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Phyloproteomics indicate common viral origin from ancient cells before archaea, bacteria, and eukaryota split and subsequent size and complexity reductions occurred. Further independent evidence for the cellular origin of viruses is reviewed for the virus order Megavirales, focusing on the family Poxviridae. Megavirales comprises giant viruses, double-stranded DNA viruses whose genomes exceed some bacterial ones and large enough to parasitize large-celled protists (amoeba). Giant viruses, virophages, and mitochondria have homologous DNA and RNA polymerases and share RNA splicing punctuation by stem-loop hairpins. Giant virus factories and amoeban mitochondria colocate, with viral proteins homologous to mitochondrial proteins specifically targeting mitochondrial inner membranes. Mitochondria share asexual budding with many bacterial endospores and membrane-enveloped viruses. These megavirus-mitochondrion similarities are not coincidental: systematic alignment analyses have detected candidate megaviral homologs of each amoeban mitogene (including ribosomal RNAs) distributed across megaviral genomes. These candidate megaviral homologs overall follow mitogene order, and megaviral-mitogenome synteny increases with viral genome reduction. This analysis is repeated within Poxviridae, whose organellar-like morphogenesis is reminiscent of mitochondria. More generally, the results confirm the patterns observed in Megavirales: synteny with amoeban mitogenomes increases with genome reduction. Parsimoniously interpreting this suggests Megavirales and mitochondria have a rickettsia-like common ancestor. Megavirales could be the missing links between bacterial-like cells and mitochondria, implying cellular-to-viral-to-subcellular macroevolution., (© 2019 New York Academy of Sciences.)

One century after the first description of rickettsiae as human pathogens, the rickettsiosis remained poorly understood diseases. These microorganisms are indeed characterized by a strictly intracellular location which has, for long, prohibited their detailed study. Within the last ten years, the completion of the genome sequences of several strains allowed gaining a better knowledge about the molecular mechanisms involved in rickettsia pathogenicity. Here, we summarized available data concerning the critical steps of rickettsia-host cell interactions that should contribute to tissue injury and diseases, that is, adhesion, phagosomal escape, motility, and intracellular survival of the bacteria.

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.

Our objective was to learn the prevalence of spotted fever group (SFG) Rickettsia detected in ticks in La Rioja, in the north of Spain. From 2001 to 2005, 496 ticks representing 7 tick species were analysed at the Hospital de La Rioja. Ticks were removed from humans with or without rickettsial syndrome (n = 59) or collected from mammals (n = 371) or from vegetation by dragging (n = 66). The presence of SFG Rickettsia in these ticks was investigated by semi-nested PCR (ompA gene) and sequencing. A phylogenetic tree using Clustal method (neighbor-joining) was constructed with these data. Only 3 of 170 Hyalomma marginatum ticks carried SFG Rickettsia. Sequencing analysis demonstrated the presence of Rickettsia aeschlimannii (1.8%). Furthermore, Rickettsia massiliae and BAR29 were found in 3 of 120 Rhipicephalus sanguineus specimens (2.5%). In contrast, 81 of 83 tested Dermacentor marginatus ticks were PCR-positive (97%). Rickettsia slovaca (40.6%) and Rickettsia sp. strains RpA4, DnS14, DnS28 and JL-02 (59.3%) were found within this tick species. No SFG Rickettsia was detected using ompA primers when Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus turanicus, Rhipicephalus eversti eversti, Hyalomma detritum scupense and Rhipicephalus sp. were analyzed. We detected 17.5% of ticks associated with different SFG Rickettsia: R. aeschlimannii, R. massiliae, BAR29, R. slovaca and Rickettsia sp. strains RpA4, DnS14, DnS28 and JL-02. Their presence has to be taken into account since most of them have been recognized as human pathogens.

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.

Rickettsia slovaca, the causative agent of TIBOLA, is transmitted by Dermacentor ticks. Dermacentor marginatus is the most widely species distributed in northeastern Spain, and the wild boar constitutes the main host. D. marginatus ticks were collected from hunter-killed wild boar and were tested by PCR/RFLP. Rickettsial DNA-positive ticks were sequenced using the ompA PCR primers. The prevalence of R. slovaca in D. marginatus ticks was 17.7%. Other spotted fever group rickettsiae were detected in ticks, but these were not definitely identified.

In southern Spain, Dermacentor marginatus ticks can be infected with several genospecies of spotted fever Group (SFG) Rickettsia. We developed a nested polymerase chain reaction assay by using a species-specific probe targeting the ompA gene to detect and differentiate between the two groups of rickettsiae previously described in D. marginatus. SFG rickettsia has been detected in 85.15% of ticks studied (26.7% of positives have been to R. slovaca, the causative agent of TIBOLA-DEBONEL, and 73.3% to SFG rickettsia closely related to strains RpA4-JL-02-DnS14-DnS28).

A total of 4701 Ixodes ricinus, collected during the summer of 2003, were analyzed for three pathogens. DNA was detected from the three pathogens. Co-detection of more than one pathogen was observed.

The extent to which rickettsiae are present in Western Australia (WA) is largely unknown. Recently there has been anecdotal evidence of a disease of unknown but possibly rickettsial origin occurring on Barrow Island, WA. Ticks were collected from people and screened using PCR. The rickettsial species was then cultured and its novelty and phylogenetic position examined. The infecting rickettsial species is divergent enough to be classified as a novel species. Sequence data suggest that the evolutionary route for Australian rickettsiae did not progress through a recent common ancestor. The pathogenic potential of the novel species is as yet unknown.

Brazilian spotted fever (BSF) is a highly lethal disease caused by Rickettsia rickettsii. In the present study, rickettsial infection was evaluated in 669 Amblyomma aureolatum adult ticks collected from naturally infested dogs in Taiaçupeba, a BSF-endemic area in the state of São Paulo. Ten (1.49%) ticks were infected with Rickettsia bellii, and 6 (0.89%) ticks were infected with R. rickettsii. Both Rickettsia species were isolated and established in Vero cell cultures. The Rickettsia isolates were characterized by molecular analyses, sequencing fragments of different rickettsial genes. Our results suggest that A. aureolatum is an important vector of R. rickettsii in Brazil.

During our more than 20 years of monitoring, we have used epidemiological, field, and experimental methods for characterization of natural foci of tick-borne rickettsioses in Russia. The main results were obtained through genetic methods (PCR sequence) at the Université de la Mediterranée (Marseille, France). We describe considerable heterogeneity of tick-borne alpha(1)-proteobacteria: 16 microorganisms the of the order Rickettsiales were detected in Russia and Kazakhstan. R. sibirica-caused North Asiatic tick-borne rickettsiosis is the main tick-borne rickettsiosis in Russia, with wide distribution in Siberia and the Russian Far East and high epidemic activity of natural foci of different landscape types. Our results show circulation of different pathogenic rickettsiae in the same endemic territories. In the Far East region, R. sibirica subsp. R. sibirica, R. sibirica subsp. BJ-90, and R. heilongjiangensis were detected; in the Altay and Krasnojarsk regions, R. sibirica subsp. R. sibirica and R. heilongjiangensis; and in the Kurgan district of West Siberia, R. sibirica subsp. R. sibirica and R. slovaca. The roles of more than 15 new genotypes of alpha(1)-proteobacteria in infectious disease in Russia and Kazakhstan are in need of further study.

The authors detected Rickettsia genus organisms using shell vial and polymerase chain reaction (PCR)/sequencing analysis in blood clots in patients suspected of having Brazilian spotted fever (BSF). DNA was detected using PCR with three sets of primers to access the gltA, ompA, and ompB genes. Sequence analysis was carried out using an automatic sequencer with Bioedit software. Seventy-five percent of the culture samples were positive and all samples amplified rickettsial gene fragments. To date, 46% of the samples have been sequenced.

Ixodes ricinus, the most commonly observed tick species in Poland, is known vector of microorganisms pathogenic for humans as TBE virus, Borrelia burgdorferi s.1., Anaplasma phagocytophilum and Babesia sp. in this country. Our study aimed to find out whether this tick can also transmit also rickettsiae of the spotted fever group (SFG). DNA extracts from 560 ticks (28 females, 34 males, and 488 nymphs) collected in different wooded areas in northern Poland were examined by PCR for the detection of Rickettsia sp., using a primer set RpCS.877p and RpCS.1258n designated to amplify a 381-bp fragment of gltA gene. A total of 2.9% ticks was found to be positive. The percentage of infected females and males was comparable (10.5% and 11.8%, respectively) and 6.6-7.6 times higher than in nymphs (1.6%). Sequences of four PCR-derived DNA fragments (acc. no. DQ672603) demonstrated 99% similarity with the sequence of Rickettsia helvetica deposited in GenBank. The results obtained suggest the possible role of I. ricinus as a source of a microorganism, which recently has been identified as an agent of human rickettsioses in Europe.

Rhipicephalus turanicus tick colony infected in the laboratory with Rickettsia massiliae showed that the rickettsia is transovarially and transdatially tramsmitted. The expression of rOmpB did not change with temperature or the stages of the tick life cycle. In contrast, rOmpA was less expressed during the larval stage.

A genetic locus named Rc-65, which is 5' adjacent to gene dksA and 3' adjacent to xerC gene, has previously been demonstrated to contain a VNTR with high discriminatory power in several rickettsial strains and thus, potentially useful for genetically similar strains identification. In this work, we present an automated molecular identification method based on capillary electrophoresis separation of VNTRs amplicons. The resulting electropherograms were in agreement with the sequence data obtained in a previous work. The presented genotyping method is fast and suitable for full automation, being a powerful tool for epidemiological surveillance in a large number of samples and enables the detection co-infected samples. The combination of other VNTR loci should improve the discriminatory capacity of this typing system, providing greater resolution and contributing to a more accurate VNTR-based assay. To our knowledge, this is the first automated assay for rickettsial strains identification.

Omithodoros moubata complex (Argasidae) ticks collected from human dwellings in central Tanzania were found to carry a novel rickettsial species that clustered among the spotted fever group. Although no evidence of human infection was evident, these ticks feed primarily on man, thus providing opportunity for zoonotic infection.

This study aims to provide information on the occurrence of spotted fever rickettsiae in Ixodes ricinus ticks in southern Germany. A total of 2,141 I. ricinus ticks was collected in Bavaria. Pools of 5-10 ticks were studied by a PCR targeting the rickettsial citrate synthase gene gltA. The average prevalence rate was 12% (257 of 2,141). Sequencing data exclusively identified Rickettsia helvetica DNA. Results and other data demonstrate the possible role of R. helvetica in I. ricinus as a source of human infections in southern Germany.

Between June 2002 and July 2003, 987 fleas, representing four species, and 1019 ticks, representing one argasid and eight ixodid species, were collected from Egyptian animals. These arthropods were tested for rickettsial agents using polymerase chain reaction. DNAs from Anaplasma and Ehrlichia spp. were detected in 13 ticks. Previously undescribed Bartonella spp. were detected in 21 fleas. Coxiella burnetii was detected in two fleas and 20 ticks. Rickettsia typhi was detected in 27 fleas from 10 cities. Spotted fever group rickettsiae were detected in both fleas and ticks and included Rickettsia aeschlimanii and an unnamed Rickettsia sp.

Ticks collected in Northern Algeria between May 2001 and November 2003 were tested by PCR for the presence of Rickettsia spp. DNA using primer amplifying gltA and OmpA genes. Three different spotted fever group rickettsias were amplified from these ticks: R. Conorii subsp. P. conorii strain Malish in Rhipicephalus sanguineus, R. aeschlimannii in Hyalomma marginatum, and R. massiliae in Rhipicephalus turanicus. Our results confirm the presence of R. conorii in ticks in Algeria and provide the first detection of R. aeschlimannii and R. massiliae in Algeria.

We present the first report of superinfection in a Dermacentor variabilis tick from nature. The single tick, collected in Ohio, was found infected with Rickettsia belli, R. nontanensis, and R. rickettsii.

Phylogenetic analysis of five rickettsial genes (17-kDa gene, gltA, ompB, ompA, and sca4) from two molecular isolates of Candidatus Rickettsia andeanae from two ticks (Amblyomma maculatum and Ixodes boliviensis) collected from two domestic horses living in two separate locations in northern Peru (Coletas and Naranjo) was conducted to more clearly characterize this recently reported novel spotted fever group (SFG) rickettsia. Following nested polymerase chain reaction (PCR) amplification of the 17-kDa gene, gltA, ompB, ompA, and sca4, amplicons were purified, sequenced, and compared to those downloaded from GenBank. Phylogenetic analyses of the Candidatus Rickettsia andeanae sequences generated from 17-kDa gene (483 bp), gltA (1185 bp), ompA (1598 bp), ompB (4839 bp), and sca4 (2634 bp) demonstrated that they aligned strongly with those of SFG rickettsiae. Moreover, the sequences of these five genes most closely aligned with the following rickettsiae: ompA: Rickettsia sp RpA4 (98.03%), R. sp DnS28 (97.90%), and R. rhipicephali and R. massiliae (97.11%); ompB: R. aeschlimannii (97.22%), R. rhipicephali (97.20%), and R. sp Bar 29 (97.10%); and sca4: R. massiliae (97.8%), R. rhipicephali, and R. slovaca (97.7%). These results from the additional phylogenetic analyses of Candidatus Rickettsia andeanae confirm its inclusion within, and distance and uniqueness from, other known SFG rickettsiae.

A truncated gene ompA was amplified from Rickettsia heilongjiangensis isolated in China and a 56-kDa truncated OmpA protein was expressed in E. coli cells transformed with the ompA-recombined expression plasmid. High levels of serum antibodies to R. heilongjiangensis and proliferation of the splenic cells were found in mice immunized with the truncated OmpA. After challenge with R. heilongjiangensis or R. rickettsii, fever and pathological damages of the guinea pigs immunized with the truncated OmpA were significantly slighter as compared with those of nonimmunized guinea pigs. These results suggest that the truncated OmpA of R. heilongjiangii is immunogenic for effectively inducing humoral and cell-mediated immune responses against homologous and heterologous species in the spotted fever group.

Signaling interactions between vascular endothelium and invading rickettsiae provide a unique way of coordinating appropriate physiologic responses, which are important determinants of the ensuing pathogenesis mechanisms such as host defense and inflammation. Two major subgroups of pathogenic Rickettsia species, namely the typhus group (TG) and the spotted fever group (SFG), exhibit marked differences in their intracytoplasmic behavior. Using in vitro infection of cultured human endothelial cells with R. rickettsii, the causative agent of Rocky Mountain SF, we previously identified activation of NF-kappaB and p38 MAP kinase as essential components of intracellular signaling events responsible for Rickettsia-induced transcriptional activation. Our data also suggest that p38 activity does not contribute to NF-kappaB response, implicating involvement of independent upstream signaling pathways. Since divergent cytopathologies suggest potentially different interactions with host cells, the aim of this study was to compare these responses after endothelial cell infection with R. conorii, the agent of Mediterranean SF, and a TG representative R. typhi, the agent of endemic typhus.

In this study we describe molecular mechanisms of resistance to several classes of antibiotics within drug targets by in silico genome comparisons for bacteria of the genus Rickettsia. Apart from the mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species previously reported by our team, we found that typhus group (TG) rickettsiae had a triple amino acid difference in the highly conserved region of the L22 ribosomal protein as compared to the spotted fever group rickettsiae (SFG), which could explain the natural resistance of SFG rickettsia to erythromycin. We found also that the genome of R. conorii contains an aminoglycoside 3'-phosphotransferase. Finally, either folA gene (encoding dihydrofolate reductase) and/or folP gene (encoding dihydropteroate synthase) was missing in the genome of rickettsial strains explaining the natural resistance to cotrimoxazole. Finally, multiple genes encoding for pump efflux were found especially in the genome of R. conorii that could be involved in resistance to antibiotics. Five specific ORFs related to antibiotic resistance have been identified in the genome of R. felis including a streptomycin resistance protein homologue, a class C beta-lactamase, a class D beta-lactamase, a penicillin acylase homologue, and an ABC-type multidrug transporter system. For the first time, using this approach, an experimental beta-lactamase activity has been shown for this bacterium. We believe that whole genome sequence analysis may help to predict several phenotypic characters, in particular resistance to antibiotics for obligate intracellular bacteria.

The complete genome sequences available for eight species of Rickettsia and information for other near relatives in the Rickettsiales including Orientia and species of Anaplasmataceae are a rich resource for comparative analyses of the evolution of these obligate intracellular bacteria. Differences in these organisms have permitted them to colonize varied intracellular compartments, arthropod vectors, and vertebrate reservoirs in both pathogenic and symbiotic relationships. We summarize some comparative aspects of the genomes of these organisms, paying particular attention to the recently completed sequence for R. canadensis McKiel strain and an estimated two-thirds of the genome sequence for a Thailand patient isolate of Orientia tsutsugamushi. The Rickettsia genomes exhibit a high degree of synteny punctuated by distinctive chromosome inversions and consistent phylogenetic relationships regardless of whether protein coding sequences or RNA genes, concatenated open reading frames or gene regions, or whole genomes are used to construct phylogenetic trees. The aggregate characteristics (number, length, composition, repeat identity) of tandem repeat sequences of Rickettsia, which often exhibit recent and rapid divergence between closely related strains and species of bacteria, are also very conserved in Rickettsia but differed significantly in Orientia. O. tsutsugamushi shared no significant synteny to species of Rickettsia or Anaplasmataceae, supporting its placement in a unique genus. Like Rickettsia felis, Orientia has many transposases and ankyrin and tetratricopeptide repeat domains. Orientia shares the important ATP/ADP translocase and proline-betaine transporter multigene families with Rickettsia, but has more gene families that may be involved in regulatory and transporter responses to environmental stimuli.

During a survey for possible rickettsial vectors in villages of the central part of the Thai-Myanmar border from September 2001 to February 2002, four species of fleas were collected from common peridomestic animals. All fleas were tested by PCR to detect DNA of bacteria of the genera Rickettsia (gltA and ompB genes) and Bartonella (ITS and ftsZ genes). Sequencing of PCR-amplified products was done using gltA fragments for Rickettsia and ftsZ fragments for BARTONELLA: Two genotypes related to Rickettsia felis were identified in three Ctenocephalides canis and one C. felis specimen. Further, the following Bartonella spp. were detected: Bartonella henselae in two C. felis specimens; Bartonella clarridgeiae in three C. felis specimens; and a new Bartonella genotype in one Nosopsylla fasciatus specimen. Rickettsia and Bartonella may be frequently detected in fleas infesting peridomestic animals from the western border of Thailand.

In 1993, a novel rickettsia was isolated from the blood of inhabitants of Flinders Island, Australia, with acute febrile illnesses. This rickettsia was found to be a new species of spotted fever group (SFG) rickettsia, eventually named Rickettsia honei. The suspected ectoparasite vector of this rickettsia has yet to be identified. The purpose of this study was to evaluate the presence of this rickettsial species in a suspected tick vector, Aponomma hydrosauri, by DNA sequencing and electron microscopy (EM). Ticks collected from an Australian blue-tongued lizard on Flinders Island and a copperhead snake in Tasmania were demonstrated to be infected with R. honei by PCR, DNA sequencing, and EM. Rickettsiae were found in ultrathin sections of salivary glands, malpighian tubules, and midgut epithelial cells. In a previous study with a R. honei-infected tick from Flinders Island, rickettsiae were found in the nuclei of midgut epithelial cells, and EM also revealed the presence of rickettsiae in the cytosol of oocytes and immature eggs, suggesting transovarial transmission. These results implicate A. hydrosauri as a possible host of R. honei on Flinders Island and Tasmania and also provide evidence favoring transovarial maintenance of R. honei.

The availability of the complete genome sequences of several organisms allows the comparative analysis of genomes, a branch of bioinformatics known as genomics. With this approach, much can be learned about the biology of organisms that are difficult to culture, even when few, if any, of their proteins have been isolated and studied directly. We have focused our interest on Rickettsia conorii, an obligate intracellular bacterium responsible for Mediterranean spotted fever, a disease endemic in southern Europe. While bioinformatic annotation of the complete genome of this bacteria has allowed identification of 1,374 genes, a large number of them remain functionally uncharacterized. The final goal of many experiments in molecular biology is to use biological systems to synthesize the protein encoded by the gene being studied. Because three-dimensional structures are more resilient to evolution and change than amino acid sequences, structure determination of some open reading frames should also exhibit structural similarity to previously described protein families. We have thus initiated a systematic expression and structure determination program for the proteins encoded by rickettsial genes of interest. We have cloned different genes of R. conorii by recombinational cloning (GATEWAY), Invitrogen) a method that uses in vitro site-specific recombination to accomplish a directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. The constructions in p-Dest17 yielded several clones able to express recombinant proteins with a C-terminal histidine tag. Expression of corresponding proteins was then performed using a cell-free protein expression system (Rapid Translation System, RTS, Roche Diagnostics). The recombinational cloning approach coupled to RTS provides an approach to rapid optimization of protein expression and is very useful to express rickettsial proteins. Moreover, this system is able to overcome some of the limitations encountered with rickettsial proteins highly toxic for E. coli or insect cells.

Ehrlichiosis and rickettsiosis are very common, widespread diseases. These diseases are present in Sardinia year round because its temperate weather permits the survival of many kinds of tick vectors. A thousand dogs were subjected to physical, hematological, biochemical examinations and serological tests. All 1,000 sera were analyzed by indirect immunofluorescence antibody test to detect antibodies against E. canis and R. rickettsii. A high seroprevalence (about 50%) was detected for both etiological agents, without differences in relation to sex, breed, or usage. A high seroprevalence, corresponding to 62.5% for ehrlichiosis and 64% for rickettsiosis, was observed in the age group of 13-60 months. The mortality was greatest in the males in the age group, which manifested the disease in the chronic phase.

A total of 360 ticks were removed from 353 asymptomatic subjects in Belluno Province, Italy and surrounding areas, from 1998 to 2001. Ticks were identified as Ixodes ricinus (357), Ixodes hexagonus (1), Rhipicephalus sanguineus (1), and Ixodes ventalloi (1). Tick DNA was investigated by PCR and subsequent sequencing of amplified products to identity associated bacterial agents. Primers targeting different genes of Rickettsia (gltA and OmpA), Borrelia (16S rDNA, rpoB), Francisella (16S rDNA), and all genera members of the Anaplasmataceae (16S rDNA), were used. DNA of bacterial agents was identified in 28 Ixodes ricinus specimens (7.8%). Rickettsia helvetica was detected in 7 ticks. Rickettsia sp. IRS4 and Borrelia afzelii was detected in 4 ticks each. B. garinii and B. valaisiana were identified in one tick each. Anaplasma phagocytophilum, the agent of human granulocytic ehrlichiosis, was identified in 1 specimen of I. ricinus. A new Ehrlichia sp. ("Candidatus Ehrlichia walkerii", sp. nov.) was identified in 10 I. ricinus specimens.

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.

During the months of October and November 2000 a total of 70 Dermacentor marginatus ticks were removed from different game pigs Sus scrofa in southern France and investigated by PCR using primers derived from the citrate synthase (gltA) and the outer membrane protein (OmpB) genes of rickettsiae. Based on sequence analysis of 1,150 bp fragment of gltA, Rickettsia slovaca was identified in 11 ticks (15.7%). These results confirm that Rickettsia slovaca, an emerging pathogen is highly prevalent in Dermacentor marginatus ticks in France. Moreover, a new Rickettsia genotype was detected in one specimen (1.4%). The pathogenic role of this new rickettsia has yet to be demonstrated.

Current tendencies and problems of molecular biology and its application in infectious diseases are discussed. Basic stages of the development of molecular epidemiology as a leading tool for epidemiological studies are reviewed. More than 30 years ago, molecular epidemiology was first used to study nosocomial infections. Special attention is given to the achievements of Russian scientists, especially in the discovery of Astrakhan spotted fever Rickettsia, noncultured forms of Vibrio cholerae, and the microorganism "Montezuma." Also, the development of PCR-based methods of identification and typing of Mycobacterium tuberculosis by Russian specialists and its significant role in strategies for diagnosing and treatment of tuberculosis are discussed. Now, the most important field of application of molecular biology methods is bacterial evolution, especially with regard to pathogenic microorganisms and emerging infections. Searching for the novel pathogenic agents, establishment of the infectious nature of diseases with unclear origin, and determining the precise mechanisms of pathogenicity are most intriguing issues. So, molecular biology should play a major role both in clinical and research fields.

Tick-borne spotted fever group rickettsiae are maintained in nature primarily through transstadial and transovarial transmission in their vector. In the tick, Dermacentor variabilis, the infection of, and persistence within, the ovaries are critical steps in the transmission cycle of rickettsiae from one generation to the next. Although ixodid ticks can experimentally maintain several species of rickettsiae, often only a single species is maintained transovarially. The molecular mechanisms underlying exclusion processes are unknown. In this study, an attempt to identify the molecules that may be involved in rickettsial adhesion to tick cells was carried out. Monoclonal antibodies to mammalian alpha and beta subunits of integrins were used to screen a lambda phage library, constructed from uninfected tick ovarian tissues. Positive plaques for both alpha and beta integrins were identified. For one integrin, beta(3), the plaque was isolated and the insert sequenced. The role of these molecules, identified by library screening, in rickettsial infection and survival are discussed.

Species of the genus Rickettsia are responsible for several human diseases, namely epidemic typhus, Rocky Mountain spotted fever, and tick-borne typhus transmitted by arthropod vectors. The rrl-rrf intergenic spacer region (rrl-rrf ITS) was sequenced for 12 Rickettsia strains, including R. typhi, 6 untested species, R. aeschlimannii, Bar29, R. helvetica, R. honei, R. massilae, and R. slovaca as well as 5 Portuguese isolates. Phylogenetic trees inferred from rrl-rrf spacer sequences using maximum-parsimony and distance methods provided largely congruent tree topologies, supported by significant bootstrap values, enabling the identification of five distinct rickettsiae clusters.

The cytoplasmic 120 kDa antigen genes of 9 isolates of Rickettsia conorii (RC), 12 isolates of R. africae (RA), and 3 isolates of Israeli tick typhus rickettsiae (ISTT) were compared for restriction fragment length polymorphisms (RFLP) present in portions of the open reading frame amplified by polymerase chain reaction (PCR). Initially, DNAs from 13 species or serotypes of spotted fever group rickettsiae were used to select restriction enzymes (RE) that detected RFLP in gene fragments amplified with primer pairs 483WF/1514R and 764F/3409R. Among the R. conorii complex isolates, Dpn II gave RFLP differentiating all three serotypes. Unique RE patterns were obtained for RC with Bsr I and Hinf I, for RA with Mwo I, Pst I and Ssp I, and for ISTT with Hpa II. While RFLP typing of the 120 kDa gene permitted rapid separation of R. conorii complex isolates into three groups corresponding to the RC, RA, and ISTT rOmp serotypes, additional intragroup genetic variation was also detected in all three serotypes.

The population structure of tick-borne rickettsiae show the following characteristics: (1) the amount of genetic differentiation between strains within subpopulations is very small. (2) The evolution of the subpopulations does not fit into models based on either host or geographic similarities, suggesting the need for more information on the frequency of vertical versus horizontal transmission of strains between hosts. (3) The species are highly clonal, with little evidence of genetic exchange between populations. (4) The dominant class of genetic change is single-nucleotide point mutation. No evidence for major rearrangements was observed. (5) Differentiation between species of the spotted fever group is equivalent to that seen between local strains of E. coli.