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Monitoring of tickborne diseases is critical for prevention and management. We analyzed 418 ticks removed from 359 patients during 2014-2021 in Marseille, France, for identification and bacteria detection. Using morphology, molecular methods, or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we identified 197 (47%) Ixodes, 136 (33%) Dermacentor, 67 (16%) Rhipicephalus, 8 (2%) Hyalomma, 6 (1%) Amblyomma, 2 (0.5%) Argas, and 2 (0.5%) Haemaphysalis tick species. We also detected bacterial DNA in 241 (58%) ticks. The most frequent bacterial pathogens were Rickettsia raoultii (17%) and R. slovaca (13%) in Dermacentor ticks, Borrelia spp. (9%) in Ixodes ticks, and R. massiliae (16%) in Rhipicephalus ticks. Among patients who were bitten, 107 had symptoms, and tickborne diseases were diagnosed in 26, including scalp eschar and neck lymphadenopathy after tick bite and Lyme borrelioses. Rapid tick and bacteria identification using a combination of methods can substantially contribute to clinical diagnosis, treatment, and surveillance of tickborne diseases.

Rickettsia species are endosymbionts hosted by arthropods and are known to cause mild to fatal diseases in humans. Here, we analyse the evolution and diversity of 34 Rickettsia species using a pangenomic meta-analysis (80 genomes/41 plasmids). Phylogenomic trees showed that Rickettsia spp. diverged into two Spotted Fever groups, a Typhus group, a Canadensis group and a Bellii group, and may have inherited their plasmids from an ancestral plasmid that persisted in some strains or may have been lost by others. The results suggested that the ancestors of Rickettsia spp. might have infected Acari and/or Insecta and probably diverged by persisting inside and/or switching hosts. Pangenomic analysis revealed that the Rickettsia genus evolved through a strong interplay between genome degradation/reduction and/or expansion leading to possible distinct adaptive trajectories. The genus mainly shared evolutionary relationships with α-proteobacteria, and also with γ/β/δ-proteobacteria, cytophagia, actinobacteria, cyanobacteria, chlamydiia and viruses, suggesting lateral exchanges of several critical genes. These evolutionary processes have probably been orchestrated by an abundance of mobile genetic elements, especially in the Spotted Fever and Bellii groups. In this study, we provided a global evolutionary genomic view of the intracellular Rickettsia that may help our understanding of their diversity, adaptation and fitness., (© 2022. The Author(s).)

Spotted fever group rickettsiae (SFGR) and Coxiella burnetii are intracellular bacteria that cause potentially life-threatening tick-borne rickettsioses and Q fever respectively. Sao Tome and Principe (STP), small islands located in the Gulf of Guinea, recently experienced a dramatic reduction in the incidence of malaria owing to international collaborative efforts. However, unexplained febrile illnesses persist. A One Health approach was adopted to investigate exposure to SFGR and C. burnetii in humans and examine the diversity of these bacteria in ticks parasitizing domestic ruminants. A cross-sectional human serological study was conducted in Agua Grande district in Sao Tome Island from January to March 2016, and ticks were collected from farmed domestic ruminants in 2012 and 2016. In total, 240 individuals varying in age were randomly screened for exposure to SFGR and C. burnetii by indirect immunofluorescence assay. Twenty of 240 individuals (8.3%) were seropositive for SFGR (4 for Rickettsia africae and 16 for R. conorii) and 16 (6.7%) were seropositive for C. burnetii. Amblyomma astrion were collected exclusively in 2012, as were A. variegatum in 2016 and Rickettsia spp. were detected in 22/42 (52.4%) and 49/60 (81.7%) respectively. Sequence analysis of multiple gene targets from Rickettsia spp. detected in ticks suggests the presence of a single divergent R. africae strain (Sao Tome). While no ticks were found positive for C. burnetii, Coxiella-like endosymbionts were detected in nearly all ticks. This is the first study in STP to provide serological evidence in humans of SFGR and C. burnetii and additional molecular evidence in ticks for SFGR, which may be responsible for some of the unexplained febrile illnesses that persist despite the control of malaria. Future epidemiological studies are needed to confirm the occurrence and risk factors associated with SFG rickettsioses and Q fever in both humans and animals., (© 2019 Blackwell Verlag GmbH.)

Over recent years, genomic information has increasingly been used for prokaryotic species definition and classification. Genome sequence-based alternatives to the gold standard DNA-DNA hybridization (DDH) relatedness have been developed, notably average nucleotide identity (ANI), which is one of the most useful measurements for species delineation in the genomic era. However, the strictly intracellar lifestyle, the few measurable phenotypic properties and the low level of genetic heterogeneity made the current standard genomic criteria for bacterial species definition inapplicable to Rickettsia species. We evaluated a range of whole genome sequence (WGS)-based taxonomic parameters to develop guidelines for the classification of Rickettsia isolates at genus and species levels. By comparing the degree of similarity of 74 WGSs from 31 Rickettsia species and 61 WGSs from members of three closely related genera also belonging to the order Rickettsiales ( Orientia , 11 genomes; Ehrlichia , 22 genomes; and Anaplasma , 28 genomes) using digital DDH (dDDh) and ANI by orthology (OrthoANI) parameters, we demonstrated that WGS-based taxonomic information, which is easy to obtain and use, can serve for reliable classification of isolates within the Rickettsia genus and species. To be classified as a member of the genus Rickettsia , a bacterial isolate should exhibit OrthoANI values with any Rickettsia species with a validly published name of ≥83.63 %. To be classified as a new Rickettsia species, an isolate should not exhibit more than any of the following degrees of genomic relatedness levels with the most closely related species: >92.30 and >99.19 % for the dDDH and OrthoANI values, respectively. When applied to four rickettsial isolates of uncertain status, the above-described thresholds enabled their classification as new species in one case. Thus, we propose WGS-based guidelines to efficiently delineate Rickettsia species, with OrthoANI and dDDH being the most accurate for classification at the genus and species levels, respectively.

Objective: The objective was to investigate fixative solutions: 3.7% formaldehyde, 4% paraformaldehyde, 4% paraformaldehyde in the cytoskeletal buffer and 4% paraformaldehyde in PHEM buffer (containing PIPES, HEPES, EGTA and MgCl 2 ), applicable for immunofluorescence assay., Results: Herein we optimized this serological technique, testing four fixative solutions, for the sensitive detection of rickettsial antigens, and preservation of intracellular structures of the host cells, particularly filamentous actin. Rickettsial antigens were presented equally well both with formaldehyde and all paraformaldehyde-based fixations, but only protocol with 4% paraformaldehyde in PHEM buffer allowed accurate imaging of actin filaments, and simultaneously allows monitoring of rickettsiae using actin-based motility during infection inside the host cells.

As a result of migrations and globalization, people may face a possible increase in the incidence of central nervous system rickettsial infections (CNS R). These diseases, caused by Rickettsia species and transmitted to humans by arthropod bites, are putatively lethal. However, the diagnosis of CNS R is challenging and often delayed due to their nonspecific clinical presentation and the strict intracellular nature of rickettsiae. Furthermore, transfer of rickettsiae to the brain parenchyma is not yet understood. The aim of this review is to analyze and summarize the features and correlated findings of CNS R in order to focus attention on these intriguing but frequently neglected illnesses. We also incorporated data on CNS infections caused by Rickettsia-related microorganisms., Competing Interests: The authors have declared that no competing interests exist.

Rickettsia species are strictly intracellular bacteria that evolved approximately 150 million years ago from a presumably free-living common ancestor from the order Rickettsiales that followed a transition to an obligate intracellular lifestyle. Rickettsiae are best known as human pathogens vectored by various arthropods causing a range of mild to severe human diseases. As part of their obligate intracellular lifestyle, rickettsial genomes have undergone a convergent evolution that includes a strong genomic reduction resulting from progressive gene degradation, genomic rearrangements as well as a paradoxical expansion of various genetic elements, notably small RNAs and short palindromic elements whose role remains unknown. This reductive evolutionary process is not unique to members of the Rickettsia genus but is common to several human pathogenic bacteria. Gene loss, gene duplication, DNA repeat duplication and horizontal gene transfer all have shaped rickettsial genome evolution. Gene loss mostly involved amino-acid, ATP, LPS and cell wall component biosynthesis and transcriptional regulators, but with a high preservation of toxin-antitoxin (TA) modules, recombination and DNA repair proteins. Surprisingly the most virulent Rickettsia species were shown to have the most drastically reduced and degraded genomes compared to closely related species of milder pathogenesis. In contrast, the less pathogenic species harbored the greatest number of mobile genetic elements. Thus, this distinct evolutionary process observed in Rickettsia species may be correlated with the differences in virulence and pathogenicity observed in these obligate intracellular bacteria. However, future investigations are needed to provide novel insights into the evolution of genome sizes and content, for that a better understanding of the balance between proliferation and elimination of genetic material in these intracellular bacteria is required., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Rickettsia species are arthropod endosymbiotic α-proteobacteria that can infect mammalian hosts during their obligate intracellular lifecycle, and cause a range of mild to severe diseases in humans. Paradoxically, during their adaptation to a bottleneck lifestyle, rickettsial genomes have undergone an evolution marked by a progressive chromosomic and plasmidic degradation resulting in a genome reduction from 1.5 to 1.1 Mb, with a coding capacity of 69-84%. A striking finding of rickettsial genomics has been that the most virulent species had genomes that were drastically reduced and degraded when compared to closely related less virulent or nonpathogenic species. This paradoxical evolution, which is not unique to members of the genus Rickettsia but has been identified as a convergent evolution of several major human pathogenic bacteria, parallels a selected loss of genes associated with transcriptional regulators, but with a high preservation of toxin-antitoxin (TA) modules and recombination and DNA repair proteins. In addition, these bacteria have undergone a proliferation of genetic elements, notably short palindromic elements, whose role remains unknown. Recent proteomic and transcriptomics analyses have revealed a differential level or degradation of gene expression that may, at least partially, explain differences in virulence among Rickettsia species. However, future investigations are mandatory to provide novel insights into the mechanisms by which genomic reductive evolution contributes to an emergence of pathogenesis., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Rickettsioses are globally distributed and caused by the family Rickettsiaceae , which comprise a diverse and expanding list of organisms. These include two genera, Rickettsia and Orientia Serology has been traditionally the mainstay of diagnosis, although this has been limited by cross-reactions among closely related members and diminished sensitivity/utility in the acute phase of illness. Other techniques, such as nucleic acid amplification tests using blood specimens or tissue swabs/biopsy specimens, sequencing, and mass spectrometry, have emerged in recent years for both pathogen and vector identification. This paper provides a concise review of the rickettsioses and the traditional and newer technologies available for their diagnosis., (Copyright © 2018 American Society for Microbiology.)

A rickettsial organism harboured by Amblyomma triguttatum ticks on Barrow Island, Western Australia, was discovered after reports of possible rickettsiosis among local workers. Subsequent isolation of this rickettsia (strain BWI-1) in cell culture and analysis of its phylogenetic, genotypic and phenotypic relationships with type strains of Rickettsia species with standing in nomenclature suggested that it was sufficiently divergent to warrant its classification as a new species. Multiple gene comparison of strain BWI-1 revealed degrees of sequence similarity with Rickettsia raoultii, its closest relative, of 99.58, 98.89, 97.03, 96.93 and 95.73 % for the 16S rRNA, citrate synthase, ompA, ompB and sca4 genes, respectively. Serotyping in mice also demonstrated that strain BWI-1T was distinct from Rickettsia raoultii. Thus, we propose the naming of a new species, Rickettsia gravesii sp. nov., based on its novel genotypic and phenotypic characteristics. Strain BWI-1T was deposited in the ATCC, CSUR and ARRL collections under reference numbers VR-1664, CSUR R172 and RGBWI-1, respectively.

Background: Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes., Results: Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events., Conclusion: Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as gene duplication and genesis. The plasmids are plastic and mosaic structures that may play biological roles similar to or distinct from their co-residing chromosomes in an obligate intracellular lifestyle.

Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group of the genus Rickettsia. These zoonoses are among the oldest known vector-borne diseases. However, in the past 25 years, the scope and importance of the recognized tick-associated rickettsial pathogens have increased dramatically, making this complex of diseases an ideal paradigm for the understanding of emerging and reemerging infections. Several species of tick-borne rickettsiae that were considered nonpathogenic for decades are now associated with human infections, and novel Rickettsia species of undetermined pathogenicity continue to be detected in or isolated from ticks around the world. This remarkable expansion of information has been driven largely by the use of molecular techniques that have facilitated the identification of novel and previously recognized rickettsiae in ticks. New approaches, such as swabbing of eschars to obtain material to be tested by PCR, have emerged in recent years and have played a role in describing emerging tick-borne rickettsioses. Here, we present the current knowledge on tick-borne rickettsiae and rickettsioses using a geographic approach toward the epidemiology of these diseases.

In order to investigate the prevalence of rickettsioses in febrile exanthemas in eastern Algeria, we conducted a prospective serological analysis of all patients presenting with this clinical picture at the Infectious Diseases Department in the Batna Hospital from January 2000 to September 2006. One hundred and eight adult patients were included in the study, 46% of whom younger than 25 years, and 72.5% were admitted from May to September. Patients were tested for antibodies to Rickettsia conorii conorii, R. conorii israelensis, R. aeschlimannii, R. felis, R. sibirica mongolitimonae, R. africae, R. massiliae, R. typhi, and R. prowazekii using microimmunofluorescence and Western blot as confirmation procedure. Both methods confirmed the diagnosis of Mediterranean spotted fever in 5 patients (4.6%), flea spotted fever in 2 patients (1.9%), and R. aeschlimannii infection in 2 patients (1.9%). In addition, 4 patients were diagnosed as having murine typhus (3.7%) and one with epidemic typhus (0.9%). To the best of our knowledge, this is the first report of R. felis infection and R. aeschlimannii infection in Algeria. In conclusion, at least 5 different kinds of rickettsiosis coexist in eastern Algeria and, being responsible for 13% of cases of febrile exanthemas, should be considered among major causes of this clinical picture in this area. In addition, we emphasize the high incidence of typhus group rickettioses, including epidemic typhus, in this area., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Rickettsia japonica strain YH, isolated in 1984 in Japan, is the type strain of R. japonica, the tick-borne agent of Japanese spotted fever. Here, we report the 1.33-Mb genome of this rickettsial species.

Rickettsia australis strain Phillips(T) was isolated in Queensland, Australia, in 1950. It is the tick-borne agent of Queensland tick typhus, a disease endemic in Australia. The 1.29-Mb genome sequence of this bacterium is highly similar to that of Rickettsia akari but contains two plasmids.

Rickettsia honei strain RB(T) was isolated from a febrile patient on Flinders Island, Australia, in 1991 and has been demonstrated to be the agent of Flinders Island spotted fever, a disease transmitted to humans by ticks. The comparison of this 1.27-Mb genome with other Rickettsia genomes provides additional insight into the mechanisms of evolution in Rickettsia species.

Rickettsia sibirica sibirica is the causative agent of Siberian or North Asian tick typhus, a tick-borne rickettsiosis known to exist in Siberia and eastern China. Here we present the draft genome of Rickettsia sibirica sibirica strain BJ-90 isolated from Dermacentor sinicus ticks collected in Beijing, China.

We report the complete and annotated genome sequence of Rickettsia helvetica strain C9P9, which was first isolated in 1979 from Ixodes ricinus ticks in Switzerland and is considered a human pathogen.

"Rickettsia sibirica subsp. mongolitimonae" is the agent of lymphangitis-associated rickettsiosis, an emerging human disease that has been diagnosed in Europe and Africa. The present study reports the draft genome of Rickettsia sibirica subsp. mongolitimonae strain HA-91.

The present study reports the complete and annotated genome sequence of the human pathogen Rickettsia slovaca strain 13-B, which was isolated from a Dermacentor tick in Slovakia in 1968. The 1.27-Mb genome provides further insights into the acquisition of virulence related to genome reduction in Rickettsia species.

Until recently, Amblyomma maculatum (the Gulf Coast tick) had garnered little attention compared to other species of human-biting ticks in the United States. A. maculatum is now recognized as the principal vector of Rickettsia parkeri, a pathogenic spotted fever group rickettsia (SFGR) that causes an eschar-associated illness in humans that resembles Rocky Mountain spotted fever. A novel SFGR, distinct from other recognized Rickettsia spp., has also been detected recently in A. maculatum specimens collected in several regions of the southeastern United States. In this study, 198 questing adult Gulf Coast ticks were collected at 4 locations in Florida and Mississippi; 28% of these ticks were infected with R. parkeri, and 2% of these were infected with a novel SFGR. Seventeen isolates of R. parkeri from individual specimens of A. maculatum were cultivated in Vero E6 cells; however, all attempts to isolate the novel SFGR were unsuccessful. Partial genetic characterization of the novel SFGR revealed identity with several recently described, incompletely characterized, and noncultivated SFGR, including "Candidatus Rickettsia andeanae" and Rickettsia sp. Argentina detected in several species of Neotropical ticks from Argentina and Peru. These findings suggest that each of these "novel" rickettsiae represent the same species. This study considerably expanded the number of low-passage, A. maculatum-derived isolates of R. parkeri and characterized a second, sympatric Rickettsia sp. found in Gulf Coast ticks.

Ticks (n = 252) were collected from five wild-caught reptile species during routine trapping in the Djukbinj National Park and Fogg Dam Reserve, Northern Territory, Australia. Pooling of ticks (one to four ticks per pool), according to sex or host animal, resulted in 187 samples used for screening for the presence of Rickettsia species via molecular methods. Rickettsia DNA was detected via the amplification of the gltA, ompA, and ompB genes in 57 (34%) of the 187 tick samples, all of which contained only the tick Amblyomma fimbriatum Koch (Acari: Ixodidae). Further amplification and sequencing of nine of the positive samples (4.8%) for the gltA, ompA, ompB, 17-kDa, and 16S rRNA genes identified a Rickettsia species sharing closest identity to Rickettsia tamurae. In addition, amplification and sequencing of the 16S rRNA gene detected in the same tick samples the presence of a Francisella species closely related to other tick-borne Francisellae identified in ticks from the Northern Hemisphere.

Background: The Rickettsia genus includes 25 validated species, 17 of which are proven human pathogens. Among these, the pathogenicity varies greatly, from the highly virulent R. prowazekii, which causes epidemic typhus and kills its arthropod host, to the mild pathogen R. africae, the agent of African tick-bite fever, which does not affect the fitness of its tick vector., Results: We evaluated the clonality of R. africae in 70 patients and 155 ticks, and determined its genome sequence, which comprises a circular chromosome of 1,278,540 bp including a tra operon and an unstable 12,377-bp plasmid. To study the genetic characteristics associated with virulence, we compared this species to R. prowazekii, R. rickettsii and R. conorii. R. africae and R. prowazekii have, respectively, the less and most decayed genomes. Eighteen genes are present only in R. africae including one with a putative protease domain upregulated at 37 degrees C., Conclusion: Based on these data, we speculate that a loss of regulatory genes causes an increase of virulence of rickettsial species in ticks and mammals. We also speculate that in Rickettsia species virulence is mostly associated with gene loss.The genome sequence was deposited in GenBank under accession number [GenBank: NZ_AAUY01000001].

We describe the characterization of a novel Rickettsia species cultivated from Dermacentor ticks collected in Russia and France, for which we propose the name Rickettsia raoultii sp. nov. Using multigene sequencing, we demonstrated that five rickettsial isolates from Dermacentor silvarum, Dermacentor reticulatus, Dermacentor marginatus and Dermacentor nuttalli ticks were classified within this novel spotted fever rickettsia species. This rickettsia also exhibited a serotype distinct from previously described Rickettsia species. The type strain of Rickettsia raoultii sp. nov. is strain Khabarovsk(T) (=CSUR R3(T) =ATCC VR-1596(T)).

Background: In order to estimate whether multi-spacer typing (MST), based on the sequencing of variable intergenic spacers, could serve for the identification of Rickettsia at the species level, we applied it to 108 rickettsial isolates or arthropod amplicons that include representatives of 23 valid Rickettsia species., Results: MST combining the dksA-xerC, mppA-purC, and rpmE-tRNAfMet spacer sequences identified 61 genotypes, allowing the differentiation of each species by at least one distinct genotype. In addition, MST was discriminatory at the strain level in six species for which several isolates or arthropod amplicons were available., Conclusion: MST proved to be a reproducible and high-resolution genotyping method allowing clear identification of rickettsial isolates at the species level and further additional differentiation of strains within some species.

Mediterranean spotted fever due to Rickettsia conorii conorii was thought, for many years, to be the only tick-borne rickettsial disease prevalent in southern and eastern Europe. However, in recent years, six more species or subspecies within the spotted fever group of the genus Rickettsia have been described as emerging pathogens in this part of the world. Tick-borne agents include Rickettsia conorii israelensis, Rickettsia conorii caspia, Rickettsia aeschlimannii, Rickettsia slovaca, Rickettsia sibirica mongolitimonae and Rickettsia massiliae. Many Rickettsia of unknown pathogenicity have also been detected from ticks and could represent potential emerging pathogens to be discovered in the future. Furthermore, a new spotted fever rickettsia, Rickettsia felis, was found to be associated with cat fleas and is an emerging human pathogen. Finally, the mite-transmitted Rickettsia akari, the agent of rickettsialpox, is also known to be prevalent in Europe. We present here an overview of these rickettsioses, focusing on emerging diseases.

The Rickettsia sibirica species is composed of isolates that are genotypically close but can be classified within two distinct serotypes, that is, R. sibirica sensu stricto and R. sibirica mongolitimonae (incorrectly named R. mongolotimonae). We investigated the possibility of classifying rickettsiae closely related to R. sibirica as R. sibirica subspecies, as proposed by the ad hoc Committee on Reconciliation of Approaches to Bacterial Systematics. For this, we first estimated the genotypic variability by using multilocus sequence typing (MLST), including the sequencing of five genes, and multispacer typing (MST) using three intergenic spacers, of five isolates and three tick amplicons of R. sibirica sensu stricto and six isolates of R. sibirica mongolotimonae. Then, we selected a representative of each MLST genotype and used mouse serotyping to estimate their degree of taxonomic relatedness. Among the 14 isolates or tick amplicons studied, 2 MLST genotypes were identified: (i) the R. sibirica sensu stricto type; and (ii) the R. sibirica mongolitimonae type. Representatives of the two MLST types were classified within three MST types and into two serotypes. Therefore, as isolates within the R. sibirica species are genotypically homogeneous but show MST genotypic, serotypic, and epidemio-clinical dissimilarities, we propose to modify the nomenclature of the R. sibirica species through the creation of subspecies. We propose the names R. sibirica subsp. sibirica subsp. nov. (type strain = 2-4-6, ATCC VR-541(T)), and R. sibirica subsp. mongolitimonae subsp. nov. (type strain = HA-91, ATCC VR-1526(T)). The description of R. sibirica is emended to accommodate the two subspecies.

A total of 395 adult ixodid ticks from three genera (Dermacentor, Haemaphysalis, and Ixodes) collected from the Urals to the Far East of Russia were tested by PCR and sequencing for the presence of spotted fever rickettsiae, anaplasmae, and ehrlichiae. Four, pathogens recognized in humans were detected in ticks: Rickettsia sibirica, R. heilongjiangensis, R. helvetica, and Anaplasma phagocytophilum. In addition, rickettsiae and ehrlichiae of unknown pathogenicity were detected, including Rickettsia sp. RpA4, Rickettsia sp. DnS14, Rickettsia sp. DnS28, "Candidatus R. tarasevichiae," a rickettsia closely related to R. helvetica, A. bovis, Ehrlichia muris, "Ehrlichia-like" "Schotti variant," and bacterium "Montezuma." Our findings indicated the distribution of rickettsiae and ehrlichiae in hard ticks in Russia.

The characterization of a novel Rickettsia species, first isolated from Ixodes ovatus ticks in Japan in 1993, is described for which the name Rickettsia asiatica sp. nov. is proposed. Using multilocus sequence comparison, five rickettsial isolates from I. ovatus collected at various locations in Japan were found to be identical but belonged to a novel species. Serotyping also demonstrated this rickettsia to be distinct from previously described Rickettsia species. The type strain of Rickettsia asiatica sp. nov. is IO-1(T) (=CSUR R2(T)). Following discussions held at the meeting of the International Committee on Systematics of Prokaryotes (ICSP) and its Judicial Commission (JC) in San Francisco, 2005, and in anticipation of the published minutes of the JC and ICSP, a committee consisting of the chairman of the ICSP, the chairman of the Judicial Commission of the ICSP and the Editor of the IJSEM has granted an exception in this case to the Rule governing the deposit of type material in two different collections in two different countries.

African tick-bite fever (ATBF) is a rickettsiosis caused by Rickettsia africae. We describe histologic features and immunodetection of R. africae in cutaneous inoculation eschars from 8 patients with ATBF, which was diagnosed by culture or association of positive PCR detection and positive serologic results. We used quantitative image analysis to compare the pattern of inflammation of these eschars with those from Mediterranean spotted fever. We evaluated the diagnostic value of immunohistochemical techniques by using a monoclonal antibody to R. africae. ATBF eschars were histologically characterized by inflammation of vessels composed mainly of significantly more polymorphonuclear leukocytes than are found in cases of Mediterranean spotted fever (p < 0.05). Small amounts R. africae antigens were demonstrated by immunohistochemical examination in 6 of 8 patients with ATBF. Neutrophils in ATBF are a notable component of the host reaction, perhaps because ATBF is a milder disease than the other rickettsioses. Immunohistochemical detection of rickettsial antigens may be useful in diagnosing ATBF.

Current data on rickettsiae and rickettsial diseases in China remain limited. Using partial ompA gene sequencing and multispacer typing, we identified 15 rickettsial isolates from China. All isolates were found to belong to Rickettsia sibirica subsp. sibirica. Four isolates from Dermacentor sinicus collected in Beijing, China, were fully identical to strain BJ-90, previously demonstrated to belong to R. sibirica subsp. sibirica despite antigenic and genotypic specificities. All 11 remaining isolates were similar to the R. sibirica subsp. sibirica type strain, 246. These were widely distributed in China in humans and different tick species. We emphasize the importance of surveying the distribution of R. sibirica in China.

Rickettsia sp. strain AT-1(T) was isolated from Amblyomma testudinarium ticks in Japan in 1993. Comparative analysis of sequences obtained from 16S rRNA, gltA, ompA, ompB and sca4 gene fragments demonstrated those from AT-1(T) to be markedly different from those of other members of the spotted fever group. Using mouse serotyping, it was also observed that Rickettsia sp. strain AT-1(T) was different from other Rickettsia species with validly published names. Such genotypic and phenotypic characteristics warrant its classification as a representative of a novel species, for which the name Rickettsia tamurae sp. nov. is proposed, with the type strain AT-1(T) (=CSUR R1(T)).

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair-long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic "melting pot" where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Thirty-one rickettsial isolates from ticks or patients in North Asian tick typhus (NATT) foci from the Ural region to the Russian Far East were obtained at the Omsk Research Institute of Natural Foci Infections between 1954 and 2001. Using citrate synthase (gltA) and outermenbrane protein a (ompA) gene sequencing, we identified these isolates as Rickettsia sibirica sensu stricto (25 isolates), R. sibirica strain BJ-90 (2 isolates), R. slovaca (1 isolate), and R. heilongjiangensis (3 isolates). We demonstrate that Ixodes persulcatus ticks should be considered potential vectors of NATT. We also demonstrate the presence of R. slovaca in Ural and R. heilongjiangensis in Siberia and Russian Far East, where they may cause human infections misdiagnosed as cases of NATT. Clinicians should be aware that several spotted fever rickettsioses with different prognoses coexist in Russia in areas where NATT was the only previously recognized rickettsiosis.

Background: Among the 17 genes encoding autotransporter proteins of the "surface cell antigen" (sca) family in the currently sequenced Rickettsia genomes, ompA, sca5 (ompB) and sca4 (gene D), have been extensively used for identification and phylogenetic purposes for Rickettsia species. However, none of these genes is present in all 20 currently validated Rickettsia species. Of the remaining 14 sca genes, sca1 is the only gene to be present in all nine sequenced Rickettsia genomes. To estimate whether the sca1 gene is present in all Rickettsia species and its usefulness as an identification and phylogenetic tool, we searched for sca1genes in the four published Rickettsia genomes and amplified and sequenced this gene in the remaining 16 validated Rickettsia species., Results: Sca1 is the only one of the 17 rickettsial sca genes present in all 20 Rickettsia species. R. prowazekii and R. canadensis exhibit a split sca1 gene whereas the remaining species have a complete gene. Within the sca1 gene, we identified a 488-bp variable sequence fragment that can be amplified using a pair of conserved primers. Sequences of this fragment are specific for each Rickettsia species. The phylogenetic organization of Rickettsia species inferred from the comparison of sca1 sequences strengthens the classification based on the housekeeping gene gltA and is similar to those obtained from the analyses of ompA, sca5 and sca4, thus suggesting similar evolutionary constraints. We also observed that Sca1 protein sequences have evolved under a dual selection pressure: with the exception of typhus group rickettsiae, the amino-terminal part of the protein that encompasses the predicted passenger domain, has evolved under positive selection in rickettsiae. This suggests that the Sca1 protein interacts with the host. In contrast, the C-terminal portion containing the autotransporter domain has evolved under purifying selection. In addition, sca1 is transcribed in R. conorii, and might therefore be functional in this species., Conclusion: The sca1 gene, encoding an autotransporter protein that evolves under dual evolution pressure, is the only sca-family gene to be conserved by all Rickettsia species. As such, it is a valuable identification target for these bacteria, especially because rickettsial isolates can be identified by amplification and sequencing of a discriminatory gene fragment using a single primer pair. It may also be used as a phylogenetic tool. However, its current functional status remains to be determined although it was found expressed in R. conorii.

The Rickettsia genus is a group of obligate intracellular parasitic alpha-proteobacteria that includes human pathogens responsible for the typhus disease and various types of spotted fevers. rOmpA and rOmpB are two members of the "surface cell antigen" (Sca) autotransporter (AT) protein family that may play key roles in the adhesion of the Rickettsia cells to the host tissue. These molecules are likely determinants for the pathogenicity of the Rickettsia and represent good candidates for vaccine development. We identified the 17 members of this family of outer-membrane proteins in nine fully sequenced Rickettsia genomes. The typical architecture of the Sca proteins is composed of an N-terminal signal peptide and a C-terminal AT domain that promote the export of the central passenger domain to the outside of the bacteria. A characteristic of this family is the frequent degradation of the genes, which results in different subsets of the sca genes being expressed among Rickettsia species. Here, we present a detailed analysis of their phylogenetic relationships and evolution. We provide strong evidence that rOmpA and rOmpB as well as three other members of the Sca protein family--Sca1, Sca2, and Sca4--have evolved under positive selection. The exclusive distribution of the predicted positively selected sites within the passenger domains of these proteins argues that these regions are involved in the interaction with the host and may be locked in "arms race" coevolutionary conflicts.

As rickettsioses may be severe diseases and Rickettsia prowazekii is a potential agent of bioterrorism, highly efficient diagnostic techniques are required to detect rickettsiae in patients. We developed a nested PCR assay using single-use primers targeting single-use gene fragments present in the genomes of both Rickettsia conorii and R. prowazekii. We used this "suicide" PCR with DNA from 103 skin biopsy specimens from patients who definitely had a rickettiosis, 109 skin biopsy specimens from patients who possibly had a rickettsiosis, and 50 skin biopsy specimens from patients with nonrickettsial diseases. The suicide PCR detected "R. conorii conorii" in 38 biopsy specimens, R. africae in 28 biopsy specimens, R. slovaca in 12 biopsy specimens, "R. sibirica mongolotimonae" in 5 biopsy specimens, R. aeschlimannii in 2 biopsy specimens, and "R. conorii caspia" and "R. sibirica sibirica" in 1 biopsy specimen each. The technique had a specificity of 100% and a sensitivity of 68%. It was 2.2 times more sensitive than culture (P < 10(-2)) and 1.5 times more sensitive than regular PCR (P < 10(-2)). The efficacy of the suicide PCR was reduced by antibiotic therapy prior to biopsy (P < 10(-2)) and was increased when it was performed with eschar biopsy specimens (P = 0.03). We propose the use of the suicide PCR as a sensitive, specific, and versatile technique for improving the diagnosis of rickettsioses, especially when it is used on eschar biopsy specimens taken prior to antibiotic therapy.

An acute tick-borne rickettsiosis caused by Rickettsia heilongjiangensis was diagnosed in 13 patients from the Russian Far East in 2002. We amplified and sequenced four portions of three rickettsial genes from the patients' skin biopsy results and blood samples and showed that the amplified rickettsial genes belong to R. heilongjiangensis, which was recently isolated from Dermacentor sylvarum ticks in nearby regions of China. This rickettsia, belonging to subgroup of R. japonica, was previously suggested to be pathogenic for humans on the basis of serologic findings. We tested serum samples with different rickettsial antigens from 11 patients and confirmed increasing titers of immunoglobulin (Ig) G and IgM to spotted fever group rickettsiae, including R. heilongjiangensis. Clinical and epidemiologic data on these patients show that this disease is similar to other tick-borne rickettsioses.

Using PCR, we screened 411 ticks from four genera collected in Russia and Kazakhstan for the presence of rickettsiae and ehrlichiae. In Russia, we detected "Rickettsia heilongjiangensis," Rickettsia sp. strain RpA4, and Ehrlichia muris. In Kazakhstan, we detected Rickettsia sp. strain RpA4 and a rickettsia closely related to Rickettsia aeschlimannii. These agents should be considered in a differential diagnosis of tick-borne infections in these areas.

We report that eight patients from France, Italy, and Thailand had serological evidence of Rickettsia helvetica infection. The infection presented as a mild disease in the warm season and was associated with fever, headache, and myalgia but not with a cutaneous rash. R. helvetica should be suspected in patients with unexplained fever, especially following a bite from an Ixodes sp. tick.

Recent seroepidemiological studies and examinations of Ixodes ricinus ticks in Europe have demonstrated the presence of an emerging tick-borne infection with Rickettsia helvetica. We conducted a serosurvey in 168 Danish patients seropositive for borreliosis reflecting their exposure to I. ricinus ticks. A total of 21 patients (12.5%) had positive antibody titres to R. helvetica including 4 cases of seroconversion. None of the samples were positive for antibodies to Ehrlichia. We conclude that in humans exposed to I. ricinus ticks in Denmark the risk of acquiring rickettsial infection is for the first time demonstrated. In the same region of Denmark we collected 570 I. ricinus ticks from various sources, and examinations by PCR for Rickettsia were performed. Positive reactions were obtained in 23 ticks (4%), and R. helvetica was identified in all 13 of those for which sequencing was performed.

We propose genetic guidelines for the classification of rickettsial isolates at the genus, group, and species levels by using sequences of the 16S rRNA (rrs) gene and four protein-coding genes, the gltA, ompA, and ompB genes and gene D. To be classified as a member of the genus Rickettsia, an isolate should exhibit degrees of rrs and gltA homology with any of the 20 Rickettsia species studied of >/=98.1 and >/=86.5%, respectively. A member of the typhus group should fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of either Rickettsia typhi or Rickettsia prowazekii of >/=99.4, >/=96.6, >/=92.4, and >/=91.6%, respectively. A member of the spotted fever group should either possess the ompA gene or fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of any member of this group of >/=98.8, >/=92.7, >/=85.8, and >/=82.2%, respectively. The existence of a distinct "ancestral" group should be questioned. To be classified as a new Rickettsia species, an isolate should not exhibit more than one of the following degrees of nucleotide similarity with the most homologous validated species: >/=99.8 and >/=99.9% for the rrs and gltA genes, respectively, and, when amplifiable, >/=98.8, >/=99.2, and >/=99.3% for the ompA and ompB genes and gene D, respectively. By use of our classification scheme, "Rickettsia heilongjiangii" belongs to a new species for which we officially propose the name Rickettsia heilongjiangensis sp. nov.

To estimate the incidence of, identify risk factors for, and describe the clinical presentation of travel-associated African tick bite fever (ATBF), a rapidly emerging disease in travel medicine, we prospectively studied a cohort of 940 travelers to rural sub-Equatorial Africa. Diagnosis was based on suicide polymerase chain reaction and the detection of specific antibodies to Rickettia africae in serum samples by multiple-antigen microimmunofluorescence assay, Western blotting, and cross-adsorption assays. Thirty-eight travelers, 4.0% of the cohort and 26.6% of those reporting flulike symptoms, had ATBF diagnosed. More than 80% of the patients had fever, headache, and/or myalgia, whereas specific clinical features such as inoculation eschars, lymphadenitis, cutaneous rash, and aphthous stomatitis were seen in < or = 50% of patients. Game hunting, travel to southern Africa, and travel during November through April were found to be independent risk factors. Our study suggests that ATBF is not uncommon in travelers to rural sub-Saharan Africa and that many cases have a nonspecific presentation.

We collected 209 Ixodes persulcatus ticks in various regions of Russia, including the southern Urals and western and eastern Siberia. Using PCR amplification and sequencing of the citrate synthase-encoding gene (gltA), we detected a new rickettsial genotype, which we named "Candidatus Rickettsia tarasevichiae." This bacterium was found in 9.27%, 10.0%, and 20.5% of the ticks collected in western Siberia, eastern Siberia, and the southern Urals, respectively. "Candidatus Rickettsia tarasevichiae" exhibited a 98% and 96% nucleotide sequence homology, with the 16S rDNA and gltA sequence, respectively, of R. canadensis, a rickettsia previously only found in Haemaphysalis leporispalustris ticks in North America. The phylogenetic analysis of "Candidatus Rickettsia tarasevichiae" and other Rickettsia species allowed the creation of a new cluster with high bootstrap values within the Rickettsia genus involving this rickettsia, R. canadensis, and three uncultured rickettsiae from plant insects.