Widespread use of real-time PCR for rickettsial diagnosis

We report 2?years of experience with rickettsial molecular diagnosis using real-time PCR at the French National Reference Center. All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R.?conorii, five Rickettsia sibirica mongolitimonae, four R.?slovaca, two R.?australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.     

Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.     

Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever

Abstract Sera from patients suffering from Mediterranean spotted fever (i.e. an infection due to Rickettsia conorii ) were studied by immunoblot to investigate cross-reactivity. A prevalence of IgM antibodies to Proteus OX 19, Proteus 0X 2, to the Rickettsia typhus group, to Legionella pneumophila serovars 4 and 5, to L. bozemanii Wiga and to L. micdadei Tatlock was found. Western blot confirmed that the antibodies were directed against the lipopolysaccharide as demonstrated by proteinase K digestion of the antigens. Cross-adsorptions showed that there is a common cross-reacting epitope among L. bozemanii Wiga, R. typhi and Proteus OX 19 but cross-reacting antibodies to L. micdadei and OX 2 were distinct and independent. This IgM cross-reaction could lead to a misdiagnosis.     

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.     

Spotted fever rickettsioses in southern and eastern Europe

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.     

Transmission electron microscopy as a tool for exploring bacterial proteins: model of RickA in Rickettsia conorii

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever, belongs to the spotted fever group of Rickettsia. It is an obligate intracellular bacterium that grows within the cytoplasm of its eukaryotic host cells. It is motile in the cytoplasm of infected cells and RickA is reported as critical protein in this aspect. However, the subcellular localization of RickA remains uncertain. We describe a simple method allowing RickA protein to be localized by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). By using IFA we showed the global expression of surface protein RickA in R. conorii organisms. The TEM results showed that RickA is widely expressed over the entire bacterial surface of R. conorii.     

Molecular detection of various rickettsiae in mites (acari: trombiculidae) in southern Jeolla Province, Korea

This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission.     

Cloning and sequencing of the gene encoding the candidate HtrA of Rickettsia typhi

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466-amino-acid protein similar to the heat-shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N-terminus, a serine protease-like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.     

Detection of Rickettsia spp. and host and habitat associations of fleas (Siphonaptera) in eastern Taiwan

Rickettsia typhi and Rickettsia felis (Rickettsiales: Rickettsiaceae) are two rickettsiae principally transmitted by fleas, but the detection of either pathogen has rarely been attempted in Taiwan. Of 2048 small mammals trapped in eastern Taiwan, Apodemus agrarius Pallas (24.5%) and Mus caroli Bonhote (24.4%) (both: Rodentia: Muridae) were the most abundant, and M. caroli hosted the highest proportion of fleas (63.9% of 330 fleas). Two flea species were identified: Stivalius aporus Jordan and Rothschild (Siphonaptera: Stivaliidae), and Acropsylla episema Rothschild (Siphonaptera: Leptopsyllidae). Nested polymerase chain reaction targeting parts of the ompB and gltA genes showed six fleas to be positive for Rickettsia spp. (3.8% of 160 samples), which showed the greatest similarity to R. felis, Rickettsia japonica, Rickettsia conorii or Rickettsia sp. TwKM01. Rickettsia typhi was not detected in the fleas and Rickettsia co-infection did not occur. Both flea species were more abundant during months with lower temperatures and less rainfall, and flea abundance on M. caroli was not related to soil hardness, vegetative height, ground cover by litter or by understory layer, or the abundance of M. caroli. Our study reveals the potential circulation of R. felis and other rickettsiae in eastern Taiwan, necessitating further surveillance of rickettsial diseases in this region. This is especially important because many novel rickettsioses are emerging worldwide.     

Proteome analysis of Rickettsia conorii by two-dimensional gel electrophoresis coupled with mass spectrometry

The availability of genome sequence offers the opportunity to further expand our knowledge about proteins expressed by Rickettsia conorii, strictly intracellular bacterium responsible for Mediterranean spotted fever. Using two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF mass spectrometry, we established the first reference map of R. conorii proteome. This approach also allowed identification of GroEL as the major antigen recognized by rabbit serum and sera of infected patients. Altogether, this work opens the way to characterize the proteome of R. conorii, to compare protein profiles of different isolates or of bacteria maintained under different experimental conditions and to identify immunogenic proteins as potential vaccine targets.     

Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii

Rickettsia conorii, a strictly intracellular and category C priority bacterial pathogen (NIAID), invades different mammalian cells. Although some signaling events involved in bacterial entry have been documented, the bacterial and host proteins mediating entry were not known. We report the identification of the Ku70 subunit of DNA-dependent protein kinase (DNA-PK) as a receptor involved in R. conorii internalization. Ku70 is recruited to R. conorii entry sites, and inhibition of Ku70 expression impairs R. conorii internalization. Bacterial invasion is dependent on the presence of cholesterol-enriched microdomains containing Ku70. R. conorii infection stimulates the ubiquitination of Ku70. In addition, the ubiquitin ligase c-Cbl is recruited to R. conorii entry foci, and downregulation of endogenous c-Cbl blocks bacterial invasion and Ku70 ubiquitination. An affinity chromatography approach identified the rickettsial protein rOmpB as a ligand for Ku70. This is the first report of a receptor-ligand interaction involved in the internalization of any rickettsial species.     

Continous alert for rickettsiosis in Sicily: molecular characterization of Rickettsia sp. obtained from ticks and human beings (1986-2001)

Several strains of Rickettsia sp. were isolated from patients in western Sicily with MSF (Mediterranean Spotted Fever) as well as ticks. Strains isolated were examined by PCR and identified as belonging to R. conorii sp. Importantly a strain of Israeli Spotted Fever Rickettsiae, obtained from a tick, was also identified. Our data prove that strains other than the classical R. conorii also circulate in Sicily.     

Development of recombinant OmpA and OmpB proteins as diagnostic antigens for rickettsial disease

In this study the diagnostic potential of Rickettsia conorii recombinant antigens was analyzed. For this, site-specific PCR primers were used to clone the OmpA and OmpB genes of R. conorii into pMAL-c2X plasmids. Six fragments of OmpA and four of OmpB were expressed as fusion proteins with maltose-binding protein in Escherichia coli. OmpA_1350-1784, OmpB_801-1269, and OmpB_1227-1634 regions from truncated proteins were selected as diagnostic candidate antigens by ELISA using control sera. ELISA results of three antigens were compared to the results obtained by using a commercial ELISA kit which contained whole OmpA and OmpB antigens from R. conorii . For this analysis, 40 serum samples taken from febrile patients and uninfected controls were tested. Of the 20 R. conorii test results which were positive with the commercial kit, 18 were shown to be positive by ELISA using OmpA_1350-1784 (a sensitivity of 90%). The specificity of the ELISA was 100%; all of the 20 samples shown to be negative using the commercial kit were also negative in our assay. The sensitivities of the ELISA using the OmpB_801-1269 and OmpB_1227-1634 were 90% and 95%, respectively. The specificities of the OmpB_801-1269 and the OmpB_1227-1634 were 100% and 95%, respectively. These results suggest that specific regions of OmpA and OmpB effectively detect antibodies against R. conorii , and the truncated recombinant antigens could be used for development of diagnostic tools for rickettsial disease.     

Some lessons from Rickettsia genomics

Sequencing of the Rickettsia conorii genome and its comparison with its closest sequenced pathogenic relative, i.e., Rickettsia prowazekii, provided powerful insights into the evolution of these microbial pathogens. However, advances in our knowledge of rickettsial diseases are still hindered by the difficulty of working with strict intracellular bacteria and their hosts. Information gained from comparing the genomes of closely related organisms will shed new light on proteins susceptible to be targeted in specific diagnostic assays, by new antimicrobial drugs, and that could be employed in the generation of future rickettsial vaccines. In this review we present a detailed comparison of the metabolic pathways of these bacteria as well as the polymorphisms of their membrane proteins, transporters and putative virulence factors. Environmental adaptation of Rickettsia is also discussed.     

The rhizome of life: the sympatric Rickettsia felis paradigm demonstrates the random transfer of DNA sequences

The intracellular flea symbiont, Rickettsia felis, may meet other organisms intracellularly such as R. typhi. We used a single-gene phylogenetic approach of the 1375 R. felis genes to look for horizontal transfers that occurred as a result of the bacterial promiscuity with other organisms. Our results showed that besides genes that are linked to the Spotted Fever Group, 165 genes have a different history and are linked to other Rickettsia such as R. bellii (107 genes), R. typhi (15 genes), or to other bacteria such as Legionella sp. and Francisella sp. or to eukaryotes. Among these genes, we identified 73 individual genes and 34 spatial clusters containing 2-4 adjacent genes, a total of 79 genes, with evidence of en bloc transfer. We described 13 chimeric genes resulting from gene recombination with sympatric R. typhi. The transferred DNA sequences present different sizes and functions, suggesting that the horizontal transfer in R. felis is random and neutral within its specific host. Our study shows that the strict intracellular bacteria R. felis exhibits a mosaic genome. We therefore developed a new representation for the evolutionary history of R. felis showing its different putative ancestors in the form of a rhizome.     

Dog tick-borne diseases in Sicily

In Sicily many tick borne diseases are endemic, in particular way those that see like main carrier ticks that prefer, for their vital cycle, climatic conditions characterized by high temperatures and a warmth-humid atmosphere. The more important pathologies transmitted by ticks causing diseases in dogs are babesiosis and ehrlichiosis. Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia conorii, Coxiella burnetii and tick transmitted encephalitis virus assume particular relevance because they are agents of zoonosis. Our centre, C.R.A.Ba.R.T, have conducted many researches and carried out many tests for diagnostic aim in order to estimate the spread of the main tick borne diseases in Sicilians' dogs. A study lead on 342 dogs has evidenced seroprevalence for Babesia canis, Ehrlichia canis and Rickettsia respective of 5.17%, 21.70% and 53.43%. A study on zoonotic agent seroprevalences in dogs gave the following percentages: C. burnetii 31.50%, R. conorii 73.60% and A. phagocytophilum 32.80%. The data carried out from IZS Sicily diagnostic service on 5,634 tests done in 2004-2005, confirm the experimental results on the presence of B. canis, E. canis, R. conorii, A. phagocytophilum, C. burnetii, Rickettsia spp., Anaplasma spp. and Ehrlichia spp. in all the Sicilian areas.     

In vitro propagation of Candidatus Rickettsia andeanae isolated from Amblyomma maculatum

Candidatus Rickettsia andeanae was identified during an investigation of a febrile outbreak in northwestern Peru (2002). DNA sequencing from two ticks (Amblyomma maculatum, Ixodes boliviensis) collected during the investigation revealed a novel Rickettsia agent with similarity to the spotted fever group rickettsiae. Since then, Candidatus R.?andeanae has been detected in A.?maculatum ticks collected in the southeastern and southcentral United States, Argentina, and Peru. To date, Candidatus R.?andeanae has not been successfully cultivated in the laboratory. We present evidence for the continuous cultivation in three cell lines of Candidatus R.?andeanae isolated from an A.?maculatum tick (Portsmouth, Virginia).     

Emerging rickettsioses

Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed. Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed.