and 23s rRNA gene sequences, we determined that R. bellii is the product of an early divergence which occurred prior to the schism between the SFG and TG. While some other workers have suggested that this species should occupy a peripheral position (10,26,27), we describe the first quantitative classification of R. bellii in which genes with known evolutionary patterns were used. In addition, our data provide insight into more extensive proteobacterial comparisons; they indicate that the genus Rickettsia is a distinct, ancient lineage of the 01 subgroup of the Proteobacteria.…”

mentioning

confidence: 99%

Ancestral Divergence of Rickettsia bellii from the Spotted Fever and Typhus Groups of Rickettsia and Antiquity of the Genus Rickettsia

Stothard

Clark

Fuerst

1994

The eubacterial genus Rickettsia belongs to the cu subgroup of the phylum Proteobacteriu. 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 rickettsia1 classification scheme. The 23s rRNA gene sequences described in this paper are the first 23s rRNA sequences reported for any member of the Rickettsiuceue. 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. A comparison of the 16s rRNA and 23s rRNA gene sequences of Rickettsia strains with other proteobacterial sequences confirmed that the genus Rickettsia is a unique deeply branching member of the cu subgroup of the Proteobacteria and that the Rickettsia species form a monophyletic cluster. While divergence of the contemporary members of the genus Rickettsia occurred recently, the unique evolutionary line represented by this genus appears to be very old.Rickettsia strains are small, gram-negative bacteria that are intimately associated with eukaryotic cells and are members of a diverse family, the Rickettsiaceae, which also includes other intracellular organisms, including the genera Ehrlichia, Wolbachia, Anaplasma, and Cowdria (4,36). Rickettsiae have natural arthropod hosts (either ticks, mites, or insects) and can be pathogenic for humans and other vertebrates. The obligately intracellular lifestyle and fastidious nature of these organisms have made them difficult to study. A number of genotypic and phenotypic characteristics indicate that the Rickettsia species are closely related, and this genus is now recognized as the sole member of a branch of the cx subgroup Proteobacteriu (8) on the basis of 16s rRNA cataloging and partial 16s rRNA gene sequence data (34).The genus Rickettsia is divided into three biotypes on the basis of immunological cross-reactivity and ecological characteristics (36). Rickettsia species are placed into the spotted fever group (SFG), the typhus group (TG), or the scrub typhus group on the basis...